key: cord-275837-2avxd80i authors: ahirwar, ashok kumar; asia, priyanka; sakarde, apurva; kaim, kirti title: covid -19 outbreak – diabetes aspect and perspective date: 2020-05-19 journal: curr med res pract doi: 10.1016/j.cmrp.2020.05.005 sha: doc_id: 275837 cord_uid: 2avxd80i nan covid-19 pandemic is a new international public health concern, threatening the life of billions of people worldwide. india's health care system is also going through tough time due to covid-19 outbreak. most of the cases have flue like symptoms but elderly and co morbid individuals have higher risk of adverse outcome. common causes of death in covid-19 are pneumonia, acute respiratory distress syndrome (ards) and multi organ failure, out of which most common is pneumonia. [1] india has been known as the "world capital of diabetes" for long. covid-19 infection and diabetes coexistence might cause concern in medical fraternity. diabetes increases the risk of pneumonia in covid-19 infected individual and it is an important risk factor for adverse outcome. [2] a study shows that diabetes mellitus worsens covid-19 infections and is associated with increased pulmonary inflammation, macrophage infiltration. [3] the problem with known diabetic patients is not increased risk of covid 19 infections, but high chances of worst outcome in these patients if infected with covid-19 virus. the patients with diabetes have much higher rate of serious complications and death than the patients without diabetes because of compromised immunity. ace inhibitor and angiotensin ii receptor blockers (arbs) are the few of drugs used for the appropriate management of diabetic patients. covid-19 virus utilizes ace -2 receptor for binding to various tissue such as endothelium, kidney, lung etc. the patient already on ace inhibitor or arbs has upregulated expression of ace -2 receptors. all these may lead to increased severity of covid-19 infection in diabetic individuals. [4] moreover, genetic polymorphism of ace receptor has been shown to be linked with diabetes and it might also facilitate covid-19 infections. a single mutation in ace receptor significantly increases the ability of covid-19 virus to bind with human ace receptor. [4] on contrary to this, some researchers say that use of ace inhibitor or arbs prevents the binding of covid-19 virus to ace receptor and thus may prevent infection. moreover, these scientists say that ace inhibitor or arbs could be used as treatment modality. [5] all these are the lacunae in the existing knowledge of ace inhibitor or arbs use in diabetic individuals. still there are some golden rules for management of diabetic patients such as maintenance of hydration especially in this current summer season, regular blood sugar monitoring, body temperature monitoring, monitoring ketone body in urine if patient is on insulin therapy, follows physician advice strictly, regular physical exercise. apart from that diabetic individuals should be aware about warning sign especially in those individuals who have poor glycemic control. avoid hypoglycemia as patient should keep biscuit, sugar candy with them, so that whenever they have sign and symptoms of hypoglycemia, they prevent this grave complication by consuming it. diabetic individuals should be vaccinated against pneumonia and influenza. appropriate attentions should be given to diet especially in terms of protein, vitamins and minerals. a review of coronavirus disease-2019 (covid-19). the indian journal of pediatrics diabetes patients with covid-19 need better care comorbid diabetes results in immune dysregulation and enhanced disease severity following mers-cov infection are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? the lancet respiratory medicine enzyme 2 and its effect on the prognosis of covid-19 key: cord-353707-3n2nji8l authors: sunden-cullberg, jonas title: chronic use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers is high among intensive care unit patients with non–covid-19 sepsis but carries a moderately increased risk of death date: 2020-04-28 journal: hypertension doi: 10.1161/hypertensionaha.120.15178 sha: doc_id: 353707 cord_uid: 3n2nji8l nan i n a letter to hypertension, esler and esler 1 suggested angiotensin ii receptor blockers (arb) to be potential contributors to severe and fatal coronavirus disease 2019 (covid-19). others have similarly implicated ace (angiotensin-converting enzyme) inhibitors. 2, 3 the underlying hypothesis is that these drugs facilitate massive infection by upregulating the ace2 enzyme, which in its membrane-bound form is used by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) to enter pulmonary cells. hypertension, coronary artery disease, and diabetes mellitus are associated with severe course in covid-19. 4 because ace inhibitors/arbs are often used to treat these conditions, the increased risk may be partly due to adverse drug effects. data will soon be out on the chronic use of ace inhibitors/arbs in covid-19 patients. to put these into perspective, it is important to know how frequently the drugs are used in similar patients and to what degree use is associated with higher mortality. we, therefore, examined the chronic use of ace inhibitors/arbs and associated mortality in a historic cohort of septic patients admitted to the intensive care unit. data were collected between january 2008 and december 2015 on a nationwide swedish cohort of 2700 patients, aged 18 years and over, with community-acquired severe sepsis and septic shock admitted to the intensive care unit within 24 hours of arrival to any of 32 emergency departments throughout the country. further details on inclusion criteria, definitions, etc are described elsewhere 5 . the database includes information concerning dispensed drugs to individual patients from the swedish prescribed drug register during 5 years preceding admittance. dispensed drug during the past 365 days was considered an indicator of chronic use. we describe fraction of patients on ace inhibitors, arbs, and ace inhibitors or arbs in different strata, including sex, focus of infection, age category, and year of admittance. χ 2 and wilcoxon rank-sum tests were used to assess the distribution of age and 30-day mortality between ace inhibitors and arb users/nonusers in the various strata. results are summarized in the table. ace inhibitors/ arb use was common among patients admitted to the intensive care unit because of sepsis. the drugs were used to a slightly higher extent in men than in women, and use increased with age. there was a temporal trend towards increased use. this is confirmed using recent official statistics from the swedish prescribed drug register, which shows that the number of patients aged 20-85+ years using ace inhibitors/arb drugs nationwide increased linearly from 144 to 183 out of 1000 people between 2010 and 2019, https://sdb.socialstyrelsen.se/if_lak/resultat.aspx. chronic use among the general population in 2020 is around 10% higher than in 2015, which likely also applies to septic intensive care unit patients. use indicates higher age and comorbidity, and the fraction of patients that died was higher, but not markedly so, among users compared to nonusers. surprisingly, there was no difference in death rates between users/nonusers in the subgroup with respiratory infection. ace inhibitors/arb use and associated mortality among critically ill covid-19 patients can be compared with this data. a high incidence of chronic use should be expected, but a major disparity in death rates between users and nonusers may indicate important underlying differences in pathophysiology, including, possibly, adverse effects of ace inhibitors/arbs. june 2020 can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid-19 china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china sex-based differences in ed management of critically ill patients with sepsis: a nationwide cohort study key: cord-296683-fjn29oal authors: sarode, sachin c; sarode, gargi s; gondivkar, shailesh; gadbail, amol; gopalkrishnan, dharmarajan; patil, shankargouda title: oral submucous fibrosis and covid-19: perspective on comorbidity date: 2020-05-21 journal: oral oncol doi: 10.1016/j.oraloncology.2020.104811 sha: doc_id: 296683 cord_uid: fjn29oal nan funding source: none declared. novel corona virus infection (covid-19) is a highly infective disease with rapid global spread since january 2020 and was declared as pandemic by world health organization. 1 angiotensin converting enzyme 2 (ace 2), present on the cell surfaces of some important organs, has been identified as target receptor for covid-19 virus entry into the host cells. 2 spike protein present in the virus contains receptor-binding region, which attaches with ace 2. the host protease tmprss2 breaks down spike protein into subunits (s1 and s2) after binding with ace 2. 1 eventually, virus fuses with the cell membrane of the host cell and gains entry via endocytosis. 1 cells of lungs, heart, kidney, intestine and arteries predominantly harbor expression of ace 2 on their surfaces and thus are the potential sites for virus entry and infectivity. 3 in addition, ace 2 has been reported to be present at various locations in the oral cavity and hence, oral cavity is considered as a potential site for the covid 19 virus. 3 ace 2 is required for maintaining tissue homeostasis and is known for its anti-apoptotic, antiinflammatory, anti-oxidant as well as anti-fibrotic role. 4 these protective effects are caused due to degradation of local ang ii and its product ang1-7 via ace2-ang1-7-mas pathway. 5 disruption of this axis is the cause of various pathologies including fibrosis of the organs. thus, down regulation of ace 2 (decreased expression) has been reported to cause activation of ang ii, which acts as proinflammatory cytokines and facilitates fibrosis in many organs. 6 oral submucous fibrosis (osmf) is an insidious chronic disease characterized by increased fibrosis in the oral mucosa leading to reduced mouth opening. numerous singling pathways causing increased collagen fiber deposition and reduced collagen degradation govern the fibrosis. 7 with aforementioned discussion in mind, it is quite conceivable to speculate downregulation of ace 2 in the oral cavity of osmf patients. in this regard, ace insertion/deletion polymorphism has been reported in osmf tissues. 8 thus, the less availability of ace 2 in the oral cavity might have compromised the binding of covid 19 virus. however, this has to be confirmed with appropriate epidemiological studies. the other way round, if osmf patient is affected with covid-19, then it may cause further exhaustion of ace 2. the exhausted ace 2 will unable to degrade the ang ii via ace2-ang1-7-mas axis and lead to accentuation of fibrosis in osmf patients. (figure 1 ) the frontline doctors and healthcare workers for management of covid-19 patients belong to the medical fraternity. for them, monitoring of vital parameters is the primary task to effectively manage the severe complications of covid-19 infection. moreover, frontline doctors and healthcare workers are under tremendous stress, anxiety and depression because of fear of contacting infection and associated morbidity. 9 under these circumstances, reporting of minor clinical details such are presence of osmf is less imperative. however, if we add 'presence or absence of osmf' in the checklist of case history format of covid-19 patients, we would then be able to draw some interesting conclusions. since diagnosis of osmf is purely clinical in nature and no imaging or laboratory procedure is needed, it can be incorporated in the case history format without causing much burden to the frontline workers. moreover, correlation of mouth opening (degree of severity of osmf) with viral load will also give a nice outlook about this comorbidity. since, majority of the frontline specialists belong to the medical field such as emergency medicine, general medicine, anesthesia etc., a brief training would be needed for clinically diagnosing and recording inter-incisal distance in covid-19 positive osmf patients. the prevalence of osmf is increasing every year with a current reported rate of 1.0-3.03% in china, 0.62-6.42% in india, 0.15-14.4% in vietnam, and 0.086-17.6% in taiwan. 10 looking at the pandemic nature and magnitude of the spread of covid-19 infection, health care workers and doctors are bound to come across the osmf patients with the infection. in fact, such comorbidity might be present in the general population, which is not yet reported. we hereby strongly recommend that case history checklist for covid-19 patients should have the osmf related data entry. in conclusion, ace 2 exhaustion is the center stage of covid-19 and osmf pathogenesis. as ace 2 is the binding site for covid-19, it may compromise the infectivity of the virus. moreover, covid-19 infection in osmf patient might aggravate the disease process by activation of ang ii. nature of this complex interaction needs exploration in future studies so as to develop effective therapeutic strategies for this unique comorbidity. physiological and pathological regulation of ace2, the sars-cov-2 receptor sars and mers: recent insights into emerging coronaviruses high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa angiotensin ii revisited: new roles in inflammation, immunology and aging a novel angiotensin-converting enzymerelated carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 loss of angiotensin-converting enzyme-2 leads to the late development of angiotensin iidependent glomerulosclerosis oral submucous fibrosis: a case-control study in chennai, south india angiotensin-converting enzyme gene insertion/deletion polymorphism is associated with risk of oral precancerous lesion in betel quid chewers factors associated with mental health outcomes among health care workers exposed to coronavirus disease oral submucous fibrosis: review on aetiology and pathogenesis figure 1. flowchart depicting the possible interactions between covid-19 and oral submucous fibrosis. conflict of interest: none declared funding source: none declared. key: cord-304742-ytf2ilw4 authors: albini, adriana; noonan, douglas mcclain; pelosi, giuseppe; di guardo, giovanni; lombardo, michele title: the sars-cov-2 receptor, ace-2, is expressed on many different cell types: implications for ace-inhibitorand angiotensin ii receptor blocker-based antihypertensive therapies—reply date: 2020-07-14 journal: intern emerg med doi: 10.1007/s11739-020-02436-7 sha: doc_id: 304742 cord_uid: ytf2ilw4 nan we read with great interest the comment by dr. de cauwer [1] in response to our point of view [2] . he raises several interesting points. chromosome x harbors the gene coding for angiotensin converting enzyme 2 (ace-2) receptor, which allows sars-cov-2 entry into host cells. this could partially explain, in turn, the increased mortality reported in males in comparison with females: indeed, deaths attributed to sars-cov-2 infection were significantly higher among male individuals, both in china and in italy [2] . males carrying rare ace-2-coding variants (polymorphisms) will express those variants in all ace-2-expressing cells, whereas females will typically express those variants in a mosaic distribution, driven by early x-inactivation events. the aggregated prevalence of the aforementioned ace-2 variants is supposed to be 3.9 per 1000 males and 8.5 per 1000 females [3] . if this holds true, it might indirectly suggest a protective role of ace-2 overexpression in the clinical course of sars-cov-2 infection in female patients. the sars virus in infected cells down-regulates the ace-2, we mentioned this in our point of view [2] . ace-2 produces ang (1-7) heptapeptide, an anti-inflammatory molecule. zoonotic human beta-coronaviruses (sars-cov-1, mers and sars-cov-2) have multiple strategies for proteolytic activation of spike (s) protein and viral entry, including cell surface transmembrane protease/serine (tmprss) proteases, endosomal cathepsins, and furin [2, 4] . the transmembrane protease serine 2 tmprss2, an androgendependent enzyme, acts in reinforcing the ace-2 receptor activity in allowing cell entry to a number of viral pathogens as well as to sars-cov-2 as reported in our point of view [2] . moreover, the expression of tmprss2 on endothelial cells together with ace-2 could account for the severity of sars-cov-2 infection via microthrombus formation beyond pro-inflammatory cytokines release causing a multiorgan failure alongside a disseminated intravascular coagulation [2] . the androgen receptor (ar) gene and ace-2 both map to chromosome x and polymorphisms of these genes could result in different biological effects. the low androgen levels in females could be inferred in reducing the synergistic activity of tmprss2 with ace-2 receptor. as a consequence, a minor load of sars-cov-2 virions could colonize the lung, heart and vascular system, as well as all the other ace-2-expressing cells and tissues throughout the human body. this could pave the way to a new therapeutic target potentially counteracting sars-cov-2 infection. it would be interesting to test the effect of currently available protease inhibitors, eventually associated with renin-angiotensin-aldosterone system (raas)-inhibitors, in an attempt to reduce, if not even inhibit viral entry into susceptible host cells [2] . this will be mitigating sars-cov-2-induced cardiovascular, respiratory, enteric, renal, hepatic and cerebral damages by means of the protective role exerted by the increased amounts of ang (1-7) [2] . the gene coding for tmprss2 is located on chromosome 21, as mentioned in the comment of dr de cauwer [1] . individuals with trisomy 21 already show, by themselves, clear signs of inflammation, along with an increased frequency of autoimmune disease conditions [5] . people with trisomy 21 syndrome are additionally known to experience many different signs of chronic immune dysregulation and a propensity to develop clinical pictures consistent with a "perfect immunologic storm", most likely resulting in a greater severity also of sars-cov-2 infection [5] . specifically, this would be driven by a sudden and rapid increase of cytokine and chemokine release, resulting in a higher susceptibility to develop clinically relevant respiratory infections, both bacterial and viral [1, 5] . the negative impact of the "cytokine storm" in sars-cov-infected subjects is driven by a cascade of pathophysiological events involving acute respiratory distress, myocardial injury and vascular damage with thromboembolism and dic. furthermore, on chromosome 21, there are four out of the six known interferon gene receptors, among which the two type i ifn receptors ifnar1 and ifnar2, along with the type ii ifn receptor ifngr2 [5] . as far as concerns the greater clinical disease severity of covid-19 in trisomy 21-affected patients, de cauwer et al. have reported 4 female subjects with down syndrome, aged between 48 and 62 years, whose clinical course of sars-cov-2 infection was particularly severe, with an acute respiratory distress syndrome warranting hospital care in three and a fatal outcome in one patient [1] . yet in trisomy 21 individuals a tmprss2 protease overexpression has been documented, as mentioned in the comment of dr de cauwer [1] , with this leading to an increased viral infection's rate of susceptible host's cells and tissues. this could be of concern also for sars-cov-2 infection, thus justifying a new therapeutic approach based upon protease inhibitors, like camostat mesylate, nafamostat mesylate or bromhexine chlorhydrate (a mucolytic agent marketed in 1963) eventually combined to raas antagonist drugs, or to anti-inflammatory drugs. however, also in the case of trisomy 21 syndrome [5] , we need adequately powered studies to compare the clinical course of sars-cov-2 infection in age and gender-matched patients in comparison to the general population. in conclusion, the interesting comment by dr de cauwer points out the complex interactions between ace-2 and tmprss2 with reference to the clinical course of covid-19 as well as to sars-cov-2 infection's pathogenic evolution and severity degree in given population segments of infected individuals, like male individuals and down syndromeaffected patients [1] . in this regard, the interplay between raas-inhibiting drugs and the various disease phenotypes of sars-cov-2 infection underscores the need of clarifying the relevance of such relationships from a clinic-pathological standpoint. we think that the hitherto available studies are, at the moment, insufficient to answer these relevant questions, which is substantially due to their "retrospective approach". prospective, randomized, case-control studies will be able to define the therapeutic relevance and the clinical influence of the different classes of raas-antagonist drugs (ace-is, arbs) on covid-19-affected patients, both in the general population as well as in subgroups stratified by age, gender and relevant cardiovascular conditions, such as diabetes, hypertension, ischemic heart disease, atrial fibrillation and finally, in patients affected by down syndrome. conflict of interest the authors declare that they have no conflict of interest. this article does not contain any studies with human participants or animals performed by any of the authors. informed consent informed consent was not required for this type of publication. the sars-cov-2 receptor, ace-2, is expressed on many different cell types: implications for aceinhibitor-and angiotensin ii receptor blocker-based cardiovascular therapies: comment the sars-cov-2 receptor, ace-2, is expressed on many different cell types: implications for ace-inhibitor-and angiotensin ii receptor blocker-based cardiovascular therapies ace 2 coding variants: a potential x-linked risk factor for covid-19 disease host cell proteases: critical determinants of coronavirus tropism and pathogenesis down syndrome and covid-19: a perfect storm? key: cord-326820-11sl17ap authors: bousquet, jean; anto, josep m.; iaccarino, guido; czarlewski, wienczyslawa; haahtela, tari; anto, aram; akdis, cezmi a.; blain, hubert; canonica, g. walter; cardona, victoria; cruz, alvaro a.; illario, maddalena; ivancevich, juan carlos; jutel, marek; klimek, ludger; kuna, piotr; laune, daniel; larenas-linnemann, désirée; mullol, joaquim; papadopoulos, nikos g.; pfaar, oliver; samolinski, boleslaw; valiulis, arunas; yorgancioglu, arzu; zuberbier, torsten title: is diet partly responsible for differences in covid-19 death rates between and within countries? date: 2020-05-27 journal: clin transl allergy doi: 10.1186/s13601-020-00323-0 sha: doc_id: 326820 cord_uid: 11sl17ap reported covid-19 deaths in germany are relatively low as compared to many european countries. among the several explanations proposed, an early and large testing of the population was put forward. most current debates on covid-19 focus on the differences among countries, but little attention has been given to regional differences and diet. the low-death rate european countries (e.g. austria, baltic states, czech republic, finland, norway, poland, slovakia) have used different quarantine and/or confinement times and methods and none have performed as many early tests as germany. among other factors that may be significant are the dietary habits. it seems that some foods largely used in these countries may reduce angiotensin-converting enzyme activity or are anti-oxidants. among the many possible areas of research, it might be important to understand diet and angiotensin-converting enzyme-2 (ace2) levels in populations with different covid-19 death rates since dietary interventions may be of great benefit. a novel strain of human coronaviruses, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), named by the international committee on taxonomy of viruses (ictv) [1] , has emerged and caused an infectious disease referred to as "coronavirus disease 2019" (covid-19) by the world health organization (who) [2] . covid-19 has aggressively spread across the globe and over 160,000 deaths have been reported. however, there appears to be high-and low-death rate countries. after the outbreak in china, covid-19 has also affected europe after becoming a pandemic. interestingly, there is large variability across european countries in both incidence and mortality, and most current debates on covid-19 focus on the differences among countries. german fatalities are strikingly low as compared to many european countries. among the several explanations proposed, an early and large testing of the population was put forward [3] . however, little attention has been given to regional differences and diet [4] . according to the johns hopkins coronavirus resource center (https ://coron aviru s.jhu.edu), one of the most important ways of measuring the burden of covid-19 is mortality. however, death rates are assessed differently between countries and there are many biases that clinical and translational allergy are almost impossible to assess. differences in the mortality rates depend on the characteristics of the health care system, the reporting method, whether or not deaths outside the hospital have been counted and other factors, many of which remain unknown. countries throughout the world have reported very different case fatality ratios-the number of deaths divided by the number of confirmed cases-but these numbers cannot be compared at all due to biases. on the other hand, for many countries, the methodology reporting death rates in the different regions is standardized across the country. we used the johns hopkins coronavirus resource center to assess death rates at the national level (https ://coron aviru s.jhu.edu). the current death rate per million people in europe shows different trends. germany has a low death rate, but austria, the czech republic, poland, slovakia, the baltic states and finland have similar or lower rates. on the other hand, belgium, france, italy, spain and the uk have higher rates (fig. 1 ). large differences exist when assessing death rates within a country. in germany, bavaria started the earliest tests but was and still is the most affected region (fig. 2) . death rates per million range from 8 in mecklenburg-vorpommern to 87 in bavaria. in switzerland, the french and italian speaking cantons have a far higher death rate than the german-speaking ones ( fig. 3 ) (office fédéral de la santé publique, switzerland, https ://www.bag.admin .ch/bag/fr/home.html). in high-rate countries such as spain, large variations also exist within the country, but the numbers range from 115 in murcia to over 1000 in madrid. most diseases exhibit large geographical variations which frequently remain unexplained despite abundant research [5] . covid-19 will not be an exception. though the more relevant factors are likely to be seasonal variations, immunity, cross-immunity, intensity, timing of measures [6] , type, onset, duration and measures of protection, other factors like environment or nutrition should not be overlooked. obesity, a risk factor of mortality in covid-19, suggests the importance of nutrition [7] . the "low-rate" european countries have used different quarantine and/or confinement times and methods and none have performed as many early tests as germany. thus, although the german testing approach is very important [3] , other factors may also be significant. although there are large differences between countries in death rates, the age-dependent severity of covid-19 is similar between asian, european and american countries. the rate of deaths is increased in the older population. globally, there are risk factors for death including obesity and type 2 diabetes. a strong relationship between hyperglycemia, impaired insulin pathway, and cardiovascular disease in type 2 diabetes is linked to oxidative stress and inflammation [8] . lipid metabolism has an important role to play in obesity, diabetes and its multi-morbidities, and the ageing process [9] . dietary fatty acids have a significant role in immune responses [10] . many foods have an antioxidant activity [11] [12] [13] . resveratrol, present in many foods [14] , is an inhibitor of mers-coronavirus infection [15] . the angiotensin-converting enzyme (ace2) has multiple physiological roles: a negative regulator of the reninangiotensin system, facilitator of amino acid transport, and the sars-cov and sars-cov-2 receptor [16] . ace converts angiotensin i to angiotensin ii but ace2 catalyses the conversion of angiotensin ii to angiotensin and is also the main entry point for coronavirus 2 into cells. differences between countries in ace have been associated with genetic patterns. the ace d allele increased risk of vasculitis [17] or hypertension [18] . the ace i/d polymorphism is involved in the onset of type 2 diabetes [19] and might be associated with susceptibility to peripheral vascular diseases in the asian population [20] . however, dietary patterns have a strong effect on ace levels. a high-saturated fat diet increases ace [21] . many foods have an ace-inhibitory activity [22] [23] [24] . antioxidant activities and ace inhibition have been largely found in many foods [25] . moreover, ace levels in blood are highly and rapidly sensitive to food intake [26] . identifying whether countries with high or low ace activity have different death rates would be of great interest in understanding the clinical importance of interventions. however, the available evidence, in particular from human studies, does not seem to support the hypothesis that inhibitors of ace or renin-angiotensin-aldosterone (acei/arb) drugs increase the ace2 expression and the risk of covid-19 [27] . this might suggest that changes in ace expression (inhibition/stimulation) might not be as relevant as previously thought and other diet-related changes might be more (or equally) important. germany, austria, croatia, the czech republic, poland, slovakia, the baltic states and german-speaking swiss cantons exhibit lower covid-19 mortality rates than france, italy, spain, and the french and italian speaking swiss cantons. among many factors, diet differs considerably between these low-or high-mortality countries. it appears that death rates in germany are higher in the two southern regions as well as in saarland than elsewhere. baden-wurttemberg and saarland are in close contact with alsace (france), and the higher infection rate may be due to the high cross-border traffic of the french. however, this was not the case for rhineland-palatinate (lower death rate), possibly because the east region of france was contaminated later. in addition, saarland is a special case as half of the deaths, unlike in the other german states, occurred in only a few longterm care facilities where a high number of people were infected in a short time and all deaths during the episode were attributed to corona without autopsies being made. this potential french-based contamination does not apply for bavaria (earliest german region to be contaminated and highest death rate). diet differs within germany, the southern states traditionally having a higher fat-rich diet. diet is not normally distributed within country/region, which can be an additional argument in favour of the uneven distribution of mortality. nutrition may therefore play a role in the immune defense against covid-19 and may explain some of the differences seen in covid-19 across europe. it will be needed to test dietary differences between low and highrate countries. foods with potent antioxidant or anti ace activity-like uncooked or fermented cabbage [28] [29] [30] are largely consumed in low-death rate european countries, korea and taiwan, and might be considered in the low prevalence of deaths. although it is difficult to compare health systems and death reporting across european countries, bulgaria, greece and romania have very low death rates. this might also be associated with diet since cabbage (romania) and fermented milk (bulgaria and greece) are common foods. the latter food is a known ace natural inhibitor [31] . turkey, another apparently low-death rate country, also consumes a lot of cabbage and fermented milk products. another example may be the food supply chain. the increasing availability of foods from big retail is a revolutionary event that has impacted crops (favouring those that have the best ratio of effectiveness over costs of production) and health at a population-size level. in particular, such a change in food availability has altered alimentary habits-promoting sugar-enriched, vitamindepauperated foods-and has become one of the causes of the obesity epidemic, especially among adolescents. these foods come from centralized farms in selected areas of the world that are distributed around the planet, elongating the supply chain of food. the impact of long supply chain of food on health is measurable by an increase in metabolic syndrome and insulin resistance [32] . therefore, rural areas that are more prone to short supply food may have been able to better tolerate the covid-19 pandemia, with a lower death toll. these considerations may be partly involved in lower death rates in southern italy compared to the northern part. understanding the within and between country differences in covid-19 will be of paramount importance in understanding covid-19 risk and protective factors, and will eventually help to control the epidemics. we acknowledge that many factors may play a role in the extension and severity of covid-19, such as trained immunity of 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influence of variation in the ace gene on the prospective risk of type 2 diabetes in middle-aged men modified by obesity? ethnic differences in the association between angiotensin-converting enzyme gene insertion/deletion polymorphism and peripheral vascular disease: a meta-analysis high-saturated-fat diet increases circulating angiotensin-converting enzyme, which is enhanced by the rs4343 polymorphism defining persons at risk of nutrient-dependent increases of blood pressure food-originating ace inhibitors, including antihypertensive peptides, as preventive food components in blood pressure reduction. comprehens rev food sc food safety food-derived bioactive peptides and their role in ameliorating hypertension and associated cardiovascular diseases molecular interactions, bioavailability, and cellular mechanisms of angiotensin-converting enzyme inhibitory peptides association of angiotensin-converting enzyme insertion/deletion polymorphism with susceptibility to systemic lupus erythematosus: a meta-analysis angiotensin converting enzyme (ace): a marker for personalized feedback on dieting renin-angiotensin-aldosterone system inhibitors and risk of covid-19 in vitro and in vivo studies on the angiotensin-converting enzyme inhibitory activity peptides isolated from broccoli protein hydrolysate antioxidant components of brassica vegetables including turnip and the influence of processing and storage on their anti-oxidative properties kimchi and other widely consumed traditional fermented foods of korea: a review anti-hypertensive peptides released from milk proteins by probiotics we are what we eat: impact of food from short supply chain on metabolic syndrome publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-301546-yck1t3pp authors: kozaki, toshinori; kimmelblatt, brian a.; hamm, ronda l.; scott, jeffrey g. title: comparison of two acetylcholinesterase gene cdnas of the lesser mealworm, alphitobius diaperinus, in insecticide susceptible and resistant strains date: 2007-12-28 journal: arch insect biochem physiol doi: 10.1002/arch.20229 sha: doc_id: 301546 cord_uid: yck1t3pp two cdnas encoding different acetylcholinesterase (ache) genes (adace1 and adace2) were sequenced and analyzed from the lesser mealworm, alphitobius diaperinus. both adace1 and adace2 were highly similar (95 and 93% amino acid identity, respectively) with the ace genes of tribolium castaneum. both adace1 and adace2 have the conserved residues characteristic of ache (catalytic triad, intra‐disulfide bonds, and so on). partial cdna sequences of the alphitobius ace genes were compared between two tetrachlorvinphos resistant (kennebec and waycross) and one susceptible strain of beetles. several single nucleotide polymorphisms (snps) were detected, but only one non‐synonymous mutation was found (a271s in adace2). no snps were exclusively found in the resistant strains, the a271s mutation does not correspond to any mutations previously reported to alter sensitivity of ache to organophosphates or carbamates, and the a271s was found only as a heterozygote in one individual from one of the resistant a. diaperinus strains. this suggests that tetrachlorvinphos resistance in the kennebec and waycross strains of a. diaperinus is not due to mutations in either ache gene. the sequences of adace1 and adace2 provide new information about the evolution of these important genes in insects. arch insect biochem physiol. © 2007 wiley‐liss, inc. the lesser mealworm, alphitobius diaperinus, is a manure-breeding beetle that is the primary structural pest of the poultry industry in the united states (axtell, 1999; hinton and moon, 2003) . the lesser mealworm is also a reservoir of salmonella typhimurium, escherichia coli, tapeworms, avian leucosis virus, turkey coronavirus, turkey enterovirus (avincini and ueta, 1990; axtell and arends, 1990; despins et al., 1994; goodwin and waltman, 1996; mcallister et al., 1996; watson et al., 2000) , and may serve as a source of campylobacter contamination of poultry (bates et al., 2004) . high beetle populations consume significant amounts of bird feed (savage, 1992) . under dry conditions in the broiler house, beetles bite the skin of birds resting at night. to prevent these bites, birds will rest for short periods and then move (despins et al., 1987; vaughan and turner, 1984) . this can affect the weight gain of chicks. organophosphate and carbamate insecticides have served as effective tools for control of the lesser mealworm, and tetrachlorvinphos continues to be used for this purpose in the united states. however, a recent study indicated that there were some populations of lesser mealworm in which a substantial portion of the population was highly resistant to tetrachlorvinphos (hamm et al., 2006) . the mechanism responsible for this resistance has not been determined. organophosphate and carbamate insecticides exert their toxic effects via inhibition of acetylcholinesterase (ache). recent studies have discovered archives of insect biochemistry and physiology march 2008 that some insect species have a single ace gene (drosophila melanogaster and musca domestica), while many other species have two ace genes (culex pipiens, bombyx mori, myzus persicae, among others). beetles (coleoptera) are the most evolutionarily successful metazoans, contributing 25% of all known animal species, far more than any other taxonomic order. despite the diversity and economic importance of coleoptera, ace genes have been reported from only two beetles: leptinotarsa decemlineata (say) (zhu et al., 1996) and tribolium castaneum (http://www.hgsc.bcm.tmc/edu/projects/ tribolium/). evidence has accumulated that indicates a limited number of mutations in ace are asfig. 1 . the nucleotide and deduced amino acid sequences of adace1 (drosophila ace orthologous) cdna (accession no. eu086056). the residues that make intra-disulfide bonds are marked with *, oxianion hole with §, catalytic triad with +, acyl binding site with ¶, and anionic subsite with ‡. the locations of the primers used for genotyping are indicated as **. sociated with resistance (i.e., mutations that code for an organophosphate and/or carbamate insensitive ache) in insects (fournier, 2005; kono and tomita, 2006; oh et al., 2006) . in this study we examined if there was one or two ace genes in a. diaperinus, and if mutations in ace could be correlated with tetrachlorvinphos resistance. three strains of lesser mealworm were used. the denmark-s (susceptible) strain was obtained from saturnia (bjerringbrovej 48 2610 rødovre, denmark). two strains (from kennebec, co., me, and waycross, ga) that contain high proportions of tetrachlorvinphos resistant individuals (hamm et al., 2006) were also used. a. diaperinus colonies were maintained at 28°c with 60-70% rh, and provided a diet of cracked corn:wheat bran (95:5) ad libitum. adult beetles from the kennebec and waycross strains were exposed to tetrachlorvinphos using a residual contact bioassay method as described previously (hamm et al., 2006) . beetles that survived exposure to a concentration of tetrachlorvinphos that was 350-fold greater than the susceptible strain lc 99 for 48 hr (i.e. resistant individuals) were used in genotyping. five denmark-s adult beetles (84 mg) were used to isolate mrna using a quickprep micro mrna purification kit (ge healthcare, waukesha, wi,), and cdna was synthesized with 500 ng of mrna using superscript iii reverse transcriptase (invitrogen, carlsbad, ca). a fragment that encoded the ace cdna, orthologous drosophila ace (fournier et al., 1989) , was amplified by s11ace (taygar-tayttycciggitt), s1iace (garatgtggaaycci-aayac), and as17ace (ccicciccrtaiaycca). a fragment that encoded the ace-paralogous gene was amplified using dl3 (gciaciatgtgga aycciaa) and dr98 (ggytticcigtyttigcraa) with the following thermal cycler program 95°c for 3 min, 35 cycles (95°c for 30 sec, 40°c for 30 sec, 72°c for 1.5 min) and a final extension at 72°c for 7 min. we obtained a fragment of 151 bp for the drosophila ace orthologous gene and a fragment of 825 bp for the paralogous gene. gene specific primers for 5′-and 3′-race were designed based on these sequences. race was performed using the bd smart race cdna amplification kit (bd biosciences, mountain view, ca). to compare the ace alleles in the susceptible and tetrachlorvinphos-resistant beetles, the ace cdnas were sequenced from individual beetles. the mrna was prepared using a polyatract system 1000 (promega, madison, wi), and was concentrated with a microcon ym-100 (millipore, billerica, ma). one fourth of the mrna from a beetle was used in the rt-pcr (50°c for 30 sec, 94°c for 2 min, 40 cycles (95°c for 30 sec, 50°c 30 sec, and 72°c for 1 min) using the superscript iii one-step rt-pcr system and platinum taq (invitrogen). gene-specific primers, s54adace (aagctgcccaattcttgcta), s84adace (tct-acctcaacatctgggtgcctcagc), and as53adace (aagctagggccatccttttc) were used for the amplification of the drosophila ace-orthologous gene. primers s44adace (gctgaacaccaccac-catgc), s43adace (gacacggtgttcggggactt), and as51adace (gcgaactcgttgacgttaca) were used to amplify the drosophila ace paralogous gene. dna sequencing was performed with s84adace and as53adace for the drosophila ace orthologous gene and with s43adace and as51adace for the paralogous gene at the cornell biotechnology resource center. we obtained the nearly complete orfs for adace1 and adace2 (figs. 1 and 2) . both genes show a high similarity (95 and 93% amino acid identity, respectively) with the predicted ace genes of t. castaneum (xp_970774.1, xp_973462.1) . kyte-doolittle hydropathy plots indicated the c-terminal of both adace1 and adace2 were hydrophobic (data not shown), and thus potentially exchanged for glycolipids. the cdna sequence of adace1 (the drosophila ace orthologous gene, eu086056) was 2,123 bp; archives of insect biochemistry and physiology march 2008 encoding 636 amino acid resides of an immature ache (fig. 1) . the deduced amino acid sequence had the characteristic features of ache, including the residues for the intra-molecular disulphide bonds (c110(67)-c137(94), c312(254)-c329(265), c465(402)-c582(521)), catalytic triad (s260(200), e389(327), h503(400)), protein dimerisation (c602), anionic subsite (w127(86)), oxianion hole (g172(118), g173(119), a261(201), and acyl binding site (w293(233), f352(290), f393(329)) (number in parentheses indicates the corresponding amino acid in torpedo ache). we could not unambiguously identify the translation start site because no stop codon was found in frame in the 5′ upstream region. if this transcript is similar in size to the ace gene in the colorado potato beetle, l. decemlineata (zhu and clark, 1995) , it will be more than 13 kb in size. however, adace1 has an initiation codon that is identical to the one tentatively identified in l. decemlineata. given that adace1 does not have any of the mutations associated with organophosphate and/or carbamate resistance in drosophila (mutero et al., 1994) , lucilia cuprina (chen et al., 2001) , or m. domestica aches (kim et al., 2003; kozaki et al., 2001; walsh et al., 2001) , we conclude that adace1 encodes an organophosphate-sensitive ache (characterized by m126, v205, g287, f352, and g390). this is consistent with the denmark-s strain being insecticide susceptible. we sequenced 1,895 bp encoding 591 amino acids of adace2 (the d. melanogaster ace paralogous ache, eu086057) (fig. 2) . as also found for adace1, fig. 4 . alignment of the deduced amino acid sequences from the drosophila ace orthologous genes in coleoptera. ad, tc, and ld represent a. diaperinus, t. castaneum, and l. decemlineata, respectively. archives of insect biochemistry and physiology march 2008 the residues for the intramolecular disulphide bonds (c76(67)-c103(94), c275(254)-c295(265), c410(402)-c532(521)), catalytic triad (s208(200), e334(327), h448(440)), protein dimerisation (c558) anionic subsite (w93(86)), oxianion hole (g126(118), g127(119), a209(201)), and acyl binding site (w241(233), f298(290), f338(331)) were found in adace2. we were unable to complete the 5′-race for adace2, although we tried multiple variations of the protocol given by the manufacturer, including increased or decreased cation concentration, increasing the viscosity of the reaction mix by bsa or by using an alternative cation (mg2 + to mn 2+ ). the alignment of this gene with the drosophila ace paralogous aches showed that, as expected for an insecticide-susceptible strain, beetles from the denmark-s strain had an organophosphate and carbamate sensitive type. a partial cdna, covering the amino acid residues found to be responsible for insecticide resistance in other species, was sequenced from individual adults for both alphitobius ace genes to ascertain if resistance was due to a change in one or both genes. if resistance was due to a mutation in adace1 or adace2, all resistant individuals should have a unique allele (i.e., different from the susceptible strain). the drosophila ace orthologous gene, adace1, was sequenced from two susceptible denmark-s, four waycross (tetrachlorvinphos-resistant), and two kennebec (tetrachlorvinphos-resistant) adults. the deduced amino acid sequences from all individuals were the same. there were six synonymous polymorphisms detected (data not shown). the ace paralogous gene, adace2, was sequenced from three susceptible (denmark-s), five waycross (tetrachlorvinphos-resistant), and five kennebec (tetrachlorvinphos-resistant) adults. the sequences from all individuals were highly similar. one of the denmark-s and one of the waycross beetles had a a271(261)s mutation (detected as a heterozygote in both individuals). there were an additional 10 synonymous polymorphisms identified (data not shown). given that neither adace1 nor fig. 5 . alignments of the deduced amino acid sequences from the drosophila ace paralogous genes in coleoptera. ad and tc represent a. diaperinus and t. castaneum, respectively. manure breeding insects responsible for cestodiasis in caged layer hens poultry integrated pest management: status and future ecology and management of arthropod pests of poultry relationship of campylobacter isolated from poultry and from darkling beetles in new zealand the acetylcholinesterase gene and organophosphorus resistance in the australian sheep blowfly, lucilia cuprina transmission of enteric pathogens of turkeys by darkling beetle larvae (alphitobius diaperinus) construction profiles of high rise caged layer houses in association with insulation damage caused by the lesser mealworm, alphitobius diaperinus (panzer) in virginia mutations of acetylcholinesterase which confer insecticide resistance in insect populations drosophila melanogaster acetylcholinesterase gene structure, evolution and mutations transmission of eimeria, viruses, and bacteria to chicks: darkling beetles (alphitobius diaperinus) as vectors of pathogens resistance to cyfluthrin and tetrachlorvinphos in the lesser archives of insect biochemistry and physiology alphitobius diaperinus, collected from the eastern united states arthropod populations in highrise caged-layer houses after three manure cleanout treatments cloning, mutagenesis, and expression of the acetylcholinesterase gene from a strain of musca domestica; the change from a drug-resistant to a sensitive enzyme amino acid substitutions conferring insecticide insensitivity in ace-paralogous acetylcholinesterase fenitroxon insensitive acetylcholinesterases of the housefly, musca domestica, associated with point mutations reservoir competence of alphitobius diaperinus (coleoptera: tenebrionidae) for escherichia coli (enterobacteriales: enterobacteriaciae) resistanceassociated point mutations in insecticide-insensitive acetylcholinesterase expression of the ace-paralogous acetylcholinesterase of culex tritaeniorhynchus with an amino acid substitution conferring insecticide insensitivity in baculovirus-insect cell system reducing darkling beetles residual and topical toxicity of various insecticides to the lesser mealworm (coleoptera: tenebrionidae) identification and characterization of mutations in housefly (musca domestica) acetylcholinesterase involved in insecticide resistance limited transmission of turkey coronavirus (tcv) in young turkeys by adult lesser mealworms, alphitobius diaperinus panzer (tenebrionidae) comparisons of kinetic properties of acetylcholinesterase purified from azinphosmethyl-susceptible and resistant strains of colorado potato beetle a point mutation of acetylcholinesterase associated with azinphosmethyl resistance and reduced fitness in colorado potato beetle we thank d.a. rutz for the beetles and c. leichter, j. briddell, and c. reasor for technical as-sistance. the daljit s. and elaine sarkaria professorship and a research fellowship (to t.k.) from the japanese society for the promotion of science for young scientists supported this project. adace2 were different between resistant and susceptible beetles, we conclude that the mechanism of tetrachlorvinphos resistance in these strains of a. diaperinus is not due to mutations in the ace genes (i.e., is not an altered acetylcholinesterase).alignments of the deduced amino acid sequences from the drosophila ace orthologous and paralogous genes in coleoptera are shown in figures 4 and 5, respectively. as expected, the ace orthologous sequences of the two tenebrionidae, a. diaperinus and t. castaneum, were more similar to each other than to the leptinotarsa decemlineata (fig. 4) . these three coleopteran sequences differed primarily at the n-and c-terminal regions, but showed expected conservation at most functionally important residues. similarly, the ace paralogous sequences from a. diaperinus and t. castaneum were highly similar, with the greatest number of differences found in the c-terminal region (fig. 5) .the phylogenic tree of the arthropod aches (fig. 3) shows there are two major groups, with the acari aches being intermediates. each group is further divided into the subgroups, primarily by order. adace1 and adace2 clustered with the other coleoptera genes in both. this is consistent with the idea that beetles have two ace genes. the mutations related to organophosphate resistance were first studied in d. melanogaster and m. domestica. however, studies of drosophila ace orthologous genes failed to identify mutations responsible for organophosphate resistance in other species. subsequently, mutations in the drosophila ace paralogous genes were found to be associated with the resistance in some mosquitoes. the increasing number of insect genome sequences reveal that the ancestral condition, at least in pterygota, is two copies of ace. it also appears that mutations on the drosophila and musca ace paralogous genes are more important than the mutations on the drosophila orthologous genes, in terms of conferring organophosphate resistance, at least in many species. key: cord-314868-ei2b8oqn authors: leung, j. m.; yang, c. x.; tam, a.; shaipanich, t.; hackett, t. l.; singhera, g. k.; dorscheid, d. r.; sin, d. d. title: ace-2 expression in the small airway epithelia of smokers and copd patients: implications for covid-19 date: 2020-03-23 journal: medrxiv doi: 10.1101/2020.03.18.20038455 sha: doc_id: 314868 cord_uid: ei2b8oqn introduction: coronavirus disease 2019 (covid-19) is a respiratory infection caused by the severe acute respiratory syndrome coronavirus2 (sarscov-2). this virus uses the angiotensin converting enzyme ii (ace2) as the cellular entry receptor to infect the lower respiratory tract. because individuals with chronic obstructive pulmonary disease (copd) are at increased risk of severe covid19, we determined whether ace2 expression in the lower airways was related to copd and cigarette smoking. methods: using rnaseq, we determined gene expression levels in bronchial epithelia obtained from cytologic brushings of 6th to 8th generation airways in individuals with and without copd. we externally validated these results from two additional independent cohorts, which used microarray technologies to measure gene expression levels from 6th to 12th generation airways. results: in the discovery cohort (n=42 participants), we found that ace2 expression levels were increased by 48% in the airways of copd compared with non-copd subjects (copd=2.52 (0.66) log2 counts per million reads (cpm) versus non-copd= 1.70 (0.51) cpm , p=.000762). there was a significant inverse relationship between ace2 gene expression and fev1% of predicted (r=negative 0.24; p=0.035). current smoking also significantly increased ace2 expression levels compared with never smokers (never current smokers=2.77 (90.91) cpm versus smokers=1.78 (0.39) cpm, p=0.024). these findings were replicated in the two external cohorts. conclusions: ace2 expression in lower airways is increased in patients with copd and with current smoking. these data suggest that these two subgroups are at increased risk of serious covid19 infection and highlight the importance of smoking cessation in reducing the risk. the world health organization (who) has declared coronavirus disease 2019 (covid-19) as a pandemic [1] . covid-19 is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2). covid-19 displays symptoms ranging from mild to severe (pneumonia) that can lead to death in some individuals [2] [3] [4] . as of march 13, 2020, there have been 144,064 laboratory-confirmed cases of covid-19 worldwide with 5,397 deaths worldwide [5] . sars-cov-2 uses the angiotensin converting enzyme ii (ace-2) as the cellular entry receptor [6] . while the virus can infect individuals of any age, to date, most of the severe cases have been described in those over the age of 55 years and with significant comorbidities such as chronic obstructive pulmonary disease (copd) [7] . here, we determined whether patients with copd have increased expression of ace-2 in bronchial epithelial cells in lower respiratory tract. patients undergoing bronchoscopy at st. paul's hospital (sph), vancouver, canada for clinical purposes were enrolled (table 1 ). the protocol was approved by the university of british columbia/providence health care ethics board (ubc/phc reb h15-02166). all patients were required to be 19 years of age or older, who underwent spirometry according to international guidelines [8] . patients with copd were defined as those having a clinical diagnosis of copd made by a boardcertified respiratory physician and either a forced expiratory volume in 1 second (fev1)/forced vital capacity (fvc) <70% or a clear evidence of emphysema on computed tomographic (ct) imaging on visual inspection. cytologic brushings were obtained in subsegmental airways (6 th -8 th generation) of the lung that were unaffected by the patient's underlying clinical indication for bronchoscopy. total rna was extracted from cytologic brushings using the rneasy mini kit (qiagen, hilden, germany). transcriptome sequencing was performed on the novaseq 6000 (illumina, san diego, ca) at a sequencing depth of 55 million reads. raw sequencing reads were quality controlled with fastqc [9] and aligned to the gencode (version 31) grch37 genome reference using star (spliced transcripts alignment to a reference) [10] . after alignment, the data were quantified using rsem (rna-seq by expectation maximization) to obtain the read counts. limma voom [11] was applied to normalize the counts to log2 counts per million reads (cpm), which was used in the downstream analysis. two cohorts were used for validation; the details of which are provided in a previous publication [12] . first, we used 16 datasets obtained from bronchial brushings of 10 th -12 th generation bronchi collected at a single center using the u133 plus 2.0 microarray (denoted as the cornell . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03. 18.20038455 doi: medrxiv preprint dataset) [13] . second, we used dataset gse37147 consisting of bronchial brushings from the 6 th -8 th generation airways with gene expression profiles generated from the genechip human gene 1.0 st microarray [14] . this dataset was denoted as british columbia cancer agency (bcca) cohort. we also determined protein expression of ace-2 in resected lung tissue specimens. these samples were obtained from 10 current smokers with copd (mean±sd, fev1/fvc 61±7%), 9 non-smoker controls (fev1/fvc 85±2%), and 8 healthy current smokers (fev1/fvc 78±6%). human lung tissue samples were obtained with informed consent from patients undergoing thoracic surgery as part of the james hogg lung registry (ubc/phc reb protocol h00-50110). formalin-fixed paraffin-embedded human lung tissues were stained with antibody against ace-2 (ab15348; abcam) using the bond polymer refine red detection kit on the leica bond autostainer as previously described [15] . airway epithelial-specific ace-2 protein intensity was quantified using the aperio imaging system with normalization to the length of the basement membrane (leica biosystem; concord, ontario). for the primary study population, log2 cpm of ace-2 was the principal outcome of interest. robust linear models were used to determine whether 1) ace-2 was differentially expressed in patients with copd and in smokers after adjustment for age and sex and 2) ace-2 expression was significantly correlated with lung function. all analyses were performed in r (version 3.5.0). in the immunohistochemistry dataset, kruskal-wallis test with dunn's multiple comparisons test was used. table 1 displays the demographic and clinical characteristics of the sph cohort. ace-2 expression in the epithelial cells was significantly increased in copd versus non-copd subjects (mean±sd of non-copd=1.70±0.51 versus copd=2.52±0.66, p=7.62×10 -4 ; figure 1a ). there was a significant inverse relationship between ace-2 gene expression and fev1% of predicted (r=-0.24; p=0.035; figure 1b) . interestingly, smoking status was also significantly related to ace-2 gene expression levels in airways of these participants with current smokers having a significantly higher gene expression than never smokers (never smokers=1.78±0.39 versus current smokers=2.77±0.91, p=0.024). former smokers had gene expression levels in-between that of never and current smokers (former smokers=2.00±1.23; figure 1c ). conditional on the smoking status, the association between ace-2 expression and copd was still significant (adjusted mean±se of non-copd: 0.90±0.65 versus copd: 1.75±0.82, p=0.016). next, we validated the above findings in: 1) the cornell cohort (n= 211) and 2) british columbia cancer research agency (bcca) cohort (n=238). the average age of the cornell cohort was 43.6 (sd=10.5) years with 33.2% of the cohort being females. there were 32.2% who were never smokers and 67.8% who were current smokers at the time of the bronchoscopy. the average age of the bcca . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03. 18.20038455 doi: medrxiv preprint cohort was 64.5 (sd=5.9) years with 43.3% of the cohort being females. all were heavy smokers with at least 30 pack-years of smoking. of these, 41.6% were current smokers at the time of the bronchoscopy and the remaining were former smokers. in both the cornell and bcca cohorts, current smokers had increased ace-2 gene expression levels in the airways compared with never smokers (in the cornell cohort; mean±sd of never smokers=4.15±0.36 versus current smokers=4.34±0.45, p=1.92×10 -3 ) and with former smokers (in the bcca cohort; mean±sd of former smokers=5.57±0.37 versus current smokers=6.05±0.53, p<2×10 -16 ). in the bcca cohort, pre-bronchodilator fev1 was measured and it was significantly related to ace-2 gene expression level (r=-0.10;p=0.037). representative images of epithelial-specific ace2 protein expression in non-smokers, healthy smokers and smokers with copd are shown in figure 1d . ace-2 expression in the human small airway epithelium was significantly increased in copd compared to non-smokers but not in healthy smokers ( figure 1d ). there is a worldwide outbreak of covid-19 coronavirus. although most patients infected and diagnosed with cvoid-19 disease have mild symptoms, approximately 20% of individuals have demonstrated severe or critically severe disease including symptoms and signs of pneumonia, respiratory failure, septic shock and multi-organ failure. the estimated case-fatality rate is 1-2% [2, 3] . importantly, nearly all deaths have occurred in those with significant underlying chronic diseases including copd, and cardiovascular diseases [4] . the reason for this observation is largely unknown. one possibility is differential expression of the ace-2, which is the main receptor used by sars-cov-2 to gain entry into the host mucosa and cause active infection. here, we investigated gene expression levels of ace-2 in the airways of individuals with and without copd and found that copd and current smokers had significantly increased expression of ace-2. importantly, gene expression levels of ace-2 were inversely related to individual's fev1, suggesting a dose-dependent response. these findings were observed in 3 different cohorts, indicating their generalizability and robustness. ace-2 is a type i transmembrane metallocarboxypeptidase with homology to angiotensin converting enzyme (ace). in contrast to ace, which converts angiotensin i to the active vasoconstrictor, angiotensin ii, ace-2 breaks down angiotensin ii to its metabolites including angiotensin-(1-9) and angiotensin-(1-7), which are potent vasodilators, and thus may be a negative regulator of the renin-angiotensin system [16] . ace-2 is expressed in a variety of different tissues including both the upper and lower respiratory tract, myocardium and the gastrointestinal mucosa [17] . although its role in human health and disease has not been fully elucidated, it appears to have an . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.18.20038455 doi: medrxiv preprint important regulatory role in blood pressure and cardiac function. the physiologic role of ace-2 in the airways is largely unknown. however, in mice, ace-2 has been shown to protect animals from severe lung injury related to aspiration and sepsis [18] . to our knowledge, our study is the first to demonstrate increased ace-2 expression in airways of current (but not former) smokers and those with copd. these results are also consistent with previous observations in small animals wherein smoke exposure has been shown to upregulate both the expression and activity of ace-2 in the airways [19, 20] . while the up-regulation of ace-2 may be useful in protecting the host against acute lung injury, chronically, this may predispose individuals to increased risk of coronavirus infections, which uses this receptor to gain entrance into epithelial cells. this may in part explain the increased risk of viral respiratory tract infection in active smokers and virus-related exacerbations in those with copd. there were limitations to the study. first, the study was cross-sectional and as such, we could not determine whether interventions such as inhaled corticosteroids or bronchodilators (for those with copd) could modulate ace-2 gene expression in the airways. second, as receptor expression is one of many host factors that govern infection risk among individuals, the precise attributable risk (for coronavirus infections) imposed by cigarette smoking and copd is uncertain. third, although the airway epithelia is the major source of entry for covid-19, the virus can gain host entry through other ports including gastrointestinal mucosa, which was not evaluated in this study. fourth, we did not have access to upper airway tissues, which may also become infected with sars-cov-2. in summary, active cigarette smoking and copd up-regulate ace-2 expression in lower airways, which in part may explain the increased risk of severe covid-19 in these sub-populations. these findings highlight the importance of smoking cessation for these individuals and increased surveillance of these risk subgroups for prevention and rapid diagnosis of this potentially deadly disease. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.18.20038455 doi: medrxiv preprint the red box indicates the median and the interquartile range. the p-value was obtained from the robust linear model abbreviations: ace-2, angiotensin converting enzyme ii; copd, chronic obstructive pulmonary disease; cpm, counts per million reads . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03. 18.20038455 doi: medrxiv preprint abbreviations: ace-2, angiotensin converting enzyme ii; copd, chronic obstructive pulmonary disease; cpm, counts per million reads ace-2 gene expression in airway epithelia is inversely related to fev1% predicted (p=0.0348) . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03. 18.20038455 doi: medrxiv preprint figure 1c . a violin plot of ace2 expression in small airways of never, former and current smokers in the st. paul's hospital cohort. the red box indicates the median and the interquartile range. the p-value was obtained from the robust linear model. abbreviations: ace-2, angiotensin converting enzyme ii; cpm, counts per million reads . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03. 18.20038455 doi: medrxiv preprint figure 1d . protein staining of ace2 airways of individuals with and without copd human kidney slide was the positive control for ace-2. in airways, most of the protein expression was noted in the epithelial layer, most pronounced in those with copd. abbreviations: ace-2, angiotensin converting enzyme ii; ns, non-smoker non-smoker 100µm human kidney healthy smoker copd . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03. 18.20038455 doi: medrxiv preprint clinical characteristics of coronavirus disease 2019 in china feng z. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in standardization of spirometry fastqc: a quality control tool for high throughput sequence data star: ultrafast universal rna-seq aligner voom: precision weights unlock linear model analysis tools for rna-seq read counts widespread sexual dimorphism in the transcriptome of human airway epithelium in response to smoking threshold of biologic responses of the small airway epithelium to low levels of tobacco smoke a dynamic bronchial airway gene expression signature of chronic obstructive pulmonary disease and lung function impairment. american journal of respiratory and critical care 15 angiotensin-converting enzyme 2 is an essential regulator of heart function quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme angiotensin-converting enzyme 2 protects from severe acute lung failure alternative roles of stat3 and mapk signaling pathways in the mmps activation and progression of lung injury induced by cigarette smoke exposure in ace2 knockout mice role of angiotensin-converting enzyme (ace) and ace2 in a rat model of smoke inhalation induced acute respiratory distress syndrome key: cord-319022-1twsxzcd authors: desai, antonio; voza, giuseppe; paiardi, silvia; teofilo, francesca ilaria; caltagirone, giuseppe; pons, marta ripoll; aloise, monia; kogan, maria; tommasini, tobia; savevski, victor; stefanini, giulio; angelini, claudio; ciccarelli, michele; badalamenti, salvatore; de nalda, ana lleo; aghemo, alessio; cecconi, maurizio; boneschi, filippo martinelli; voza, antonio title: the role of anti-hypertensive treatment, comorbidities and early introduction of lmwh in the setting of covid-19: a retrospective, observational study in northern italy() date: 2020-09-25 journal: int j cardiol doi: 10.1016/j.ijcard.2020.09.062 sha: doc_id: 319022 cord_uid: 1twsxzcd background: there is a great deal of debate about the role of cardiovascular comorbidities and the chronic use of antihypertensive agents (such as ace-i and arbs) on mortality on covid-19 patients. of note, ace2 is responsible for the host cell entry of the virus. method: we extracted data on 575 consecutive patients with laboratory-confirmed sars-cov-2 infection admitted to the emergency department (ed) of humanitas center, between february 21 and april 14, 2020. the aim of the study was to evaluate the role of chronic treatment with ace-i or arbs and other clinical predictors on in-hospital mortality in a cohort of covid-19 patients. results: multivariate analysis showed that a chronic intake of ace-i was associated with a trend in reduction of mortality (or: 0.53; 95% ci: 0.27–1.03; p = 0.06). increased age (ors ranging from 3.4 to 25.2 and to 39.5 for 60–70, 70–80 and > 80 years vs < 60) and cardiovascular comorbidities (or: 1.90; 95% ci: 1.1–3.3; p = 0.02) were confirmed as important risk factors for covid-19 mortality. timely treatment with low-molecular-weight heparin (lmwh) in ed was found to be protective (or: 0.36; 95% ci: 0.21–0.62; p < 0.0001). conclusions: this study can contribute to understand the reasons behind the high mortality rate of patients in lombardy, a region which accounts for >50% of total italian deaths. based on our findings, we support that daily intake of antihypertensive medications in the setting of covid-19 should not be discontinued and that a timely lmwh administration in ed has shown to decrease in-hospital mortality. in late december 2019, a novel coronavirus emerged in wuhan, the capital city of hubei. the enveloped rna betacoronavirus has been soon named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) due to its phylogenetic similarity to sars-cov 1 . on february 20, 2020, the first case of novel coronavirus disease 2019 (covid-19) was detected in italy 2 , and 20 days later who declared covid-19 outbreak a global pandemic 3 . italy has one of the highest rates of sars-cov-2 infection worldwide, with 397.1 cases per 100.000 people, as well as the fourth highest mortality rate, 14.5% vs. a global average value of 5.1% (as of june 26 th , 2020; https://coronavirus.jhu.edu/map.html). the lombardy region, with 10.6 million people living in highly populated areas, has been the epicenter of the infection in italy, with 39.1% of total cases and almost half of total deaths (http://www.salute.gov.it/portale/home.html). the reason behind the high mortality rate in italy, and mostly in lombardy, can have several explanations: 1) the characteristics of the population (italy has the second oldest population in europe; https://ec.europa.eu/eurostat/data/database); 2) the organization of the health system in lombardy where most of the medical care is provided at hospital level rather than locally with general practitioners. in addition, a low number of intensive care units is present compared to those needed for lombardy's population; 3) the possible existence of different virus strains; 4) differences in genetic and environmental factors (e.g. air pollution); 5) overuse of antibiotics and antihypertensive agents, in italy as compared to other countries. as for today, there are discordant results regarding the use of either angiotensin converting enzyme inhibitors (ace-i) or angiotensin ii receptor blockers (arbs) as for their possible impact on covid-19 mortality. both ace-i and arbs act on the renin-angiotensinaldosterone system (raas) and are widely prescribed for primary hypertension 5 , heart failure, diabetic nephropathy and secondary prevention of myocardial infarction (mi) 6 . ace-i exerts its effect by inhibiting the angiotensin converting enzyme (ace), a specific protease found primarily in the vascular endothelium of the lungs and kidneys which catalyzes the conversion of angiotensin i to angiotensin ii, which in turn causes vasoconstriction 7 . arbs act by antagonizing the effects of angiotensin ii on its at 1 and at 2 receptors 8 , expressed in kidney 9,10 , adrenal cortex 11 , arterioles 12 , and brain 13,14 . the carboxypeptidase angiotensin-converting enzyme 2 (ace2), which is highly expressed in the lower airways, degrades angiotensin ii into its active form angiotensin 1-7, which in turn dilates blood vessels, reduces inflammation, and inactivates angiotensin ii before it could interact with its receptors 15 . sars-cov-2 binds to ace2 with its spike glycoprotein which allows it to gain entry into the host cell and by that to potentially cause an acute respiratory distress syndrome (ards) and respiratory failure 16 . in addition, hypertension has been found to be an important risk factor for mortality with a hazard ratio (hr) of 3.05 for in-hospital mortality in 191 covid-19 patients and of 1. the secondary endpoint of our study was to investigate the role of other clinical predictors on in-hospital mortality. this single-center, retrospective, observational study was conducted at the emergency department (ed) of humanitas clinical and research center. the study was approved by the ethical committee and performed in accordance with the principles of the helsinki declaration 19 . we analyzed 575 consecutive patients who were admitted to the ed with fever and cough complaints from february 21, 2020, to april 14, 2020, and had been diagnosed with covid-19, according to who interim guidance 20 . laboratory confirmation of sars-cov-2 infection was done by rt-pcr performed on a nasopharyngeal swab in all subjects. patients were discharged from the hospital or transferred to a health care facility if they had stable oxygen levels (in room air > 95%) for more than 48 hours as measured by a pulse oximeter. clinical electronic medical records were reviewed for all patients tested positive for sars-cov-2 infection. data were collected and stored in a dedicated database built solely for this purpose. any missing or uncertain records were clarified through direct communication with the relevant health-care providers and family members. we collected data on age, gender, comorbidities, chronic treatment, time interval between symptoms onset and ed admission, treatment provided during ed stay (antiviral agents, j o u r n a l p r e -p r o o f journal pre-proof antibacterial agents, hydroxychloroquine and low molecular weight heparin (lwmh) therapy), days of hospitalization, and on outcome in terms of survival. the data were available only to authorized personnel, stored on a local server and retrieved for this analysis. the primary endpoint of the study was the occurrence of in-hospital mortality. the secondary outcome was the survival time, which was defined as the time between ed admission and either death, patient transfer to another health care facility or hospital discharge. analyses tested the role of ace-i and arbs and were adjusted for confounders with potential impact on survival, these including age, gender, comorbidities, time interval between onset of symptoms and ed admission, and treatments provided in the ed. categorical variables were represented in terms of frequency distributions, while quantitative variables were described as means and standard deviations (sd) or median and interquartile range (iqr). we performed univariate analyses using chi-square test for independence for categorical variables and unpaired t-test (or, if assumptions were not met, mann-whitney's test) for quantitative variables. multivariate analysis was performed with in-hospital mortality as dependent variable and as predictors a priori selected variables (age, gender and chronic use of ace-i and arbs) and other variables which were significant with p<0.1 at univariate testing with a backward selection based on likelihood ratio to find the most parsimonious as of april 14, 2020, 1247 consecutive patients presented to our ed were isolated as suspected covid-19 cases, of whom 672 tested negatives for sars-cov-2 on rt-pcr performed on a nasopharyngeal swab the later not included in this study. the remaining 575 adult patients who were found positive for covid-19 (46.1% of total) represented the study cohort. among those found positive for sars-cov-2 infection, the male to female ratio was 1.94 with a mean age of 64.8 years (sd 14.6, range 27-93 years), 361 subjects (62.7%) were older than 60 years (table 1) . while a limited number of patients (7.1%) had mild symptoms and were discharged within few hours, the majority required hospitalization and 10.9% were transferred to an intensive care unit (icu). chronic diseases were reported in 329 patients (57.1%), these most between arbs-users vs non-users (30.9% vs 25.4%). multivariate analyses showed that the main reason of these findings was due to the age difference between the two groups. moreover, a longer hospital stay was observed in arbs-users as compared to non-users (11 vs 9 days; p=0.02). in hospital mortality was 20.9%, while 55.5% of patients were discharged, 3.8% moved to a long-term care facility and 19.8% still hospitalized at time of data analysis. mean age at time we used survival analyses to further explore significant findings identified in the logistic regression. results confirmed that the use of ace-i was a significant protective factor for covid-19 in-hospital mortality (hazard ratio: 0.61; 95% ci: 0.38-0.98; p=0.04) with a highly significant model (p=9.99e-22; n=444) ( table 3) . the study describes predictors of mortality in covid-19 patients in one of the largest singlecenter cohort of italian patients reported so far, admitted to one of the biggest hospitals in the city of milan, which is located in the epicenter of the covid-19 emergency with the highest mortality in italy. in order to understand the reasons behind the high mortality rate in this metropolitan area, we performed a multivariate statistical model to assess the role of clinical predictors evaluated at ed admission and of ace-i and arbs chronic use. in-hospital mortality was found to be 20.9%. ace-i and arbs act on the raas system, and they are widely used to treat primary hypertension 5 , heart failure, diabetic nephropathy and secondary prevention of myocardial infarction (mi) 6 . the two drugs have indeed a different mechanism of action on the raas cascade. while ace-i inhibits the conversion from angiotensin i to angiotensin ii, arbs antagonize the effects of angiotensin ii on its at i and at ii receptors 8, 9 . there are different hypotheses on the effect of these two drugs on ace2, which is the protein involved in the host cell entry of the sars-cov-2. whether they are detrimental or protective for disease severity of covid-19 is still a matter of discussion. ferrario and co-workers proved that after a treatment with lisinopril (ace-i) alone, ace2 mrna expression levels were increased without a consensual increase in ace2 activity 21, 22 . in this regard, increased levels of ace2 may possibly lead to an increase in the number of binding sites for sars-cov-2, raising the risk of covid-19 21, 23 . on the other end, other authors hypothesize that ace-i and arbs may interfere with the integrity of the ace2/angiotensin (1-7)/mas pathway resulting in a decreased expression of ace2 and host cell entry of the virus 24 . we found that ace-i, which acts by inhibiting the conversion from angiotensin i to angiotensin ii, showed a trend in protecting from mortality from covid-19 and was significant in delaying mortality as shown by multivariate cox regression analysis unlike arbs, which antagonize the effects of angiotensin ii on its receptors 2,3 . the mechanisms underlying this phenomenon are still unclear and further investigations are required in order to shed light on the subject. as demonstrated in a recent paper, another hypothesis is that soluble human ace2 inhibitors can inhibit sars-cov-2 infections, and in this regard it will be useful to investigate whether ace-i or arb are able to interfere and increase ace2 levels 25 . our data partly confirmed the results of a larger retrospective, multi-centric study performed on 1128 covid-19 adults from the hubei province in china, which showed that an inpatient use of either ace-i or arbs was associated with a lower risk of all-cause mortality when compared with ace-i/arbs non-users 26 of note, two recent papers with a large sample size were performed in lombardy and in the new york state using data from electronic health records, both of which did not confirm the protective role of ace-i towards disease risk but not towards disease severity 27, 28 . advantages of single-center studies as compared to multicentric studies or to studies using health registries lie on the availability of complete data on patients and on the possibility to follow-up affected individuals despite it is usually associated with a smaller sample size. as for today we can only say that a daily intake of antihypertensive medications (ace-i and arbs) in the setting of covid-19 should not be discontinued. the replacement of a raas inhibitor with an antihypertensive agent of a different class may require careful monitoring to avoid the rebound effect of blood pressure, as even short periods of blood pressure instability after a therapeutic change have been associated with an increase in cardiovascular risk 29, 30 . as of the role of other potential predictors, we confirmed the significant role of age and cardiovascular comorbidities on mortality rate, as shown by other papers, while males were not confirmed to have a higher mortality differently from other studies 31 . on the contrary, the early administration of lmwh in ed was shown to have a protective effect on mortality in the same patients. a prophylactic dose of lmwh was administered at ed admission starting with 0.4 ui/24h. the dose was adjusted accordingly to renal function and body weight. thromboembolic events which, according to a previous paper performed in our hospital on a series of covid-19 patients which partially overlap with ours, occurred in 7.7% of them. the paper underlies the importance of timely treatment in particular among patients with non-modifiable risk factors such as age and cardiovascular comorbidities as it can potentially improve survival rates in these patients. it is worthwhile to consider developing tools for early identification of such patients, to maximize potential outcomes. nonetheless, rigorous adherence to state based recommendations are also advisable (e.g. quarantine, social distancing), especially among high-risk patients. in conclusion, despite the limited nature of the study, identification of predictors of mortality will allow a better management of patients in the face of future disease recurrence. clinical characteristics of coronavirus disease 2019 in china coronavirus disease 2019 (covid-19) in italy who declares covid-19 a pandemic ace2 and tmprss2 variants and expression as candidates to sex and country differences in covid-19 severity in italy angiotensin converting enzyme (ace) inhibitors versus angiotensin receptor blockers for primary hypertension determinants of angiotensinconverting enzyme inhibitor (acei) intolerance and angioedema in the uk clinical practice research datalink molecular genetics of the renin-angiotensin system: implications for angiotensin ii receptor blockade angiotensin ii receptor blockers (arb, arb). in: statpearls statpearls publishing copyright © 2020 angiotensin ii receptor blockers angiotensin (at1a) receptor-mediated increases in transcellular sodium transport in proximal tubule cells role of angiotensin ii receptor subtypes on the regulation of aldosterone secretion in the adrenal glomerulosa zone in the rat the angiotensin ii type 2 receptor: what is its clinical significance? frontiers in neuroscience. diverse roles of angiotensin receptor intracellular signaling pathways in the control of water and salt intake statpearls publishing. copyright © 2020 hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase potential harmful effects of discontinuing aceinhibitors and arbs in covid-19 patients covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: what is the evidence? jama the 2019 coronavirus: learning curves, lessons, and the weakest link world medical association declaration of helsinki: ethical principles for medical research involving human subjects world health o. when covid-19 disease is suspected: interim guidance effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 antihypertensive treatment with acei/arb of patients with covid-19 complicated by hypertension outcomes in patients with covid-19 infection taking acei/arb inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 inhibitors of the renin-angiotensin-aldosterone system and covid-19 outcomes in hypertensive patients at high cardiovascular risk treated with regimens based on valsartan or amlodipine: the value randomised trial indication, timing, and surgical treatment of spontaneous intracerebral hemorrhage: systematic review and proposal of a management algorithm risk factors associated with mortality among patients with covid-19 in intensive care units in lombardy, italy anti-coagulant and anti-platelet therapy in the covid-19 patient: a best practices quality initiative across a large health system venous and arterial thromboembolic complications in covid-19 patients admitted to an academic hospital in milan, italy -thromb res we would like to thank and dedicate this paper to all the professional key: cord-290148-6cxndab8 authors: rossi, gian paolo; sanga, viola; barton, matthias title: potential harmful effects of discontinuing ace-inhibitors and arbs in covid-19 patients date: 2020-04-06 journal: elife doi: 10.7554/elife.57278 sha: doc_id: 290148 cord_uid: 6cxndab8 the discovery of angiotensin converting enzyme-2 (ace-2) as the receptor for sarscov-2 (severe acute respiratory syndrome coronavirus-2) has implicated the renin-angiotensin-aldosterone system in acute respiratory distress syndrome (ards) and respiratory failure in patients with coronavirus disease-19 (covid-19). the angiotensin converting enzyme-1–angiotensin ii–angiotensin at(1) receptor pathway contributes to the pathophysiology of ards, whereas activation of the ace-2–angiotensin(1-7)-angiotensin at(2) receptor and the ace-2–angiotensin(1-7)–mas receptor pathways have been shown to be protective. here we propose and discuss therapeutic considerations how to increase soluble ace-2 in plasma in order for ace-2 to capture and thereby inactivate sars-cov-2. this could be achieved by administering recombinant soluble ace-2. we also discuss why and how aceis and arbs provide cardiovascular, renal and also pulmonary protection in sars-cov-2associated ards. discontinuing these medications in covid-19 patients may therefore potentially be harmful. the covid-19 (coronavirus disease 2019) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infecting one million and killing more than 50'000 people worldwide as of april 2, has fueled enormous interest in the mechanisms whereby this new coronavirus causes acute respiratory distress syndrome (ards) and multiorgan failure. the estimated 79% infection rate from undocumented cases in covid-19 patients , and the high lethality of the infections, along with its enormous socio-economic impact, emphasize the importance of fully understanding these mechanisms for developing effective treatment strategies. early in 2020 reports of the full rna sequence of the sars-cov-2 virus highlighted its remarkable similarity with the sars-cov virus, which was responsible for a global outbreak that killed 774 people in 2003 (xu et al., 2020; zhou et al., 2020) . as the processes whereby the sars-cov virus infects the lung cells had been already identified , and it was held that sars-cov-2 uses identical mechanisms, these discoveries allowed an unprecedented acceleration of knowledge. acid sequence of the spike (s) protein of the envelope that both viruses use to infect mammalian cells. with a 4 amino acid residue difference the sars-cov and the sars-cov-2 s protein share an almost identical 3-d structure of the receptor binding domain (xu et al., 2020) and, moreover, binds to ace-2 with even higher affinity than sars-cov (wrapp et al., 2020) , which may explain its virulence and predilection for the lungs. upon binding to ace-2, both sars-cov and sars-cov-2 activate the transmembrane serine protease-2 (tmprss2), which is highly expressed in the lungs. through fusion of its envelope with the cell membrane, the virus penetrates into the cells (figure 1 , panel a) (heurich et al., 2014; hoffmann et al., 2020) . of note, sars-cov-2 entry can be prevented by sars convalescent sera containing neutralizing antibodies, or by tmprss2 inhibitors such as camostat (hoffmann et al., 2020) and nafamostat mesylate, both approved in japan for clinical use for other indications (yamamoto et al., 2016) . these seminal discoveries suggested several potential therapeutic strategies to prevent sars-cov-2 entry into pulmonary cells (figure 1, panel a) . what is the role of ace-1 and ace-2 in infections caused by sars-cov and sars-cov-2? are there similarities between ace-1, the target of aceis, and ace-2, the target of sars-cov and sars-cov-2? this is obviously the essential question for physicians using aceis or angiotensinreceptor blockers (arbs), which are frequently prescribed in a multitude of patients. both ace-1 and ace-2 cleave angiotensin peptides ( figure 1 , panel b). however, they differ markedly: ace-1 cleaves the dipeptide his-leu from angiotensin i, thus generating angiotensin (ang) ii, which causes vaso-and broncho-constriction, increases vascular permeability, inflammation, and fibrosis and thereby promotes the development of ards and lung failure in patients infected with the sars-cov and sars-cov-2 (yang et al., 2015) (figure 1, panel b) . compelling evidence from animal models of ards, lung fibrosis, asthma, and chronic obstructive lung disease indicate that these effects are essential for ards to develop and that both aceis and arbs block the disease-propagating effect of ang ii (dhawale et al., 2016; imai et al., 2005; kaparianos and argyropoulou, 2011) . ace-2, which is expressed more abundantly on the apical than the basolateral side of polarized alveolar epithelial cells (jia et al., 2005) , shares only 42% amino acid sequence homology with ace-1 (harmer et al., 2002) . it cleaves only one amino acid residue (leu or phe) from ang i and ang ii, respectively, to generate ang (1-9) and ang(1-7) ( figure 1, panel b) . importantly, ang(1-7) counteracts the at 1 r-mediated aforementioned detrimental effects induced by ang ii in the lungs. accordingly, genetic deletion of ace-2 worsens experimental ards , while ang(1-7) and aceis or arbs administration improve it wö sten-van asperen et al., 2011) . thus, blunting the ace-1-ang ii-at 1 r axis while enhancing the ace-1-ang ii-at 2 r, the ace-2-ang(1-7)-at 2 r or the ace-2-ang(1-7)-masr receptor axes ( figure 1 , panel b) likely protects from ards triggered by infectious pathogens, including coronaviruses (dhawale et al., 2016; imai et al., 2005; kaparianos and argyropoulou, 2011; meng et al., 2014) . after ace-2 was identified as the sars-cov-2 receptor (hoffmann et al., 2020; yan et al., 2020) , unexpectedly, and almost immediately, it was contended that treatment with aceis and arbs would be harmful for covid-19 patients. this hypothesis was quickly spread in the public, causing confusion and fear in patients taking these drugs, who started asking themselves, and their doctors if they should discontinue these medications and replace them with of antihypertensive drugs of other classes. the confusion caused in the medical community and the public was due to the publication of two commentaries containing simple hypotheses in the absence of supporting evidence. in one commentary ace-2 was suggested to be secreted at higher amounts in patients with cardiovascular disease than in healthy individuals, and in another, it was also stated that 'ace-2 levels can be increased by the use of aceis' , albeit no evidence of this occurring in the lungs mechanisms of covid-19 by which the sars-cov-2 virus infects the lower airway cells and modalities to increase circulating soluble ace-2 for therapeutic use. (a) by binding to endothelial and type 2 alveolar epithelial cells that express ace-2 at high levels, the virus activates proteases, such as tmprss2. this allows fusion with the virus' envelope to the cell membrane facilitating the virus to enter and infect the cell. of note, type 2 alveolar epithelial cells are well equipped with a molecular machinery that allows rapid replication of the viruses thus enhancing pulmonary spreading of the infection. once infected by sars-cov-2 the lung cells downregulates expression of ace-2. therefore, the lungs remain exposed to, and are unprotected from, the detrimental actions of angiotensin ii acting via the at 1 r. increasing circulating soluble ace-2 levels represents a potential new therapeutic principle to treat sars-cov-2 infection. this can be achieved using different strategies: either by increasing adam-17-dependent shedding of ace-2 facilitating its removal from tissue (strategy a) or by intravenous administration of recombinant soluble ace-2 to capture and thereby inactivate sars-cov-2 in plasma and preventing it from entering the cell (strategy b). (b). the renin-angiotensin system in the pathophysiology of sars-cov-2-associated ards. ang ii -via the at 1 r -promotes inflammation, vasoconstriction, cell proliferation, and vascular leakage and eventually, pulmonary fibrosis. these effects are counteracted by ace-2 dependent formation of ang(1-7) activating the at 2 r, masr, and mrgd and formation of ang(1-9) activating the at 2 r. the potential beneficial effects of aceis and arbs entail rescuing the downregulated ace-1-ang ii-at 2 r and the ace-2-ang(1-7)-at 2 r and ace-2-ang(1-7)-masr pathways in the lungs and capturing the virus in the circulation, thus impeding its binding to the lung cells and preventing damage to the lungs. abbreviations used: ace-1, angiotensin converting enzyme-1; ace-2, angiotensin converting enzyme-2; aceis, angiotensin converting enyzme inhibitors; arbs, angiotensin at 1 receptor blockers; at 1 r, angiotensin ii type 1 receptor; at 2 r, angiotensin ii type 2 receptor; nep, neutral endopeptidase/neprilysin; mrgprd, g-protein-coupled receptor mrgd; rhace-2, recombinant soluble human ace-2; soluble rbd of ace-2, soluble receptor-binding domain of ace-2; tmprss2, transmembrane serine protease-2. exists. these hypothetical phenomena were put forward to suggest and enhanced susceptibility to sars-cov-2 infection, and thus to warn patients about taking these drugs. correspondence to the lancet respiratory medicine suggested that patients with cardiac diseases, as hypertension and/or diabetes treated with 'ace-2-increasing drugs' would be at higher risk for severe sars-cov-2 infection, because treatment with aceis and arbs would raise ace-2 (fang et al., 2020) . to support their contention, the authors quoted a review article that however did not report such evidence (li et al., 2017) . to the contrary, a search of the literature revealed that no data that would support such notion exist. in fact, evidence of ace-2 upregulation applies to the heart, likely as a compensatory phenomenon to underlying conditions, for example myocardial infarction (burrell et al., 2005; ishiyama et al., 2004; ocaranza et al., 2006) , rather than to the drug treatment per se. moreover, in neither commentary the authors considered the fact that increased plasma levels of ace-2 (generated by delivering soluble forms of rhace-2 and/or increasing shedding of ace-2 from the cell membrane) can capture the s protein of sars-cov-2 (and sars-cov) in plasma, thus preventing the virus from binding to lung cells, two strategies that have been suggested to protect against sars-cov-2 infection of the lungs (kruse, 2020; zhang et al., 2020) (figure 1, panel a) . nonetheless, the publication of simple hypotheses unsubstantiated by any data spread so fast in public and news portals that scientific societies, including the european society of hypertension (esh), the italian society of cardiology and the italian society of arterial hypertension were required to release statements to confirm that there is no evidence that aceis and arbs could jeopardize covid-19 patients, and there is no need to recommend discontinuing treatment. to date, italy is the country with the highest number of sars-cov-2-positive individuals in the european union and the highest official number of deaths in the world. on march 17 th , 2020 a joint statement of the presidents of the hfsa/acc/aha (bozkurt et al., 2020) , followed by one of the european medicines agency (2020), and several experts' opinion articles reported and affirmed that there is no evidence to support discontinuing aceis and arbs (danser et al., 2020; greene et al., 2013; perico et al., 2020) , a notion also shared by the editors of the new england journal of medicine (rubin et al., 2020) . in our view these neutral recommendations could even be an understatement. in fact, in two large meta-analysis, and a case-control study involving over 21,000 patients in several high-risk categories of patients, including stroke survivors (shinohara and origasa, 2012) , in an asian population (caldeira et al., 2012; liu et al., 2012) , and also in patients with parkinson's disease , aceis were superior to other antihypertensive agents in pneumonia prevention. the experimental data obtained for the sars-cov virus also show that these drugs can be protective rather than harmful, which lead to the proposition of specifically enhancing the protective arm of the renin-angiotensin system as a novel therapeutic strategy for pulmonary diseases (tan et al., 2018) . moreover, abrupt withdrawal of raas inhibitors in high-risk patients, including those who have stage 3 arterial hypertension, heart failure or who had myocardial infarction, may result in clinical instability and adverse health outcomes as pointed out recently (vaduganathan et al., 2020) . the at 1 r-mediated detrimental effects of ang ii were demonstrated in several models of ards sars-cov-induced acute respiratory failure kuba et al., 2006; kuba et al., 2005) . moreover, with its vasodilatory, anti-inflammatory, anti-proliferative and antifibrotic effects, activation of the ace-2-ang(1-7)-at 2 r ace-2-ang(1-7)-masr pathways counterbalances the harmful effects of the ace-1-ang ii-at 1 r pathway on the lungs. a reduced ratio of ace-1/ace-2 has been documented in ards; furthermore, experimentally ards and lung fibrosis can be prevented by administration of ang(1-7) (cao et al., 2019 ), or arbs (wö sten-van asperen et al., 2011 , indicating that ace-2 activation limits pulmonary disease progression. this implies not only that aceis and arbs are unlikely to be detrimental in covid-19 patients, but that they likely will be protective. whether the same applies to drugs that block the mineralocorticoid receptor and antagonize aldosterone, another downstream mediator in the ace-1-ang ii-at 1 r pathway, remains unknown. in endothelial and lung type 2 alveolar epithelial cells, sars-cov-2 downregulates ace-2 and thereby the ace-2-ang(1-7)-masr pathway (imai et al., 2008) . this would also suggest that aceis and arbs can be beneficial by blunting the ace-1-ang ii-at 1 r pathway and counterbalancing the down-regulation of ace-2 ( figure 1) . administration of recombinant soluble human ace-2 (rhace-2) to capture sars-cov-2 in the bloodstream may prevent its binding to lung cells, and enhance ace-2 activity in lung tissue (figure 1, panel a) , which could be beneficial for covid-19 patients with ards, possibly even at a late stage of the infection for patients in intensive care requiring assisted ventilation. along these lines, in 2017 a pilot trial in patients with ards conducted in ten u.s intensive care units supports the value of this strategy in that rhace-2 increased levels of both ang(1-7) and alveolar surfactant protein d levels, and tended to lower the concentrations of the proinflammatory cytokine interleukin-6 (khan et al., 2017) . in summary, a disbalance between the ace-1-ang ii-at 1 r and the ace-1-ang ii-at 2 r and the ace-2-ang(1-7)-at 2 r and the ace-2-ang(1-7)-masr pathways contributes to the pathogenesis of ards and acute lung failure which likely is also relevant for covid-19 patients. therefore, it seems reasonable to conclude that rebalancing the system by blunting the deleterious effects of ang ii using aceis and arbs while enhancing the ace-2 axis is a valuable strategy to minimize the harmful effects of sars-cov-2 on the lungs. in the majority of patients with cardiovascular diseases, mainly hypertension, heart failure, or ischemic heart disease, who are on aceis or arbs and at risk of becoming infected, or have been infected by sars-cov-2 but do not need mechanical ventilation, there is no evidence for deleterious effects of acei or arbs. in fact, discontinuing these life-saving medications potentially can be harmful. competing interests matthias barton: senior editor, elife. the other authors declare that no competing interests exist. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. gian paolo rossi, conceptualization, supervision, methodology, writing -original draft; viola sanga, resources, data curation; matthias barton, supervision, writing, validation hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19: american college of cardiology myocardial infarction increases ace2 expression in rat and humans risk of pneumonia associated with use of angiotensin converting enzyme inhibitors and angiotensin receptor blockers: systematic review and meta-analysis ang-(1-7) treatment attenuates lipopolysaccharide-induced early pulmonary fibrosis renin-angiotensin system blockers and the covid-19 pandemic: at present there is no evidence to abandon renin-angiotensin system blockers activation of angiotensin-converting enzyme 2 (ace2) attenuates allergic airway inflammation in rat asthma model ema advises continued use of medicines for hypertension, heart or kidney disease during covid-19 pandemic: ema are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? haemoconcentration, renal function, and post-discharge outcomes among patients hospitalized for heart failure with reduced ejection fraction: insights from the everest trial tissue distribution of ace2 protein, the functional receptor for sars coronavirus a first step in understanding sars pathogenesis quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin-converting enzyme 2 protects from severe acute lung failure the discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice upregulation of angiotensinconverting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia local renin-angiotensin ii systems, angiotensin-converting enzyme and its homologue ace2: their potential role in the pathogenesis of chronic obstructive pulmonary diseases, pulmonary hypertension and acute respiratory distress syndrome a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 in lung diseases the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) angiotensin-converting enzyme inhibitor/angiotensin ii receptor blockers and pneumonia risk among stroke patients angiotensin-converting enzyme 2/ angiotensin-(1-7)/mas axis protects against lung fibrosis by inhibiting the mapk/nf-kb pathway enalapril attenuates downregulation of angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat should covid-19 concern nephrologists? why and to what extent? the emerging impasse of angiotensin blockade new research on possible treatments for covid-19 post-stroke pneumonia prevention by angiotensin-converting enzyme inhibitors: results of a meta-analysis of five studies in asians targeting the renin-angiotensin system as novel therapeutic strategy for pulmonary diseases renin-angiotensinaldosterone system inhibitors in patients with covid-19 angiotensin-converting enzyme inhibitors and bacterial pneumonia in patients with parkinson disease acute respiratory distress syndrome leads to reduced ratio of ace/ace2 activities and is prevented by angiotensin-(1-7) or an angiotensin ii receptor antagonist cryo-em structure of the 2019-ncov spike in the prefusion conformation evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay structural basis for the recognition of the 2019-ncov by human ace2 angiotensin-converting enzyme 2 (ace2) mediates influenza h7n9 virus-induced acute lung injury angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target covid-19 and the cardiovascular system a pneumonia outbreak associated with a new coronavirus of probable bat origin key: cord-300850-59j1m2tm authors: peron, jean pierre schatzmann; nakaya, helder title: susceptibility of the elderly to sars-cov-2 infection: ace-2 overexpression, shedding, and antibody-dependent enhancement (ade) date: 2020-05-11 journal: clinics (sao paulo) doi: 10.6061/clinics/2020/e1912 sha: doc_id: 300850 cord_uid: 59j1m2tm the world is currently facing a serious sars-cov-2 infection pandemic. this virus is a new isolate of coronavirus, and the current infection crisis has surpassed the sars and mers epidemics that occurred in 2002 and 2013, respectively. sars-cov-2 has currently infected more than 142,000 people, causing 5,000 deaths and spreading across more than 130 countries worldwide. the spreading capacity of the virus clearly demonstrates the potential threat of respiratory viruses to human health, thereby reiterating to the governments around the world that preventive health policies and scientific research are pivotal to overcoming the crisis. coronavirus disease (covid-19) causes flu-like symptoms in most cases. however, approximately 15% of the patients need hospitalization, and 5% require assisted ventilation, depending on the cohorts studied. what is intriguing, however, is the higher susceptibility of the elderly, especially individuals who are older than 60 years of age, and have comorbidities, including hypertension, diabetes, and heart disease. in fact, the death rate in this group may be up to 10-12%. interestingly, children are somehow less susceptible and are not considered as a risk group. therefore, in this review, we discuss some possible molecular and cellular mechanisms by virtue of which the elderly subjects may be more susceptible to severe covid-19. toward this, we raise two main points, i) increased ace-2 expression in pulmonary and heart tissues in users of chronic angiotensin 1 receptor (at1r) blockers; and ii) antibody-dependent enhancement (ade) after previous exposure to other circulating coronaviruses. we believe that these points are pivotal for a better understanding of the pathogenesis of severe covid-19, and must be carefully addressed by physicians and scientists in the field. the world is facing a major public health crisis due to the pandemic caused by a recently-described coronavirus, named sars-cov-2 (1) (2) (3) . reaching proportions that far surpass those of sars and mers, the sars-cov-2 epidemic started in wuhan, china in december 2019, but has now spread to more than 130 countries worldwide and has infected approximately 142,000 people, with more than 5,000 deaths being attributed to it (who, march 13 th 2020) (4) . sequencing analysis of the viral genome has revealed mutations in the spike protein-which is essential for sars-cov-2 attachment and invasion into host cells-may have favored the spill over from bats to humans (1) . most patients infected with coronaviruses develop a mild flu-like disease, in which the most common symptoms are fever and cough. however, in a study of 1,099 patients from 552 hospitals from 30 provinces of china in 2020, guan w et al. (5) , revealed that 15.7% of the patients who develop severe disease have increased difficulty in breathing because of pneumonia. radiological imaging of the lungs revealed opacity in 56.4% of the patients. approximately 2.7% of the patients needed assisted ventilation, and 1.4% died (1) . however, coronavirus disease (covid-19) may rapidly develop into severe acute respiratory syndrome (sars) in elderly subjects (460 yr), especially in those with comorbidities, such as hypertension, diabetes, and pulmonary diseases (1, 4, 6) . what is intriguing is that, unlike in the case of influenza (7), children are not included in the risk group, as very few cases of severe covid-19 in children have been reported, and there have been no reports of death in children under the age of 9. this raises questions regarding the cellular and molecular mechanisms associated with the severity of covid-19. understanding and elucidating such mechanisms may greatly improve our knowledge of the pathogenesis of the disease, and thus guide health professionals as to how to better treat the elderly population. toward this, we raise two main points of discussion, i) the increased angiotensin-converting enzyme-2 (ace-2) expression in pulmonary and heart tissues of hypertensive patients with chronic use of at1r blockers and ii) antibody-dependent enhancement (ade) after previous exposure to other circulating coronaviruses. after entering the host-usually through aerosolized viral particles or contact with contaminated surfaces-the virus needs to undergo its biological cycle. spike proteins-that are coded by the s gene in one of the open reading frames of the viral genome-need to interact with viral receptors on the surface of host cells. sars-cov-2 spike proteins bind to angiotensin-converting enzyme-2 (ace-2), which is expressed in the epithelial cells of the lungs (8, 9) . this is the main reason why coronaviruses often cause respiratory disease. notably, ace-2 may also be highly expressed in intestinal tissues (9) , leading to diarrhea, as observed in 60% of the patients during the sars-cov epidemic in 2002. only a few patients with sars-cov-2 infection had diarrhea, although viral particles may be detected in the stool (10) . after attaching to the ace-2 through the receptor-binding domain (rbd) of the s1 and s2 domains of the spike protein, the viral envelope fuses with the host cell membrane and is further internalized. genetic material, a positive rna strand of approximately 20-32 kb, is released into the cytoplasm for replication (1) . thus, the importance of ace-2 expression dynamics for viral infectivity, tropism, and pathogenicity is evident. ace-2, or ace-related carboxypeptidase, is an 805 amino acid transmembrane protein which is an important member of the renin-angiotensin system that plays a pivotal role in the regulation of blood pressure (11) . ace converts angiotensin i (ang i) into angiotensin ii (ang ii), whereas ace-2 converts ang ii into angiotensin 1-7 (ang 1-7) or angiotensin 1-9 (ang 1-9) (12). ang ii and ang 1-7 have antagonizing effects, as ang ii binds to angiotensin 1 receptor (at1r), inducing vasoconstriction and increase in blood pressure, whereas ang 1-7 binds to at2r, leading to vasodilatation and decrease in blood pressure (11, 12) . as the renin-angiotensin system affects blood pressure and kidney function, ace inhibitors and at1r blockers are widely used in hypertensive and cardiac patients (13) . in this context, the chronic use of ace inhibitors or at1r antagonists may be of particular relevance for patients infected with sars-cov-2, as they may alter the dynamics of ace-2 expression and thus increase susceptibility to sars-cov-2 infection. it has already been demonstrated by using hypertensive rat models that at1r blockade elevates ace-2 expression. treatment with losartan and lisinopril, either alone or in combination, significantly increases ace-2 mrna in cardiomyocytes of rats. this is associated with higher ang 1-7 plasma concentrations (14) . this was corroborated by analyzing the heart tissue from rats with myocardial infarction that were treated with losartan (15) . concordantly, olmesartan, a more effective at1r antagonist, significantly increased both cardiac and renal expression of ace-2 in wistar-kyoto rats (16) . this is in agreement with the use of perindopril, an ace inhibitor, in rats (17) . another interesting feature of the dynamics of ace-2 expression in tissues is its ability to be cleaved from the cell surface by a metalloproteinase called adam17 (tace) (18) . the ace-2 ectodomain gets cleaved, releasing soluble ace-2 (sace-2), whose role has not been fully elucidated. however, it has been shown that a higher concentration of sace-2 in the plasma correlates with a poorer prognosis after heart failure (19) . notably, sace-2 may also be detected at higher concentrations in the cerebrospinal fluid in hypertensive patients. this has been corroborated in nefh cre x at1ar flox/flox mice, indicating that ace-2 and sace-2 levels in the brain are associated with the etiology of neurogenic hypertension (20) . further, it was demonstrated that ace-2 may be cleaved by other mechanisms. for instance, the sars spike protein may modulate adam17 expression, which in turn cleaves ace-2 into its soluble form. this was found to be dependent on the cytoplasmic domain of ace-2, as sirna against adam17 or ace-2 lacking intracellular domains abrogated this phenomenon (21) . interestingly, increased shedding of ace-2 correlates with worsening of the disease, probably because of an increase in ang ii instead of ang 1-7. this leads to increased vascular permeability and local inflammation. interestingly, ace-2 cleavage from the cell surface may also occur in lung epithelial cells (22) . thus, cleavage of ace-2 into sace-2 would compromise the effect of ang 1-7 on at2r, thereby reducing pulmonary hypertension and inflammation. altogether, as depicted in figure 1 , these data show that modulating the renin-angiotensin axis alters ace-2 expression in several tissues, especially in the lung and heart tissues. the use of ace inhibitors and at1r antagonists seems to upregulate ace-2 expression, facilitating viral attachment and entry. the further presence of the virus itself or some cytokines, including tnf-a, leads to ace-2 release from the cell membrane, abrogating its function to counteract ang ii. altogether, ace-2 overexpression may facilitate viral replication in lung tissue and promote lung vascular permeability, a common feature of severe sars-cov-2 infection. in summary, this may greatly impact the outcome of sars-cov-2 infection in elderly and hypertensive patients, as ace-2 is the putative attachment and invasion receptor for coronaviruses (8, 23) . in fact, the use of tace inhibitors as sars antiviral agents has already been proposed in experimental models (2, 3, 24) . ' antibody-dependent enhancement ade is a phenomenon by which viruses use preexisting non-neutralizing antibodies from previous exposure to invade host cells through fc receptor-mediated internalization. this is most commonly observed during secondary dengue virus (denv) infection, causing severe hemorrhagic disease (25) . notably, the possibility of ade between zika virus (zikv), the causative agent of zikv congenital syndrome (26) , and denv has been intensely debated recently (27) (28) (29) . moreover, ade has been the focus of debate for ebola (30) and hiv (31) infections. during ade, preexisting antibodies elicited during a previous viral exposure are not able to neutralize viral particles during a secondary infection with any antigenicrelated virus. instead, igg-opsonized viral particles then target fcgr expressed on endothelial and immune cells, facilitating viral internalization into host cells. further, after intense viral replication, endothelial cells may respond by increasing vascular permeability and allowing exudate extravasation and bleeding. however, monocytes become highly activated and may initiate a cytokine storm (32) . in the context of sars-cov-2, it is plausible to think that ade occurs. as mentioned in the introduction section, elderly (460 yr) patients are more susceptible to infection for unknown reasons. as coronaviruses in general are highly prevalent in the global population, causing mild infection and flu-like symptoms, seroconversion into previous circulating coronaviruses is probably widespread. thus, it is reasonable to infer that elderly patients, for obvious reasons, have been exposed to previous infections more times than younger subjects. this would imply a vaster repertoire of antibodies against coronavirus epitopes produced by longliving plasma cells, which in fact has recently been shown to expand during sars-cov-2 infection. however, whether these antibodies are either neutralizers or enhancers must be further addressed. it is worth mentioning that, concerning the new sars-cov-2, sequencing analysis of the viral genome isolated in wuhan, china indicated that mutations mainly occurred within the coding sequence of the spike protein, which has less than 40% sequence identity with that of previously circulated coronaviruses (1). these mutations may be responsible not only for the spill over from bats to humans, but also for inducing ade, as changes in spike epitopes may result in interactions with non-neutralizing antibodies. corroborating this, a novel epitope, which was lacking in previous isolates, was mapped (33) . as covid-19 is not a hemorrhagic disease, it is probable that ade, if present, is not mediated by endothelial cells. however, it has already been shown that lung epithelial cells express high levels of fcgriia (34) . moreover, immune cells, including monocytes and dendritic cells, highly express this receptor. these populations, especially monocytes, greatly account for the inflammatory infiltrate in the lungs during pneumonia, which is consistent with the transient lymphopenia observed in patients as circulating cells may migrate to the lungs. conversely, lung imaging of patients with severe covid-19 shows a great degree of lung opacity, which is consistent with edema and cellular infiltrate (5) . thus, it is feasible that lung-infiltrating monocytes expressing fcgr greatly favor sars-cov-2 replication in the lung tissue, accounting for the greater susceptibility of the elderly patients. corroborating this hypothesis, young individuals and children do not represent the risk group for severe disease, which greatly differs from the case of influenza (7), for instance. in the context of ade, it is plausible to think that children-as they had less or no exposure to previous circulating coronaviruses-carry a very restricted repertoire of igg-or only low-affinity igm-which is not capable of inducing ade. in this context, mapping complementarity determining regions (cdrs) in iggs from young and elderly individuals would be of great importance not only to find neutralizing antibodies but also to address this hypothesis. additionally, previous studies on mers and sars have already highlighted the possibility of ade. a recent report published by wan y et al. elucidated the mechanism by which monoclonal antibodies (mabs) induce ade in human cells. interestingly, mabs that target the rbd of sars and mers spike proteins induced conformational changes in the protein that favors an interaction with dipeptidyl peptidase 4 (dpp4), the receptor for mers (38) . moreover, immunocomplexes also promoted viral entry. however, increasing concentrations of antibodies block viral invasion, as rbds become inaccessible. this is consistent with previous findings by wang q et al. (39) who infected the promonocytic cell line hl-cz-that expresses both ace-2 and fcgr-with sars-cov-2 in the presence of increasing concentrations of anti-sera. their data demonstrated that high concentrations of antibodies neutralized the virus, whereas lower concentrations induced ade. more interestingly, the anti-sera also recognized spike protein-related antigens. another study on sars-cov-2 using rhesus monkeys showed that immunization with full-length spike glycoprotein led to increased disease severity, mostly to because of an increase in neutralizing antibodies (nab) (35) . the study demonstrated that nabs switched the phenotype of lunginfiltrating macrophages to a pro-inflammatory m1 profile, instead of the tissue-healing profile m2. this aggravated lung injury and greatly contributed to its pathology. conversely, previous studies using samples from deceased patients suffering from sars-cov infection indicated that nab titers reached higher levels earlier in these patients, compared to that in patients who survived (36) . this may indicate that ade and the role of preexisting antibodies are in fact very relevant to the overall outcome of the infection. thus, as indicated in figure 2 , circulating antibodies, instead of neutralizing the current circulating sars-cov-2, may bind to viral particles and thus promote fc-mediated internalization by lung epithelial cells and infiltrating monocytes, contributing to the worsening of covid-19. these are the most recent and mechanistic studies on ade of coronaviruses thus far. although the data are consistent, whether the phenomenon of ade is observed in patients with severe covid-19 is yet to be determined. noteworthy, however, is the fact that immune complexes of low-avidity antibodies at sub-optimal concentrations were also responsible for the worsening of the pulmonary disease caused by h1n1 during the 2009 epidemic (37) . ' conclusions sars-cov-2 is the newest threat to human health and needs emergency action from governments and public health agencies worldwide, especially because of its pandemic potential as recently declared by the world health organization (who). in this context, it is essential to rapidly and deeply address all the possibilities concerning the severity of infection, especially in the elderly population that accounts for approximately 10-12% of the mortality rate. here, we present several relevant aspects that may contribute to the increased susceptibility of the aforementioned population to covid-19. we believe that i) increased expression of ace-2 in hypertensive patients being treated with ace inhibitors and at1r blockers and ii) previous exposure to circulating coronaviruses with low neutralizing capacity to sars-cov-2 may greatly contribute to the increased susceptibility of the elderly patients to covid-19. to determine whether these hypotheses are correct, further investigations are needed, not only to better understand the etiology of the current sars-cov-2 infection, but also to be better prepared for future epidemics. ' author contributions peron jps conceived and wrote the manuscript. nakaya h helped editing the manuscript. a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical characteristics of coronavirus disease 2019 in china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease global burden of respiratory infections associated with seasonal influenza in children under 5 years in 2018: a systematic review and modelling study angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore angiotensin-converting enzyme 2 is an essential regulator of heart function angiotensinconverting enzyme 2 and angiotensin 1-7: novel therapeutic targets anti-hypertensive medication combinations in the united states effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors olmesartan is an angiotensin ii receptor blocker with an inhibitory effect on angiotensin-converting enzyme upregulation of angiotensin-converting enzyme (ace) 2 in hepatic fibrosis by ace inhibitors tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndromecoronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) detection of soluble angiotensin-converting enzyme 2 in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensinaldosterone system clinical relevance and role of neuronal at 1 receptors in adam17-mediated ace2 shedding in neurogenic hypertension modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace2 induces tnf-alpha production and facilitates viral entry ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus tace antagonists blocking ace2 shedding caused by the spike protein of sars-cov are candidate antiviral compounds inhibition of dengue and chikungunya virus infections by rig-i-mediated type i interferon-independent stimulation of the innate antiviral response the brazilian zika virus strain causes birth defects in experimental models enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity dengue virus sero-cross-reactivity drives antibodydependent enhancement of infection with zika virus viral load and cytokine response profile does not support antibody-dependent enhancement in dengue-primed zika virus-infected patients antibody-dependent enhancement of ebola virus infection by human antibodies isolated from survivors extensive complement-dependent enhancement of hiv-1 by autologous non-neutralising antibodies at early stages of infection highlights for dengue immunopathogenesis: antibody-dependent enhancement, cytokine storm, and beyond functions of tam rtks in regulating spermatogenesis and male fertility in mice anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection antibody responses against sars coronavirus are correlated with disease outcome of infected individuals severe pandemic 2009 h1n1 influenza disease due to pathogenic immune complexes dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates key: cord-266755-y2lf7ssp authors: yehualashet, awgichew shewasinad; belachew, teshome fentik title: aceis and arbs and their correlation with covid-19: a review date: 2020-09-16 journal: infect drug resist doi: 10.2147/idr.s264882 sha: doc_id: 266755 cord_uid: y2lf7ssp although some animal studies suggested that the use of aceis/arbs could contribute for the prevention and treatment of the effects of the covid-19 infection, there are also contradictory scenarios indicating that their use may exacerbate the deleterious conditions of the infection. as a result of the paradoxical issue of using aceis/arbs during covid-19, it is still an area requiring extended investigation to prove. additionally, a trial evidence of their efficacy and the possible benefit risk analysis of these conventional drugs during covid-19 in connection with other comorbidities like hypertension, heart failure, and renal disease associated with diabetes should also be addressed. coronaviruses (covs) of the family coronaviridae and subfamily corovirinae are positive-sense, single-stranded rna viruses that affect a wide range of hots with diseases ranging from common cold to severe/fatal illnesses. the novel virus was initially termed as 2019-ncov which later changed to "sars-cov-2" by the coronavirus study group (csg) of international committee on taxonomy of viruses (ictv) because of the higher resemblance of the virus with severe acute respiratory syndrome coronavirus (sars-cov). 1 the continued coronavirus infection that was emerged in china has rapidly spread to each corner and led a declaration to be considered as a global health emergency by the world health organization (who). although there are many efforts for the provision of suitable therapeutic approaches, there are no vaccines or direct-acting antiviral delivers to those infected with the virus. 2 right after the first reported case of death on the first week of january 2020 was confirmed as a result of the 2019 novel coronavirus (2019-ncov; later renamed severe acute respiratory syndrome coronavirus 2 [sarscov-2]), the end of this month it was declared as an outbreak by who and subsequently named as coronavirus disease . globally, as of 5 august 2020, there have been 18,318,928 confirmed cases of covid-19, including 695,043 deaths, reported to who. 3, 4 findings revealed that the genome sequence identity of sars cov 2 and bat cov is about 96.2%, and based on virus genome sequencing results and evolutionary analysis, bat has been blamed as natural host of the virus sarscov-2 to transmit from bats via unknown intermediate hosts to infect humans. it was identified that sars-cov-2 uses angiotensin-converting enzyme 2 (ace2), the same receptor as sars-cov to infect humans. 5, 6 direct contact with intermediate host animals or consumption of wild animals was suspected to be the main route of sars-cov-2 transmission and the sources and mode of transmission is still open for further investigation. 6 virus-host interactions affect viral entry and replication. sars-cov-2 is an enveloped positive single-stranded rna (ssrna) coronavirus. about two-thirds of viral rna, mainly located in the first open reading frame (orf 1a/b), encode 16 non-structure proteins (nsps). the remaining part of the virus genome encodes four essential structural proteins, including spike (s) glycoprotein, small envelope (e) protein, matrix (m) protein, and nucleocapsid (n) protein, and also several accessory proteins. the s glycoprotein of sars-cov-2 binds to host cell receptors, ace2 which is assumed to be a critical step for the entry of the virus. however, the possible molecules facilitated membrane invaginations for sars-cov-2 endocytosis are still unclear and other virus proteins may contribute to pathogenesis. 6 to date, the 2019 novel coronavirus (2019-ncov) outbreak is being a major concern of the scientific community to treat with potential medications. becuase there is lack of effective and scientifically proven antiviral therapy against covid-19, the current treatment approaches mainly focused on the symptomatic and respiratory support based up on the diagnosis and treatment of pneumonia caused by oxygen therapy is almost applied to all patients with the covid-19 and who also recommended extracorporeal membrane oxygenation (ecmo) to patients with refractory hypoxemia. to certain critical case immunoglobulin g and convalescent plasma are used to save the life of the patients as per their condition. 3, 8 according to literature searched on web, few drugs other than vaccines are enumerated as unproven optional and adjuvant drugs for covid-19 and other coronaviruses. these include antivirals (opinavir/ritonavir, remdesivir), chloroquine/hydroxychloroquine, and interferon, tocilizumab, corticosteroids, antibiotic therapy, nsaids/ ibuprofen, and some others. 9 previous studies reported that chloroquine and hydroxychloroquine possess a broad spectrum of antiviral effects on a variety of viruses as diverse as human immunodeficiency virus (hiv), marburg virus, zika virus, dengue virus, ebola virus, and sars-cov-1, etc. in addition, chloroquine and hydroxychloroquine can regulate immune system by affecting cell signaling and production of pro-inflammatory cytokines. [10] [11] [12] [13] chloroquine and hydroxychloroquine have been shown by several studies to reduce the sars-cov-2 viral load and shorten the duration of viremia. although the immunomodulatory effect of these drugs also plays a role in the treatment of covid-19, there is still a huge need for further investigation. for coronaviruses, the potential therapeutic benefits of chloroquine were notably reported for sars-cov-1. in vitro, chloroquine can prevent sars-cov-1 from infecting the glycosylation of a virus cell surface receptor, ace2. 7, 13 remdesivir has been reported to treat the first us case of covid-19 successfully. results obtained from the recently conducted in vitro study against covid-19 are promising since the drugs remdesivir and chloroquine were found to be highly effective in controlling the infection. even though in vitro studies showed that remdesivir and chloroquine are highly effective in the control of 2019-ncov infection, these compounds have been used in human patients with a safety track record and shown to be effective against various ailments. 14,15 the pathophysiology and virulence mechanisms of sars-cov-2 have a linkage with the function of the nsps (nonstructural proteins) and structural proteins. nsp is able to block the host innate immune response. among the structural elements of covs, there are the spike glycoproteins which have two subunits; one subunit, s1, binds to the receptors on the cell surface; the other subunit, s2, fuses with the cell membrane. for many covs, s is cleaved at the boundary between the s1 and s2 subunits, which remain non-covalently bound in the prefusion conformation. 16, 17 the s1 subunit comprises the receptorbinding domain(s) and contributes to stabilization of the prefusion state of the membrane-anchored s2 subunit that contains the fusion machinery. for all covs, s is further cleaved by host proteases at the so-called s20 site located immediately upstream of the fusion peptide. 16, 18 this cleavage proposed to activate the protein for membrane fusion via extensive irreversible conformational changes. 19 the entry of coronavirus into susceptible cells is something which is complicated and requires the great effort in understanding the action of receptor-binding and proteolytic processing of the s protein to promote virus-cell fusion. sars-cov and several sars-related coronaviruses (sarsr-cov) interact directly with ace2 via s b to enter to the target. studies showed that ace2 is highly expressed in the mouth and tongue, facilitating viral entry in the host. in human lungs, ace2 is expressed in lower lungs on type i and ii alveolar epithelial cells. after infection, sars-cov-2 entry starts with the attachment of the spike glycoprotein expressed on the viral envelope to ace2 on the alveolar surface. the attachment of sarscov-2 to ace2 modulates the clathrin-dependent endocytosis of the sars-cov-2 and ace2 complex, inducing fusion at the cell membrane. once enter the cells, sars-cov-2 exploits the endogenous transcriptional machinery of alveolar cells to replicate it and spreads through the entire lung. 20 ace2 could assist sars-cov-2 s-mediated entry into cells, thereby establishing it as a functional receptor for this newly emerged coronavirus, covid-19. the sars-cov2 s b engages human ace2 (hace2) with higher affinity than to sars-cov s b. 21, 22 both ace-1 and ace-2 cleave angiotensin peptides in that ace-1 cleaves angiotensin i and generating angiotensin (ang) ii, which causes vasoconstriction, bronchoconstriction, increases vascular permeability, inflammation, and fibrosis and enhance the development of acute respiratory disease syndrome (ards) and lung failure in patients infected with sars-cov-2. the effect of ace1 signaling is by the gpcrs; at1 and at2 receptors. ace1-generated at1 receptors function as the key mediator of ang ii actions and the opposing actions of ace2-derived peptides. ace2 is a carboxypeptidase (zinc metalloprotease), which is responsible for ang ii degradation to ang (1-7). 23 the conversion of ang ii to ang (1-7) by the enzyme ace2 produces effects that oppose the action of ang ii mediated by at1. the sars-cov-2 virus infects alveolar pneumocytes by binding to ace2 (as in figure 1 ), leading to a decrease in ang ii conversion to ace2-derived peptides, for example, a reduction in ang (1-7) and its actions that counteract effects of ang ii. higher imbalance between the action of ace1 and ace2 aggravates pathology, making it more likely that the immune response will be overcome. blunting the ace-1-ang ii-at1r enhances the action of ace-1yehualashet and belachew infection and drug resistance 2020:13 submit your manuscript | www.dovepress.com ang ii-at2r, the ace-2-ang (1-7)-at2r or the ace-2-ang (1-7) which likely protects from ards triggered by infectious pathogens, including coronaviruses. 24, 25 ace2 is expressed at apical plasma membranes of epithelial cells, including those of respiratory origin, the primary location of sars-cov infection. 26 during the infection of sars-cov, the cell surface expression of ace2 down-regulated through internalization of the receptor-ligand complex or activation of tace-mediated ectodomain shedding of soluble ace2. after attachment of viral spike proteins with ace2, the amount of cell surface-expressed ace2 is reduced. ace2 receptor downregulation provokes a worsening effect of lung failure. 27, 28 in addition to the alveolar cells in the lungs, ace2 is expressed in other organs, including the kidney, the heart, and the gut. whether robust ace2 expression in these organs affects sars-cov-2 infectivity remains illdefined. acute kidney injury (aki), cardiac damage, and abdominal pain are the most common co-morbidities of covid-19. 20 angiotensin-converting enzyme inhibitors (aceis) and angiotensin receptor blockers (arbs) have a different mechanism in the pathogenesis of covid-19. as depicted in figure 1 aceis in clinical use do not directly affect ace2 activity 23 and the intestinal messenger rna level of ace2 increased with acei but not observed with arbs. sars-cov-2 down-regulates ace2 expression that decreases protective effects of ace2 on different organs. 29 aceis increased the entrance more than twice the entrance with arbs. this cannot definitely conclude risks or benefits of these therapies with patients confounding variables like age, hypertension, and impact of yet-unidentified comorbidities on outcome with the covid-19 pandemic. 30, 31 in the other way, aceis or arbs could prevent covid-19 viral entry by stabilizing ace2-at1r interaction and preventing viral protein ace2 interaction and internalization. the interaction between viral protein and ace2 decreased in the presence of stabilized ace2-at1r complexes. 31 aceis, the first drugs targeting the raas (renin angiotensin aldosterone system), are used for the treatment of a wide range of indications related to hypertension, cardiovascular disease, and renal disease for over 30 years. aceis and arbs not only lower blood pressure but also may possess unique cardioprotective properties. 32 they improve endothelial function and regress both left ventricular hypertrophy and arterial mass. they also reduce rates of death, myocardial infarction, stroke, cardiac arrest, and revascularization procedures. 33 aceis have been shown to protect against oxidative stress and prevent glycosylation of proteins, which may confer cardiovascular benefit. acei or arb also used for conditions associated with insulin resistance, such as metabolic syndrome, hypertension, impaired fasting glucose, family history of diabetes, obesity, congestive heart failure, or other risks for the development of type 2 diabetes. [33] [34] [35] the renin-angiotensin system (ras) is a homeostatic regulator signaling pathway participated in the control of vascular function. the regulation of blood pressure, natriuresis, and blood volume recruits this signaling pathway. this system also has a local role in the regulation of regional blood flow and controlling our body's responses to a range of stimuli. 36 following a rapid increase in the cases of covid-19, different observational studies are undertaken to identify the possible risk factors for infection and related poor outcomes. with that yang et al, 17% diabetes cases were reported among 52 critically ill patients with covid-19 37 and 16.2% dm and 23.7% hypertension cases were identified from 1099 patients by guan et al. 38 similarly, out of 140 hospitalized patients, it was reported by yang et al that 12% dm cases and 30% htn cases were observed in critically ill patients with covid-19. 37 the large percentage of patients with hypertension undertaking aceis or arbs was a remarkable characteristic among those presenting severe covid-19 manifestations. these are classes of anti-hypertensive massively used as first-line therapy due to their additional kidney and cardiologic protections that occur regardless of their use for blood pressure control. 39 ace2 is established as a functional receptor for the entry of sars-cov-2 s-into cells. considering the pathophysiologic mechanisms and possible drug targets the scientific community is intensively striving to develop therapeutics for the current pandemic covid-19. therefore, the driving force of this review is to discuss the correlation between aceis/ arbs with covid-19 considering the existing scientific evidence. ace2 is a type 1 integral membrane glycoprotein which is expressed and identified to be active in most tissues. the highest expression of ace2 is observed in the kidney, the endothelium, the lungs, and in the heart. 36 the probable rational proposed for the possible relation between the use of aceis/arbs, and progression to ards in covid-19 is the increased availability of ace-2 attached to surface in the lung endothelium, an inherent effect of these two classes, leading to enhanced coupling of sars-cov2 to ace-2 and its consequent cell entry. indeed, the receptor-binding domain (rbd) of the sars-cov2 has a stronger interaction with ace-2, compared to other viruses from the same family, and any increase of ace-2 expression may potentially amplify the virus capacity to entry the cells. 40, 41 in the case of acei's, the reduced angiotensin ii levels increase the proportion of ace-2 uncoupled to this molecule, which would act transforming angiotensin ii into angiotensin 1-7. the overcompensation of the ace-2 action of transforming angiotensin i into angiotensin 1-9, which may be enhanced under the use of acei's, since angiotensin i is increased, does not fully compensate the lack of coupling of the attached ace-2. distinctly, arbs increase angiotensin ii availability since this class blocks its coupling with at2 receptor, leading to compensatory up-regulation of ace-2 in the membrane. studies differentiating the risk of ards and related pulmonary complications between acei's and arbs lack, precluding from a comparative analysis of the clinical outcomes between these two classes. 40, 42 while expression and availability of attached ace-2 is directly correlated with covid-19 severity, the free circulating form of ace-2 may inactivate sars-cov2, and preclude from its entry in the pulmonary endothelium, and has been proposed that recombinant human soluble ace-2 could act as a potential molecule to protect from the development of severe manifestations, ards, and death in covid-19. hence, a speculative ratio between attached ace-2 availability and expression, and freely circulating ace-2 could predict the lung pathogenicity of covid-19. 43, 44 according to yang et al (2020), higher prevalence of hypertension was observed in patients with severe covid-19; however, a concluding remark has not been made in that the severity of the infection has a link with hypertension. similarly, no data were reported with respect to the use of aceis and arbs for the infection. guo et al (2020) , in evaluating cardiovascular implications of covid-19 infection, the use of aceis/arbs did not show any association with mortality. 37, 45 but previous animal studies showed that aceis and arb increase ace2 activity. based on prior animal studies, it was suggested that proposed aceis and arbs can enhance ace2 activity and thereby increase infectivity of covid-19 virus. other studies in mice and humans showed in contrary to the above animal studies. ace2 mrna expression in rat heart cells treated with an acei 7 and ace2 activity in the presence of either acei or arbs in humans, in both scenarios the level of ace2 does not show any change. 46 as per fang et al (2020), it was hypothesized that the use of ace2 receptor increasing drugs is at higher risk for severe covid-19 infection. acei initially inhibits ace leading to decreased angiotensin i levels, causing a possible negative feedback loop that ultimately upregulates more ace2 receptor to be able to interact with the decreased angiotensin i substrate available. this ace2 receptor up-regulation results in increased binding sites for sars-cov-2, leading to preferential covid-19 infection. this is particularly observed in patients with diabetes and/ or hypertension since they are usually taking aceis/arb. it was, therefore, suggested that patients with cardiac diseases, hypertension, or diabetes, who are treated with ace2-increasing drugs, are at higher risk for severe covid-19 infection and, therefore, should be monitored for ace2-modulating medications, such as aceis/ arbs. 47 on the contrary, some studies indicated that aceis/ arb use may be beneficial in covid-19 infection prevention. because of a proposed mechanism that acei inhibition of ace may stimulate a negative feedback (given the lack of angiotensin ii, up-regulating ace2 receptors and decreasing overall inflammation). 48 in severe lung injury animal models, preclinical studies have showed that ace2 is significantly downregulated and it has been shown that the inhibition of the angiotensin type 1 receptor by arb like losartan reduces severe acute lung injury in mice administered with the spike glycoprotein of sars-cov. 49, 50 the above complementary approach reflects that ace2 is protective in lung injury during infection of coronavirus. based on this, there are ongoing trials studying the effect of losartan (an arb) in patients with covid-19 in outpatient and inpatient settings. 51, 52 in order to determine the influence of the ras system and active treatments on sars-cov-2 infection and on depending on the current evidence available in the web, it is difficult to confirm the co-relation of aceis/arb and their use during covid-19 either to be beneficial or harmful. therefore, much more research is highly in need to better elaborate the correlation of the raas with sars-cov2 infection. it is also very challenging to suggest on the consequences of using aceis/arbs in patients infected with covid-19 because of the complexity of the ras system and lack of evidence in human to support the hypothesis. 54 so that whether the benefits of ace-1 inhibitors or arbs during an episode of infection with sars-cov-2 outweigh the potential risk or not, require further investigation. although some animal studies suggested that their use could have value in preventing and treating the effects of the covid-19, there are also contradictory scenarios suggesting that the use of aceis/arbs may exacerbate the deleterious conditions of the infection. as a result of the paradoxical issue of using aceis/arbs during covid-19, it is still an area requiring investigation to prove. additionally, a trial evidence of their efficacy and the possible benefit-risk analysis of these conventional drugs during covid-19 in connection with other comorbidities like hypertension, heart failure, and renal disease associated with diabetes mellitus should also be addressed. both authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. an update on sars-cov-2/ covid-19 with particular reference on its clinical pathology, pathogenesis, immunopathology and mitigation strategies-a review clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment clinical management of severe acute respiratory infection when novel coronavirus (ncov) 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covid-19 pandemic? drug resistance is an international, peer-reviewed openaccess journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. the journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. the manuscript management system is completely online and includes a very quick and fair peerreview system the authors report no conflicts of interest for this work. key: cord-332680-zfn81hew authors: chan, chieh-kai; huang, yu-shan; liao, hung-wei; tsai, i-jung; sun, chiao-yin; pan, heng-chih; chueh, jeff s.; wang, jann-tay; wu, vin-cent; chu, tzong-shinn title: renin-angiotensin-aldosterone system inhibitors and risks of severe acute respiratory syndrome coronavirus 2 infection: a systematic review and meta-analysis date: 2020-09-10 journal: hypertension doi: 10.1161/hypertensionaha.120.15989 sha: doc_id: 332680 cord_uid: zfn81hew the viral spike coat protein of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) engages the human ace (angiotensin-converting enzyme)2 cell surface receptor to infect the host cells. thus, concerns arose regarding theoretically higher risk for coronavirus disease-19 (covid-19) in patients taking ace inhibitors/angiotensin ii type 1 receptor antagonists (angiotensin receptor blockers [arbs]). we systematically assessed case-population and cohort studies from medline (ovid), cochrane database of systematic reviews pubmed, embase, medrxiv, the world health organization database of covid-19 publications, and clinicaltrials.gov through june 1, 2020, with planned ongoing surveillance. we rated the certainty of evidence according to cochrane methods and the grade approach. after pooling the adjusted odds ratios from the included studies, no significant increase was noted in the risk of sars-cov-2 infection by the use of aceinhibitors (adjusted odds ratio, 0.95 [95% ci, 0.86–1.05]) or arbs (adjusted odds ratio, 1.05 [95% ci, 0.97–1.14]). however, the random-effects meta-regression revealed that age may modify the sars-cov-2 infection risk in subjects with the use of arbs (coefficient, −0.006 [95% ci, −0.016 to 0.004]), that is, the use of arbs, as opposed to ace inhibitors, specifically augmented the risk of sars-cov-2 infection in younger subjects (<60 yearsold). the use of ace inhibitors might not increase the susceptibility of sars-cov-2 infection, severity of disease, and mortality in case-population and cohort studies. additionally, we discovered for the first time that the use of arbs, as opposed to ace inhibitors, specifically augmented the risk of sars-cov-2 infection in younger subjects, without obvious effects on covid-19 outcomes. sars-cov-2 test positivity. [7] [8] [9] [10] [11] mancia et al. found that ace inhibitors and arbs were more frequently taken by patients with covid-198 than by controls because of their higher prevalence of cardiovascular disease, but there was no evidence that ace inhibitors or arbs affected the risk of covid19 . the effect of raasi on the risk of sars-cov-2 infection could complicate clinical patient management. a possible scenario is that in patients with hypertension a raasi might be no longer organ-protective, and could even be potentially harmful due to the activation of ace2 during the covid-19 pandemic. carefully curated and reliable data from an all-inclusive systemic review may shed light on this important concern timely when a well-designed clinical trial is not available. in this study, we aimed to clarify the association between the use of ace inhibitors or arbs and the risk of sars-cov-2 infection. the medical literature regarding the impact of the use of ace inhibitors or arbs on sars-cov-2 infection was systematically reviewed, and a meta-analysis was performed to provide comprehensive evidence of the effect of raasi on the susceptibility to sars-cov-2 infection. data, analytic methods, and study materials are given below. further details are available in the data supplement and on reasonable request from the corresponding author. we followed the recommendations of the preferred reporting items for systematic reviews and meta-analyses statement 12 and used cochrane methods. 13 we prospectively submitted the systematic review protocol for registration on prospero (https://www.crd. york.ac.uk/prospero/; crd42020190666; appendix pp 20-29). the systematic search was performed by searching all publications from january 1, 2020 to june 1, 2020, in medline (ovid), cinahl(ovid), cochrane database of systematic reviews pubmed, embase, medrxiv, cnki.net, world health organization database of covid-19 publications, and clinicaltrials.gov to june 1, 2020, with planned ongoing surveillance that investigated the association between the use of raasi and the risk of sars-cov-2 infection. two investigators (c.-k. chan and y.s. huang) who performed the literature search also independently extracted the data from the included studies using a standardized data spreadsheet. discrepancies were resolved through consensus or referral to a third reviewer (v.-c. wu). the following variables were extracted: author, journal, publication year, study design, geographic location, participants' details (number, study population, age, sex, and comorbidities, including hypertension, diabetes mellitus, heart failure, and chronic kidney disease), use of antihypertensive drugs, such as ace inhibitors, arbs, calcium-channel blockers, beta-blockers, diuretics, outcomes (including positive sars-cov-2 test results and disease prognosis/severity, if available). study authors were not contacted for additional information. the quality of the included studies was assessed independently by 2 investigators (c.-k. chan and i.-j. tsai) using the newcastle-ottawa quality assessment scale scoring system for comparative nonrandomized studies corresponding with all studies design. 14 studies with a newcastle-ottawa quality assessment scale score of ≥7 were regarded as high quality. two investigators (h.-c. pan and i.-j. tsai) independently assessed the risk of bias of each included study with the updated version of the cochrane risk of bias tool. 15 we used the gradepro app to rate evidence and present it in grade evidence profiles and summary of findings tables. 15 we formally assessed the credibility of potential effect-modifiers using grade guidance. 16 in addition, we included the studies extracted from the medrxiv (under peer review but also visible in pubmed) because we want to explore the whole picture of the raasi with all available data. the primary outcome was the positive sars-cov-2 infection. secondary outcomes were the severe infection (including ventilator use or intensive care unit admission) or mortality of covid-19. crude odds ratio and the 95% ci were directly calculated when the 2 by 2 cross-table was provided in all of the included studies. in contrast, adjusted odds ratio (aor) and the 95% ci was also extracted. the pooled aor for binary outcomes (ie, sars-cov-2 infection) was calculated in this meta-analysis. the data from individual studies were pooled using the dersimonian and laird random-effect model. inconsistency across studies was assessed using the i 2 statistics in which a value >50% indicated a substantial heterogeneity. the quantitative meta-analysis was conducted using comprehensive meta-analysis version 3.3.070 (biostat). the preferred reporting items for systematic reviews and meta-analyses flow diagram of the meta-analysis is shown in figure s1 in the data supplement and the preferred reporting items for systematic reviews and meta-analyses checklist is shown in appendix pp 10-12. the search strategy identified 945 studies, of which 419 articles were duplicates. a total of 491 articles were excluded after reading their titles and abstracts. the remaining 88 articles were evaluated for this meta-analysis. in total, 7 articles, 7-11,17,18 including 4 cohort studies and 3 case-control studies, were eligible for this meta-analysis according to the criteria mentioned above. the respective use of ace inhibitors or arbs records could be separately collected within all the 7 articles. among them, 6 articles 7-11,17 provided the aor of the risk of sars-cov-2 infection, and 4 articles 7-10 provided the aor of the severity or mortality of covid-19. the list of excluded studies and the reasons for exclusion are listed in figure s1 . the patient characteristics of the included studies are shown in table 1 , and the outcomes of included comparative studies are shown in table 2 . the 7 studies were performed in italy, spain, israel, south korea, and the united states. all of the selected studies were published in 2020 with different sample sizes that ranged from 3789 to 65 149 patients. the average or median age of 3 studies 8, 9, 18 was >60 years and that of 4 studies 7,10,11,17 was <60 years. clinical outcomes were defined as the risk of sars-cov-2 infection and the severity/mortality of covid-19 with crude odds ratio and aor in all studies. the results of the quality assessment of the included studies are presented in (table s1 ). the initial crude results showed that either the users of ace inhibitors or arbs had higher risks of sars-cov-2 infection than nonusers (crude odds ratio, 1.21 [95% ci, 1.01-1.45] for ace inhibitors; crude odds ratio, 1.25 [95% ci, 0.99-1.57] for arbs), but with a substantial heterogeneity (both i 2 statistics >75%; figure s2 ). after adjusting for possible confounders, the risk of sars-cov-2 infection in the users of ace inhibitors was similar to that of nonusers (aor, 0.95 [95 % ci, 0.86-1.05]; i 2 statistics=34.7%). although the risk of sars-cov-2 infection in the users of arbs was numerically slightly higher than that of the nonusers, no statistically significant difference was noted (aor, 1.05 [95 % ci, 0.97-1.14]; i 2 statistics=34.9%; figure 1 ). it was noted that the heterogeneity substantially decreased when we pooled the aors. additionally, the sensitivity analysis revealed that the results were not altered when removing anyone's study (data not shown). we first separated the included studies by the mean or median age of 60 years. the results showed that either the older users of ace inhibitors or older users of arbs had similar risks of sars-cov-2 infection than those of the nonusers. the younger users of ace inhibitors also had similar risk of sars-cov-2 infection than that of the nonusers. noticeably, the result indicated that the users of arbs <60-year-old had a mild but statistically higher risk of sars-cov-2 infection than that of nonusers (aor, 1.09 [95% ci, 1.01-1.18]; i 2 statistics=0%; figure 2 ). furthermore, we conducted a meta-regression analysis by treating the continuous mean or median age as an effect modifier. the result also suggested that the age did not modify the effect of ace inhibitors on risks of sars-cov-2 infection (coefficient, −0.006 [95% ci, −0.016 to 0.004]; table s2 and figure 3a) . however, the result showed age significantly modified the effect of arbs on risks of sars-cov-2 infection (coefficient, −0.006 [95% ci, −0.012 to 0.0002]; p=0.042; table s2 and figure 3b ). in addition to age, we also investigated the possible effect modification of other characteristics, including the proportions of male sex, the existence of hypertension, diabetes mellitus, heart failure, or chronic kidney disease. the results showed that these characteristics did not modify the effect of ace inhibitors on the risk of sars-cov-2 infection. regarding the impact of the use of arbs, it was shown that the proportion of hypertension patients might play a role table s2 ). the overall analysis included 4 articles. 7 figure 4 ). in the subgroup analysis, we did not see the effect of the use of ace inhibitors or arbs on the outcomes of covid-19 in older and younger subjects ( figure s5 and figure s6 ). the risks of bias should be addressed as low to moderate in these observational study designs ( table 1) . as seen in figure s3 and figure s4 , the funnel plots showed symmetrical distribution of the adjusted odds ratios, which suggested an absence of publication bias among the included studies. the egger tests also confirmed the absence of publication bias among the included studies for the use of ace inhibitors (p=0.545) and the use of arbs (p=0.265). the systematic review findings of the 7 high-quality studies (with comparative data on the controls) on sars-cov-2 infection provide the best available evidence proving that therapy with ace inhibitors or arbs is not associated with an increase of positive sars-cov-2 test result and the severity of covid-19 disease or overall population mortality as a whole in case-population and cohort studies. however, we also showed for the first time that the usage of arbs, as opposed to ace inhibitors, specifically augmented the adjusted odds ratio of sars-cov-2 infection in younger subjects (<60 years old); thus, we demonstrated that ace inhibitors and arbs might lead to different susceptibility to sars-cov-2 infection in various individual groups. ace indicates angiotensin-converting enzyme; arbs, angiotensin receptor blockers; bmi, body mass index; ckd, chronic kidney disease; dm, diabetes mellitus; htn, hypertension; icu, intensive care unit; n/a, not applicable; oha, oral hypoglycemic agents; raasi, renin-angiotensin-aldosterone system inhibitors; and sars-cov-2, severe acute respiratory syndrome coronavirus 2. the higher susceptibility to sars-cov-2 infection via the usage of arbs seems more obvious in younger subjects, whereas the usage of ace inhibitors with the younger subjects did not show increased risk of sars-cov-2 infection. for younger subjects who require raasi therapy, caution needs to be executed regarding arbs prescribed during the covid-19 era, as arbs might aggravate their risk of sars-cov-2 infection. although the study is observational in nature, this finding was identified from multiple population-based, case-control studies with stringent meta-analysis methodology, and consistent across different populations, sex, and patients with various comorbidities. given that ace2 is the receptor that allows coronavirus entry into cells, pretreatment with raasi, such as ace inhibitors or arbs, might modulate sars-cov-2 infection or compound organ damage. there is no solid data to support the notion that ace inhibitors or arbs administration facilitates coronavirus entry by increasing ace2 expression in either animals or humans. nonetheless, preclinical analysis showed inconsistent findings regarding the effects of raasi on ace2 expression. 19 ace inhibitors could decrease 20 or not affect the activity of ace2, 21 whereas arbs have been shown to augment ace2 expression more consistently at both the mrna and protein level. 19, 22 the increased levels of angiotensin ii occurring after arbs treatment, but not after ace inhibitors, would impose an increased substrate load on ace2, thus leading to its upregulation 23 ; as manifested in patients with hypertension who had been treated with olmesartan (an arb) their urinary level of ace2 was shown higher than those treated with ace inhibitors and other antihypertensives. 24 our finding intriguingly suggests that the effects on ace2 should not be assumed uniform across various subcategories of raasi, especially regarding their respective relationship in affecting sars-cov-2 infection susceptibility. intriguingly, a recently geospatial study from the united states showed that the use of arbs (compared with no-use), but not the use of ace inhibitors, was associated with a higher covid-19 confirmed case rate, but they did not conduct sub-analysis with different age groups. they suggest that long-term use of arbs might facilitate sars-cov-2 entry and increase infectivity. 25 this article was not included in this meta-analysis because it did not provide the use of raasi information in the covid-19 negative cases and no sars-cov-2 testnegative controls. hypertension as the most frequently coexisting comorbidity in patients with covid-19 increases susceptibility to sars-cov-2 infection, particularly, in elderly individuals. 26, 27 however, from our meta-regression (included no patients with hypertension) adjusted with recorded status of lower hypertension prevalence, the use of arbs was linked to more sars-cov-2 infection (table s2 ). speculation arose that the susceptibility might be based on the use of antihypertensives rather than the comorbidity caused by high blood pressure. age has consistently been reported to be a common risk attributing to covid-19 infection and its related mortality. older patients usually have more significant comorbidities, higher prevalence of hypertension, or use multiple drugs, in which there are already higher risks of sars-cov-2 infection, and thus masking off the clear effect of infection susceptibility attributed to arbs alone. ace2 gene is located on the x chromosome, and there is a possibility that the dosage effect of sex chromosome may impact ace2 activity due to escape from x-inactivation. 28 however, our analysis did not show the sex difference contributed to the detrimental effect of raasi. diabetes mellitus 29 or patients with underlying cardiovascular diseases, especially high prevalence of heart failure, 30 is associated with a higher risk of severity and fatality of covid-19. however, our meta-regression did not show this comorbidity integrated the effect of ace inhibitors or arbs. our meta-analysis on the aors of various secondary end points, that is, the severity of covid-19 (including admission to intensive unit, respirator usage, or mortality) revealed that the users of raasi had similar secondary end point outcomes as compared to those of the nonusers. we also did not see the influence of the outcomes among various age subgroups of various users of raasi which further confirms the safety of using raasi during this era of covid-19 pandemic. interestingly, some data suggested that hypertensive covid-19 patients receiving ace inhibitors/arbs treatment were found to have a lower rate of disease severity 31 or similar risk as non-ace inhibitors/arbs usage in patients with covid-19, 32 a trend toward reduced blood levels of interleukin-6 and reduced viral load and increased counts of cytotoxic t cells. 33 some animal data supported elevated ace2 expression could confer potential protective pulmonary and cardiovascular effects 34 and argue the biologic plausibility, thus suggesting that arbs may be helpful in managing covid-19. 18 possible induction of ace2 in lungs by raasi would augment soluble ace2 and may have a beneficial effect of having protection from membrane-anchored ace2mediated injury with sars-cov-2. 35 in addition, liu et al 36 showed that the level of angiotensin ii in patients with sars-cov-2 infection was strongly associated with viral load and lung injury which was suggesting that covid-19 could cause the imbalanced raas and drugs of ace inhibitors and arbs balancing raas may have the potential benefit on the lung protection in covid-19. there still remains a lot we do not know about covid-19 versus ace inhibitors/arbs/raasi. increased ace2 expression by preexisting raasi treatment may affect the virus susceptibility in certain sub-population, but could eventually be protective in the course of sars-cov-2 infection. in clinical practice, raasi are typically used to treat cardiovascular diseases, diabetes mellitus, and chronic kidney disease. on the contrary, discontinuing these agents could lead to the risk that they may not be resumed after the intervention and lead to withdrawal effect. 37 simply suspending or abandoning antihypertensive agents is strongly dissuaded and should not be a choice, considering the widespread use of raasi worldwide. our results further scientifically coincide and confirm the mainstream recommendations of various medical societies, which suggest not to discontinue medication in patients who have a reliance on these drugs. 31 their therapeutic benefit, most likely, outweighs any potential risk of being predisposed to sars-cov-2 infection. there are still many potential study limitations to be discussed. first, the available clinical database from the pandemic to date is still insufficient and heterogeneous, for example, race and screening policy of covid-19 to provide sufficient detail on all the variables of interest. these factors might account for some of the residual statistical heterogeneity seen for some outcomes, in light of our results, the albeit i 2 is commonly inflated in meta-analyses of observational data. 33 however, the evidence we presented was based on 7 large methodologically sound observational studies. second, some drug indications among those with the use of ace inhibitors/arbs were not elucidated, which could confound the risk. the use of raasi may indicate a subgroup of patients who are especially vulnerable to the virus. in such a case, the use of arbs is not a cause, but a marker of risk. third, further studies are required to define the mechanisms through which raasi modulate ace2 and exert beneficial or harmful effects among patients with covid-19. however, caution should be admonished regarding these associations because of potential unmeasured confounding interactions given the observational design of the studies and the small numbers of patients in the secondary investigative analysis. fourth, although we revealed higher susceptibility to sars-cov-2 infection for younger patients taking arbs, we do not imply to withdraw raasi during covid-19 pandemic period, as the second end point of covid-19 outcomes showed no difference among the users of raasi versus nonusers. fifth, we included the studies extracted from the medrxiv which were the unpublished articles before certification by peer review to explore the whole picture of the raasi with all available data. our comprehensive systematic review provided the best available information on treatment with raasi and was not associated with a higher likelihood of a positive test for sars-cov-2 infection, disease severity, or mortality. however, in younger subjects, the use of arbs could increase the risk of sars-cov-2 infection. nonetheless, when international collaborative and well-conducted studies, including randomized trials, of different raasi usage strategies and susceptibility of covid-19, are potentially unethical regardless of the challenges, this systematic appraisal of the currently best available evidence could be considered to inform interim guidance. world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) world health organization. world health organization (who) coronavirus disease (covid-19) dashboard epidemic update of covid-19 in hubei province compared with other regions in china china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? renin-angiotensin-aldosterone system inhibitors and risk of covid-19 reninangiotensin-aldosterone system blockers and the risk of covid-19 use of renin-angiotensinaldosterone system inhibitors and risk of covid-19 requiring admission to hospital: a case-population study association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease 2019 (covid-19) angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers are not associated with increased risk of sars-cov-2 infection the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration renovascular hypertension by two-kidney one-clip enhances endothelial progenitor cell mobilization in a p47phox-dependent manner robins-i: a tool for assessing risk of bias in non-randomised studies of interventions grade guidelines: 1. introduction-grade evidence profiles and summary of findings tables grade guidelines: 7. rating the quality of evidence-inconsistency association of previous medications with the risk of covid-19: a nationwide claims-based study from south korea covid-19 testing, hospital admission, and intensive care among 2,026,227 united states veterans aged 54-75 years effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 differential regulation of renal angiotensin-converting enzyme (ace) and ace2 during ace inhibition and dietary sodium restriction in healthy rats evaluation of angiotensin-converting enzyme (ace), its homologue ace2 and neprilysin in angiotensin peptide metabolism the effects of different angiotensin ii type 1 receptor blockers on the regulation of the ace-angii-at1 and ace2-ang mas axes in pressure overload-induced cardiac remodeling in male mice can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? urinary angiotensinconverting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin ii receptor blocker the association between angiotensin-converting enzyme inhibitors and angiotensin receptor blockers and the number of covid-19 confirmed cases and deaths in the united states: geospatial study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study association of hypertension with the severity and fatality of sars-cov-2 infection: a meta-analysis sex differences in renal angiotensin converting enzyme 2 (ace2) activity are 17β-oestradiol-dependent and sex chromosome-independent influence of diabetes mellitus on the severity and fatality of sars-cov-2 infection. diabetes, obes metabolism characteristics and outcomes of 21 critically ill patients with covid-19 in washington state antihypertensive drugs and risk of covid-19? acei/arb use and risk of infection or severity or mortality of covid-19: a systematic review and meta-analysis use of grade for assessment of evidence about prognosis: rating confidence in estimates of event rates in broad categories of patients angiotensin-converting enzyme 2 protects from severe acute lung failure renin-angiotensin-aldosterone system inhibitors in patients with covid-19 clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury discontinuation of and changes in drug therapy for hypertension among newly-treated patients: a population-based study in italy we thank the staff of the second core lab in the department of medical research at national taiwan university hospital for technical assistance. this study was supported by taiwan what is new?• the results of our meta-analysis, based on case-population studies and cohort studies, support the theory that treatment with raasi (reninangiotensin-aldosterone system inhibitors), especially ace (angiotensinconverting enzyme) inhibitors, was not associated with a higher likelihood of a positive test for severe acute respiratory syndrome coronavirus 2 infection, disease severity, or mortality. however, the younger subjects with angiotensin receptor blockers (arbs) treatment might have increased risk of severe acute respiratory syndrome coronavirus 2 infection with arbs treatment.what is relevant?• severe acute respiratory syndrome coronavirus 2 engages the human angiotensin-converting enzyme2 cell surface receptor to infect the host cells. thus, concerns arose regarding theoretically higher risk for coronavirus disease 2019 (covid-19) in patients taking ace inhibitors/arbs. to the best of our knowledge, our study is the first meta-analysis to holistically evaluate whether independent the use of ace inhibitors or arbs is associated with increased risk of severe acute respiratory syndrome coronavirus 2 infection or not. the use of either ace inhibitors or arbs treatment was not associated with an increased risk of positive severe acute respiratory syndrome coronavirus 2 test for the population as a whole; however, we showed for the first time that the use of arbs, as opposed to ace inhibitors, specifically augmented the risk of severe acute respiratory syndrome coronavirus 2 infection in younger subjects. in addition, the use of either ace inhibitors or arbs treatment did not aggravate disease severity or mortality of covid-19 in the whole population and subgroup analysis in our study. key: cord-291146-f3e5ynhu authors: sarangarajan, rangaprasad; winn, robert; kiebish, michael a.; bountra, chas; granger, elder; narain, niven r. title: ethnic prevalence of angiotensin-converting enzyme deletion (d) polymorphism and covid-19 risk: rationale for use of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers date: 2020-09-08 journal: j racial ethn health disparities doi: 10.1007/s40615-020-00853-0 sha: doc_id: 291146 cord_uid: f3e5ynhu rationale: hypertension, obesity and diabetes are major risk factors associated with morbidities underlying covid-19 infections. regression analysis correlated presence of ace insertion/deletion (i/d) polymorphism to covid-19 incidence and mortality. furthermore, covid-19 prevalence correlated to allele frequency of angiotensin-converting enzyme (ace) deletion (d) polymorphism within the european population. objective: homozygous ace deletion polymorphism is associated with increase in ace and angiotensin ii (ang-ii), sustained levels can result in inflammation, fibrosis and organ damage. the ace dd polymorphism is also associated with hypertension, acute respiratory distress and diabetic nephropathy, all considered high risk for covid-19 infection and outcomes. the study objective was to describe a biological framework associating ethnic prevalence of ace deletion polymorphism to covid-19 comorbidities providing rationale for therapeutic utility of ace-i/arbs to improve outcomes. method and results: the allele frequency database (alfred) was queried for frequency of rs4646994 representing ace i/d polymorphism. in a total of 349 worldwide population samples, frequency of ace d allele was higher in european, asian, and africans cohorts. in the usa, the frequency of ace d allele was higher in non-hispanic black compared with non-hispanic white and mexican americans. conclusion: covid-19 binding mediated reduction/inactivation of ace-ii can increase ace/ang-ii signalling pathway and related pathologies. the presence of ace dd polymorphism with covid-19 infection likely augments ace/ang-ii activities, increasing severity of covid-19 morbidities and impacts outcomes. thus, ethnic prevalence of ace dd polymorphism can explain in part the severity of covid-19 morbidity providing rationale for the use of ace-i/arbs to improve outcomes. the sars-cov-19 (covid19) infection has infected in excess of seventeen million individuals around the globe and is designated as a pandemic by the world health organization. the global efforts are focused on understanding the disease onset, progression and to identify causal linkage for differences in observed outcomes among the affected population and within specific demographics. despite worldwide spread of the covid-19 infections, european countries and the usa appear to have experienced higher incidence and mortality rates [1] [2] [3] . hypertension, obesity, and diabetes were identified as the most common comorbidities associated with covid-19 infection; higher severity of disease and mortality was generally reported in the elderly (> 50 years) population. angiotensin-converting enzyme 2 (ace2) is the predominant receptor for sars-cov viral entry and infection, resulting in the reduction of expression of ace2 [4, 5] . ace2 is an enzyme component of the renin-angiotensin system (ras), a complex integrated network of peptides-enzyme combination, generating catalytically active peptides with prominent influence on the vascular, renal, cardiac, and immune system [6] . in this report, we describe a framework of the pathophysiological consequence of covid-19-induced reduction in ace2, i.e., overactivation of the ras pathway with the potential to have deleterious effect on organ functions including the lungs, kidneys, heart, and immune system. the deleterious activities of ras within the covid-19-infected cohorts can be further amplified by the presence of genetic polymorphism in the angiotensin-converting enzyme (ace). increased prevalence in frequency of the ace polymorphism within ethnic groups, in part, is likely responsible for the observed severity of covid-19 comorbidities and mortality in this population. this is substantiated by recent regression analysis linking presence of ace-1 i/d (insertion/deletion) polymorphism with incidence and mortality with covid-19 infection [7] . renin-angiotensin system: ace, ang-ii, and inflammation the ras system has a prominent role in the regulation of vascular dynamics; its components directly or indirectly influence functions of the lung, heart, kidney, brain and the immune system [6] . in addition to central ras components, i.e., renin (kidney), ace (lungs), and angiotensinogen (liver), tissue-specific localized systems including the kidney, heart, and lungs have been identified [6, 8] . within ras, the canonical angiotensin-converting enzyme (ace) is responsible for conversion of angiotensin-1 (ang-i) to angiotensin-2 (ang-ii) (fig. 1a) . subsequently, ang-ii mediates its effects through activation of at-1 and at-2 receptors, resulting in distinct intracellular signalling pathways [9] [10] [11] . activation of at-1 receptors is associated with the well-characterized physiological actions of ang-ii in various organs including the lung, heart, kidney, and the vascular system [10] . in addition to its hemodynamic effect, ang-ii has significant pro-inflammatory effects, promoting generation of reactive oxygen species (ros), cell proliferation, extracellular matrix remodelling, and regulation of gene expression via signalling pathways leading to tissue injury [8, 12] . ang-ii promotes expression of proinflammatory chemokines in the kidneys, heart, and vasculature to induce inflammation [13] . several studies have characterized key inflammatory processes influenced by ang-ii on macrophages, dendritic cells, and mesangial cells resulting in mobilization and activation of cytokines, chemokines, and pro-inflammatory factors resulting in tissue damage and progressive organ failure [14] . due to profound influence of ang-ii signalling pathways that are predominantly adverse when unmitigated, the potency of ang-ii is tightly regulated via proteolytic activities of enzymes to generate various angiotensin peptide fragments with physiological activities different from ang-ii [14] (fig. 1a) . ace2 is an enzyme component of ras, with proteolytic activities different from the canonical ace. ace2 is responsible for cleaving angiotensin i to ang (1-9) and angiotensin-2 to ang (1-7) peptides respectively (fig. 1) , of which the latter is a potent vasodilator [15, 16] . several studies support a major role for ang (1) (2) (3) (4) (5) (6) (7) in providing the counterbalance to the physiological effects of ang-ii [17] [18] [19] . thus, the pro-inflammatory effects of ace/ang-ii axis are balanced by activation of anti-inflammatory pathways by ace2 and other systems. two recent publications reported that ace insertion/ deletion polymorphism correlated to infectivity and mortality associated with covid-19 infections [7, 20] . in humans, the gene encoding ace is located on chromosome 17 and exhibits an insertion/deletion polymorphism that is characterized by an insertion (allele i) or deletion (allele d) of a 287 base pair marker in intron 16 that results in three different genotypes, i.e. dd or ii homozygotes or id heterozygotes. it is reported that the deletion (d) allele occurs in 55% of the population and associated with increased ace activity, implicating the presence of d allele with disease pathologies associated with ras activity [21] . the allele frequency database (alfred; https://alfred. med.yale.edu/alfred/index.asp; rrid:scr_001730) was queried for frequency of rs4646994 representing ace i/d polymorphism, one of the best studies of all ace polymorphisms. the allelic frequencies of the insertion (i, +) and deletion (d, −) genotypes within various geographic regions from 349 population samples were obtained from alfred and are summarized in table 1 . inclusion of data from all european studies demonstrated almost equal distribution of the ace (i) or ace (d) allele, with italians, ashkenazi jews and canarians demonstrating slightly higher prevalence compared with the population averages. in contrast to europe, among the african population, the frequency of d allele was almost twice compared with the i allele among 2126 population samples with highest levels observed in pygmies, ethiopian jews, moroccan, nigerian and tunisian populations. these are consistent with other studies reporting significant increase in the frequency of deletion polymorphism of ace observed in individuals of african descent and associated with disease pathology [22] . specifically, a prevalence of the d allele of 60% has been reported in individuals of african descent [22] . in the usa, the non-hispanic black population has higher frequency of the d allele (table 2 ) compared with non-hispanic white and mexican american population [23] . the frequency of the d allele was increased compared with the i allele within the middle eastern population with higher values observed in both arab and saudi arabia sample populations. in contrast to africa and middle east, increased frequency of the i allele was observed in sample populations from asia (india, pakistan nepalese, tajik regions and sri lanka), oceania (new zealand, papua new guinea and micronesia), east asia (china, japan, korea, taiwan, cambodia, vietnam, philippines and malaysia) and south american countries. fig. 1 a overview of the reninangiotensin system. the figure describes the basic components of the renin angiotensin system with focus on the impact of ace and ace2 in the generation of angiotensin peptides, the respective cognate receptor(s) and corresponding physiological consequence of receptor activation. b influence of ace deletion (dd) polymorphism on reninangiotensin system. the figure describes the consequence of the ace deletion polymorphism, the increase in levels of ace and angiotensin ii resulting in activation of at-1 receptor and downstream pathophysiological effects. c consequence of covid-19 infection and ace deletion (dd) polymorphism on reninangiotensin system. the figure describes the increased activation of ace and generation of ang-ii as a consequence of covid-19-mediated reduction in ace2 in the presence of ace deletion polymorphism. the result is disruption of physiological balance of the ace/ace2 axis resulting in overactivation of at1-r signalling and associated pathological consequence ace deletion (d) polymorphism and disease-increased susceptibility and severity to co-morbidities associated with covid-19 although the ace i/d polymorphism is located in a noncoding region, its presence is directly linked to regulation of renin-angiotensin system and associated pathological conditions. a positive association between d allele and high blood pressure, atherosclerosis, coronary artery disease, stroke, diabetic nephropathy and alzheimer's disease has been extensively reviewed [24] . the molecular underpinning of these diseases is multi-factorial and complex, and the presence of the ace deletion polymorphism may contribute to influence disease pathology. indeed, to date, there is distinct lack of consensus studies linking the presence of ace deletion polymorphism to disease causality. nevertheless, the increase in levels of ace in individuals with the id and dd genotypes and potential augmentation of the ras system and associated signalling cascades can influence pathways to influence disease pathology [25] (fig. 1b) . indeed, increased levels of ace and ang-ii have been implicated in the pathophysiology of [26, 27] ) and kidney disease (chronic kidney disease, diabetic nephropathy [28, 29] ). in the african american population, the deletion polymorphism is associated with increase in systolic blood pressure, hypertension and altered vascular reactivity with potential impact on cardiovascular disease [30] [31] [32] . a subset of individuals with a positive diagnosis of covid-19 infection have rapid progression of lung dysfunction leading to acute respiratory distress with potential need for ventilatory support [2, 3] . presence of ace insertion/ deletion (i/d) polymorphism is associated with susceptibility and is an independent risk factor for mortality in patients with acute respiratory distress syndrome (ards) [33, 34] . of the three ace polymorphisms, there is positive association with frequency of the dd allele and incidence of ards, increased fatality and a prognostic factor of outcomes [35] [36] [37] . further, the dd genotype is usually associated with higher ace levels relative to other genotypes and with increased mortality in acute lung injury (ali)/ards patients [38, 39] . elevated levels of ace have been observed in the bronchoalveolar fluid of individuals with ards [28] . although decreases in circulating ace have been reported in ards patients [40] , this might be a consequence of the progressive damage to lung tissue as increased levels of ace are evident in the bronchoalveolar fluids of individual with ards [40] . the positive relationship between dd genotype and ali/ards and the corresponding increase in ace levels suggest the potential involvement of increased ang-ii in the etiopathology of ards. during the avian (h7n9) flu infections, approximately 70% of patients developed ards [41] . in a subset of infected patients, increase in plasma ang-ii levels was linked to severity and fatal outcomes [41] . within the covid-19-infected population, there is increased incidence of kidney injury associated with higher mortality rates [42, 43] . chronic kidney disease (ckd) is associated with severity of covid-19 infection [44] . interestingly, both ace and ace2 expressions in the kidneys are predominant in the proximal tubules with minor expression in the glomerular apparatus [45] . the balance between ang-ii and ang (1-7) affects renal ras to maintain balance of kidney functions; imbalance of the ratio results in kidney disease [46] [47] [48] . chronic kidney disease is characterized by decreases in cardiac and renal ace2 in human [49] . diabetic nephropathy (a ckd) is characterized by decrease in ace2, increased ace and ang-ii-mediated tubular and glomerular damage as a result of renal ras activation [28, 29] . based on these studies, the ability of covid-19 to bind and decrease ace2 in target tissues is most likely responsible for the observed increase in blood urea nitrogen, proteinuria and hematuria associated with kidney damage [49] . thus, covid-19-associated decrease in ace2 most likely results in disruption of the ace/ace2 balance in the kidney leading to sustained activation of ace and ang-ii activities and kidney damage. ace insertion/deletion polymorphism is also associated with diabetic kidney disease, the frequency of dd and id genotype distribution being higher compared with non-diabetic kidney disease cohorts, leading to functional decline [50, 51] . the above observations suggest that presence of the dd genotype of ace in patients with covid-19 infection may be associated with severe respiratory distress compared with the other genotypes. multiple studies have reported on the prevalence of ace i/ d polymorphism, specifically the id and dd polymorphism in increasing levels of ace and ang-ii, which could in part influence susceptibility to underlying pathologies considered high risk for covid-19 infections, progressive organ dysfunction and poor outcomes. thus, presence of id and dd polymorphism by itself is a potential underlying risk factor associated with severity and outcomes in individuals with positive diagnosis of covid-19 infection [20, 21] . the proteolytic cleavage of ang-ii by ace2 to generate ang (1-7) represents a major event leading to the physiological inactivation of ang-ii function. thus, in patients with active covid-19 infections, decrease in ace2 expression/activity should most likely lead to sustained ace-mediated generation of ang-ii and downstream signalling deleterious to organ functions including that of lung, kidney and heart [52] . although the status of circulating and lung ace levels in covid-19 patients is unclear, the ability of sars-cov-2 binding specifically to ace2 decreases its expression and activity suggesting upregulation of ace/ang-ii-mediated activities. this is consistent with the observation that knockdown of ace2 is associated with severe ards in multiple rodent models compared with corresponding wild-type controls [18] . loss of ace2 expression in mutant mice is associated with worse lung function and characterized by increases in vascular permeability, lung oedema and neutrophil accumulation [18] . interestingly, reduced plasma levels of ace2 are also observed within populations of african descent including african americans, specifically in individuals with pre-hypertensive status, diabetes and renal disease [53, 54] . administration of a catalytically active recombinant ace2 protein improved symptoms of acute lung injury in ace2 knockout and wild-type mice [55] . in a pilot clinical investigation, administration of recombinant human ace2 (apn311) in patients with acute respiratory distress was associated with rapid decrease in ang-ii level and did not significantly influence oxygenation indices in the treated population compared with placebo-controlled group [56] . the recombinant human ace2 is undergoing renewed clinical testing in the covid-19 patient population to investigate clinical outcomes [52] . sars-cov-2 binding to ace2 results in reduction of protein expression, activity and ability to generate anti-inflammatory signalling, all of which contribute to a pro-inflammatory phenotype due to presence of ace activity and ang-ii signalling (fig. 1c) . presence of ace d polymorphism increases ace levels and ang-ii leading to pro-inflammatory phenotype and is associated with disease susceptibilities considered high risk for covid-19 infections. recently, it was proposed that reduced plasma levels of ace2 in individuals of african descent most likely lowers potential for covid-19 infection [57] ; the overall outcomes in individuals with presence of ace deletion polymorphism after infection with covid-19 most likely leads to exacerbation of comorbidities and overall deleterious outcomes. based on the described biological consequence of covid-19 infections on the ras system, treatment with ace-i and arbs should be associated with improved outcomes within the overall covid-19 patient cohorts. indeed, several meta-analyses provide preliminary support for the potential benefits of the use of ace-i/arbs in management of covid-19 infections. in a multicenter study of 1128 adult patients with hypertension with positive covid-19 diagnosis, in-patient use of ace-i/arb was associated with reduced risk of mortality from all causes when compared with patients not treated with the medications [58] . recent publications further highlight the use of ace-i and arbs in providing cardiovascular and renal benefits to patients with covid-19 diagnosis [59, 60] . in a metaanalysis, patients treated with ace-i/arbs had 44% reduction in odds of developing severe disease and death compared with patients not treated with ace-i/arbs [61] . these studies provide rationale for investigation into the utility of ace-i/arbs in the ethnic population with known prevalence of ace deletion polymorphisms in an effort to mitigate severity and improve outcomes in response to covid-19 infections. ace is a multi-functional, relatively non-specific peptidase enzyme with a wide range of substrate specificities that impact physiological pathways in influencing blood pressure, haematopoiesis, hormone regulation, renal function and immune responses. the specificity of hypertension and cardiovascular disease as underlying causes for severity of covid-19 infection, the inherent role of ace-mediated generation of ang-ii and downstream signalling to potentially exacerbate inflammation and organ damage along with genotypic impact on ace status provide compelling support of the use of ace-i and arbs in the clinical management of patient with positive diagnosis of covid-19. the biological impact of the presence of deletion polymorphism of ace in individuals with covid-19 infection provides a significant rationale for serious consideration of shortterm use of ace-i and/or arbs in patients without underlying issues with blood pressure or cardiovascular disorder. the guidance statement issued by the heart failure society of america (hfsa), the american college of cardiology (acc) and american heart associated (aha) states that in the absence of favourable or detrimental effects of ace-i and arbs in the covid-19 setting, the recommendation is to not arbitrarily or pre-emptively discontinue these agents in patients currently on the medication as standard of care (acc. org). indeed, both ace-i and arbs have been extensively used in conditions ranging from hypertension, congestive heart failure, prevention of kidney failure and other indications. both classes of drugs have extensive use history, understanding of safety, tolerability, efficacy, adverse events profile and drug interactions. the significant genetic, scientific and clinical data supporting a potential role for increased ace levels and associated ang-ii effect in target organs provides compelling argument for use of ace-i and arbs in the clinical management of patients with covid-19 infections to improve outcomes. high salt sensitivity-associated low plasma renin activities are responsible for the attenuated blood pressure-lowering response of ace-i in the african american population [62] . however, this particular phenomenon might be of potential advantage in dosing and management of severity of covid-19-associated morbidities in african american and other ethnic populations with ace deletion polymorphism. in summary, this study describes the biological relevance of genetic polymorphism of ace deletion with higher prevalence in certain ethnic populations including african americans in context of covid-19 infection and rationale for the use of ace-i/arbs for therapeutic management of severity of morbidity and improving outcomes associated with covid-19. author contributions rs is responsible for concept; mak, cb, eg, nrn contributed to the structure and content of the manuscript. funding mr. carl e. berg supported this work. data availability all data obtained from public resources and referenced in the manuscript. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. covid-19 in europe: the italian lesson coronavirus disease 2019 (covid19) in the eu/eea and the uk -ninth update hospitalization rates and characteristics of patients hospitalized with laboratoryconfirmed coronavirus disease 2019 -covid-net, 14 states sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor tissue 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syndrome effect of angiotensin converting enzyme gene i/d polymorphism and its expression on clinical outcome in acute respiratory distress syndrome ace insertion/deletion polymorphism in sepsis and acute respiratory distress syndrome compared evolution of plasma fibronectin and angiotensin-converting enzyme levels in septic ards angiotensin ii plasma levels are linked to disease severity and predict fatal outcomes in h7n9-infected patients kidney impairment is associated with in-hospital death of covid-19 patients caution on kidney dysfunctions of covid-19 patients chronic kidney disease is associated with severe coronavirus disease 2019 (covid-19) infection expression of ace and ace2 in individuals with diabetic kidney disease and healthy controls high-salt diet increases glomerular ace/ace2 ratio leading to oxidative stress and kidney damage ace2 alterations in kidney disease synergistic expression of angiotensin-converting enzyme (ace) and ace2 in human renal tissue and confounding 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mortality among patients with hypertension hospitalized with covid-19 potential harmful effects of discontinuing ace-inhibitors and arbs in covid-19 patients angiotensin-converting enzyme 2 and anti-hypertensives (angiotensin receptor blockers and angiotensin converting enzyme inhibitors) in coronavirus disease 2019 (covid-19) the effect of angiotensin converting enzyme inhibitors and angiotensin receptor blockers on death and severity of disease in patients with coronavirus disease 2019 [covid-19]: a meta analysis. medrxiv. 2020; his version posted a review of ace inhibitors and arbs in black patients with hypertension publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-346811-gorp9n1g authors: hippisley-cox, julia; young, duncan; coupland, carol; channon, keith m; tan, pui san; harrison, david a; rowan, kathryn; aveyard, paul; pavord, ian d; watkinson, peter j title: risk of severe covid-19 disease with ace inhibitors and angiotensin receptor blockers: cohort study including 8.3 million people date: 2020-07-31 journal: heart doi: 10.1136/heartjnl-2020-317393 sha: doc_id: 346811 cord_uid: gorp9n1g background: there is uncertainty about the associations of angiotensive enzyme (ace) inhibitor and angiotensin receptor blocker (arb) drugs with covid-19 disease. we studied whether patients prescribed these drugs had altered risks of contracting severe covid-19 disease and receiving associated intensive care unit (icu) admission. methods: this was a prospective cohort study using routinely collected data from 1205 general practices in england with 8.28 million participants aged 20–99 years. we used cox proportional hazards models to derive adjusted hrs for exposure to ace inhibitor and arb drugs adjusted for sociodemographic factors, concurrent medications and geographical region. the primary outcomes were: (a) covid-19 rt-pcr diagnosed disease and (b) covid-19 disease resulting in icu care. findings: of 19 486 patients who had covid-19 disease, 1286 received icu care. ace inhibitors were associated with a significantly reduced risk of covid-19 disease (adjusted hr 0.71, 95% ci 0.67 to 0.74) but no increased risk of icu care (adjusted hr 0.89, 95% ci 0.75 to 1.06) after adjusting for a wide range of confounders. adjusted hrs for arbs were 0.63 (95% ci 0.59 to 0.67) for covid-19 disease and 1.02 (95% ci 0.83 to 1.25) for icu care. there were significant interactions between ethnicity and ace inhibitors and arbs for covid-19 disease. the risk of covid-19 disease associated with ace inhibitors was higher in caribbean (adjusted hr 1.05, 95% ci 0.87 to 1.28) and black african (adjusted hr 1.31, 95% ci 1.08 to 1.59) groups than the white group (adjusted hr 0.66, 95% ci 0.63 to 0.70). a higher risk of covid-19 with arbs was seen for black african (adjusted hr 1.24, 95% ci 0.99 to 1.58) than the white (adjusted hr 0.56, 95% ci 0.52 to 0.62) group. interpretation: ace inhibitors and arbs are associated with reduced risks of covid-19 disease after adjusting for a wide range of variables. neither ace inhibitors nor arbs are associated with significantly increased risks of receiving icu care. variations between different ethnic groups raise the possibility of ethnic-specific effects of ace inhibitors/arbs on covid-19 disease susceptibility and severity which deserves further study. the first cases of infection caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (covid19) in the uk were confirmed on 24 january 2020. since then the disease has spread rapidly through the population. there are no vaccines, preventative or curative treatments for covid-19 disease and only one possible diseasemodifying treatment 1 so the government has used social distancing as a population-level intervention to limit the rate of increase in cases. case series of confirmed covid-19 have identified age, 2 sex, 3 comorbidities 2 4 and ethnicity 5 as potentially important risk factors for susceptibility to infection, hospitalisation or death due to infection. in addition, chronic use of some medications at the time of exposure has been suggested as a potential risk factor for infection or severe adverse outcomes due to infection, 6 although the evidence is currently too limited to confirm or refute these concerns. 7 understanding this chronic medication use is important because medications could be modified in individuals or at a population scale to alter the likelihood of infection or adverse outcomes. furthermore, associations between medications and improved outcomes, if confirmed from large cohorts, could provide a basis for rapid prioritisation in prospective randomised clinical trials, and might provide important insights into disease mechanisms and pathogenesis. sars-cov-1 and sars-cov-2, which have been responsible for the sars epidemic and for the covid-19 pandemic, respectively, interface with the renin-angiotensin-aldosterone system (raas) through ace2, an enzyme that modulates the effects of the raas but is also the primary receptor for both sars viruses. the interaction between the sars viruses and ace2 may be one determinant of their infectivity, and there are concerns that raas inhibitors may change ace2 expression and hence covid-19 virulence. this hypothesis has been extensively reviewed. 7 ace inhibitors and angiotensin receptor blocker (arb) drugs are recommended by the national institute for health and care excellence as firstline treatment for patients under 55 years of age with hypertension and second-line treatment for those over 55 years of age and for those of african descent. 8 ace inhibitors are also widely used to treat congestive cardiac failure. uncertainty around possible associations of these drugs with covid-19 disease, and the subsequent risk that patients might special populations stop taking these drugs of proven effectiveness, has led to regulatory and professional bodies issuing statements urging patients to keep taking their regular medications. 9 although several studies have considered the effect in hospitalised patients of drugs acting on the renin-angiotensin on disease course, 6 10 11 none has looked at population use of these drugs to determine if they modulate susceptibility. we report a large, population-based study where we examined the drug histories of approximately 20% of all patients tested positive for coronavirus in england to determine if there was an independent association between ace inhibitor and arb drug prescription and severe covid-19 disease susceptibility and progression. we undertook a large, open cohort study of all patients aged 20-99 years registered with 1205 general practices in england contributing to the qresearch database (v.44, uploaded 24 march 2020) linked to covid-19 rt-pcr test records (updated until 26 april 2020) and with intensive care records (updated until 27 april 2020). the protocol is published. 12 we included general practices in england contributing to the qresearch database from which current data were available. qresearch is a high-quality research database established in 2002, which has been extensively used for pharmaco-epidemiological research. 13 qresearch is the largest and most representative general practitioner (gp) practice research database nationally. 14 two national databases were linked to qresearch. the first was the national registry of covid-19 rt-pcr test positive results held by public health england (phe). since covid-19 is a notifiable disease, laboratories in england are required to send results of all tests to phe. at the time of analysis, 106 529 positive covid-19 test results were available from 106 507 individuals in england, until 26 april 2020, of whom 104 665 were aged 20-99 years. of these, 19 486 (18.6%) were linked to qresearch patients. the second linked database was the intensive care national audit and research centre (icnarc) case mix programme (cmp) database. this is a high-quality, clinical research database which includes contemporaneous data from 285 icus in england, wales and northern ireland and is widely used for cohort studies, comparative audit and outcome data ascertainment for randomised clinical trials. 15 16 as of 28 april 2020, there were 6968 patients admitted for icu care with covid-19 disease, of whom 6963 were aged 20-99 years. of these, 1286 (18.5%) were linked to qresearch. we identified a cohort consisting of all patients aged 20-99 years who were fully registered with the gp practices on the start date (1 january 2020). patients entered the cohort on this date and were censored at the earliest of the date of death, leaving the gp practice, the study end date (27 april 2020) or occurrence of the relevant outcomes of interest. we used all the relevant patients on the pooled database to maximise power and to enhance generalisability of the results. during our study period, over 98.6% of all covid-19 rt-pcr tests in england were undertaken in a hospital setting for symptomatic patients sufficiently unwell to warrant hospital assessment and admission. our main outcomes for these analyses were: 1. covid-19 rt-pcr test positive disease. 2. covid-19-related admission for icu care. we had two main exposures of interest: 1. ace inhibitors. 2. arbs. we classified a patient as having had exposure to either medication if they had three or more prescriptions, including a prescription issued in the 90 days preceding cohort entry. we extracted data from the gp record for explanatory and potential confounding variables including variables with some evidence of being risk factors for covid-19 disease or severe disease as measured by icu admission and variables likely to influence prescribing of ace inhibitors and arb medications. we used the latest information recorded in the gp record on or before study entry as follows: 1. where quintile 1 is the most affluent and 5 is the most deprived). 4. geographical region within england, categorised into 10 groups. 5. body mass index (kg/m 2 ), categorised into five categoriesunderweight (<20 kg/m 2 ); normal weight (20-24.9 kg/m 2 ); overweight (25-29.9 kg/m 2 ); obese (30-34.9 kg/m 2 ); severely obese (>35 kg/m 2 ). 6. smoking status in five categories-never-smoker; exsmoker; light smoker (1-9 cigarettes/day); moderate smoker (10-19 cigarettes/day); heavy smoker (20+ cigarettes/ day). 7. gp recorded diagnosis of type 1 or type 2 diabetes. 8. gp recorded diagnosis of cardiovascular disease. 9. gp recorded diagnosis of congestive cardiac failure. 10 . gp recorded diagnosis of hypertension. 11. gp recorded diagnosis of atrial fibrillation. 12. gp recorded diagnosis of asthma. 13. gp recorded diagnosis of chronic obstructive pulmonary disease. 14. gp recorded diagnosis of chronic kidney disease (ckd stage 3, 4 or 5). we also extracted medication use for the following classes of drugs as potential confounding variables. we focused on classes of drugs rather than individual drugs to ensure adequate power. we classified patients as exposed using the same definitions as ace inhibitors and arbs. 1. drugs to treat type 2 diabetes including sulfonylureas, biguanides and other drugs (thiazolidinediones, gliptins, sodium glucose co-transporter 2 inhibitors, glucagon-like peptide-1 receptor agonists, meglitinides). 2. anticoagulant drugs (warfarin and direct oral anticoagulants). 3. antiplatelet drugs. 4. calcium channel blocking drugs. 5. thiazides. 6. potassium-sparing diuretics. 7. statins. after conducting univariable analyses, we conducted a multivariable analysis based on patients with complete data. we then used multiple imputation with chained equations to replace missing values for ethnicity, body mass index and smoking status and used these values in our main analyses. 17 we included all exposure and explanatory variables in the imputation model, along with the nelson-aalen estimator of the baseline cumulative hazard, and the outcome indicator. we carried out five imputations. we used cox's proportional hazards models to estimate adjusted hrs for ace inhibitors and arbs adjusting for the confounders. we tested for interactions between ace inhibitors, arbs and ethnicity. we undertook several sensitivity analyses. to further reduce indication biases, additional analyses restricted to patients with hypertension or heart failure to directly compare risks for ace inhibitors and arbs with other antihypertensive drugs. we also undertook analyses adjusted for the number of antihypertensive drugs as a proxy for severity of hypertension (untreated hypertension; monotherapy; dual therapy; triple or more therapy). lastly, we changed the definition of exposure to requiring a prescription within the last 30 days prior to cohort entry. we used p<0.01 (two-tailed) to determine statistical significance, to take account of multiple testing. patient representatives from the qresearch advisory board have advised the whether to undertake this research, on the data linkage, public interest and likely public benefit resulting from the study, dissemination of studies using qresearch data, including the use of lay summaries describing the research and its findings. one thousand two hundred five qresearch practices were included in our analysis. of the 10 594 500 patients registered on 1 january 2020, 8 275 949 were aged between 20 and 99 years. of these, 19 486 (0.24%) had a covid-19 rt-pcr positive result and 1286 were admitted to an icu. table 1 shows the baseline characteristics of the overall cohort consisting of 8 275 949 patients. the median age was 47 years (iqr 33-62); self-assigned ethnicity was recorded in 6 691 660 (80.9%). a total of 645 577 patients (7.8% of 8 275 949) were currently prescribed an ace inhibitor and 308 881 (3.7%) were currently prescribed an arb drug. table 2 shows the proportions of patients prescribed ace inhibitors and arbs by ethnicity and other characteristics. figure 1a and b show adjusted hrs for each outcome based on the multiply-imputed data. figure 2a and b show the same for the complete case analysis. ace inhibitors were associated with a significantly reduced risk of covid-19 disease requiring hospital admission (adjusted hr 0.71, 95% ci 0.67 to 0.74) but were not significantly associated with risk of icu care (adjusted hr 0.89, 95% ci 0.75 to 1.06) after adjusting for a wide range of confounders. adjusted hrs for arbs were 0.63 (95% ci 0.59 to 0.67) for covid-19 disease and 1.02 (95% ci 0.83 to 1.25) for icu care. the results there were significant interactions with ethnicity for ace inhibitors and arb (both p<0.001) for the covid-19 rt-pcr diagnosed disease outcome. table 3 shows the adjusted hrs for ace inhibitor and arb use for each of the ethnic groups. for ace inhibitors the risks of covid-19 disease were significantly higher in the caribbean and black african groups than the white group, with a significantly increased risk in the black african group (adjusted hr 1.31, 95% ci 1.08 to 1.59), although the cis were wide in the non-white ethnic groups. the risks associated with arb use were significantly higher in the other asian, black african, chinese and other ethnic group than the white group. while men were at no greater risk of having covid-19 diagnosed disease requiring hospital admission than women (adjusted hr 1.02, 95% ci 0.99 to 1.05), they had a threefold increased risk of icu admission despite adjustment for confounders ( figure 1b) had an increased risk of covid-19 disease and icu admission. there were regional variations in the risk of covid-19 disease and icu admission, the south west had the lowest risk of both outcomes, the north east had the highest risk of covid-19 disease and london had the highest risk of icu admission. overall, compared with the white ethnic group, all other ethnic groups except chinese and bangladeshi groups were associated with a significantly increased risk of covid-19 disease. highest risks were found for the other asian group who had a 2.1-fold increased risk; black african (1.8-fold increased risk); black caribbean (1.71-fold increased risk) and indian (1.61-fold increased risk) compared with the white group. the comparative risk of icu admission in these ethnic groups was even higher. compared with the white group, all other ethnic groups had twofold to threefold higher risks of icu admission, but smaller numbers of people in these groups led to some imprecise estimates. the risks of covid-19 disease and of icu admission were higher in those with increasing bmi. the most pronounced gradient was for icu admission, where being obese was associated with a 2.6-fold increased risk and severe obesity with a 4.4fold increased risk compared with the normal weight group. this was after adjustment for all other variables shown in figure 1b. there was a small increased risk of both adverse outcomes among ex-smokers compared with never-smokers. we observed a markedly decreased risk of both covid-19 disease and icu admission in smokers. the apparent protective association was greatest for heavy and moderate smokers and most markedly on the risk of icu admission which was 88% lower in heavy smokers compared with non-smokers ( figure 1b) . each of the comorbidities included in the analysis was associated with an increased risk of covid-19 disease. however, only ckd, hypertension, type 1 and type 2 diabetes were significantly associated with an increased risk of icu admission. figure 1a shows significantly increased risks of covid-19 disease associated with anticoagulants, antiplatelets, other diabetes drugs; significantly decreased risks of 10% for statins, 30% for thiazides and 8% for calcium channel blockers and no significant association for biguanides, beta-blockers or sulfonylureas. for icu admission there was a significantly increased risk for calcium channel blockers, but no significant associations with the other drugs (at p<0.01). in this very large population-based study, ace inhibitor and arb prescriptions were associated with a reduced risk of covid-19 rt-pcr positive disease, having adjusted for a wide range of demographic factors, potential comorbidities and other medication. there was no evidence of an increased or reduced risk of icu admission with either drug. there were marked variations in risk of covid-19 disease and of requiring icu admission by ethnic group, with highest rates among black, asian, and minority ethnic (bame) groups. this association is important and adds to existing knowledge 18 since it is not explained by age, sex, deprivation, geographical region or several comorbidities and intercurrent medications included in our analysis. to date, published studies reporting associations between chronic medication with ace inhibitor or arb drugs and covid-19 infections are limited to hospitalised patients 6 10 11 19 or those attending a hospital clinic. 20 this allows the study of drug treatment effects on the in-hospital disease course but not effects on disease susceptibility since there is no information on medication use in the uninfected or less severely infected population. most in-hospital studies are relatively small containing low numbers of patients or ace inhibitors of arbs in comparison to our study. however, two 6 19 were able to correct for the confounding effects of age, gender, comorbidities and in-hospital medications. in one study of 1128 patients with hypertension of whom 188 were taking ace inhibitors/arb, 6 in-hospital use of ace inhibitor or arb medication was associated with a lower risk of all-cause in-hospital mortality (adjusted hr 0.42; 95% ci 0.19 to 0.92; p=0.03). in the larger 8910 patient study (770 taking ace inhibitors and 556 arbs), ace inhibitors were associated with reduced in-hospital mortality (2.1% vs 6.1%; or 0.33; 95% ci 0.20 to 0.54) but arbs were not (6.8% vs 5.7%; or 1.23; 95% ci 0.87 to 1.74). 19 conversely, there was no evidence of reduced risk in outcomes in patients receiving ace inhibitor and arb drugs in initial reports from new york. 11 in our study, prior prescription of ace inhibitor and arb drugs did not have a significant effect on the risk of patients developing covid-19 disease severe enough to require icu care. in contrast, we found that previously prescribed ace inhibitor and arb drugs are associated with the likelihood of an individual testing positive for covid-19 in a hospital setting. the effect was similar for both drug classes. this may indicate that drug treatment at the time of exposure altered susceptibility to covid-19 infection and/or altered the likelihood of an infection progressing to the point where testing is sought. it is also possible that this reflects a 'healthy user' selection bias. there are no other population-based studies of ace inhibitor/arb use and covid-19 infection. losartan is already being tested in a clinical trial as a treatment of covid-19 infection. 21 its efficacy may depend on the context in which it is tested. since the recommendations for treatment of hypertension differ according to ethnic groups and age, we considered the possibility that these factors might contribute to the observed association between ace inhibitor or arb use and covid-19 disease or severity. ace inhibitors are recommended as first-line treatment for hypertension, whereas calcium channel blockers are recommended in patients of black ethnic origin. 8 indeed, there were significant interactions between ethnicity, ace inhibitor and arbs for covid-19 disease. arbs were significantly less protective in the other asian, black african, chinese and other ethnic group than the white group. ace inhibitors appeared less protective in the caribbean than the white group and were associated with an increased risk of covid-19 disease in the black african group. this raises the possibility of ethnic-specific effects of ace inhibitors/arbs on covid-19 disease susceptibility and severity or unmeasured confounding. however, as numbers were relatively small in the non-white ethnic groups so cis were wide, caution is needed in interpreting these results. studies of patients hospitalised with covid-19 have noted a greater than expected number of patients with hypertension, 2 and hypertension appears to be a risk factor for more severe covid-19 disease across many studies. 4 in our study, hypertension was a risk factor for being tested positive for covid-19 in a hospital setting independent of ace inhibitor and arb treatment, but was only modestly associated with likelihood of icu admission. we found an expected association with obesity, with those who are obese or severely obese having higher risk of covid-19 disease and icu admission. however, we have reported a counterintuitive finding for smoking, with light, moderate and heavy smokers having a lower risk for both covid-19 disease and icu admission. one systematic review concluded on the basis of limited evidence either there is no difference in risk by smoking status or that there is an increased risk in smokers. 22 however, our data are consistent with very low rates of smoking seen in patients presenting with covid-19 in wuhan 23 and similar data from the usa 24 and with the findings of a more limited analysis of patients with covid-19 in france. 25 this may reflect a general immunomodulatory effect, a mechanism that is thought to explain the lower incidence of sarcoidosis, extrinsic allergic alveolitis and ulcerative colitis in current smokers. 26 27 alternatively, smoking may cause increased ace2 mrna expression in human lung much as ace inhibitors or arbs are believed to, suggesting a possible common protective mechanism for severe covid-19 disease. 28 additional possible mechanisms include a direct protective effect of nicotinic receptor stimulation 29 or an association of smoking with another protective factor. this finding arose when including smoking status as a confounder and should be interpreted cautiously. further studies are required to verify the apparent protective association, determine whether it is independent of other risk factors, and investigate potential mechanisms. we have used two high-quality, established large validated research databases (qresearch and icnarc cmp) and linked them to the national register of covid-19 test results. our study is observational with strengths and inherent limitations since the data were collected as part of routine nhs care. key strengths include the use of highquality, established validated databases, size, representativeness, lack of selection, recall and respondent bias. uk general practices have good levels of accuracy and completeness in recording clinical diagnoses and prescriptions and provide the ability to update analyses as data change over time. 30 it is therefore likely to be representative of the population of england. it has good face validity since it has been conducted in the setting where most patients in the uk are assessed, treated and followed up. we have been able to adjust for a wide range of confounders based on detailed coded information recorded in the patients' electronic medical record. we restricted the sample for these analyses to only include patients with hypertension or heart failure so that all patients, whether treated with ace inhibitors/arbs or not, had the same indication for treatment. this is an important additional analysis as hypertension and heart failure themselves are associated with adverse covid-19 outcomes, and this restricted analysis reduces their confounding effect and allows for a more direct comparison of the antihypertensive drugs in people with indications for their use. we also accounted for ethnicity and other confounding variables in this restricted analysis which could influence the selection of an antihypertensive treatment and also be associated with covid-19 outcomes. some systematic differences are still likely between patients who are treated and those who are not, such as severity of hypertension. we have carried out an additional analysis where we adjusted for a proxy measure of severity. there may be some over-ascertainment of exposure to medication since our definition was based on issued prescriptions rather than dispensed medication. our analyses focused on drug classes rather than individual drugs as there were insufficient cases to support an analysis at individual drug level. we have not investigated the relationship between the intensity and duration of exposure and the risk of disease in this early analysis. we investigated the more mechanistically likely and therefore immediately clinically important drug associations. other drug classes can be investigated as numbers accrue. data on community and care home deaths or deaths occurring within hospital but not in icu are not yet available from hospital episode statistics and civil registrations. linkage of the gp data to national registries of outcome data, updated in near real time, will have minimised ascertainment bias relating to laboratory confirmed cases. however, there will be underascertainment of total covid-19 cases due to the current absence of widespread systematic testing strategy in the uk, and due to false negative tests. as uk health policy during the study period confined testing for covid-19 to hospitalised patients, our data focus on the incidence of more severe disease, rather than all cases, as most people with probable covid-19 are not admitted to hospital. some patients deemed to be at high risk of adverse outcomes of covid-19 will have self-isolated during our study period to reduce their risk of contracting the virus and if effective, may result in a selection bias with such patients less likely to be become infected and subsequently admitted to hospital or icu. not all acutely unwell patients in hospital are admitted to icu and this may result in a selection bias. admission to icu is limited to those who might benefit from this treatment and so varies on the basis of patient demographic and medical characteristics. data on deaths in icu were available to us but a significant proportion of patients admitted to an icu were still being treated in an icu and this varied by region as the pandemic spread. for this reason, icu deaths were not included in the analysis. further analyses of mortality will be undertaken once the relevant data (including out-of-hospital deaths) become available. we have undertaken two new novel data linkages by linking qresearch to both covid-19 test results and outcomes recorded on the icnarc cmp data. this new linked data asset is a valuable resource for future research projects. in this very large population-based study, ace inhibitor and arb prescriptions were associated with a reduced risk of covid-19 rt-pcr positive disease in a hospital setting adjusting for a wide range of demographic factors, potential comorbidities and other medication. there was no evidence of an increased or decreased risk associated with either drug for icu admission. there are marked variations in risk of covid-19 disease and icu admission by ethnic group, with highest rates among bame groups. the strength of this association is greater with the more severe outcome and is not explained by age, sex, deprivation, geographical region or several comorbidities and intercurrent medications included in the analysis. the counterintuitive finding of smokers having a lower risk of covid-19 disease requiring hospital admission and icu admission deserves further study. what is already known on this subject? ► there is uncertainty about the interaction of ace inhibitor and angiotensin receptor blocker (arb) drugs with covid-19 disease susceptibility and disease severity. what might this study add? ► in this very large population-based study, treatment with ace inhibitor and arb prescriptions is associated with a reduced risk of covid-19 rt-pcr positive disease after adjusting for a wide range of variables. ► neither ace inhibitors nor arbs are associated with increased risks of receiving icu care for covid-19 disease. ► there are significant interactions with ethnicity for ace inhibitors and arbs for covid-19 disease with higher risks among the non-white ethnic groups particularly black african patients compared with the white group, although the confidence intervals for some analyses are wide; this finding is important and adds to existing knowledge. how might this impact on clinical practice? ► neither ace inhibitors nor arbs are associated with increased risks of covid-19 rt-pcr positive disease or of receiving icu care for covid-19 disease. ► variations between different ethnic groups raise the possibility of ethnic-specific effects of ace inhibitors/arbs on covid-19 disease susceptibility and severity which deserves further study. hopes rise for coronavirus drug remdesivir hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease clinical presentation and initial management critically ill patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection in brescia, italy does comorbidity increase the risk of patients with covid-19: evidence from meta-analysis ethnicity and covid-19: an urgent public health research priority association of inpatient use of angiotensinconverting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 renin-angiotensin-aldosterone system inhibitors in patients with covid-19 hypertension in adults: diagnosis and management nice guideline european society of cardiology. position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan, china presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area anticholinergic drug exposure and the risk of dementia: a nested case-control study spatial distribution of clinical computer systems in primary care in england in 2016 and implications for primary care electronic medical record databases: a cross-sectional population study admission patterns and survival from status epilepticus in critical care in the uk: an analysis of the intensive care national audit and research centre case mix programme database development and validation of the new icnarc model for prediction of acute hospital mortality in adult critical care imputation is beneficial for handling missing data in predictive models disparities in the risk and outcomes of covid cardiovascular disease, drug therapy, and mortality in covid-19 association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease 2019 (covid-19) losartan for patients with covid-19 not requiring hospitalization covid-19 and smoking clinical characteristics of coronavirus disease 2019 in china preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease 2019 -united states low incidence of daily active tobacco smoking in patients with symptomatic covid-19 nicotine treatment for ulcerative colitis is smoking beneficial for granulomatous lung diseases? tobacco smoking increases the lung gene expression of ace2, the receptor of sars-cov-2 a nicotinic hypothesis for covid-19 with preventive and therapeutic implications sources, uses, strengths and limitations of data collected in primary care in england key: cord-289144-d6fgs8qg authors: sieńko, jerzy; kotowski, maciej; bogacz, anna; lechowicz, kacper; drożdżal, sylwester; rosik, jakub; sietnicki, marek; sieńko, magdalena; kotfis, katarzyna title: covid-19: the influence of ace genotype and ace-i and arbs on the course of sars-cov-2 infection in elderly patients date: 2020-07-21 journal: clin interv aging doi: 10.2147/cia.s261516 sha: doc_id: 289144 cord_uid: d6fgs8qg since the beginning of 2020, the whole world has been struggling with the pandemic of coronavirus disease 2019 (covid-19) caused by a novel coronavirus sars-cov-2. the sars-cov-2 infection depends on ace2, tmprss2, and cd147, which are expressed on host cells. several studies suggest that some single nucleotide polymorphisms (snps) of ace2 might be a risk factor of covid-19 infection. genotypes affect ace2 structure, its serum concentration, and levels of circulating angiotensin (1-7). moreover, there is evidence that ace genotype affects the outcomes of acute respiratory distress syndrome (ards) treatment, the most severe consequence of sars-cov-2 infection. covid-19 morbidity, infection course, and mortality might depend on ace d allele frequency. the aim of this narrative review was to analyze and identify the mechanisms of ace-i and arbs with particular emphasis on angiotensin receptors and their polymorphism in the light of covid-19 pandemic as these medications are commonly prescribed to elderly patients. there is no direct evidence yet for ace-i or arbs in the treatment of covid-19. however, for those already taking these medications, both the european society of cardiology and the american college of cardiology recommend continuing the treatment, because at present, there is no clear clinical or scientific evidence to justify the discontinuation of ace-i or arbs. individualized treatment decisions should be based on the clinical condition and co-morbidities of each patient. since the beginning of the year 2020, the whole world has been struggling with the epidemic of the new sars-cov-2 virus causing coronavirus disease 2019 . it was first detected in the chinese province of hubei at the end of 2019, and in march 2020 the world health organization (who) recognized covid-19 as a global health threat by declaring it a global pandemic. 1 sars-cov-2 is a novel virus from the coronaviridae family, mainly found as a pathogen in animals (birds and mammals). 1, 2 history has already seen human infections with other viruses from this family. 3, 4 both sars-cov-1 causing severe acute respiratory syndrome (sars) and mers-cov causing middle east respiratory syndrome (mers) show similarity to sars-cov-2 causing covid-19 by using receptors for angiotensin ii (angii) for viral entry into the cell during infection. these receptors are found throughout the human body, with large numbers occurring in the lungs, especially on type 2 pneumocytes, which explains why the symptoms are mainly associated with the respiratory system. 3, 4 the main source of infection spread is droplet and direct contact, but fecal-oral route and direct intraneural entry through olfactory nerves have been suggested. [5] [6] [7] usually, the sars-cov-2 infection is asymptomatic, while symptoms include fever, weakness, myalgia, cough, difficulty breathing, sometimes abdominal discomfort and diarrhea or anosmia. 6-,8 it has also been suggested that neurological presentation, including delirium, may precede respiratory symptoms. 9, 10 it is important to identify laboratory abnormalities associated with covid-19, including leuko-and lymphopenia, thrombocytopenia, elevated lactate dehydrogenase level, increased liver enzymes or d-dimers. 11, 12 people with symptoms often develop rapidly progressing infections of the lower respiratory tract, which can lead to viral pneumonia and severe acute respiratory infection (sari), as well as sepsis and death. 8, 13 the aim of this narrative review was to analyze and identify the mechanisms of ace-i and arbs with particular emphasis on angiotensin receptors and their polymorphism in the light of covid-19 pandemic as these medications are commonly prescribed to elderly patients. the selected literature review is based on an in-depth analysis and selection of articles in terms of their credibility and relevance to the topic. this is only a small part of the available literature on the topic, but we chose the most reliable individual articles for the purpose of this review. in estimating the course of the disease, the most common patient profile is important. the current meta-analyzes determine the average age of the patient to be between 30 and 79 years of age. 13 concomitant diseases and multimorbidity are serious problems of modern times. one of the main civilization diseases is hypertension. 14 the possibility of a coexistence of hypertension and susceptibility for sars-cov-2 infection raises many questions; therefore, it is important to know the pathogenesis and mechanisms of both diseases. we chose several, most recent articles describing these mechanisms. changes in ras activity are related to the pathogenesis of hypertension and inflammatory lung disease. targeting ras is an effective antihypertension therapeutic strategy. aceis and arb, which inhibit the ace/ang ii/at1r system, are commonly used drugs for hypertensive patients. 13 in a study on a rat model in 12-day treatment with drugs that either inhibit the synthesis of circulating ang ii or block the at1 receptor, the therapy induced an increase in ace2 heart mrna, accompanied by an increase in ace2 activity in the heart membrane of rats treated with any of losartan or both losartan and lisinopril. although the dominant effect of ace inhibition may be due to the combined effect of reduced ang ii formation and ang 1-7 metabolism (1-7), the antihypertensive effect of at1 antagonists may be partially due to increased ang ii metabolism by ace2. 15 the current study confirmed that increased age was associated with death in patients with covid-19. previous studies in macaques inoculated with sars-cov found that older macaques had stronger host innate responses to virus infection than younger adults, with an increase in differential expression of genes associated with inflammation, whereas expression of type i interferon beta was reduced. the age-dependent defects in t-cell and b-cell function and the excess production of type 2 cytokines could lead to a deficiency in the control of viral replication and more prolonged proinflammatory responses, potentially leading to poor outcome. 16 more than half of these people suffer from associated diseases, mainly hypertension -the most common chronic disease in the elderly. [17] [18] [19] the most commonly used drug groups in antihypertensive therapy are angiotensinconverting enzyme inhibitors (ace-i) and angiotensin receptor blockers (arbs). 20 due to the mechanism of development of covid-19 infection, questions arise about the legitimacy of the use of these drugs in the treatment of patients and the risks or therapeutic options associated with it should be identified. it has been proven that sars-cov-2 infection depends on ace2 interaction with the virus (figure 1 ). this indicates that the role of the renin-angiotensin (ra) system in the physiology and pathophysiology of the cardiovascular system is of major importance. in the lungs, angiotensin i (ang i) is being converted to angiotensin ii (ang ii). further, ang ii binds to either at1 receptor (at1r) causing vasoconstriction, hypertension, promoting inflammation, and may be converted to angiotensin (ang iv) via at4 receptor (at4r) causing thrombosis. the role of ace2 is to inactivate ang ii by converting it to angiotensin (1-7) (ang (1-7)) that binds to mas receptor causing vasodilation and hypotension. thus, in a healthy individual, ace2 negatively regulates the renin-angiotensin (ra) system and attenuates vasoconstriction, fibrosis, and hypertrophy induced by it. 21 when the spike protein of sars-cov-2 binds with ace2 this leads to the internalization of the complex and further to ace2 shedding by enzyme adam17. in 2005, studies appeared on the effect of tumor necrosis factor-α convertase (adam17) on the penetration of sars virus into the cells. adam17 overexpression has been shown to lead to increased virus penetration. 22 palau et al analyzed data on this metalloproteinase in the context of covid-19 infection. 23 adam17 has been shown to compete with tmprss2 for ace2, which, in light of the reports regarding the effect of tmprss2 on viral penetration, forces further research into the mechanisms of viral penetration to prevent infection. 23, 24 decreased availability of ace2 causes less ang ii degradation. excessive amounts of ang ii lead not only to overstimulation of at1r but to conversion to ang iv which promotes thrombosis via at4r. this leads to abnormalities seen in covid-19, namely acute lung injury with local vasoconstriction facilitating ards, myocardial injury, and thrombosis. 25 the potential effect of ace-i/arbs treatment may be seen in upregulation of ace2, leading to the increased amounts of free ace2 after viral binding. ace-inhibitors (ace-i) cause less synthesis of ang ii and arbs prevent angii from binding on the at1r. this leads to less at1r stimulation and persistent interaction with ace2, avoiding ace2 internalization. ace-2 is available for the transformation of angi i into angiotensin (1-7), causing less at1r and at4r stimulation. sars-cov-2 infection depends not only on ace2, but also on tmprss2, a cellular protease and cd147 (extracellular matrix metalloproteinase inducer -emmprin) which are expressed on host cells. 24,26-28 ace2, the homolog of ace, is composed of a single zinc metalloprotease active site, identical to ace only in 42%, and a transmembrane domain. 29-31 its expression is not so unique in organs as ace. ace2 transcripts are found in the renal tubular epithelium, testis, type 2 pneumocytes, endothelium of coronary and intrarenal vessels. 29, 32, 33 ace2 is present in serum after cleavage of the transmembrane domain. 29 this soluble form might limit covs infection 34, 35 and if combined with fc fragment of the antibody, it neutralizes the virus. 36 the aforementioned findings suggest that the course of infection might depend on the ace2 expression. ace2 activity is negatively correlated with blood pressure and bmi. 37 it is hypothesized that ace2 activity is a predictor of cardiac dysfunction in patients with hypertension. 37 however, chen et al found that circulating ace2 levels are positively associated with serum creatinine, a marker of kidney disease. 38 ace2 is located on a short arm of a chromosome x (xp22.2) and comprises 19 exons. 29, 39 since its identification as a potentially functional for cardiovascular diseases, there have been many trials verifying the hypothetical influence of single nucleotide polymorphisms (snps) located within ace2 gene. 39 ace2 might influence cardiovascular diseases even before birth. a study on 474 samples of umbilical cord blood connected rs2074192 (t allele) with 40 many ace2 variants are related to common diseases, whose incidence depends on the balance in the reninangiotensin-aldosterone pathway, as depicted in table 1 . hypertension is associated with rs1514283, rs2074192, rs233575, rs4646155, rs4646176, rs2285666, rs879922, rs2106809, rs4646188, rs4240157, rs4830542, rs2158083, and rs879922. 37, 41, 42 some of these variants affect the response to ace inhibitors. 43, 44 moreover, rs2285666 (a allele) is associated with lower probability of cardiovascular death in female population (hr = 0.3, p<0.05). 41, 45, 46 amongst people with hypertension minor alleles: rs2106809 and rs2074192 are associated with left ventricular hypertrophy. 47 lieb et al found the association between rs4646156, rs879922, rs4240157, rs233575 and higher septal wall thickness or left ventricular hypertrophy in male population. 48 this association might be a result of lower levels of circulating angiotensin (1-7) in this group. 38 wang et al conducted a study on 265 with atrial fibrillation (af) and 289 healthy people. researchers found that rs2106809 (t allele) is af risk factor. 49 ace2 snps might influence patients' lipid profiles. ldl concentration is associated with rs1978124, rs2106809, rs233575, rs4646188, and rs879922. hdl concentration might be decreased in individuals with rs2106809, rs2285666, rs4646142, rs4646155, and rs4646188. 41 snps: rs2106809 and rs4646188 are risk factors for hypertriglyceridemia. 41 some projects failed to find any association between ace2 variants and diseases, whose frequency is correlated with blood pressure. wu et al did not find any association between snps and recurrent strokes. 50 the aforementioned results suggest that some snps might be a risk factor of covid-19. genotypes affect ace2 structure, its serum concentration, and levels of circulating angiotensin (1-7) . 38, 51 moreover, there is evidence that ace genotype affects the outcomes of acute respiratory distress syndrome (ards) treatment. in a prospective study on 84 patients with ards and 200 healthy people, ace polymorphisms were genotyped. every study participant was caucasian. researchers found that being homozygous for ace d (deletion in intron 16) allele was associated with increased mortality. 52 geographic distribution of ace i (insertion in intron 16) allele was summarized by saab et al its frequency increases eastwards and westwards from the middle east. 53 it was proven that ace2 decrease the risk of lung failure. 33 nevertheless, chiu et al did not find any significant association between ace2 variants and sars. 54 another interesting study by goulter et al proved the connection between heart failure and ace2 expression. ace2 is probably upregulated due to idiopathic dilated cardiomyopathy and ischemic cardiomyopathy. 55 it needs to be verified if these diseases affect the likelihood of covid-19. covid-19 morbidity, infection course, and mortality might depend on ace d allele frequency. 56 delanghe et al's findings are unanimous with the role of ace in covs infections. 57 stawiski et al performed genomic datasets analysis and identified multiple ace2 variants, which might affect covid-19 susceptibility. 58 however, none of these snps is common in the general population. this finding might help in the recognition of people less and more prone to covid-19. 58 cao et al performed a genetic dataset analysis of various populations for polymorphisms in the ace2 structure. the differences between populations and higher tissue expression of ace2 in east asian populations were determined, which may suggest other susceptibility to infection. however, the studies did not show genetic evidence confirming the existence of the ace2 mutation resistant to coronavirus protein. 59 co-morbid conditions, including hypertension, diabetes, heart disease, and progressive age have been found to contribute to more severe disease in patients with covid-19. in addition, these patients were more often admitted to the intensive care unit, received mechanical ventilation, or died compared to patients with mild disease. 60 due to the fact that in these diseases, groups of drugs that are often used are drugs that have an inhibitory effect on the raas system, there are concerns that they may have contributed to some degree of adverse health effects or more severe course of this disease. 16, 60 ace2 is a key enzyme that breaks down angiotensin ii into angiotensin (1-7) , thus reducing its effect on vasoconstriction, sodium retention, water retention, and fibrosis. although angiotensin ii is the basic substrate for ace2, this enzyme also degrades angiotensin i to angiotensin (1-9) and is involved in the hydrolysis of other peptides. 61 the balance of these vasoactive peptides has a profound effect on organs and systems and is altered by both ace inhibitors (which block the action of ace-1) and arbs (which block the action of angiotensin ii on at1 receptors). studies of human tissue samples from 15 different organs have been shown to generally express ace2, including in the heart and kidneys, as well as in major sars-cov-2 target cells (and the site of the dominant injury)follicular epithelial cells. 32 in addition, circulating soluble ace2 levels are relatively low, and the functional role of ace2 in the lungs appears to be relatively minimal under normal conditions but might be elevated in some specific clinical conditions. 62 there are many hypotheses about the pathomechanism and impact of using ace-i and arbs. one of them says that acei initially inhibits ace, which leads to a decrease in the level of angiotensin i, resulting in a possible negative feedback loop, which ultimately regulates more of the ace2 receptor to be able to interact with the available substrate of angiotensin i. 63 this upregulation of the ace2 receptor causes an increase in sars-cov-2 binding sites, which can lead to covid-19 infection. this can be especially seen in patients with diabetes or hypertension, as they are usually treated with ace-i or arb. 63 an april 2020 a study including patients hospitalized in the hubei province in china found a death rate of 3.7% for hospitalized patients who had hypertension and were on ace-i or arbs versus 9.8% for hospitalized patients with hypertension not on such drugs, suggesting that the drugs are not harmful and may help against the coronavirus. 64 otherwise, some researchers believe that using ace-i or arbs may be beneficial in preventing covid-19 infection. li et al proposed that ace1 inhibition by ace-i may stimulate negative feedback. given the lack of angiotensin ii, an increase in ace2 receptors and a reduction in general inflammation. 65 probably the effect on the renin-angiotensin system by both ace-i and arbs leads to increased expression of ace2. theoretically, this may help alleviate some of the harmful effects of angiotensin ii on the human body. it is believed that the increased level of soluble ace2 may act as competitive to sars-cov-2 and may slow down the penetration of the virus into cells and protect against damage to lung tissue. 30 there is some evidence that ace-i/arbs may be beneficial in patients with acute lung injury (ali) or ards as a complication after covid-19 infection. in a meta-analysis of 37 studies, the use of ace-i and arbs was associated with a reduced risk of pneumonia and mortality from pneumonia compared to control treatment. 66 in one randomized, double-blind, placebo-controlled clinical trial in 61 patients, 8 patients randomly received enalapril up to 10 mg intravenously (within 24 hours after a blood pressure regimen) had more days without assisted ventilation (12.3 vs. 8.7 days; p = 0.18) and days spent outside of the intensive care unit (8.9 vs. 4.9 days; p = 0.09) compared to those randomly assigned to placebo. this study has not yet been completed due to the slow registration of patients. 67 in another korean retrospective study of 132 patients with ards, 9 patients receiving ace-i/arbs showed better survival compared to the control group, although other determinants could influence the results. 68 sun et al proposed that ace-i use implies the ace receptor/angiotensin ii/angiotensin-1 receptor pathway and thus interferes with the integrity of ace2/angiotensin 1-7/mas (mas-associated g-protein coupled receptor). ace2/angiotensin disruption route 1-7/mas may lead to a decrease in ace2 production, reducing the chance of sars-cov-2 getting into the cell. 69 one study to date has looked at the effects of ace-i and arb on the covid-19 population. according to peng et al among 112 patients, cardiovascular disease led to worse outcomes, with most deaths following fulminant inflammation, lactic acidosis and thrombotic conditions. the use of ace-i and arb did not affect morbidity or mortality. 70 receptor polymorphisms have been inclining towards genetic research and phenotyping for years. the main reason for consideration is the lack of response to the treatment of hypertension by ace-i and arbs. in 2007, danser et al proposed ace phenotyping to explain 69 due to many variables such as age, gender and comorbidities, they were unable to determine the effect of these drugs on the development of infection, but they proved that patients using ace-i had an increase in the number of ace2 receptors. therefore, more research is needed because of the popularity of these drugs. 72 zhixin liu et al conducted a large genetic analysis comparing the structure of the virus and receptors in many animals to detect potential carriers of the virus. animals possessing an ace receptor with human-like properties may constitute a reservoir other than bats. 73 thanks to such research, we are able to determine where the virus can exist as a potential source of another infection despite the removal of an infectious focus in humans. this type of research, based on the analysis of receptor structure, may be the key to predicting the severity of patient infections, detecting particularly sensitive patients, and maybe even discovering a treatment by preventing the virus from entering the cells. constant uncertainty, guesswork, and lack of knowledge about the harmfulness of ace inhibitors and arbs in patients with covid-19 contributed to the creation of population analyzes in this group of patients. mancia et al conducted a field study on a group of 6272 infected patients, comparing them with 30,759 controls with similar age, gender, and comorbidities. the results showed that patients with the sars-cov-2 virus were more likely to take these drugs; however, this was due to worse health and cardiovascular disease. ace-i and arbs have not been shown to affect the risk of covid-19 infection. 74 mehra et al analyzed patients from 169 european, asian, and north american hospitals for mortality and risk factors. the analysis showed that an increased mortality can be observed in patients with chronic obstructive pulmonary disease (or=2.96), coronary artery disease (or=2.7), heart failure (or=2.48), arrhythmia (or=1,95), age>65 (or=1.93), and smokers (or=1.79). there was no statistical significance in increasing the risk of hospital mortality associated with the use of ace inhibitors (or=0.33) and arbs (or=1.23). 75 reynolds et al also assessed the increased risk of drug use. in patients subjected to covid-19 diagnostics, the relationship between the use of different classes of hypertensive drugs and the occurrence and severity of the disease itself was evaluated. again, no statistical significance was demonstrated between the use of any group of drugs for hypertension and the onset of the disease and its severity. 76 there is no direct evidence yet for ace or arb inhibitors in the treatment of covid-19. however, for those patients already taking these medications, the european society of cardiology recommends that doctors and patients continue treatment because there is no clinical or scientific evidence to suggest that ace-i or arbs should be discontinued due to covid-19 infection. 77 the american college of cardiology recommends continuing treatment in conditions such as heart failure, hypertension or ischemic heart disease, and that if covid-19 occurs, "individualized treatment decisions should be made based on the hemodynamic condition and clinical presentation of each patient". 78 according to clinicaltrials.gov webpage, there are currently 26 clinical trials regarding drugs targeting ras in covid-19 (summarized in table 2 ), e.g.: nct04312009, nct04335786, which aim to verify the effects of initiation of the treatment with drugs interfering with ras in patients with covid-19 and nct04338009, nct04364893, which verify outcomes of discontinuation of ace-i/arbs therapy. 79 nct04359953 aims to include 1600 participants and the results are expected in december 2020. this trial is designed to compare one of the arbstelmisartan with hydroxychloroquine and azithromycin in covid-19 treatment. 80 the sars-cov-2 infection depends on ace2 which is expressed on host cells. there is no direct evidence for the role of ace-i or arbs in the treatment of patients with covid-19. however, for those patients who are already taking these medications, both the european society of cardiology and the american college of cardiology recommend continuing the treatment. currently, there is no clear clinical or scientific evidence to justify the discontinuation of ace-i or arbs. individualized treatment decisions should be based on age, clinical condition, and co-morbidities of each patient, weighing the benefits of 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gene polymorphism and enzymatic activity with essential hypertension in different gender: a case-control study relationship between genetic variants of ace2 gene and circulating levels of ace2 and its metabolites the ace2 gene: its potential as a functional candidate for cardiovascular disease fetal but not maternal angiotensin converting enzyme (ace)-2 gene rs2074192 polymorphism is associated with increased risk of being a small for gestational age (sga) newborn association of ace2 polymorphisms with susceptibility to essential hypertension and dyslipidemia in xinjiang, china ace2 gene polymorphism and essential hypertension: an updated meta-analysis involving 11 polymorphisms of ace2 gene are associated with essential hypertension and antihypertensive effects of captopril in women impact of ace2 gene polymorphism on antihypertensive efficacy of ace inhibitors a polymorphism in ace2 is associated with a lower risk for fatal cardiovascular events in females: the morgam project the association between the angiotensin-converting enzyme-2 gene and blood pressure in a cohort study of adolescents hypertension and hypertensive left ventricular hypertrophy are associated with ace2 genetic polymorphism association of angiotensin-converting enzyme 2 (ace2) gene polymorphisms with parameters of left ventricular hypertrophy in men. results of the monica augsburg echocardiographic substudy polymorphisms of angiotensin-converting enzyme 2 gene confer a risk to lone atrial fibrillation in chinese male patients the association between ace2 gene polymorphism and the stroke recurrence in chinese population association between circulating levels of ace2-ang-(1-7)-mas axis and ace2 gene polymorphisms in hypertensive patients ace i/d but not agt (−6)a/g polymorphism is a risk factor for mortality in ards the geographic distribution of the ace ii genotype: a novel finding ace2 gene polymorphisms do not affect outcome of severe acute respiratory syndrome ace2 gene expression is 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hypertension hospitalized with covid-19 the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases risk of pneumonia associated with use of angiotensin converting enzyme inhibitors and angiotensin receptor blockers: systematic review and meta-analysis ace inhibitor for lung protection during mechanical ventilation for acute lung injuryresults of the double-blind, placebo controlled, randomised acemevent pilot study effect of renin-angiotensin system blockage in patients with acute respiratory distress syndrome: a retrospective case control study inhibitors of ras might be a good choice for the therapy of covid-19 pneumonia clinical characteristics and outcomes of 112 cardiovascular disease patients infected by 2019-ncov ace phenotyping as a first step toward personalized medicine for ace inhibitors. why does ace genotyping not predict the therapeutic efficacy of ace inhibition? coronavirus disease 2019 (covid-19): do angiotensin-converting enzyme inhibitors/angiotensin receptor blockers have a biphasic effect? composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 dovepress sie nko et al clinical interventions in aging 2020:15 submit your manuscript | www renin-angiotensin-aldosterone system blockers and the risk of covid-19 cardiovascular disease, drug therapy, and mortality in covid-19 renin-angiotensinaldosterone system inhibitors and risk of covid-19 position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers. european society of cardiology hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 american college of cardiology available from efficacy of hydroxychloroquine, telmisartan and azithromycin on the survival of hospitalized elderly patients with covid-19 (covid-aging). 2020. available from: clinicaltrials medline, cas, scopus and the elsevier bibliographic databases. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use key: cord-306739-4lokd97u authors: gesierich, wolfgang title: sind ace-hemmer eher negativ oder doch von vorteil bei covid-19? date: 2020-07-14 journal: pneumo news doi: 10.1007/s15033-020-1857-7 sha: doc_id: 306739 cord_uid: 4lokd97u nan sars-cov-2 bindet mit seinem spike-protein an ace2 und führt nach endozytose des komplexes zu einer herunterregulation von ace2 auf der zelloberfläche [1] . dementsprechend kommt es zu einer lokalen enthemmung des raas mit entsprechender begünstigung gewebsschädigender effekte (▶abb. 1). bei covid-19 wird neben der meist vorherrschenden akuten lungenschädigung (ards) auch eine relevante rate an myokardialen schäden (anstieg von troponin) und akutem nierenversagen beobachtet. bereits aus der zeit von sars gibt es experimentelle daten, die eine entsprechende interaktion zwischen sars-cov-1 und dem raas belegen [2] . ebenso gibt es experimentelle studien, die einen krankheitsfördernden effekt einer raas-aktivierung bei ards nicht infektiöser genese nahelegen [3] . auch gibt es erste versuche, diese erkenntnisse klinisch-therapeutisch zu nutzen. eine pilotstudie zu einer lungenprotektiven therapie mit ace-hemmern bei mechanisch beatmeten patienten mit ards aus 2017 zeigte einen trend zur einer verkürzung der beatmungszeit [4] . die dargestellten britischen daten dürfen als erstes signal gewertet werden, dass ace-hemmer auch bei covid-19 einen günstigen effekt auf den krankheitsverlauf haben könnten. es handelt sich aber um eine relativ kleine, monozentrische kohorte mit kurzem follow-up. weitere klinische studien sind also dringend erforderlich, bevor den ace-hemmern ein therapeutischer effekt bei covid-19 zugeschrieben werden kann. entsprechende projekte sind in deutschland in vorbereitung. vorerst darf folgendes fazit gezogen werden, das von nationalen und internationalen fachgesellschaften unterstützt wird: eine vorbestehende therapie mit einem ace-hemmer, die für patienten mit kardiovaskulären erkrankungen und diabetes mellitus einen relevanten organprotektiven und damit prognostischen wert hat, sollte in der aktuellen pandemie-situation und insbesondere bei diagnose einer sars-cov-2-infektion nicht abgesetzt werden. renin-angiotensin-aldosterone system inhibitors in patients with covid-19 a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure ace inhibitor for lung protection during mechanical ventilation for acute lung injury-results of the double-blind, placebo controlled international conference 2017 abstracts, session b16. critical care: new insights from clinical trials key: cord-306755-9q1mawfs authors: rivière, guillaume; michaud, annie; corradi, hazel r.; sturrock, edward d.; ravi acharya, k.; cogez, virginie; bohin, jean-pierre; vieau, didier; corvol, pierre title: characterization of the first angiotensin-converting like enzyme in bacteria: ancestor ace is already active date: 2007-09-01 journal: gene doi: 10.1016/j.gene.2007.05.010 sha: doc_id: 306755 cord_uid: 9q1mawfs angiotensin-converting enzyme (ace) is a metallopeptidase that converts angiotensin i into angiotensin ii. ace is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. in vertebrates, both transmembrane and soluble ace, containing one or two active sites, have been characterized. so far, only soluble, single domain aces from invertebrates have been cloned, and these have been implicated in reproduction in insects. furthermore, an ace-related carboxypeptidase was recently characterized in leishmania, a unicellular eukaryote, suggesting the existence of ace in more distant organisms. interestingly, in silico databank analysis revealed that bacterial dna sequences could encode putative ace-like proteins, strikingly similar to vertebrates' enzymes. to gain more insight into the bacterial enzymes, we cloned the putative ace from the phytopathogenic bacterium, xanthomonas axonopodis pv. citri, named xcace. the 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. in vitro expression and biochemical characterization revealed that xcace is a functional 72 kda dipeptidyl-carboxypeptidase. as in mammals, this metalloprotease hydrolyses angiotensin i into angiotensin ii. xcace is sensitive to ace inhibitors and chloride ions concentration. variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ace. xcace characterization demonstrates that ace is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution. angiotensin-converting enzyme (ace, dipeptidyl-peptidase a, kininase ii, e.c. 3.4.15.1, dcp1) is a dipeptidyl-carboxypeptidase that belongs to the m2-metalloprotease family. in mammals, this zinc-dependent metalloprotease participates in blood pressure elevation (review in (corvol and eyries, 2004) ), generating the vasopressor angiotensin ii (ang ii) from the inactive angiotensin i (ang i), and by inactivating the vasodilator bradykinin. in vertebrates, somatic (sace) and testicular (germinal) ace (tace) have been characterized. these two separate isoforms are transcribed from two alternate promoters within a single gene (howard et al., 1990) , which arose from duplication of an ancestral gene (hubert et al., 1991) . consequently, sace possesses two domains (termed n-domain and c-domain, respectively) bearing the highly conserved gluzincin motif hexxh, whereas tace possesses only one, corresponding to the c-terminal domain of sace. crystal structures of both the human n-domain (corradi et al., 2006) and tace (natesh et al., 2003) have shown them to be homologous helical ellipsoids harbouring two large cavities connected by a narrow catalytic channel containing the catalytic zinc and the hexxh motif. somatic ace n-and c-domain active sites have distinct enzymatic specificities with respect to in vivo and/or synthetic substrates. indeed, the c-domain is highly selective for hippuryl-histidyl-leucine (hhl) (wei et al., 1991a) whereas the n-domain efficiently cleaves the hemoregulatory peptide ac-ser-asp-lys-pro (acsdkp) (rousseau et al., 1995) and ang 1-7 (deddish et al., 1998) . in addition, ace inhibitors (dive et al., 1999) and chloride ion dependence (jaspard et al., 1993) can discriminate between the two domains. both sace and tace possess a c-terminal transmembrane domain and are membrane-anchored isoforms, though a post-translational shedding releases active solubilized ace into the extracellular milieu (eyries et al., 2001; oppong and hooper, 1993) . a single domain ace homologue, ace2, has also been characterized in the human (donoghue et al., 2000; tipnis et al., 2000) and its structure determined (towler et al., 2004) . ace2 is an essential regulator of heart function (crackower et al., 2002) and has also been identified as a functional receptor for the sars coronavirus (li et al., 2003) . in invertebrates, however, functional ace-related enzymes exist in insects and in annelids (which belong to the ecdysozoa and lophotrochozoa groups, respectively). interestingly, and though in silico evidence suggest the presence of a two-domain ace-related protein in mosquitoes (burnham et al., 2005) , all the cloned genes encode soluble, single active site enzymes (tatei et al., 1995; taylor et al., 1996; wijffels et al., 1996) . in the fruit fly drosophila melanogaster, two homologues have been cloned and named ance (cornell et al., 1995) and acer (tatei et al. 1995) (or race) , of which ance has had its structure determined (kim et al., 2003) . ance and acer show about 40% amino-acid sequence identities with vertebrate aces, share similar enzymatic properties (coates et al., 2000) , acting on a broad range of substrates and play a key role in development (crackower et al., 2002) and reproduction (ekbote et al., 1999; hurst et al.., 2003) . molecular and biochemical data from the leech ace homologue have related it to the n-domain of mammalian ace (riviere et al., 2004) . however, no functional ace is present in the genome of the nematode caenorhabditis elegans (c. elegans), despite acn-1, a homologue that lacks ace-like proteolytic activity, is crucial in development (brooks et al., 2003) . recently, a dicarboxypeptidase, lddcp, was cloned in a pathogenic unicellular eukaryote, leishmania donovani (goyal et al., 2005) . lddcp, like ace, is able to cleave a carboxyterminal dipeptide from hhl and is inhibited by captopril, suggesting that ace may have already been present before the appearance of multicellular organisms. however, lddcp was not reported to hydrolyse angi and does not display sequence homology with ace enzymes. these observations raise questions about the presence of a true ace in more distant species. very surprisingly, in silico databank analysis revealed that dna sequences in bacterial genomes have been annotated as encoding potentially functional ace-like enzymes. all these sequences display a great similarity despite phylogenetic distance (for references see http://tolweb.org and (delong and pace, 2001)). the striking sequence conservation in the active region of these putative ace-like peptidases suggests that the protease activity would be conserved in these ancestral, prokaryotic species. among them, xanthomonas axonopodis pv. citri (x. citri) is a phytopathogen gamma-proteobacteria in whose genome an ace-like encoding sequence has been annotated. in order to gain more insight into stages in ace evolution and to investigate its putative activity in unicellular organisms, we report the cloning and biochemical characterization of the expressed recombinant enzyme. we have also generated a homology-based model of xcace using the structure of tace to understand the interactions of residues in the active site. this ace-like enzyme in x. citri is, to our knowledge, the first bacterial ace-like enzyme ever characterized. the protein sequence of the leech ace (ttace, genbank accession no. 45272589), the most distant known ace characterized at the time of the present study, was submitted to similarity searches using the blast program suite (tblastn; default parameters) (http://www.nih.nlm.ncbi.gov/blast) against all bacterial genomic sequences available at the ncbi server at the time of the study. alignments were performed using the clustalw (default parameters) software (http://www.ebi.ac.uk/ clustalw/). the distance matrix generated into the tree file of clustalw was used in the drawtree program of the phylip suite and an unrooted tree diagram was generated (http:// bioweb.pasteur.fr/seqanal/interfaces/drawtree-simple). x. citri strain n1 was from the culture collection of the laboratory of plant pathology, faculty of agriculture, shizuoka university, shizuoka, japan (talaga et al., 1996) . bacterial dna was obtained as previously described (davis et al., 1980) . total rna was extracted using the sv total rna isolation system (promega). 2.5. protein extraction x. citri was cultured in yp medium under agitation at 30°c until an optical density of 0.3 at 620 nm and centrifuged (5000 ×g, 30 min). cells were washed in water, centrifuged, and the pellet resuspended in water in one hundredth of the culture volume. the resulting suspension was passed twice through a french pressure cell at 15,000 lb/in. 2 (10 8 pa) and unbroken cells and debris were removed by 5 min centrifugation at 5000 ×g. to assess the presence of a messenger rna correponding to the xcace gene, total rnas (30-40 ng) were treated with rnasefree dnase i and reverse transcribed with superscript iii reverse transcriptase (invitrogen). the same amount of nucleic acids (170 ng) was further used in pcr reactions containing 2% dmso using xcint-s1 (5′-gctgcagcagatcccacaga-3′) and xcint-as1 (5′-ctattgcgtgggcgtaggc-3′) as primers. water was used instead of cdna as a negative control. pcr products were resolved on an agarose gel stained with ethidium bromide. 2.7. cloning of xcace, construction of the pxcace vector and expression in mammalian cell cultures 2.7.1. cloning full-length xcace cdna sequence (np 641553; genbank accession no. gi: 21241971) was obtained by pcr on x. citri dna (see section 2.4). sense and antisense primers (xcacefl-s1 5′-ctgacctattcggatgcgctcaaggat-3′ and xca-cefl-as2 5′-ctctagccttcggcggacttcactg-caaaggac-3′) were used (2 pmol each) in a pcr reaction containing 400 ng dna, 2% dmso, 20 μm dntps, 5 u pfu dna polymerase (promega) in 50 μl total volume. reaction cycling parameters were 94°c, 3 min; 94°c, 40 s, 61°c, 1 min, 72°c, 4 min for 35 cycles. the expected 2.2 kb product was bluntend subcloned into the ecorv site of pcdna3.1 (invitrogen) and sequenced with xcace and pcdna3 specific primers. heterologous expression of xcace was carried out using mammalian cho-k1 cells (invitrogen). because the start codon is gtg for xcace, it was necessary to change it into the standard atg, a sequence recognized as an initiation codon by cho-k1 cells. this substitution mutation (t11a) was realised by pcr using the xcflas2.2 (5′-gct cta gcc ttc ggc gga ctt caa tgc aag-3′) antisense oligonucleotide and resulted in an in-frame atg initiation codon. the pcr product was subcloned in pcdna3.1 and the obtained pxcace plasmid sequenced as described above (see section 2.7.1). cho-k1 cells were stably transfected with pxcace using lipofectamine 2000 reagent (invitrogen), and were selected for resistance to geneticin. individual resistant colonies producing large amounts of xcace were cloned by limiting dilution techniques as described (wei et al., 1991b) . both native and recombinant xcace were partially purified by ion exchange chromatography. briefly, native xcace was obtained from bacterial protein extract, and secreted recombinant xcace from concentrated culture medium of transfected cho cells (fraction a). the fraction a samples were first desalted on a hiprep fast desalting column (amersham pharmacia biotech), then fractionated onto a fplc monoq anion-exchange column system (pharmacia). the resulting fractions showing specific ace activity were pooled and concentrated. the resulting fractions were named native xcace (nxcace) and recombinant xcace (rxcace), respectively. for nxcace purification steps, each pooled fractions containing specific ace activity were analysed by sds-page. both nxcace and rxcace (see section 2.8.1) were submitted to western blot experiment using a polyclonal antibody (hkce) raised against the human full-length sace. ace activity was assayed using various substrates: hhl (5 mm), as a human ace c-domain specific substrate, acsdackp (acetyl-serine-aspartyl-acetyl-lysine-proline) (2.5 mm), as an n-domain specific substrate, ang i (100 μm), as both an n-and a c-domain ace substrate. assays were performed at 37°c for 30 or 60 min. hydrolysis of [leu5]enkephalinamide ([leu5]-enk-nh2) (0.8 mm, incubated for 330 min), as an amidated substrate and [leu5]-enkephalin ([leu5]-enk) (0.8 mm, incubated for 30 min), were compared to determine whether xcace also exhibits endopeptidase activity. the rate of hydrolysis of all substrates used was determined and quantified using reverse phase hplc (waters co, milford, ma, usa) as previously described (azizi et al., 2000; houard et al., 1998; wei et al., 1991b) . the specificity of the reaction was assessed in the presence of 10 μm captopril. the effect of chloride ions (nacl concentrations from 0 to 1.0 m) on hhl hydrolysis was determined under standard conditions. the inhibition potentials of various tight binding ace inhibitors (captopril, delaprilat, fosinoprilat, lisinopril, perindoprilat, rxp 407 and enalaprilat) were assessed by determining the ic 50 values for the fluorogenic substrate mca-ala-ser-asp-lys-dpaoh hydrolysis in standard conditions as described previously (dive et al., 1999) . compounds were pre-incubated with nxcace (see section 2.8.1) for 1 h and reactions initiated by the addition of 12 μm substrate. a model of xcace was generated by the esypred3d server (http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/ esypred/) (lambert et al., 2002) using testis ace (proteinda-tabank pdb code 1o8a) as a model. this model was compared with the testis ace structures (natesh et al., 2003; natesh et al., 2004) and the n-domain structure (corradi et al., 2006) and ance (houard et al., 1998) to examine xcace localization, ace activity in medium and in bacterial extracts was monitored along during bacterial growth using hhl as substrate as described above. among 465 bacterial strains genomes screened, 26 sequences produced significant similarity (e value b 0.5) with leech ttace. further analysis revealed that at least 22 sequences within 19 different bacterial strains within 8 generas potentially encode putative ace-like enzymes. these species are ecologically as well as phylogenetically distant, since they are found amongst cyanobacteria (gloeobacter violaceus, gi: 37522712, gi: 35213714), acidobacteria (solibacter usitatus, gi: 67858770, gi: 66769433) and proteobacteria of the subdivision alpha (erythrobacter litoralis, gi: 61100845, gi: 60736774), delta (anaeromyxobacter dehalogenans, gi:66855040) and gamma (x. citri, gi: 21241971, gi: 21107345; xanthomonas oryzae, gi: 37931607, gi: 58583162; xanthomonas campestris pv campestris, gi:21230574, gi: 21112152; shewanella oneidensis, gi: 24374038, gi: 24348510) proteobacteria. all these predicted proteins share between 38 and 98% primary sequence identity between them, indicating a wide variety among the potential bacterial aces ( fig. 1 and supplementary data) . nevertheless, all these sequences exhibit key ace features such as the presence of two highly conserved consensus vchasawdfy and ganpgfhea motifs surrounding a putative m2-catalytic site hexxh (data not shown). we cloned an ace homologue in the phytopathogenic bacterium x. citri corresponding to the sequence xac1217 (gi: 21107356, gene id 1155288). the transcription of the xcace gene was suggested by the sequence conservation upstream the start codon and is assessed by the presence of a specific 437-bp rt-pcr product in dnase-treated, reverse-transcribed total rna (fig. 2) . the protein encoded by this gene, referred to as xcace (xanthomonas citri angiotensin-converting enzyme), is a 672 amino-acid protein. hydrophobicity profile (signalp v1.1 at www.cbs.dtu.dk/services/signalp) indicates cleavage of a putative signal peptide between residues 26 and 27 of the native protein (…rda↕ap…). there does not appear to be a motif encoding a transmembrane domain at the c-terminus or elsewhere in the mature molecule, suggesting that xcace should exist only in a soluble form. because of the mutation inserted, the recombinant proenzyme is slightly different from the native one in the n-terminal end (rxcace: mqgppvnprll vs nxcace: mnprll). however, this difference does not change predictions about the signal peptide (hydrophobicity profile and cleavage site) (data not shown), indicating that the mature recombinant and native proteins have identical primary sequences. the mature enzyme has a theoretical molecular weight of 72 kda and an isoelectric point of 5.72. its primary structure includes all the residues implied in zinc (h 429 (natesh et al., 2004) binding. thus the soluble, secreted enzyme should possess a single active site like the other aces found in invertebrates. whereas two chloride atoms were observed bound to the human tace, xcace appears to be more similar to the n-domain in this respect, as it only has the chloride ii binding site conserved (y 230 and r 567 ), and only one amino acid among the three implicated in the binding of the first chloride atom (w 530 ). the xcace primary sequence has between 31 and 37% identity with the other known aces (human, drosophila and leech) and 26% with c. elegans acn-1 ( fig. 1 and supplementary data). the semi-purification of xcace procedure using fplc (fig. 3a ) resulted in a fraction containing a protein band at the expected size for xcace (∼75 kda), as revealed by sds-page analysis of the different fractions obtained after each purification step (data not shown). both native and recombinant xcace were analysed by western blotting and migrated with an apparent molecular weight of 72 kda, matching theoretical predictions (fig. 3b) , and have a similar mobility on sds-page to deglycosylated human tace (yu et al., 1997) . xcace shows dipeptidyl-carboxypeptidase activity, cleaving a carboxyterminal dipeptide from peptidic substrates. it generates angii from angi (fig. 4a) , hippuric acid from hhl (fig. 4b) , ackp from acsdackp (fig. 4c) , and tyrosylglycyl-glycine (ygg) from leu-enkephalin (data not shown). xcace also displays an endopeptidase activity, as shown by its ability to hydrolyse the amidated substrate, leu-enk-nh2 (fig. 4d) . as for angi hydrolysis (fig. 4e) , the same results were obtained with recombinant xcace (data not shown). xcace activity depends on zinc binding. indeed, hydrolysis of mca-sdkp-dpaoh was abolished after zinc chelation with edta and dialysis but was restored by zinc addition (data not shown). native xcace is already active in the absence of chloride ions. however, its activity in the presence of increasing chloride ion concentrations increases 3-fold. in this respect, xcace cl − activation presents intermediate characteristics when compared to both n-and c-domain human ace (riviere et al., 2004) . (fig. 4f ). fosinoprilat is the most potent inhibitor of xcace activity (ic 50 : 3.0·10 − 8 ±1.0·10 − 8 m), whereas captopril is less efficient (1.6 · 10 − 7 ±0.1·10 − 7 m). rxp 407 (1.9 · 10 − 6 ±1.5·10 − 6 m) and enalaprilat (3.8 · 10 − 6 ±2.8·10 − 6 m) displayed ic 50 values in the micromolar range. perindoprilat (1.3 · 10 − 4 ±1.3·10 − 4 m), delaprilat (3.7 · 10 − 5 ± 1.7 · 10 − 5 m) and lisinopril (4.7 · 10 − 5 ± 4.3 · 10 − 5 m), all containing carboxylate zinc-binding groups, were the least effective inhibitors of xcace. values are means ±standard error from three independent experiments. as the crystal structures of human ace (corradi et al., 2006; natesh et al., 2003) , ace2 (towler et al., 2004) and the drosophila ance (kim et al., 2003) all have the same fold, the 34% sequence identity of human tace and bacterial xcace allowed us to model with confidence the overall fold of xcace based on the tace structure (pdb code 1o8a). we identified active site residues that could account for the differences in inhibitor affinities (see section 4.2) (fig. 5) . only background ace activity was detected in the culture medium, which remains constant over time. in contrast, ace activity in bacterial extract correlates bacterial growth indicat-ing that xcace is constituvely expressed and is likely to be secreted and further localised in the periplasmic space (fig. 6) . databank analysis revealed the presence of putative aceencoding genomic dna sequences in a very wide range of bacterial species, corresponding to a broad range of ecological lifestyles and terminal electron acceptor for growth (delong and pace, 2001) , suggesting that ace is an ancestral enzyme. characterization of xcace constitutes the first evidence of the presence of an ace-like enzyme in more distant organisms than cellular metazoans. indeed, in contrast to xcace, the amino-acid sequence of l. donovani dicarboxypeptidase (lddcp) (goyal et al., 2005) does not show any significant alignment with all other known aces, was not reported to hydrolyse ang i to ang ii, and lacks conserved consensus motifs found in almost all ace primary sequences (for references see the merops database available at http://merops.sanger.ac.uk/). however, in contrast with leishmania, none of the bacterial species putatively possessing an ace mentioned in this study is known to be a human pathogen, whereas xanthomonadacaea are phytopathogens among which x. citri was chosen for technical reasons. the xcace possesses a single m2 catalytic site and a signal peptide but no membrane anchor, like the ace characterized in invertebrates. despite the fact that the recombinant enzyme was expressed in a heterologous system, both native and recombinant forms display almost identical features, indicating a satisfactory purity of xcace and strongly suggesting that the ace in x. citri is a functional ace enzyme in vivo. xcace, like drosophila ance (data not shown), is active in the absence of chloride ions and less sensitive to variations in chloride ion concentration than human c-domain ace (houard et al., 1998) . this characteristic is likely to be due to the absence in the primary sequence of xcace, as in ance, of two key amino acids observed to bind cli in human tace (xcace : r186h and r489k; ance : r1867yand w485f, when compared to tace). xcace hydrolyses a broad range of substrates, including human n-and c-domain ace specific substrates (acsdackp and hhl, respectively). in addition to this dicarboxypeptidase activity, xcace also acts as an endopeptidase, hydrolysing the c-terminal blocked amidated substrate [leu5]-enkephalinamide. western blot results using polyclonal antibody raised against the full-length human sace protein show a unique band corresponding to xcace, indicating that xcace shares common epitopic features with the human enzyme. molecular modelling allowed us to visualise the structural differences in the catalytic site. key active site residues such as the zinc-binding motif, his 513 and tyr 526 (tace numbering) are conserved in xcace and many other ace-like enzymes. however, in line with the assay results, the non-conserved active site residues are neither identical to the c-or n-domain of human ace, and are the presumed cause of the different substrate selectivity and inhibition profile of xcace. comparison of the xcace model with either the human tace or n-domain shows that lisinopril fits in the xcace active site and retains most of the same binding interactions. the exception is in the lysyl pocket, which has features in common with ance or the human n-domain including a positively charged residue, lysine (equivalent to arg 356 in ance and arg 350 in the human n-domain) that appears to affect the overall charge of the pocket (fig. 5) . indeed, similar to xcace, neither ance nor the n-domain have as high affinity for lisinopril as tace (michaud et al., 1997; williams et al., 1996) . furthermore, the difference in affinity of xcace and tace for inhibitors with hydrophobic p1 groups (i.e. lisinopril, delaprilat and perindoprilat) could be attributed to the difference in hydrophobicity of the s1 subsite. xcace has a threonine and tyrosine in contrast to phe 512 and val 518 in tace (fig. 5) . the n-terminal aspartate, n-acetyl groups and the cterminal amide of rxp407 are the reported determinants required for its remarkable n-domain specificity (dive et al., 1999) . of the four possible residues suggested for n-domain selective recognition of rxp 407 (corradi et al., 2006; tzakos and gerothanassis, 2005) , xcace only retains the threonine (t 496 in the n-domain) and has a lysine which replaces arg 500 of the n-domain. this, combined with the hydrophobic p1 group, may be the reason that rxp407 is not a strong inhibitor for xcace. the affinity of xcace for fosinoprilat is harder to rationalise as it has been modelled in different conformations for the n-and c-domains of human ace (tzakos and gerothanassis, 2005) . however, xcace retains all the residues suggested to be involved in inhibitor binding with both domains of human ace. the biological functions of xcace remain unclear but could constitute the original, ancestral function of ace. xcace is soluble and its sequence exhibits a signal peptide indicating, along with the absence of ace activity in the culture medium, that xcace is a secreted enzyme which is trapped into the periplasmic space. the constitutive expression could indicate an important function for ace in x. citri, as a possible role for the enzyme in peptidoglycan remodelling, a critical parameter in environmental adaptation. this is in line with the characterization in the periplasm of other gram negative bacteria of vanx dalanyl-d-alanine dipeptidase, a regulated carboxypeptidase that is responsible for vancomycin resistance (lessard and walsh, 1999) . xcace could also participate in x.citri pathogenicity within the cocktail of degradative enzymes required during plant infection. interestingly, in this regard, ace inhibitors are found in hydrolysates of plants sensitive to the "black rot" caused by xanthomonas, including rice and soybean (he et al., 2005; wu and ding, 2001) . the large number of substrates of xcace does not exclude a possible function of protein degradation that might generate dipeptides for protein biosynthesis. to this extent, it is interesting to note that ace is highly expressed in the digestive tract of all metazoan species presenting an active ace. one of the most relevant questions raised by this study deals with phylogenetic considerations and evolution of ace. its presence in eukarya as well as in bacteria indicates that the enzyme could have appeared before the divergence between these two groups. this strongly suggests that xcace is the most ancestral present-day representative of ace characterized to date. however, as all completely sequenced genomes of plants, algaes and mycetes lack ace, caution should be exercised with this hypothesis. the presence of an ace-like enzyme in bacteria could also be the result of a horizontal gene transfer from eucaryotic to bacterial species. however, conservation in sequences surrounding the ace gene, suggestive of mobile genetic elements, is not observed within 30 kb of the gene in seven distinct bacterial strains (data not shown). furthermore, the parasitic bacterial species with an ace-like gene are all hosted by organisms lacking the enzyme. in addition, not all bacterial species with a putative ace gene are parasitic. thus, the presence of ace in bacteria, like the situation encountered in annelids, is unlikely to be related to parasitism, suggesting that bacterial ace, represented herein by xcace, corresponds to the ancestral, original enzyme. xcace exhibits a similar primary sequence identity with both n-and c-domains and, although xcace cleaves the n-domain substrate acsdackp, the n-domain specific inhibitor rxp407 is not a potent inhibitor of xcace activity. in contrast, lisinopril, a c-domain-related inhibitor, is less efficient than captopril, related to the n-domain, though most of the residues for binding of both are conserved. thus, from the biochemical and the structural properties of xcace it is not possible to ascribe this bacterial enzyme to either the mammalian n-or c-domain of human ace. the primitive ace would then not be closer to either domain of the mammalian enzyme, in contrast to what was previously suggested (coates et al., 2000; hubert et al., 1991; lattion et al., 1989) . this work describes the cloning, expression, purification, biochemical characterization, molecular modelling and localization of xcace, the first ace-like enzyme in a bacterium, x. citri. taken together, our results bring the evidence of the presence of a functional ace in prokaryotes, in contrast to the previous hypothesis about the latter appearance of ace during phylogeny. it also appears that sequence/structure conservation of ace enabled preservation of dipeptidase activity during evolutionary specialisation. xcace is the most ancestral ace present-day representative and has features in common to both the n-and the c-domain of mammalian somatic ace. it is possible that this ancestral ace was a poorly specialised protease which has acquired its present specialisation over a long evolutionary period. however, the biological function(s) as well as the endogenous substrates and/or inhibitors of bacterial ace remain to be characterized. to clarify these issues, the use of ace inhibitors in vivo and the generation of a mutant bacterial strain lacking ace may be helpful. some of these functions of ace might also be conserved in humans, and their characterization could lead to a better understanding of the evolution of this crucial enzyme. in vitro and in vivo inhibition of the 2 active sites of ace by omapatrilat, a vasopeptidase inhibitor an essential role in moulting and morphogenesis of caenorhabditis elegans for acn-1: a novel member of the angiotensin-converting enzyme family that lacks a metallopeptidase active site the angiotensin-converting enzyme (ace) gene family of anopheles gambiae functional conservation of the active sites of human and drosophila angiotensin i-converting enzyme cloning and expression of an evolutionary conserved single-domain angiotensin converting enzyme from drosophila melanogaster crystal structure of the n domain of human somatic angiotensin iconverting enzyme provides a structural basis for domain-specific inhibitor design peptidyl dipeptidase a: angiotensin i converting enzyme angiotensin-converting enzyme 2 is an essential regulator of heart function advanced bacterial genetics. manual for genetic engineering n-domain-specific substrate and c-domain inhibitors of angiotensinconverting enzyme : angiotensin-(1-7) and keto-ace the pymol molecular graphics system environmental diversity of bacteria and archaea rxp 407, a phosphinic peptide, is a potent inhibitor of angiotensin i converting enzyme able to differentiate between its two active sites a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a mosquito (anopheles stephensi) angiotensin i-converting enzyme (ace) is induced by a blood meal and accumulates in the developing ovary coot: model-building tools for molecular graphics increased shedding of angiotensin-converting enzyme by a mutation identified in the stalk region cloning and characterization of angiotensin converting enzyme related dipeptidylcarboxypeptidase from leishmania donovani optimization of angiotensin i-converting enzyme (ace) inhibition by rice dregs hydrolysates using response surface methodology the drosophila melanogaster-related angiotensin-iconverting enzymes acer and ance-distinct enzymic characteristics and alternative expression during pupal development transcription of testicular angiotensin-converting enzyme (ace) is initiated within the 12th intron of the somatic ace gene structure of the angiotensin i-converting enzyme gene. two alternate promoters correspond to evolutionary steps of a duplicated gene the drosophila angiotensin-converting enzyme homologue ance is required for spermiogenesis differences in the properties and enzymatic specificities of the two active sites of angiotensin i-converting enzyme (kininase ii). studies with bradykinin and other natural peptides crystal structure of drosophila angiotensin i-converting enzyme bound to captopril and lisinopril esypred3d: prediction of proteins 3d structures the testicular transcript of the angiotensin i-converting enzyme encodes for the ancestral, non-duplicated form of the enzyme vanx, a bacterial d-alanyl-d-alanine dipeptidase: resistance, immunity, or survival function? angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus substrate dependence of angiotensin i-converting enzyme inhibition: captopril displays a partial selectivity for inhibition of n-acetyl-seryl-aspartyl-lysylproline hydrolysis compared with that of angiotensin i structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin i-converting enzyme crystal structure of the human angiotensin-converting enzyme-lisinopril complex characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane characterization of the first non-insect invertebrate functional angiotensin-converting enzyme (ace): leech ttace resembles the n-domain of mammalian ace the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensin-converting enzyme cell-associated glucans of burkholderia solanacearum and xanthomonas campestris pv. citri: a new family of periplasmic glucans race: a drosophila homologue of the angiotensin converting enzyme the acer gene of drosophila codes for an angiotensin-converting enzyme homologue a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis domain-selective ligand-binding modes and atomic level pharmacophore refinement in angiotensin i converting enzyme (ace) inhibitors the two homologous domains of human angiotensin i-converting enzyme are both catalytically active expression and characterization of recombinant human angiotensin i-converting enzyme. evidence for a c-terminal transmembrane anchor and for a proteolytic processing of the secreted recombinant and plasma enzymes cloning and characterisation of angiotensin-converting enzyme from the dipteran species, haematobia irritans exigua, and its expression in the maturing male reproductive system drosophila melanogaster angiotensin i-converting enzyme expressed in pichia pastoris resembles the c domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats identification of n-linked glycosylation sites in human testis angiotensinconverting enzyme and expression of an active deglycosylated form supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gene.2007.05.010. key: cord-351567-ifoe8x28 authors: rabi, firas a.; al zoubi, mazhar s.; kasasbeh, ghena a.; salameh, dunia m.; al-nasser, amjad d. title: sars-cov-2 and coronavirus disease 2019: what we know so far date: 2020-03-20 journal: pathogens doi: 10.3390/pathogens9030231 sha: doc_id: 351567 cord_uid: ifoe8x28 in december 2019, a cluster of fatal pneumonia cases presented in wuhan, china. they were caused by a previously unknown coronavirus. all patients had been associated with the wuhan wholefood market, where seafood and live animals are sold. the virus spread rapidly and public health authorities in china initiated a containment effort. however, by that time, travelers had carried the virus to many countries, sparking memories of the previous coronavirus epidemics, severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), and causing widespread media attention and panic. based on clinical criteria and available serological and molecular information, the new disease was called coronavirus disease of 2019 (covid-19), and the novel coronavirus was called sars coronavirus-2 (sars-cov-2), emphasizing its close relationship to the 2002 sars virus (sars-cov). the scientific community raced to uncover the origin of the virus, understand the pathogenesis of the disease, develop treatment options, define the risk factors, and work on vaccine development. here we present a summary of current knowledge regarding the novel coronavirus and the disease it causes. coronaviruses, named for the crown-like spikes on their surface (latin: corona = crown), are positive-sense rna viruses that belong to the coronvirinae subfamily, in the coronaviridae family of the nidovirales order [1] . they have four main subgroups-alpha, beta, gamma, and delta-based on their genomic structure. alpha-and betacoronaviruses infect only mammals, usually causing respiratory symptoms in humans and gastroenteritis in other animals [2, 3] . until december of 2019, only six different coronaviruses were known to infect humans. four of these (hcov-nl63, hcov-229e, hcov-oc43 and hku1) usually caused mild common cold-type symptoms in immunocompetent people and the other two have caused pandemics in the past two decades. in 2002-2003, the severe acute respiratory syndrome coronavirus (sars-cov) caused a sars epidemic that resulted in a 10% mortality. similarly, the middle east respiratory syndrome coronavirus (mers-cov) caused a devastating pandemic in 2012 with a 37% mortality rate. in late 2019, a cluster of pneumonia cases in wuhan city, hubei province, china were identified as with a novel betacoronavirus, first called the 2019 novel coronavirus (2019-ncov) and often referred to as the wuhan coronavirus. when the genomics of the 2019-ncov was sequenced, it shared 79.5% of the genetic sequence of the sars-cov that caused the 2002-2003 pandemic [4] and the international committee on taxonomy of viruses renamed the 2019-ncov as sars-cov-2 [5] . patients began to present in november and december with various degrees of respiratory distress of unknown etiology and treated at the time as possible influenza infections. as it became apparent that most cases had a shared history of exposure to the huanan seafood wholesale market (the so-called "wet market"), the wuhan local health authority issued an epidemiologic alert on 30 december 2019 and the wet market was closed. about a week later, on 9 january 2020, chinese researchers shared the full genetic sequence of the novel coronavirus, now called sars-cov-2 [6] . since the novel coronavirus was recognized, the disease it caused was termed coronavirus disease 2019 (covid19) , and several reports on the clinical presentation, epidemiology, and treatment strategies have been published [7] [8] [9] [10] . in addition, several websites have been setup to track the epidemic and the case detection rate, which are being updated as often as hourly [11] [12] [13] [14] . on 30 january 2020, the world health organization (who) declared the covid-19 outbreak to be a global public health emergency, sixth after h1n1 (2009), polio (2014), ebola in west africa (2014), zika (2016) and ebola in the democratic republic of congo (2019), and on 11 march 2020, the who characterized covid-19 as a pandemic [15] . the timeline of events is summarized in figure 1 . all coronaviruses that have caused diseases to humans have had animal origins-generally either in bats or rodents [16] . previous outbreaks of betacoronaviruses in humans involved direct exposure to animals other than bats. in the case of sars-cov and mers-cov, they were transmitted directly to humans from civet cats and dromedary camels respectively ( figure 2 ). animal origins of human coronaviruses. severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) and were transmitted to humans from bats by civet cats and dromedary camels, respectively. the 2019 sars-cov-2 was likely transmitted to humans through pangolins that are illegally sold in chinese markets [16, 17] . the sars-related coronaviruses are covered by spike proteins that contain a variable receptor-binding domain (rbd). this rbd binds to angiotensin-converting enzyme-2 (ace-2) receptor found in the heart, lungs, kidneys, and gastrointestinal tract [18] thus facilitating viral entry into target cells. based on genomic sequencing, the rbd of sars-cov-2 appears to be a mutated version of its most closely related virus, ratg13, sampled from bats (rhinolophus affinis) [19] . it is, therefore, believed that the sars-cov-2 also originated from bats and, after mutating, was able to infect other animals. the mutation increased the rbd affinity to ace-2 in humans, but also other animals such as ferrets and malayan pangolins (manis javanica; a long-snouted, ant-eating mammal sold illegally for use in traditional chinese medicine), but also decreased the rbd affinity to ace-2 found in rodents and civets. the pangolin is believed to be the intermediate host of sars-cov-2 [17] . there was some early speculation that sars-cov-2 emerged from a manmade manipulation of an existing coronavirus, but there is no evidence to support such a theory. in fact, anderson et al. suggest that the particular mutation that was found in the rbd of sars-cov-2 is different to what would have been predicted based on previously used genetic systems. the authors, however, stated that "it is currently impossible to prove or disprove the other theories of [the sars-cov-2] origin [19] ". since sars-cov and sars-cov-2 are so similar, the biochemical interactions and the pathogenesis are likely similar. binding of the sars-cov to the angiotensin-converting enzyme 2 (ace-2) receptors in the type ii pneumocytes in the lungs triggers a cascade of inflammation in the lower respiratory tract [20] . it has been demonstrated that when the sars spike protein binds to the ace-2 receptor ( figure 3a ), the complex is proteolytically processed by type 2 transmembrane protease tmprss2 leading to cleavage of ace-2 and activation of the spike protein ( figure 3b ) [21, 22] , similar to the mechanism employed by influenza and human metapneumovirus, thus facilitating viral entry into the target cell ( figure 3c ). it has been suggested that cells in which ace-2 and tmprss2 are simultaneously present are most susceptible to entry by sars-cov [23] . early indications are that sars-cov-2 virus also requires ace-2 and tmprss2 to enter cells [24] . in the first published review of the clinical presentation of 41 patients admitted to hospital with covid-19 [9] , 98% of patients had a fever, 76% had a cough, and 55% had shortness of breath on admission. however, those admitted may have had less severe symptoms for 2 to 14 days prior to presentation, during which they were likely contagious. by the time patients developed shortness of breath, they had been sick for an average of eight days. once admitted to the hospital, all patients developed clinical pneumonia supported by chest ct findings, and 13 of the 41 patients (32%) developed hypoxic respiratory failure necessitating icu admission. four patients (10%) required mechanical ventilation, two of which received extracorporeal membrane oxygenation due to refractory hypoxia. in total, six patients died, giving a case fatality rate (cfr) of 15% and triggering panic that quickly spread worldwide. while early media reports suggested that deaths were more likely in patients with comorbid conditions, of the 41 patients described in the chinese review, only 38% had comorbid conditions and the average age was 49. as of march 16, 0700 gmt, there were 169,930 confirmed cases, about half of which (80,860 cases, 47.6%) were within mainland china. about 18% of ill people had severe disease, and 82.0% had mild disease and a total of 889 tested-positive cases were asymptomatic [13, 25] . while initially confined to china among those who visited the wuhan wet market, over the course of about 3 months the sars-cov-2 has to date been confirmed in 157 countries and one cruise ship [13] . the chinese cdc published the epidemiologic characteristics of the covid-19 outbreak as of 11 february 2020 (table 1 ) [26] . initial data suggests that the majority of patients (73%) were over age 40 years, and that the risk of death increases with age. no deaths were reported in patients younger than 10 years old, and only 2.6% of the total fatalities were in patients younger than 40 years of age. [26] . [11, 13] . due to aggressive containment strategies in china, including a mass quarantine of the entire 11 million population of wuhan, the acceleration of new cases in china has slowed whereas that outside of china has increased. as of march 2nd, the number of daily new cases outside of china was nine times higher than those within china. many countries have instituted travel bans and/or quarantine procedures for incoming travelers. closures of public schools and social gatherings have been instituted in many countries in an effort to contain the spread of covid-19 and decrease the public health burden [27, 28] and the cdc has released recommendations on school closure criteria [29] . in comparison, the 2002 sars pandemic, which also originated in china, resulted in 8096 people infected and 774 deaths (9.6%). on the other hand, the 2012 mers pandemic infected 2494 people causing 858 deaths (34.4%). therefore, although mers and sars had higher mortality, the much larger number of people infected with sars-cov-2, and the rate at which the number is increasing, raises red epidemiologic flags. to assess the magnitude of the risk posed by the sars-cov-2, we review four parameters that we believe important: the transmission rate, the incubation period, the case fatality rate (cfr), and the determination of whether asymptomatic transmission can occur. the reproduction number, or "r naught" (r0), is a mathematical term that defines contagiousness [30] . specifically, it is the number of people that one sick host can infect. if the r0 is less than one the disease will disappear. if the r0 ≥ 1 then the disease will spread between people. estimates of the r0 of sars-cov-2 have ranged from 2.24 to as high as 3.58 [31] although the world health organization estimates it is between 1.4 and 2.5 [32] . for the purposes of comparison, the mean r0 for seasonal influenza is between 1.1 and 2.3 (variable by region and immunization rates), whereas for sars was between 1 and 2.75. the slightly higher r0 for sars-cov-2 may be because it has a longer prodromal period, increasing the period during which the infected host is contagious. coronaviruses are generally thought to be spread most often by respiratory droplets, not to be confused with airborne transmission [33] . droplets are larger and tend to fall to the ground close to the infected host and only infect others if the droplet is intercepted by a susceptible host prior to landing. droplet transmission is typically limited to short distances, generally less than 2 m. however, the airborne route involves much smaller droplets that can float and move longer distances with air currents. under certain humidity and temperature environments, airborne droplets can remain in flight for hours. generally, pathogens that are transmissible via the airborne route have higher r0, because infected particles can remain in the air long after the infected individual has left the premises. this airborne route occurs, for example, in measles (r0 between 12 and 18 [34] ) and chicken pox (r0s between 3.7 and 5.0 [35] ). once infected droplets have landed on surfaces, their survivability on those surfaces determines if contact transmission is possible. based on our current understanding from other betacoronaviruses, including sars and mers, coronaviruses can survive, and remain infectious, from 2 h up to 9 days on inanimate surfaces such as metal, glass, or plastic, with increased survival in colder and dryer environments [36] [37] [38] . for this reason, the chinese government has been reported to be disinfecting and even destroying cash in an effort to contain the virus [39] . reassuringly, cleansing of surfaces with common biocidals such as ethanol and sodium hypochlorite is very effective at inactivation of the coronaviruses within 1 min of exposure [36] . the timing of maximum infectivity is currently being assessed. a small study of 17 patients showed that nasal viral load peaks within days of symptom onset, suggesting that transmission of disease is more likely to occur early in the course of infection [40] . understanding incubation periods is very important as it allows health authorities to introduce more effective quarantine systems for suspected cases. the best current estimates of the sars-cov-2 infection range from 2 to 14 days. analysis of the first 425 cases of covid-19 in wuhan a mean incubation period of 5.2 days [41] . a later report, based on 1324 cases, reported a mean incubation period of 3.0 days [42] . yet another report, on 88 cases who traveled to wuhan between 20 and 28 january, had incubation period ranges from 2.1 to 11.1 days, with a mean of 6.4 days [43] . to calculate the case fatality rate (cfr) of an infection, one must divide the mortality number (m) by all those who were infected. the total number of those infected includes those who were infected and recovered without presentation (i r ), infected and presented to a health care facility (i p ), and infected and died (i d ). the cfr would be m/(i r + i p + i d ). clearly, one must have an accurate estimation of each of these parameters to accurately determine the cfr of covid-19. while the (m) is generally easier to count, and a focus of media, the denominator can take much longer to calculate. during the early phases of a deadly epidemic, the number of those who were infected and recovered (i r ) is not yet known, since only those who were infected and became seriously ill are recognized and tested. in addition, because this is a novel virus, there were no existing detection methods, so early deaths due to clinical entities such as influenza, for example, may have been mis-attributed to covid-19. the viral genome was published about 2 weeks after the start of the outbreak, and pcr analysis was quickly used to diagnose suspected cases [6] . public health officials can now test suspected cases, especially close contacts of known cases, and others with mild symptoms, but the testing capabilities can become saturated, potentially limiting the ability to get an accurate estimation of i p . for example, the initial ability of the wuhan health authority was limited to 200 tests per day, but that number has grown to 4196 tests per day [44] . the combination of these factors leads to a gross underestimation of the denominator of the cfr calculation, and thus an exaggeration of the mortality. until we are able to accurately represent i r and i p , it is currently impossible to precisely estimate the cfr of sars-cov-2. however, during the course of a potentially fatal pandemic, an accurate estimation of cfr is important. while it is tempting to estimate the cfr by dividing the number of known deaths by the total number of confirmed cases, the resulting number may be off by orders of magnitude, especially since infected individuals at one point in time may die x days later. using the lag period approach and dividing the current number of deaths to the number of cases x days ago may be a more acute estimator of cfr. nucleuswealth.com applied this method by using the number of deaths at any particular day and dividing by number of cases 4, 8, or 12 days prior. as seen in figure 4 , as time progresses, whether whichever number of days is used for x, the cfr seems to converge at just under 5% for cases within hubei, and about 0.8% for cases outside of hubei [14] . the higher mortality in wuhan may be overestimated because early in the course of this epidemic, viral testing was limited to only the severe cases. however, the china national health commission admits that wuhan has a relative lack of medical resources, which may have contributed to the higher mortality rate. pathogens 2020, 9, x for peer review 8 of 15 and dividing the current number of deaths to the number of cases x days ago may be a more acute estimator of cfr. nucleuswealth.com applied this method by using the number of deaths at any particular day and dividing by number of cases 4, 8, or 12 days prior. as seen in figure 4 , as time progresses, whether whichever number of days is used for x, the cfr seems to converge at just under 5% for cases within hubei, and about 0.8% for cases outside of hubei [14] . the higher mortality in wuhan may be overestimated because early in the course of this epidemic, viral testing was limited to only the severe cases. however, the china national health commission admits that wuhan has a relative lack of medical resources, which may have contributed to the higher mortality rate. infection transmission by asymptomatic individuals can make control of disease spread challenging. since late january, sars-cov-2 transmission from infected but still asymptomatic individuals has been increasingly reported [45, 46] . assessment of the viral loads in symptomatic individuals not only showed that the viral loads peak within the first few days of symptoms, but also that asymptomatic patients can have a similarly high viral load without showing symptoms [40] . it was suggested that viral testing should no longer be limited to symptomatic individuals, but also include those who have traveled to affected areas [47] . at such an early phase of the covid-19 pandemic, it is difficult to accurately describe the populations most at risk, especially when teasing out risk factors for infection from risk factors for death from disease. early on, it became clear that those who have visited the wuhan wet market were most at risk of infection, but the population visiting the market is not an accurate reflection of the general population. the chinese cdc published the epidemiologic characteristics of the covid-19 outbreak along with associated risk factors for death [26] . the largest risk factor for death is age. other risk factors include male sex and the presence of comorbid conditions (table 2 ). however, in addition to real age-specific mortality, the age-based risk could reflect underlying comorbidities among the elderly and the distribution of the underlying population in wuhan, where the outbreak initiated. infection transmission by asymptomatic individuals can make control of disease spread challenging. since late january, sars-cov-2 transmission from infected but still asymptomatic individuals has been increasingly reported [45, 46] . assessment of the viral loads in symptomatic individuals not only showed that the viral loads peak within the first few days of symptoms, but also that asymptomatic patients can have a similarly high viral load without showing symptoms [40] . it was suggested that viral testing should no longer be limited to symptomatic individuals, but also include those who have traveled to affected areas [47] . at such an early phase of the covid-19 pandemic, it is difficult to accurately describe the populations most at risk, especially when teasing out risk factors for infection from risk factors for death from disease. early on, it became clear that those who have visited the wuhan wet market were most at risk of infection, but the population visiting the market is not an accurate reflection of the general population. the chinese cdc published the epidemiologic characteristics of the covid-19 outbreak along with associated risk factors for death [26] . the largest risk factor for death is age. other risk factors include male sex and the presence of comorbid conditions (table 2) . however, in addition to real age-specific mortality, the age-based risk could reflect underlying comorbidities among the elderly and the distribution of the underlying population in wuhan, where the outbreak initiated. table 2 . fatality rate by age, sex, and pre-existing medical conditions. the death rate represents the probability (%) of the corresponding group of dying from sars-cov-2 [26] . with what we know about the pathogenesis of the sars-cov virus, it seems reasonable to assume that those with higher levels of ace-2 receptors may be at greatest risk. there was some speculation that the expression of ace-2 receptors may be linked to race, specifically after an early report suggested that asian males had higher ace-2-expressing cell ratios than white and african americans [48] . however, the sample size contained only eight different individuals (five african americans, two whites, and one asian) and extrapolating those findings to a whole race is impractical. yet, in another study assessing ace-2 receptor expression in tissues of 224 patients with lung cancer, there were no significant disparities in ace-2 gene expression between racial groups (asian vs. caucasian), age groups (older or younger than 60 years old), or gender groups (male vs. females) [49] . ace-2 gene expression was, however, significantly elevated in smokers suggesting that smoking history should be considered in identifying susceptible populations. since smoking in china is predominantly a male attribute (54% of men, 2.6% of women) [50] , this may help to explain the gender difference seen in the hospitals in china. early in the covid-19 epidemic, it appeared that children were a protected group, but this may have been because they were less likely to have frequented the wuhan wet market, or because they were more likely to have asymptomatic or mild disease and thus less likely to have been tested. covid-19 has affected infants as young as 1 month of age [51] , most with mild or asymptomatic disease. there have been no reported cases of adverse infant outcomes for mothers who developed covid-19 during pregnancy. second to the hubei population, the other population at increasing risk is healthcare workers. as of february 17, 2020, total of 1716 healthcare workers in china have been infected, five of whom fatally [25] . the current best strategy of treatment of patients with covid-19 is purely supportive. clinicians and intensive care specialists are applying much of what they have learned during the sars epidemic to guide current therapy of covid-19. recommendations for admission to critical care units, guidelines for infection control, and procedures to minimize nosocomial transmission are being established [52] . however, there are several fronts that are being studied to develop targeted treatments. the most efficient approach to the treatment of covid-19 is to test whether existing antiviral drugs are effective. in previous betacoronavirus epidemics, several antiviral drugs, such as ribavirin, interferon, lopinavir-ritonavir, and darunavir/cobicistat (prezcobix) were tested, with some showing promising in vitro results [53] . remdesivir, an adenosine analog used against rna viruses (including sars and mers-cov), was a candidate ebola treatment with promising in vitro results but disappointing in vivo effects against ebola [54, 55] . there is currently in vitro evidence that remdesivir may be effective in controlling sars-cov-2 infection [56] . in fact, compassionate use of remdesivir was employed in the treatment of the first covid-19 case in the united states, during a period of rapid clinical deterioration, and within one day there was dramatic improvement of the clinical condition [57] . randomized double-blinded, placebo-controlled clinical trials are currently underway in china and usa to evaluate the efficacy of remdesivir and initial results are expected by the end of april 2020 [58, 59] . other existing drug candidates include chloroquine and camostat mesylate. chloroquine is a widely used anti-malarial drug that is known to block virus-cell fusion and has been shown to interfere with the glycosylation of sars-cov and ace-2 cellular receptors, rendering the ace-2-sars-cov interaction less efficient [60] . there is also in vitro evidence that chloroquine may be effective in preventing sars-cov-2 cellular entry [56] . camostat mesylate, also known as foy 305 [61] , was initially developed and currently approved for the treatment of chronic pancreatitis in japan [62, 63] . camostat mesylate targets the tmprss2 protease, theoretically preventing viral entry. researchers in germany showed that camostat mesylate reduced the amount of sars-cov-2 viral replication [64] . a simple but very effective treatment modality is the use of convalescent plasma, or serum from patients who have recovered from the virus, to treat patients. patients with resolved viral infection will have developed a specific antibody response which may be helpful in neutralizing viruses in newly infected individuals. this modality was successfully employed during the 2014-2015 ebola outbreak [65, 66] . however, the use of convalescent sera is of limited benefit in an outbreak situation since the exponential growth of infected patients exceeds the ability of previous patients to provide donor plasma. the recent finding that sars-cov-2 binds to the same ace-2 receptors targeted by the 2002 sars-cov [24] opens up the possibility of using the previous research on the 2002 sars epidemic and applying it to covid-19. the first strategy would be to employ either a small receptor-binding domain (rbd) or a neutralizing antibody targeting the ace-2 receptor, thus blocking the binding of s protein and preventing virus entry into cells. initial in vitro results have shown promising results [67, 68] and specific monoclonal antibodies are being contemplated as candidates for treatment [69, 70] . the main limitation of using rbds or antibodies is that the treatment must be given within a specific time window, before the initiation of viral replication [20] . in addition, the side effects of ace-2 blockade, especially since ace-2 is also present in non-pulmonary tissue, must be understood and minimized before implementation. in addition, finally, the turnover of ace-2 receptors would influence how often the therapeutic rbd or antibody would have to be administered. a second strategy is to create an ace-2-like molecule that would bind to the s protein of the coronavirus itself. again, research in to the 2002 sars virus demonstrated that soluble ace-2 proteins blocked the sars virus from infecting cells in vitro [68, 71] . the additional benefit to using this strategy lies in the possible prevention of s protein-mediated ace-2 shedding that has been shown to induce the pulmonary edema characteristic of sars [72, 73] . a phase ii clinical trial of recombinant ace-2 in ards reported significant modulation of inflammatory proteins, but no significant differences in respiratory parameters [74] . further research is necessary to assess if the animal studies will translate to clinical benefit. there are currently more than 80 clinical trials to test a variety of potential sars-cov-2 treatments [75] . the long-term goal of sars-cov-2 research is developing an effective vaccine to yield neutralizing antibodies. the national institutes of health in the us, and baylor university in waco, texas, are working on a vaccine based on what they know about the coronavirus in general, using information from the sars outbreak. in 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and immunogenicity study of 2019-ncov vaccine (mrna-1273) to treat novel coronavirus-full text view-clinicaltrials.gov this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. pathogens 2020, 9, 231 key: cord-342786-dl8vjwfn authors: sattar, yasar; ullah, waqas; rauf, hiba; ul hassan virk, hafeez; yadav, sunita; chowdhury, medhat; connerney, michael; mamtani, sahil; pahuja, mohit; patel, raj d.; mir, tanveer; almas, talal; moussa pacha, homam; chadi alraies, m title: covid-19 cardiovascular epidemiology, cellular pathogenesis, clinical manifestations and management date: 2020-07-14 journal: int j cardiol heart vasc doi: 10.1016/j.ijcha.2020.100589 sha: doc_id: 342786 cord_uid: dl8vjwfn abstract coronavirus disease 2019 (covid-19) is a rapidly progressing global pandemic that may present with a variety of cardiac manifestations including, but not limited to, myocardial injury, myocardial infarction, arrhythmias, heart failure, cardiomyopathy, shock, thromboembolism, and cardiac arrest. these cardiovascular effects are worse in patients who have pre-existing cardiac conditions such as coronary artery disease, hypertension, diabetes mellitus, and coagulation abnormalities. other predisposing risk factors include advanced age, immunocompromised state, and underlying systemic inflammatory conditions. here we review the cellular pathophysiology, clinical manifestations and treatment modalities of the cardiac manifestations seen in patients with covid-19. coronavirus disease 2019 (covid-19) is a rapidly progressing global pandemic that may present with a variety of cardiac manifestations including, but not limited to, myocardial injury, myocardial infarction, arrhythmias, heart failure, cardiomyopathy, shock, thromboembolism, and cardiac arrest. these cardiovascular effects are worse in patients who have pre-existing cardiac conditions such as coronary artery disease, hypertension, diabetes mellitus, and coagulation abnormalities. other predisposing risk factors include advanced age, immunocompromised state, and underlying systemic inflammatory conditions. here we review the cellular pathophysiology, clinical manifestations and treatment modalities of the cardiac manifestations seen in patients with covid-19. severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the virus causing the coronavirus disease 2019 (covid-19) pandemic, has involved more than 7 million cases worldwide. the us has the highest number of infected patients with more than 2 million cases and 100,000 deaths by the second week of june, 2020 [1, 2] . the respiratory symptoms including acute respiratory distress syndrome (ards) are well discussed in the literature. however, the extrapulmonary manifestations with likely cellular cytotoxicity is not well studied [3] . the cardiovascular sequela of covid-19 can cause contractility disorders, arrhythmias, pericardial disease, vascular insufficiency, and sudden cardiac arrest. we sought to review cellular cytotoxicity, clinical symptoms, diagnosis and management of cardiovascular complications in covid-19. shortly after the outbreak of covid-19 pneumonia in wuhan, china covid-19, its causative agent of sars-cov-2 was first reported in january, 2020 [4] . this outbreak has rapidly spread across china and globally through person to person transmission. the mean incubation period of this virus ranges between 5-7 days, therefore the travelers and suspected contacts are advised to quarantine for 14 days. the basic reproduction number ranges from 2.24 to 3.58 and can be as high as 6.47 in intensive social contacts [5] . the most common symptoms at the disease onset include fever, sore throat, cough and myalgia. the infected patients may also present with cardiovascular disease (cvd) like acute coronary syndrome(acs) and congestive cardiac failure(chf) [6] . a study of 5700 patients have reported hypertension (56.6%), coronary artery disease (11.1%) and congestive cardiac failure(6.9%) as common underlying co-morbidities in confirmed covid-19 cases [7] . another study comprising 44,672 cases reported five-fold increase in case fatality rates in patients with underlying cvd as compared to patients without cvd (10.5% vs 2.3%) [8] . the impact of covid-19 on the cardiovascular system is evidenced through multiple studies which report myocarditis in 7-17%, heart failure in 24%, arrhythmias in 17% and thrombotic complications in 31% of hospitalised covid-19 cases [9, 10] . the covid-19 infection is initiated through binding of s-protein of sars-cov-2 with the host receptor angiotensin-converting enzyme 2 (ace2) which mediates its entry into the cells. ace-2 is highly expressed on the pulmonary epithelial cells, cardiac myocytes and vascular endothelial cells which is responsible for extensive cardiopulmonary symptoms [11] . upon binding with ace-2, s-protein cleaves at dibasic arginine site by host protease tmprss2 to generate s1 and s2 subunits. the s2 subunit induces membrane fusion and viral endocytosis in the cell. after viral entry into the cell, the viral rna is released in the cytoplasm where it replicates and processed into virion-containing particles which fuses with the cell membrane to be released for widespread infection. sars-cov-2 also internalizes and downregulates the expression of ace-2 on the cell surface [11] . since ace-2 primarily converts angiotensin i and ii to cardioprotective peptides, angiotensin 1-9 and angiotensin 1-7; its loss on cell surface may potentiate cardiac damage. additionally, the loss of ace-2 on vascular endothelium may exacerbate endothelial dysfunction, inflammation and thrombosis [6, 12] . the ace-2 expression in vascular endothelial cells is linked to underlying pathological state, age and gender. it's activity is reduced in vessels with established atherosclerotic plaques and diabetes whereas it is increased in women and young adults due to a potential role of estrogen [13, 14] . since the ace-2 levels are downregulated in covid-19, any underlying factor that diminishes ace-2 expression compromises the cardioprotective action of ang 1-7/ang 1-9, further promoting the vascular damage. the reduced ace-2 also induces cytokine release through dysregulating renin-angiotensinaldosterone system, depressing mas receptor (ace2/masr axis) and activating ace2/bradykinin b1r/dabk axis [15] . these cellular effects are translated into exacerbation of underlying cardiovascular disease or new onset of cardiac symptoms. the cardiac complications of covid-19 can be divided into electrical and mechanical dysfunction. the electrical aberrance is seen in arrhythmias whereas pericardial, myocardial, valvular and vascular complications arise due to mechanical dysfunction. arrhythmia in covid-19 can be secondary to electrolyte imbalance, pulmonary disease, medication side effects, activated protein kinase c (pkc), or direct oxidized ca 2+ /calmodulindependent protein kinase ii (camkii) [16, 17] . in particular, hypokalemia and hypomagnesemia are commonly seen in covid-19 due to angiotensin ii-mediated urinary excretion and gastrointestinal losses [18] . the hypoxia in ards can increase the risk of arrhythmias as evidenced in an experimental study by clark et al. which reported arrhythmias in 61% of the hypoxic rats [19, 20] . the widely used drugs chloroquine, hydroxychloroquine, azithromycin, and lopinavir/ritonavir can also trigger arrhythmias by prolonging qt [21] . the upregulated angiotensin ii and angiotensin ii receptor type 1 axis (angii-at1r) activates the pkc which interferes with initiation and propagation of action potentials leading to arrhythmias [22, 23] . another probable mechanism is that ang ii activates nadph oxidase via phosphorylation and translocation of p47phox [24] . the activated nadph oxidase increases the reactive oxygen species (ros) which oxidizes the camkii into ox-camkii. the ox-camkii in turn phosphorylates ryanodine receptor 2 (ryr2) leading to increased diastolic sarcoplasmic ca 2+ leak which triggers delayed after depolarizations and induces atrial and ventricular arrhythmias [17, 24] . the systemic inflammation in covid-19 may also dysregulate the post-translational modification of cardiac ion channels resulting in arrhythmia [25, 26] it is also noteworthy that viral proteins of sars-cov-2, orf3 and orf8, activate nlrp3 inflammasomes which inturn promotes atrial fibrillation [27, 28] . elevated angiotensin ii levels facilitate an inflammatory cascade via nf-kb, elevated il-6, tgf-β and vascular endothelial growth factor (vegf). these cytokines promote serosal inflammation and fibrosis that may present as pericarditis [29] [30] [31] [32] . sars-cov-2 can cause pericarditis that may progress to pericardial effusion. pericarditis associated with pericardial effusion creates physical stress and can further complicate takotsubo cardiomyopathy [33] . the combined cases of myocarditis and pericarditis cases due to covid-19 have also been reported [29, 34] . the clinical presentation may overlap with other covid-19 symptoms like fever, chest pain, and severe hypotension. the diagnosis is made through a combined diagnostic approach including electrocardiography, echocardiography, and chest radiographs. myocardial dysfunction in the setting of covid-19 can present as acs, myocarditis, heart failure, stress cardiomyopathy, and shock. several mechanisms have been suggested for the myocardial injury associated with covid-19. sars-cov-2 can induce systemic inflammatory response syndrome (sirs) through a combination of cytokine release syndrome (crs) and dysregulated immune activity that leads to myocardial dysfunction [29, 35] . the immune system recognizes sars-cov-2 via recognition of intrinsic viral rna patterns, or the pathogen-associated molecular patterns (pamps). this identified pathogen attaches to the pattern recognition receptor (prrs), which initiate host-immune defense against the virus. the interaction of pamps with prrs in combination with a dysregulated inflammatory response in the setting of sepsis leads to cytokine storm. cytokines contribute to myocardial damage by facilitating the release of reactive oxygen species (ros), endogenous nitric oxide, and superoxide anion ( figure 1 ). eventually, damage-associated molecular proteins (damps) like heat-shock proteins, highmobility group box 1 (hmgb1), histones, and oxidized lipoproteins are released from the damaged myocardium which further activate the inflammation and injure myocytes. these excessive damps create an ongoing vicious inflammatory cycle resulting in septic/covid-19 cardiomyopathy [36, 37] . the notch signaling pathway also promotes inflammatory cytokines, facilitates the sars-cov-2 infection, and plays a major role in structural cardiac remodeling therefore a therapy directed at modulation of notch pathway can limit the covid-19 infection and limit the inadvertent remodeling [38] . the sars-cov-2 may cause direct myocardial cytotoxicity via 3c proteinase-mediated apoptosis, disruption of host protein translational mechanisms, loss of cellular homeostasis, and dysregulated host-immune response [39, 40] . after viral infection of cardiomyocytes, the immune system triggers the infiltration of natural killer cells, macrophages, and lymphocytes. these cells initially confer protective effects, but hyper-activation can worsen the severity of myocarditis and lead to cardiomyopathy [41] . biopsy specimens in myocarditis have demonstrated infiltration of mononuclear cells in the cardiac myocytes causing focal or diffuse damage [15] . in addition, the hypoxemia in covid-19 can induce increased intracellular calcium levels which can result in cardiomyocyte damage and eventual cardiomyopathy [42] . covid-19 can also cause stress cardiomyopathy due to emotional or physical stress on cardiac myocardium in the setting of anxiety and widespread inflammation [43] .the myocardial stunning in the setting of catecholamine surge and coronary microvascular dysfunction may be exacerbated by covid-19 from ras dysregulation and plaque disruption [44] . increased catecholamines further worsens the microvascular dysfunction through increasing cytokine production of il-6 via alpha-1 adrenergic receptor signaling in immune cells [44] . the excessive catecholamines may also worsen the transient hypokinesia through beta2-coupling from gs to gi [45] . the combined effects of catecholamine surge and microvascular dysfunction in covid-19 may result in hypercontraction of the left ventricular apex and outflow tract resulting in stress cardiomyopathy. the increased cardiometabolic demand in systemic infection coupled with hypoxia secondary to pulmonary dysfunction can create myocardial oxygen supply/demand mismatch. these events when accompanied by inflammation induced hypercoagulation, microthrombi and plaque rupture can lead to cardiac ischemia and acute coronary syndrome [46] . it is unclear whether the sars-cov-2-mediated myocardial damage will have long-term implications on cardiac function and survival therefore follow up data on survivors is required to better assess the long-term outcomes. the symptoms of acute coronary syndrome (acs) include chest pain, dyspnea, and sinus dysfunction which can overlap with covid-19 symptoms [47] . a case series of 18 covid-19 patients with stemi reported that 33% of cases manifested chest pain at the time of stelevation. among these patients, 22% died of myocardial infarction whereas 50% died of non-coronary myocardial injury [48] . the diagnosis of acs requires elevated troponin along with either echocardiographic or electrocardiographic abnormalities [49] . the ventricular dysfunction in covid-19 may present with features of heart failure, cardiogenic shock, and myocarditis [50] . their clinical presentation will overlap with the symptoms of covid-19 including respiratory distress and hypotension. the diagnosis can only be ascertained by utilizing various diagnostic modalities such as cardiac markers, echocardiography, mri, and biopsy. a study reports that heart failure is a common manifestation of covid-19 patients, seen in 23% of affected patients [47] . cardiogenic shock was reported in a few cases with depressed left ventricular ejection fraction, left ventricular hypokinesis, and elevated cardiac markers [33, 37] . a case of myocarditis showed similar diagnostic features but presented without the typical respiratory features of covid-19 [51] . cardiovascular complications may present in isolation without respiratory symptoms in covid-19. the ace-2 is widely expressed on the stromal fibroblasts of cardiac valves, particularly aortic valves. it is evidenced from the rt-pcr that the ace-2 expression is downregulated in stenotic valves. the depressed ace2/angiotensin-(1-7)/mas receptor axis may potentiate inflammation, fibrosis and valvular sclerosis [52] . simultaneously, upregulated angii-at1r axis induces inflammatory cytokines like tnf-α and il-6 amplifier (il-6 amp) which further induces pro-inflammatory cytokines and recruits macrophages to create a positive feedback loop of inflammation [53] . these downstream effects of sars-cov-2 infection result in valvular damage which may not present acutely due to the slowly progressive nature of cardiac valvular disease. however, periodic follow-up of cardiac function is essential in covid-19 survivors to timely identify this pathology. hypertension is frequently seen in covid-19 patients however it is unclear whether it is a risk factor for acquiring covid-19. the unregulated angiotensin ii stimulates vasoconstriction and systemic inflammation which may worsen the underlying hypertension through oxidative stress and endothelial dysfunction. the reactive oxygen species (ros) like superoxide, interacts with nitric oxide to form peroxynitrite which reduces the availability of no and inhibits the enos activity [54] . the elevated levels of inflammatory markers like crp and tnf also destabilize enos and limit no production. since no has vasodilatory and anti-inflammatory effects, its reduced bioavailability can exacerbate hypertension. the ros can also impair the vascular structure and function by directly inducing cell damage [54] . hypercoagulation is another commonly seen manifestation of the disease [29] [30] [31] . diffuse cytokine storm, elevated angiotensin ii, endothelin-1, plasminogen activator inhibitor 1 (pai-1), tissue factor, ros, and extrinsic clotting cascade are all interrelated pathophysiological components of hypercoagulation [55] (figure 1 ). the endothelial cells line the intima of the vasculature and are closely connected by adherents, tight and gap junctions which maintain the endothelial integrity [56] . in sars-cov-2, excessive cytokines, ros and proteases from the activated neutrophils may damage these junctional proteins, resulting in trans-endothelial migration of neutrophils [57] . these neutrophils degranulate and form neutrophil extracellular traps which release damps (dna and neutrophil elastase) that further damage the junctional proteins and endothelial layer [58] . this leads to greater serum contact with subendothelial tissue, which activates von-willebrand factor, platelet adhesion, and the coagulation cascade. inflammation leads to increased pai-1, which increases endothelial fibrin deposition and reduced thrombolysis, ultimately contributing to the hypercoagulable and prothrombotic state of covid-19 ( figure 1 ). the coagulation profile in covid-19 patients may show thrombocytopenia, elevated ddimer, prolonged prothrombin time, international normalized ratio (inr), and short activated partial thromboplastin time (aptt) [59] . this coagulation disarray predisposes patients to thromboembolic complications. a case study reports an incidence of acute pulmonary embolism with right ventricular failure in a covid-19 patient which demonstrated elevated d-dimers at 1280 ng/ml [60] . the increased risk of thrombosis further predisposes the risk of disseminated intravascular coagulation (dic), venous thromboembolism (vte) and multi-organ failure. there are multiple risk stratification tools to ascertain the risk of vte in covid19 [61] . another study has shown that 71.4% of the non-survivors of covid-19 had dic with elevated d-dimer [47] . given the high mortality in patients who develop dic, there is an expert consensus as reviewed by song et al. regarding the management of coagulopathy in these patients. current recommendations emphasize the importance of routine monitoring of coagulation parameters and the implementation of scores to diagnose covid-19 related coagulopathy. a variety of different drug treatments are being investigated in covid-19 infection despite the lack of definitive evidence about their efficacy. the proposed drug regimens include hydroxychloroquine, azithromycin, lopinavir/ritonavir, tocilizumab, remdesivir and ribavirin [62] . the role of hydroxychloroquine in treating covid-19 patients with cvd is controversial. it inhibits binding of sars-cov-2 with ace-2 receptor by blocking tmprss-2, and inhibits viral entry by altering endosomal ph [63] . this drug has shown positive outcomes in terms of viral clearance however its safety in patients with cardiovascular disease is not clearly established due to potential side effects of polymorphic ventricular tachycardia, long qt-syndrome and increased risk of sudden death [64] . a clinical trial of 81 patients reported high dose of chloroquine is associated with severe arrhythmias and deaths and was therefore halted [65] . since different drugs used in covid-19 have a certain risk of pro-arrhythmic side effects, it should be balanced with the anticipated benefit of therapy and administered under close monitoring. arrhythmias in covid-19 are managed based on etiology including repletion of electrolytes, discontinuation of medications causing arrhythmia, managing volume status, or suppressing catecholamine surge in covid-19 [44, [66] [67] [68] . brugada syndrome can be treated by alleviating the underlying triggers including fever, or pneumonia, or icd placement in cases of brugada-1 [69] [70] [71] . furthermore, in the setting of shock in covid-19, the use of vasopressor such as dobutamine, epinephrine or pde-inhibitors such as milrinone should be avoided due to synergistic proarrhythmic activity of these medications. in hemodynamically stable arrhythmias, beta-blockers should be used and epinephrine should be avoided especially during the catecholaminergic surge in covid-19 [72] . the pericarditis in covid-19 is treated with steroids and supportive care however pericardiocentesis may be required in pericardial effusion. the collected fluid should be checked for cytology, autoimmune reactions, cultures and tested for sars-cov-2 [51] . the choice of treatment in covid-19 patients with stemi depends on the availability of a pci facility, or fibrinolytic and institutional policy of covid-19 stemi cases. anticoagulant therapies, such as unfractionated heparin or enoxaparin, may also be considered in covid-19 with stemi and nstemi [73] . in covid-19 with nstemi, the decision to perform pci depends on the global registry of acute clinical events (grace) score >140, or thrombolysis in myocardial infarction (timi) >3 or hemodynamic instability [74] . as per the protocol by one chinese medical center, for stable patients who tested positive for covid-19 with stemi, it is reasonable to administer thrombolytics if onset of symptoms is <12 hours, pending no contraindications. elective pci can be considered once the patients are treated and have recovered from covid-19. however, in unstable patients and those who do not have severe pneumonia, the recommendation assessed the risks and benefits of early pci [75] . since dual antiplatelet therapy (dapt) after pci in covid-19 patients has increased bleeding risk, the new strategy is to reduce the duration of aspirin to 1-3 months and allow prolonged use of p2y 12 inhibitors. the early antiplatelet therapy with p2y 12 antagonists like ticagrelor, is beneficial after pci in covid-19 due to the inhibitory effects on platelet activation and intravascular thrombus formation [76] . furthermore, the management of cardiovascular cardiomyopathy is based on type of manifestation. the treatment of hemodynamically stable heart failure involved optimal medical regimen including aldosterone antagonists, diuretics, and beta-blockers [51, 77] . in patients with cardiogenic shock, iatrogenic agents such as milrinone, a phosphodiesterase 3-inhibitor (pde-3i) can be used under close observation [51] . there is widespread controversy about the use of acei, arbs, and neprilysin inhibitors (sacubitril) in covid-19 exacerbated heart failure. acei/arbs can worsen covid-19 by increasing ace2 expression which may amplify the chances of viral invasion [78, 79] (figure 1 ). on the contrary, ace inhibitors can be beneficial by increasing ace-2 and further opposing the action of angiotensin ii receptor type 1 (at1r) through increasing ang 1-7 and, ang 1-9 downstream effects [80] (figure 1) . a case-control study by mancia et al. showed no association of risk of covid-19 with the use of acei in overall case patients (odds ratio, 0.96 [95% ci, 0.87 to 1.07]) and those with fatal outcomes (odds ratio, 0.91 [95% ci, 0.69 to 1.21) [81] . however, we still need evidence from different randomized clinical trials to support the use of acei/arbs in these patients. the summary of balance between at1r and ang 1-7, ang 1-9 is shown in figure 1 . in patients with refractory cardiogenic shock in the setting of covid-19, veno-arterial (vav) extracorporeal membrane oxygenation (ecmo) can be helpful [82, 83] . many covid-19 patients present with acute respiratory distress syndrome and pulmonary embolism which can increase the right ventricular afterload resulting in impaired right ventricular function. echocardiogram and other advanced imaging modalities can play a significant role in determining the right ventricular dysfunction and ascertain the prognosis [84] . covid myocarditis can be treated with immunosuppressants including steroids, cyclosporine, azathioprine, and immunoglobulins [85, 86] . il-6 antagonists can be efficacious as they inhibit cytokine storms in covid-19 [87] . the steroid use is controversial as chen et al. suggested that steroids might not be helpful in viral myocarditis however few reports have shown favorable outcomes in fulminant covid-19 cases [88, 89] . in addition, a placebocontrolled trial has shown safety of intravenous immunoglobulin (ivig) in idiopathic dilated cardiomyopathy or biopsy-proven viral myocarditis with no added benefit in improvement of lv function [90] . ivig can be considered a salvage therapy in fulminant myocarditis with hemodynamic instability [83] . preliminary studies about the use of convalescent plasma have also demonstrated efficacy in critical covid-19 infection [91] . statins also have a potential benefit in limiting cardiovascular complications in covid-19 through inhibiting toll-like receptors (tlr)-nf-κb pathway. it's use may also counter the hyperlipidemic sideeffects of other antiviral drugs used in covid-19 [92] . multiple studies have supported the use of acei and arbs in hypertension due to their potential to improve the clinical symptoms and reduce mortality. a study reported a lower rate of severe disease and il-6 levels in covid-19 patients with hypertension who were being treated with acei /arbs. hence, these drugs may prevent the worsening of blood pressure due to inhibition of inflammatory response [93] . the international society on thrombosis and haemostasis (isth) recommends vte prophylaxis in hospitalised covid-19 patients. in non-icu hospitalised cases, standard or intermediate dose of low molecular weight heparin (lmwh) is preferred for thromboprophylaxis however in icu patients, a combined approach of prophylactic or intermediate-dose lmwh with mechanical compression device is advised after assessing the bleeding risk. the duration of postdischarge thromboprophylaxis in covid-19 patients ranges between 14 to 30 days [94] . a retrospective study has shown reduced 28-day mortality in covid-19 patients with elevated d-dimers who used heparin as compared to non-heparin users. (p=0.017). hence, prophylaxis with low molecular weight heparin may have a role in preventing covid-19 associated thrombotic complications and mortality [95] . in cases of diagnosed vte, parenteral anticoagulation with lmwh is preferred in an in-patient setting whereas direct oral anticoagulants (doac) are given after hospital discharge. the total duration of treatment in diagnosed vte is at least 3 months [94] . there is no evidence that antiplatelets increase the risk of severe covid-19 hence it should be continued for conditions like recent mi or pci within the last 3 months. the decision for antiplatelet therapy is individualised on a case to case basis depending on the platelet count, bleeding risk and drug-drug interaction [96] . covid-19 infection has been linked to myocardial injury, leading to severe disease progression. although incredible efforts are being done to accurately comprehend the mechanism of myocardial injury, it is prudent to expand the randomized controlled 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with ards in patient on durable lvad support corticosteroids for viral myocarditis controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy treatment of 5 critically ill patients with covid-19 with convalescent plasma statin therapy in covid-19 infection covid-19, hypertension and cardiovascular diseases: should we change the therapy? scientific and standardization committee communication: clinical guidance on the diagnosis, prevention and treatment of venous thromboembolism in hospitalized patients with covid-19 anticoagulation in covid-19 covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review increased ros can also cause camk-ii activation causing early depolarization and arrhythmias. increased et1 also leads to increased thrombosis by increased endothelial dysfunction by oxidizing low density lipoprotein and pai-1. lipopolysaccharide binds to receptor tlr-4 increasing nfkbeta ang 1-9 have a positive role in our body, and can fight against covid 19 but their levels, and downstream pathways are decreased in covid-19 patients due to low ace-2 r levels. enos, npr a/b r, at 2r, masr normally counteracts the negative effects of at1r abbreviations: sars-cov-2: severe acute respiratory syndrome coronavirus-2 il-6: interleukin 6; ang ii: angiotensin ii ang 1-7: angiotensin 1-7; ace: angiotensin converting enzyme; at1r: angiotensin ii receptor type 1; at2r: angiotensin ii receptor type 2 natriuretic peptide receptor a/b guanylate cyclase; tnfα: tumor necrosis factor alpha; inos: inducible nitric oxide synthase; enos: endothelial nitric oxide synthase; tgf-β: transforming growth factor beta; ros: reactive oxygen species; camk-ii: calmodulin dependent protein kinase ii; cam: calmodulin; cgmp: cyclic guanosine monophosphate atrial/brain natriuretic peptide tlr4: transmembrane lipopolysaccharide receptor 4; icam: intercellular adhesion molecule; vcam: vascular cell adhesion molecule; nf-κb: nuclear factor kappa-light-chain-enhancer of activated b cells; pge2: prostaglandin e; masr: mitochondrial assembly g protein coupled receptor; nepa: neprilysin a receptor; nepb: neprilysin b receptor; npr a/b: neprilysin receptor a/b; hdac: histone deacetylase; gata: globin transcription factor; nfat: nuclear factor of activated t-cells; ppar-γ: peroxisome proliferatoractivated receptor gamma sodium ion; ncx: sodium-calcium exchanger stemi: st-segment elevation myocardial infarction color coding: green frames resemble the protective and anti-inflammatory mechanisms, which are imbalanced by the pro-inflammatory covid-19 state depicted in red causing overt inflammation and cell destruction; brown color depicts the potential therapeutic targets that can help balance this inflammatory state; yellow color shows all enzymes involved in raas or covid key: cord-346912-o09qmp7x authors: bayraktar, e.; cohen, a.; nellis, a. title: a macroeconomic sir model for covid-19 date: 2020-06-23 journal: nan doi: 10.1101/2020.06.22.20137711 sha: doc_id: 346912 cord_uid: o09qmp7x the current covid-19 pandemic and subsequent lockdowns have highlighted the close and delicate relationship between a country's public health and economic health. macroeconomic models which use preexisting epidemic models to calculate the impacts of a disease outbreak are therefore extremely useful for policymakers seeking to evaluate the best course of action in such a crisis. we develop an sir model of the covid-19 pandemic which explicitly considers herd immunity, behavior-dependent transmission rates, remote workers, and indirect externalities of lockdown. this model is presented as an exit time control problem where the lockdown ends when the population achieves herd immunity, either naturally or via a vaccine. a social planner prescribes separate levels of lockdown for two separate sections of the adult population those who are low-risk (ages 20-64) and those who are high-risk (ages 65 and over). these levels are determined via optimization of an objective function which assigns a macroeconomic cost to the level of lockdown and the number of deaths. we find that, by ending lockdowns once herd immunity is reached, high-risk individuals are able to leave lockdown significantly before the arrival of a vaccine without causing large increases in mortality. additionally, if we incorporate a behavior-dependent transmission rate which represents increased personal caution in response to increased infection levels, both output loss and total mortality are lowered. lockdown efficacy is further increased when there is less interaction between lowand high-risk individuals, and increased remote work decreases output losses. overall, our model predicts that a lockdown which ends at the arrival of herd immunity, combined with individual actions to slow virus transmission, can reduce total mortality to one-third of the no-lockdown level, while allowing high-risk individuals to leave lockdown well before vaccine arrival. the current covid-19 global pandemic has led to massive lockdowns to slow the spread of the virus. now, policymakers face a dilemma -extended periods of lockdown have put strain on the economy, but returning to "normal" too quickly could result in an equally troubling second wave of infections. the task is therefore to find the optimal balance between public health and economic growth. models such as those proposed by alvarez et al. [aal20] and acemoglu et al. [ace+20] have used a macroeconomic approach and variations on the susceptible-infectious-recovered (sir) epidemic model proposed by kermack et al. [kmw27] to solve an optimization problem determining the lockdown policy that minimizes both loss of life and effects on output. we consider a variation on these models which incorporates several new concepts and gives a wider picture of the overall situation. • we formulate an exit time control problem where lockdown measures are lifted when the population reaches herd immunity, even if this occurs before a vaccine is developed. • we incorporate a transmission rate that captures how individuals reacts to current infection levels, as discussed in [coc] . this "behavior-dependent" transmission rate seeks to model individual behaviors that occur independently of lockdown. for example, individuals might wear masks, practice social distancing, and take other precautions to reduce their risk as infection numbers go up, even in the absence of official lockdown measures. for the full 1.5 years until the vaccine). additionally, lockdowns for the low-risk group are 6 weeks shorter. • the addition of a behavior-dependent virus transmission rate contributes to these shorter lockdowns and decreases mortality. in an extreme situation where individuals can take measures that decrease transmission by 95% when infections reach 30%, less than a month of lockdown is prescribed for the low-risk group. in the more moderate benchmark case, where individuals are able to reduce their transmission by 25% when infections reach 30% of the population, herd immunity arrives a month earlier than in a situation with a constant disease transmission rate. in both cases, we also observe lower output loss due to shorter lockdown and fewer deaths due to slower transmission. • increasing the level of remote work reduces the impact of covid-19 by decreasing both mortality and output loss, even though a longer lockdown is imposed. this supports the intuitive idea that increased remote work reduces infection risk without sacrificing economic activity. • increasing the predicted length of future unemployment and the predicted rate of lockdownrelated deaths both decrease lockdown length in a similar manner, but also have negative impacts on outcomes. adjusting the length of future unemployment and the predicted number of indirect deaths due to lockdown lead to trade-offs between output and mortality. running the model with different initial conditions shows that higher pre-lockdown infection levels lead to earlier onset of herd immunity but higher death tolls, highlighting the risks of infection spikes. future impacts of current missed health screenings and a penalty for overfull icus are revealed to have little impact on the optimal lockdown policy, at least in our formulation. the main body of the paper presents our model and its numerical results. in section 2, we lay out the sir dynamics used to model the transmission of the virus and discuss certain model additions, especially the addition of deaths indirectly caused by lockdown and a behavior-depending transmission rate. in section 3, we introduce the exit time control problem that ends when the population reaches herd immunity and discuss the terms in the objective function. in section 4, we discuss our numerical model, which discretizes the problem and is solved through value iterations. we calibrate it with the results of [ace+20] and [aal20] and compare these results to our augmented model using death rates on the same scale. then, we update death rates to match more recent data from [cdc] and adjust the non-pecuniary value of life. we present and discuss our results and perform some parameter robustness analysis. these experiments serve to illustrate the general mechanisms of the model and to present planners with an idea of our model's potential. if a planner wishes to use our model, parameter values can be changed in our code, found at 1 , to accurately reflect a specific planner's current situation. as in [ace+20], we consider policies which assign different lockdown strategies to population groups with different responses to infection and lockdown. influenced by their results, we divide adults into one group aged 20-64, called"low-risk" and indexed by j = 1, and one group aged 65 and over, called "high-risk" and indexed by j = 2. we will only consider adults older than 20, so the low-risk group makes up 82% of the population of interest, while the high-risk group makes up 18% [hm] . the second group can also include individuals of any age who are more likely to contract severe cases of covid-19 and experience complications due to immunodeficiency, respiratory weakness, or other preexisting conditions. these individuals are considered separately from the general working population. we denote the population of group j as a proportion, n j , of 1 https://github.com/april-nellis/covid19-bsir all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint the total population and lay out the following relationship: individuals move from susceptible (s j (t)) to infected (i j (t)) to recovered (r j (t)), with additional flows from all three to death (d j (t)). the dynamics are given bẏ (2.1) the number of new infections depends on the size of the susceptible and infected populations, as well as the lockdown levels, l j (t), and transmission rates between groups, β ij . as others have suggested ([ace+20], [aal20] ), a portion of the population will disregard lockdown orders. this level of obedience is represented by θ. as in [ace+20] , we set θ = 0.75, though this parameter is difficult to quantify exactly. patients move out of the infected category with rate γ in accordance with the expected recovery time of 18 days. deaths due to covid-19 occur at rate φ(i j (t)) and other deaths occur at rate ξ(l j (t)). for convenience, the parameters that appear in (2.1) and in the objective function (3.1), along with their levels, are listed in table 1 of the appendix. 2.1. deaths. one unique element of covid-19 is its significantly different death rates for different groups. therefore, the base death rate δ 0 j for each group is set individually. in addition, as the number of infected individuals increases and hospitals become more crowded, death rates increase as a function of the total number of infected patients as in [aal20] . we represent this increase in the death rate as δ 1 j and follow [ace+20] in assuming that an infection level of 30% increases the death rate by a factor of five. therefore, the death rate due to viral infection is additionally, as lockdowns stretch on concerns have been raised regarding "deaths of despair" due to the impacts of lockdowns on mental health ([zha+20], [ems20] ). hospitals have also shut down many departments to accommodate the increased need for icu units for covid-19 patients. many non-elective surgeries and routine health checks have also been cancelled or rescheduled ( [sor+20] ). this neglect of health maintenance is also very likely to have repercussions on public health. to encompass all this, we add the term ξ(l j (t)), which is written as this represents the number of deaths indirectly caused by the lockdown, and scales with l j (t). we argue that, in the absence of any way to verify immunity, indirect deaths occur in both the susceptible and recovered populations. so, the total number of deaths is given by j φ(i j (t))i j (t) + ξ(l j (t))(s j (t) + r j (t)). behavior-dependent disease transmission. the basic transmission rate of covid-19 is approximately 0.2 ([aal20], [ace+20] ). this means that about 20% of those who come in contact with an infected individual will become infected themselves. however, we incorporate a transmission rate that decreases as infections increase due to increased caution between people, as discussed in [coc] . in addition, we consider an inter-group interaction factor, ρ, as in [ace+20] . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. this reflects a lower rate of interactions between groups. for example, working people aged 20-64 will interact more with their peers than with those in the high-risk group. combining these two ideas, we represent the transmission rate as the scale factor α i is chosen such that the rate of transmission can be decreased by a factor of e −0.3α i when 30% of the population is infected with the virus. the questions of how long and how severely to lock down the population during a pandemic can be thought of as a planning problem. given the above dynamics, we model the optimization problem that must be solved by a social planner using the following objective function, which represents the overall societal costs of a given lockdown policy: 3.1. attainable lockdown levels. there are certain jobs that must be done even during a pandemic, preventing the population from attaining full lockdown. these essential professions include healthcare workers, grocery store employees, delivery workers, and the postal service, among others. because of this, we set an upper limit on the possible lockdown level, denotedl j , and the set of possible lockdown policies is written as λ = [0,l 1 ] × [0,l 2 ]. we setl 1 at 0.7 to account for essential workers in the low-risk group. on the other hand,l 2 is set at 1 since we assume that the high-risk group does not work. 3.2. herd immunity. previous models considered either an infinite time horizon with stochastic vaccine arrival or a fixed horizon with deterministic vaccine arrival when formulating their objective function. we contribute a new approach that sets reaching herd immunity as the end of the problem. this can be reached either naturally via infection spread and recovery (which confers immunity) or via the arrival of a vaccine. we assume that those who have been infected with covid-19 once will remain immune for the rest of the outbreak. additionally, we assume that if a vaccine is approved for distribution, vaccination levels will be high enough to produce herd immunity. we define σ as the time at which herd immunity is reached. we assume a herd immunity threshold of 60% recovered 2 , so we set 3.3. output loss. the most noticeable result of lockdown measures is economic slowdown. many workers who are not deemed essential and cannot work remotely have found themselves jobless as companies lose revenue. as in [ace+20] , we take the average wage of a full-time worker and normalize it to 1 and assume that on average, those in the high risk group do not earn any wages. we do not assume the existence of an "immunity passport" given to those who have recovered and are immune, so we set a flag parameter p = 1 (this can be set to 0 to be consistent with the cases 2 in [cdc] table 1 (scenario 5: current best estimates), the basic reproduction number of covid-19 is r0 = 2.5. herd immunity is calculated as 1 − 1/r0 = 0.6. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint of [aal20] and [ace+20] ). on the other hand, we do consider some proportion h of the workforce who are able to work from home. therefore, we denote the purely salary-based cost of lockdown as when presenting our numerical results, we refer to the output loss due to lockdown. this is not the value of the objective function presented in (3.1), but rather the losses in output caused by requiring people to stay home and not work. the output loss is represented by and is compared to annual "normal" output. this baseline output is calculated as the amount of output produced until the expected vaccine arrival time, 1/nu, if there is no lockdown, annualized using the expected vaccine arrival time. this is given by 3.4. cost of death. we calculate the cost of a covid-19 death in group j in the same manner as in [ace+20] . here, χ is the non-pecuniary cost of life, which we consider as a measure of the public impact of deaths due to covid-19. this can be thought of as a measure of the planner's priorities. lower values of χ lead to prioritizing output loss minimization, while higher values are chosen to encourage longer lockdowns and decrease mortality at the expense of output. to ensure that this cost is on the same order of magnitude as wages, we scale by the interest rate when we choosing χ, similarly to [aal20] . note that χ = 0.2/r is consistent with [ace+20] where χ = 20 and r = 0.01. ∆ j is the number of years left in an average individual's career. we set ∆ 1 = 20 and ∆ 2 = 0. therefore, the cost per death due to covid-19 is given by deaths indirectly caused by the lockdown are not explicitly categorized and counted, and so can be considered "invisible deaths". for this reason, we do not include χ in the cost of these deaths and only count lost productivity. we also account for similar future deaths due to lack of preventative healthcare using a constant f (the number of indirect deaths in the future relative to those that occur during lockdown). these deaths do not appear in the dynamics, as they have not yet occurred, but they are considered when calculating the costs of lockdown. for this reason, f appears in (3.1) but not in (2.1), and the total cost of indirect deaths is given by 3.5. future loss of employment. another addition to the model acknowledges the long-lasting economic impacts of a period of economic slowdown. already some large corporations like jcpenney and hertz have filed for bankruptcy ( [mon] ), and there are certainly more companies, both large corporations and small business, who are under financial strain. federal stimulus measures may alleviate some of this burden, but they cannot completely compensate for current drops in consumption. effects may manifest in a variety of ways, but we choose to express them as a "future loss in employment", in which 1 day in lockdown results in some α e days of lost employment (on average) after lockdown ends. we set this to be 0.42 (reflects current 14.7% unemployment [bls] and average 3 days of unemployment for one day of lockdown based on median unemployment duration of 25.2 weeks (6 months) in 2010 [cun] ). the cost of future unemployment is modeled by all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint 3.6. icu overcapacity. a major incentive for lockdown measures is "flattening the curve"slowing the spread of the virus so that hospitals and icus will not get overwhelmed by a flood of patients in need of ventilators and other specialized medical equipment. this is already reflected in the death rate which increases as infections increase, but we add an additional penalty on top of that. we assume that a fixed proportion of infected patients, ι j , require icu care. we set this level to be 2.6% for people without underlying conditions and 7.4% for high-risk groups [cdc] . then, we incorporate a penalty η (representing a daily penalty scaled by the level of overcapacity) for hospitalizations exceeding the estimated average icu capacity, which is 30 beds per 100,000 people [pw12] . this is done via the function 4.1. numerical method. we used value iterations, first introduced in [bel57] , to solve the optimization problem presented by our model. the model is discretized using first-order taylor approximations and the value function is calculated over a regular grid. because the change in population due to deaths is very small, we follow the precedent set in [aal20] and iterate over a four-dimensional (s 1 , s 2 , i 1 , i 2 ) grid to determine optimal lockdown policy instead of the larger and more computationally expensive (but more accurate) six-dimensional grid (s 1 , s 2 , i 1 , i 2 , r 1 , r 2 ). by this, we mean that instead of separately keeping track of the recovered and dead populations, they are considered together as one unit when determining the optimal lockdown policy. since the vast majority of this "non-susceptible" group is recovered, this simplification, which removes two state variables, has a small effect on accuracy but a large effect on computational complexity. and, when determining the pandemic trajectory for given initial conditions and a given lockdown policy, total deaths can still be calculated via the population dynamics shown in (2.1). we choose ∆s = 0.0714 and ∆i = 0.0357 in our discretization, and set days as the unit of time. for all models, we take the initial conditions to be uniform across groups (if applicable) and set them at the level of 98% susceptible, 1% infected, 1% recovered unless otherwise specified. to test the validity of our numerical models, we use the parameter values of [aal20] and [ace+20] and compare our model's recommendations to their results. a full list of the parameter values used in this section is presented in table 1 . in figure 1 , we compare a one-group version of our model with the one presented in [aal20] , whose recommended optimal lockdown reaches 70% lockdown after about one month, and then slows reduces in intensity until lockdown is lifted approximately 140 days (4.5 months) after the outbreak begins. our version of this model maintains the maximum lockdown of 70% for slightly longer and ends slightly later. interestingly, the ending of lockdown nearly coincides with the population reaching herd immunity, though we did not add any such considerations when running this example. in figure 2 , we set up our model to mimic the semi-targeted policy from [ace+20] and find similar levels of output loss, total deaths, and general lockdown recommendations. namely, the optimal strategy keeps the high-risk group in lockdown until the arrival of a vaccine, while the low-risk group is able to emerge and return to work after approximately 200 days of lockdown have elapsed. in this figure, note that the population reaches herd immunity well before the arrival of a vaccine, implying that the lockdown on the high-risk group could have been ended earlier. now, we investigate the results of our new model using comparable parameter levels. we incorporate herd immunity, deaths indirectly due to lockdown, ability to work remotely, and behaviordependent transmission rates. additionally, we consider the possibility of lost employment after the end of the pandemic, as well as the costs of missed health screenings and a monetary penalty for exceeding icu capacity. to allow comparisons with previous works, we use death rates of a all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint similar magnitude to those in [aal20] and [ace+20] , but we change some parameters to better fit the current situation. interest rates have dropped significantly, so we use a 0.001% interest rate, instead of the 5% used by [aal20] or the 1% used by [ace+20] . note that since the interest rate is extremely low, there is little to no discounting applied to wages. we also lengthen the projected average career length in the low-risk group. finally, we adjust the population distribution slightly from 21% high-risk to 18% high-risk, based on data from the 2010 united states census [hm] . the results of our model using the parameters listed in table 1 is shown in figure 3 . most noticeably, lockdown rates for both groups fall to 0 after the entire population reaches herd immunity, which is explicitly imposed by our model. additionally, the lockdown for the low-risk group is slightly shorter but more intense. unsurprisingly, incorporating deaths of despair increases the total number of deaths due to the epidemic, but this effect is kept small by the shorter lockdowns. long-term costs of lockdown (an additional penalty for icu overcrowding, future deaths due to current health negligence, and future unemployment beyond the lockdown) increase output loss while not directly contributing to deaths during lockdown. however, these output losses are offset by the proportion of the population that is able to work remotely from home and the shorter lockdown periods. rates. recent cdc reports [cdc] indicate that the death rates are much lower than those used in subsection 4.2. to increase the realism of our model, we update the model death rates according to this newer data 3 and use them for all subsequent results. these death rates are listed in table 2 and the result, shown in figure 4 , predicts total mortality of 0.4464% and total output loss of 0.0013%. the negligible output loss is due to the negligible lockdown for the low-risk group. however, lockdowns have already been imposed for both groups (and indeed we might desire a death rate lower than 0.4464%), so we increase the non-pecuniary value of life, χ, and observe how the model changes. by increasing χ from 0.2/r to 10/r, figure 5 shows that both groups experience levels of lockdown similar to that of figure 3 , but with an output loss of 4.8984% and a lower total death toll of only 0.3266%. we designate this the benchmark situation, which uses death rates from table 2 and χ = 10/r but keeps all other parameter values consistent with those in table 1 . we also compare the results of the optimal lockdown to those generated by an uncontrolled scenario with the same parameters, shown in figure 6 and table 4 . without lockdown there is no output loss, but final mortality numbers are approximately twice as high. 4.4. varying initial conditions. since we are currently in the middle of the pandemic, we investigate at how different initial conditions change the recommended lockdown levels. we model a situation where the pandemic is ongoing and lockdown measures have been lifted, but a sudden spike in infections occurs which prompts new lockdown measures. we consider a case where 20% of the population has recovered and 0.2% has died, similar to estimates of the current situation in new york city [sta] . in figure 7a , a small infection spike affects 5% of the population before lockdown measure are put in place. in this case, we see additional deaths of 0.2452%. in figure 7b , a large infection spike affects 25% of the population, causing 0.3346% additional deaths. note that the lockdown is actually shorter for larger infection spikes, since the larger infection level (which occurs before lockdowns are imposed) moves the population closer to herd immunity. the price of this shorter lockdown, though, is higher mortality rates. 4.5. parameter robustness. it is natural to ask how changes in other parameters affect the optimal controls. in general, changes in parameters create the expected changes in lockdown length and intensity, output loss, and mortality. the more interesting question asks about the level 3 we use the data in [cdc] [hm] and multiply by 0.65, since the cdc estimates that 35% of cases are asymptomatic. for the same reason, we multiply the symptomatic case fatality ratio for the 65+ group by 0.65 to determine δ 0 2 . we set δ 1 j such that a 30% infection level causes a five-fold increase in deaths, as in [ace+20] . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint of impact of various parameters. the effects of non-pecuniary value of life (χ) have already been discussed and are displayed in figures 4 and 5 and table 3 , but now we discuss other parameters. in table 5 , we list the lockdown levels, mortality, and output loss for various other configurations of parameter choices. there are some elements of the model which do not have large impacts on the results. the icu overcapacity constraint barely affects results, likely due to a combination of low infection rates and a sufficiently high number of icu beds (on average) in the united states. this allows icu admittance rates to remain at or below the threshold. the expected vaccine arrival date also does not have much effect on the optimal lockdown levels, since the population is expected to reach herd immunity well before its introduction. based on the current situation, it seems extremely unlikely that a vaccine will be developed less than 8 months after the start of the outbreak, so we considered expected vaccine arrival times of 1 year (ν = 1) and 8 months (ν = 1.5) and found that neither adjustment had much effect. it can also be seen from the table that removing f , the cost representing future deaths due to current lack of health maintenance, lengthens the lockdown only slightly, and has little effect on output loss and mortality. in contrast, loss of future employment, ability to work remotely, indirect death rate in lockdown, inter-group interaction, and behavior-dependent infection transmission have significant effects on lockdown length and severity. adding output savings from remote work, future employment loss due to lockdown, and indirect deaths of lockdown affects the wider population and so produce similar changes in outcomes. the model produces uniformly better outcomes when the level of remote work, h, is increased, though lockdowns do last longer as seen in figure 8 . intuitively, this follows from the idea that more people working remotely helps to maintain economic activity without increasing risk of infection. changing α i and α l in the opposite direction of remote work creates similar effects on the optimal lockdown policy, though outcomes move differently. increasing the length of projected future unemployment, α e , leads to a shorter lockdown and less output loss, while deaths increase. this suggests that α e influences the trade-off between output loss and mortality. and, when we look at varying values of α l , we can see what happens when the optimization tries to minimize deaths due to covid-19 while also trying to avoid deaths due to lockdown. when α l = 0 and the model doesn't take indirect deaths into account, the lockdown extends for longer and has a larger effect on output, but we see much lower mortality levels. however, when α l is increased to 50 deaths per 100,000 individuals at full lockdown, the model dramatically shortens the lockdown, which decreases output loss and indirect deaths but which leads to higher deaths due to covid-19. we now discuss the effects of behavior-related parameters. interestingly, these are the only parameters we discuss which have also an effect on the uncontrolled outcomes. if the level of interaction between groups, ρ, is lowered, there is less interaction between the low-risk and highrisk groups and so lockdowns are more effective. in figure 10 , when ρ goes from 0.75 to 0.5, the low-risk group is able to begin easing the lockdown earlier since there is less worry about transmission to high-risk individuals. however, the lockdown lasts longer overall, since it is harder to reach herd immunity. this increase in output is offset by a drop in mortality. with the optimal lockdown, mortality decreases to 0.2552% compared to the benchmark of 0.3166% when ρ = 0.75. with no lockdown, mortality decreases to 0.5268% from 0.6189%. the opposite occurs when ρ is increased to 1, meaning that the groups mix freely. herd immunity arrives sooner due to increased inter-group transmission, however mortality increases to 0.3599% with lockdown and to 0.6891% without it. this suggests that it is beneficial for high-risk individuals to exercise extra caution in their interactions with members of the low-risk group. the other parameter that reflects individual behavior, α i , also has a notable impact on optimal lockdown policies. this parameter determines the efficacy of personal actions taken to slow transmission of covid-19. in the benchmark case, α i = 1. if transmission rates are constant (α i = 0), then the lockdown lasts longer due to the increased likelihood of transmission and mortality increases to 0.3394%. in the uncontrolled case, all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint the mortality increases too, to 0.7586%. but, if α i is set very high (say 10, which implies that personal caution can reduce the transmission rate by 95% when infections reach 30%), then a lockdown is barely necessary, as shown in figure 9 . in this case, uncontrolled mortality is a mere 0.2581%. this second scenario is perhaps too optimistic, but it demonstrates the potential power of social distancing. if we examine α i = 6 and α i = 8, we see that the lockdowns increase as α i decreases. if we consider the more modest change from α i = 0 to α i = 1 and decrease the interaction level between groups from ρ = 0.75 to ρ = 0.5, then imposing the optimal lockdown decreases overall mortality from 0.7586% to 0.2552%. if the population is less obedient and disregards lockdown measures, for example if θ decreases from 0.75 to 0.6, we see that the lockdown is shorter because herd immunity is reached sooner. this decreases output loss, but leads to more deaths. if the population is more obedient, however, for example if θ = 0.85, the effect of a given level of lockdown is larger with respect to the same level of output loss so lockdowns are less intense but last longer. this slows the arrival of herd immunity, leading to higher output loss, but has the benefit of lower mortality rates. finally, in figure 11 we investigate the effect of increasing the herd immunity threshold σ(x) = min{t ≥ 0 : r t ≥ x}. in all of our examples, the low-risk group is able to leave lockdown before the arrival of herd immunity, so moving this threshold does not have much impact on output loss. however, this parameter has important implications for the high-risk group. from figure 11 , we see that there is a clear change in dynamics for thresholds of 75% and above, where the lockdown policy becomes comparable to that of [ace+20] . the length of lockdown for the low-risk group increases by about 20 days, but the high-risk group remains in lockdown until the vaccine arrives -an increase of almost 300 days. this abrupt change in strategy arises because eventually the population reaches a steady state with very low infections. the number of susceptible individuals decreases very slowly and is driven largely by deaths due to lockdown, so will not achieve thresholds of 75% and over before the vaccine is expected to arrive. in this case, the pandemic has been neutralized even though the population has not reached the threshold prescribed by the parameters. these experiments demonstrate that there is not much benefit to be found from a planner overestimating the herd immunity threshold. if the herd immunity threshold is 100%, this is equivalent to removing the herd immunity exit time. the impact of σ can therefore be observed as an decrease in output loss and an increase in mortality, while decreasing lockdown length for the high-risk group by 340 days and for the low-risk group by 45 days. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. table 1 output loss: 13.4232%, total deaths: 1.1754% figure 2 . recreation of [ace+20] model (two groups and no herd immunity), parameters from table 1 output loss: 8.9676%, total deaths: 1.3121% all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint figure 3 . our model (two groups, herd immunity), parameters from table 1 herd immunity: 211 days, output loss: 7.8767%, total deaths: 1.8873% (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. table 2 . death rates based on [cdc] , γ = 1/18 is recovery rate as listed in table 1 figure table 3 . a comparison of mortality rates for different non-pecuniary value of life, death rates from table 2 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. table 5 . parameter robustness results (note: lockdown for 65+ ends at herd immunity) benchmark parameters: χ = 10/r, r = 0.001%, α e = 0.42, h = 0.4, α l = 10 −5 , α i = 1, ρ = 0.75, f = 1, θ = 0.75, ν = 0.67, η = 10, death rates from table 2 , herd immunity threshold = 60% all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint figure 6 . comparison of optimal lockdown policy to no lockdown, using benchmark parameters, optimal lockdown deaths: 0.3266% vs uncontrolled deaths: 0.6189% all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. figure 7 . results for varied initial conditions using benchmark parameters all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. figure 10 . robustness results for ρ (inter-group interaction level) χ = 10/r, r = 0.001%, ν = 0.67, α l = 10 −5 , α i = 1, α e = 0.42, η = 10, f = 1, h = 0.4 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint (a) herd immunity = 65%, output loss: 7.8084%, total deaths: 0.2948% (b) herd immunity = 70%, output loss = 7.8496%, total deaths = 0.2724% (c) herd immunity = 75%, output loss: 7.967%, total deaths: 0.2732% figure 11 . robustness results for σ (arrival of herd immunity) ρ = 0.75, χ = 10/r, r = 0.001%, ν = 0.67, α l = 10 −5 , α i = 1, α e = 0.42, η = 10, f = 1, h = 0.4 (cont.) all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 23, 2020. . https://doi.org/10.1101/2020.06.22.20137711 doi: medrxiv preprint a simple planning problem for covid-19 lockdown. working paper 26981. national bureau of economic research optimal targeted lockdowns in a multi-group sir model. working paper 27102 a markovian decision process the employment situation cdc. covid-19 pandemic planning scenarios an sir model with behavior great recession, great recovery? trends from the current population survey students under lockdown: assessing change in students social networks and mental health during the covid-19 crisis age and sex composition a contribution to the mathematical theory of epidemics beware of bankrupt stocks like jcpenney and hertz international comparisons of intensive care: informing outcomes and improving standards immediate and long-term impact of the covid-19 pandemic on delivery of surgical services. advance online publication amid ongoing covid-19 pandemic, governor cuomo announces results of completed antibody testing study of 15,000 people showing 12.3 percent of population has covid-19 antibodies unprecedented disruption of lives and work: health, distress and life satisfaction of working adults in china one month into the covid-19 outbreak key: cord-327697-80msva10 authors: sarı, cenk; şimşek, ersin çağrı; özdoğan, öner title: the outcomes of the postulated interaction between sars-cov-2 and the renin-angiotensin system on the clinician’s attitudes toward hypertension treatment date: 2020-11-09 journal: j hum hypertens doi: 10.1038/s41371-020-00436-w sha: doc_id: 327697 cord_uid: 80msva10 concern has arisen about the role played in coronavirus disease 2019 (covid-19) infection by angiotensin-converting enzyme (ace) inhibitors and angiotensin ii receptor blockers (arbs). this study was designed to assess the practice behaviors of physicians toward hypertension treatment with ace-i or arbs during the covid-19 pandemic. a self-administered survey questionnaire consisting of 26 questions about current hypertension treatment with ace-i/ arbs was applied to cardiologists, internists, and family physicians in central and western turkey, between 01 and 19 may 2020. a total of 460 physicians were approached, and 220 (47.8%) participated in the study. of the total respondents, 78.7% reported that they had not changed their antihypertensive medication prescribing pattern, 8.6% of clinicians had changed ace-i/ arbs medicine of patients during the covid-19 pandemic and 12.7% of them were undecided. the median (±interquartile range) score indicating general reliance level of physicians in ace-i/arbs therapy was 8 ± 4 (range, 1–10). in multiple comparison analyses, the general reliance level in ace-i/arbs, reliance level when starting a new acei/arbs and changing behavior in heart failure patients were significantly different with regard to the specialties (p:0.02, p:0.009, p:0.005 respectively). although most of the physicians found the publications about ace-i/ arbs during the covid-19 pandemic untrustworthy, there were variable levels of knowledge and reliance among different physicians and specialty groups. in general, the ace-i/ arbs prescribing habits were not affected by safety concerns during the covid-19 pandemic in turkey. in the first half of 2020, it was speculated that angiotensinconverting enzyme inhibitors (ace-i)/angiotensin receptor blockers (arbs) may make patients more susceptible to covid-19 disease and lead to worse outcomes. this early hypothesis led to concerns among patients taking these medications and among physicians prescribing them for long periods. angiotensin-converting enzyme 2 (ace2) is a crucial component of the renin-angiotensin-aldosterone system (raas), which metabolizes a potent vasoconstrictor angiotensin ii to vasodilator peptide angiotensin [1] . this hypothesis was based on the fact that coronavirus 2 (sars-cov-2) that causes covid-19 disease gains entry into cells by binding to the ace2 receptor [2] [3] [4] . moreover, some previous studies have suggested that ace-i/arbs blocker treatment up-regulates ace expression and the use of these drugs may increase susceptibility to infection by sars-cov-2 [5] [6] [7] [8] . observational studies have reported that patients taking ace-i/arbs treatment are at increased risk of becoming infected with sars-cov2 and developing severe forms of covid-19 disease. during the pandemic, contradictory studies and opinions have suggested that local inactivation of the reninangiotensin aldosterone system may have protective effects against the development and progression of acute lung failure [3, 9] . professional societies have navigated this ambiguity by recommending that patients should continue to use ace inhibitors or arbs [10] . however, these recommendations have not been supported by medical data. in consequence, the clinical decision-making process has become even more difficult for physicians until this interaction is clarified with robust clinical data. the aim of this study was to assess the behaviors of physicians toward prescribing ace-i/arbs treatment during the covid-19 pandemic to determine if physicians had changed their routine treatment algorithms and how they had been affected by these speculations. this cross-sectional survey included cardiologists, internists, and family physicians responsible for the treatment of hypertension or prescribing antihypertensive drugs. the study was administered in six metropolitan areas located in central and western turkey, between 1 may and 19 may 2020. the hospitals and family health centers from the mentioned cities were selected according to their covid-19 patient loads. the questionnaire was developed in turkish by an expert committee including the manuscript authors. although the surveys were not formally validated, the development process involved comprehensive reviewing and piloting of the surveys. the survey consisted of 26 questions about current hypertension treatment behavior with ace-i and arbs during the covid-19 pandemic. physicians who were responsible for the treatment of at least 30 patients with hypertension per month were invited to participate in the survey by phone or e-mail. the survey was delivered as an online form and responses were submitted online. the study was approved by the ethics committee of izmir tepecik training and research hospital (approval number 2020/ 6-11). the sample size was calculated to be 213 to provide 80% power with an effect size of 0.5 at an alpha of 0.05 using open source software, open-epi version 3.01. a convenience sample of 460 physicians was approached by phone or e-mail at hospital and family health centers in central and western turkey. a total of 232 physicians completed the survey, presenting a response rate of 50.4%. data obtained in the study were analyzed statistically using spss for windows version 15.0 software (spss inc., chicago, il, usa). descriptive statistics were presented using tables of frequencies for the ordinal variables and median and interquartile range values for the appropriate variables. the primary outcome was the proportion of subjects changing the prescribing pattern of raas blockers. the pearson χ2 test was used to identify significant relationships between categorical variables. to determine whether physician specialist training contributed to the behaviors of hypertension treatment, the physicians were separated into three groups based on their specialist area (family medicine, internal medicine, and cardiology). the kruskal-wallis test was used to assess differences between specialists in attitudes and behaviors about hypertension treatment and to assess multiple group comparisons for ordinal variables. bonferroni correction was performed to adjust for multiple group comparisons. a value of p < 0.05 was considered statistically significant. a total of 232 physicians completed the survey in the defined study period and 220 physicians (142 male, 78 female) gave permission for the data retrieved from the questionnaire to be analyzed within the framework of the study. a total of 12 physicians were excluded from the study as they managed <30 patients with hypertension per month. most of the participants (63.2%) were aged <40 years and 12.8% of them were >50 years of age. nearly half of the physicians participating in the survey were cardiologists (n = 88, 40%) 71 (32.3%) were family medicine/ general practitioners and 61 (27.7%) were internal medicine specialists. of the physicians, 111 (50.4%) were working in tertiary level healthcare centers (university/training and research hospital). in respect of experience, half of the participants (53%) had >10 years of experience in practice ( table 1) . of the total physicians in the study, 65% described themselves as having a moderate to high level of knowledge about the relationship between covid-19 infection and ace-i/arbs treatments and 16% of the study population stated that they did not have enough information on this subject. the sources of information showed variety but 83.9% of the physicians were informed from at least one reliable academic document such as reports of scientific societies and articles of medical journals. when the respondents were asked to evaluate the reliability of the evidence of these sources, 84% stated that the reports were suspicious /contradictory and emphasized that new studies are needed. the opinions of the physicians about the relationship between covid-19 infection and ace-i/arbs treatments are shown in fig. 1a . requests from patients to change ace-i/arbs treatment during the pandemic were reported to have been received by 65% (143/220) of the physicians. of the total physicians, 78.7% (173/220) advised continuing the ace-i/arbs medications, 12.7% (28/220) left the decision to the patient, having explained that there were conflicting views on this issue, and 8.6% (19/220) discontinued the treatment and switched to another drug. the respondents were asked to score their level of reliance of ace-i/arbs treatments in different clinical conditions on a 10-point likert scale from 1 to 10. questions were then asked to ascertain the probability of the physician beginning and repeating ace-i/arbs treatment during the covid-19 pandemic. these items were also scored using a likert scale. the median (±interquartile range) score indicating the general reliance level of physicians in ace-i/arbs therapy was 8 ± 4 and for the trend to start new ace-i/arbs therapy was 8 ± 4. the median score indicating withholding the current acei/arbs therapy was 1 ± 1 and for the continuation of the therapy was 9 ± 1 (fig. 2) . the tendency for changing the prescribing patterns of ace-i/arbs treatment in different clinical conditions, including coronary artery disease, heart failure, hypertension alone, and multiple comorbidities is shown with box-plot graphs in fig. 3 . although more than half of the specialists did not believe there was any relationship between ace-i/arbs use and risk of having covid-19 disease, only one-third of the assistant doctors/general practitioners held these beliefs. nearly 40% of the assistant doctors believed that ace-i/ arbs users had more-severe covid-19 disease. the effect of experience on knowledge level and on the prescribing pattern was evaluated. there was determined to be a close interaction with every 5 years increase of experience in practice showing a steady increase in the high level of knowledge ratio about ace/arb treatment and covid-19 disease. (fig. 4) . the percentage of physicians who thought they had a high level of knowledge about ace-i/arb treatment and covid -19 interaction was 48.7% in cardiologists, 23.7% in internal medicine, and 11.5% in family medicine (fig. 5a) . the percentage of physicians who considered that ace-i/arbs therapy may be harmful during the covid-19 pandemic was 44.2% of the family medicine specialists, 34.8% of the internal medicine specialists, 33.3% of the general practitioners, and 15.4% of the cardiologists. conversely, 27% of cardiologists, 13% of internal medicine specialists, 8% of general practitioners, and 6% of family medicine physicians had positive opinions that these medications could be associated with less severe disease and could be protective against infection of covid-19 disease (fig. 5b) . more than two-thirds (76.2%) of the physicians did not change their mind, 27.6% of cardiologists changed their opinion in favor of ace-i/arbs treatment and 16.6% of general practitioners were affected negatively by the environment of uncertainty during the covid-19 pandemic. calcium channel blockers (ccb) were the most preferred agents in the case of ace-i/arbs cessation (fig. 5c) . it was observed that 14% of all physicians, 19% of cardiologists and 24% of internal medicine doctors considered changing the ace-i/arbs therapy in the case of hypertension and multiple comorbidities respectively. the general reliance level was highest among cardiologists (8.0 ± 4) and lowest (5.0 ± 3) among the family medicine/general practitioners (p < 0.05). the strategy for repeating therapy was the same in all physicians, and the practice of starting new ace-i/arbs reported by the cardiologists was statistically different from that of both internal medicine and family medicine/general practitioners (8.0 ± 4 vs 8.0 ± 4 vs 9.0 ± 3, p < 0.05) ( table 2) . the institutions where the respondents worked were categorized in three levels as primary, secondary, and tertiary in order to evaluate the effects of the type of healthcare facility on physician's behavior while starting or stopping acei/ arb treatments. the level of the healthcare institution and the duration of working were found to have no impact on repeating/starting/general practice about ace-i/arbs (table 3 ). by mid-june 2020, at the time of writing this study, 8.5 million people have been infected with sars-cov-2 and over 470,000 have died worldwide. more and more patients are still becoming infected every day. as covid-19 disease is caused by the sars-cov-2 virus, some speculations have been put forward linking aminopeptidase ace2 with this disease [11] . based on these speculations, arguments and concerns about these medications have arisen in the community. despite the supporting reports of scientific societies against discontinuation of these drugs, the lack of information at the beginning of the pandemic might have had an impact on the behaviors of physicians in respect of whether or not to prescribe [12] . early reports, which mainly came from china, raised suspicion and encouraged doctors to stop prescribing acei/arbs [13] . these suggestions come from observational studies of large patient populations from different countries [14] [15] [16] . in these trials, researchers observed that hypertensive patients had more-severe covid-19 disease than other patients, so concluded that, hypertension could be a risk factor for a severe disease course [17] . nevertheless, the hypertensive patients with severe covid-19 in those studies had poorer general health as they had additional risk factors such as older age and comorbidities (heart failure, diabetes, chronic kidney disease).these preliminary results not taking these potential confounding factors into account may have caused cessation of these important beneficial and more recently, at later stages of the pandemic, more sophisticated studies were published, which mainly aimed to clarify whether ace-i/arbs were harmful and to propose risk factors for severe covid-19 disease with multivariate logistic-regression analysis. however, some researchers have argued that ace-i/arbs may have some protective effects against covid-19 disease. it has been suggested that ace2 upregulation by ace-i/arbs inhibitors may decrease unopposed angiotensin ii activity, which contributes to facilitating neutrophil infiltration and causes organ injury in response to bacterial endotoxin [18, 19] . in a small study from china, liu et al. found that elevated levels of plasma angiotensin ii were correlated with viral load and degree of lung injury [20] . prior to that study, there had been studies of other viruses showing that recombinant ace2 administration reversed lung injury [21, 22] . in the current study, 16.7% of the respondents believed that acei/arbs treatment was associated with less severe disease or a low likelihood of being infected. a recent study conducted in italy clearly showed that neither ace inhibitors nor arbs were harmful during the covid-19 outbreak [23] . in the current study, while physicians expressed many concerns regarding acei/arbs, nearly half believed that ace-i/arbs treatment may worsen the clinical scenario and increase the probability of getting infected. however, two-thirds of the physicians had not changed their antihypertensive medication prescribing pattern. the results suggest that the physician's behavior toward hypertension treatment had not changed easily based on speculations or concerns during the pandemic in turkey. with the argument regarding hypertension as a risk factor for covid-19, studies have been conducted to determine which antihypertensive medications are the safest. reynolds et al. assessed the relationship between five common classes of antihypertensive medications (ace-i/ arbs, beta-blockers, calcium-channel blockers, thiazide diuretics) and covid-19 [24] . there was reported to be no association for any of the mentioned drug classes. in the current study, majority of respondents stated that they continued as before and had made no change in their treatment regimen, and only a small number of participants emitted that they were hesitant to start new ace-i/arbs therapies. ca channel blockers were considered a safe alternative in cases where ace-i/arbs drugs were discontinued or changed, and 12.9% of physicians reported that they preferred ccb instead of ace-i/arbs. between internal medicine and cardiologist p < 0.05. table 3 effect of duration on practice and working center on reliance level of ace-i/arbs. general reliance the data are expressed as median ± interquartile range. from the results of this study, it appears that practicing physicians found less risk in changing their prescribing behavior in cases with hypertension alone. nevertheless, they considered continuing with raas inhibitors, which are cornerstone therapies in heart failure patients. sars-cov-2 virus is a cardiotropic virus than can cause severe myocarditis in up to 40% of critically ill patients [25] . cessation of ace-i/arbs treatment may lead to decompensation in those with previous heart failure and results in sars-cov-2 infected patients being more susceptible to myocardial damage. an interesting new hypothetical study from england investigated the connection of influenza infection with sars disease via the shared mechanism of both for entering the human cells by ace2 receptors [26] . the use of ace-i/arbs therapies was found to be associated with either no effect on the incidence of influenza or possibly a much lower incidence, depending on the duration of use. based on this trial, it was suggested that the same relationship can be seen in sars-cov-2. this study had several limitations. the overall sample size was small and the subgroups were not normalized to the practicing population of family medicine, internal medicine, cardiologist, and general practitioner. in addition, the questionnaire that was used had not been previously validated. in the literature, there are no other studies that have utilized a questionnaire to measure physicians' attitudes regarding sars-cov-2 and hypertension treatment interactions. the study population was sampled at hospitals and family health centers in six major cities in turkey, so the results might not be reflective of physicians practicing in the whole of turkey. the physicians interested in this issue might have agreed to participate in the survey, which could have led to the high rate of physicians who did not change the treatment strategy. the present survey is the first study to have provided a snapshot showing behaviors of turkish physicians toward prescribing ace-i/arbs treatment during the covid-19 pandemic. an important finding of this study is that the majority of physicians were not affected by the concerns, which had arisen at the beginning of the pandemic and continued to prescribe ace-i/arbs treatments. however, there was a statistically significant difference between different physician specialties in the reliance level in different clinical situations. given the probability of emerging new evidence on this matter, additional studies should be conducted to clarify the changes in prescribing habits of physicians of antihypertensive medications during the course of the covid-19 pandemic. what is known about topic • in the first half of 2020 covid-19 infection became pandemic. sars-cov-2 virus enters into cells by binding to the ace2 receptor. • hypertension is the most common comorbity in covid-19 patient. in early studies, it was speculated that ace/arb users may be more susceptible to covid-19 disease and have worse outcomes. • concerns about ace/arb use have arisen among healthcare professionals. scientific societies against discontinuation of these drugs. what this study adds • in pandemic era, general reliance level of in ace-i/ arbs therapy was high. 78.7% of the physicians advised continuing the ace-i/arbs medications. • calcium channel blockers were the most preferred agents in the case of ace-i/arbs cessation due to safety concerns. the strategy for repeating therapy was the same in all specialities. the practice of starting new ace-i/arbs and reliance level in different clinical situations showed significant difference between physician specialties. funding this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. conflict of interest the authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. informed consent all subjects gave informed consent for participation. the vasoprotective axes of the reninangiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases covid-19 and the cardiovascular system renin-angiotensin-aldosterone system inhibitors in patients with covid-19 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus enalapril attenuates downregulation of angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat upregulation of angiotensin-converting enzyme (ace) 2 in hepatic fibrosis by ace inhibitors upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 raas inhibitors do not increase the risk of covid-19 managing hypertension during the covid-19 pandemic covid19 and increased mortality in african americans: socioeconomic differences or does the renin angiotensin system also contribute? joint hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? lancet clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region covid-19 patients with hypertension have more severe disease: a multicenter retrospective observational study angiotensin receptor blockers as tentative sars-cov-2 therapeutics attenuation of pulmonary ace2 activity impairs inactivation of des-arg9 bradykinin/bkb1r axis and facilitates lps-induced neutrophil infiltration clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury angiotensinconverting enzyme 2 inhibits lung injury induced by respiratory syncytial virus angiotensinconverting enzyme 2 protects from lethal avian influenza a h5n1 infections renin-angiotensin-aldosterone system blockers and the risk of covid-19 renin-angiotensin-aldosterone system inhibitors and risk of covid-19 characteristics and outcomes of 21 critically ill patients with covid-19 in washington state association between angiotensin blockade and incidence of influenza in the united kingdom key: cord-289477-cjm7qhr4 authors: mueller, sylvia; gothe, rita; siems, wolf-dieter; vietinghoff, gabriele; paegelow, inge; reissmann, siegmund title: potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide bpp(9α) date: 2005-04-25 journal: peptides doi: 10.1016/j.peptides.2005.03.046 sha: doc_id: 289477 cord_uid: cjm7qhr4 synthetic analogues of the bradykinin potentiating nonapeptide bpp(9α) indicate significantly different structural requirements for potentiation of the bradykinin (bk)-induced smooth muscle contraction (gpi) and the inhibition of isolated somatic angiotensin i-converting enzyme (ace). the results disprove the ace inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected cos-7 cells as molecular mechanism of potentiation. our results indicate a stimulation of inositol phosphates (ip(n)) formation independently from the b(2) receptor. furthermore, the results with la(3+) support the role of extracellular ca(2+) and its influx through corresponding channels. the missing effect of calyculin on the gpi disproves the role of phosphatases in the potentiating action. these experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. the chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor. hormone actions can be potentiated by different factors interacting with the receptor, by enzymatic degradation or by signal pathways. however the entire overall process has been studied in detail for only very few hormones [30, 69, 45] . at least 40 years ago a potentiating action was observed for the nonapeptide hormone bradykinin (bk). indeed the history of bk isolation and characterization has long been closely related to the use of potentiating factors. werle and coworkers [74] used snake venoms to trigger the formation of bk from plasma and to describe this tissue hormone functionally. immediately after the isolation, chemical characterization, synthesis and functional characterization, certain snake venoms were described as bradykinin potentiating compounds. kato and suzuki [39, 40] , ferreira et al. [26, 27] , and ondetti et al. [13, 59] isolated different oligopeptides with bradykinin potentiating activity from the venoms of the two snakes agkistrodon halys blomhoffii and bothrops jararaca, including the bradykinin potentiating nonapeptide bpp 9␣ (trade name teprotide). bradykinin potentiating peptides have also been isolated from other snakes [8, 9, [23] [24] [25] 35] or other toxins [3, 21, 22, 44, 52, 73] as well. very recently new potentiating peptides have been isolated from the venom of b. jararaca [36] . surprisingly peptides with potentiating activity have also been formed by the partial hydrolysis of proteins taken from serum [80] , hemoglobin [38, 66, 81] , milk [34, 46] , or wheat germ [51] . also degradation fragments of angiotensin such as the heptapeptide 1-7 were found to potentiate the bk action [64] . in addition linear bk analogues, partial sequences [7] , certain active and inactive side chain and backbone cyclic agonists are able to potentiate the bk action on gpi [68] . the angiotensin i-converting enzyme (ace) cleaves dipeptides from the c-terminus of angiotensin i and bradykinin resulting on the one hand in the formation of the highly hypertensive hormone angiotensin ii and on the other hand in the inactivation of the hypotensive bk. this enzyme has been extensively studied because of these important functions in the blood pressure regulation. ace contains a n-terminal as well as a c-terminal catalytic domain, described in most publications as having only slight differences in their structural requirements [14, 79] . more recently cotton et al. demonstrated in an excellent investigation using domain-specific substrates and inhibitors, affinity differences between n-and c-terminal catalytic domains of about three orders of magnitude [11] . the membrane bound form of this enzyme seems to play an important role in the potentiation of the bk action. inhibitors of this enzyme are used as drugs for treatment of different forms of hypertension and heart failure. for therapeutically used hormones this knowledge about potentiating compounds and their action mechanisms is very important. this knowledge can help to improve the therapeutic effect, to prevent not only an excessive dose, but also interaction with other drugs and side effects. on the other hand the therapeutic use of potentiating compounds requires the knowledge about the interaction with the hormone action on the molecular basis as well. as shown by li et al. ace may also act as a receptor for sars coronavirus [47] . with extensive therapeutical application of bradykinin potentiating compounds such as captopril [12] , enalaprilat [64] , ramiprilat [75] , quinaprilat [41] and lisinopril [6] , studies of the molecular action mechanism have become more and more important. many other proteases are also able to inactivate bk including the neutral endopeptidase (nep, neprolysin; e.c. 3.4.24.11) [42, 71] [72] . potentiation of bk action has also been studied in in vivo models by pharmacological tests on isolated organs and on the cellular level by biochemical methods. in the in vivo models, potentiation of bk action has been measured on the pressor response to intravenous bk in conscious rabbits [68] or on the hypotensive effect in freely moving wistar rats [65] . isolated organs such as the guinea pig ileum (gpi) [54] , rat heart [34] , rabbit jugular vein [17, 33] , cerebral microvasculature (estimation of permeability) [58] and porcine coronary arteries [76] have been used for in vitro tests. yet even at the onset of the search for the molecular mechanism of bk potentiation, certain contradictory findings have been observed. the inhibition of ace by various peptide and nonpeptide compounds did not correlate well with the potentiating activity [5, 10] . furthermore, the maximum of the bk-induced contraction of guinea pig ileum can be enhanced by potentiating compounds [77] . also the action evoked by enzymatically stable bk agonists can be potentiated in some test systems [78] . repeated exposure of porcine coronary arteries to bk has led to receptor desensitization. the addition of the potentiating compounds quinaprilat or angiotensin 1-7 fully restored the relaxant effect at a point when bk alone was no longer able to induce relaxation [76] . at the molecular level the co-immunoprecipitation of ace and the b 2 receptor with an anti receptor antibody clearly indicates an interaction of both partners on the cell membrane [50] . despite all these contradictory and to some degree confusing findings regoli and coworkers [29] and dendorfer et al. [17] have demonstrated that in their test systems (rabbit isolated aorta and venoconstriction) the potentiation by therapeutically used ace inhibitors results exclusively from the inhibition of enzymatic bk degradation. nevertheless, the group of regoli found under influence of ace inhibitors, a resensitization of the rabbit jugular vein [31] and a change in the density of b 2 receptors in rat spinal cord [60] . to understand the molecular basis for these discrepancies many approaches have been undertaken to elucidate the influence of potentiating compounds on the different bradykinin destroying enzymes, on the bradykinin receptors (bkr) and on signal pathways. direct interaction of the potentiating compounds was postulated with the b 1 receptor [37] . phosphorylation of the receptor by protein kinase c leads to internalization [19] (desensitization) and dephosphorylation by phosphatase shp-2 to resensitization [18, 49] . bradykinin potentiating factors can influence both desensitization and resensitization, as well as the hetero-oligomerization of the b 2 receptor including the interaction with ace [54] and other degrading enzymes [15] . at the level of signal pathways, a crosstalk with other pathways induced by other hormones [43] or nonreceptormediated intracellular reactions [28] have been observed. as initial experimentation on the potentiation of the bradykinin action was primarily performed on isolated smooth muscle organs, in the last decade the potentiating activity was mainly investigated on the affinity and density of the receptor [54] , the intracellular mobilization of ca 2+ [49, 54] , the release of arachidonic acid [49, 54] , of inositol phosphates [54] , and of nitric oxide [37] . later studies on the molecular basis of bradykinin-evoked actions have been performed more and more on cell cultures instead of smooth muscles. these studies have used primarily chinese hamster ovary cells (cho cells) cotransfected with the b 2 receptor and ace, ace-mutants or neutral endopeptidase (nep) [15] . endothelial cells have been used because they constitutively express the b 2 receptor and ace [49] . the aim of our experiments was to contribute to a better understanding of the potentiating mechanisms, and to prove or disprove postulated action mechanisms. for these studies we used three different systems: the smooth muscle contraction, transiently transfected cos-7 cells and polymorphonuclear leucocytes. we began to find differences between ace inhibition and potentiation of the bk action. we were interested in determining the role of ca 2+ -uptake from extracellular sources and its release from sarcoplasmatic reticulum for the potentiating action. we tested the direct interaction with the b 2 receptor in cos-7 cells and checked the influence of potentiating peptides on receptor dephosphorylation and selected signal transduction reactions. since bk plays an important role in inflammatory processes we studied the potentiation on immunocompetent polymorphonuclear leucocytes. however it was not only our aim to verify or negate the contradictory opinions regarding the mechanism of bradykinin potentiation, we would also like to show that there simultaneously exist different mechanisms, depending on the assay and on the potentiating factors used. boc-, fmoc-and ddz-amino acids were purchased from bachem (switzerland) and orpegen-pharma (germany). boc-pro-ocr-oh was synthesized according to the general procedure published by gothe et al. [32] . to form the hycram tm -linker the hydroxycrotonic ester was coupled to the aminomethyl resin tentagel-s-nh 2 . the used resins were purchased from the following companies: amino methyl polystyrene, tentagel-s-nh 2 and wang resin from rapp polymere (germany), and chlorotrityl resin from nova-biochem (germany). the peptides were synthesized on a pss-80 automatic synthesizer (applied protein technologies, usa) or on the semiautomatic synthesizer sp-650 (bachem, switzerland). each step was monitored by the kaiser test. couplings were carried out after neutralization by repeated washings (5-10 times) of the resin with 5% diea in dcm in a two-fold excess of n ␣ -protected amino acid and diisopropylcarbodiimide (dic) in dcm for 4 h; 2-(1hbenzotriazol-1-yl) 1,1,3,3-tetramethylguanidinium tetrafluoroborate (tbtu)/1-hydroxybenzotriazole (hobt): boc-or fmoc-protected amino acids were used in a four-fold excess, solved in dmf or dcm/ dmf 1:1. tbtu and hobt were applied in the same excess, diisopropylethylamine (diea) in a six-fold excess. the reaction time was between 4 and 12 h. deprotecting procedures: boc-deprotection was performed with tfa/dcm 1:1 without any scavenger in two steps, 5 min treatment followed by washing with dcm and second treatment for 20 min; ddz-deprotection was carried out with 5% tfa in dcm in two steps, 5 and 20 min; fmocdeprotection was achieved with 20% piperidine in dmf in two steps, 5 and 20 min. the ileum (1.5 cm) of the guinea pigs was suspended in a 5 ml organ bath containing tyrode solution at 37 • c. isotonic contractions were recorded under a resting tension of 500 mg. bk was applied in a concentration of 1 × 10 −8 m that corresponded to an ed 25 to ed 30 of bk under these experimental conditions. generally this effect was increased twice by bpp 9␣ (20 nm) that was used as standard. the bradykinin potentiating peptides or the ace inhibitors were given 5 min before application of bk. the potentiating effects of the analogues were calculated as per cent of the bk-induced contraction and compared with the effect caused by bpp 9␣ (=1.0 or 100%) in the same experiment. to investigate the influence of ca 2+ on the bkpotentiating action of bpp 9␣ (20 nm bk, 20 nm bpp 9␣ ), la 3+ (1.8 mm) was used as an inhibitor of the ca 2+ influx in ca 2+ -free or ca 2+ -containing tyrode solution. lacl 3 was always given 1 min before or after the bk-induced contraction and before the application of bpp 9␣ (see fig. 3 ). calyculin (100 nm) was used to estimate the involvement of bk-induced (20 nm bk) and potentiated (20 nm bk, 20 nm bpp 9␣ ) contraction of the guinea pig ileum. calyculin was applied before bpp 9␣ was given at the maximum of the bk-induced contraction or 1 min before the bk-and bpp-induced effect was elicited (see fig. 6 ). inhibitory activities were determined with an enzyme preparation from the guinea pig lung, using benzoyl-gly-his-leu as the substrate. this method is described elsewhere [70] . cell culture and transfection of cos-7 cells have been described in detail [2] . cos-7 cells (atcc) were routinely grown in dulbecco's modified eagle's medium (dmem) containing 10% (v/v) fetal calf serum and antibiotics, and were kept in a humidified 5% co 2 , 95% air atmosphere. subconfluent cells were transfected in 24-well plates with bkr-b 2 -cdna (1 g cdna/well) by the deae-dextran technique, and cells were used 48-72 h after transfection. preparation of cos-7 membranes was done as described previously [57] . competition binding studies were performed using . thereafter, the samples were filtered through whatman gf/b glass fiber filters pretreated with 0.1% (w/v) aqueous polyethylenimine. the filters were washed three times with 5 ml of ice-cold binding buffer, transferred into scintillation vials, dried and analyzed for bound radioactivity by scintillation counting. cos-7 cells were transfected and labeled with 0.4 ci/ml [ 3 h]arachidonic acid (60-100 ci/mmol, nen life science products, usa) for 24 h as described previously [57] . the cells were washed three times with label-free dmem containing 0.5% (v/v) fetal calf serum and then stimulated in 0.5 ml in the same medium with 3 × 10 −8 m bk for 30 min in the absence or the presence of j526 (0.1 m) or j527 (0.1 m) at 37 • c. assays were terminated by removing the medium from the cells. the medium was measured for [ 3 h]arachidonic acid release by scintillation counting. heparinized venous blood from healthy volunteers was mixed with 1% dextran sulphate in 0.9% nacl at a ratio of 1:1 (v/v) and left for 50 min at 37 • c for erythrocyte sedimentation to occur. the leukocyte-rich plasma was layered over an equal volume of histopaque-1077, and the gradient was centrifuged for 30 min at 700 × g. contaminating erythrocytes were eliminated by lysis with ammonium chloride (0.98%), and the pure pmn were washed twice with eagle's minimal essential medium (mem) and resuspended (1 × 10 6 pmn/ml). chemotaxis was quantified using the boyden chamber technique as described previously [61] . pmn were suspended in eagle's mem containing 20 mm hepes (ph 7.3) and 5 × 10 5 /0.5 ml, pipetted into the top of boyden chamber which were separated by a 3 m pore filter (sartorius sm 11302) from the lower part. peptides were put into the lower stimulus compartment. inhibitors or potentiating peptides were applied to the pmn for 15 min at 37 • c and then to the upper compartment of the chamber. after an incubation period of 3 h at 37 • c in an atmosphere of 5% co 2 , 95% air at high humidity, the filters were fixed and stained with methylene blue. using an image analyzer (chemotaxis analyzer hca-100, ha-sotec, rostock, germany), the pmn were counted at every 10 m interval from the original monolayer to the distal surface in 10 high power fields of each duplicate filter. an average locomotion index (li m+ ) was quantified and expressed as the migration index in comparison to the control (control = 1.0). mostly the values are presented as means ± s.e.m. statistical analysis was evaluated with the student's t-test for independent samples or for paired samples, depending on the experimental protocol. details are described in the legends for each figure. bpp 9␣ and a variety of analogues were synthesized according to the three different strategies in scheme 1 on the solid phase. to prevent dioxopiperazine formation, chlorotrityl resin or hycram tm resin was used. in the case of cl-trityl resin, cyclization of the c-terminal dipeptide was avoided by sterical hindrancy at the resin. using the bocstrategy with the hycram tm resin prevents ring formation and thereby loss of loading. syntheses on wang resin provided very low yields. after removal from the resins all the peptides were purified by semipreparative hplc and chemically characterized. some of the analogues are prepared for photoaffinity labeling of the binding protein. the inhibitory activities of all analogues listed in table 1 were determined on isolated ace from guinea pig lungs. as most organs, particularly guinea pig ileum, contain different bk degrading enzymes, we did not use an organ homogenate to estimate the inhibition of bk degradation. we used the isolated ace to test the inhibition of hydrolysis of a synthetic tripeptide substrate by the potentiating peptides. as shown in table 1 , distinct differences exist between potentiation of the bk-induced contraction of the guinea pig ileum and the inhibition of the isolated ace. to quantify this difference we calculated a quotient from both activities. for the analogue [1-pro]-bpp 9␣ this quotient was accounted to 1. most distinct differences were found by nonapeptide analogues and partial sequences labeled with azidosalicylic acid (asa). some quotients reach 10 5 . thus the compound j526 shows about 160% potentiation compared to bpp 9␣ , but to enrich the same inhibition of ace as the 1-pro analogue a 10 5 higher concentration is required, resulting in a quotient of 10 5 . one of the earliest pharmacological tests used for bradykinin was the contraction of the isolated guinea pig ileum (gpi). in our test the contraction of gpi can be potentiated by bpp 9␣ and by the ace inhibitor ramiprilat as well. both potentiators enhance the isotonic measured contraction after 5 min preincubation (fig. 1) . the pd 2 -values for bk were shifted in the presence of 20 nm bpp 9␣ from 7.15 ± 0.11 (n = 8) to 7.84 ± 0.19 (n = 4) and by ramiprilat (20 nm) to 7.87 ± 0.22 (n = 4). both potentiators are also able to shift the cumulative gpi contraction curves for the enzymatically more stable bradykinin analogue darg-arg-pro-hyp-gly-thi-ser-pro-abu(ßph)-arg [16] to higher affinities. the pd 2 -value of this bk analogue was shifted by bpp 9␣ (20 nm) from 6.65 ± 0.14 (n = 6) to 7.15 ± 0.18 (n = 3) and by ramiprilat to 7.16 ± 0.15 (n = 3). to study the influence of bradykinin potentiating peptides on the bradykinin b 2 receptor cos-7 cells were transfected scheme 1. strategies for synthesis of bpp 9␣ analogues using different linkers and resins (wang resin, chlorotrityl resin, hycram tm resin) and different protecting group combinations. table 1 analogues of the bradykinin potentiating peptide bpp 9␣ (teprotide) with distinct differences between potentiation of the bk-induced contraction of the isolated guinea pig ileum (gpi) and inhibition of the isolated angiotensin i-converting enzyme (ace) aminoacid sequence potentiation (%) ace-inhibition: ic 50 with the gene of the human receptor. expression, chemical and functional characterization of the expressed receptor is described in a preceding publication [56] . radioligand binding studies with tritium labeled bradykinin [ 3 h]bk were performed on intact cells. fig. 2 lanthanium ions (la 3+ ) act as inhibitor of the uptake of ca 2+ from extracellular space. we used la 3+ to estimate the influence of bpp 9␣ on ca 2+ uptake. bk induces the contraction of the gpi only in the presence of extracellular calcium ions. the lowest extracellular concentration of ca 2+ necessary for a detectable contraction is 1.8 mm. in a ca 2+ containing tyrode buffer, bk induces the contraction of the isolated gpi, which can also be potentiated in the presence of 1.8 mm la 3+ (fig. 3) . bradykinin alone is unable to contract the gpi in the presence of la 3+ . the ileum contracts only after addition of bpp 9␣ , as shown in part a of fig. 3 . part b of this figure, reflecting the results in the ca 2+ free tyrode buffer, clearly underlines the important role of extracellular ca 2+ for the bk-induced smooth muscle contraction. part a also shows evidence that mobilization of intracellular ca 2+ is necessary for the potentiation by the peptides. bradykinin induces the formation of inositol phosphates in cos-7 cells transiently expressing the bk receptor. both analogues of the potentiating nonapeptide are not able to augment the bk-induced intracellular concentration of inositol phosphates. rather it seems that j527 reduces the bk-induced ip n formation. the most striking result from fig. 4 is the significant enhancement of the basal level by both potentiating peptides in the absence of bradykinin, possibly indicating a nonreceptor-mediated pathway. bradykinin triggers the release of [ 3 h]-labeled arachidonic acid from labeled phospholipids, presumably through activation of phospholipase a 2 by a g ␣ -protein. the level of arachidonic acid is significantly enhanced by bk as shown in fig. 5 . both potentiating peptides j526 and j527 slightly, but significantly, enhance the bk-mediated release of labeled arachidonic acid. without bk both peptides have no significant influence on the basal level. desensitization and resensitization are processes at the receptor level elicited by cytosolic receptor phosphorylation, followed by internalization or dephosphorylation, which is followed by reintegration into the cell membrane. dephosphorylation of cytosolic ser-and thr-residues of the bk receptor results from activated protein phosphatases. calyculin is known as a potent inhibitor of the protein phosphatases 1 and 2a. we used this inhibitor to check the involvement of these phosphatases in the potentiation of bradykinin action. as fig. 6 illustrates calyculin has no influence on the potentiation of the bk-induced contraction. application of calyculin, neither before nor after bk administration, changes the potentiating effects of bpp 9␣ or ramiprilat. bradykinin stimulates the migration of pmn corresponding to its concentration gradient. this effect could be characterized as true chemotaxis. these cells contain both types of bk receptors, bkr-b 1 and brk-b 2 , as demonstrated using bk agonists and antagonists in the migration assay [62] . they also contain the complete system for synthesis and release of bioactive kinins. degradation of bk proceeds in pmn mainly by the neutral endopeptidase nep (e.c. 3.4.24.11). the bk-induced accelerated migration of pmn can be potentiated after preincubation (5 min) of the cells with the nep inhibitor phosphoramidon (10 −7 m). the migration index (mi = 1.12 ± 0.03) of bk (10 −8 m) is significantly (p < 0.5; t-test for paired samples; n = 12) enhanced in the presence of phosphoramidon (mi = 1.19 ± 0.03) (not shown). the migratory capacity of bk for pmn can also be potentiated by the ace inhibitor ramiprilat. the migation index of bk (10 −7 m) without ramiprilat is mi = 1.30 ± 0.06, in the presence of ramiprilat (10 nm) mi = 1.69 ± 0.26, measured in six experiments (not shown). as shown in fig. 7 bpp 9␣ can enhance the migration of pmn induced as well by bk and by the enzymatically stable bk agonist d-arg-arg-pro-hyp-gly-thi-serpro-abu(␤ph)-arg [16] , but is unable to enhance the migratory activity of the nonpeptide agonist fr190997 [4] and the bkr-b 1 agonist desarg 9 -bk. bpp 9␣ (10 nm) is able to potentiate the dose-response curve of the bk-induced migration of the pmn (fig. 8a) . it potentiates the bk-induced effect in a concentration-dependent way (fig. 8b ). the potentiating action, despite many long years of experimental research and investigation, remains a phenomenon not fully understood. beginning with studies on the potentiation of smooth muscle contraction more than 30 years ago, the search has been extended to other organs such as the vascular system, to different bradykinin degrading enzymes, to immunocompetent cells, and in the last decade to molecular mechanisms at the level of the bradykinin receptors and signal pathways, primarily studied on cell cultures. other potentiating peptides and peptidomimetics besides the oligopeptides isolated from snake venoms have been used in these studies. generally, some of the contradictory findings and therefore explanations in the literature seem to result not only from the complexity of the system, but also from: the use of enzymatically not fully stable bradykinin agonists e.g. [hyp 3 ,tyr(me) 8 ]-bk [31, 53] , the species dependency of the bradykinin receptor, the very different protease compositions of the tissues and cell lines used in the different studies, and the different structural requirements for both catalytic centers of ace. furthermore, we have to keep in mind the different density of the bk receptors, their localization in microdomains in the plasma membrane, the presence of certain other hormone receptors, the different signal pathways in the used tissues and cells, a possible influence on g-protein independent signal transduction, on g-protein trafficking pattern in the cells and on activators or regulators of g-protein signaling [48] . additionally the potentiating effect can be differentiated into an unspecific and specific part. consequently, the search for potentiation of hormone actions is strongly related to the very recent research on multifunctional signal proteins. the isolation, chemical and functional characterization of oligopeptides taken from the venoms of different snakes provided lead structures for a number of potentiating peptides. long ago these oligopeptides gave some evidence of different structural requirements for potentiation of smooth mus-cle contraction and inhibition of ace. our synthesized analogues and fragments of the highly active nonapeptide bpp 9␣ show in some cases differences between potentiation and enzyme inhibition of more than five orders of magnitude. this result indicates strongly different structural requirements for both biological activities, and is inconsistent with the exclusive reduction of potentiating action to the inhibition of ace, as discussed by regoli and his coworkers [31] and dendorfer et al. [17] . we might draw this conclusion despite the different sources for the isolated organs and the ace. since the bk receptor is species dependent, the human bkr-b 2 shares about 80% sequence homology with that of the guinea pig [20] , whereas the ace seems to be nearly species independent. interestingly, the potentiating activity is much less influenced by amino acid replacements or modifications of the lead peptide bpp 9␣ than the inhibition of ace. from the analogues listed in table 1 , it is clearly evident that the potentiation of gpi contraction is less sensitive to amino acid replacements and modifications in the peptide sequence than the inhibition of ace. thus potentiating activities vary between 160% and 20%, whereas the ic 50 values for ace inhibition vary between 5 × 10 −10 and 7 × 10 −5 m. both activities show no correlation. the estimated values indicate that the potentiating activity allows beside amino acid replacements, a shortening of the sequence as well as substitutions of the n-terminus or side chains by aromatic moieties. the inhibitory activity seems to depend on the whole sequence. the replacement of the basic amino acid arg at position 4 by neutral aliphatic or aromatic amino acids influences the ace inhibition only slightly, on the other hand the acylation of the lys side chain at the same position by asa reduces the ic 50 value by four orders of magnitude. according to cotton et al. [11] position 4 seems to be crucial for inhibitory activity. but, our results did not provide clear structure activity relationships. discussing the potentiation of the bk-induced contraction of gpi we have to keep in mind that this tissue also contains other proteases than ace involved in bk degradation. but this finding is not able to explain the strong differences between both activities as expressed in the ratio in table 1 . it is reasonable to assume an interaction of the potentiating peptides with the bk receptor. for this interaction certain possible mechanisms exist. at first, the potentiating factors could act like allosteric effectors stabilizing the active receptor conformation. secondly, these factors could influence the phosphorylation and dephosphorylation of cytosolic parts of the receptor, resulting in desensitization and resenzitization of the receptor. a third possible mechanism consists in the influence of potentiating factors on receptor heterooligomerization. abdalla et al. [1] found that both the active high affinity angiotensin receptor at 1 and the bkr-b 2 are able to form heteromeric complexes in smooth muscle cells. most importantly, this heterodimerization evokes a signal en-hancement, a further possible useful explanation of the potentiating effect. for checking these possibilities we used cos-7 cells transiently transfected with the human bkr-b 2 . the displacement curves show no differences between the displacement of [ 3 h]-bk by bk alone nor in the presence of potentiating peptides. this finding clearly demonstrates that the analogues of bpp 9␣ used in this study do not act as allosteric effectors stabilizing the active receptor conformation. because cos-7 cells do not contain ace, a heterodimerization of the b 2 receptor with somatic ace as demonstrated by erdos and marcic [19] is impossible in our system. our radioligand binding curves show no influence of the potentiating peptides on the receptor capacity, indicating that under the test conditions the number of intramembranal receptors was not changed. this result excludes an internalization or reintegration of the receptor under the influence of potentiating peptides. but, we would not exclude these processes after heterodimerization with ace or with the angiotensin receptor. the contraction of smooth muscles requires calcium ions. consequently the potentiation of bk-induced contraction of gpi depends on extracellular and intracellular ca 2+ . in a variety of publications the enhancement of the intracellular ca 2+ -level is used as a qualitative or quantitative proof of the potentiating action. our results with the inhibition of ca 2+ -uptake from extracellular sources by la 3+ indicates that the potentiation of bk-action requires mobilization of ca 2+ from intracellular stores. on the other hand, in the beginning phase of the bk-evoked gpi contraction extracellular calcium is needed. thus, our results with la 3+ agree well with the finding of marcic et al. [49] , who described the inhibition of resensitization of the bk receptor in cho cells by la 3+ . the inositol phosphates (ip n ) formed in transiently with the b 2 receptor transfected cos-7 cells opens the ca 2+ channels of the endoplasmatic reticulum leading to an increase of cytosolic ca 2+ . but, both potentiating nonapeptides j526 and j527 are unable to enhance the bk-induced formation of ip n . surprisingly, both potentiating peptides increase the basal level of ip n in the absence of bk. possibly this may be due to an unspecific, nonreceptor-mediated part of the potentiating action. contrary to the absent effect of both potentiating peptides on the bk-induced enhancement of inositol phosphates, the bk-induced release of arachidonic acid is significantly increased. furthermore, contrary to the ip n formation, no influence of either potentiating peptides on the basal level could be found. we continue to be unable to explain the potentiation of arachidonic acid release because no effect on the receptor has been detected. the missing effect of calyculin on the potentiation might indicate that the inhibited phosphatases are not involved into resensitization. besides gpi and cos-7 cells the pmn represent our third system for studies about the potentiating mechanism. these cells constitutively express b 1 as well as b 2 receptors and have a cell specific set of proteases involved in bk degradation. in this system the potentiating nonapeptide bpp 9␣ and ramiprilat accelerate the bk-induced migration. in contrast to ignjatovic et al. [37] , who found on other cells a concentration-dependent direct activation of the b 1 receptor in the absence of kinins, we could not detect any activity of the potentiating nonapeptide without administration of b 1 or b 2 agonists. though the migratory activity of the proteolytic stabilized bk analogue j725 [16] is potentiated, the action of the nonpeptide agonist fr190997 [4] could not be accelerated by bpp 9␣ and ramiprilat. it is difficult to explain this finding. in agreement with the weaker acceleration of the stabilized bk analogue, the effect of the nep inhibitor phosphoramidon and the concentrationdependent potentiation induced by bpp 9␣ , we might suppose that in this test system the potentiation occurs possibly exclusively by inhibition of the enzymatic bk degradation. but, we also have to consider a different mechanism for the peptide and nonpeptide agonist. this assumption is supported in that test by the strongly reduced intrinsic activity of the nonpeptide agonist fr190997 compared to bk itself. during the last three decades many different and controversial explanations on the potentiating mechanism have been derived from experiments performed in vivo, in vitro and at the cellular level. the distinct and in some cases very significant differences between the inhibitory and potentiating activity of the synthesized bpp 9␣ analogues clearly indicate, in our opinion, that on guinea pig ileum this mechanism cannot be exclusively reduced to enzyme inhibition. the observed differences even exceed the different structural requirements for n-and c-terminal catalytic centers of ace. the experiments with la 3+ gave evidence that the potentiation of smooth muscle contraction requires extracellular ca 2+ which is taken up through opened channels. the increase of the intracellular level of inositol phosphates, triggered by potentiating peptides, leads to an additional enhancement of the intracellular ca 2+ concentration. application of potentiating peptides to cos-7 cells which were transiently transfected with the b 2 receptor and did not contain endogenous ace, shows no increase in receptor affinity or density. these results disprove the described suggestion of direct interaction of bpp with the bk receptor. the described receptor resensitization by potentiating peptides could result from dephosphorylation of the activated receptor. our results obtained with the phosphatase inhibitor calyculin disprove the influence of bpp 9␣ on the calyculin sensitive phosphatase. no difference between potentiation neither in the absence nor in the presence of the inhibitor could be observed. the release of arachidonic acid is one of the prerequisites for inflammatory processes. the potentiating peptides have no effect on the basal level but they significantly increase the bk-induced release in cos-7 cells. this result indicates the possible involvement of potentiating peptides in inflammatory processes without affecting ace. contrary to that mechanism the potentiation of the migratory activity of polymorphonuclear leucocytes seems to be nearly exclusively evoked by the ace inhibition. on these b 1 and b 2 receptor containing cells no direct interaction with the b 1 receptor could be observed. our results together with those from the literature may lead to the general conclusion that different potentiating factors can act on different organs, tissues and cells by different mechanisms. furthermore, bpp can evoke both receptor-mediated and nonreceptor-mediated intracellular reactions. in our opinion the contradictory explanations describing the potentiation mechanism result on the one hand from the very high complexity of the kallikrein-kinin and renin-angiotensin systems and on the other hand from the different in vivo and in vitro tests used. at1-receptor heterodimers show enhanced g-protein activation and altered receptor sequestration bradykinin b(2) receptor-mediated mitogen-activated protein kinase activation in cos-7 cells requires dual signaling via both protein kinase c pathway and epidermal growth factor receptor transactivation isolation and characterization of biological properties of inhibitors angiotensin-1-converting enzyme from the spider venom latrodectus tredecimguttatus pharmacological characterization of a nonpeptide bradykinin b 2 receptor antagonist, fr165649, and agonist fr190997 on the potentiation of kinins by sulfhydrylic compounds effect of a new angiotensin converting enzyme inhibitor mk 421 and its lysine analogue on the components of the renin system in healthy subjects bradykinin analogs as inhibitors of angiotensinconverting enzyme structure-function studies on the bradykinin potentiating peptide from chinese snake venom (agkistrodon halys pallas) primary structure and biological activity of bradykinin potentiating peptides from bothrops insularis snake venom potentiation of some bradykinin effects by thiol compounds selective inhibition of the c-domain of angiotensin i converting enzyme by bradykinin potentiating peptides design of potent competitive inhibitors of angiotensin-converting enzyme. carboxyalkanoyl and mercaptoalkanoyl amino acids inhibition of angiotensin-coverting enzyme by analogs of peptides from bothrops jararaca venom n-domain-specific substrate and c-domain inhibitors of angiotensin-converting enzyme: angiotensin-(1-7) and keto-ace neprilysin inhibitors potentiate effects of bradykinin on b2 receptor structural requirements for b 2 -agonists with improved degradation stability potentiation of kinin analogues by ramiprilat is exclusively related to their degradation a novel protein-protein interaction between a g protein-coupled receptor and the phosphatase shp-2 is involved in bradykinin-induced inhibition of cell proliferation kinins, receptors, kininases and inhibitors-where did they lead us? cloning, sequencing and functional expression of a guinea pig lung bradykinin b2 receptor peptide t, a novel bradykinin potentiator isolated from tityus serrulatus scorpion venom isolation and characterization of a bradykinin potentiating peptide (bpp-s) isolated from scaptocosa raptoria venom isolation: analysis and properties of three bradykinin-potentiating peptides (bpp-ii, bpp-iii, and bpp-v) from bothrops neuwiedi venom a new bradykinin-potentiating peptide (peptide p) isolated from the venom of bothrops jararacussu (jararacucu tapete, urutu dourado) structure and effects of a kinin potentiating fraction f (appf) isolated from agkistrodon piscivorus piscivorus venom a bradykinin-potentiating factor (bpf) present in the venom of bothrops jararaca isolation of bradykininpotentiating peptides from bothrops jararaca venom g protein-dependent activation of mast cell by peptides and basic secretagogues do angiotensin-converting enzyme inhibitors directly stimulate the kinin b1 receptor? dopamine receptor modulation by a highly rigid spiro bicyclic peptidomimetic of pro-leu-gly-nh 2 studies on the angiotensinconverting enzyme and the kinin b2 receptor in the rabbit jugular vein: modulation of contractile response to bradykinin combination of allyl protection and hycram tmlinker technology for the synthesis of peptides with problematical amino acids and sequences potentiation of the biological efficacy of bradykinin by ace inhibitors: a shift in the affinity of the b2 receptor? bradykinin potentiating peptides isolated from alpha-casein tryptic hydrolysate bradykinin-potentiating peptides and c-type natriuretic peptides from snake venom identification of five new bradykinin potentiating peptides (bpps) from bothrops jararaca crude venom by using electrospray ionization tandem mass spectrometry after a two-step liquid chromatography activation of bradykinin b1 receptor by ace inhibitors hemoglobin as a source of endogenous bioactive peptides: the concept of tissue-specific peptide pool bradykinin-potentiating peptides from the venom of agkistrodon halys blomhoffii bradykinin-potentiating peptides from the venom of agkistrodon halys blomhoffi. isolation of five bradykinin potentiators and the amino acid sequences of two of them, potentiators b and c synthesis of novel angiotensin converting enzyme inhibitor quinapril and related compounds. a divergence of structure-activity relationships for non-sulfhydryl and sulfhydryl types neutral endopeptidase 24.11: its physiologic and possibly pathophysiologic role in inflammation with special effect on respiratory inflammation insulin potentiates bradykinin-induced inositol 1,4,5-triphosphate in neonatal rat cardiomyocytes yukel'son ly. bradykinin-potentiating peptides from echis multisquamatus venom analysis of structural requirements for trh-potentiating peptide receptor binding by analogue design isolation and characterization of a new bradykinin potentiating octapeptide from gamma-casein angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus g protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts enhancement of bradykinin and resensitization of its b2 receptor replacement of the transmembrane anchor in angiotensin i-converting enzyme (ace) with a glycosylphosphatidylinositol tail affects activation of the b2 bradykinin receptor by ace inhibitors preparation and characterization of novel bioactive peptides responsible for angiotensin i-converting enzyme inhibition from wheat germ a bradykinin-potentiating peptide (peptide k12) isolated from the venom of egyptian scorpion buthus occitanus potentiation of the effects of bradykinin on its receptor in the isolated guinea pig ileum potentiation of the actions of bradykinin by angiotensin i-converting enzyme inhibitors. the role of expressed human bradykinin b2 receptors and angiotensin i-converting enzyme in cho cells liver bradykinin-inactivating-endopeptidase is similar to the metalloendopeptidase expression and functional characterization of a phistagged human bradykinin b2 receptor in cos-7 cells intramolecular signal transduction by the bradykinin b2 receptor metalloendopeptidases ec 3.4.24.15/16 regulate bradykinin activity in the cerebral microvasculature angiotensin-converting enzyme inhibitors from the venom of bothrops jararaca. isolation, elucidation of structure, and synthesis chronic effects of angiotensin-converting enzyme inhibition on kinin receptor binding sites in the rat spinal cord nachweis chemotaktischer faktoren. jena: gustav fischer verlag migratory responses of polymorphonuclear leukocytes to kinin peptides human aortic endothelial cell aminopeptidase n a new class of angiotensin-converting enzyme inhibitors potentiation of the hypotensive effect of bradykinin by angiotensin-(1-7)-related peptides isolation and characterization of a bradykinin-potentiating peptide from a bovine peptic hemoglobin hydrolysate aminopeptidase p is disposed on human endothelial cells synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues c-type natriuretic peptide (cnp) increases [ 125 i]anf binding to frtl-5 rat thyroid cells by increasing anf receptor affinity procedure for the determination of angiotensin converting enzyme angiotensin converting enzyme (ace) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies membrane anchoring and release of carboxypeptidase m: implications for extracellular hydrolysis of peptide hormones bradykinin-potentiating peptides from the spider latrodectus tredecimguttatus-inhibitors of carboxycathepsin and of a preparation of karakurt venom kininase werle foundation of the henning l. voigt family award ceremony synthesis of a highly active angiotensin converting enzyme inhibitor: 2 octane-3-carboxylic acid (hoe 498) bradykinin potentiation by angiotensin-(1-7) and ace inhibitors correlates with ace c-and n-domain blockade the mechanism of action of two bradykinin-potentiating peptides on isolated smooth muscle structure-activity relationships of bradykinin potentiating peptides the two homologous domains of human angiotensin i-converting enzyme are both catalytically active the thiol enzyme from rat spleen that produces bradykinin potentiating peptide from rat plasma opioid peptides derived from hemoglobin: hemorphins we thank i. agricola and h. rohde for skilful technical assistance. this work was supported by the deutsche forschungsgemeinschaft (re 853/2-1 and collaborative research center 436, jena). key: cord-318327-9sh2eksm authors: garg, m.; angus, p. w.; burrell, l. m.; herath, c.; gibson, p. r.; lubel, j. s. title: review article: the pathophysiological roles of the renin–angiotensin system in the gastrointestinal tract date: 2012-01-05 journal: aliment pharmacol ther doi: 10.1111/j.1365-2036.2011.04971.x sha: doc_id: 318327 cord_uid: 9sh2eksm background: the renin‐angiotensin system (ras) is a homeostatic pathway widely known to regulate cardiovascular and renal physiology; however, little is known about its influence in gastrointestinal tissues. aim: to elicit the anatomical distribution and physiological significance of the components of the ras in the gastrointestinal tract. methods: an extensive online literature review including pubmed and medline. results: there is evidence for ras involvement in gastrointestinal physiology and pathophysiology, with all the components required for autonomous regulation identified throughout the gastrointestinal tract. the ras is implicated in the regulation of glucose, amino acid, fluid and electrolyte absorption and secretion, motility, inflammation, blood flow and possibly malignant disease within the gastrointestinal tract. animal studies investigating the effects of ras blockade in a range of conditions including inflammatory bowel disease, functional gut disorders, gastrointestinal malignancy and even intestinal ischaemia have been encouraging to date. given the ready availability of drugs that modify the ras and their excellent safety profile, an opportunity exists for investigation of their possible therapeutic role in a variety of human gastrointestinal diseases. conclusions: the gastrointestinal renin‐angiotensin system appears to be intricately involved in a number of physiological processes, and provides a possible target for novel investigative and therapeutic approaches. the renin-angiotensin system (ras) is a homeostatic pathway widely known to regulate cardiovascular and renal physiology; however, little is known about its influence in gastrointestinal tissues. to elicit the anatomical distribution and physiological significance of the components of the ras in the gastrointestinal tract. an extensive online literature review including pubmed and medline. there is evidence for ras involvement in gastrointestinal physiology and pathophysiology, with all the components required for autonomous regulation identified throughout the gastrointestinal tract. the ras is implicated in the regulation of glucose, amino acid, fluid and electrolyte absorption and secretion, motility, inflammation, blood flow and possibly malignant disease within the gastrointestinal tract. animal studies investigating the effects of ras blockade in a range of conditions including inflammatory bowel disease, functional gut disorders, gastrointestinal malignancy and even intestinal ischaemia have been encouraging to date. given the ready availability of drugs that modify the ras and their excellent safety profile, an opportunity exists for investigation of their possible therapeutic role in a variety of human gastrointestinal diseases. the gastrointestinal renin-angiotensin system appears to be intricately involved in a number of physiological processes, and provides a possible target for novel investigative and therapeutic approaches. the renin-angiotensin system (ras) plays a central role in regulating cardiovascular and renal physiology. the contemporary view of the ras has evolved from that of a simple linear pathway involving the conversion of angiotensinogen to angiotensin ii (ang ii) via a two-step process facilitated by renin and angiotensin converting enzyme (ace), to a much more complex system involving homologues of ace and multiple angiotensin peptides which play supplementary and counter-regulatory roles ( figure 1 ). the ras was, for many years, thought of as an endocrine system with enzymes and peptides released into the systemic circulation to act on target organs. more recently, it has been recognised that most organs including the brain, kidney, heart, liver, pancreas, reproductive organs, skin and the gastrointestinal tract constitutively express all the components required to allow autonomous function of a local intra-organ ras, where it performs both paracrine and autocrine functions. table 1 summarises the current view of the ras, the key components and their physiological and clinical effects. essentially, the relative activity of two counterbalancing pathways determines the predominant tissue effect. the proinflammatory, profibrotic pathway includes the classical ras components ace and ang ii, and renin, prorenin, chymase and neutral endopeptidase (nep, also known as neprilysin). renin, a glycoprotein derived predominantly from the juxtaglomerular apparatus in the kidney, is an aspartyl protease that cleaves the liver-derived angiotensinogen to angiotensin i. both renin and its proenzyme prorenin, which was previously considered physiologically inactive, 1 have now been demonstrated to have independent pro-inflammatory and pro-fibrotic effects via signalling through the pro(renin) receptor (prr). 2 the classical ras comprising the zinc metalloproteinase ace and ang ii induces vasoconstriction, salt and figure 1 | the contemporary renin-angiotensin system (ras). ace, angiotensin converting enzyme; nep, neutral endopeptidase; am, aminopeptidase; at1r, angiotensin type 1 receptor; at2r, angiotensin type 2 receptor; at4r, angiotensin type 4 receptor; prr, (pro)renin receptor. water retention, thirst response, cardiac hypertrophy, tissue inflammation and fibrosis through the g-protein coupled seven-transmembrane domain receptor angiotensin type i receptor (at1r). ang ii also stimulates adrenal gland secretion of aldosterone resulting in renal sodium and water retention. inhibition of this pathway with either ace inhibitors or at1r antagonists has beneficial effects in hypertension, cardiac failure, ischaemic heart disease, diabetic nephropathy and renal fibrosis. chymase expressed in the heart and vascular wall and secreted by activated mast cells, acts as an alternative enzyme to ace to generate ang ii from ang i. 3-6 nep, a membrane bound zinc metalloproteinase with a structure distinct from ace, was discovered in the 1970s as a key enzyme involved in the cleavage of bradykinin. 7, 8 in recent years, it has been shown to also have a role in the formation of ang (1-7) from ang i, as an inactivator of atrial natriuretic peptide 9 and in the degradation of amyloid b peptide, 10 a protein involved in the pathogenesis of alzheimer's disease. the net effect of nep inhibition is vasodilatation and natriuresis, a property encompassed by vasopeptidase inhibitors that target both ace and nep and may have additional anti-hypertensive effects to ace inhibitors. 11 in contrast, the alternative ras, comprising ace2 and ang (1-7), acting via the g-protein coupled seventransmembrane receptor mas, 12, 13 has vasodilatory, antihypertensive, anti-thrombotic, cardioprotective, antiinflammatory and anti-fibrotic effects in a variety of tissues. 14-18 ace2 is a zinc metalloproteinase and homologue of ace, which cleaves a single amino acid from ang ii to form the heptapeptide ang (1-7). indeed, part of the clinical benefit attributed to ace inhibitors and at1r blockers (arbs) may be through the diversion of the classical ras components towards ang (1-7) with subsequent mas receptor activation. [18] [19] [20] the complexity of the ras is further highlighted by recent findings regarding the actions of other angiotensin peptides including angiotensin iii [ang iii, also denoted as ang (2) (3) (4) (5) (6) (7) (8) ], angiotensin iv [ang iv, also known as ang (3) (4) (5) (6) (7) (8) ] and the at2 and at4 receptors. ang iii is formed by cleavage of ang ii by aminopeptidase a, and ang iv results from further conversion by aminopeptidase b or n ( figure 1 ). the at2 receptor (at2r) has affinity for ang ii, ang iii, ang iv and ang (1) (2) (3) (4) (5) (6) (7) , and is also thought to have effects counteracting the at1r and analogous to those of the mas receptor, with vasodilatory, anti-inflammatory and anti-proliferative downstream actions. previously recognised largely for an important role in foetal development, more recently, the at2r has been shown to be upregulated in atherosclerotic disease, 21 cutaneous wounds 22 and pancreatic fibrosis, 23 and to stimulate neurite outgrowth, a marker of neuronal regeneration. 24, 25 ang iii is believed to have actions analogous to ang ii. ang iv appears to have opposing effects to ang ii, and acts predominantly via the at2r and the at4r, formerly known as insulin regulated aminopeptidase (irap). 26 the greatest role of ang iv is in the central nervous system (cns), where it has a positive effect on neuronal development, learning and memory. 27, 28 concept of local ras there is considerable evidence that most or all of the components of the ras are present in a variety of organs, supporting the theory that local expression and modulation of the ras play important roles in tissue homeostasis. these roles may be summarised as involving (1) fluid and electrolyte transport, (2) regional blood flow regulation and (3) promoting the wound healing response, including cell proliferation, inflammation and fibrosis. some of the regional effects of the ras are listed below: (i) in the kidney, local angiotensinogen is converted by renin to ang i, which in turn is cleaved by tubular brush border ace to ang ii to facilitate sodium and fluid absorption via luminal at1r. this may influence blood pressure independent of systemic ang ii levels and vascular tone. 29, 30 (ii) the heart expresses renin, prr, ace, chymase, angiotensinogen, at1r and at2r, and these components modulate myocyte proliferation and cardiac remodelling. 6, [31] [32] [33] [34] [35] [36] [37] (iii) the brain has been shown to express renin, angiotensinogen, ang ii, ang iii, ang iv, ang (1-7), at1r, at2r and at4r, with these components regulating blood pressure, fluid and electrolyte balance, thirst, maintenance of the blood-brain barrier and neuronal development including learning and memory processes. [38] [39] [40] [41] [42] [43] [44] (iv) the liver expresses renin, angiotensinogen, ang ii, ace, at1r, ang (1-7), ace2 and mas receptors, all of which are upregulated in the diseased liver. 45, 46 furthermore, arbs and ang (1-7) have been demonstrated to reduce liver fibrosis in animal models. [47] [48] [49] (v) in the pancreas, ang ii has been shown to inhibit glucose stimulated insulin secretion, and via at1r and at2r, regulates exocrine enzyme secretion and the microcirculation. [50] [51] [52] (vi) a local ras has been identified and shown to be involved in tissue homeostasis in the reproductive organs, skin and even adipose tissue. [53] [54] [55] [56] [57] [58] detailed reviews of these and other local ras effects are published elsewhere. 12, 30, 50, 54, [59] [60] [61] localisation and functionality of the ras in the gastrointestinal tract our understanding of the involvement of the ras in the gastrointestinal tract has gradually evolved over the past five decades since the formulation of the hypothesis that ang ii had a direct effect on intestinal smooth muscle in addition to an indirect effect via myenteric plexus cholinergic neurons. [62] [63] [64] since then, many of the components of the ras have been identified throughout the gastrointestinal tract. an overview of the current state of knowledge is illustrated in figure 2 . the regions of the gut are addressed separately here. most attention has been paid to the small intestine (figure 2a) , as outlined below: (i) ace, ace2 and neutral endopeptidase: ace has been shown in humans to be located in abundance on the brush border of epithelial cells and in the mesenteric microvascular endothelium. 65 ace2 mrna and protein is present in large amounts in small intestinal epithelial brush border, muscularis mucosa and muscularis propria, as well as microvascular endothelium and vascular smooth muscle cells. 66 remarkably, the highest tissue concentrations in the human body of ace and ace2 mrna are found in the terminal ileum, duodenum and colon. 67, 68 expression of nep has been demonstrated in the rat intestinal wall, and is suppressible by administration of the combined ace/nep inhibitor omapatrilat. 69 (ii) angiotensin receptors: at1r has been localised to the epithelial brush border. 70 the circular and longitudinal muscle layers and the myenteric plexus also strongly express at1r, but the at2r appears to be largely restricted to the myenteric plexus. 71, 72 small vessels in the muscularis propria also express at1r. 71, 72 in early studies in the rat intestine, ang ii binding sites were reported to be confined to the muscularis, 73 but subsequent reports have identified expression of at1r and a lesser amount of at2r in the muscularis mucosa and mucosa, including in epithelial cells. 74, 75 (iii) renin: mrna for renin has been detected in the human small intestine. 76 (iv) angiotensin peptides: ang ii has been detected in the crypt and crypt-villus junction epithelial cells. 70 to date, the expression of angiotensinogen, ang i or ang (1) (2) (3) (4) (5) (6) (7) in the human small intestine has not been reported. however, angiotensinogen has been widely localised in the rat brush border, epithelial cells, lamina propria, muscularis mucosa, submucosal blood vessels and muscularis propria. 77 angiotensinogen mrna has been isolated in concentrations of over one-third that of the liver in the rat mesentery, 78 and a high level of proangiotensin-12, a precursor of ang i, has been located in the rat intestine. 79 thus, all of the required components for local production and action of ang ii appear to be present in small intestine. there is now evidence of important roles for the ras in a variety of intestinal processes: (i) bicarbonate secretion: this is stimulated by ang ii via at1r and at2r in the duodenum. 80 (ii) sodium and water absorption: in the jejunum and ileum, this process appears to be modulated by ang ii in conjunction with the enteric sympathetic nervous system. [81] [82] [83] when applied in low dose to rat jejunum, ang ii stimulates sodium and water absorption through at2r, but in high dose, it unexpectedly inhibits absorption through at1r. 84 both ang ii and ang iii may also increase sodium and water absorption via stimulation of release of noradrenaline from sympathetic neurons, which in turn may act through adrenergic receptors on the basal surface of epithelial cells. 83, 85, 86 (iii) glucose absorption: ang ii has also been shown to inhibit rat jejunal sodium-dependent glucose transporter (sglt1)-mediated glucose uptake in vitro. 77 (iv) digestion and absorption of peptides: both brush border ace and ace2 are thought to function as peptidases, allowing for mucosal digestion and absorption of peptides. 87, 88 ace2 increases the activity of the neutral amino acid transporter b 0 at1, which is mutated in a rare amino acid deficiency disorder, hartnup disorder, clinically manifested by cerebellar ataxia and pellagra-like skin rash. 89 (v) secretion: a role for ace2 in active secretion has been suggested by the observation that ace2 is the target for the coronavirus mediating severe acute respiratory syndrome, sars-cov. some patients with this infection suffer from watery diarrhoea, but the exact mechanism remains to be determined. 66 there have been limited studies of ras components in the colonic wall (figure 2b) . by a combination of rt-pcr and immunohistochemistry, renin was found in the surface epithelium, lamina propria mesenchymal cells, microvascular walls and muscularis mucosa. at1r was detected on surface epithelial cells and in crypt bases, lamina propria macrophages, myofibroblasts and mucosal vessel walls, and weak expression of at2r has been found on surface epithelium, in crypts and in some mesenchymal cells. 90 ace was also weakly expressed in parts of the surface epithelium, and more prominently in mesenteric microvascular walls, lamina propria and submucosal mesenchymal cells. 90 ace2 appears to be localised to the mesenteric microvascular endothelium in the colon and is not present in the epithelium. 66 angiotensinogen mrna has also been isolated in homogenised rat colon, 78 but its expression has not been examined in the human colon. a more limited range of functional roles has been attributed to components of the ras in the colon than in small intestine. ang ii has been shown to increase sodium and water reabsorption in rats through nacl coupled transport. 91 the response of circular and longitudinal muscle contraction to ang ii also suggests a role in normal colonic motility. 92 as detailed below, the ras may also be involved in the inflammation associated with ibd, as mucosal levels of ang i and ang ii are higher in patients with active crohn's colitis compared with normal controls and patients with ulcerative colitis. 93 components of the ras are present in the mucosal biopsy specimens of gastric antrum and body from healthy adults (figure 2c ). 94 renin and angiotensinogen were both seen in lamina propria mesenchymal cells and vascular endothelial cells. at1r and at2r were both observed in gastric epithelium (mainly in the basal surface), lamina propria mesenchymal cells and vascular endothelium. at1r were noted in a subgroup of endocrine cells in the base of antral mucosal glands, and ace and nep in vascular endothelial cells, but not in other parts of the mucosa. other investigators, however, have noted ace in fundic chief cells and mucin secreting cells of the antrum. 95 longitudinal and circular muscle of the stomach has been demonstrated to respond in vitro to ang ii, suggesting the presence of appropriate receptors on gastric myocytes. 96 to date, few functional or pathogenic roles have been attributed to the ras in the stomach. a role of local ras in gastric inflammation has been suggested by higher expression of at1r expression in helicobacter pylori positive than h. pylori negative patients 94 and the potentiation of ulceration in animal models by ang ii. 97, 98 oesophagus immunoreactive ace, at1r and at2r have been found in the lamina propria microvascular walls, and at1r and at2r were identified in the superficial stratified epithelium 99 and circular and longitudinal muscle of the oesophagus 100 (figure 2d ). ang ii caused contraction of isolated oesophageal smooth muscle in vitro, and the at1r antagonist candesartan inhibited swallow-induced peristaltic contractions in the distal oesophagus. 100 the expression of other ras components has not been reported. the presence of the various components of the ras in the gastrointestinal tract raise the possibility that modification of this system locally may be a potential therapeutic target in a myriad of gastrointestinal diseases where current strategies are suboptimal. these include inflammatory bowel disease (ibd), gastrointestinal cancer, gut motility disorders and mesenteric ischaemia. although clinical data are sparse, results from animal models and pre-clinical studies provide support for further investigation. information relevant to ibd has arisen from studies in crohn's disease (cd) and ulcerative colitis (uc) and animal models of both ibd and other chronic inflammatory conditions. studies in patients with crohn's disease and ulcerative colitis. two components of the ras have been studied. the first is ace, which was subject to intense interest from the 1980's in its role in sarcoidosis and other granulomatous conditions. studies of serum ace concentrations yielded largely conflicting findings in ibd, with many studies showing reduced levels [101] [102] [103] [104] and some finding no difference. [105] [106] [107] many of these studies have been limited by relatively small numbers of patients. serum ace levels are associated with ace gene insertion/deletion (i/d) polymorphisms, with higher levels seen with the dd polymorphism than id or ii. 108 matsuda et al. showed that the ace gene polymorphism variation was similar in 39 patients with cd and 43 patients with uc to 341 controls, but that serum ace levels were lower in patients with ibd after adjusting for polymorphisms. 101 furthermore, they demonstrated that ace levels significantly increased in all of nine patients with active cd when they achieved clinical remission. a larger study involving 124 uc patients and 108 cd patients also found no difference in ace gene polymorphisms when compared with normal controls, but a subgroup analysis revealed a higher proportion of dd genotype in 25 uc patients with extra-intestinal manifestations, with an odds ratio (or) of 4.08. 109 indeed, it is difficult to reconcile these findings into a unifying hypothesis regarding the role of ace in ibd pathogenesis. it is possible that inflammatory cytokines such as tnf-a and il-1 downregulate systemic endothelial ace production, 110 whereas ang ii produced via local intestinal ace contributes to tissue inflammation. tissue ace levels have not been measured in active ibd. the other major area of investigation has focussed on angiotensin peptide levels in cd. genotype analyses have revealed a significant association of angiotensinogen-6 aa genotype (or 2.38) in a cohort of 235 patients with ibd. this genotype results in increased production of angiotensinogen via a substitution in its gene promoter. 111 indeed, mucosal levels of ang i and ang ii are elevated in rectosigmoid biopsies in patients with crohn's colitis compared with patients with uc or normal controls, 93 and a significant correlation was noted between these levels and endoscopic grade of colitis. although these findings demonstrate that components of the local tissue ras probably play a part in inflammation, they do not prove any causal link in the pathogenesis of ibd. nonetheless, they suggest that inhibition of the local ras provides a potential avenue for targeting inflammation and fibrosis. studies in animal models. angiotensin converting enzyme inhibition or angiotensin receptor antagonism have been shown to produce a number of beneficial anti-inflammatory effects in rodent models of intestinal inflammation. ang ii and at1a receptor expression are both upregulated in dextran sodium sulphate (dss)induced colitis, a widely studied mouse model of colitis, and inflammation was significantly ameliorated in at1a receptor-deficient mice. 112 the ace inhibitor enalaprilat, given parenterally, reduced inflammation and tnf-a, 113 and topical enalaprilat reduced tgf-b expression and fibrosis in mice with dss-colitis. 114 administration of the arb, valsartan, significantly reduced macroscopic inflammation, tnf-a, tgf-b and il-18 in mice with trinitrobenzene sulphonic acid (tnbs) induced colitis, reduced microscopic inflammation and raised il-10 (an anti-inflammatory cytokine secreted by regulatory t cells) in dss-colitis. 115, 116 the ace inhibitor captopril reduced macroscopic and microscopic inflammation, fibrosis and tgfb mrna expression in mice with tnbs-induced colitis. 117 homozygous deficiency of angiotensinogen protects against tnbs-induced colitis, with reduced il1-b, ifn-c and greater il-4 and il-10 production than wild-type mice. 116 animal studies have not been restricted to the classical ras pathway. nep knockout mice have been shown to have more severe colitis in response to dinitrobenzene sulphonic acid than wild-type mice, which is prevented by the administration of recombinant nep. 118 other evidence to suggest that ras dysfunction may potentiate immune-based diseases such as ibd and provide a target for therapy comes from recent studies involving models for multiple sclerosis (ms). t helper type 1 (th1) and type 17 (th17) cells are strongly implicated in the pathogenesis of ibd, especially cd, and lisinopril and candesartan have been demonstrated to suppress th1 and th17 cytokine expression and induce foxp3+ regulatory t cells in experimental autoimmune encephalitis (eae), a mouse model of ms. 119 other groups have shown a crucial role for ang ii and at1r in eae 59, 120 and murine autoimmune nephritis. 121 the possible involvement of perturbed ras components in solid organ malignancies represents a fascinating expansion of our insight into local tissue ras. early epidemiological studies demonstrated a reduced risk of incident and fatal cancer in patients on ace inhibitors versus those on other anti-hypertensive medication. 122, 123 in contrast, a recent meta-analysis reported a higher risk of lung cancer in patients taking arbs 124 ; however, two large meta-analyses since then have reported no increased risk. 125, 126 all these studies have been limited by their retrospective design. increasing in vitro and pre-clinical data suggest a protective effect of at1r inhibition against cancer cell proliferation, invasion and metastasis in a variety of solid organs. 127 in gastric cancer, at1r and ang ii are expressed to a greater extent than in adjacent normal tissue. 128 the at1r and ace gene polymorphism d allele increase risk of nodal metastasis and tumour stage in patients with intestinal-type gastric cancer. 129 in gastric cancer cell cultures, ang ii stimulates map kinase, nfjb and survivin activation, increasing proliferation. 128 the most likely mechanism by which the ras may influence cancer biology is through increasing angiogenesis, 130, 131 via increased expression of vascular endothelial growth factor (vefg) signalling. [130] [131] [132] in mouse models, ace inhibition and arbs reduced colorectal cancer liver metastases 133 and prolonged survival in peritoneal carcinomatosis. 128 indeed, ace inhibitor use independently protected against distal metastasis in a single centre retrospective review of patients with colorectal carcinoma. 134 also, patients treated with ace inhibitors had a non-significant trend towards reduction in risk of oesophageal adenocarcinoma in a retrospective study of the uk general practice research database. 135 recently, there have been intriguing insights into the involvement of the ras in peutz-jeghers syndrome (pjs). shorning and colleagues have shown that in mice lkb1 gene deletion, which is associated with pjs, results in marked transcriptional upregulation of the renin gene ren1, and also increased ace expression and ang ii production. 70 in human pjs tumour tissue, at1r was noted to be increased in stromal tissue, but reduced in the epithelium. 70 gut motility disorders the functional role of the ras in smooth muscle contraction suggests that it might be a target in motility dysfunction. for example, the ability of ang ii blockade to inhibit contraction of oesophageal body and lower oesophageal sphincter (les) smooth muscle, together with the demonstrated reduction in the amplitude of contraction of primary peristaltic oesophageal waves and les on manometry in vivo, 100 suggests a possible role in treatment of hypercontractile oesophageal disorders such as diffuse oesophageal spasm, nutcracker oesophagus and achalasia. furthermore, selective at1r-mediated contraction of the les may be an option for treatment of gastro-oesophageal reflux disease, a condition that affects up to 15-20% of the population. 136 the contribution of at1r to small and large intestinal muscle contractility also provides an opportunity to intervene in functional intestinal and motility disorders through at1r agonism or blockade. furthermore, the role of ace and ace2 in intestinal fluid and electrolyte absorption suggests a potential mechanism for modulating fluid shifts across the brush border, with subsequent effects on stool consistency and frequency. the ras plays an important role in regulation of the smooth muscle tone of the mesenteric vasculature. in acute hypovolaemia and systemic sepsis, splanchnic vasoconstriction occurs as a homeostatic response to preserve cerebral and renal blood flow, predisposing the gut to ischaemia. this splanchnic response has been shown to correlate with a markedly increased expression of ang ii. 137 furthermore, lower-body negative pressure induction in normal human volunteers has also been shown to raise serum ang ii and reduce intestinal mucosal nitric oxide production. 138 the administration of candesartan maintained jejunal and mucosal perfusion during severe hypovolaemia in pigs and reduced mortality. 139, 140 a further porcine study reported improved mucosal oxygen delivery, but not an improvement in intestinal mucosal acidosis in pigs administered candesartan during endotoxic shock. 141 these results were replicated by tardos et al. in pigs with burn and endotoxin induced gut ischaemia. in this model, intestinal permeability and bacterial translocation were reduced with the administration of the ang ii inhibitor dup753. 142 the obvious limiting factor in applying ang ii blockade in humans at risk of mesenteric ischaemia is the potential for current arbs and ace inhibitors to cause further hypotension and kidney injury, although no adverse renal consequences were noted in one porcine study. 139 manipulation of local gastrointestinal tract ras -potential targets and limitations as a ubiquitous system with a wide array of homeostatic roles, investigation into therapies that manipulate the ras has been extensive. apart from the well-established roles of at1 receptor blockade and ace inhibition in the treatment of hypertension, cardiovascular and kidney disease, new drugs targeting the mas receptor, at2 receptor, at4r, renin and nep, as well as a recombinant ang (1-7), are under trial for various applications ( table 2) . there is limited knowledge of the effect of currently available ace inhibitors and arbs on gastrointestinal function at doses employed for cardiovascular and renal effect at a molecular level. most of these drugs may result in adverse effects including nausea, diarrhoea or constipation in an idiosyncratic manner in between 1 and 3 per cent of patients, often noted in similar numbers of patients in the placebo arm in randomised con-trolled trials. it is likely that gastrointestinal tissue concentrations achieved by these medications at current doses are insufficient to note clinical effect. furthermore, the effect of these drugs in pathological states, like functional gut disorders or ibd, has not been described. for applicability to human gastrointestinal disease, drugs manipulating the ras will need to target the relevant areas of the gut to obtain satisfactory tissue effect without systemic adverse reactions. ideally, this will comprise delivery to the mucosa, absorption and binding of cellular receptors and sufficient first pass hepatic conversion to inactive metabolites to limit systemic effect. there is now a significant body of literature demonstrating the existence and pathophysiological relevance of local tissue renin-angiotensin systems. given the current evidence of involvement of the ras in gastrointestinal fluid and electrolyte homeostasis, smooth muscle control, inflammation and malignancy, it follows that manipulation of this system could be of benefit in a range of gi pathologies. therapies targeting the gastrointestinal ras are attractive, given their excellent safety and tolerability profile and confirmed benefits in other organs and diseases. it is hoped that the recent emergence of further experimental evidence supporting a role for the local ras in intestinal disorders will provide greater impetus for the 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candesartan in the porcine splanchnic and renal circulation specific angiotensin ii receptor blockage improves intestinal perfusion during graded hypovolemia in pigs angiotensin ii receptor antagonism increases gut oxygen delivery but fails to improve intestinal mucosal acidosis in porcine endotoxin shock angiotensin ii inhibitor dup753 attenuates burnand endotoxin-induced gut ischemia, lipid peroxidation, mucosal permeability, and bacterial translocation increased plasma inactive renin in diabetes mellitus. a marker of microvascular complications angiotensin ii stimulates interleukin-6 release from cultured mouse mesangial cells signal transduction mechanisms of the angiotensin ii type at-receptor: looking beyond the heterotrimeric g protein paradigm potential roles of angiotensin receptor-activating autoantibody in the pathophysiology of preeclampsia angiotensin ii regulates the synthesis of proinflammatory cytokines and chemokines in the kidney angiotensin receptor blockers and angiogenesis: clinical and experimental evidence recent advances in the angiotensin-converting enzyme 2-angiotensin(1-7)-mas axis angiotensin iii increases mcp-1 and activates nf-kappab and ap-1 in cultured mesangial and mononuclear cells angiotensin iii stimulates aldosterone secretion from adrenal gland partially via angiotensin ii type 2 receptor but not angiotensin ii type 1 receptor stimulation of different subtypes of angiotensin ii receptors, at1 and at2 receptors, regulates stat activation by negative crosstalk direct angiotensin ii type 2 receptor stimulation acts anti-inflammatory through epoxyeicosatrienoic acid and inhibition of nuclear factor kappab angiotensin ii stimulates thick ascending limb no production via at receptors and akt1-dependent nitric-oxide synthase 3 (nos3) activation the angiotensin ii at2 receptor is an at1 receptor antagonist new approaches to blockade of the reninangiotensin-aldosterone system: chymase as an important target to prevent organ damage design, synthesis, and biological evaluation of the first selective nonpeptide at2 receptor agonist development of selective non-peptide angiotensin ii type 2 receptor agonists prophylactic and therapeutic treatments with at 1 and at 2 receptor antagonists and their effects on changes in the severity of pancreatitis protective effect of candesartan in experimental ischemic stroke in the rat mediated by at2 and at4 receptors beneficial effects of angiotensin (1-7) in diabetic rats with cardiomyopathy the angiotensin-(1-7)/mas receptor axis is expressed in sinoatrial node cells of rats impairment of cardiac function and remodeling induced by myocardial infarction in rats are attenuated by the nonpeptide angiotensin-(1-7) analog ave 0991 anti-inflammatory effects of the activation of the angiotensin-(1-7) receptor, mas, in experimental models of arthritis the nonpeptide ave0991 attenuates myocardial hypertrophy as induced by angiotensin ii through downregulation of transforming growth factor-beta1/smad2 expression ave 0991-angiotensin-(1-7) receptor agonist, inhibits atherogenesis in apoeknockout mice angiotensin-converting enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction recombinant human angiotensinconverting enzyme 2 as a new reninangiotensin system peptidase for heart failure therapy effects of ace2 inhibition in the post-myocardial infarction heart comparison of vasopeptidase inhibitor, omapatrilat, and lisinopril on exercise tolerance and morbidity in patients with heart failure: impress randomised trial comparison of omapatrilat and enalapril in patients with chronic heart failure: the omapatrilat versus enalapril randomized trial of utility in reducing events (overture) blood-pressure reduction with lcz696, a novel dual-acting inhibitor of the angiotensin ii receptor and neprilysin: a randomised, doubleblind, placebo-controlled, active comparator study inhibition of diabetic nephropathy by a decoy peptide corresponding to the "handle" region for nonproteolytic activation of prorenin involvement of receptor-bound prorenin in development of nephropathy in diabetic db/db mice prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type 1a receptordeficient mice prorenin receptor blockers: effects on cardiovascular complications of diabetes and hypertension pathologic roles of prorenin and (pro)renin receptor in the eye role of nonproteolytically activated prorenin in pathologic, but not physiologic, retinal neovascularization suppression of ocular inflammation in endotoxin-induced uveitis by inhibiting nonproteolytic activation of prorenin key: cord-302316-raf5rlkq authors: brüssow, harald title: covid‐19: from pathogenesis models to the first drug trials date: 2020-06-23 journal: microb biotechnol doi: 10.1111/1751-7915.13611 sha: doc_id: 302316 cord_uid: raf5rlkq the number of people infected with sars‐cov‐2, and sadly dying from covid‐19, has exploded, and so the amount of literature on the novel coronavirus and the disease it causes has increased proportionately. the case numbers in some countries are beyond the epidemic peak, but the uncertainty about a second wave keeps politicians and societies under pressure. appropriate decision‐making and winning support from the population depends on precise scientific information rather than leaving the field to scaremongers of all proveniences. this mini‐review is an update of earlier reports (brüssow, microb biotechnol 2020a;13:607; brüssow, microb biotechnol 2020b; https://doi.org/10.1111/1751-7915.13592). the number of people infected with sars-cov-2, and sadly dying from covid-19, has exploded, and so the amount of literature on the novel coronavirus and the disease it causes has increased proportionately. the case numbers in some countries are beyond the epidemic peak, but the uncertainty about a second wave keeps politicians and societies under pressure. appropriate decision-making and winning support from the population depends on precise scientific information rather than leaving the field to scaremongers of all proveniences. this mini-review is an update of earlier reports (br€ ussow, microb biotechnol 2020a; 13:607; br€ ussow, microb biotechnol 2020b; https://doi.org/10.1111 /1751 ). autopsies can provide valuable insight into new diseases which complement or correct observations made by clinicians. surprisingly, it took a while until the first pathology reports were published for covid-19 patients. this delay is explained by the emergency situation of a pandemic, the limited number of complete sections that can be conducted by a pathology institute, biosafety issues and a decreasing role of pathology in medical research. in a us covid-19 patient, the lungs were of firm consistency and heavy with oedema. upon histological analysis, diffuse alveolar damage (dad) was diagnosed. thrombi were seen in small lung arteries, and there was congestion in capillaries. alveoli showed hyaline membrane formation. the lung tissue displayed chronic inflammation with invasion of t lymphocytes. the patient died of cardiac arrest (barton et al., 2020) . a chinese covid-19 patient showed, likewise: dad, hyaline membranes and pulmonary oedema together with inflammatory infiltrates and multinuclear syncytia, typical cytopathic effects of coronaviruses. the patient showed overactive t cells with a marked increase in th17 cells and high cytotoxicity of cd8 t cells, potentially explaining the severe immune injury seen in the lung of this patient . the first larger series of autopsies was published for 12 german covid-19 patients. their average age was 73 years, and most were obese. deep venous thrombosis was found in 7 patients that had not been suspected before death. pulmonary embolism was the cause of death in 4 patients. the thrombi were derived from deep veins in the lower extremities. in all cases, the cause of death was found in the lungs which were congested and heavy, weighing 2 kg instead of 800 g. at a histological level, diffuse alveolar damage was seen in 8 patients. viral rna was detected with high titres in liver, heart and kidney. clinical chemistry showed increased levels of lactate dehydrogenase, d-dimer and c-reactive protein, suggesting coagulopathy as a complication in these severe covid-19 cases (wichmann et al., 2020) . interestingly, obesity is increasingly seen as a risk factor for severe disease. in a retrospective study with 3615 covid-19 patients from new york city, clinicians had described obesity as a risk factor for intensive care need (or 1.8 to 3.6) in patients < 60 years (lighter et al., 2020) . clinicians had already elaborated concepts for covid-19 pathogenesis that concur with these observations from the autopsies. in one model, covid-19 patients show a diffuse pulmonary intravascular coagulopathy which isin contrast to disseminated intravascular coagulation (dic)restricted to the lung vascular bed. the increased d-dimer concentrations reflect the pulmonary thrombosis followed by fibrinolysis, and the elevated cardiac enzymes reflect the stress imposed on the heart ventricle by pulmonary hypertension. according to this model, it is not the viral infection but the vascular thrombosis induced by the infection which underlies the severe pathology. the coagulopathy might be caused by an activation of an immune mechanism involving macrophages as observed in 'macrophage activation syndrome' (mas), suggesting targets for drug intervention (mcgonagle et al., 2020) . another popular hypothesis for severe covid-19 pathology is based on observations of acute respiratory distress syndrome (ards) where pneumonia, sepsis or aspiration pneumonia lead via a release of pro-inflammatory cytokines from immune cells ('cytokine storm') to severe lung damage. in covid-19, the recruitment of the cellular virus receptor by the infecting virus causes disappearance of the angiotensin-converting enzyme-2 (ace-2) from the cell surface and by its absence results in an increase in unprocessed angiotensin 2 (ang ii). via a cascade of reactions involving the metalloproteinase adam17, increased ang ii concentrations induce the cytokines tumour necrosis factor alpha (tnf-a) and interleukin 6 (il-6) which activate the il-6 amplifier (il-6 amp), leading to the release of inflammatory cytokines. in parallel, the virus induces inflammatory cytokines by activating the nf-jb pathway via pattern recognition receptors (prrs). this hypothesis explains much of the observed pathology in covid-19 and suggests a number of targets for pharmacological intervention (hirano and murakami, 2020) . the expression of immune genes was studied in 3 covid-19 patients with severe, moderate and mild disease. elevated plasma levels of pro-inflammatory cytokines (il-1b, il-8 and tnf-a) were observed, although they occurred after the peak of respiratory failure (mehta et al., 2020; ong et al., 2020) . only 5% of covid-19 cases show severe infections that lead to death in about 1.4 % of cases from severe lung injury and multiorgan dysfunction. chinese clinicians observed that critically ill patients showed signs of shock with cold extremities, weak peripheral pulse, metabolic acidosis and microcirculation dysfunction. this combination of symptoms suggests septic shock, but in 76% of these patients, sars-cov-2 was the only pathogen. these researchers propose 'viral sepsis' as the cause for severe covid-19, where the virus infects the lymphocytes, induces detrimental immune reactions and infects vascular epithelia leading to disseminated intravascular coagulation . these models of covid-19 pathogenesis are not mutually exclusive. sars-cov-2 infections have a variable presentation ranging from causing: no symptoms; only mild symptoms; or severe disease necessitating hospitalization, if not intensive care. in addition, the clinical presentation differs with severe covid-19 depending whether diffuse alveolar damage, coagulopathy, cytokine overproduction or viral sepsis dominates the disease. viral sepsis and cytokine storm might, in fact, be connected. greek researchers compared immune activation and dysregulation in patients with pneumonia caused by bacteria, influenza virus or sars-cov-2. at the moment of hospitalization, covid-19 patients were clinically less affected than patients with bacterial pneumonia. however, a common observation in covid-19 patients is that one week after hospitalization they progressed from a relatively good clinical state into sudden deterioration. these researchers observed low expression of the human leucocyte antigen (hla)-dr on cd14 monocytes in covid-19 patients in need of mechanical ventilation. the patients showed a unique combination of defective antigen presentation and lymphopenia. interleukin-6 (il-6) and c-reactive protein (crp) were significantly increased in severe cases. il-6 is known to inhibit hla-dr expression. an inverse correlation between these markers was, in fact, observed in severe cases. the authors of this study suggest a clinical trial exploring a specific blocker of the il-6 pathway such as tocilizumab to restore the expression of hla-dr, and to alleviate the immune-paralysis seen in severe covid-19 cases. (giamarellos-bourboulis et al., 2020) data from wuhan concur with these observations. when investigating 48 covid-19 patients with distinct disease severity, the clinicians observed viral rna in the blood ('rnaaemia') of five critically ill patients, two of whom died of respiratory failure. interestingly, all 5 patients also showed sharply increased il-6 levels. viral rna in the blood and high il-6 were biomarkers of severe disease . defence against viral infection begins at the cellular level. cells detect a replicating virus by pattern recognition receptors that signal the presence of unusual rna structures. these receptors, when bound to these aberrant rna molecules, activate the transcription factors irf and nf-jb, which launch two antiviral programmes: (i) induction of type i and iii interferon (inf-i, inf-iii), which upregulate interferon-stimulated genes (isg); (ii) recruitment of specific leucocytes by chemokine secretion. us researchers studied the viral and cellular transcriptional response upon infection of cell cultures and in animal models with different respiratory viruses including influenza a virus and sars-cov-2. in cell culture, viral rna represented 50% or more of all cellular messenger rna (mrna). the external addition of inf-i reduced sars-cov-2 replication dramatically. however, infection of cells with sars-cov-2 did not induce an interferon response, but a strong chemotactic and inflammatory response instead. in nasal washes from infected ferrets, viral rna represented < 2% of all mrna. sars-cov-2 induced a different cellular transcription response compared to the influenza virus: a cytokine response was followed by gene transcripts of cell death and leucocyte activation. postmortem lung samples from covid-19 patients showed no inf-i and inf-iii, but a robust chemokine transcription instead. the observations were confirmed in serum samples of covid-19 patients compared with samples from control subjects, showing no interferon, but a cytokine response. the data suggest that sars-cov-2 has learned to suppress interferon induction and to disarray the antiviral response (blanco-melo et al., 2020) . another impressive us study, so far only available as a preprint, investigated the expression of ace-2, the viral receptor of sars-cov-2, and tmprss2, the protease activating viral fusion with cell membranes leading to cell entry, in a large set of 695 healthy and asthmatic children from puerto rico (gala ii study). ace-2 expression was highly correlated with the expression of a gene network associated with cytotoxic t cells, induced in virally infected epithelia and correlated with a network of interferon and epithelial viral response genes. notably, 78% of subjects showing high interferon levels were asymptomatic respiratory virus carriers (not with sars-cov-2, the study preceded the epidemic) compared with 10% asymptomatic infections in subjects displaying only low interferon levels. eighteen children were infected with seasonal coronaviruses, and they demonstrated an increased ace-2 expression. the researchers correlated nasal ace-2 and tmprss2 gene expression with genome wide genetic variation data for these children. they identified a variant locus downstream of the transcription start site of the ace-2 gene which was associated with a large decrease in ace-2 expression. they also found variants that were associated with increased or decreased tmprss2 expression. the distribution of these variants showed clear differences in diverse populations worldwide. it will be interesting to study whether distinct expression pattern and the observed genetic variants explain the strikingly different clinical symptomatology between individuals and whether these genetic markers can explain geographical differences in the worldwide prevalence of the pandemic (sajuthi et al., 2020) . data on the tissue tropism of sars-cov-2 are important in order to understand the clinical aspects of covid-19. while both sars-cov-2 and sars-cov (the coronavirus associated with the 2002 sars epidemic) use the same cellular receptor ace-2, sars-cov-2 causes much less diarrhoea (10% of patients) than sars-cov, but it has more neurological manifestations (confusion, 9%). such distinct dissimilarity in organ tropism was also reflected in the differences in their capacity to infect pulmonary, intestinal and neurological cell lines . ex vivo studies with human tissues obtained during surgery demonstrated that sars-cov-2 infects ciliated and mucus-producing goblet cells and club cells in the bronchi, pneumocytes in the lung and the conjunctiva of eyes (hui et al., 2020) . organoids, 3-d cell structures grown from stem cells that recapitulate key aspects of the organs, were obtained from human small intestine and could also be successfully infected with sars-cov-2. enterocyte progenitors were the primary viral target cells, but also differentiated enterocytes were infected and no other intestinal cell types. the viral receptor ace-2 was abundantly expressed on the brush border membranes of enterocytes. viral replication occurred in intracellular 'viral factories' surrounded by a double membrane. viruses were mainly released apically from the enterocytes. sars-cov-2 induced the transcription of cytokines and interferon-stimulated genes in gut organoids . an impressive approach to tissue tropism was published by the us lung biological network. the consortium used large single-cell rna sequencing (scrnaseq) data sets from primates and humans, asking which cellular subsets coexpressed ace-2 and tmprss2. doublepositive cells are the likely support for in vivo viral replication. seventeen cell types were distinguished in the lungs of rhesus monkeys, but only type ii pneumocytes expressed both ace-2 and tmprss2. in lung resection material from humans, type ii pneumocytes and ciliated cells showed this double expression. a striking observation both in rhesus monkey and in humans was that interferon-induced genes were upregulated in these double-positive cells. absorptive enterocytes from the ileum and jejunumboth from monkeys and from the biopsies of childrencoexpressed both genes, explaining the viral tropism in the gut. in the upper respiratory tract of humans, apical and ciliated cells of the ethmoid sinus and secretory goblet cells of the inferior turbinate of the nose showed this doubly positive expression pattern, and, again, showed a concomitant upregulation of an interferon alpha-stimulated gene set. in primary human upper airway epithelial cells, the authors tested whether interferon plays an active role in upregulating ace-2 expression. this was indeed the case for interferon alpha, but not for interferon gamma. when screening the rnaseq database, the authors found ace-2 upregulation in human lung tissues infected ex vivo with influenza virus. these observations led to the hypothesis that coronaviruses exploit the antiviral interferon response to their advantage by increasing the expression of their cell receptor, allowing a greater number of cells to be infected. the authors caution against the use of interferon alpha in clinical trials with covid-19 patients (ziegler et al., 2020) . testing animals sars-cov-2 is suspected to have originated from bats, but the intermediate host for the transfer to humans is currently unknown. chinese scientists inoculated a number of animals with both an environmental virus isolate from the wet food market in wuhan, from where the epidemic started, and an isolate from a patient from wuhan early in the epidemic. ferrets have been infected and developed an upper respiratory tract infection with fever. outbred domestic cats have also been infected and then have developed respiratory tract symptoms and specific antibodies. cats can transmit infection via the airborne route to other cats. viral rna, but not infectious virus, was detected in the intestine of infected cats. young cats were found to be more susceptible to infection than adult cats. dogs that have been infected showed viral rna in the intestine, but not in the lung. no infectious virus was found in the gut of dogs, indicating low susceptibility of dogs for this virus. pigs, chickens and ducks could not be infected with sars-cov-2 . animal models that reproduce the human infection are not only important for the understanding of the pathology of covid-19 infections but are also crucial for the preclinical testing of antiviral drugs and vaccine candidates. ferrets, a popular model for respiratory viral infections, developed fever after intranasal inoculation with a virus from a covid-19 patient. ferrets showed moderate titres of virus in the nose and low viral titres in the lung and intestine and displayed lung tissue pathology. ferrets recovered after 2 weeks and seroconverted with neutralizing antibodies. infected ferrets transmitted the infection efficiently in the presymptomatic stage to na€ ıve ferrets not only via close contact, but also, albeit less efficiently, to ferrets in separate cages (kim et al., 2020) . hamsters, infected with a virus from a hong kong covid-19 patient, showed weight loss, lethargy and rapid breathing. high virus titres were found in the nose, the lung and in the intestine. histopathological changes were seen in the nose, the trachea and the lung displaying alveolar destruction, but also in the gut demonstrating epithelial necrosis. interferon and pro-inflammatory cytokines were rapidly induced, reached peak titres and dropped after a week with the development of neutralizing antibodies and recovery from disease. na€ ıve contact hamster were efficiently infected, but showed less weight loss . macaques have been infected with sars-cov-2 but showed no clinical symptoms, except for moderate titres of virus excreted from the nose and throat. maximal viral titres were seen early after infection, and titres were higher in older animals. upon autopsy, pulmonary damage presented as the diffuse alveolar damage that is associated with replicating virus. the animals seroconverted (rockx et al., 2020) . also, rhesus monkeys showed viral replication in the pharynx and severe interstitial pneumonia . lung tissue from 17 of 25 malayan pangolins from a wildlife rescue centre in china yielded a positive result for sars-cov-2-related sequences. pangolins are suspected to have been intermediate hosts at the wet food market from wuhan, where the epidemic started. fourteen of the pangolins died, showing lung consolidation and diffuse alveolar damage similar to human covid-19 victims. a complete coronavirus genome has been constructed from rna sequencing of the lung tissues obtained from trafficked malayan, but not chinese, pangolins which represent two distinct species. the malayan pangolin coronavirus is not a direct precursor to sars-cov-2, because its genome differs too much from the human isolates, but may have contributed the receptor binding domain of the spike protein gene by genetic recombination with a bat coronavirus. pangolins and bats are both nocturnal animals that both eat insects and share overlapping ecological niches, rendering contact likely (xiao et al., 2020) . to fulfil koch's postulates, transgenic mice expressing the human ace-2 were infected with a sars-cov-2 isolate from a chinese patient. transgenic mice, but not wild-type mice (which lack the viral cell receptor ace-2), showed symptoms of disease (interstitial pneumonia) thatpartiallyreproduced the disease in humans. the virus replicated in the organ where the pathology was observed, and -finally the virus could be re-isolated from this organ. one overt clinical sign was weight loss in mice. also, viral replication produced moderate peak titres and was limited to the lung and bronchi . after much media hype, political support and emergency use authorization by the fda, the initial high hopes placed in the repurposed malaria drug hydroxychloroquine administered to hospitalized covid-19 patients were not validated by clinical data, which had shown only mixed results. chloroquine increases endosomal and lysosomal ph and interferes with some viral infections in cell culture. a small chinese trial enrolling 62 covid-19 patients on hydroxychloroquine, or placebo, showed, in fact, a shortened period of recovery from fever and cough in the treatment group . a french study randomizing 181 covid-19 patients with pneumonia on hydroxychloroquine or placebo, observed, however, no significant effect of treatment on transfer to icu, mortality, or in the prevention of development of acute respiratory distress syndrome (mah evas et al., 2020). eighty-one adult brazilian patients with severe covid-19 were randomized on a high or a low dose of chloroquine plus azithromycin and oseltamivir. a placebo group was considered to have been unethical due to media support for chloroquine use. overall mortality was 27%, which was not lower than in historical controls. mortality was substantially higher in the high-dose group compared with the lowdose chloroquine group (39 vs. 15%), and the high-dose regime was stopped in this trial by the data safety monitoring board. on day 4 of hospitalization, more than 80% of the patients in both groups showed viral rna in nasopharyngeal secretions excluding an antiviral effect (borba et al., 2020) . one hundred and fifty covid-19 patients from china were treated with oral hydroxychloroquine (hcq), and this was compared with standard treatment alone in an open clinical trial. the treatment did not result in an accelerated clearance of viral rna in upper and lower respiratory tract secretions. clinical symptom alleviation within 28 days was not different between the two groups. a more rapid symptom alleviation was seen for hcq, over the control group during the second week of treatment, but it was not statistically significant. this trend became even more visible when in a post hoc analysis only cases of patients without other antiviral agent treatment were investigated. hcq led to a more rapid normalization of c-reactive protein levels compared with those in the controls. hcq addition led to a higher rate of adverse events over the controls (30% vs 9%), which consisted mostly of diarrhoea (tang et al., 2020b) . from 1376 consecutive covid-19 patients hospitalized in new york city, 811 were treated with a high dose of hcq on day 1, and a low dose during the following 4 days. half of them received the high dose within 24 h of hospitalization, and the follow-up course was for three weeks. there were 565 covid-19 patients who did not receive hcq. the primary end-point was intubation or death, which occurred in 25% of the patients. in crude, unadjusted analysis of this observational study, hcq-treated patients were 2.4 times more likely to reach the detrimental primary end-point than non-treated patients. since hcq-treated patients were, at baseline, more severely ill than non-treated patients (by oxygenation index), the 811 cases of hcq patients were compared in a second analysis to those of 274 better matched non-treated patients. neither benefit nor harm has been associated with hcq use (geleris et al., 2020) . in an open-label and compassionate trial, 53 patients with severe covid-19, in which 34 of them were on invasive ventilation and received remdesivir, a nucleotide analogue developed against ebola virus. after 3 weeks of follow-up, 68% of them showed clinical improvement; 57% of patients on mechanical ventilation were extubated, while 13 % died. this fatality rate is a relatively better result when compared with a 66% mortality rate in chinese patients receiving invasive ventilation. due to a small study size, short length of follow-up and the lack of a randomized control group, the outcome must be interpreted with caution (grein et al., 2020) . in hubei, there were 237 chinese patients with severe covid-19 who were subsequently randomized on a 2:1 basis for intravenous remdesivir, or placebo, in a multicentre clinical trial. the time it took to show clinical improvement in the remdesivir group was statistically not significantly different from that of the control group (median 21 vs. 23 days). the time from onset of symptoms to start of treatment was 10 days. patients receiving the treatment during the first 9 days showed a numerically greater difference for clinical improvement (18 vs. covid-19: pathogenesis and drugs 5 23 days), but this difference is not statistically significant. the length of time of viral excretion in upper and lower respiratory tract samples did not differ between the two groups, demonstrating a lack of in vivo antiviral activity of this nucleoside analog. the mortality rate was 15% in the remdesivir group, and 13% in the placebo group. this otherwise well-conducted clinical trial was underpowered because it could not attain the enrolment of the planned 453 patients, since the epidemic stopped in hubei as a consequence of containment measures. both groups did not differ in the types of, or number of adverse events, demonstrating the safety of intravenous remdesivir . on 1 may, the fda granted an emergency use authorization for remdesivir to physicians. the decision was based on the outcome of a clinical trial, which showed an accelerated recovery in more than 1000 enrolled patients treated with either remdesivir or placebo (11 vs. 15 days). there were fewer deaths in the group with treatment than in the placebo group, but the difference was not statistically significant. details of the trial have not yet been published (ledford, 2020) . a total of 86 covid-19 cases of patients from china with mild/moderate disease were randomized on the antiviral lopinavir (an inhibitor of hiv protease combined with ritonavir, which prolongs the presence of drugs in the body) or the antiviral arbidol (an influenza virus fusion inhibitor only registered in russia) or in a control group in a 2:2:1 ratio. the primary end-point was positive-to-negative conversion for viral rna in pharyngeal swabs. no difference was seen between the groups. secondary outcomes were represented by clinical parameters: fever, cough and lung ct improvement. again, no difference was seen between the groups. adverse events were not observed in the controls, but in 35% and 14% of the lopinavir and arbidol group, respectively, mainly manifested as diarrhoea. thirteen patients progressed to severe disease during hospitalization: namely 8, 3 and 2 in lopinavir, arbidol and control group, respectively. the study is small and was only conducted at a single centre, but it still confirms the lack of efficacy of lopinavir/ritonavir treatment in severe covid-19 cases (li et al., 2020b) . in a retrospective cohort study with 33 chinese patients suffering from moderate covid-19 showing lung ct abnormalities, 16 patients were treated with an arbidol/lopinavir/ritonavir combination while 17 patients received only lopinavir/ritonavir. the group including arbidol in the combination showed a significantly shorter nasopharyngeal and stool excretion of sars-cov-2, and an accelerated normalization of lung ct, compared with the control group. the positive outcome must, however, be interpreted with caution since the two groups differed significantly for glucocorticoid treatment (6% vs. 41% in control). the better outcome in the arbidol arm could therefore be due to less use of detrimental glucocorticoid . triple therapy: lopinavir, ribavirin, interferon beta-1b in a multicentre trial, 127 covid-19 patients from hong kong were enrolled into a prospective, randomized, open-label phase 2 clinical trial. eighty-six patients received a combination treatment of the lopinavir (plus ritonavir), the nucleoside analog ribavirin and interferon beta-1b, while the control group was only treated with lopinavir (plus ritonavir). at baseline, both groups were well matched and showed mild-to-moderate disease. the combination group demonstrated, in comparison with the controls, a significantly reduced time (7 vs. 12 days) to clearance of viral rna in the nasopharynx (the primary end-point), as well as a shortening of oropharynx, throat and stool virus excretion. the combination therapy was also associated with significant clinical improvement compared with controls, and a shorter hospitalization stay. there were no fatalities. the treatment group also displayed lower il-6 values than the controls. a post hoc subgroup analysis comparing 76 patients (52 in combination and 24 in control group), who started treatment less than 7 days after symptom onset, showed again a significant difference between the two groups. in contrast, no difference was seen between the two groups when the treatment was initiated after 7 days. out of concern for safety, patients in the combination group did not receive interferon at later stages of the infection. the therapeutic effect in the combination group could be attributed to either earlier ribavirin treatment or interferon inclusion (hung et al., 2020) . anakinra is a recombinant interleukin il-1 receptor antagonist. high-dose intravenous anakinra has fda and ema off-label approval for the treatment of hyperinflammatory conditions such as macrophage activation syndrome (see above coagulopathy). a chinese study used anakinra in severe covid-19 patients who could not receive intensive care due to overwhelming patient numbers. low-dose subcutaneous anakinra (the standard application) had no effect on c-reactive protein levels or clinical parameters, compared with controls. high-dose intravenous anakinra application suppressed hyperinflammation, as documented by a decrease in creactive protein, and resulted in a significantly higher rate of survival at 3 weeks compared with controls (90 vs. 56%). the retrospective nature and the small number of patients treated limit positive conclusions (cavalli et al., 2020) . according to a popular pathogenesis model, interleukin il-6 production induces immunopathology in severe covid-19 cases. tocilizumab, a recombinant humanized monoclonal antibody, which binds the il-6 receptor and inhibits its signal transduction, has been previously used in the treatment of a special form of juvenile arthritis associated with increased il-6 production. this monoclonal antibody led to a rapid increase in blood lymphocytes in six covid-19 patients (giamarellos-bourboulis et al., 2020). clinicians from china gave tocilizumab to 21 patients with severe or critical covid-19 who had failed to respond to standard therapy. one day after the injection of tocilizumab, the fever disappeared in all patients, c-reactive protein returned to normal in most of them, peripheral oxygen saturation improved, ct-documented lung damage ameliorated, and the virus was cleared. adverse effects were not observed, and all patients were discharged (xua et al., 2020) . three weeks after the onset of disease, 6 severely ill covid-19 patients received plasma from convalescent covid-19 patients, while 11 covid-19 patients received non-convalescent plasma as a control. convalescent plasma application was linked to a significantly higher viral clearance over controls (100 vs 21%) but did not affect mortality which was high in both groups (5/6 and 10/11 died) . in a retrospective study from china, 20% of 449 severely ill covid-19 patients were treated with the coagulation inhibitor heparin. mortality was 30%, irrespective of whether heparin was given or not. however, when patients were stratified with respect to coagulopathy at baseline (using d-dimer concentration sixfold higher than normal as threshold), a 20% reduction in mortality was associated with heparin use. the data need to be interpreted with caution, since they come from a retrospective subgroup analysis . hydroxycytidine a promising prodrug is the nucleotide analog hydroxycytidine. in cell culture infections, this drug is active against a wide range of human and bat coronaviruses thus potentially offering inhibitory activity against future spill-over infections from animals (zoonosis). the drug increases the mutation rate of the virus during replication resulting in infectivity losses. in mouse infection models for sars-cov and mers virus, it had good oral bioavailability, ameliorated lung function, reduced virus titre and diminished weight loss caused by the viral infection. the drug demonstrated therapeutic effects up to 24 h after infection, which is substantial since mice show a compressed disease course compared to that of humans. coronaviruses that had become resistant to remdesivir showed enhanced susceptibility to inhibition by hydroxycytidine (sheahan et al., 2020) . hydroxycytidine causes mutagenesis in the bacterium escherichia coli and the fungus neurospora crassa. cell protease inhibitor sars-cov-2 needs, after the docking of its spike protein to the cell receptor ace-2, a proteolytic cleavage of the spike protein at a polybasic site separating the s protein into two protein fragments s1 and s2, where s2 mediates the fusion of the viral and cell membranes, which leads to the entry of the viral genome into the infected cell. cell culture infection tests demonstrated that sars-cov-2 uses two proteases for this proteolytic processing, either the lysosomal cathepsin catb/l or the transmembrane protease tmprss2. the serine protease inhibitor camostat, which is a registered drug in japan for gastroenterology problems, inhibits tmprss2 and confers partial resistance to infection with sars-cov-2, and total protection when combined with e-64d, an inhibitor of catb/l (hoffmann et al., 2020) . chinese scientists have targeted the sars-cov-2 main protease for inhibition. after expressing the protease and designing a fluorescence-labelled substrate, they used, as an inhibitor, compound n3 which was active against sars-cov. in silico docking verified that it could fit into the predicted structure of sars-cov-2 main protease. kinetic analysis revealed a two-step inactivation process resulting in n3 covalent binding to the catalytic site. the crystal structure for the protease-bound n3 was solved and was followed by a virtual screening of a chemical database for better inhibitors. the next step was a highthroughput screening of 10'000 compounds. one of the best inhibitors was an organo-selenium compound that inhibited the infectivity of sars-cov-2 in a cell culture test. since this compound has already been investigated for the treatment of several diseases, where it showed a high safety profile, this repurposed drug could be re-entered into clinical trials relatively quickly . us researchers have expressed in a cell culture system all of the sars-cov-2 proteins that were tagged with a recognition peptide, which allowed the isolation of cellular proteins interacting with the viral bait protein. the protein complexes were collected by affinity chromatography, and the copurified cellular proteins were identified by mass spectrometry. this approach yielded 332 highconfidence protein-protein interaction partners. the captured proteins showed high expression in lung tissue, and many of them also interacted with another lung pathogen, mycobacterium tuberculosis. the viral proteins interacted with proteins from multiple innate immune pathways, the host translation machinery, the ubiquitin ligase complex and proteins involved in transcriptional regulation of antiviral responses. from a search of chemical databases, the scientists identified 69 ligands, including fda-approved drugs and compounds currently used in clinical trials that interacted with the identified proteins. in cell culture, several compounds showed inhibitory activity against sars-cov-2. the candidate inhibitors included the following: antihistamines, antitussives, antipsychotics and interestingly also hydroxychloroquine (gordon et al., 2020) . parallel to these state-of-the-art biochemical approaches, one must also mention the traditional approaches promoted in china. according to chinese state media, jinhua qinggan herbal granules accelerated, in a clinical trial with covid-19 patients, conversion to a negative virus test within 2 days. xuebijing, an extract of five herbs, reduced mortality in covid-19 by 8.8% via removing blood stasis. details of these trials are not yet available (cyranosky, 2020) . in the emergency situation of the expanding pandemic and in an effort to save lives, severe covid-19 cases were frequently treated with many different drugs. this situation leads to complex drug interaction, potential detrimental side effects for the patients and difficulty to disentangle the effect of tested drugs in clinical trials from concomitant treatment. the extent of the pandemic also led to questions about whether the drug treatment for existing morbidities of the patients increased the risk for sars-cov-2 infections, or even led to a more severe clinical course. from the large selection of literature about treatment of other diseases during the covid-19 epidemic, i have selected one which was cited as a major risk for developing severe covid-19, namely hypertension. ace-2 is the cellular receptor of sars-cov-2, and it is the target for a class of antihypertensive drugs that block this enzyme, which leads to its increased expression. since hypertension has been frequently reported as a risk factor in severe covid-19 infections, the safety of antihypertensive drugs during the covid-19 epidemic has been questioned. cardiologists studied this question in new york city, an epicentre of this pandemic. more than 12'000 cases were investigated, nearly half of them had a positive viral test. from these 6'000 cases, 1'000 had severe covid-19, 726 were in intensive care, and 447 died. the researchers ruled out a significant effect from any of five common antihypertensive drugs favouring infection (i.e. from being tested positive for sars-cov-2) or that they increase the likelihood of inducing severe covid-19. patients taking beta-blockers even had a significant, although marginally lower, risk of testing positive for the virus (reynolds et al., 2020) . the same question was addressed in another epicentre of the pandemic, in lombardy, with a large case-control study involving 6'272 covid-19 patients and 30'000 controls, matched for age, sex and municipality. angiotensin ii receptor blockers (arb) and ace inhibitors (acei), common antihypertensive drugs, were more frequently used by covid-19 patients than in the control group (arb: 22 vs 19%; acei: 24 vs. 21%). after multivariate analysis, neither arb, acei nor other common antihypertension drugs had a significant association with covid-19. the italian study confirmed the chinese observations that the covid-19 patients were in poorer health than the general population matched for age, particularly with respect to previous hospitalization for cardiovascular diseases (mancia et al., 2020) . these conclusions were corroborated in another study in which 8910 hospitalized covid-19 patients were compared for outcome, namely death (n = 515) versus survival to discharge (n = 8395 patients). the cases were from hospitals in north america, europe and asia. in multivariate analysis: age greater than 65 y, coronary artery disease, congestive heart failure, cardiac arrhythmia, chronic obstructive pulmonary disease and current smoking were all associated with a higher risk of in-hospital death. ace inhibitor use was associated with a significantly lower risk of death, but might be a coincidental observation for multiple comparisons. however, the data do not provide evidence for a detrimental effect of antihypertensive drugs on covid-19 mortality (mehra et al., 2020) . until now no prospective, sufficiently powered, controlled clinical trial that has been reported in a peer-reviewed publication has reported an impact of drug interventions on covid-19 mortality, despite more than 1'000 trials registered on clinicaltrials.gov, including 600 intervention studies. technical problems, such as the lack of true control groups, concomitant other treatments, make it difficult to disentangle the effects. many trials were started before the insight into covid-19 pathogenesis, which are now accumulating, became available. probably, reliable biomarkers for different pathological types of covid-19 forms are needed in order to allow more precisely targeted therapeutic interventions (bauchner and fontanarosa, 2020) . many approaches are with repurposed drugs, developed for other infections, in order to cut down the time needed for drugs to reach patients in an emergency situation. these approaches need to be complemented by trials specifically tailored to the covid-19 pathology. a. fauci reminded us that drug trials of hiv infections progressed in small steps before effective drug combinations could be developed. the pathogenicity of sars-cov-2 in hace2 transgenic mice covid-19 autopsies, oklahoma, usa randomized clinical trials and covid-19: managing expectations imbalanced host response to sars-cov-2 drives development of covid-19 effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study china is promoting coronavirus treatments based on unproven traditional medicines arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study development of an inactivated vaccine candidate for sars-cov-2 observational study of hydroxychloroquine in hospitalized patients with covid-19 complex immune dysregulation in covid-19 patients with severe respiratory failure a sars-cov-2 protein interaction map reveals targets for drug repurposing compassionate use of remdesivir for patients with severe covid-19 ª 2020 the authors. microbial biotechnology published by john wiley & sons ltd and society for applied microbiology. covid-19: pathogenesis and drugs 9 covid-19: a new virus, but a familiar receptor and cytokine release syndrome sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor tropism, replication competence, and innate immune responses of the coronavirus sars-cov-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures epub ahead of print triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial structure of m(pro) from sars-cov-2 and discovery of its inhibitors infection and rapid transmission of sars-cov-2 in ferrets sars-cov-2 productively infects human gut enterocytes hopes rise for coronavirus drug remdesivir epub ahead of print sars-cov-2 and viral sepsis: observations and hypotheses efficacy and safety of lopinavir/ritonavir or arbidol in adult patients with mild/moderate covid-19: an exploratory randomized controlled trial no evidence of clinical efficacy of hydroxychloroquine in patients hospitalised for covid-19 infection and requiring oxygen: results of a study using routinely collected data to emulate a target trial renin-angiotensin-aldosterone system blockers and the risk of covid-19 immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia cardiovascular disease, drug therapy, and mortality in covid-19 silencing the cytokine storm: the use of intravenous anakinra in haemophagocytic lymphohistiocytosis or macrophage activation syndrome a dynamic immune response shapes covid-19 progression renin-angiotensin-aldosterone system inhibitors and risk of covid-19 comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model type 2 and interferon inflammation strongly regulate sars-cov-2 related gene expression in the airway epithelium an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy hydroxychloroquine in patients with covid-19: an open-label, randomized, controlled trial remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial autopsy findings and venous thromboembolism in patients with covidª 2020 the authors isolation of sars-cov-2-related coronavirus from malayan pangolins pathological findings of covid-19 associated with acute respiratory distress syndrome effective treatment of severe covid-19 patients with tocilizumab effect of convalescent plasma therapy on viral shedding and survival in covid-19 patients sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues i thank jacqueline steinhauser, and kenneth timmis and lutz br€ ussow for critical reading of the manuscript. the author consults nestl e, his former employer, on the scientific aspects of the covid-19 epidemic, but he does not consider this as a conflict of interest. key: cord-256020-wrui3i2l authors: fadaka, adewale oluwaseun; sibuyi, nicole remaliah samantha; adewale, olusola bolaji; bakare, olalekan olanrewaju; akanbi, musa oyebowale; klein, ashwil; madiehe, abram madimabe; meyer, mervin title: understanding the epidemiology, pathophysiology, diagnosis and management of sars-cov-2 date: 2020-08-26 journal: j int med res doi: 10.1177/0300060520949077 sha: doc_id: 256020 cord_uid: wrui3i2l the emergence of coronavirus disease 2019 (covid-19) in december 2019 has resulted in over 20 million cases and 741,808 deaths globally, affecting more than 200 countries. covid-19 was declared a pandemic on 11 march 2020 by the world health organization. the disease is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2). there is limited information on covid-19, and treatment has so far focused on supportive care and use of repurposed drugs. covid-19 can be transmitted via person-to-person contact through droplet spread. some of the recommended precautionary measures to reduce the rate of disease spread include social distancing, good hygiene practices, and avoidance of crowded areas. these measures are effective because the droplets are heavy and can only travel approximately 1 meter in the air, settling quickly on fixed surfaces. promising strategies to combat sars-cov-2 include discovery of therapeutic targets/drugs and vaccines. in this review, we summarize the epidemiology, pathophysiology, and diagnosis of covid-19. we also address the mechanisms of action of approved repurposed drugs for therapeutic management of the disease. covid-19 has become one of the most dangerous pandemics in recent history. the pandemic has claimed more than 740,000 lives, with more than 20 million reported cases since the original outbreak. the disease is caused by sars-cov-2, a zoonotic pathogen that acquired mutations as it crossed the species barrier from bat to pangolin enabling it to infect humans. 1 sars-cov-2 was confirmed as a novel coronavirus using molecular methods and initially named 2019 novel coronavirus (2019-ncov). 2 the disease caused by this virus was later renamed covid-19 by the world health organization (who). 3 sars-cov-2 is highly infectious and has spread to more than 200 countries in all continents. hence, the virus was declared a global threat (pandemic) by the who. sars-cov-2 has a single-stranded positive sense rna genome (þssrna) approximately 30 kb long. the sars-cov-2 genome is organized similarly to those of sars-cov-1 and middle east respiratory syndrome (mers)-cov. 4 using phylogenetic analysis, the sars-cov-2 genome was demonstrated to share 96% sequence similarity with a bat cov genome. sars-cov-2 belongs to the b-coronavirus genus that includes sars-cov-1 and mers-cov. 5 the clinical symptoms of covid-19 include fever, cough, and pneumonia, which makes the disease enormously dangerous with a high case fatality rate. 6, 7 in contrast to other b-coronaviruses, many sars-cov-2 deaths have resulted from multiple organ dysfunction syndrome (msof) rather than respiratory failure. 8 this could be attributed to the widespread distribution of angiotensin converting enzyme-2 (ace-2), the primary receptor for sars coronaviruses, in multiple organs. 9,10 ace-2 is expressed as a cellsurface molecule in the respiratory tract (epithelium, arterial and venous endothelium), the small intestinal epithelium, and arterial smooth muscle cells. 11 sars-cov-2 is morphologically spherical and is characterized by presence of a spike protein, lower pathogenicity and higher transmissibility between humans. 12 some of the primary measures taken to reduce the number of infections and prevent community transmission are to avoid crowds and gatherings and to practice good hygiene. interestingly, countries with the highest reported prevalence and mortality such as the united states, spain, italy, the united kingdom, russia, germany, brazil, france, turkey, iran and china, are more concerned with flattening the curve through early case detection, isolation, and development of therapeutic drugs and vaccines. because of the novel nature of this disease, there is limited information regarding risk factors for severe outcomes. specific factors such as the serial interval, viral lifespan, incubation period, pathogenic mechanisms, clinical features and optimal disease management remain unclear. therefore, this review aimed to summarize the epidemiology and pathophysiology of sars-cov-2 as well as the use of repurposed food and drug administration (fda) approved drugs for therapy. the entry of this virus into host cells and possible downstream complications deserve closer attention. covs are members of the subfamily othocoronavirinae of the family coronaviridae. the subfamily consist of four genera: alpha, beta, gamma and delta covs. 13 both alpha-and beta-covs can infect mammals, including humans, while the gamma-and delta-covs only infect birds. 14 about seven covs have been isolated from humans ( figure 1 ). these include two alpha-covs, human coronavirus 229e (hcov-229e) and human coronavirus nl63 (hcov-nl63), and five beta-covs: human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), sars-cov-1, mers-cov, and sars-cov-2. sars-cov-1, mers-cov, and sars-cov-2 are pathogenic and cause severe infections in humans following contact with the respective intermediate hosts (bats) (figure 2 ). however, hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1 do not appear to cause severe infections in humans. 14 covs are enveloped viruses with þssrna genomes. they have the largest genomes (approximately 26-33 kb) among rna viruses. all covs possess nonsegmented genomes with similar organization. 15 generally, about two-thirds of the genome consists of two large and overlapping open reading frames (orf1a and orf1b), which are translated into polyproteins pp1a and pp1ab and subsequently processed to yield 16 non-structural proteins (nsp1 to nsp16). 16 the remaining one-third of the genome consists of orfs encoding structural proteins including the spike (s) glycoprotein embedded in the envelope and the envelope (e), matrix (m), and nucleoproteins (n). 17 there are short untranslated regions at both the 3 0 and 5 0 ends. the s protein plays a role in receptor-binding and entry of virus into host cells and is thus considered a major therapeutic target. the m and e proteins are important for viral assembly, while the n protein is necessary for synthesis of rna. 18 overview of the sars-cov family sars-covs belongs to a global family of viruses causing respiratory disease and influenza-like symptoms such as fever, muscle pain, sore throat, headache, and cough. 19 figure 3 highlights the onset and progression of sars-cov-1, mers-cov, and sars-cov-2 infection. the first case of sars-cov-1, reported in china, resulted in an outbreak that caused hundreds of deaths and thousands of infected cases in the early 2000s. 20 a pneumonia-like syndrome (mers) was first discovered in saudi arabia and then spread to several countries, where it incurred a mortality rate of about 3% to 6%. marra et al (21) observed that the mers mortality rate increased with age and was as high as 43% to 55% in people older than 60 years. in december 2019, sars-cov-2, caused an outbreak in china and then spread worldwide. the resulting disease (covid-19) is a serious threat mostly in people with compromised immune systems or underlying conditions such as lung disease, diabetes mellitus, and human immunodeficiency virus infection. 22 sars-cov-1. sars-cov-1 causes a viral respiratory disease and belongs to lineage b of the beta-covs. 11 of uncooked meat, milk or urine, as shown in figure 2 . human-to-human infection also occurred through nosocomial transmission. 11 symptoms of human sars-cov-1 infections include headache, fever and respiratory complications such as cough, dyspnea, and pneumonia. 23 the sars-cov-1 genome is 29,727 nucleotides in length. the 5 0 end of the genome contains orf1a and orf1b. the polyproteins encoded by these orf are auto-catalytically processed to yield a number of viral proteases as well as the rna-dependent rna polymerase (rdrp). the remainder of the genome encodes the viral structural proteins (s, e, m and n) as well as several accessory proteins. 18 the receptor for sars-covs is ace-2, a surface molecule found on cells of the respiratory tract, the small intestinal epithelium, and smooth muscle. in the respiratory tract, ace-2 is expressed on epithelial cells of the alveoli, bronchi, trachea, and bronchial serous glands, as well as on alveolar monocytes and macrophages. the ace-2 enzyme plays an important role in protection against lung failure. 11 mers-cov. mers-cov causes a viral respiratory disease and belongs to lineage c of the beta-covs. 11 the first cases of human infection by mers-cov were reported in saudi arabia in 2012. cases were subsequently reported in other countries including qatar, egypt, and the republic of korea following contact with infected camels (figure 3b ). cases of mers-cov were identified in about 27 countries between 2012 and 2018. 18 mers-cov rna in camels showed more than 99% genomic sequence similarity to human mers-cov. 23 bats are the natural hosts of mers-cov (figure 2) , and a high prevalence of mers-cov infections in dromedary camels (intermediate hosts) was confirmed in the middle east, spain, and africa. mers-cov infections were transmitted to humans following contact with camels infected with the virus. 23 the mers-cov genome is about 30,119 nucleotides in length and has a 5 0 terminal cap structure and a poly (a) tail at the 3 0 end. the genome encodes 16 non-structural proteins (nsp1-nsp16) at the 5 0 end, four structural proteins (s, e, m, and n) at the 3 0 end, 24 and five accessory proteins in orf3, orf4a, orf4b, orf5, and orf8. 18 risk factors for severe mers-cov include age and the presence of underlying conditions such as diabetes, obesity, hypertension, chronic renal disease, and lung diseases. 25 the receptor for mers-cov is dipeptidyl peptidase 4 (dpp4 or cd26). dpp4 is a multifunctional cell surface protein and is expressed on the epithelial cells of the respiratory tract, liver, kidney, small intestine, and prostate, as well as on activated white blood cells. dpp4 also plays a vital role in activation of t cells and providing co-stimulatory signals for immune responses of t cells. 11, 18 consequently, mers-cov causes acute pneumonia and renal dysfunction with associated clinical symptoms such as cough, chest pain, sore throat, fever, diarrhea, vomiting, and abdominal pain. mers-cov can infect human dendritic cells and macrophages in vitro, thereby contributing to dysregulation of the immune system. 18 sars-cov-2. sars-cov-2, a newly discovered coronavirus, has a þssrna genome and a spherical morphology when observed under the electron microscope. 26,27 sars-cov-2 encodes a richly glycosylated spike protein responsible for binding to the ace-2 receptor. 28 the virion's shape and the ability of spike proteins to form a crownlike structure gave coronaviruses their name. 29, 30 the sars-cov-2 genetic material is surrounded by a lipid-bilayer envelope. other structural components include the nucleocapsids, membrane, envelope, and hemagglutinin. unlike the envelope, the membrane exists in higher quantities within the virions. 31 among other functions, the envelope serves to release viral particles from the host cells. 32 the nucleocapsid assist in rna packaging during virion assembly. 33, 34 hemagglutinin enhances the entry and pathogenesis of coronaviruses. 33 some of the characteristics of sars-cov-2 are summarized in table 1 . many of these features are shared with sars-cov-1. more information on the epidemiology and general characteristics of sars-cov-1 can be found later in the review. other important human covs include alpha-covs (hcov-229e and hcov-nl63) and two beta-covs (hcov-oc43 and hcov-hku1). the receptor for hcov-229e is human aminopeptidase n (cd13), a cell-surface metalloprotease present on cells of the kidney and lung, as well as on epithelial and intestinal cells. the receptor for hcov-nl63 is also ace-2. the receptor for hcov-oc43 is 9-o-acetylated sialic acid, while no receptor has been identified for hcov-hku1. 42, 43 generally, the most common diagnostic tools for human covs are molecular detection techniques such as reverse transcription-polymerase chain reaction (rt-pcr) using rna extracted from respiratory tract samples as template. other methods include serological tests and viral cultures. 11 although several agents against covs, including antibodies, antiviral peptides, and cell or viral protease inhibitors, have been shown to be effective both in vitro and/or in vivo, clinical trial outcomes have not been reported. 11 therefore, clinical treatments for covs are still lacking. nevertheless, supportive and symptomatic therapy are used for at present, 215 countries and territories are affected by the sars-cov-2 outbreak, including the united states, italy, germany, south africa, and nigeria. the distribution of sars-cov-2 confirmed cases, including mortality and recovery, is shown in figure 4 . the united states, china, and some european countries have high case numbers and mortality rates. recovery rates are increasing worldwide with higher numbers reported in china. the cfr is defined as the percentage of deaths recorded among confirmed cases. as of 14 april 2020, the number of deaths among confirmed cases was estimated at 126,066 to 1,992,189, resulting in a cfr of 6.33%. this differs from the cfr of 3.70% calculated on 15 march 2020. however, several factors can prevent the accurate determination of the cfr. we compared the global cfr to the african cfr. the highest cfr was observed in italy, followed by spain and the netherlands (figure 5a ). in africa, countries such as egypt, algeria, burkina faso, and morocco had cfrs above 5% (figure 5b) . a major challenge in the accurate calculation of the cfr is the denominator (the number of confirmed cases). asymptomatic cases of covid-19, patients with mild symptoms, or individuals who are misdiagnosed may be left out of the denominator, leading to underestimation or overestimation of the cfr. 45 high cfrs reflect limited access to health care for the most vulnerable patients and limitations in health-care systems, including limited capacity of surveillance systems to trigger a timely response (who, 2020). to date, over 20 million cases of covid-19 have been reported globally. it is important to estimate the reproductive number for this virus to enable accurate predictions. two primary factors, the reproductive ratio (r o ) and the serial interval (s i ), are essential to estimate the rate of transmission of this disease. serial interval (s i ). to understand the case turnover and transmissibility of covid-19, the serial interval (s i ) from the onset of illness in a primary case to the onset of illness in a secondary case is important. 49 recent studies estimated the average s i for covid-19 as 3.77 (2.23-4.82) days. 49-51 a shorter s i makes covid-19 harder to contain and more likely to rapidly transmit within populations ( figure 6 ). taking the r 0 and s i of covid-19 into consideration, it can be inferred that approximately 4 days are required for an infected person to transmit covid-19. it is highly likely that the individual can infect approximately two or three other persons, making the spread of covid-19 extremely rapid and dangerous ( figure 7a ). the extent of spread is totally dependent on the r o value (figure 7b ). if r 0 < 1, each existing infection causes less than one new infection. in this case, the disease will decline and eventually die out. if r o ¼ 1, each existing infection causes one new infection. the disease will remain stable in the population, but will not result in epidemic spread. if r 0 > 1, each existing infection causes more than one new infection. the disease will spread between individuals and eventually lead to an outbreak. covid-19 was regarded as an outbreak (30 january 2020) with a r 0 > 1 (figure 7b) . importantly, the r 0 value of a disease only applies when everyone in a population is completely susceptible. this means that no one has been vaccinated or had the disease previously, and there is no way to control the spread of the disease using interventions such as drugs or vaccines. there are currently two known modes of covid-19 transmission: the fecal-oral route and respiratory droplets. 6, [52] [53] [54] [55] [56] droplets have potential to come into contact and infect a healthy person within 3 to 6 feet (1 meter). droplets that stick to surfaces can survive for more than 24 hours, remaining infectious. the virus can remain airborne for about 3 hours, long enough to permit transmission. upon infection with sars-cov-2, the virus infects type ii pneumocytes of the alveoli. 57 these pneumocytes are responsible for surfactant production. surfactant decreases the surface tension within alveoli and reduces the collapsing pressure. the spike protein of the virus binds to ace-2 on the pneumocytes (figure 8 ) permitting virion entry into the host cell. 58 the virus hijacks the host cell's machinery (ribosomes) to enable translation of its þ ssrna genome into different protein molecules. the virus can also use its rdrp to produce additional copies of its þ ssrna genome (figure 9 ). the translated polyproteins are further processed into different individual components within the host cell. these processes give rise to multiple virions, which are then released upon pneumocyte damage. in response to this process, type ii pneumocytes releases specific inflammatory mediators that instruct macrophages to secrete interleukins 1 and 6 (il-1 and il-6) and tumor necrosis factor-alpha. these cytokines cause the endothelial cells lining blood vessels to dilate, leading to increased capillary permeability. in response, fluids accumulate in the alveoli leading to edema. 59 as surface tension increases, the collapsing pressure of the alveoli increases. a decrease in gas exchange is also observed through this process, which in turn leads to hypoxia and difficulty breathing (dyspnea). this can progress to a critical condition such as acute respiratory distress syndrome (ards). inflammatory mediators further stimulate neutrophils, which release reactive oxygen species and proteases. this process damages the alveoli (both type 1 and 2), leading to consolidation and alveolar collapse. high levels of il-1 and il-6 travel through the blood to the central nervous system, instructing the hypothalamus to release prostaglandins and causing fever. severe lung inflammation leads to systemic inflammatory respiratory syndrome. progression can lead to increased capillary permeability. the overall blood volume decreases, and through a series of processes involving hypotension and decreased perfusion of multiple different organs, multiple system organ failure (msof) can occur. during msof, elevated levels of blood urea, nitrogen, and creatinine accrue in the kidney. the liver also releases specific inflammatory and acute phase response biomolecules (aspartate transaminase, alanine transaminase, bilirubin, c-reactive protein [crp], fibrinogen and il-6) that can serve as biomarkers for patients with covid-19. there is continued research by multiple groups into the mechanism of transmission of sars-cov-2 through the eyes. investigations were necessary because health care providers, including a perking university physician, may have contracted the virus while not wearing eye protection when treating patients. 61 some researchers have asserted that avoidance of touching the eyes, nose or mouth with unwashed or unsterilized hands can reduce covid-19 transmission. the mucous membranes that line various cavities in the body are generally most susceptible to viral transmission. 62 ocular symptoms such as viral conjunctivitis can result from sars-cov2 upper respiratory tract infections. 63 this was confirmed in 9 of 1,099 patients in china. 64 other research also found that 1 of 30 patients hospitalized with covid-19 were diagnosed with conjunctivitis. 65 in a study conducted by the american optometric association, covid-19 was linked to ocular signs and symptoms including photophobia, irritation, conjunctival infection and ocular discharge. 66 thus, the superficial blood vessels of the conjunctiva are an alleged route of exposure and transmission of sars-cov-2. the clinical manifestations of sars-cov-2 are uncertain and change frequently. some infections are asymptomatic. symptoms can include respiratory distress syndrome, pneumonia of different levels of severity, 67 and sometimes death. according to the who, the most common symptoms of covid-19 are fever, fatigue, dry continuous cough, and shortness of breath. 68, 69 some patients may have a runny nose, sore throat, nasal congestion, aches and pains, and diarrhea. 70 some patients report a loss of sense of smell and taste. 71 in some cases, symptoms are mild and similar to those of the common cold; in such patients, recovery can occur without any treatment. 72 the least commonly observed symptoms include nausea or vomiting, coughing up blood or bloody mucus, and viral conjunctivitis causing red eyes, watery discharge from the eyes, swollen eyelids and light sensitivity. 3 occasional upper respiratory and gastrointestinal symptoms, accompanied by changes vital signs such as increased respiration (heart rate) and low blood pressure may also be observed, especially in the elderly and among individuals suffering from heart disease, chronic respiratory conditions and diabetes. 73 additionally, patients critically ill with covid-19 may present with increased venous thromboembolism including thrombocytopenia, elevated d-dimer, prolonged prothrombin time and disseminated intravascular coagulation (dic). these coagulation abnormalities are associated with a systemic inflammatory response and an imbalance between pro-coagulant and anticoagulants homeostasis mechanisms. and increase the risk of mortality. 74 some of these clinical features are also observed in cases of dic observed in septic patients. these features are very distinct in covid-19 patients as their levels are higher than the standards for sepsis. 75, 76 diagnosis, prognosis, treatment, and management of sars-cov-2 sars-cov-2 causes various complications ranging from fever, dry cough, and pneumonia to decreased organ perfusion leading to msof. early symptoms are similar to those of influenza, and the first step to differentiate covid-19 from flu and pneumonia is a nasopharyngeal swab test (viral testing for influenza a/b). 77 quantitative polymerase chain reaction-based (qpcr) methods are the major diagnostic tests for sars-cov-2 using nasal swab, aspirate, sputum, or blood as samples. these method have some limitations as they are time consuming and have variable sensitivity (30%-80%). [78] [79] [80] another diagnostic method is the newly approved nucleic acid test, which is carried out based on the principle of fluorescence pcr. 81 the main goal of sars-cov-2 diagnosis is to accurately detect the virus and to minimize further transmissions by timely isolation and treatment of infected patients. other tests (not specific for sars-cov-2) used in conjunction with the methods above are based on clinical manifestations. these include blood tests such as complete blood count, comprehensive metabolic panels, basic metabolic panels, and assessment of liver/kidney markers and procalcitonin levels (for bacterial infections). inflammatory markers may also be assessed including crp, erythrocyte sedimentation rate, il-6, lactate dehydrogenase, d-dimer, ferritin, troponin and creatine kinase-mb. imaging investigations are typically computed tomography scans: in covid-19 patients these often show glass opacities, areas of consolidation, and paving patterns in cases of severe and progressive disease. ground glass opacities can also be observed on chest x-ray. finally, ultrasound can show b-lines, pleural line thickening, and lung consolidation. air bronchograms can also be used for assessment. these tests are non-specific but helpful to determine patients' health status. covid-19 patients with severe ards could potentially present with pneumonia. pneumonia may be severe, leading to ards and msof. it is therefore imperative to mechanically ventilate the lungs to avoid ards and msof. although the risk factors for covid-19 remain unclear, some risk factors put patients at a significantly higher risk of mortality, especially in individuals with underlying conditions. 6, 35, 40, 82 some medical disorders are correlated with higher risk of mortality in covid-19 patients. these include, cardiovascular diseases mortality risk 10.6%), 41 lung disease (mortality risk 7.3%), type 1 and 2 diabetes (mortality risk 6.3%), immunosuppression (e.g., cancer patients; mortality risk 5.6%), [83] [84] [85] [86] and age ( figure 10 ). 87 covid-19 mortality rates increase with advancing age, and are especially high in those aged >60 years. elevated inflammatory markers in response to tissue damage (elevated levels of d-dimer, ferritin, creatine kinase-mb, and troponin) have been associated with higher mortality rates. the first line of treatment for patients presented with the symptoms of covid-19 (fever, dry cough, and shortness of breath) is self-quarantine for at least 14 days. cases are monitored for progression of symptoms such as increased fever (>40 c), significant difficulty breathing or shortness of breath, mouth breaks, constant coughing, and dehydration. if there is no significant improvement in symptoms, it is advisable to consult a clinician for confirmation of the diagnosis and to avoid further spread of the virus. the main treatment currently available is supportive care. although there is limited information on covid-19, it has been linked to ards. for patients with high fevers that could potentially lead to dehydration, intravenous fluids such as normal saline or lactated ringer's fluid can be administered sparingly to prevent lung overload. to reduce fever, antipyretic drugs such as paracetamol or acetaminophen can be administered. drugs such as remdesivir, chloroquine, ritonavir, tocilizumab, corticosteroids have been repurposed for the treatment of covid-19 (figure 11 ), although their clinical effectiveness has not yet been confirmed. [88] [89] [90] [91] [92] [93] unfortunately, the use of chloroquine and derivatives such as hydroxychloroquine, alone or in combination with other drugs, resulted in cardiac toxicity. investigations of these drugs were recently suspended by the who. 94 mechanism of action of selected repurposed drugs for treatment of covid-19. the elucidation of potential targets could lead to covid-19specific treatments (figure 11 ). approaches to anti-sars-cov-2 drug development include (a) inhibition of sars-cov-2 entry, (b) disruption of sars-cov-2 þ ssrna synthesis after entry, (c) inflammatory response suppression, and (d) disruption of sars-cov-2 translation. an fda-approved immunosuppressive and anti-malarial parasite drug (chloroquine) can inhibit the entry of covid-19 into the endosome after binding to ace-2 receptors, thereby preventing the release of þ ssrna for translation. 89, 93, 95, 96 studies also showed that hydroxychloroquine, an analog of chloroquine, was more potent and could be used in place of chloroquine. 97 remdesivir, a novel antiviral drug and nucleotide analog used to treat ebola virus infection, is currently under clinical development. the mechanism of the drug is reported to be at the post viral entry stage. 89 its mode of action is to inhibit the rdrp, preventing synthesis of the viral þ ssrna (figure 11 (2)). this drug is currently in phase 2 clinical trials. the protein inhibitor ritonavir has also been proposed for the treatment of covid-19. this drug interferes with the protease enzymes (proteinases) by inhibiting the conversion of polyproteins into the mature components (spike proteins, nucleocapsids) needed by the virus for multiplication (figure 11 (3)). another immunosuppressive drug, tocilizumab, has also been repurposed for covid-19 because of its ability to block il-6 and inhibit inflammatory responses. corticosteroids can also decrease inflammation by inhibiting phospholipases such as phospholipase a 2 , and thus suppress the excessive production of prostaglandins (figure 11 (4) ). sars-cov-2 appears unfamiliar to the human innate immune system. coupled with its emergence and its spread globally via human-to-human transmission, the development of vaccines for prevention is no longer a debate but a necessity. several vaccine platforms are being developed and some have entered clinical trials. however, approval by regulatory agencies such as the fda and manufacturing may take 12 to 18 months. 98 studies of the antiviral activity of host-directed drugs and compounds have identified two classes of molecules (protein biogenesis inhibitors and ligands of the sigma1 and sigma2 receptors) as effective inhibitors of viral infectivity. molecules that target the sigma1 and sigma2 receptors perturb the virus through different mechanisms from translation inhibitors, potentially including modulation of cellular stress responses. 99 the ligands haloperidol, pb28, pd-144418, and hydroxychloroquine are currently undergoing clinical trials in covid-19 patients. 100 these molecules exert their antiviral effects during viral replication by inhibiting nucleoprotein expression after viral entry has occurred. the lack of selectivity of chloroquine and hydroxychloroquine, including off-target effects on the human ether-a`-go-go-related gene (herg) and other molecules, may be related to the adverse cardiac reactions that have limited their use. 101 a recent study by gordon et al identified 332 highconfidence sars-cov-2-human protein interactions connected to multiple biological processes, including protein trafficking, translation, transcription and ubiquitination regulation. 102 the study identified 69 ligands, including fda approved drugs, compounds in clinical trials, and preclinical compounds that might theoretically have therapeutic effects as host-directed interventions against covid-19. to date, no known antiviral drugs nor vaccines against sars-cov-2 with proven clinical efficacy have been identified. part of the reason for the absence of such agents is limited information regarding the molecular details of the infection. to develop therapeutic interventions against covid-19, it is crucial to understand how the virus interactions with the host during infection. ace-2, a potential target for covid-19 treatment. ace-2 has been identified as the functional receptor for sars-cov-2. it is an outer membrane enzyme expressed in vascular endothelial cells, the renal tubular epithelium, and leydig's cells of the testes, lungs, heart, kidney, and gastrointestinal tract. 59, 103 it is a type-ii transmembrane metallocarboxypeptidase with its enzymatically active domain exposed at the cell surface. 104 ace-2 is a key player in the renin-angiotensin system (ras) and a target for treatment of hypertension. 105 ace-2 catalyzes the cleavage of angiotensin ii, a vasoconstrictor, into angiotensin 1-7, a vasodilator, thereby lowering blood pressure by negatively regulating ras. 106 ace-2 is a promising drug target for treating cov infection as well as other cardiovascular diseases. 5 ace-2 confers a protective effect against lung injury induced by viruses by increasing the production of angiotensin 1-7. the virus presumably causes lung damage by reducing ace-2 levels on cells through the process of degradation and internalization. 107, 108 because studies have shown that ace inhibitors and angiotensin ii receptor blockers could be used to up-regulate the expression and activity of ace-2 in hypertensive patients, similar strategies might reduce the severity of covid-19. 109, 110 recent studies have shown that the expression of human ace-2 is associated with sars-cov infection and that genetic variations of this receptor may contribute to susceptibility and/or resistance against infection. 111 for instance, a single-cell rna sequencing analysis of ace-2 revealed that type ii alveolar cells had higher expression of ace-2 in eight individual lung tissues obtained from normal donors. ace-2 expression was higher in the two male samples compared with the six female samples. additionally, ace-2 expression was higher in the only asian male in the study compared with caucasian and black americans, 59 which might explain why the four german cases showed mild clinical symptoms with no severe illness. 112 this implies that variation in ace-2 expression in covid-19 patients is likely to affect susceptibility, symptoms and intervention outcomes following sars-cov-2 infection. the degree of spread of covid-19 is currently about 5.3% and could potentially increase if precautionary measures are not considered. global prevention of the spread of covid-19 is therefore a crucial and urgent goal. to prevent further spread of this disease, detection and isolation of individuals with covid-19 is of the utmost priority. examples of measures to curb spread include self-quarantine, isolation of infected individuals, social distancing, good personal hygiene (frequent hand washing with soap and water/alcohol-based sanitizers and avoiding touching the eyes, nose and the mouth), and use of personal protective equipment. certain classes of compounds, called surfactants, are contained in soap and have the ability to neutralize microbes such as sars cov-2. 113 this is because soap can assemble into bubblelike structures called micelles that trap viral matter and other biomaterials. 114 surfactants in soap lather have their hydrophilic parts pointing outwards and interacting with solvent and their hydrophobic heads pointing inwards. this opens the coronavirus outer membrane and encapsulates viral molecules within micelles, thus making insoluble viral molecules easily soluble in water and effectively removing them from hands, surfaces or other areas after about 20 s. 115 therefore surfactants in soap can disrupt and sequester viruses and other contaminants while sanitizer and disinfectants are designed to kill sars-cov-2. 116 the outbreak of sars-cov-2 has become a global threat. however, information regarding this virus remains limited. the available information is inconsistent and there are constant data updates, which may contribute to variation between study results. for more consistent and accurate results, well-annotated data from clinical patients and subclinical subjects in normal populations could help to better understand the pandemic. the information provided in this review is based on data provided by the center for systems science and engineering (csse) at johns hopkins university during specific date ranges. key insights into the prevalence, pathophysiology, diagnosis, and potential treatment of sars-cov-2 are herein summarized. research efforts are being intensified to address the current challenges in the quest for adequate treatments, diagnostics, and vaccines. clinical studies into the genetic variation of receptors such as ace-2 in tissues and across populations will remain an active area of research until relevant targets and therapies are found. while the development of adequate treatments and vaccines for covid-19 is underway, it is advisable that good hygiene practices including washing of hands and social distancing should be practiced, and government guidance/guidelines should be followed. this will help to reduce the spread of the disease. we hope that the insights gained from this review will enable researchers to help patients develop accommodating lifestyles and improve the efficiency of health care practitioners. data are freely available at the following sources. centers for disease control and prevention (https://www.cdc.gov/). center for systems science and engineering (csse) at johns hopkins university (https://coronavirus.jhu. edu/map.html). worldometers (https://www. worldometers.info/coronavirus/?). zoonotic origins of human coronaviruses detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 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pathway and execute protective function in er stress outcomes related to covid-19 treated with hydroxychloroquine among in-patients with symptomatic disease cardiotoxicity of antimalarial drugs a sars-cov-2 protein interaction map reveals targets for drug repurposing angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target substernal electrical stimulation system compensation of ace2 function for possible clinical management of 2019-ncov-induced acute lung injury potent neutralization of 2019 novel coronavirus by recombinant ace2-ig evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin receptor blockers as tentative sars-cov-2 therapeutics structural variations in human ace2 may influence its binding with sars-cov-2 spike protein comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations cationic gemini-surfactants based on waste cooking oil as new 'green' inhibitors for n80-steel corrosion in sulphuric acid: a combined empirical and theoretical approaches the effect of surfactant concentration, salinity, temperature, and ph on surfactant adsorption for chemical enhanced oil recovery: a review the micelle thermodynamics and mixed properties of sulfobetaine-type zwitterionic gemini surfactant with nonionic and anionic surfactants managing neonates with respiratory failure due to sars-cov-2-authors' reply the authors declare that there is no conflict of interest. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. adewale oluwaseun fadaka https://orcid. org/0000-0002-3952-2098 nicole remaliah samantha sibuyi https:// orcid.org/0000-0001-7175-5388 ashwil klein https://orcid.org/0000-0002-5606-886x key: cord-334490-42gykxdx authors: kammerlander, andreas a.; mascherbauer, julia title: covid-19: frequently asked questions to the cardiologist date: 2020-07-24 journal: wien klin wochenschr doi: 10.1007/s00508-020-01696-9 sha: doc_id: 334490 cord_uid: 42gykxdx nan in december 2019, a previously unknown betacoronavirus was identified in a cluster of patients with pneumonia in wuhan, china [1] . the pathogen was later named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), sharing similarities with already known sars strains. the disease caused by sars-cov-2 was named coronavirus disease 2019 (covid-19) [2] . the main symptoms of covid-19 are cough and fever, although a broad spectrum of symptoms have been described, including the absence of both fever and cough in patients admitted to the intensive care unit (icu) with acute respiratory syndrome (ards) in need of mechanical ventilation [2] . given the presumably high number of undetected mild covid-19 cases, exact case fatality rates are currently unknown [3, 4] . identification of patients at highest risk, hence, is crucial to optimize patient triage, particularly with respect to icu capacities. covid-19 is considered primarily a respiratory disease; however, patients with cardiovascular disease represent a vulnerable population during this pandemic. furthermore, cardiac injury in patients with covid-19, defined as a rise of highly sensitive troponin levels, has been identified as a strong predictor of morbidity and mortality in covid-19 patients. the prevalence of cardiac injury in hospitalized patients was reported to range between 20% and 30% [5, 6] . the specific causes of troponin rise in covid-19 in patients without cardiac conditions, such as acute coronary syndrome (acs), aortic stenosis, hypertrophic cardiomyopathy, and tachycardia a. a. kammerlander, md, phd · ao. univ. prof. dr. j. mascherbauer ( ) division of cardiology, department of medicine ii, medical university of vienna, waehringer guertel 18-20, 1090 wien, austria julia.mascherbauer@meduniwien.ac.at arrhythmia are incompletely understood but two main theories prevail. firstly, elevated troponin levels are often observed in patients with critical illnesses and have been linked to the systemic inflammatory state (cytokine storm), which can involve damage to any organ, including the heart [7] . secondly, viral diseases are known to potentially cause myocardial inflammation/myocarditis [8] . indeed, several cases of fulminant myocarditis in patients with covid-19 have been reported [9] ; however, comprehensive clinical and imaging work-up of these patients in prospective cohorts is lacking. frequently, cardiologists face questions by patients as well as other healthcare professionals about cardiovascular-specific aspects of covid-19. the intention of this editorial is to provide useful sources with up to date information and discuss some of the most frequently asked questions. the sars-cov-2 enters human cells by binding to the angiotensin-converting enzyme 2 (ace2) receptor. concerns exist that ace inhibitors (ace-i) and angiotensin receptor blockers (arb) may be harmful due to an upregulation of ace2, which could facilitate infections with sars-cov-2 or increase disease severity [10] . given the frequent use of ace-i and arb in patients suffering from hypertension, coronary artery disease, heart failure, and chronic kidney disease, such considerations lead to great uncertainty among patients and physicians. in addition, early observational data reported that patients taking ace-i and arb were at higher risk for a dismal outcome in covid-19 [11] ; however, these early reports did not take into account the higher risk profile which we expect in patients taking ace-i and arb as compared to those having no previous medication. conversely, some animal models sug-gested a protective effect of ace-i and arb in acute lung injury [12, 13] . several retrospective studies reported that patients with covid-19 taking ace-i or arb were at lower risk for mortality compared to patients without ace-i or arb in adjusted models [14, 15] . dozens of prospective trials randomizing patients with covid-19 to either ace-i and arb or placebo are currently ongoing, which will augment our knowledge on potential benefits and harms of ace-i and arb in covid-19. as of june 2020, all major cardiovascular scientific organizations and official health regulatory bodies recommend not to discontinue ace-i and arb in patients with covid-19 [16, 17] . a beneficial effect, however, remains uncertain and ace-i and arb should not be used as treatment for covid-19 outside of the setting of clinical trials [18] . updated information may be found under: https:// www.covid19treatmentguidelines.nih.gov/concomita nt-medications/. (2) my patient with covid-19 has elevated troponin levels-what now? cardiac injury, defined as elevated troponin levels, is frequently observed in patients with covid-19. potential mechanisms include type i and type ii myocardial infarction (mi), takotsubo syndrome, shock as part of covid-19, myocarditis with direct viral and cytopathic effects, and cytokine release syndrome [19] . the european association of percutaneous cardiovascular interventions (eapci) issued a position statement on invasive management in patients with acs during the covid-19 pandemic [19] . the eapci recommends that in cases of mild troponin elevation (<2-3 times the upper limit of normal), particularly in older patients with pre-existing cardiac conditions, a work-up for type 1 mi is not indicated, unless strongly indicated by clinical presentation and electrocardiograph (ecg) findings. marked elevations in cardiac troponin levels (e.g. >5 times the upper limit of normal) may be found in acute respiratory syndrome, tachycardia, systemic hypoxemia, shock as part of covid-19, myocarditis, takotsubo syndrome, or type 1 mi triggered by covid-19. if symptoms and ecg findings are not suggestive of type 1 mi, the eapci recommends that echocardiographic work-up should be considered in order to help diagnose the underlying cause [19] . percutaneous coronary intervention (pci) centers have incorporated covid-19-specific triage systems, in accordance with local regulations and advisory statements. concerns exist that patients with acs may not attend medical services due to fear of an infection with sars-cov-2. indeed, studies demonstrated a 40% drop in st-elevation myocardial infarction (stemi) activations in pci centers [20] . patients and healthcare professionals should be encouraged to not delay consultation of pci centers if symptoms are suggestive of acs. of note, several antiviral drugs investigated for treatment of covid-19 are known to potentially cause myocardial damage but limited data on the clinical significance are available at this time [21] . updated information may be found under: https:// www.escardio.org/education/covid-19-and-cardiol ogy. (3) should i use anticoagulation in patients with covid-19? current literature suggests that patients with covid-19 are at increased risk for venous thromboembolism (vte). in critically ill patients, vte is frequent but seems to occur much more frequently in patients with covid-19, with reported incidence rates of up to one third in icu patients [22] . the us national institutes of health (nih) provide regularly updated recommendations on anticoagulation strategies in patients with covid-19 [21] . in nonhospitalized patients there are no data to support any use of anticoagulation. patients receiving antiplatelet or anticoagulant treatment for underlying conditions should continue their medications irrespective of a covid-19 diagnosis [21] . in patients hospitalized with covid-19, both nih and the anticoagulation forum recommend pharmacologic vte prophylaxis [21, 23] ; however, irrespective of laboratory findings, there are currently insufficient data to support routine screening for vte in patients without clinical signs suggestive of vte. patients with confirmed thromboembolic events who have been diagnosed with covid-19 should receive standard of care anticoagulation treatment [21] . updated information may be found under: https:// www.covid19treatmentguidelines.nih.gov/antithromb otic-therapy/. (4) how long can invasive procedures for cardiac conditions be postponed? as healthcare systems around the globe experience unprecedented challenges, clinical management of all chronic cardiac conditions has changed dramatically. the european society of cardiology (esc) issued a guidance document on how to prioritize management in patients with cardiac conditions [24] . patients with acs, left main pci, battery replacement in case of battery end of life and pacing dependency, and valvular heart disease, who are hemodynamically unstable should be considered emergency and urgent priority and invasive procedures should not be postponed. symptomatic patients with severe aortic stenosis should undergo valve replacement within 3 months in cases of advanced disease state (aortic valve area <0.6 cm 2 , mean transvalvular gradient >60 mm hg). all cases should be discussed by the interdisciplinary heart team and indications for transcatheter aortic valve implantation (tavi) extended to intermediate and selected low-risk patients. increased use of transfemoral tavi (when feasible) may allow optimal utilization of resources by avoiding general anesthesia covid-19: frequently asked questions to the cardiologist k main topic and intubation, shortening (or preventing) icu stay and accelerating hospital discharge and recovery. updated information may be found under: https:// www.escardio.org/education/covid-19-and-cardiol ogy/esc-covid-19-guidance. a novel coronavirus from patients with pneumonia in china clinical characteristics of coronavirus disease 2019 in china a novel coronavirus emerging in china-key questions for impact assessment epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study association of cardiac injury with mortality in hospitalized patients with covid-19 in wuhan, china cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) myocardial injury in critically ill patients. a frequently unrecognized complication coronavirus fulminant myocarditis saved with glucocorticoid and human immunoglobulin receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus vital surveillances: the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china, 2020. china cdc weekly angiotensin-converting enzyme 2 (ace2) mediates influenza h7n9 virus-induced acute lung injury risks and impact of angiotensin-converting enzyme inhibitors or angiotensinreceptor blockers on sars-cov-2 infection in adults association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan, china association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 ema advises continued use of medicines for hypertension, heart or kidney disease during covid-19 pandemic hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 considerations for certain concomitant medications in patients with covid-19. nih covid-19 treatment guidelines eapci position statement on invasive management of acute coronary syndromes during the covid-19 pandemic reduction in stsegment elevation cardiac catheterization laboratory activations in the united states during covid-19 pandemic antithrombotic therapy in patients with covid-19. nih covid-19 treatment guidelines incidence of thrombotic complications in critically ill icu patients with covid-19 thromboembolism andanticoagulanttherapy during thecovid-19 pandemic: interim clinical guidance from the anticoagulation forum esc guidance for the diagnosis and management of cv disease during the covid-19 pandemic conflict of interest a.a. kammerlander and j. mascherbauer declare that they have no competing interests. key: cord-311099-59pnm4fn authors: lubel, john s; herath, chandana b; burrell, louise m; angus, peter w title: liver disease and the renin–angiotensin system: recent discoveries and clinical implications date: 2008-06-28 journal: j gastroenterol hepatol doi: 10.1111/j.1440-1746.2008.05461.x sha: doc_id: 311099 cord_uid: 59pnm4fn the renin–angiotensin system (ras) is a key regulator of vascular resistance, sodium and water homeostasis and the response to tissue injury. historically, angiotensin ii (ang ii) was thought to be the primary effector peptide of this system. ang ii is produced predominantly by the effect of angiotensin converting enzyme (ace) on angiotensin i (ang i). ang ii acts mainly through the angiotensin ii type‐1 receptor (at(1)) and, together with ace, these components represent the ‘classical’ axis of the ras. drug therapies targeting the ras by inhibiting ang ii formation (ace inhibitors) or binding to its receptor (angiotensin receptor blockers) are now in widespread clinical use and have been shown to reduce tissue injury and fibrosis in cardiac and renal disease independently of their effects on blood pressure. in 2000, two groups using different methodologies identified a homolog of ace, called ace2, which cleaves ang ii to form the biologically active heptapeptide, ang‐(1–7). conceptually, ace2, ang‐(1–7), and its putative receptor, the mas receptor represent an ‘alternative’ axis of the ras capable of opposing the often deleterious actions of ang ii. interestingly, ace inhibitors and angiotensin receptor blockers increase ang‐(1–7) production and it has been proposed that some of the beneficial effects of these drugs are mediated through upregulation of ang‐(1–7) rather than inhibition of ang ii production or receptor binding. the present review focuses on the novel components and pathways of the ras with particular reference to their potential contribution towards the pathophysiology of liver disease. most of us can recall the schema of the renin-angiotensin system (ras) taught in physiology lectures (fig. 1) . the system is often depicted as a simple enzyme cascade starting with the degradation of angiotensinogen (derived from the liver) by circulating renin (secreted from the juxtaglomerular apparatus of the kidney) to form angiotensin i (ang i). subsequent enzymatic action by angiotensin converting enzyme (ace) in the capillaries of the lung yields the predominant effector peptide of the system, angiotensin ii (ang ii). [1] [2] [3] two receptors for ang ii have been cloned and characterized, the angiotensin ii type-1 receptor (at1) is the abundant receptor in adult life, whereas the angiotensin ii type-2 receptor (at2) is present in the fetus and persists in the central nervous system of adults. [4] [5] [6] binding of ang ii to the at1 receptor mediates a number of diverse effects including vasoconstriction and sodium hemostasis. ang ii also participates in inflammation and wound healing through the release of critical cytokines and production of extracellular matrix. 7 the effect of ang ii on vascular tone and systemic blood pressure has been extensively studied and is mediated through direct effects on vascular smooth muscle cells or indirectly by increasing vascular sympathetic tone. sodiumconserving effects occur via reabsorption of sodium by the renal tubules as well as stimulating the adrenal gland to secrete aldosterone. the effect of ang ii to stimulate thirst is mediated through at1 receptors in the brain. 8 figure 1 illustrates the conventional view of the 'classical' ras. this schema is useful as it clarifies how drugs like ace inhibitors or at1 receptor blockers (arb) produce their beneficial therapeutic effects in cardiovascular and renal disease. however, there have been a number of major advances in our understanding of the ras which have made it clear that the system is far more complex than this 'classical' view would suggest (fig. 2) . one key point of understanding is that ang ii is just one member of a family of angiotensin peptides produced by the ras. ang ii consists of eight amino acids, which, like other peptides, has a free amino group at one end (n-terminus) and a free carboxyl group (c-terminus) at the other. ang ii can also be denoted as ang(1) (2) (3) (4) (5) (6) (7) (8) , where the first amino acid is at the n-terminus and the eighth amino acid is at the c-terminus. therefore, cleavage of amino acids from either end of the ang ii molecule can generate smaller peptide fragments (fig. 3) . for example, removal of the n-terminus amino acid results in the generation of a peptide consisting of seven amino acids starting from the second amino acid of ang ii, and is denoted by ang-(2-8) (historically also known as ang iii). of the fragments that can be generated from ang ii, only three are known to be physiologically relevant. 9 two are derived from n-terminus cleavage, ang-(2-8) (ang iii) and ang-(3-8) (ang iv) and one formed by cleavage of a single amino acid from the c-terminus, angiotensin-(1-7) (ang-(1-7)). ang iii is formed following cleavage of the aspartate-arginine bond of ang ii by aminopeptidase a, and ang iv can be formed by further cleavage of ang iii by aminopeptidase b or n. ang iii shares many of the properties of ang ii with 40% of the pressor activity and 100% of the aldosterone stimulating activity. ang iv has its own distinct receptor (at4) and has central nervous system effects together with some opposing actions to ang ii. 10 ang-(1-7) is generated from cleavage of either ang ii or ang i and has been the focus of much research since the discovery that it has biological functions that oppose those of ang ii. for decades, the 'classical' arm of the ras was recognized as being the only system of biological relevance. however, this interpretation was challenged in the late 1980s with the discovery of ang-(1-7) and description of its diverse biological functions. 11 this was followed by studies which clearly demonstrated new components of the ras, such as ace2 12, 13 and the ang-(1-7) receptor, mas. 14 these new components, together with the effector molecule ang-(1-7), form the axis which we now recognize as the 'alternative' arm of the ras. the discovery of these new ras components provided some missing connections to the hitherto complex biochemical pathways of the ras. the new components of the alternative arm of the ras are reviewed below with a major emphasis on their potential contribution towards the pathophysiology of liver disease. interest in alternative components of the ras was re-ignited in the year 2000 when two groups independently discovered an enzyme similar to ace in human tissue. 12, 13 this homolog of ace was initially called hace but has subsequently been named angiotensin converting enzyme 2 (ace2). although structurally similar to ace, ace2 has different substrate affinities and resists inhibition by ace inhibitors. ace2 is a zinc-metalloproteinase and, like ace, is a type-1 transmembrane protein. it consists of 805 amino acids with a single transmembrane alpha-helical portion, an external n-terminus portion containing the catalytically active enzyme and an internal inactive c-terminus section. a transmembrane proteinase, adam 17 acts as a 'sheddase' releasing the active enzyme into the extracellular environment (fig. 4) . 15 the released ace2 (soluble ace2) is a carboxypeptidase, capable of cleaving a single amino acid from the c-termini of its various substrates, including, ang ii, ang i, des-arg9-bradykinin, neurotensin 1-13 and kinetensin (see review by burrell and colleagues). 16 importantly, ace2 can generate ang-(1-7) directly from ang ii or indirectly by cleaving ang i into an inactive intermediate fragment, ang-(1-9), which is then cleaved by ace to produce ang-(1-7) (fig. 2 ). of these two ace2 pathways, the conversion of ang ii into ang-(1-7) is kinetically favoured 400-fold compared to the conversion of ang i to ang-(1-9). 17, 18 in addition to its role in the 'alternative' ras, the ace2 transmembrane protein has, interestingly, been identified as a receptor site for spike proteins of the severe acute respiratory syndrome (sars) coronavirus, thereby facilitating infection of target cells. 19 much of the work on ang-(1-7) has been carried out in animals and, to date, this peptide has been shown to have antihypertensive, anti-arrhythmic, and cardioprotective properties [20] [21] [22] as well as anti-trophic properties in vascular endothelial cells, smooth muscle cells, cardiac myocytes and cardiac fibroblasts. [23] [24] [25] [26] in contrast to ang ii, ang-(1-7) also has anti-inflammatory, antifibrotic 27 and anti-thrombotic properties. 28, 29 as a result of these studies, ang-(1-7) has been proposed to represent the effector peptide of a counterbalancing arm of the ras, capable of opposing the deleterious actions of ang ii. the putative receptor for ang-(1-7) is the g protein-coupled receptor encoded by the mas proto-oncogene, 14 although other receptors may well exist. 30 thus, ace2 together with ang-(1-7) and the mas receptor represent an 'alternative' arm or axis of the ras which may present a counterfigure 1 'classical' renin-angiotensin system (ras). the ras is depicted here as a linear cascade leading to the generation of angiotensin ii (ang ii) through the enzymatic action of renin on angiotensinogen and angiotensin converting enzyme (ace) on angiotensin i (ang i). there are two known receptors for angiotensin ii, angiotensin ii type-1 receptor (at1) and angiotensin ii type-2 receptor (at2). the at1 receptor is thought to play a more important role than the at2 receptor in human disease. balancing system to the deleterious ace/ang ii/at1 axis (fig. 5) . clearly, ace2 holds a central role in the ras influencing both axes, as it is capable of simultaneously degrading ang ii and generating ang-(1-7) (fig. 2) . angiotensin converting enzyme is known to participate actively in the kallikrein-kinin system by degrading bradykinin (fig. 2) . 31 inhibitors of ace can therefore lead to the accumulation of bradykinin, which may contribute to the antihypertensive properties of these drugs, as well as to some of the observed side-effects, such as chronic cough and angioedema. in the liver, bradykinin binds to the b2 receptor and causes increases in hepatic resistance and elevation of portal pressure. 32 in other vascular beds, bradykinin induces vasodilatation on binding to the b2 receptor, and ang-(1-7) has been shown to induce bradykinin-mediated relaxation in porcine coronary arteries. 33 a possible explanation for this is that ang-(1-7) has ace inhibitory properties that prevent acemediated degradation of bradykinin. 34 in recent years, scientists have departed from the traditionally held view of the ras being exclusively a circulating endocrine system and have realized that many organs, such as the heart, kidney, liver and pancreas, constitutionally express all the 'classical' ras components required for a functioning, autonomous intra-organ contemporary renin-angiotensin system (ras). angiotensin converting enzyme (ace) 2 has a central role in the ras influencing both the 'classical' and 'alternative' axes, as it degrades angiotensin ii (ang ii) while simultaneously generating ang-(1-7). ace is important in generating ang ii, but is also responsible for the degradation of ang-(1-7) into the inactive peptide fragment ang(1) (2) (3) (4) (5) . the ras interacts with the kinin system through ace degradation of bradykinin. the two axes of the ras and the kinin system are shaded grey. enzymes are shown in yellow boxes and peptides in blue boxes. aminopeptidase a (apa) and aminopeptidase n (apn) sequentially cleave ang ii to form angiotensin iii and angiotensin iv, respectively. neprilysin (nep) is involved in both the ras and the kinin system. possible peptide-receptor interactions are shown by dashed lines. peptide structure and fragments of angiotensin i. angiotensin i is a decapeptide (ang-(1-10)) which can be fragmented by various enzymes into four peptides with biological activity; angiotensin ii (ang-(1-8)), angiotensin iii (ang-(2-8)), angiotensin iv (ang-(3-8)) and angiotensin 1-7 (ang-(1-7)). further enzymatic degradation of ang 1-7 yields the inactive fragment angiotensin 1-5 (ang-(1-5)). aminopeptidases are shown in blue and cleave amino acids from the n-terminus, whereas carboxypeptidases are shown in red and cleave amino acids from the c-terminus. amino acids are given numerical values, where 1, aspartic acid; 2, arginine; 3, valine; 4, tyrosine; 5, isoleucine; 6, histidine; 7, proline; 8, phenylalanine; 9, histidine; 10, leucine. ras. 35, 36 these locally generated angiotensin peptide fragments have been demonstrated to have a multitude of actions, being implicated in cell growth, cell proliferation, apoptosis, reactive oxygen species generation, inflammation, and fibrogenesis. although conceptually separate, the local intra-organ ras and the systemic ras must interact and the final peptide products will depend on the interplay between the two. despite the discovery of ang-(1-7) and the recognition that many of its actions oppose ang ii, the importance of this heptapeptide fragment of ang ii remained elusive until recently. it is now clear that in the diseased liver, not only are the 'classical' ras components such as renin, ace, ang ii and the at1 receptor overexpressed, but, importantly, components of the 'alternative' ras, such as ace2, ang-(1-7) and the mas receptor are also upregulated. 37, 38 the implication from these studies is that the 'classical' components contribute to the fibrotic process whereas the 'alternative' components may be upregulated in an attempt to restore the status quo. in liver disease, architectural changes to the microscopic structure of the liver occur as a result of inflammation and fibrosis. these changes lead to capillarization of the hepatic sinusoids, increased extracellular matrix (ecm) formation and elevated hepatic resistance; the latter impedes liver blood flow and leads to portal hypertension. 39 stretching of the portal vein (as with increased hepatic resistance to blood flow) and oxidative stress together cause release of vasodilators, including nitric oxide, which induce a number of compensatory mechanisms important for restoring the functional blood volume. these mechanisms are effected via sodium and water preservation and stimulation of the sympathetic nervous system, which together contribute to the development of ascites, edema, hepatorenal syndrome, and a hyperdynamic circulation, all of which are typically seen in patients with advanced liver disease. the ras is involved with all these processes. as the result, manipulation of the ras with either antagonists of the 'classical' pathway, or agonists of the 'alternative' pathway could have potential therapeutic benefits. balanced against the possible benefits are the potential side effects of such therapy, as the compensatory mechanisms activated by the systemic ras are necessary to maintain an adequate circulation. counterbalancing effects of the two axes of the renin-angiotensin system (ras). the ras can be thought of as two counterbalancing axes. the angiotensin converting enzyme (ace)/angiotensin ii/at1 receptor axis causes vasoconstriction, salt retention, inflammation, fibrosis and thrombosis, whereas the ace2/angiotensin 1-7/mas receptor axis has opposing effects. hepatic stellate cells (hsc) are thought to play a pivotal role in fibrogenesis within the liver, 40 and there is a large body of evidence to support the hypothesis that ang ii promotes activation, and dedifferentiation of these cells into myofibroblasts. furthermore, ang ii encourages myofibroblast contraction, proliferation and promotes release of inflammatory cytokines as well as the deposition of extracellular matrix (ecm). although both of the ang ii receptors (at1 and at2) are expressed in the liver, the at1 receptor is far in abundance and is thought to be responsible for most of the ang ii-mediated effects. studies using gene-deletion mice have demonstrated that at1a receptor-deficient mice 41 are protected from hepatic fibrosis whereas at2 receptor-deficient mice have worse fibrosis. 42 a great deal of evidence supporting the role of the ras in hepatic fibrosis has come from animal studies using ace inhibitors and angiotensin receptor blockers (arb). numerous studies using a variety of animal models have demonstrated antifibrotic effects of these drugs. [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] however, there appear to be some conflicting observations reported in the literature. for example, losartan treatment failed to influence either liver injury or progression of fibrosis in an animal model of non-alcoholic steatohepatitis (nash). 54 [editor's note: a detailed review of animal models of nash has been written by larter and yeh for a later article in this basic science miniseries.] in contrast, a study with a similar model of nash but using the arb olmesartan, demonstrated a 70% reduction in fibrosis in the arb-treated group. 55 the avid interest in ras-blocking drugs is, in part, related to their relative safety in humans and widespread use in cardiovascular and renal medicine. despite the large number of animal studies, there is a relative paucity of human data to support the use of these drugs in human liver disease. in part, this could be due to the need to perform multiple liver biopsies to histologically confirm resolution of fibrosis, which, outside the setting of posttransplantation recurrent hepatitis c, is rarely indicated in 2008. in addition, the slow progression of fibrosis in most diseases such as hepatitis c and non-alcoholic fatty liver disease (nafld) make it difficult to detect possible beneficial effects of antifibrotic therapy, unless studies are conducted over a number of years. a pilot study examining the effects of 6 months of losartan treatment on liver fibrosis in chronic hepatitis c demonstrated a significant decrease in fibrosis stage in the treated group compared to control patients. 56 in support of this, a study using candesartan for 48 weeks in 24 compensated child a and b cirrhotic patients demonstrated a significant reduction of plasma hyaluronic acid levels, a surrogate marker for fibrogenesis. however, in this study, two of three serum markers of fibrosis used showed no improvement, and there were no histological data provided; this makes it difficult to evaluate any effects on architectural changes. 57 a number of other studies have reported possible antifibrotic effects of ras blockers in patients with hepatitis c. in one study, 30 hepatitis c virus (hcv)-infected patients with mild fibrosis were treated with losartan 50 mg/day and ursodeoxycholic acid 600 mg/day whereas controls received ursodeoxycholic acid alone. there were significant reductions in serum markers of hepatic fibrosis such as transforming growth factor b1 (tgf-b1) and type iv collagen in the losartan and ursodeoxycholic acid group, but no significant changes in fibrosis score between the groups. 58 another report described outcomes in patients with hepatitis c treated with low-dose interferon (ifn alpha 3 ¥ 10 6 iu 3 times a week for 12 months) in combination with the ace inhibitor, perindopril (4 mg/day). treatment was accompanied by significant improvement in serum markers of fibrosis (hyaluronic acid, type iv collagen 7s and procollagen iii-n-peptide), but histological analysis was not carried out. 59 although this study did not have a perindopril monotherapy group, a subsequent study by the same group demonstrated that perindopril alone decreased serum fibrosis markers in patients with chronic hepatitis c. the addition of interferon significantly augmented the effect of perindopril monotherapy. finally, a retrospective review compared liver histology in liver transplant patients with recurrent hepatitis c who were taking ras-blocking drugs (n = 27) with those who were not (n = 101). the group taking ras blockers were less likely to develop severe hepatic fibrosis (bridging fibrosis or cirrhosis) at 1 and 10 years after transplantation than were the control group (15% vs 35% at 1 year [p < 0.05], and 35% vs 70% at 10 years, respectively). 60 only small studies have looked at ras blockers and nash. one such study (n = 7) found that giving losartan (50 mg/day for 48 weeks) in hypertensive patients with nash reduced serum tgf-b1, serum ferritin and aminotransferase levels. five patients showed improvement in the grade of hepatic necroinflammation. 61 the study design could have been improved had the investigators examined pre-and post-treatment histology and biochemical markers in a placebo group. in a subsequent study, the pre-and post-treatment biopsies of seven patients with nash treated with losartan (50 mg/day for 48 weeks) were compared with eight patients with nafld who acted as a control group. the treatment group showed a significant improvement in necroinflammatory grade, stage of fibrosis, significantly fewer activated hsc and a mild increase in quiescent hsc at the end of 48 weeks. 62 however, the lack of a proper randomized control group is a particular problem in studies of patients with nash, as the disease can improve in response to changes in lifestyle. fixed changes in hepatic architecture account for approximately 70% of the total resistance to portal blood flow in the cirrhotic liver. 63 the remaining 30% results from a reversible or 'dynamic' resistance caused by the contraction of activated myofibroblasts positioned around the sinusoidal endothelial cells within the space of disse. as portal resistance increases, a number of factors, including distension of the portal venous system, endotoxemia and oxidative stress result in the release of mediators, including nitric oxide, which dilate the mesenteric and systemic vasculature. activation of compensatory mechanisms designed to restore functional blood volume results in sodium and water retention, stimulation of the sympathetic nervous system and the development of a hyperdynamic circulation. this cascade of events contributes to many of the key features and complications of advanced liver disease including development of ascites, edema and the hepatorenal syndrome. the ras is involved with all these processes. manipulation of the ras with either antagonists of the 'classical' pathway, or agonists of the 'alternative' pathway therefore has potential for therapeutic benefit. variceal bleeding is one of the most important causes of morbidity and mortality in patients with portal hypertension. a number of pharmacological approaches have been developed for the prevention and treatment of this problem. non-selective ß-adrenergic antagonists (beta-blockers) lower portal pressure by decreasing cardiac output and constricting the mesenteric vascular bed but have no direct effect on intrahepatic resistance to portal flow. these drugs have become the mainstay of treatment for the prevention of variceal bleeding. however, only 36% of patients achieve the target reduction in portal pressure of 20%, as measured by hepatic venous pressure gradient (hvpg), and they are poorly tolerated in patients with severe liver disease. 64 as a result, there is a major interest in the development of other pharmacological therapies which can lower portal pressure. interestingly, betablockers interact with the ras by inhibiting renin release, but have not been shown to impact on the development or progression of hepatic fibrosis. in contrast, the use of either ace inhibitors or arb to reduce portal pressure is an attractive proposition, as these drugs have the additional potential benefit of slowing the progression of hepatic fibrosis. ang ii is a potent vasoconstrictor, and myofibroblasts derived from hsc express the at1 receptor and contract in response to ang ii. 35, 65 additionally, cirrhotic rat livers are hyperresponsive to ang ii with an increased portal pressure compared to those from healthy rats as a result of increased expression of at1 receptors. 66 this finding is of interest given that the relative importance of ang ii as a mediator of increased portal resistance has been questioned, 67 based on a study of hepatic hemodynamics in isolated perfused cirrhotic rat livers which suggested that ang ii-mediated vasoconstriction is attenuated in the cirrhotic liver. 68 following some persuasive animal studies, 53,69 the effects of at1 blockade on portal hypertension have been examined in a number of human studies. 70 despite some encouraging initial studies showing a significant reduction of portal pressure by arb, subsequent well-designed studies have failed to confirm these findings. schneider and colleagues reported a dramatic reduction in hvpg with losartan in both moderate and severe portal hypertensive patients, but with only a 3 mmhg drop in mean arterial pressure (map). 71 these findings were markedly different to a subsequent randomized controlled trial comparing the hemodynamic effects of losartan with propanolol 72 given for 6 weeks following an index variceal bleed. losartan failed to reduce hvpg, yet resulted in a significant reduction of map by 8%. treatment tolerance was equivalent. the hemodynamic effect of losartan was further corroborated by a recent small study of 12 pre-ascitic patients which also found that losartan had no affect on hvpg, but did cause a drop in map of 7.8%. 73 irbesartan, another arb, produced only modest reduction in portal pressures (12% ϯ 6.6%, p < 0.05) in a randomized, placebo-controlled, double-blind study. importantly, however, this was associated with significant arterial hypotension and significant renal impairment in 22% of patients. in this study, plasma renin activity before treatment was a predictor of patients that would not tolerate treatment. 74 the explanation for this adverse effect is that the ras is known to play a central role in the homeostatic response to vasodilatation in patients with portal hypertension. the ras, together with other compensatory systems, the posterior pituitary (through vasopressin secretion) and the sympathetic nervous system, endeavors to restore circulatory volume and organ perfusion by inducing vasoconstriction and sodium and water retention. in patients with advanced cirrhosis, plasma renin, ang ii, ace and aldosterone levels are all increased 75 and, within the kidney, ang ii is critical for maintenance of renal perfusion pressure and an adequate glomerular filtration rate (gfr). as liver disease progresses, the decrease in effective circulatory volume results in vasoconstriction of the glomerular afferent circulation, renal hypoperfusion and a fall in gfr. in response to renal hypoperfusion, ang ii selectively constricts the efferent glomerular arterioles; this restores glomerular perfusion pressure and gfr. the maintenance of adequate renal perfusion is therefore ace dependent. furthermore, ace inhibition results in a rapid fall of gfr. 76, 77 this adverse effect of ras inhibition on renal function in patients with advanced cirrhosis represents a major disadvantage for the use of this class of drug for the treatment of portal hypertension. a recent study by debernardi-venon and colleagues examined the effects of candesartan treatment for 48 weeks on 24 compensated child a and b cirrhotic patients. treatment was well tolerated, with a mild but significant reduction in hvpg in more than 70% of those treated. furthermore, 25% of patients treated achieved a 20% reduction in their hvpg. interestingly, the changes in hvpg correlated well with those observed for plasma hyaluronic acid. however, the treatment group was preselected in that patients were excluded from analysis if they had large varices, evidence of significant arterial hypotension or renal impairment. 57 angiotensin receptor blockers have also been studied in portal hypertensive gastropathy; at least one study has reported a positive benefit from their use. 78 the effects of ace inhibitors on portal pressure have also been examined in a few small studies, but the results generally have been disappointing, with poor agreement between studies. 79-82 a number of explanations have been proposed to explain the lack of uniformity in results from clinical studies investigating the benefits and adverse effects of ras inhibitors. there are known genetic polymorphisms for the at1 receptor gene and genes responsible for cleaving angiotensin i, including ace; 83 these may confer patient-to-patient variations in response to these drugs. this has led to the suggestion that genetic testing may help determine which patients are likely to have a positive response to therapy. 84 in addition, chronic ace inhibition may not lead to sustained ang ii suppression because of increased renin activity and upregulation of alternative enzymes, such as hepatic chymase, which is capable of generating ang ii from ang i. 85, 86 furthermore, chronic use of arb also results in hyper-reninemia and elevated ang ii levels; the latter increasingly compete with the at1 receptor antagonist for binding sites on the at1 receptor molecule. 87, 88 finally, it has also been claimed that there is tissuedependent responsiveness to ace inhibitors and arb and, at current therapeutic dosing, both classes of drug may not completely inhibit their respective targets. 88 to date, no studies have examined the effects on portal pressure of combined therapy with an ace inhibitor and arb; theoretically, this may overcome some of the possible issues of ang ii reactivation with use of ace inhibitors alone. in summary, the use of ras inhibitors (other than betablockers) to reduce portal pressure has been disappointing. at the doses used in clinical trials, these drugs appear to have only minor effects on portal pressure but very significant side-effects, includ-ing systemic hypotension and renal impairment. these complications are a useful reminder of the homeostatic role the ras plays in maintaining map and gfr in the vasodilated patient with severe liver disease. [89] [90] [91] based on the current available evidence, the use of either ace inhibitors or arb for reducing portal pressure remains controversial and cannot be recommended outside clinical trials. as outlined above, there is increasing evidence that both the 'classical' and the 'alternative' ras are upregulated in chronic liver disease. 38, 66 it has recently been suggested that the progression of liver fibrosis may be influenced by a balance between ace and ace2 activation. 92 in both an animal model of secondary biliary fibrosis and in humans with hepatitis c, ace2 gene and activity are upregulated. 38, 66 as fibrosis worsens, the progressive rise in ace and at1 gene expressions coincide with an increase in ace2 and mas expression, together with increased plasma levels of both ang-(1-7) and ang ii. 66, 93 cirrhotic livers have a greater capacity than healthy livers to convert ang ii to ang-(1-7) because of upregulated ace2 gene and protein expression (fig. 6 ). in addition, the hepatic production of ang-(1-7) from ang ii is augmented by ace inhibition. 38, 66 this increased ang-(1-7) production in the presence of an ace inhibitor can be explained by the fact that ang-(1-7) is cleaved by ace to produce the inactive peptide ang-(1-5) (fig. 2) . inhibition of ace therefore increases ang-(1-7) half-life, leading to an increase in net production and accumulation of ang-(1-7). [94] [95] [96] evidence for a beneficial role of ang-(1-7) in hepatic fibrosis has been provided by a study examining the effects of the mas receptor antagonist [7-d-ala]-ang-(1-7) (a779). treatment with a779 worsened experimental liver injury with increases in tgf-b1 and hydroxyproline levels; this infers that mas receptor stimulation plays a protective role in liver fibrosis. 93 further compelling evidence for a beneficial role of ang-(1-7) has come from a recent rat study presented at aasld 2007 by our group. 97 we demonstrated that ang-(1-7) infusion in bile duct-ligated rats attenuated fibrosis as quantified using metavir fibrosis score, hydroxyproline content, and type 1 collagen mrna expression. alpha-smooth muscle actin (a-sma) gene and protein expression were also reduced, indicating that hepatic stellate cell activation was inhibited by ang-(1-7). interestingly, ang-(1-7) infusion also inhibited ace gene and protein expression, and resulted in downregulation of mas receptor gene expression. the ang-(1-7) infusion group also showed decreased mrna expression levels for connective tissue growth factor (ctgf, also known as ccn2) and vascular endothelial growth factor (vegf), two critical growth factors implicated in fibrosis and tissue repair. this is the first direct evidence showing that ang-(1-7) can ameliorate hepatic fibrosis. 97 evidence from studies in ace2 deletion mice further supports a central role of ace2 in regulating fibrosis in liver disease. 98 despite a number of reports that ang-(1-7) is a vasodilator, 99 experiments on rat isolated perfused livers have failed to demonstrate any vasodilatory effect in normal or cirrhotic livers. 66, 100, 101 likewise, experiments in isolated vessels from normal and cirrhotic rats also failed to show any direct vasodilatory effect of this peptide. 38 conversely, ang-(1-7) has been shown to enhance acetylcholine-mediated vasodilatation in aortic rings from cirrhotic rats. 38 the vasodilatory effects of ang-(1-7) are thought to be mediated through increased production of nitric oxide (no). 34, 102 hence, the absence of a vasodilatory effect by ang-(1-7) in the cirrhotic rat liver could be explained by the known general impairment of no-dependent vasodilatation in the cirrhotic liver due to endothelial dysfunction. 103, 104 in summary, there is considerable evidence supporting the concept that opposing axes of the ras are involved in the pathogenesis of chronic liver injury. on one side, the ace/ang ii/at1 receptor axis promotes liver injury and deposition of extracellular matrix, on the other, ace2/ang-(1-7)/mas receptor promotes collagen degradation and resolution of inflammation. both axes are upregulated in liver disease, but presumably the balance between the two systems is critical in determining the net effect. 92 for many years researchers in the field of ras have concentrated on blocking components of the 'classical' system in an attempt to reduce fibrosis. however, both ace inhibitors and arb have an impact on other components of the 'classical' ras apart from ang ii, as plasma renin activity and ang i levels increase following chronic therapy. 105 this, in part, explains the phenomenon of 'angiotensin ii reactivation' and 'aldosterone escape' whereby chronic administration of an ace inhibitor fails to completely suppress either plasma ang ii or aldosterone production. [106] [107] [108] the actual mechanism underlying this phenomenon remains elusive, figure 7 effects of angiotensin converting enzyme (ace) inhibitors and angiotensin type-1 receptor (at1) receptor blockers (arb) on the two axes of the renin-angiotensin system (ras). the ras is shown as a balance with 'classical' and 'alternative' axes counterbalancing each other. ace inhibitors (acei) cause an initial reduction in angiotensin ii (ang ii), but after chronic administration increases in plasma renin activity and plasma angiotensin i (ang i) levels occur. both ang ii and aldosterone levels can subsequently rise as a consequence of non-ace-dependent pathways facilitated by enzymes such as chymase. both acei and arb result in elevated levels of ace2 and ang-(1-7) which possibly contribute to the effects of these drugs. manipulations of the ras aimed at tipping the balance in favour of 'alternative' components represents a potential target for antifibrotic therapies. liver and the renin-angiotensin system js lubel et al. although non-ace-dependent pathways involving enzymes like chymase, which is capable of generating ang ii, may play an important part. 85, 109 interestingly, ace inhibitors and arb have a profound impact on the 'alternative' system by causing significant increases in ang-(1-7). 94, 95, 110, 111 it has been postulated that some of the beneficial effects observed with arb and ace inhibitors are mediated through ang-(1-7). 28, 29, [112] [113] [114] [115] [116] in support of this, ace2 activity and gene expression are both increased in the heart by arb or ace inhibitors. 112, 117 the elevated ace2 activity in such tissues would result in both diminished levels of ang ii and simultaneous elevations in tissue ang-(1-7), thus tipping the ras balance in favor of the 'alternative' axis. interestingly, our own studies and those of others have shown that ang-(1-7) can inhibit ace activity and gene expression; this would further tend to alter the balance of the two axes towards the 'alternative' axis ( fig. 7) . 34, 97 conclusions and future directions our understanding of the ras has considerably expanded since the discovery of ace2. emerging evidence supports the hypothesis that the ras consists of two opposing axes. manipulation of the ras, by either blocking the 'classical' ras or by stimulating the 'alternative' ras represents a potential target for antifibrotic and portal hypertension therapy. limitations to treatment may be the side-effects of such drugs, particularly their impact on arterial blood pressure and renal function. current therapies such as ace inhibitors and arb used in cardiovascular and renal fibrosis have been shown to impact on both the 'classical' and 'alternative' pathways. the elevated ang-(1-7) plasma levels caused by these drugs may represent a mechanism by which these drugs exert some of their effects. new drugs which mimic the effects of ang-(1-7) have been developed. this represents a novel set of agents that could be used for the treatment of hepatic fibrosis or portal hypertension. one such drug, ave0991 is a non-peptide analog of ang-(1-7). this orally active ang-(1-7) receptor agonist represents an entirely new class of drug 118 spawn from the latest insights into the complexities of the contemporary ras and has possible novel therapeutic applications in liver disease. purification and properties of angiotensin-converting enzyme from hog lung biochemistry and kinetics of the renin-angiotensin system renin-angiotensin system: a dual tissue and hormonal system for cardiovascular control isolation of a cdna encoding the vascular type-1 angiotensin ii receptor cloning and expression of a complementary dna encoding a bovine adrenal angiotensin ii type-1 receptor molecular cloning of a novel angiotensin ii receptor isoform involved in phosphotyrosine phosphatase inhibition signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin ii in 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rats vasodepressor actions of angiotensin-(1-7) unmasked during combined treatment with lisinopril and losartan evidence that prostaglandins mediate the antihypertensive actions of angiotensin-(1-7) during chronic blockade of the renin-angiotensin system upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors pharmacological effects of ave 0991, a nonpeptide angiotensin-(1-7) receptor agonist angiotensin-converting enzyme 2 and new insights into the renin-angiotensin system dr john lubel is a recipient of an australia national health and medical research council (nhmrc) scholarship, and peter angus and louise burrell hold an nhmrc project grant (509315). key: cord-315754-dq2empne authors: hasan, anwarul; paray, bilal ahamad; hussain, arif; qadir, fikry ali; attar, farnoosh; aziz, falah mohammad; sharifi, majid; derakhshankhah, hossein; rasti, behnam; mehrabi, masoumeh; shahpasand, koorosh; saboury, ali akbar; falahati, mojtaba title: a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin date: 2020-04-22 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1754293 sha: doc_id: 315754 cord_uid: dq2empne the widespread antigenic changes lead to the emergence of a new type of coronavirus (cov) called as severe acute respiratory syndrome (sars)-cov-2 that is immunologically different from the previous circulating species. angiotensin-converting enzyme-2 (ace-2) is one of the most important receptors on the cell membrane of the host cells (hcs) which its interaction with spike protein (sp) with a furin-cleavage site results in the sars-cov-2 invasion. hence, in this review, we presented an overview on the interaction of ace-2 and furin with sp. as several kinds of covs, from various genera, have at their s1/s2 binding site a preserved site, we further surveyed the role of furin cleavage site (fcs) on the life cycle of the cov. furthermore, we discussed that the small molecular inhibitors can limit the interaction of ace-2 and furin with sp and can be used as potential therapeutic platforms to combat the spreading cov epidemic. finally, some ongoing challenges and future prospects for the development of potential drugs to promote targeting specific activities of the cov were reviewed. in conclusion, this review may pave the way for providing useful information about different compounds involved in improving the effectiveness of cov vaccine or drugs with minimum toxicity against human health. communicated by ramaswamy h. sarma at the end of december 2019, chinese public health officials announced to the world health organization (who) that a new and unknown virus caused a disease with symptoms similar to pneumonia in wuhan (khan & fahad, 2020) . they immediately recognized that the virus was from the coronavirus (cov) family and was rapidly spreading out of wuhan. the who is seeking to identify, track and restrict a new disease from the cov family called cov disease 2019 , which is still affecting many peoples in china and outbreaking to other countries. this type of cov is also spreading in other countries such as iran, italy and south korea (memberships & join, 2020) . with the number of covs currently increasing, the researchers are trying to figure out what is the main cause of the virus to spread easily and widely. some genetic and structural analyzes have identified the main characteristic of the virus, which could increase the efficiency and speed of virus transmission between human cells (belouzard et al., 2012; jaimes & whittaker, 2018) . also, some groups are investigating a receptor on cell membranes that introduces the fusion of new cov into human hcs (veljkovic et al., 2020; zheng & perlman, 2018) . both the cellular and protein receptors of the virus provide potential targets for development of drugs against pathogen (kaufmann et al., 2018; papadopoulos et al., 2017) . however, some welldeveloped experiments and promising data are required to ensure such a mechanism. indeed, exploring the transmission mechanism of the virus is important to prevent its future outbreaks. the covid-19 spreads much more easily than the sars virus and infecting easily the people have been already infected with sars lai et al., 2020; liu et al., 2020) . to infect a cell, viruses use spike protein (sp) to bind the cell membrane, a process activated by specific cellular enzymes such as trypsin, furin, and cathepsin l (jaimes & whittaker, 2018; millet & whittaker, 2015) . genomic analysis of the new cov has shown that its sp differs from that of other viruses (du et al., 2017; li, 2016) , indicating that the protein has a site activated by a hc enzyme called furin (millet & whittaker, 2015) (figure 1 ). this enzyme normally cleaves and activates a wide range of substrates in different organisms ( figure 2a ) (braun & sauter, 2019) . this enzyme is found in many human tissues, including the lungs, liver and small intestine, which means that the virus has the potential to invade different organs (belouzard et al., 2012; heald-sargent & gallagher, 2012; scamuffa et al., 2006) . the furin activation site (fas) makes the new cov much different in cell entry than sars, and probably affects the stability of the virus and, consequently, the transmission process (li et al., 2015; millet & whittaker, 2014; yamada & liu, 2009) . several other groups have also identified the site of activation to investigate how the virus spreads among humans (basak et al., 2007; kim et al., 2016; kleine-weber et al., 2018; porter et al., 2014) . they pointed out that other viruses including influenza family viruses contain these sites that are easily spread among people (huang et al., 2011; menachery et al., 2014; tang et al., 2020; vlasak et al., 1988 ) . but in these viruses, the site of activation is found on a protein called haemagglutinin, not on the sp (bernstein et al., 2020; koopman et al., 2019; lai et al., 2019) . however, some other researchers are more cautious about the significant role of the activation site in helping cov and facilitating its spread (amer et al., 2018; van doremalen et al., 2013) . other scientists are also skeptical of comparing the influenza viral activation sites with the new cov transmission channels (heymann & shindo, 2020; yang et al., 2005) . the haemagglutinin protein at the surface of influenza viruses is not the same as the sp in covs (yin et al., 2016; zmora et al., 2018) . in other hand, the influenza virus that has caused the deadliest recorded pandemic lacks even a fas (coutard et al., 2020) . therefore, some studies on cellular or animal models are required to assay the function of the activation site. indeed, covs are unpredictable and some experiments should be developed to assess how to modify the activation site. some other experiments have done to explain why the new covs successfully infects human cells (huang et al., 2020; letko & munster, 2020) . their experiments have shown that the sp binds to the human cell receptor (hulswit et al., 2019) . for example, a drug that blocks the receptor may make cov entry into cells difficult. interaction of furin and angiotensin converting enzyme-2 (ace-2) with sp furin and furin-like proteases belong to the group of proprotein convertases. scientist have pointed out that among several targets, the sp as fusogenic envelope glycoprotein of sars cov, is a promising site for treatment of infectious diseases (li et al., 2018; licitra et al., 2013) . sps mediate cov fusion and the entry of the cov genetic material into the human cell (senathilake et al., 2020; struck et al., 2012) . afterwards, a sp-selective strategy, when used at the initial phase of cov infection is probably considered as a potential approach to mitigate infection inside the body. the furin induces the cleavage of sps at the rntr761#ev site (bergeron et al., 2005) and results in the production of two segments: s1 and s2 ( figure 2b ); each segment shows its own biological activity. the s1 segment known as the globular section of sp mediates attachment of cov to the receptor, ace-2 with dominance of a-helix structures (li et al., 2003) . a 193-resdiue fragment of sp has been determined as the protein-binding site (wong et al., 2004) . s2, known as the biomembrane-anchored stalk domain and leads to cov-human cell fusion (prabakaran et al., 2004) . some other experiments also exhibited that other biomembrane-anchored proprotein convertases (prcos), namely poco5b and poco7 cleave sps . in this regard, basak et al. (2007) employing intramolecularly quenched fluorogenic (iqf) proteins relied on sars-cov sp claimed that poco5b and poco7 beside furin may result in the cleavage of sps. kinetic studies showed that the cleavage of peptide occurred potentially by furin as well as recombinant poco5b, but with a limited amount by poco7. furthermore, they demonstrated that the cleavage activity could be inhibited by a poco-inhibitor, a1-pdx. circular dichroism intensities revealed that the content of b-sheet conformation in poco increases after cleavage by furin ( figure 2c ). 1h nuclear magnetic resonance (nmr) spectroscopy displayed that this peptide has a turn motif at its c-terminal part, close to the cleavage site. coutard et al. (2020) revealed that the sp of the sars-cov-2 possesses a furin-like cleavage site absent in cov of the same clade. the genome sequence revealed that sars-cov-2 is included in lineage b of b-cov ( figure 3a ). chen et al. (2020) presented that sars-cov-2 as a newly detected cov shows a high level of similarity to sars-cov. the structural analysis of the receptor binding domain (rbd) of sp from the two viruses show 72% similarity in residue sequences ( figure 3b ). molecular modeling studies depicted that sars-cov-2 rbd provides a stronger attachment with ace-2. they found that a distinct phe residue plays a key role in the in the binding site due to its interaction with ace-2. several kinds of cov, from various genera, have at their s1/ s2 binding site a furin cleavage site (fcs) (belouzard et al., 2012 ). an important phase in the life cycle of the cov is the human cell entry and fusion derived from proteolytic activity of the relative fusion protein (fp) by respective proteases (jaimes & whittaker, 2018) . a conventional approach to explore the cov life cycle is to clone the fp gene into an appropriate vector, transfect the cells, incubate with the related protease, purify the protein and carry out the protein assays (cong et al., 2019; li et al., 2019) . this approach shows a number of drawbacks: the need of viral genome, expensive synthesizing process, availability of monoclonal antibodies, and time-consuming process. therefore, jaimes et al. (2019) reported the fluorogenic peptide cleavage analysis to assay the proteolytic activity of recombinant fp which can be applied in the case of cov sp in a less labor and time intensive way. in another study, xi et al. (2020) isolated one strain of sars-cov-2 (zj01) in mild covid-19 patient and reported the presence of more than 30 specific gene mutation. the theoretical analysis of fcs and the case alignment of cov family determined that fcs may be a crucial site of cov evolution. zj01 showed mutations close to fcs (f1-2), which led to alterations in the conformation and the charge distribution on the surface of the sp. they employed adaptive poisson-boltzmann solver (apbs) analysis and exhibited that the binding site of furin was covered with a number of negative residues (figure 4a ). the f1 site of sars-cov-2, namely zj01, wuhan-hu-1 and ratg13 were almost covered with positive moieties while sars was covered by both mixed negative and positive residues. f1 site of zj01 showed more positive charge distribution in its head and more negative residues in its basal site in comparison with wuhan-hu-1. (basak et al., 2007) . (c) cd signals of qsars-4 peptide after incubation with different concentrations of recombinant furin (basak et al., 2007) . reprinted with permission from refs. (basak et al., 2007; braun & sauter, 2019) . the f2 site of gz02 had negative charge distribution while f2 site of wuhan-hu-1 and ratg13 demonstrated a limited level of positive moieties. zj01 displayed more positive charge distribution in f2 site than the other species, most likely derived from gene deletion. gz02 presented a number of negative charge distribution in f3 site while limited negative charge distribution was observed in sars-cov-2-associated virus (figure 4a) . hence, they deduced that, the mutation close to fcs site lead to a significant change in the protein conformation and surface charges, which further affected its interaction with the ligands (xi et al., 2020) . hoffmann et al. (2020) depicted that sars-cov-2 cell entry relied on ace-2 and tmprss2 and is limited by a protease inhibitor. indeed, exploring the cellular factors employed by sars-cov-2 for entry might result in providing useful information about viral outbreak and a number of therapeutic approaches. hoffmann et al. (2020) exhibited that sars-cov-2 utilizes the sars-cov receptor ace-2 for specific cellular 2) (i), code alignment and residue sequences of the s-protein from cov-zxc21 and sars-cov-2 (2019-ncov) at the s1/s2 site (coutard et al., 2020) . (b) comparison of the structures sars-cov rbd and its interaction with ace-2 by the obtained molecular models of sars-cov-2 rbd . reprinted with permission from refs. coutard et al., 2020) . internalization and the tmprss2 as a serine protease for sp cleavage. a tmprss2 inhibitor can be developed in clinical application to block the cellular internalization and might result in the advancement of a therapeutic approach. in general, they demonstrated crucial similarities between sars-cov-2 and sars-cov infection and reported a promising target for development of anti-viral platforms (figure 4b ) (hoffmann et al., 2020) . the design of enzyme inhibitors as therapeutic platforms against cov require the control of multiple pharmacologic features beyond the interaction and ace-2-and furin-targeting drugs (baron et al., 2020; kong et al., 2020) . a number of these pharmacologic characteristics show their molecular underpinning in chemical pathways within the biological systems. examples of this include drug transport pathways, blood circulation of drug, drug metabolism, and side effects derived from the interactions of drug with a wide range of enzymes (zumla et al., 2016) . thus, some thermodynamic and kinetic data in the pharmacological development of drugs during preclinical studies are required. as we overviewed so far, reducing the levels of ace-2, might provide a great deal of interest in fighting the cov. in a point of fact, ace-2 can induce a protective impact against cov-stimulated lung damage by enhancing the formation of the vasodilator angiotensin . indeed, the attachment of the sp of the cov to the ace-2 triggers a reduction in the levels of ace-2 , most likely stimulating lung injury. xu et al. (2017) reported that vitamin d can be used as a potential candidate to mitigate lipopolysaccharide-induced acute lung damage via control of the renin-angiotensin system. therefore, it could be suggested that vitamin d can control the outbreak of cov through inhibition of ace-2. gurwitz (2020) claimed that angiotensin receptor blockers can be used as promising sars-cov-2 therapeutics. also, it has been suggested that probable way of fighting the cov could be the injection of ace-2 which will result in the preventing the interaction of the cov to off-infected cells and replenishing ace-2 in infected cells . some other studies have indicated a close correlation between hypertension and heart disease and cov infection which may be associated with the prescription of ace-2 inhibitors (fang et al., 2020) . indeed, treatment of cov infection with ace-2 inhibitors leads to an upregulation of this receptor especially by epithelial cells of the lung followed by facilitation of infection with different kinds of sars-cov . based on these reports, some attention has been given to the development of furin inhibitors as potential therapeutics platform against sars-cov-2 infection. although, a great deal of research is required to exhibit experimentally this assertion, the inhibition of furin or furin-like enzymes may display a promising anti-viral platform. actually, it has been recently demonstrated that hcs infected by several kinds of viruses stimulate an interferon-based activity to block the enzymatic activity of furin-like enzymes (lodermeyer et al., 2013) . it was also revealed that virus infection triggers the upregulation of some receptors (braun & sauter, 2019; kim et al., 2015) that inhibit the furin trafficking in post-golgi compartments. also, based on the crystal structure of furin, some potent inhibitors like 2,5-dideoxystreptamine-mediated inhibitor (xi et al., 2020) . the similarity between sars-cov and sars-cov-2 (hoffmann et al., 2020) . reprinted with permission from refs. (hoffmann et al., 2020; xi et al., 2020) . were developed to be used in clinical trials (dahms et al., 2017) . because, furin-like enzymes contributed in a several pathways, one crucial point would be to limit the systemic inhibition that may lead to some adverse effects. consequently, it is most likely that such small molecule or other active agents as promising drugs, probably administered by inhalation and presenting a strong interaction with furin to stimulate a prolonged inhibition, deserve to be quickly analyzed to examine their anti-viral impact against sars-cov-2 . in general, these details disclosed that inhibitors of furin or furin-like enzymes may play a key role in blocking virus outbreak. ongoing challenges and future perspective ace-2 exhibited the same primary structures patterns in vertebrates' lineages . structural investigations indicated that ace-2 from these systems can efficiently interact with rbd of sars-cov-2, inducing them all to serve as promising hosts for the virus infection (mathewson et al., 2008; poon & peiris, 2020) . furthermore, it can be deduced that small molecular and ligands inhibitors that can limit the interaction of ace-2 with rbd should be developed to combat the spreading cov epidemic senathilake et al., 2020) . isolating and cultivating cov in vitro may not usually be practical or demand particular facilities that are not accessible in every bioresearch laboratory. therefore, there is a necessity for developing some strategies to assay the human health response to spread of emerging cov that can be performed in normal laboratory systems. apart from viruses targeting humans, several animal viruses also have identical fps, hence, exhibiting the comparable features than their human sites. purifying these kinds of viruses can display some inevitable biological challenges, making the application of pioneering devices to examine them unavoidable. current drugs have limited efficacy in treating cov in different populations and species. given the high incidence of cov resistance, especially in immunocompromised patients, the design of new drugs that target specific activities of the virus and stop one or more stages of its infection cycle is essential (prajapat et al., 2020; yang et al., 2020; zhou & zhao, 2020) . in recent years, most research has focused on blocking virus transmission to the hc, rna polymerase activity of the virus, and hc-virus interactions (schaack & mehle, 2019) . genetic changes, reappearance and emergence of antigenic variants and transmission of cov to humans require extensive measures to control globalization. vaccination, drug follow-up, and immediate protection are important tools for dealing with viral infections (ahmed et al., 2020) . due to the possible genetic modification of cov, producing a suitable vaccine against this disease is difficult (kim et al., 2016) . any changes in the antigenic sites of surface proteins, especially sp, which is the most important surface antigen of the virus, give rise to appearance of new strains (du et al., 2017; kleine-weber et al., 2018) . ; changes in these regions affect the antibodies produced against the former strains, and therefore have no role in the immunity against this disease (stebbing et al., 2020) . the emergence of resistant strains under drug selective pressure and their limited availability in high-risk cases further exacerbates the need for new therapeutic strategies (zu et al., 2020) . in recent years, compounds affecting different stages of the virus's life cycle have been introduced and a wide range of anti-viral strategies have been proposed, including inhibiting the entry and stopping of viral replication or targeting intracellular signal transduction pathways (peeri et al., 2020) . in recent decades, targeting viral proteins inducing humoral and cellular immune responses have received a great deal of attention in development of antiviral compounds (zumla et al., 2016) . the ability of biomolecular systems such as cytokines, interleukins, and bacterial derivatives to improve immunogenicity and xenografts is being evaluated as a novel strategy, although immune system regulatory proteins have received more attention. covs are rna viruses replicating in the cytoplasm of hcs. to transfer their genetic materials into the human hcs, they are dependent on the interaction of their envelope with the human hc biomembrane. the sp mediates cov entry and conducts the interaction of cov with receptor (ace-2) on the hcs as well as mediating the fusion of hc biomembrane and viral envelope. also, sars-cov-2 furin substrate site can facilitate the cleavage of the sp. this review discussed an overview on the role of ace-2 and furin in the binding of cov with hc biomembrane mediated by sp. also, we surveyed the contribution of fcs on the cov outbreak. moreover, we considered the ability of small molecular inhibitors on the limiting the interaction of ace-2 and furin with sp to be used as promising 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neutralizing antibodies against the novel coronavirus sars-cov-2 tmprss11a activates the influenza a virus hemagglutinin and the mers coronavirus spike protein and is insensitive against blockade by hai-1 coronavirus disease 2019 (covid-19): a perspective from china. radiology, 1 coronaviruses-drug discovery and therapeutic options key: cord-330093-asba80bi authors: leung, janice m.; sin, don d. title: smoking, ace-2 and covid-19: ongoing controversies date: 2020-07-16 journal: eur respir j doi: 10.1183/13993003.01759-2020 sha: doc_id: 330093 cord_uid: asba80bi smoking increases severity of covid-19 https://bit.ly/2ywp3jb and cardiovascular disease: a viewpoint on the potential influence of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers on onset and severity of severe acute respiratory syndrome coronavirus 2 infection. j am heart assoc 2020; 9: e016219. copyright ©ers 2020. this version is distributed under the terms of the creative commons attribution non-commercial licence 4.0. to the editor: we have read with great interest the paper by leung et al. [1] published in the european respiratory journal, the correspondence by russo et al. [2] , and also the subsequent comment by the first group [3] . both research teams are reporting increased angiotensin-converting enzyme 2 (ace-2) expression in airways of current smokers and those with copd, with important implications for coronavirus disease 2019 (covid-19) patients. since ace-2 has been shown to be the main receptor utilised by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) to enter the host cells [2] , the authors conclude that nicotine is a risk factor for covid-19. russo et al. [2] have shown that nicotine upregulates ace-2 through α7-nachrs which are present in neuronal and non-neuronal cells. leung et al. [3] provided further evidence in support of this hypothesis and propose the repurposing of α7-nachr antagonists for the pandemic (e.g. methyllycaconitine, α-conotoxin), expecting that such treatment will alter ace-2 expression and prevent sars-cov-2 entry. while this hypothesis is based on laboratory experiments, it is not supported by clinical data. recent observations on the prevalence of smoking among hospitalised covid-19 patients have raised some important issues. many studies, while based on preliminary data and subject to several limitations (e.g. lack of adjustment for confounding factors, possibility for inability to report, inaccurate recording or under-reporting of the smoking status), suggest that the proportion of hospitalised covid-19 patients who are current smokers is by far lower than expected based on population smoking rates [4, 5] . in one study, smoking was associated with lower odds of hospitalisation for covid-19 after adjusting for covariates [6] . to further address this issue, we calculated the pooled prevalence of current smoking in 11 published case series (table 1) , nine from china and two from the usa [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] , and compared it to the expected prevalence based on gender-adjusted and gender-and age-adjusted population smoking rates in each country by estimating the prevalence odds ratio (por) using random effects meta-analysis. due to the lack of data on patients' age distribution, the age-adjustment for the expected smoking prevalence was calculated by assuming that all patients were aged ⩾65 years, since lower smoking prevalence is observed in the elderly compared to younger adult age groups. population smoking prevalence information was derived from the world health organization 2018 global adult tobacco survey [16] for china, and from the us centers for disease control and prevention (for gender adjustment) [17] and statista (for gender and age adjustment) [18] . the pooled prevalence of smoking was 5.3% (95% ci 3. in conclusion, the observations of a low smoking prevalence among hospitalised covid-19 patients, despite the important limitations, together with the hypothetical links between dysfunction of the nicotinic cholinergic system and clinical manifestations of the disease raise some important research questions, considering that nicotine is a cholinergic agonist. the interaction between sars-cov-2 and the nicotinic cholinergic system should be further examined and any proposal for the repurposing of α7-nachr antagonists should be approached with caution, since it could potentially propagate the cytokine storm and adversely affect the prognosis. obviously, smoking cannot be considered protective for covid-19 (or any other disease), but pharmaceutical nicotine products are widely available and their role in covid-19 should be explored. covid-19 and vaping: risk for increased susceptibility to sars-cov-2 infection? to the editor: with great interest we read and commend the study done by russo et al. [1] , highlighting their findings that nicotine induces an increase in angiotensin-converting enzyme 2 (ace-2) expression in human bronchial epithelial cells (hbepc) and is mediated by α7-subtype nicotinic receptors (α7-nachr). it raises the concern that all electronic nicotine-delivery systems may put users at greater risk of succumbing to coronavirus disease 2019 (covid-19). we [2] , along with leung et al. [3] , have shown that ace-2 expression is upregulated in the small airway epithelia of smokers and patients with copd. in particular, we observed increased ace-2 expression in type-2 pneumocytes and alveolar macrophages along with the small airway epithelium of smokers compared to healthy never-smokers [2] . similar studies are yet to be done in the context of electronic cigarettes (e-cigarettes), heat-not-burn devices (iqos) or waterpipe exposure to human airways. ace-2 is the binding site for severe acute respiratory syndrome coronavirus 2 (sars-cov-2), mediating entry of the virus into cells [4] . binding affinity between the spike proteins of the virus and ace-2 on respiratory cells has been identified to be much higher than any previously identified human coronavirus. the significance of such overexpression of ace-2 in smokers should not be ignored. covid-19 and progression of severe pneumonia may be more likely to occur in smokers, particularly in those that have smoking-related comorbidities [5] . we are beginning to elucidate the role of traditional cigarette smoking and nicotine-driven changes to the lungs in the context of coronavirus transmission and susceptibility. cigarette smoke has been identified and linked to increasing expression of the binding site for the cause of the 2020 pandemic (sars-cov-2) via mediating nicotine receptors. with this, an avoidable and potentially gigantic risk-factor has emerged for covid-19, as the pandemic continues to claim ultimate grasp over the year of 2020. here, we bring to the discussion whether the increased susceptibility and virulence of sars-cov-2 via α7-nachr and the upregulation of small airway ace-2 expression may also be relevant for those who vape using nicotine-based e-cigarettes. e-cigarette vapour studies, although in their infancy, have already shown that they can enhance the virulence and inflammatory profile of pathogens such as streptococcus pneumoniae, among other deleterious biological effects [6] . vaping intensifies pneumococcal adherence through an increase in platelet-activating factor receptor expression, ultimately rendering those who vape with an increased risk of pneumonia [7, 8] . we, among others, have previously shown that e-cigarettes and iqos are not "safer", as having a vast pro-inflammatory response [9] . we compared cigarette smoke versus e-cigarette and iqos on airway epithelial and smooth muscle cells [9] . all tested pathological biomarkers were elevated in cells exposed to e-cigarette aerosols and iqos, which included chemokine cxcl8, extracellular matrix proteins and markers of mitochondrial dysfunction. we found these products toxic to the cells, evident from decreased cellular viability and integrity. more devastatingly, vaping also interfered with cellular energetics. our results further substantiate current research that e-cigarettes and @erspublications absolute cessation of any tobacco product in any form: implications for covid-19 https://bit.ly/ 3cyk9ra ioqs are indeed detrimental with increases in oxidative stress, inflammation, infections and airway remodelling in the lungs of these device users. as the scientific evidence mounts, confirming the fears that e-cigarettes and iqos are strongly associated with the development and progression of debilitating lung diseases [10] , now may be the prime time to include all electronic nicotine delivery systems in the vocalisation of concerns concerning tobacco-related death and disease. we recirculate the simple notion that the lungs are not designed for the chronic inhalation of anything but air and that the indication for a smoking-and nicotine-induced increase in ace2 is more evidence to the stacking weight of toxicity that tobacco is for humanity. given the role of the nicotine receptor, vaping may also lead to the upregulation of ace-2. research in this area will be invaluable in the development of e-cigarette research and providing trusted, peer-reviewed and real evidence for the youth of the 2020s. we strongly recommend that the world health organization and countries act to advance their efforts to reduce smoking, vaping and waterpipe use. during a pandemic it is difficult to focus on anything other than the immediate threat. the "primacy of rescue" has overwhelmed preventive action. additional research into the relationship of smoking, and all electronic nicotine delivery systems to the infection, transmission and progression of covid-19 is required. progress towards easily identifying those susceptible to severe disease or capable of asymptomatic transmission are important goals for managing the disease at a community level. covid-19 is a dress rehearsal for the next pandemic, and the next, and the one after that: the new norm. from the authors: the three letters from d. lutchman, k.d. mcalinden and co-workers, and k. farsalinos and co-workers together capture the divergence in opinion on the impact of smoking on coronavirus disease 2019 (covid-19) and whether the angiotensin-converting enzyme 2 (ace-2) receptor mediates this relationship. at the heart of this controversy is whether smoking reduces or increases the risk of contracting covid-19. k. farsalinos and co-workers, through analysis of the pooled prevalence of current smoking across 11 case series determined that current smoking status was significantly lower than expected gender-and age-adjusted prevalence in covid-19 patients. that smoking could potentially be protective against covid-19 has not gone unnoticed by the public. since late april, multiple media outlets have reported on this possibility, prompting the world health organization (who) to release a warning on 4 may, 2020, on tobacco use during this pandemic [1] . while we do not dispute that the prevalence of smoking in covid-19 cases has been surprisingly low across the world, we would echo who's advice, based on emerging evidence that outcomes in covid-19 are worse in patients who do smoke. an analysis conducted by killerby et al. [2] , of 220 hospitalised and 311 nonhospitalised patients with covid-19 patients across six acute care hospitals and associated outpatient clinics in metropolitan atlanta, georgia, for instance, demonstrated that smoking was an independent risk factor for covid-19 hospitalisation, carrying an odds ratio of 2.3 (95% ci 1.2-4.5). a recent meta-analysis has also shown that smokers have a relative risk of 1.34 (95% ci 1.07-1.67) of having more severe disease or experiencing refractory or progressive disease [3] . while smoking may not necessarily increase one's risk for contracting covid-19, the biological and inflammatory cascade that occurs upon severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection may be particularly devastating for a smoker. k.d. mcalinden and co-workers raise the possibility that a similar effect could be occurring in patients who vape. certainly, the risks of significant pulmonary injury with vaping are now well-described in the literature [4] , and the multiple ways that vaping can cause cellular damage and impede the lung's response to infection are clearly delineated by the authors. the theoretical possibility that vaping could prime the lung for sars-cov-2 infection is still hypothetical, given that to date none of the epidemiological studies have reported on vaping prevalence amongst their covid-19 patients. several demographic factors, however, make such estimates unlikely to be obtained with much precision. for instance, consider the landscape of e-cigarette use in china, the first epicentre of covid-19. a 2018 survey of 10 233 individuals in five chinese cities found that only 0.9% had used e-cigarettes within the past 30 days [5] . only 0.2% of those 65 years and older reported e-cigarette use within the past 30 days compared to 1.5% of those in the 15-24 year age range. similarly, in 2016, of 32 931 adults included in the us national health interview survey, 1.0% of those over 65 years reported current e-cigarette use compared to 4.6% of the 18-44 year age group [6] . older age groups, the ones more likely to have severe covid-19, present to a hospital, and therefore be captured by epidemiologists in their surveys, are therefore less likely to report current vaping. on the other hand, it may be difficult to ascertain the prevalence of vaping in younger age groups who are much more likely to vape, but also much more likely to have mild or asymptomatic covid-19 infections that are not captured, either for their failure to present to a healthcare provider or the constraints placed on available tests in resource-limited settings. nonetheless, we would argue for hospitals to capture these data as best they can and hope that data for mild cases in younger outpatients begin to be reported from around the world. similar to smoking, it is possible that vaping may still be associated with worse outcomes, if not necessarily being a risk factor for contracting infection in the first place. finally, as d. lutchman notes, if the culprit player for worse outcomes in smokers in this pandemic is the heightened ace-2 receptor in the airway epithelium, soluble ace-2 might be a therapeutic option. indeed, we would agree with the excitement for this approach as this was the subject of a recent study by monteil et al. [7] , which showed that human recombinant soluble ace-2 (hrsace-2) reduced sars-cov-2 viral loads in infected vero-e6 cells by a factor of 1000-5000. hrsace-2 also inhibited sars-cov-2 infections of kidney and vascular organoids. hrsace-2 is now under phase 2 investigation in europe as a therapeutic agent for covid-19 (clinicaltrials.gov: nct04335136). whether such a therapy will be helpful for the smokers and patients with copd who display higher levels of ace-2 in their airways and may suffer worse outcomes from covid-19 remains to be determined. ace-2 expression in the small airway epithelia of smokers and copd patients: implications for covid-19 covid-19 and smoking: is nicotine the hidden link? covid-19 and nicotine as a mediator of ace-2 systematic review of the prevalence of current smoking among hospitalized covid-19 patients in china: could nicotine be a therapeutic option? preliminary estimates of the prevalence of selected underlying health conditions among patients with 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infections in engineered human tissues using clinical-grade soluble human ace2 this version is distributed under the terms of the creative commons attribution non-commercial licence 4 key: cord-295041-5vpawtef authors: jakhmola, shweta; indari, omkar; chatterjee, sayantani; jha, hem chandra title: sars-cov-2, an underestimated pathogen of the nervous system date: 2020-09-28 journal: sn compr clin med doi: 10.1007/s42399-020-00522-7 sha: doc_id: 295041 cord_uid: 5vpawtef numerous clinical studies have reported neurological symptoms in covid-19 patients since the spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), apart from the atypical signs of pneumonia. angiotensin-converting enzyme-2 (ace-2), a potential receptor for sars-cov-2 entry, is expressed on various brain cells and cerebral parts, i.e., subfornical organ, paraventricular nucleus, nucleus of the tractus solitarius, and rostral ventrolateral medulla, as well as in non-cardiovascular areas such as the motor cortex and raphe. the resident cns cells like astrocytes and microglia also express ace-2, thus highlighting the vulnerability of the nervous system to sars-cov-2 infection. additionally, transmembrane serine protease 2 (tmprss2) and furin facilitate virus entry into the host. besides, the probable routes of virus entry into the nervous system include the hematogenic pathway, through the vagus, the olfactory nerve, or the enteric nervous system. however, the trajectory of sars-cov-2 to the brain needs investigation. furthermore, a th17-mediated cytokine storm is seen in covid-19 cases with higher levels of il-1β/2/7/8/9/10/17, gm-csf, ifn-γ, tnf-α, cxcl-10, mcp1, and mip1α/β. some cytokines can cross the blood-brain barrier and activate the brain’s immune cells to produce neural cytokines, leading to neuronal dysfunctions. nonetheless, most of the neurological conditions developed due to viral infections may not have effective and registered treatments. although, some antivirals may inhibit the virus-mediated pathogenesis and prove to be suitable in covid-19 treatment. therefore, clinicians’ and researchers’ collective expertise may unravel the potential of sars-cov-2 infection to prevent short-term and long-term cns damage. the initial cases of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection appeared in december 2019 in hubei province, china [1] . since then, it has become a global threat. besides systemic and respiratory ailments, 36 .4% of coronavirus disease of 2019 (covid19) patients developed neurological symptoms [2] . additionally, taste, smell, and visual impairments are reported in several cases of covid-19 [2] . sars-cov-2, a human cov (hcov) belongs to β-coronaviruses, and various clinical and pre-clinical studies have reported potential neurovirulent properties of these viruses [3] . furthermore, the presence of sars-cov-2 in cerebrospinal fluid (csf) of covid-19 patients is confirmed through genome sequencing [4] ; however, experimental evidence is needed to validate virusmediated neurological damage. moreover, acute necrotizing hemorrhagic encephalopathy (ane) was observed in brain computed tomography and magnetic resonance imaging of a covid-19 patient [5] . this rare complication is often associated with intracranial cytokine storms and points towards the indirect mode of sars-cov-2 influence on the brain [5] . also, a detailed study of brain tissue distribution of angiotensin-converting enzyme-2 (ace-2), a potential receptor for sars-cov-2 entry [6] , may shed light on potential sars-cov-2-induced neurological alterations. elaborate ace-2 expression studies state that the receptor is preferentially expressed in the endothelium, vascular smooth muscle cells, and on the surface of a variety of the central nervous system (cns) and peripheral nervous system (pns) cells [7] [8] [9] . additional plausible entry routes to the brain may include the hematogenic pathway, transmission through the vagus, the olfactory nerve, or the enteric neuron ( fig. 1a) [10] . in brief, here we recapitulate varied aspects of covid-19-related neurological manifestations. this article is part of the topical collection on clinical outcomes of virus-mediated brain dysfunction: more prevalent than acknowledged? the association of viruses with neural disorders is widely popular, although the relativity is still disputed. neurodegenerative diseases, affecting approximately 37 million people worldwide, include degenerative ailments of the nervous system-the brain, spinal cord, and nerves [11] . numerous genomic and proteomic studies unravel the similarities between virusmediated and classical neurodegeneration or neuropathies [12, 13] . viruses introduce alterations in the functioning of neurons directly or indirectly. the neurotropic viruses afflict neurons through cell lysis, necrosis, or apoptosis [14] . indirectly, the viruses damage the neurons by manipulating or attacking the host immune responses. in the cns, the virus can activate both the adaptive and innate immune responses [15] . common pathways involved in the activation of the immune responses include the tlr mostly 3, 7, and 8 mediated damage, the release of free radicals, and inflammation [15] . although, not always does the cns immune response lead to detrimental outcomes as they usually assist in repair and regeneration [15] . multiple studies mention the corroboration of infectious respiratory organisms as causative agents of various neurological diseases [16] . respiratory syncytial virus (rsv) is known to infect the lower respiratory tract, cause infections in the immunocompromised patients, and target the cns [16] . often the virus is detected in the csf samples of the patients exhibiting symptoms like seizures and convulsions, along with signs of ataxia, hormonal dysfunction, and encephalopathies [16] . also, in vivo studies demonstrate the movement of the virus intranasally to the cns [10] . another respiratory virus that affects the infants and has neurovirulent abilities is the human metapneumovirus (hmpv) [17] . the virus is substantially detected in encephalopathic patients' csf samples, although studies demonstrating the virus' neuroinvasive properties are to be conducted [18] . furthermore, hendra virus (hev) and nipah virus (niv) affect humans and cause lung damage, pneumonia, along with hemorrhagic and necrotizing alveolitis [19] . typical signs of neurological disturbance, including convulsions, seizures with motor deficits, and febrile encephalitic syndrome, are observed due to infection caused by these zoonotic viruses [19] . animal studies show the olfactory nerve to be the main route to the cns [20] . also, the flu-causing influenza viruses account for numerous seasonal epidemics with a severe lethality rate, approximately a million cases per year [21] . additionally, the viruses also affect the brain and are linked to encephalitis, febrile seizure, acute necrotizing encephalopathy, and syndromes like the reye syndrome and guillain-barré syndrome [22] . according to some animal studies, the influenza virus can alter the brain homeostasis by traveling to the brain through the vagus nerve or the olfactory route [23, 24] . intriguing, its association with parkinson's disease (pd) and multiple sclerosis (ms) is also mentioned [25] . many encephalitis lethargica and postencephalitic parkinsonism cases followed by the 1918 "spanish" flu pandemic, caused by influenza a (h1n1), make the involvement of the flu virus evident [26] . viruses like the enteroviruses polioviruses (pv), coxsackieviruses (cv), echoviruses, and human rhinoviruses (hrv) are known to invade the cns [27] . studies describe hrv-induced meningitis and cerebellitis [27] . ev-a71 (hand-foot-mouth disease (hfmd)) and d68 outbreaks are associated with neurological complexities like myelitis (afm), meningitis, and encephalitis [27] . the hcovs can aggravate various neuropathologies. hcovs are related to the neuroinvasive animal covs like porcine hemagglutinating encephalomyelitis virus, feline cov, and the mouse hepatitis virus, which is used to generate ms models [28, 29] . furthermore, a study conducted to demonstrate the relation between the hcovs (229e and oc43) with ms and other neurological disorders involves identifying viral rna in human brain autopsies [30] . importantly, cov-oc43 and cov-229e are found in the csf of pd patients [31] . however, detailed studies are needed to differentiate the mere presence and virus-associated disease alterations. in addition, association of sars-cov is not just limited to the virus in the bloodstream may infect the peripheral immune cells. these infected leukocytes may traverse the blood-brain barrier (bbb) composed of specialized tight junctions, endothelial cells, pericytes, and astrocytes. in addition, the virus may also cross the bbb which could be severed due to the action of the cytokines or may enter the cerebrospinal fluid (csf) by direct interaction with the brain microvascular endothelium cells. both the mechanisms result in alterations in the brain homeostasis and aggravate cytokine production within the cns (ii) several viruses like hsv and influenza viruses are known to infect the olfactory epithelial membrane. sars-cov-2 may also infect and damage olfactory sensory neurons (osns) in the epithelium lining. the damage may be direct or due to the production of cytokines produced by the accessory cells in the olfactory system. the virus may anterogradely reach the olfactory bulb through the cribriform plate. finally, the virus may potentially gain entry into the cns through the mitral cells along the olfactory tract. (iii) alpha herpesviruses (e.g. hsv-1, prv) and polio virus (pv) along with rabies viruses (rv) may migrate to the cns through the peripheral nerves. (i) viruses may infect the mucosal epithelium following infection of the axonal termini of the peripheral nerves. the virus may spread to the spinal cord through retrograde axonal transport. (ii) viruses infect the smooth muscle cells and spread through the neuromuscular junctions (nmj) from muscles into the sensory/motor neurons of pns ganglia. (iv) the gastrointestinal epithelium expresses ace-2 receptors. therefore, the cells may be easily infected by the virus. the virus may directly invade the enteric nervous system or indirectly it may prime the immune cells which may result in delayed neurological impairment. b sars-cov-2-mediated cytokine storm. after attachment and entry into the epithelial cells through ace-2 receptor, the virus may activate the pro-inflammatory pathway through tlr or nf-κb signaling followed by the formation of inflammasome. various pro-inflammatory cytokines and chemokines released due to this autonomous intrinsic defense mechanism include ccl-2, ccl-4, cxcl-10, and il-6. these proteins attract various immune cells in the circulation like the monocytes, macrophages, t cells, and neutrophils at the site of infection. additionally, the situation is worsened by production of tnf-β, il-6, il-4, il-12, and il-23 by the t lymphocytes, which further accumulate the immune cells establishing a pro-inflammatory feed-back loop. these cytokines may damage the bbb and activate astrocytes and microglia, the cns resident immune cells. in response, the activated microglia and astrocytes produce il-1β, il-6, tnf-α, and il-8. elevated levels of these inflammatory cytokines can impart neurotoxic effects leading to neuronal dysfunction and various cns disease-associated pathologies lungs; instead, it is known to infect many organs, including the cns [7, [32] [33] [34] . the real-time quantitative pcr assay targeting the polymerase (orf1ab) and nucleocapsid region of the sars-cov confirmed the presence of sars-cov in csf and serum of the infected patients [35, 36] . a report suggests the association of status epilepticus with sars [35] . hospitalized children with the acute encephalitis-like syndrome were positive for anti-sars-cov igm [37] . sars-cov is associated with demyelinating pathology and found in the brain parenchyma of ms patients [5, 28] . neurological symptoms are also associated with mers-cov [38] . these examples impress on the connection of hcovs with neurological dysfunctions. therefore, the association of neurological complications with sars-cov-2 is not surprising. according to a case report of sars-cov-2 infection, virus rna was determined in the patient's csf; however, the nasopharyngeal swabs tested negative [4] . currently, evidence to state the neuropathogenesis of the sars-cov-2 in covid-19 remains scarce. nevertheless, reports suggest that sars-cov-2 can cause meningitis and encephalitis [4] . variable neurological symptoms are displayed by the covid-19 patients like pns symptoms, including hypogeusia, hyposmia, hypoplasia, and neuralgia vertigo, and cns dysfunctions like cephalgia, impaired consciousness, seizures, ataxia, and acute cerebrovascular disease, with headache and dizziness being the most common [39, 40] . neurological manifestations are common in many covid-19 patients like anosmia, an early covid-19 symptom [2, [41] [42] [43] . though seizures are seldom reported in covid-19 patients, and usually indicate an ischemic stroke, meningitis, or cerebral hypoxia, its association with comorbidities like hypocalcemia or drugs remains elusive [44] . the neurological alterations caused by the virus may result from direct cns/pns attack or indirect influence on various organs that later affect the nervous system. for example, hypertension, common covid-19 comorbidity, results in blood-brain barrier (bbb) impairment and may enhance the risk of covid-19-related cerebral complexities [45, 46] . a hypothesis relates neuronal damage to the respiratory stress from deteriorated lung conditions [47] . the oxygen deprivation may result in multiple organ failure and may affect the brain [47] . besides, patients considered during the earlier studies of the sars-cov pandemic displayed axonal motor sensory neuropathy and myopathy [48] . however, it remains unclear if the illness was virus-mediated or an outcome of high drug doses [48] . nevertheless, the effect of the sars-cov-2 on pns is noteworthy as guillain-barre, miller-fisher syndrome, and polyneuritis cranialis are reported in covid-19 fig. 1 (continued) [49] [50] [51] [52] [53] [54] . development of rhabdomyolysis, neuralgia, and myalgia in sars-cov-2-infected patients further support the virus' ability to affect pns [52] [53] [54] . a study reported elevated creatinine kinase and muscle pain in 10.7% of patients with severe covid-19 [2] . furthermore, some covid-19 patients with neurological symptoms might have a prior history of neurological complications or maybe treated for viral infections. hence, it is necessary to treat such cases using drugs with properties of high bioavailability in the brain. we have summarized information like the mode of action and brain or csf/plasma ratio of a few antivirals, which have shown promising outcomes in covid-19 treatment (table 1) [70, 71] . the use of efficient bbb penetrating drugs may be preferred during this pandemic to minimize the onset of neurological consequences of sars-cov-2 infection. indirectly the viruses may damage the neurons by manipulating or attacking the host immunity [15] . in the cns, sars-cov-2 can activate both the adaptive and innate immunity [15] . t helper cell 17 (th17)-mediated cytokine storm, evident in virus infections, is seen in covid-19 with neurological manifestations (fig. 1b) [72, 73] . clinical studies report systemic inflammation involving enhanced cytokines, particularly il-1β, il-6, il-10, granulocyte colony-stimulating factor, granulocyte-monocyte colony-stimulating factor, c-x-c motif chemokine ligand 10 (cxcl10), mcp-1, macrophage inflammatory proteins 1-α, and tumor necrosis factor α in covid-19. additionally cd4+ and cd8+ t cell lymphopenia and decreased secretion of ifn-γ in severe cases of covid-19 are reported (fig. 1a) [28, 37, 72] . intriguingly, a study suggests that an ms patient undergoing ocrelizumab (an immunosuppressive drug) therapy diagnosed positive for covid-19 does not display serious complications [74] . the increased levels of cytokines may escalate vascular and bbb permeability and inflammation [74, 75] . this information supports the hypothesis that increased bbb permeability allows virus entry into the cns, leading to covid-19-related neurological complexities. some cytokines released in the circulation can cross the bbb and activate the resident brain immune cells like microglia and astrocytes to produce neural cytokines, further worsening the condition (fig. 1b) [76] . astrocytes regulate a wide variety of functions, which may aggravate neuroinflammation. microglia mature into macrophages and may engulf the neighboring neurons on activation [77, 78] . furthermore, microglia are the primary source of pro-inflammatory cytokines, nitric oxide, prostaglandin e2, and reactive oxygen and nitrogen species [77] . microglia express ace-2, along with ace and at1 [79] . these receptors play a significant role in microglia activation and balance the pro-inflammatory or antiinflammatory effects [80] . more specifically, sars-cov-2 infection can hamper the ace-2-mediated signaling, creating a glitch in the at1 receptor-mediated path, thereby inducing a pro-inflammatory response [80] . in vivo studies suggest induction of pro-inflammatory cytokines in microglia and the mouse brain and spinal cord [81] . the situation becomes dreadful when the pro-inflammatory substances produced by astrocytes and microglia fenestrate the bbb [77, 78] . besides, sars-cov infects the myeloid cells and manipulates the innate immune system to ease its propagation to other tissues (fig. 1a) [82] . these persistently infected leukocytes act as reservoirs of the neuroinvasive hcov and can be held responsible for long-term neurological sequelae [83] . therefore, [68] , pd [69] *csf/serum ratio. hand hiv-associated neurocognitive disorders, pd parkinson's disease, rsv respiratory syncytial virus. the brain to plasma ratio or csf to plasma ratio has been denoted for each drug assuming that brain penetration is similar between rodents, non-human primates, and human patients the possibility of such cases of persistent sars-cov-2 infection may appear in the future. notably, peripheral inflammatory reactions observed in covid-19 may result in symptoms of neurological disorders [84] . cytokine storms may influence the cns and enhance the severity of covid-19 patients to develop ane, meningitis, and hemorrhage [5, 85] . therefore, it is necessary to identify the mechanism behind sars-cov-2induced cytokine storms and the course of release of the cytokines during the infection. the contribution of the proinflammatory cytokines alone and the direct tissue damage caused by the virus needs to be addressed. the indirect influence of systemic inflammation on the cns by targeting the proinflammatory mediators will be worth investigating. it is found that ace-2 is expressed in various brain regions, like the subfornical organ, the nucleus of the tractus solitarius, and rostral ventrolateral medulla, as well as in non-cardiovascular areas such as the motor cortex and raphe [86] . according to a spatial distribution analysis, ace-2 is expressed in substantia nigra and brain ventricles [87] [88] [89] . the protein's cell type distribution revealed both excitatory and inhibitory neurons, pericytes and endothelial cells, and glial cells like astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex express ace-2, unlike the cells in the region of the prefrontal cortex [9] . additionally, the hippocampus has few ace-2 expressing cells [9] . studies report that angiotensin ii downregulates the expression of ace-2 in neonatal rat cerebellar or medullary astrocytes [90] . therefore, the predominant expression of ace-2 in the brain hints towards the virus's potential to infect the cns. furthermore, brain endothelial and smooth muscle cells of the blood vessels express ace-2 [7] . the virus may enter into the cns through the hematogenic pathway, subsequently crossing the bbb [91] . a post-mortem study of the frontal lobe of a covid-19 patient reports virus presence in neurons and capillary endothelial cells [92] . infection of endothelial cells may allow the virus to pass from the respiratory tract to the blood. the virus in the peripheral system can move into the cerebral circulation, where the blood's sluggish movement may facilitate the viral s protein interaction with the ace-2 expressed on the endothelial lining of the brain (fig. 1a) [93] . another speculated entry route for sars-cov-2 may be through the enteric nervous system upon infection of enterocytes [94, 95] . enterocytes express high magnitudes of ace-2 [7] . once inside the brain, the virus can infect the neural cells, astrocytes, and microglia. these cells express ace-2, thus initiating the viral budding cycle followed by neuronal damage and inflammation (fig. 1a) [96] . moreover, multiple transcriptome studies show and validate ace-2 expression levels in various non-neuronal cells of olfactory mucosa [97] . studies support the viral susceptibility of the mucosal cells, sustentacular cells, bowman's cells, and olfactory stem cells [98, 99] . loss of smell in covid-19 is marked by potential deterioration of olfactory stem cells and other accessory cells [98] . also, a high-throughput single-cell expression study mentioned no ace-2 expression in olfactory covering glia, microvillar cells, and immature or mature olfactory sensory neurons [100] . it is speculated that sars-cov-2 on binding may stimulate olfactory receptor neurons (orns) to exert an exaggerated immune response. earlier studies with sars-cov have established infection of the brain through orns [101] . studies describing the transneuronal/transsynaptic movement of the sars-cov already exist. rabies viruses can take over the vesicular axonal transport machinery to disseminate in the brain (fig. 1a) [102] . human herpesvirus-6 (hhv-6) propagates in olfactory endothelial (oe) cells before invading the brain [93] . these studies enable to predict and support the movement of sars-cov-2 through the vesicular axonal pathway in an anterograde fashion through the olfactory nerve and facilitate brain infection [102] (fig. 1a) . also, the virus may directly reach the csf around the olfactory nerve fibers from oe cells [82] . a probable trajectory of sars-cov-2 to the brain may be via high-ace-2-expressing non-neuronal oe cells to low-ace-2-expressing mature orns along the olfactory axons. this mechanism highlights the ace-2 independent process of virus spread. lastly, the expression of transmembrane serine protease 2 (tmprss2) in human olfactory mucosa may further worsen the case of sars-cov-2 infection [97] . a study demonstrates that respiratory epithelial cells express tmprss2 without ace-2 [103] . the mosaic distribution of tmprss2 in mature orns is reported [104] . therefore, the virus can preferentially gain entry into the pns through one of the two epithelial cell types in the nose, either the goblet cells or the ciliated cells. tmprss2, in collaboration with furin, accelerates sars-cov-2 entry [105] . furin, a host serine endoprotease, is particularly of neurological relevance. in general, furin can activate neuronal growth factors and influence cns homeostasis [106] . however, upon attachment of sars-cov-2 with ace-2, the enzyme generates an active s protein through irreversible cleavage of the precursor protein [105] . the protein s1/s2 subunits separate, which subsequently facilitate virus entry into the host [105] . thus, exploring the possible avenues of sars-cov-2 entry and impact on cns is the need of the hour. various clinical reports have made the association of sars-cov-2 with neurological dysfunction prominent. covid-19-associated neurological severity is primarily associated with cytokine storms. the earlier identified sars-cov is already known to suppress the host antiviral response and activate the pro-inflammatory pathways. briefly, it would be crucial to analyze the ifn-antagonizing and inflammasome-activating properties of sars-cov-2. furthermore, the interaction of sars-cov-2 and ace-2-expressing neuronal/glial cells may facilitate virus entry into the nervous system through different routes. thus, the nervous system's involvement in covid-19 may be more than the current situation apprehends, therefore referring to the virus as an underestimated pathogen. medical expert clinicians and researchers' collaboration may address the enhanced incidents of neural dysfunctions in infected individuals. after identifying initial neurological damages, careful monitoring of covid-19 patients in the long term is also necessary. acknowledgments we thank the ministry of human resource and development and department of biotechnology, govt. of india for fellowship to shweta jakhmola and omkar indari, respectively, in the form of a research stipend. we appreciate our lab colleagues for insightful discussions and advice. we gratefully acknowledge the indian institute of technology indore for providing facilities and support. authors' contributions conceptualization, data curation, formal analysis, investigation: sj, oi, sc. methodology, project administration, resources, supervision: sj, hcj. validation: hcj. visualization, roles/ writing -original draft: sj. writing-review and editing: sj, hcj, oi. conflict of interest the authors declare that they have no conflict of interest. disclaimer the funding organization has not played any role in the study design or the preparation of the manuscript. a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china neuroinfection may contribute to pathophysiology and clinical 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human lung cells furin at the cutting edge: from protein traffic to embryogenesis and disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-006087-hynkb0a8 authors: acharya, k. ravi; sturrock, edward d.; riordan, james f.; ehlers, mario r. w. title: ace revisited: a new target for structure-based drug design date: 2003 journal: nat rev drug discov doi: 10.1038/nrd1227 sha: doc_id: 6087 cord_uid: hynkb0a8 current-generation angiotensin-converting enzyme (ace) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of us $6 billion. however, the use of these ace inhibitors, which were developed in the late 1970s and early 1980s, is hampered by common side effects. moreover, we now know that ace actually consists of two parts (called the nand c-domains) that have different functions. therefore, the design of specific domain-selective ace inhibitors is expected to produce next-generation drugs that might be safer and more effective. here we discuss the structural features of current inhibitors and outline how next-generation ace inhibitors could be designed by using the three-dimensional molecular structure of human testis ace. the ace structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry. the neural activity of the sympathetic nervous system, regulating (through adrenergic receptors) cardiac and vascular function. the resistance to blood flow, which is directly proportional to the extent of vasoconstriction, is one of the primary determinants of blood pressure. angiotensin i (ang i) and an octapeptide called angiotensin ii (ang ii) 15 . two years later a chloride-dependent, metalloenzyme that could convert ang i to ang ii was purified from horse plasma 16 , and was later referred to as ace. ace, also known as peptidyl-dipeptidase a (ec 3.4.15.1), belongs to the m2 family of the ma clan (a protein clan contains all the modern-day polypeptides that have arisen from a single evolutionary progenitor) of zinc metallopeptidases 17 . it is a dipeptidyl carboxypeptidase that catalyses the hydrolytic cleavage of dipeptides from the carboxyl terminus of a wide variety of oligopeptides in vitro. its best known function is the in vivo conversion of ang i (drvyihpfhl), which circulates in plasma, into the potent vasopressor ang ii by removal of the c-terminal his-leu (fig. 1) . ang i is generated primarily by the renin-catalysed hydrolysis of the leu10-val11 peptide bond of angiotensinogen, a liver-derived 55-kda plasma protein. ace also affects blood pressure by cleaving bradykinin (bk, rppgfspfr), thereby abolishing its vasodilating activity (because of this, ace is sometimes referred to as kininase ii (kininase i is carboxypeptidase n)). bk is generated from a kininogen precursor by the action of plasma kallikrein (a serine proteinase) in a process analogous to that of ang i formation (fig. 1) . human ace is a monomeric zinc metalloenzyme that is synthesized as a 1,306-amino-acid polypeptide, is processed to a 1,277-residue mature form, is heavily glycosylated (30% by weight), and is localized to the plasma membrane of endothelial and absorptive epithelial and neuroepithelial cells 18 . a sequence of 22 hydrophobic amino acids located near the carboxyl terminus of the protein serves as a transmembrane domain that anchors ace to the cell surface. this creates a 28-residue cytosolic domain and a 1,227-residue glycosylated extracellular domain. its cellular orientation defines ace as a type i transmembrane ectoprotein and, in the case of endothelial cells, positions it optimally for interaction with its circulating substrates. the amino acid sequence of human endothelial ace provides clear evidence for a gene duplication event in its evolutionary history. it consists of an n-terminal domain of about 612 amino acids, a 15-residue interdomain sequence and a 650-residue c-terminal domain. a 357-aminoacid segment of the n-domain has more than 60% sequence identity to the corresponding segment of the c-domain (fig. 2) . each domain contains a five-residue sequence of amino acids, hemgh, which is characteristic of catalytic zinc sites found in a large family of neutral endoproteinases. detailed kinetic and mutational analyses have established that both of the zinc sites have catalytic activity 19, 20 . the physiological consequence of such tandem active sites in an enzyme is unknown. some differences in catalytic properties have been observed for these two sites: the n-domain site is notably 50-times more active toward the haemoregulatory peptide n-acetyl-ser-asp-lys-pro (acsdkp) 21 , 1000-times here, we provide an overview of ace and the ras, current ace inhibitors and their clinical utility, insights from the tace crystal structure, and the rationale and prospects for developing second-generation, domainselective inhibitors by structure-guided design. the recognition that an elevated basal blood pressure can lead to a shortened life expectancy and higher morbidity (due to cardiovascular complications such as kidney failure, heart failure and stroke) only evolved during the first half of the twentieth century, when numerous clinical surveys demonstrated a normal distribution of blood pressures and a 10-20% prevalence of hypertension in an apparently healthy population 9 . hypertension occurs in millions of people worldwide, the large majority of whom are unaware of their condition; a recent report indicated that prevalence rates are even higher than previously suspected, at 28% in north america and 44% in europe 10 . many long-term studies were required before the increased morbidity and mortality associated with hypertension became accepted and it was classified as a disease in need of treatment 9 . the underlying cause of hypertension in most cases was, and still is, unknown. therapy was initially directed at reducing blood volume by the use of diuretics, sympathetic tone by the use of adrenergic-blocking agents, or vascular resistance by the use of vasodilators. a major breakthrough in our understanding of blood pressure regulation came with the discovery of the ras and ace (reviewed in ref. 11 ). on the basis of a suspected relationship between kidney and blood pressure, goldblatt, houssay, page and others identified the renal enzyme renin and the pressor substance angiotensin in the 1930s and 1940s [12] [13] [14] . in the mid-1950s, skeggs and co-workers purified angiotensin and found that it existed in two forms: a decapeptide called is much less so 19, 24 . however, given the relative constancy of plasma chloride concentration, the significance of this chloride dependency is not obvious. human endothelial ace, also known as somatic ace, is encoded by a single gene that consists of 26 exons, more sensitive to inhibition by the phosphinic peptide rxp407 (ref. 22) , and more than 3000-times less sensitive to inhibition by rxpa380 (ref. 23 ace is released from the endothelial cell surface by the action of a so-called ace-secretase, or sheddase, that cleaves the arg1203-ser1204 peptide bond near the transmembrane region 32, 33 . soluble ace is a minor component of total ace activity, and its physiological function is unknown. activators of protein kinase c stimulate the release of ace from endothelial and other cells in culture, and markedly elevated concentrations of plasma ace are observed in certain diseases, particularly sarcoidosis. besides its role in blood pressure regulation, ace also has been postulated to participate in 'local' or 'tissue' rass. the ang ii arising from such systems is thought to act as a paracrine growth factor 4 . this activity has been implicated in the development of left ventricular dysfunction that can occur after a major heart attack, and seems to account for the beneficial effects of ace inhibitor therapy in such situations. the structural and functional conservation of the gene encoding ace are indications of its widespread evolutionary importance as a key metallopeptidase involved in the metabolism of regulatory peptides 34 . the drosophila melanogaster genome contains six genes (acer, ance, ance-2, ance-3, ance-4 and ance-5) that encode ace-like proteins. two of these, angiotensinconverting enzyme (ance) and angiotensin-converting enzyme-related (acer), are enzymatically active and share 36% sequence identity with human ace 35 . ance displays properties very similar to those of the human c-domain, whereas acer is inhibited by the n-domainselective inhibitor rxp407 (ref. 36 ). ace2 (also known as aceh) is an ace homologue found in humans and rodents that functions as a carboxypeptidase with a preference for c-terminal hydrophobic or basic residues (reviewed in refs 3, 37) . it is expressed mainly in the heart, kidney and testis and is important for the regulation of blood pressure and cardiac function. interestingly, ace2 is not inhibited by ace inhibitors such as lisinopril, captopril and enalaprilat. one of the main impediments to determining the structure of ace by x-ray crystallography was the production of diffraction-quality crystals. the c-domain of ace has seven potential n-linked glycosylation sites, six of which are located on the surface of the protein and the seventh in the juxtamembrane region. two concurrent approaches succeeded in paving the way for the crystallization 38 and successful x-ray structure determination of tace 5 . five of the n-linked sites were disrupted by substituting glutamines for each of the asparagine residues in the glycosylation sequences and a truncated form was expressed in the presence of a glucosidase inhibitor yielding crystals suitable for x-ray diffraction. the latter protein was used for the subsequent threedimensional structure determination of tace. highresolution crystal structures of the human tace and its complex with the widely used inhibitor lisinopril at 2.0 å resolution were recently reported 5 . all but one of which, exon 13, is transcribed into the corresponding messenger rna 25 . a form of ace found only in adult testis (tace) is encoded by the same gene but its mrna begins before exon 13 and continues through exon 26. it is transcribed by the interaction of a promoter with a site present in intron 12 that is active only in adult male germinal cells 25 . translation of this mrna results in a 701-amino-acid version of ace that, except for the first 36 residues, is identical to the c-terminal domain of somatic ace 26 . testis or germinal ace is found in developing sperm cells and mature sperm. sperm lacking ace are deficient in transport within the oviduct and in binding to the zonae pellucidae, and male ace -/mice have markedly diminished fertility 27 . a polymorphism involving a 287-base-pair insertion corresponding to an alu repetitive sequence has been found in intron 16 of the human ace gene 28 . the insertion is all or none: in one study the (i)/(d) frequency ratio was 44:56. the deletion (d) allele of the ace gene lacks the sequence that is present in the insertion (i) allele. the d allele is associated with a higher plasma and tissue ace activity. the dd genotype is positively correlated with plasma ace activity and numerous attempts have been made to search for associations between the dd genotype and specific cardiovascular diseases, but the conclusions remain controversial 29 . the association of allele i with athletic performance is equally controversial. some studies have shown that this polymorphism is associated with endurance performance, whereas others have shown the d allele to be associated with that of elite shortdistance athletes 30,31 . notable differences in hydrophobicity and charge are observed in the lid-like structure comprising helices α1, α2 and α3 (using tace nomenclature; fig. 2 ). this part of the structure seems to affect the substrate specificity of the n-or c-domain, as tace mutants which had this region replaced by the corresponding n-domain sequence showed a preference for n-domain substrates 43 (z. l. woodman et al., unpublished data). second, the zn 2+ -binding site with the canonical hexxh motif is conserved. third, it has been shown that the c-domain has greater chloride dependence than the n-domain, both in terms of substrate hydrolysis and inhibitor binding 19, 24 . from the model we can predict that arg186 (a key residue for binding one of the two chloride ions in tace) is replaced by his164 in the n-domain. so, in the n-domain only one chloride-ion-binding pocket is plausible, involving arg500 and tyr202. fourth, positioning of the lisinopril molecule in the active site of the n-domain model revealed that the full complement of structurally conserved residues was found as observed in the tace structure, confirming that the n-domain of somatic ace could also bind lisinopril with similar affinity, as previously reported 24, 44 . fifth, the s 2 sub-site is formed by asn494 and thr496, which replace a serine and a valine, respectively, in the c-domain. furthermore, the asparagine occurs in an n-glycosylation sequon (nvt) that is unique to the n-domain; the attachment of any glycan to this residue would occlude the s 2 pocket. this, in part, might explain how the bulky aromatic ring of the benzamido group in keto-ace (an ace inhibitor; see fig. 5a) is accommodated by the s 2 site in the c-domain (tace), making keto-ace more c-domain selective. sixth, the ace inhibitor rxp407 has an n-acetyl group that confers a certain degree of n-domain specificity 22 . the model of rxp407 and the n-domain reveals two residues, arg381 and tyr369, that form van der waals interactions with the n-acetyl carbonyl/methyl groups (fig. 5d) . however, a glutamate and phenylalanine are substituted for these two residues in the c-domain and might exert repulsive forces in the binding of the inhibitor to the c-domain active site. homology modelling of ace2 using the atomic coordinates of the tace structure revealed differences in the ligand-binding pockets of the two homologues that account for their substrate and inhibitor selectivity 105 . first, the accommodation of the s 2 ′ sub-site in tace increases the substrate-binding cavity and permits the binding of an additional amino acid to the obligatory binding site; second, gln281, which interacts with the carboxyl terminus of lisinopril in tace, is replaced by an arginine in ace2. this represents an elongation of the side chain of residue 281, which causes steric conflict with the p 2 ′ residue of lisinopril when it is docked in the active site of the ace2 model. in addition, the substitution of the leucine and phenylalanine for the hydrogen-bonding lys511 and tyr520, respectively, and replacement of thr282 in tace by the more bulky phe274 residue, probably account for the changes in substrate specificity. there are no striking differences in the large s 1 ′ sub-sites of tace and ace2 except for a proline in ace2 which corresponds to ala354 in tace. drosophila ace (ance) contains a single domain similar to tace. however, it has only three potential n-linked glycosylation sites, which are not required for secretion and enzymatic activity 39 . recently, the crystal structures of this homologue (which has considerable similarity to the tace structure), bound to captopril and lisinopril, were reported 40 . the wild-type protein was expressed in a baculovirus expression system and the oligosaccharides did not hamper crystallization. however, it is unlikely that this will be the case with the ace2 homologue, the ace n-domain or the full-length somatic ace structures -ace structural milestones that are still eagerly awaited. prominent features of the tace structure include an abundance of α-helices and a deep central cavity that divides the molecule into two halves, which pack together into an overall ellipsoid shape (fig. 3) . the active site, identified by the catalytic zn 2+ ion bound to the hexxh sequence (and to lisinopril in its complex with the enzyme), is located in the deep cavity, some 10 å from its entrance. the amino-terminal helices (α1-3) form a lid-like extension that partially covers the activesite channel, which limits the access of substrates and inhibitors. in fact, the aperture of the channel opening is approximately 3 å in diameter, which is too small for most peptide substrates, indicating that tace must undergo some conformational change, possibly associated with its unique chloride ion activation, for the substrate to enter the channel. from the structure of the lisinopril-tace complex (fig. 4) it is evident that the phenyl ring of the inhibitor interacts with the s 1 sub-site in the active site, the lysine with the s 1 ′ sub-site, and the proline occupies the s 2 ′ sub-site. the carboxyl group, located between the phenylpropyl group and the lysine, binds to the zn 2+ in the active site and also forms a hydrogen bond with the side-chain carboxylate of glu384. other key interactions occur between the sidechain amino group of the inhibitor lysine and glu162 of tace, and between the c-terminal proline carboxyl group with lys511 and tyr520. the binding of the inhibitor to the s 1 , s 1 ′ and s 2 ′ pockets and its zinc coordination form the basis for the structure-guided design of improved domain-selective ace inhibitors. in addition, two buried chloride ions that are important for the activation of the enzyme were identified in the crystal structure (outside the active site), both distant (20.7 å and 10.4 å, respectively) from the catalytic zn 2+ ion. the first is bound to arg186, arg489 and trp485, whereas the second is bound to arg522 and tyr224. the structure is indicative of an indirect mechanism for chloride activation, possibly through effects on active-site structure. this study also revealed that the structure of tace (ma sub-clan of m clan of peptidases (m clan peptidases are metalloenzymes and the metal is involved in catalysis)) resembles that of rat neurolysin 41 and a newly identified carboxypeptidase from the hyperthermophilic archaeon pyrococcus furiosus 42 -both of which are members of the ma clan -despite low sequence similarity. structure-based modelling of the n-domain of somatic ace (using the three-dimensional structure of tace 5 , which has ~60% amino-acid sequence identity to somatic ace) reveals some interesting features. first, glycosylation sequon consensus sequence asn-x-ser/thr whose core glycosylation generally occurs at the asn residue. nevertheless, the use of cpa as a model led to key conceptual insights in inhibitor design, because cpa was much better understood and its crystal structure was known. cushman and ondetti reasoned that ace was an exopeptidase like cpa, with the difference that ace cleaved the penultimate peptide bond to release a dipeptide product. the second major breakthrough in inhibitor design derived from an earlier observation that an extract from the south american pit viper bothrops jararaca, known as bradykinin potentiating factor (bpf), could inhibit ace [56] [57] [58] . bpf was a mixture of peptides 59 , which were shown to be potent and specific inhibitors of ace (table 1), and structure-activity studies indicated that the optimal c-terminal inhibitory sequence was phe-ala-pro 60 . this work led to the proposal that the venom peptides were substrate analogues that bound competitively to the obligatory substrate-binding sites in the ace active site (fig. 5a) . what was needed were orally active, non-peptide analogues of bpf. the third key insight derived from work by byers and wolfenden 61 describing a new design concept for inhibitors of cpa based on benzylsuccinic acid, referred to as by-product analogues. cushman and ondetti recognized that part of the binding affinity of benzylsuccinic acid derived from coordination of the active-site zinc by the carboxyl group, and predicted that a similar succinylamino acid derivative would inhibit ace if its structure was analogous to the dipeptide product of ace activity. on the basis of the phe-ala-pro sequence derived from the bpf peptides, they synthesized methylsuccinyl-pro (analogous to carboxy-ala-pro) and indeed found it to be a specific inhibitor, with an ic 50 of 22 µm 53 . cushman and ondetti then searched for a superior zinc-binding group and the potency breakthrough was achieved by replacing the carboxyl with a sulphydryl group, yielding captopril with an ic 50 of 23 nm 53, 54 (fig. 5a) . captopril became the first ace inhibitor in clinical use (first approved in 1981) and rapidly established itself as a powerful new therapeutic agent in the treatment of hypertension and heart failure. reports of captoprilrelated side effects, such as loss of taste and skin rash, prompted patchett and colleagues to focus on the design of non-sulphydryl ace inhibitors, by reverting to carboxyl compounds and introducing additional functionalities that would complete the by-product design. captopril did not make use of at least two potential binding sites: the s 1 binding site and a hydrogen-bonding site for the amide nitrogen of the (substrate) scissile bond (fig. 5a) . so, structure-activity studies were performed on a series of n-carboxyalkyl dipeptides of the general formula r-chco 2 h-a 1 -a 2 . the r group, occupying the s 1 pocket, was best served by benzylmethylene, whereas a 1 -a 2 was either ala-pro (as in the bpf peptides) or, surprisingly, lys-pro, leading to enalaprilat and lisinopril, respectively (fig. 5a) 62 , which show nanomolar inhibition constants. enalaprilat and lisinopril are essentially tripeptide analogues with a zn 2+ -coordinating carboxyl group substituting for the substrate scissile amide carbonyl. enalaprilat closely resembles the phe-ala-pro sequence the story of the design and synthesis of the first orally active, potent inhibitors of ace is one of the great success stories of modern medicinal chemistry. it has been described as one of the first examples of true 'rational drug design' 3 , and although this might not be true in the sense that we understand that term today, the design of the ace inhibitors in the late 1970s and early 1980s was certainly based on a series of brilliant insights that, together with a sprinkling of serendipity, constituted what might now be called 'rational intuition' . it is not our intention to provide a comprehensive review of the events leading to the design and synthesis of captopril, enalaprilat and lisinopril, the original group of potent ace inhibitors that formed the basis for all subsequent compounds of what can now be termed first-generation ace inhibitors. several excellent reviews of this history have been published during the past two decades, especially by the inventors themselves [45] [46] [47] [48] [49] [50] . however, it is instructive to consider some of the key insights that led to these drugs, especially in the context of the crystal structure that is now available and the structure-guided drug design of second-generation ace inhibitors that can now be undertaken. a role for a metal in the catalytic mechanism had been suspected since the discovery of the enzyme 16 and was confirmed in the 1970s 51,52 , leading to the proposal that ace was mechanistically similar to carboxypeptidase a (cpa) 53, 54 . we now know that ace is unrelated to the cpa class of enzymes, but instead falls into the group of metallo-endopeptidases characterized by the hexxh zinc-binding motif 55 (cpa possesses hxxe) . has been attributed to a direct vascular protective and anti-atherogenic effect of ace inhibitors, because it has been observed even in normotensive individuals 69 . a major debate concerns the mechanism by which cardiovascular benefits are conferred by ace inhibitors. it is generally accepted that ang ii not only has direct pressor effects and stimulates salt and water retention (via aldosterone release), but also stimulates myocyte proliferation and exerts pro-atherogenic effects via the induction of oxidative stress, endothelial dysfunction and vascular inflammation 71, 72 . however, there is also considerable evidence that some of the benefits of ace inhibition derive from potentiation of bk signalling, which stimulates release of the vasodilator nitric oxide and of the fibrinolytic protein tissue plasminogen activator 72, 73 . indeed, co-administration of the specific bk-receptor antagonist icatibant significantly attenuated the hypotensive effect of captopril in both normotensive and hypertensive subjects 74 . further complicating this debate is the recent appreciation that the ras is more complex than originally thought. there are multiple ang peptides and at least three or four ang receptors, some of which have opposing activities (fig. 1) . for instance, the principal ang ii receptor, the at 1 receptor, mediates the well-known effects of ang ii described above, but the at 2 receptor, which has a more limited tissue distribution, mediates largely opposing effects. similarly, ang 1-7 , which is derived from both ang i and ang ii by the action of various peptidases including ace and ace2, opposes the actions of ang ii 3, 37 . moreover, as already discussed, ace also acts on non-ang peptides, including bk, substance p, luteinizing hormone-releasing hormone (lh-rh) and the haemoregulator acsdkp; with the exception of bk, the importance of these peptides for the cardiovascular effects of ace inhibitors is unknown. some insights might derive from comparisons of the efficacy and side-effect profiles of ace inhibitors and at 1 -receptor blockers (arbs). ace inhibitors are generally well tolerated, but certain class-specific side effects have emerged, in particular cough and angioedema 70 . the incidence of cough has been estimated at 5-20% of patients and might result in the discontinuation of treatment 70, 75 . angioedema affects 0.1-0.5% of patients and can be life-threatening 76 . both cough and angioedema have been attributed to alterations in levels of non-ang peptides, especially raised bk concentration. recently, an association has been found between aceinhibitor-related angioedema and low plasma levels of aminopeptidase p, an enzyme that is also involved in the metabolism of bk, indicating that these individuals are at risk for developing angioedema when treated with ace inhibitors 76 . the potentiation of bk signalling by ace inhibitors (fig. 1) seems to result not only from reduced bk degradation but also from inhibition of desensitization of the b 2 bk receptor, possibly by inducing crosstalk between ace and the b 2 receptor [77] [78] [79] . therefore, both the benefits and side effects arising from increased bk signalling by ace inhibitors seem to be mechanistically complex and have implications for the design of n-or c-selective inhibitors (see below). that was found to be the optimal c-terminal sequence among the venom peptides. the structure of the ace-lisinopril complex confirms that lisinopril makes extensive contacts with the active site, including occupation of the s 1 , s 1 ′ and s 2 ′pockets, binding of a lysine by the c-terminal carboxylate, a hydrogen bond involving the (substrate) scissile amide nitrogen, and coordination of the active-site zn 2+ by the carboxyalkyl carboxylate 5 (fig. 4) . lisinopril binds with twofold greater affinity than enalaprilat 63 , probably because the lysine side chain makes better contacts with the deep s 1 ′ pocket, including a weak hydrogen bond between the lysyl ε-amine and glu162 (ref. 5 ). the design of captopril, enalaprilat and lisinopril was later extended by others, and a total of 17 ace inhibitors have been approved for clinical use 4 . the later compounds are all variations on the original theme, with most of the differences residing in the functionalities that bind the active-site zinc and the s 2 ′ pocket 48 . the different types of zinc-coordinating groups are of interest, because these have also found application in the design of inhibitors for other metalloproteases, such as the matrix metallopeptidases. on the basis of work first described by holmquist and vallee 64 , phosphonates have proven useful 22, 23 , as have hydroxamates 65 , ketones 66 and silanediols 67 . since their introduction in 1981, ace inhibitors have been studied extensively (for recent reviews, see refs 4, 68, 69) . ace inhibitors are first-line therapy for hypertension, congestive heart failure, left ventricular systolic dysfunction and myocardial infarction, and are recommended for slowing the progression of diabetic and non-diabetic nephropathy 68, 70 . more recently, ace inhibitors have also been shown to slow the progression of atherosclerotic vascular disease. this vascular benefit conditions 4, 75, 81, 82 . this difference might be due to the additional effect of ace inhibitors on bk, although this comes at the cost of increased side effects 75, 81 . interestingly, and contrary to earlier assumptions, angioedema has also been reported with arb therapy 75, 83 , which might be related to unopposed activation of the at 2 receptor leading to increased bk concentrations 73 . in light of the enormous importance of the ras in cardiovascular pathophysiology, there is continued interest in novel compounds that target this system 4 . eplerenone, the first selective aldosterone antagonist, the arbs were introduced more recently but have also been studied extensively 4, 80 . as is the case for ace inhibitors, arbs have been shown to be effective in the treatment of hypertension and heart failure, in reducing cardiovascular morbidity and mortality, and in slowing the progression of nephropathy. studies in myocardial infarction and heart failure have indicated a trend towards lower mortality in patients treated with ace inhibitors versus arbs, leading to the recommendation that ace inhibitors remain the first-line agents in these c-domain site will allow some level of bk degradation to continue, catalysed by the n-domain. this could be sufficient to prevent the excessive bk accumulation that has been observed during attacks of angioedema 86 . second, bk potentiation by b 2 receptor resensitization is maximal when both the n-and c-domains are inhibited 95 , indicating that a pure c-selective inhibitor will have a lower propensity for excessive bk stimulation. third, the multiple ang and non-ang peptides known to be vasoactive are not hydrolysed equally by the two domains 3, 96 , making it likely that the ratio of vasopressor to vasodilator peptides will differ between c-selective and mixed inhibitors. so, a highly selective c-domain inhibitor has the potential for effective blood pressure control with reduced vasodilator-related side effects. in contrast to a c-selective inhibitor, an n-selective inhibitor might open up novel therapeutic areas. as discussed, the n-domain seems to play a minor role in blood pressure control in vivo. at least three physiologically important peptides are hydrolysed preferentially or exclusively by the n-domain: lh-rh, ang 1-7 and acsdkp 21, 44, 96 . the contribution of ace to the metabolism of lh-rh and ang 1-7 in vivo is unclear, but there is increasing evidence that ace is the principal metabolizing enzyme for acsdkp, a natural haemoregulatory hormone 97 . acsdkp has antiproliferative and antifibrotic activities, and might have utility in protecting haematopoietic stem cells against chemotherapyinduced injury 97 and in limiting cardiac fibrosis 98 . administration of ace inhibitors results in a four to sixfold elevation of acsdkp plasma levels 94, 97 . this might be the basis for the observed association between ace inhibitors and anaemia, and the effective treatment of altitude polycythaemia by the ace inhibitor enalaprilat 99 . current-generation ace inhibitors in clinical use are essentially mixed n-and c-domain inhibitors 48 . although a modest degree of domain selectivity can be observed in some cases, this is not likely to be clinically significant. nevertheless, these differences might be instructive and can guide future attempts to develop highly domain-selective inhibitors. captopril has been noted to be modestly n-selective, depending on clconcentration, whereas lisinopril and enalaprilat are more c-selective 24, 44 . more recently, keto-ace, originally described in 1980 (ref. 66 ), was found to exhibit a 40-50-fold c-selectivity 96 ; one of the bpp peptides, bppb, was shown to be 300-fold more c-selective 100 ; and the phosphinic tetrapeptide rxpa380 is 3,000-fold more c-selective 23 . by contrast, bpp-12b is 30-fold, and the phosphinic tetrapeptide rxp407 1,000-fold, more n-selective 22, 101 (table 1) . examination of these compounds reveals a number of features (table 1 and fig. 5a) . captopril is the smallest inhibitor and can be viewed as an n-thioalkyl derivative of the dipeptide ala-pro, whereas enalaprilat, lisinopril and keto-ace are tripeptide analogues of phe-ala-pro, phe-lys-pro and phe-gly-pro, respectively. this might indicate that a bulky p 1 group -present in enalaprilat, lisinopril and keto-ace, but absent in was approved for the treatment of hypertension in september 2002 and will probably also find use in the treatment of severe heart failure 7 . an orally active renin inhibitor, aliskiren, is now in clinical development for hypertension 84 . of particular interest are the vasopeptidase inhibitors, which are dual ace-neutral endopeptidase (nep) inhibitors, of which omapatrilat is the most advanced in clinical development. nep, also a zinc-metallopeptidase, is the principal enzyme responsible for the degradation of natriuretic peptides, which are vasodilatory and diuretic peptides that reduce volume loading and are therefore beneficial in both hypertension and heart failure 85 . as expected, omapatrilat was equivalent or superior to ace inhibitors in clinical trials, but was found to be associated with a significantly higher incidence of angioedema, which has delayed its approval 86 . the higher incidence of angioedema is probably related to the fact that nep also inactivates bk (fig. 1) , and its inhibition might result in even higher plasma bk levels, which together with raised concentrations of natriuretic peptides (and potentially other vasodilatory peptides) might promote vasodilation-related adverse events 4, 86 . nep might also be required for the removal of the neurotoxic amyloid β-peptide, and so chronic nep inhibition could lead to neurodegeneration 87 . the clinical results with omapatrilat have had a sobering effect on the field 88 and have indicated that broad inhibition of vasoregulatory peptides should be approached with caution 89 . indeed, overly vigorous inhibition of neuro-hormonal activation during heart failure, by simultaneous treatment with an ace inhibitor, an arb and a β-blocker was associated with excess mortality in the valsartan heart failure trial (val-heft) 90 . so, efforts to develop triple inhibitors of ace, nep and endothelin-converting enzyme-1 (ece-1) 91 might need to be re-evaluated. ece-1, which is structurally and functionally related to nep, is the principal activating enzyme for the potent vasopressor peptides endothelin-1 and -3 (ref. 92 ), and so triple inhibitors might show even greater efficacy than single ace or dual ace/nep inhibitors. however, there is clearly an even greater potential for adverse events such as angioedema. in light of these developments, highly specific, singledomain inhibitors of ace offer an attractive alternative. as we have attempted to show in this review, our understanding of the ras (and related vasoregulatory systems) has come a long way since the introduction of the first ace inhibitors. the n-and c-domain sites of ace hydrolyse ang i and bk at comparable rates in vitro, but in vivo it seems that the c-domain is primarily responsible for regulating blood pressure 93, 94 . this might indicate that a c-selective inhibitor would have a profile comparable to current mixed inhibitors, but this is not necessarily the case. first, whereas ang i is hydrolysed predominantly by the c-domain in vivo 94 , bk is hydrolysed by both domains 23 and therefore selective inhibition of the be predicted that this sub-site is significantly different in the n-domain active site. modelling of the s 2 sub-site in the n-domain has also revealed differences, as expected (see earlier), confirming its potential utility for conferring domain selectivity (fig. 5c,d) . these considerations can form the starting point for the structureguided design of domain-selective inhibitors, which will be refined further once the n-domain structure becomes available. an important caveat in considering the design and pharmacological utility of domain-selective ace inhibitors is the potential for conformational effects that have not yet been observed in the tace crystal structure. for instance, it is unknown whether chloride binding and dissociation result in significant movement of the n-terminal 'lid' formed by helices α1, α2 and α3, and thereby restrict substrate access 5 . even more importantly, the physical orientation of the n-and c-domains in somatic ace is unknown, as is whether there is any significant degree of domain interaction or cooperativity. inhibitor titrations in vitro 44 and studies with domainselective inhibitors in vivo 23 have provided indirect evidence for some form of domain interaction, which could have significant effects on the pharmacological profile of domain-selective inhibitors. the past decade has seen major advances in structurebased drug design approaches, including technologies such as mass spectrometry, x-ray crystallography and nuclear magnetic resonance. these are important tools in structural proteomics and to some extent have eliminated the scepticism about the feasibility and value of the structure-based approach. in particular, high-throughput structure-based drug design using protein crystallography has become a very attractive proposition for the pharmaceutical industry. many examples exist today in which a combination of the three-dimensional structure of the target protein, computer-aided drug design (in silico or virtual screening), and a rational approach using highthroughput screening have produced important lead compounds that are now being evaluated in clinical trials (for recent reviews see refs 1, 2, 103, 104) . in addition to mining the untapped riches of the human proteome, the application of modern structure-based drug design methods to existing drug targets will generate more selective compounds for known disease targets, such as ace. we expect that next-generation, domain-selective ace inhibitors will be a result of such endeavours. captopril -confers c-selectivity, and that a larger p 1 ′ side chain also promotes c-selectivity (fig. 5b) , because lisinopril is more c-selective than enalaprilat. interestingly, trandolaprilat, although a potent inhibitor for both domains, was tenfold more c-selective 24 (table 1) . trandolaprilat contains a c-terminal hexahydroindoline group, which also indicates that a bulky p 2 ′ group confers c-selectivity. this is confirmed by results from radioligand-binding studies, which indicated that both perindoprilat and quinaprilat, which contain hexahydroindoline and tetrahydroisoquinoline groups, respectively, in the p 2 ′ position, were 45-180-fold more c-selective 102 . similarly, the highly c-selective tetrapeptide rxpa380 also contains a bulky methylindole group in the p 2 ′ position. these studies also indicated that lisinopril and 351a (a hydroxybenzamidine analogue of lisinopril) were 110-146-fold more c-selective, reinforcing the importance of a bulky p 1 ′ group 103 . on the other hand, structure-activity studies performed with a series of phosphinic tetrapeptides indicated that a phenylalanine in the p 1 position did not confer c-selectivity. instead, the single most important determinant for n-selectivity was an amidated c-terminal carboxyl in the p 2 ′ position, followed by an acidic group in the p 2 position (fig. 5d) 22 . the bradykinin potentiating peptides (bpps) confirm the conclusion regarding the p 2 group: bppa, bppb, bppc and bpp2 all end with the sequence ile-pro-pro, yet only bppc is unselective. bppc has a proline in the p 2 position versus a lysine in the most c-selective peptide, bppb 100 . similarly, the c-selectivity of keto-ace can probably be ascribed to a bulky p 2 benzyl group (fig. 5c) ; the selectivity of rxpa380 is also consistent with the proposed importance of a phenyl p 2 group (fig. 5a) . taken together, these data indicate that c-selectivity is conferred by bulky p 1 ′ and p 2 ′ groups and a large, neutral or basic p 2 group, whereas n-selectivity is conferred by a blocked c-terminal carboxyl and an acidic p 2 group. the ace c-domain crystal structure revealed that the s 1 ′ pocket was surprisingly deep, easily accommodating the lysyl group of lisinopril, with a hydrogen bond between glu162 and the ε-amine (figs 4 and 5b) 5 . additional modifications to the p 1 ′ group will potentially further enhance c-selectivity. moreover, the c-terminal carboxylate of lisinopril was found to bind to lys511, explaining the importance of a free c-terminal (p 2 ′) carboxyl for binding to the c-domain active site 22 . binding to lys511, instead of to an arginine (as originally predicted), might prompt investigation of functionalities other than carboxylates in this position. since n-selectivity is conferred by a blocked p 2 ′ carboxylate, it can also high-throughput crystallography and lead discovery in drug design the genesis of high-throughput structure-based drug discovery the angiotensin-converting enzyme gene family: genomics and pharmacology outstanding review of the ras and of the landmark studies that established the clinical utility of ace inhibitors crystal structure of the human angiotensinconverting enzyme-lisinopril complex first report on the three-dimensional structure of human testis ace and its interactions with the potent ace inhibitor lisinopril at the molecular level the development of cox2 inhibitors comparative pharmacology of h 1 antihistamines: clinical relevance hypertension: pathophysiology, diagnosis, and management hypertension prevalence and blood pressure levels in 6 european countries, canada, and the united states hypertension: pathophysiology, diagnosis, and management studies on experimental hypertension. i. the production of persistent elevation of systolic blood pressure by means of renal ischemia secretion hipertensora del rinon isquemaido a crystalline pressor substance (angiotonin) resulting from the interaction between renin and renin activator the purification of hypertensin i references 14-16 are a classic series of papers describing the isolation and characterization of ang i handbook of proteolytic enzymes first report of the molecular cloning of somatic ace and the surprising finding of a two-domain structure the two homologous domains of human angiotensin i-converting enzyme are both catalytically active differences in properties and enzymatic specificities between the two active sites of angiotensin 1-converting enzyme the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensin-converting enzyme rxp 407, a phosphinic peptide, is a potent inhibitor of angiotensin i converting enzyme able to differentiate between its two active sites roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin i and bradykinin the two homologous domains of human angiotensin i-converting enzyme interact differently with competitive inhibitors structure of the angiotensin i-converting enzyme gene. two alternate promoters correspond to evolutionary steps of a duplicated gene molecular cloning of human testicular angiotensinconverting enzyme: the testis isozyme is identical to the c-terminal half of endothelial angiotensin-converting enzyme angiotensin-converting enzyme and male fertility an insertion/deletion polymorphism in the angiotensin 1-converting enzyme gene accounting for half of the variance of serum enzyme levels angiotensin converting enzyme gene insertion/deletion polymorphism and cardiovascular disease: therapeutic implications human performance: a role for the ace genotype? endurance and the ace i/d polymorphism proteolytic release of membranebound angiotensin-converting enzyme: role of the juxtamembrane stalk sequence shedding of somatic angiotensinconverting enzyme (ace) is inefficient compared with testis ace despite cleavage at identical stalk sites insect angiotensin-converting enzyme. a processing enzyme with broad substrate specificity and a role in reproduction the acer gene of drosophila codes for an angiotensin-converting enzyme homologue functional conservation of the active sites of human and drosophila angiotensin i-converting enzyme just the beginning: novel functions for angiotensin-converting enzymes deglycosylation, processing and crystallization of human testis angiotensin converting enzyme a major breakthrough using protein-engineering tools on testis ace, making it amenable for structural study drosophila melanogaster angiotensin iconverting enzyme expressed in pichia pastoris resembles the c domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity crystal structure of drosophila angiotensin i-converting enzyme bound to captopril and lisinopril structure of neurolysin reveals a deep channel that limits substrate access crystal structure of a novel carboxypeptidase from the hyperthermophilic archaeon pyrococcus furiosus effects of the n-terminal sequence of ace on the properties of its c-domain first biophysical demonstration that somatic ace indeed contains two zinc-binding active sites and that lh-rh enzymes of the renin-angiotensin system and their inhibitors the design and properties of n-carboxyalkyldipeptide inhibitors of angiotensin-converting enzyme design of angiotensin converting enzyme inhibitors angiotensin-converting enzyme inhibitors natural products and design: interrelated approaches in drug discovery the discovery of captopril spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung pulmonary angiotensin-converting enzyme. structural and catalytic properties design of potent competitive inhibitors of angiotensinconverting enzyme. carboxyalkanoyl and mercaptoalkanoyl amino acids design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents landmark paper describing the brilliant series of insights leading to the first clinically useful ace inhibitor, captopril zinc coordination, function, and structure of zinc enzymes and other proteins conversion of angiotensin i to angiotensin ii fate of angiotensin i in the circulation conversion of angiotensin i to angiotensin ii by cell-free extracts of dog lung activity of various fractions of bradykinin potentiating factor against angiotensin i converting enzyme angiotensin-converting enzyme inhibitors from the venom of bothrops jararaca. isolation, elucidation of structure, and synthesis binding of the by-product analog benzylsuccinic acid by carboxypeptidase a important extension of the cushman-ondetti model, laying the groundwork for a series of additional ace inhibitors inhibition of rabbit lung angiotensinconverting enzyme by n α (s)-1-carboxy-3-phenylpropyl]llysyl-l-proline metal-coordinating substrate analogs as inhibitors of metalloenzymes dipeptidehydroxamates are good inhibitors of the angiotensin iconverting enzyme synthesis and biological activity of a ketomethylene analogue of a tripeptide inhibitor of angiotensin converting enzyme siliconbased metalloprotease inhibitors: synthesis and evaluation of silanol and silanediol peptide analogues as inhibitors of angiotensin-converting enzyme the renin-angiotensin system: a review of trials with angiotensin-converting enzyme inhibitors and angiotensin receptor blocking agents mechanisms of cardiovascular risk reductions with ramipril: insights from hope and hope substudies using ace inhibitors appropriately the at 1 -type angiotensin receptor in oxidative stress and atherogenesis. part i: oxidative stress and atherogenesis inflammation in early atherogenesis: impact of ace inhibition contribution of bradykinin to the cardioprotective effects of ace inhibitors effect of bradykinin-receptor blockade on the response to angiotensin-converting-enzyme inhibitor in normotensive and hypertensive subjects effects of losartan and captopril on mortality and morbidity in high-risk patients after acute myocardial infarction: the optimaal randomized trial aminopeptidase p in individuals with a history of angio-oedema on ace inhibitors potentiation of the actions of bradykinin by angiotensin i-converting enzyme inhibitors. the role of expressed human bradykinin b 2 receptors and angiotensin-converting enzyme in cho cells enhancement of bradykinin and resensitization of its b 2 receptor angiotensin-converting enzyme inhibitor ramiprilat interferes with the sequestration of the b 2 kinin receptor within the plasma membrane of native endothelial cells angiotensin blockade for hypertension: a promise fulfilled effect of losartan compared with captopril on mortality in patients with symptomatic heart failure: randomized trial -the losartan heart failure survival study elite ii comparison of candesartan, enalapril, and their combination in congestive heart failure. randomized evaluation of strategies for left ventricular dysfunction (resolvd) pilot study can angiotensin receptor antagonists be used safely in patients with previous ace inhibitor-induced angioedema? angiotensin ii suppression in humans by the orally active renin inhibitor aliskiren (spp100): comparison with enalapril vasopeptidase inhibition and angio-oedema β-amyloid catabolism: roles for neprilysin (nep) and other metallopeptidases? omapatrilat -the story of overture and octave vasopeptidase inhibition: a double-edged sword? a randomized trial of the angiotensinreceptor blocker valsartan in chronic heart failure n-[2-(indan-1-yl)-3-mercapto-propionyl] amino acids as highly potent inhibitors of the three vasopeptidases (nep, ace, ece): in vitro and in vivo activities hydrolysis of peptide hormones by endothelin-converting enzyme-1. a comparison with neprilysin the critical role of tissue angiotensinconverting enzyme as revealed by gene targeting in mice rxp 407, a selective inhibitor of the n-domain of angiotensin i-converting enzyme, blocks in vivo the degradation of hemoregulatory peptide acetyl-ser-asp-lys-pro with no effect on angiotensin i hydrolysis bradykinin potentiation by angiotensin-(1-7) and ace inhibitors correlates with ace c-and n-domain blockade n-domain-specific substrate and c-domain inhibitors of angiotensin-converting enzyme. angiotensin-(1-7) and keto-ace angiotensin i-converting enzyme and metabolism of the haematological peptide n-acetyl-seryl-aspartyl-lysyl-proline effect of n-acetyl-seryl-aspartyl-lysylproline on dna and collagen synthesis in rat cardiac fibroblasts angiotensin-converting-enzyme inhibition therapy in altitude polycythaemia: a prospective randomised trial selective inhibition of the c-domain of angiotensin i converting enzyme by bradykinin potentiating peptides the c-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme structural constraints of inhibitors for binding at two active sites on somatic angiotensin converting enzyme integration of virtual and high-throughput screening structure-based screening and design in drug discovery angiotensin-converting enzyme-2 (ace-2): comparative modelling of the active site, specificity requirements and chloride dependence the ace research in k.r.a.'s and e.d.s.'s laboratories is supported by the wellcome trust, uk. we would like to acknowledge the help of r. natesh and s. iyer in the preparation of this manuscript. the following terms in this article are linked online to: locuslink: http://www.ncbi.nlm.nih.gov/locuslink/ ace | ace2 | acer | angiotensinogen | at 1 receptor | at 2 receptor | bk | ece-1 | endothelin-1 | endothelin-3 | lh-rh | renin access to this interactive links box is free online. key: cord-338417-7kw9lws0 authors: singh, awadhesh kumar; gupta, ritesh; misra, anoop title: comorbidities in covid-19: outcomes in hypertensive cohort and controversies with renin angiotensin system blockers date: 2020-04-09 journal: diabetes metab syndr doi: 10.1016/j.dsx.2020.03.016 sha: doc_id: 338417 cord_uid: 7kw9lws0 background and aims: covid-19 is already a pandemic. emerging data suggest an increased association and a heightened mortality in patients of covid-19 with comorbidities. we aimed to evaluate the outcome in hypertensive patients with covid-19 and its relation to the use of renin-angiotensin system blockers (rasb). methods: we have systematically searched the medical database up to march 27, 2020 and retrieved all the published articles in english language related to our topic using mesh key words. results: from the pooled data of all ten available chinese studies (n = 2209) that have reported the characteristics of comorbidities in patients with covid-19, hypertension was present in nearly 21%, followed by diabetes in nearly 11%, and established cardiovascular disease (cvd) in approximately 7% of patients. although the emerging data hints to an increase in mortality in covid-19 patients with known hypertension, diabetes and cvd, it should be noted that it was not adjusted for multiple confounding factors. harm or benefit in covid-19 patients receiving rasb has not been typically assessed in these studies yet, although mechanistically and plausibly both, benefit and harm is possible with these agents, given that covid-19 expresses to tissues through the receptor of angiotensin converting enzyme-2. conclusion: special attention is definitely required in patients with covid-19 with associated comorbidities including hypertension, diabetes and established cvd. although the role of rasb has a mechanistic equipoise, patients with covid-19 should not stop these drugs at this point of time, as recommended by various world organizations and without the advice of health care provider. coronavirus disease 2019 (covid -19) has been declared as a pandemic by word health organization on march 11, 2020, as soon as it satisfied the epidemiological criteria (infection in more than 100,000 people in 100 countries) [1] . as of march 27, 2020, world has witnessed more than half a million cases of covid-19 with more than 24,000 deaths [2] . this suggests the magnitude of its spread across the world, since it was first reported on december 31, 2019 from wuhan, hubei province in china. emerging data suggests that older covid-19 patients with other comorbid conditions such as diabetes, hypertension, cardiac and pulmonary disease are in particular more susceptible, compared to general populations and have higher mortality. therefore, it is necessary to re-look into these subgroups of covid-19 patients with associated comorbidities. in this review article, we have collated all the available evidence that has emerged so far on outcomes and comorbidities in patients with covid-19. here, we have focused on outcomes in patients of covid-19 with hypertension and analyzed the controversies surrounding the use of renin-angiotensin system blockers (rasb). we have systematically searched the pubmed medical database up till march 27, 2020 using mesh key words that include covid-19, coronavirus, hypertension, diabetes, cardiovascular disease, angiotensin receptor blockers, angiotensin converting enzyme inhibitors. we have retrieved all the available literature published in english language on covid-19, that reported the outcomes in different co-morbidities. the association of hypertension and diabetes in patients with covid-19 is not unexpected, given the rising prevalence of both of these chronic diseases, globally. interestingly, in the pooled data from the ten chinese studies (n ¼ 2209) that have reported the characteristics of comorbidities in patients with covid-19; associations of hypertension, diabetes and presence of established cardiovascular disease (cvd) are larger, varying from 15 to 30% (average 21%), 5e20% (average 11%) and 2e40% (average 7%) respectively (table 1) . established cvd was also present in nearly 43% in italian study of 355 patients with covid-19. while consistent association of hypertension in patients with covid-19 across all these studies is unique, the concern which needs a serious attention is the increase in mortality. two chinese studies have worked in this direction to-date. in 191 patients with covid-19, zhou et al. found hypertension to have an odds ratio (or) of 3.05 (95% ci, 1.57 to 5.92; p < 0.006), diabetes with or of 2.85 (95% ci, 1.35 to 6.05; p < 0.001), whereas presence of coronary artery disease had an or of 21.40 (95% ci, 4.64 to 98.76; p < 0.0001) for in-hospital mortality, in an univariate analysis [8] . however, the association between these disorders and covid-19 mortality were no longer significant after a multivariate regression analysis. similarly, in an analysis of 201 patients with covid-19, wu et al. found hypertension to have a hazard ratio (hr) of 1.82 (95% ci, 1.13 to 2.95; p ¼ 0.01) for acute respiratory distress syndrome (ards) and 1.70 (95% ci, 0.92 to 3.14, p ¼ 0.09) for death. the diabetic patients had a hr of 2.34 (95% ci, 1.35 to 4.05; p ¼ 0.002) for ards and hr of 1.58 (95% ci, 0.80 to 3.13, p ¼ 0.19) for death, in a bivariate cox regression analysis [11] . it should be noted however, that neither of these studies were adjusted for all confounding variables. nevertheless, a study of 187 patients with covid-19, guo et al. reported nearly a twice increase in mortality in patients with established cvd and raised troponin t (tnt), compared to patients without cvd and raised tnt (69.4% vs. 37.5% respectively) [14] . the chinese center for disease control and prevention in a summary report of covid-19, reported a case fatality rate (cfr) of 2.3% (1023 deaths among 44,672 confirmed cases). however, cfr was elevated to 6.0% for hypertension, 7.3% for diabetes, and 10.5% for presence of cvd [15] . unsurprisingly, based on the above findings, researchers have recently proposed that the course of treatment and prognosis of covid-19 should be stratified based on the absence or presence of co-morbidities in to type a, b and c. type a denotes covid-19 patients with pneumonia but without comorbidities, type b denotes covid-19 pneumonia and comorbidities, whereas, type c denotes covid-19 patients with multi-organ dysfunction [16] . nonetheless, it still remains unclear whether these increased association of hypertension with covid-19 and heightened risk of mortality is directly related to hypertension or other associated comorbidities, or, anti-hypertensive treatment. there has been a growing concern that this association with hypertension and or cvd may be confounded by the treatment with certain antihypertensive medications such as rasb. although a recent paper has proposed to stop rasb and suggested to replace it with the calcium channel blocker, while treating hypertension in patients with covid-19 [17] , this hypothesis has been questioned by several other colleagues [18e22]. the entry of coronavirus into the cell is facilitated by the spike (s) protein. before attachment to the receptor on host cell, the s protein needs to be primed by a serine protease named tmprss2. the s proteins of different coronaviruses may utilise different receptors; mers utilises cd26 while sars cov and sars cov2 utilise angiotensin converting enzyme-2 (ace-2) [23] . the efficiency of the interaction between s-protein and ace-2 may be a key determinant of the transmissibility of the virus, viral replication and the severity of disease. in theory, this efficiency could be influenced by changes or amino acid substitutions in either the viral s-protein or [24] . the major outbreak of sars cov occurred in 2002e2003 with a minor localised outbreak with mild symptoms in 2003e2004. it was observed that the s-protein of sars cov in the 2002e2003 outbreak bound to ace-2 receptor much more efficiently than that of sars cov in the 2003e2004 outbreak, consistent with the absence of human-to-human transmission during the latter outbreak. several molecular changes in the s-protein influence the binding with human ace-2; for example, the substitution of threonine by serine at position 487 in s protein reduces the binding [25] . similarly, asparagine at position 479 increased the binding affinity [26] . methylation at position 487 has also been shown to influence binding. recently, s-protein of sars cov2 has been shown to be similar to that of sars cov, barring a few gains of function mutations. the most important of these is a glutamine at position 493 at the receptor binding domain, which explains its increased transmissibility compared to sars cov [27] . b) changes in ace-2 receptor: with regard to mers-cov, which employs cd26 (dpp-4) as its receptor for cellular entry, there are naturally-occurring polymorphisms in dpp4 that impact cellular entry of mers-cov and might thus modulate mers development in infected patients [28] . there is a possibility that similar variations or polymorphisms in ace-2 could affect the viral entry and disease course. polymorphisms of ace-2 gene have been identified; however, there is no evidence that they affect susceptibility to or severity of sars cov2 infection [29] . a recent study found differential ace-2 gene expression in human lung tissue with no racial/gender differences, but a higher gene expression in lungs of smokers compared to non-smokers, which could explain the higher risk of infection in smokers [30] . the role of ace-2 on vascular bed is opposite to that of angiotensin converting enzyme (ace). ace converts angiotensin i to angiotensin ii, which is a vasoconstrictor. ace-2 converts angiotensin ii to angiotensin (1e7) which causes vasodilatation after binding to the mas receptor in the vascular bed [31] . downregulation of ace-2 was observed in animal models of lung injury induced by sars cov [32] . recombinant ace-2 improved pulmonary blood flow and oxygenation in animals with lung injury, indicating that ace-2 may be the main determinant of lung injury caused by sars cov [33] . however, there is lack of human data except a small study in 10 patients with acute respiratory distress syndrome which showed that recombinant ace-2 was well tolerated and led to an increase in angiotensin (1e7) [34] . there has been considerable interest regarding the role of ras blockers in covid-19 infection. both benefit and harm have been postulated. figure 1 illustrates the interactions between the effect of rasb and covid-19. fig. 1 a) rationale and evidence for harm: there is evidence that ace-2 expression increases with the use of ace inhibitors and arb, especially in heart and kidney [35, 36] . this has raised a theoretical concern that by increasing ace-2 expression, aceis and arbs could facilitate the entry of virus into the host cell and increase the chances of infection or its severity [17] . also, there is increased ace-2 expression in elderly [37] . to what extent this predisposes the elderly to infection with sarscov2 is not known. in a study of 187 patients with covid-19, guo et al. reported an increased trend in mortality with those receiving rasb, compared to those not receiving. mortality was 36.8% (6 of 19) and 25.6% (43 of 168) in patients with or without rasb, respectively [14] . indeed, increased association and heightened mortality with covid-19 have been observed consistently across the studies in elderly, hypertensive, diabetics and known cvd; however, it is not exactly known whether it has any causal relation with the use of rasb or these subgroups are more on rasb due to these illness, compared to the rest of population. moreover, there is no solid evidence to back this concern either in covid-19 or in infection by other coronaviruses. b) rationale and evidence for benefit: as discussed above, increasing ace-2 levels in coronavirus infection could reduce lung injury. in an experimental study with mice, kuba et al. found that losartan showed significantly diminished lung injury and pulmonary edema after acid aspiration-induced acute lung injury (with addition of sars-cov spike protein) compared to placebo [32] . similarly, severe lung injury and pulmonary edema were prevented by both recombinant human ace-2 infusions or losartan in ace2-knockout mice [38] . mice with coronavirus induced lung injury showed improvement when treated with losartan [39] . moreover, a retrospective analysis found reduced rates of death and endotracheal intubation in patients with viral pneumonia who were continued on ace inhibitors [40] . treatment with arbs was reported to reduce mortality in ebola virus infection [41] . the exact mechanism of apparent benefit of these drugs in coronavirus infection is not yet clear. however, there could be several explanations [42] . i. increased ace-2 expression may not result in more viral entry into the cell because of the limited availability of the serine protease tmprss2. camostat mesylate, which is a tmprss2 inhibitor, has been shown to inhibit cellular entry of sars cov2 and could be a potential therapeutic option [43] . ii. increased ace-2 expression on the cell membrane may also lead to increased soluble ace-2 in blood, which may actually bind to most of sars cov2 and prevent its interaction with the membrane bound receptor. iii. ras blockers increase angiotensin ii, which is a substrate for ace-2. the interaction of ace-2 with angiotensin ii could induce a conformational change in the receptor binding domain of ace-2, limiting its ability to bind with sars cov2 [44] . iv. ace-2 receptors are present at a much higher density in lung tissue of children and young adults, compared to older individuals. thus, the upregulation of ace-2 receptors by the use of rasb over time in older people may emulate ace expression in young people. it is also possible that having more ace-2 receptors and increased ace-2 functions will likely produce more angiotensin (1e7) that might provide resilience against target-mediated destruction and development of pulmonary failure in patients with covid-19 [22] . this postulated positive effects on lung can be also protective during overwhelming infection with covid-19. v. indiscriminate discontinuation of rasb in patients with heart failure may also lead to readmission to hospital and increase in mortality [45] . collectively, relationship between ras activity and use of rasb in sars cov2 infection is very scarce. the only indirect evidence of ras activation in covid-19 is high incidence of hypokalaemia [46] . with regards to the use of rasb, a retrospective analysis of 112 covid-19 hospitalised patients with cardiovascular disease in wuhan, there was no significant difference in the proportion of acei/arb medication between non-survivors and survivors [47] . however, a study of 187 patient reported by guo et al., there was a trend of increase in mortality in patients with covid-19 receiving rasb (36.8%), compared to those not receiving (25.6%) [14] . covid-19 is increasingly associated with comorbidities that include hypertension and diabetes. special care is required in patients with covid-19 with associated comorbidities, given the heightened risk of in-hospital death. in view of lack of robust evidence for either benefit or harm, and with bulk of the experimental evidence in favour of benefit, it is reasonable for patients to continue using ace inhibitors and arb, as recommended by the european society of cardiology, hypertension canada, the canadian cardiovascular society, uk renal association, the international society of hypertension, and european society of hypertension and american heart association [48e50]. future studies reporting the outcome stratified on the basis of different anti-hypertensive agents in covid-19 may further enlighten us in this regard. we hereby declare that we have no conflict of interest related to this article. time to use the p-word? coronavirus enter dangerous new phase neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019 novel coronavirus in 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disease with generic statins and angiotensin receptor blockers. mbio should covid-19 concern nephrologists? why and to what extent? the emerging impasse of angiotensin blockade sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor ace2 x-ray structures reveal a large hingebending motion important for inhibitor binding and catalysis benefits and risks of continuing angiotensin-converting enzyme inhibitors, angiotensin ii receptor antagonists, and mineralocorticoid receptor antagonists during hospitalizations for acute heart failure hypokalemia and clinical implications in patients with coronavirusdisease 2019 (covid-19) clinical characteristics and outcomes of 112 cardiovascular disease patients infected by 2019-ncov. zhonghua xinxueguanbing zazhi position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers esh statement on covid-19 inhibitors of ras might be a good choice for the therapy of covid-19 pneumonia key: cord-266289-dkxhbmic authors: harrison, charlotte; acharya, k. ravi title: ace for all – a molecular perspective date: 2014-07-16 journal: journal of cell communication and signaling doi: 10.1007/s12079-014-0236-8 sha: doc_id: 266289 cord_uid: dkxhbmic angiotensin-i converting enzyme (ace, ec 3.4.15.1) is a zinc dependent dipeptidyl carboxypeptidase with an essential role in mammalian blood pressure regulation as part of the renin-angiotensin aldosterone system (raas). as such, it has long been targeted in the treatment of hypertension through the use of ace inhibitors. although ace has been studied since the 1950s, only recently have the full range of functions of this enzyme begun to truly be appreciated. ace homologues have been found in a host of other organisms, and are now known to be conserved in insects. insect ace homologues typically share over 30 % amino acid sequence identity with human ace. given that insects lack a mammalian type circulatory system, they must have crucial roles in other physiological processes. the first ace crystal structures were reported during the last decade and have enabled these enzymes to be studied from an entirely different perspective. here we review many of these key developments and the implications that they have had on our understanding of the diverse functions of these enzymes. specifically, we consider how structural information is being used in the design of a new generation of ace inhibitors with increased specificity, and how the structures of ace homologues are related to their functions. the anopheles gambiae genome is predicted to code for ten ace homologues, more than any genome studied so far. we have modelled the active sites of some of these as yet uncharacterised enzymes to try and infer more about their potential roles at the molecular level. angiotensin-i converting enzyme (ec number 3.4.15.1) or ace as it is more commonly known, is an extensively glycosylated integral membrane protein wei et al. 1991; ripka et al. 1993) which functions as a zinc dependent dipeptidyl carboxypeptidase to remove the c-terminal dipeptide from its peptide substrates in a zinc dependent manner (cushman and cheung 1971; das and soffer 1975) . ace is a key component of the renin angiotensin aldosterone system (raas), an essential hormone system responsible for the homeostasis of blood pressure in mammals. ace processes the precursor peptide angiotensin i to give angiotensin ii which is a potent vasoconstrictor. it also destroys the vasodilating properties of a second peptide, bradykinin (skeggs et al. 1954; zaman et al. 2002; acharya et al. 2003; gonzalez-villalobos et al. 2013) . ace therefore functions to increase blood pressure, and, as such, has long been targeted in the treatment of hypertension and other cardiovascular ailments through the use of ace inhibitors (turner and hooper 2002) . there are two isoforms of mammalian ace; somatic (sace) and testicular (tace) ace (soubrier et al. 1988 ). both are integral membrane proteins transcribed from the same twentysix exon gene. sace consists of two homologous domains (the n-domain and the c-domain) arranged in tandem, each with its own functional active site. tace expression is driven from an intragenic promoter, giving rise to a single domain protein identical to the c-domain of sace, as illustrated in fig. 1 (ehlers et al. 1989; hubert et al. 1991; wei and clauser 1992; corvol et al. 1995) . whilst sace is widely expressed, tace is expressed solely by developing spermatids and is thought to have a role in reproduction (caldwell et al. 1976; langford et al. 1993; sibony et al. 1993 sibony et al. , 1994 fuchs et al. 2005) . male mice lacking tace are infertile (krege et al. 1995; hagaman et al. 1998 ). however the mechanisms behind this are not yet clear. a 2011 study by bernstein and co-workers demonstrated that tace knockout mice produce normal amounts of motile sperm but which is unable to fertilise eggs. re-introducing tace rescues this phenotype, but, interestingly, is unable to rescue effects such as decreased blood pressure resulting from the knockout of the sace c-domain. this suggests that tace could be targeted in producing a male contraceptive without affecting the function of sace (bernstein et al. 2011) . in addition to the proposed role of tace in reproduction, it is becoming increasingly clear that sace is also involved in other physiological processes outside of blood pressure regulation . the relationship between tace and sace strongly suggests that the latter arose as the result of a gene duplication event, which would have enabled the enzyme to evolve and acquire new functions (hubert et al. 1991) . the two domains of somatic ace share greater than 60 % overall sequence identity, rising to circa 80 % in catalytic regions, yet they display some markedly different biophysical and biochemical properties. for example, the n-domain is significantly more thermally stable and resistant to proteolysis than the c-domain (sturrock et al. 1997; voronov et al. 2002) . important differences have also been observed in the catalytic properties of the two domains. the c-domain is able to process angiotensin i to angiotensin ii much more efficiently than the n-domain, whilst both domains are equally able to hydrolyse bradykinin. the c-domain alone appears sufficient for in vivo regulation of blood pressure (georgiadis et al. 2003) . a number of substrates have also been identified which are hydrolysed more efficiently by the n-domain compared to the c-domain. these include gnrh (gonadotropin-releasing hormone), a-beta42 (amyloid beta peptide), angiotensin 1-9 and acsdkp (n-acetyl-seryl-aspartyl-lysyl-proline) (rousseau et al. 1995; deddish et al. 1998; anthony et al. 2012; masuyer et al. 2012) . this is further evidence that the n-domain has, by divergent evolution, become involved in other physiological processes. the activity of the n-domain towards the peptide acsdkp is particularly interesting as acsdkp is involved in regulating the proliferation of fibroblasts in response to injury. a recent detailed study by bernstein et al. showed that in mice treated with the chemotherapy drug bleomycin to induce lung injury, the level of lung damage was much greater in mice with a mutated sace n-domain compared to in wild-type mice or those with sace c-domain mutations. there is strong evidence therefore that the n-domain plays an important role in preventing lung injury, likely due to its role in regulating the proliferation of acsdkp (bernstein et al. 2011) . the involvement of ace in processes outside of blood pressure regulation, and in particular the differences in substrate specificity of the two domains of sace highlight the growing need for a new generation of ace inhibitors. the current ace inhibitors were designed with no structural information and are instead based on peptides isolated from the venom of the brazilian pit viper bothrops jararaca (ferreira 1965; ferreira et al. 1970; ondetti et al. 1971; cushman et al. 1977; patchett et al. 1980) . most inhibit both domains of sace equally. structural studies will be crucial in understanding the reasons for these observed differences and in achieving the goal of a new generation of ace inhibitors. the initial discovery and partial purification of ace occurred in the 1950s yet it was not until 2003 that the first human ace structure, of tace, was reported . one of the main reasons for the 50 years that elapsed between the discovery of ace and the report of the first structure is the extensive glycosylation, estimated at 30 % of the weight of the enzyme ) but shown to be essential for expression and function (gordon et al. 2003; corradi et al. 2007 ). ultimately, the structure of tace was determined using a minimally glycosylated, truncated construct, lacking the transmembrane domain . the structure of tace is predominantly helical, with twentyseven α-helices and only six short β-strands, which combine to form an ellipsoid shaped molecule ( fig. 2a and b) . the structure shows six glycosylation sites, all located on the surface of the protein. key features of the molecule include a large central channel and an n-terminal lid. the channel stretches approximately 30 å across almost the entirety of the molecule, effectively dividing it into two subdomains. the channel narrows at the centre, where the catalytic zinc ion is found. as shown in fig. 2c , the n-terminal lid is formed by the three n-terminal fig. 1 schematic representation of the domain structures of sace and tace. sace consists of two homologous domains: the n-and c-domains. both contain a functional active site, illustrated here by the hexxh zinc binding motif. the two domains are joined by a short linker sequence (lr) which is unique to sace. the n-domain and most of the c-domain are extracellular. there is a transmembrane (tm) domain towards the cterminus of sace followed by the c-terminus (ct) which is intracellular. in contrast, tace is a single domain protein with only one active, shown here by a single hexxh zinc binding motif. the 36 residues at the nterminus (nt) of tace are unique, taking the place of the linker region (lr) in sace. like sace, most of tace is extracellular with a transmembrane domain (tm) and the c-terminus (ct) is positioned inside the cell helices; α1; α2 and α3 and appears to prevent the enzyme from hydrolysing large, folded substrates . in 2006, the structure of the n-domain of sace was determined, providing the first opportunity for the two domains of sace to be compared. the overall structure of the ndomain is essentially identical to that of the c-domain; a predominantly helical, ellipsoid shaped molecule with a long channel that narrows at the centre where the catalytic zinc ion is found. in total it comprises 27 helices, of which 18 are αhelices and only 6, relatively short, β-strands ( fig. 3a and b) . comparing the structures of the n and c-domains provides some insight into the observed differences in substrate specificity. the structures of both domains were solved with lisinopril, an inhibitor that is slightly selective for the cdomain, bound. although lisinopril is bound in broadly the same conformation in both structures, minor differences, mainly concentrated in the s 1 ' binding pocket help to explain the selectivity. for example, asp140 in the n-domain takes the place of glu162 in the c-domain. glu162 forms electrostatic interactions with the lysyl side chain of lisinopril, which asp140 is unable to replicate ( fig. 3c ) (corradi et al. 2006) . by studying the structures of the n-and c-domains of sace significant progress has already been made towards the design of domain specific inhibitors. rxpa380 was developed from a phosphinic peptide library and is highly selective for the c-domain, with a k i three orders of magnitude lower than for the n-domain ). the structure of the cdomain bound to rxpa380 was reported in 2007 (corradi et al. 2007 ) and provides an insight into the reasons for the specificity of this inhibitor (fig. 4) . in the c-domain phe391 in the s 2 subsite makes an aromatic interaction with phe at the p 2 position of rxpa380. in the n-domain phe391 is replaced by tyr369, which would not be able to replicate this aromatic interaction. furthermore, the structure suggests that the hydroxyl group of tyr369 would be within 2 å of the p 2 phe, preventing the inhibitor from binding in the same conformation (fig. 4d) . further relevant differences are observed in the s 2 ' subsite. during the development of rxpa380 it was suggested that pro and trp at the p 1 ' and p 2 ' positions respectively were important determinants of c-domain selectivity. the s 2 ' subsite of the cdomain is lined by two valine residues (val379 and val380) which create a hydrophobic environment for the pro and trp. in the n-domain, these valines are replaced by ser357 and thr358, destroying the hydrophobic environment (fig. 4d) . a second phosphinic peptide, rxp407, has been developed which selectively inhibits the n-domain ( fig. 5a and b). the structure of the n-domain in complex with fig. 2 the structure of human tace. (a) and (b) cartoon representation of the tace structure with helices shown in cyan, β-strands in magenta and loops in pink. the catalytic zinc ion is shown as a grey sphere at the centre of the long substrate binding channel which effectively divides the molecule into two subdomains. also visible in the active site is the inhibitor lisinopril and the two chloride ions shown as orange spheres. three n-terminal helices form a "lid" over the substrate binding channel and this is shown more clearly by viewing the molecule from the angle used in (b). the role of the lid in capping the substrate binding channel is highlighted in (c), where the three n-terminal helices which form the lid are shown in purple with the rest of the molecule shown as a cyan surface representation. the lisinopril and zinc are shown bound in the active site. in (c) the molecule is viewed from the same angle as in (b) rxp407 was reported in 2010 (anthony et al. 2010) . it shows twelve hydrogen bonds from nine residues forming between the enzyme and inhibitor (fig. 5c ). all but two of these would be conserved in the c-domain. interestingly, the two residues that differ; arg381/glu403 and tyr369/phe391 are located in the s 2 subsite (fig. 5d ), further illustrating that interactions between the enzyme and inhibitors at this site are an important determinant of domain selectivity. structural studies of inhibitor binding to ace have been able to provide essential information relating to the subtle differences in the substrate binding pockets of the two domains. this will be able to be exploited in the design of new, domain specific ace inhibitors. these may include not only c-domain specific inhibitors for use in the treatment of hypertension, but also n-domain specific inhibitors targeting the roles of ace in other physiological processes. in addition to the increasing understanding that mammalian ace is involved in a range of processes outside of blood pressure regulation, in recent years a number of other homologues of ace have been identified which further emphasise the diverse range of functions of these enzymes. . the overall fold is identical save for an additional region at the c-terminus of the n-domain structure which serves as a linker region between the two domains. at the centre of the substrate binding channel the inhibitor lisinopril is visible as well as a zinc ion (grey sphere) and a single chloride ion (orange sphere). (c) lisinopril binding to tace and the n-domain. although all of the residues forming direct interactions with lisinopril are conserved between tace and the n-domain, some differences are seen in the subsites particularly in the s 1 ' binding pocket. the subsite residues are show as cyan (tace) and magenta (n-domain) sticks and labelled following the same colour scheme. the lisinopril bound to tace is shown in light blue and from the n-domain in light pink. important differences include the orientation of glu162 in tace compared to asp140 in the n-domain which prevent interactions with the lysyl side chain of lisinopril in the ndomain at this position, and the position of the backbone at thr372/ arg350 which prevents the long, positive side chain of arginine coming into contact with and repelling the lysyl side chain of lisinopril in the year 2000, a study aiming to find novel genes involved in heart failure identified a cdna coding for a human homologue of ace (tipnis et al. 2000) . this was the first evidence of an ace homologue in humans, and the protein was appropriately named ace2 (angiotensin converting enzyme 2, ec number 3.4.17.23). like ace, ace2 is a type i integral membrane protein. it has a single catalytic domain that includes a zinc binding site. ace2 and each domain of sace share 33 % sequence identity, so it appears that the two enzymes have arisen following a gene duplication event which then allowed considerable divergence in ace2 sequence (krege et al. 1995) . in contrast to the widespread expression of sace, ace2 expression appears to be confined to the heart, kidneys and testis. furthermore, ace and ace2 have quite distinct biochemical properties. there is no evidence of ace2 having dipeptidyl carboxypeptidase activity as is routinely found in ace. instead it removes a single residue from the c-terminus of its peptide substrates, which include neurotensin and kinetensin. bradykinin is not a substrate for ace2, although it is able to cleave angiotensin i, but removes only a single c-terminal residue to give the nonapeptide ang1-9. ace2 is not inhibited by the ace inhibitors captopril, lisinopril and enalaprilat (tipnis et al. 2000; donoghue et al. 2000; crackower et al. 2002) . fig. 4 binding of the c-domain specific inhibitor rxpa380 to tace. (a) and (b) the structure of the c-domain specific inhibitor rxpa380. (c) key residues of the tace active site interacting with rxpa380. rxpa380 is shown as sticks with carbon atoms in green, the side chains of tace residues are also shown as sticks with cyan carbon atoms. the active site zinc ion is shown as a grey sphere. rxpa380 binds in a similar conformation to lisinopril (fig. 3) . (d) two key differences between tace (cyan) and the n-domain (magenta) likely to decrease the affinity of rxpa380 for the ndomain. the phe391/tyr369 substitution eliminates the interaction with the phenyl moiety of the inhibitor whilst the val379/ser357 and val380/ thr358 substitutions destroy the hydrophobic environment around the tryptophan group. (e) the s 2 ' binding pocket with side chains in close proximity to rxpa380 shown as sticks illustrates that the inhibitor does not fill this subsite and interactions here could potentially be increased to improve the efficacy of rxpa380 the structure of ace2, reported in 2004, provides explanations both for the function of ace2 as a zinc dependent carboxypeptidase and for many of the observed differences between ace and ace2 (towler et al. 2004 ). the extracellular portion of ace2 was shown to consist of two distinct domains. the first (residues 19-611) is a zinc metallopeptidase sharing 42 % sequence identity with the catalytic domains of ace. the second (residues 612-740) is 48 % identical to human collectrin. poor electron density was observed for the "collectrin-like" domain (towler et al. 2004 ). the first, "ace-like" domain is, like ace, predominantly helical. it consists of two subdomains (i and ii) forming the sides of a cleft stretching 40 å along and 15 å across the molecule, with a depth of 25 å. at the base of this cleft is the catalytic site. it is located approximately halfway along the length of the molecule towards one side, as illustrated in fig. 6a . the location of the active site is marked by the presence of the catalytic zinc ion. as in ace, and characteristic of zinc dependent proteases, the catalytic zinc ion is coordinated by two histidines and a glutamic acid: his374, his378 and glu402. in the absence of the inhibitor a water molecule completes the co-ordination of the zinc ion. the location of the catalytic site means that is shielded in the cleft that separates the two subdomains (fig. 6a) . a similar effect is achieved in ace with the catalytic site being located at the centre of the long substrate binding channel capped by the n-terminal lid. like ace, ace2 is extensively glycosylated with electron density observed at six n-linked glycosylation sites. there are three disulphide bonds in the ace2 structure, all of which are conserved in ace. shown as sticks with their carbon atoms in magenta. rxp407 is also shown as sticks, with green carbon atoms and the essential zinc is shown as a grey sphere. (d) two key differences between the n-and c-domain active sites which are likely to affect rxp407 binding. in the c-domain arg381 (magenta) is replaced by glu403 (cyan) resulting in the loss of an interaction with the inhibitor. similarly, phe391 (cyan) in the cdomain is unable to hydrogen bond with rxp407 as tyr369 (magenta) is in the n-domain an interesting feature of ace2, which has not been seen in ace, is a large conformational change on inhibitor binding. the two subdomains move relative to each other to close in around the inhibitor in the active site, illustrated in fig. 6 . differences in the subsites of ace2 compared to ace help to explain the observed differences in substrate specificity and catalytic properties. in ace2 the s 1 subsite is defined by four residues with large, bulky side chains, (tyr510, arg514, phe504 and thr347) likely to restrict the size of residues that can be accommodated at the p 1 position of substrates. bradykinin and angiotensin, both substrates of ace but not ace2, have phenylalanine and tyrosine respectively at the p 1 position the side chains of which are unlikely to be accommodated by the restricted s 1 subsite fig. 6e . another significant difference between the two enzymes is observed in the s 2 ' subsite, where a glutamine residue in ace is replaced in ace2 by arg273. the large arginine side chain effectively blocks the s 2 ' subsite, prohibiting ace2 from acting as a dipeptidyl peptidase as ace does (fig, 6e) . this also explains why ace2 is not inhibited by classical ace inhibitors (captopril, lisinopril and enalaprilat) which make contacts with the s 2 ' subsite (crackower et al. 2002) . the catalytic zinc ion also moves and its positions are also shown in green and purple. the inhibitor is shown as sticks with orange carbon atoms bound in the active site. (e) some key differences between tace (cyan) and ace2 (purple) which restrict the size of the s 1 binding pocket, probably restricting space such that ace2 can only remove the terminal residue from the c-terminus of peptide substrates whilst ace removes the c-terminal dipeptide ace2 is an exceedingly important enzyme to study. it has been shown to have an important role in heart failure (crackower et al. 2002; luft 2012; kuba et al. 2013; santos et al. 2013 ) and it has also been identified as a receptor for the coronavirus which was linked to the outbreak of severe acute respiratory syndrome (sars) (li et al. 2003) . the structural details of this recognition have been characterised and were reported in 2009 (wu et al. 2009 ). it is also important to study ace2 to improve our understanding of ace homologues. the diverged properties of ace2 highlight the range of processes that ace homologues may be involved in and the potential benefits that may come from studying them further. for many years it was thought that ace was confined to mammals, where it has a critical role in blood pressure homeostasis. more recently, it has become apparent that ace has many other substrates in addition to angiotensin i and bradykinin, and that it is involved in a large number of physiological processes outside of the raas. furthermore, the discovery of ace2 illustrates how homologues of ace could have arisen from gene duplication events and then evolved to have different, but equally important functions. in 1994, the first ace homologue outside of mammals was identified in the housefly musca domestica (lamango and isaac 1994) . since then, ace homologues have been found in every insect genome sequence to date ). this conservation among insects is indicative of an essential role for ace homologues. yet, given that insects lack any form of circulatory system resembling that found in mammals, significant questions remain as to the exact nature of this role or roles. one of the first insect ace homologues to be studied in detail was ance from drosophila melanogaster. first identified in 1995, ance shares 45 % sequence identity with human tace and, as such, has been used successfully as a model for studying the structural basis of inhibitor binding to human ace (cornell et al. 1995; tatei et al. 1995) . although ance is able to hydrolyse angiotensin i and bradykinin, two of the major substrates of mammalian ace, no insect homologues of these peptides have been identified. a lot of research has been performed to try and elucidate the physiological function of ance and thus insect ace homologues in the wider sense. ance mutant embryos develop normally but die during the early larval stages. this indicates that ance has an essential role in a physiological process critical for survival ). high levels of ance expression are observed in the gut epithelium and amniosera, suggesting that ance may be involved in processing peptides required for contraction of the heart and gut muscle (cornell et al. 1995; tatei et al. 1995) . ance expression is also concentrated around the reproductive organs of both male and female flies, suggesting a role in reproduction. further evidence for this was obtained in a 1999 study using insects with hypomorphic ance alleles. male flies homozygous for the hypomorphic ance alleles were infertile as a result of sperm failing to develop properly, suggesting that ance may be required for processing a peptide involved in spermatogenesis schoofs et al. 1998) . further investigation in 2007 showed that ance expression is concentrated in the secondary cells of the accessory glands of the testis. the accessory glands produce a number of peptides that mix with the sperm and the seminal fluid and induce physiological and behavioural changes in the female insect after mating. it is reasonable to suggest then that ance is not only involved in spermatogenesis in the male, but may also have a role in the female after mating . with this in mind, dup99b was suggested as a potential ance substrate. dup99b can induce egg laying in females, but in order to be functional the basic c-terminal dipeptide must be removed from the propeptide. in theory, this would make dup99b a good substrate for ance. as yet it has not been possible to investigate this hypothesis further, as ance is already expressed by the female reproductive organs (rylett et al. 2007 ). in order to understand any effect of ance on the female after mating tissue specific ance knockdowns in female flies would need to be performed. ance is not the only insect ace homologue which has been implicated in reproduction and it seems increasingly likely that this is a conserved function of these enzymes (wijffels et al. 1996; hurst et al. 2003; ekbote et al. 2003a , ekbote et al. 2003b macours and hens 2004; vercruysse et al. 2004) . for example, ace has been shown to be necessary for egg laying by female anopheles stephensi mosquitoes, where it has been suggested that it may be required to process a myotropic peptide needed for contractions of the oviduct . drosophila also produces a second ace homologue; acer. acer cdna was first identified in 1996, and from the outset it has been clear that ance and acer code for distinct proteins with unique physiological roles. unlike ance, acer is found neither in the amniosera nor the midgut, but it is found in developing heart cells where it has been shown to be necessary for heart development (taylor et al. 1996; houard et al. 1998 ). additionally, acer is involved in heart function in adult flies. rnai was used to specifically knockdown acer in adult drosophila and resulted in age-associated changes in heart contraction, leading to a decreased life span. as yet, the mechanisms behind this remain to be determined (liao et al. 2014) . acer has also been implicated in the regulation of circadian rhythms, with the suggestion that its expression is temporally regulated by the expression of clock. flies lacking active acer display decreased night-time sleep and increased fragmentation of the sleep that they do get. the mechanisms behind this are unclear, but it has been suggested that acer processes peptides involved in metabolism, with the resulting metabolic changes upon losing acer causing behavioural changes leading to the observed effects on sleep, rather than acer being involved in circadian rhythms per se (carhan et al. 2011; ishimoto et al. 2012 ). based on the evidence obtained so far, it is clear that ance and acer have distinct physiological roles; it is therefore not surprising that they have different substrate and inhibitor specificities. for example, unlike ance and mammalian ace, acer is unable to cleave angiotensin i and also has a much lower affinity for bradykinin. on the basis of these differences, one would predict that there must be some variations between the active sites of these two enzymes (taylor et al. 1996; coates et al. 2000; bingham et al. 2006) . the structure of ance was first published in 2003 at 2.4 å resolution (kim et al. 2003) . this was followed in 2010 by higher resolution (~2.0 å) structures, in a different crystal form, of the native enzyme and a number of enzyme inhibitor complexes (akif et al. 2010 (akif et al. , 2012 . the overall architecture of ance is conserved from human ace and the molecule is also heavily glycosylated, with three n-linked glycans visible in the structure at asn53, asn196 and asn311. no structure has yet been determined for acer, however in 2005 bingham and co-workers proposed a model for the three dimensional structure of acer based on the ance structure reported by kim et al. in 2003 (kim et al. 2003 bingham et al. 2006) . by comparing this model with the ance structure, some explanations have been offered as to the observed differences in substrate specificity of these two homologous enzymes. in ance the negatively charged side chains of asp360 and glu150 are likely to form electrostatic interactions with the cterminal arginine of bradykinin. based on the acer model, asp360 is replaced by his368, which would be unable to replicate this interaction, explaining the decreased affinity for and rate of hydrolysis of bradykinin by acer compared to ance. ance and acer also behave differently towards the cdomain selective inhibitor rxpa380. acer is inhibited by rxpa380 with a k i of 4.8 μm whereas no inhibition is observed for ance. the c-domain selectivity of rxpa380 has been partially attributed to two valine residues in the s 2 ' pocket. one of these is conserved in acer but replaced by a threonine in ance. this mutation would decrease the hydrophobicity of this pocket which accommodates the tryptophan moiety of rxpa380 hence decreasing the affinity of the inhibitor for ance compared to acer and the c-domain (kim et al. 2003; bingham et al. 2006) . these are just some of the potentially interesting features of the acer active site proposed based on the modelled structure (bingham et al. 2006 ). such small differences can have significant implications on substrate and inhibitor specificity. whilst there remains a great deal more to be learnt about the in vivo functions of acer, and indeed ance, it is clear that like mammalian ace, both of these enzymes are involved in a number of important physiological processes. it follows then, that studying these enzymes, particularly from a structural perspective, can only increase our understanding of the diverse range of functions of ace homologues. anopheles gambiae is one of the mosquito vectors responsible for the transmission of plasmodium falciparum, a causative agent of malaria. this makes a. gambiae an exceedingly important organism to study. it transpires that a. gambiae is also a particularly interesting organism to study in terms of ace. there are ten a. gambiae ace homologues (anoaces); the largest number to be found in any insect genome so far (holt et al. 2002) . the reasons for there being so many anoace homologues are not yet clear, but understanding the different functions of these enzymes will surely help us to understand more about the biological roles of other insect ace homologues, and indeed of mammalian ace outside of blood pressure regulation. furthermore, one of these, anoace9, appears to consist of two catalytic domains arranged in tandem, as seen in sace. this is the first instance of a two domain ace homologue to be reported in insects (burnham et al. 2005) and indicates that this two domain arrangement has been selected for on more than one occasion and so must surely be in some way advantageous. studying anoace9 may help us to understand the benefits of such an arrangement. in 2005 burnham and co-workers investigated the gene expression patterns of the different anoace homologues to try and infer more about their potential physiological roles. one of their key observations was that the expression of anoace3, 7 and 9 was upregulated for 48 h following a blood meal (burnham et al. 2005) . a similar observation was previously reported for the a. stephensi mosquito , suggesting that insect ace homologues may have a conserved role in processing peptides involved in metabolism. the anoace expression levels were also observed to alter in response to challenging the immune system of the mosquitoes. anoace7 was upregulated on infection with salmonella typhimurium and anoace1 on infection with staphylococcus aureus. conversely, anoace9 expression was reduced when the mosquitoes were challenged with beauveria bassiana (burnham et al. 2005) . these observations alone are of course not enough to conclude that anoace homologues have an important role in the immune response, but they do indicate that this area should be investigated further. given the evidence for mammalian tace and other insect ace homologues being involved in reproduction, the likelihood of some or all of the anoace homologues having a similar role cannot be ignored. at this early stage there is no indication as to which, if any of the anoace proteins may fulfil this role and further studies, likely knock down studies, will be required to investigate this further. identifying an enzyme with such a role could be highly significant, with the possibility of designing a. gambiae specific ace inhibitors for use in insecticides. of the nine anoace genes, six (anoace2, 3a, 3b, 4, 5, 6 and 7) are clustered together on the same chromosome and are likely to have arisen from relatively recent gene duplication events. indeed anoace5 and 6 are predicted to share 97 % sequence identity whilst anoace3a and 3b are coded for by the same gene and are identical save for the first exon (burnham et al. 2005) . anoace2-6 share many common features with each other and d. melanogaster ance and acer. all six of these anoace proteins were successfully modelled based on the structure of ance (pdb code 2x8y) (akif et al. 2010 ) using the automated mode of swiss-model (arnold et al. 2006; kiefer et al. 2009 ). as would be predicted given the high sequence identity, the crucial active site residues from ance were conserved in all of the models. there were though differences in the subsites, indicating probable differences in substrate preference. these differences are summarised in table 1 . the s 2 subsite has been identified as a crucial region conferring inhibitor and substrate binding specificity on the n-and c-domains of human sace (hubert et al. 1991; gordon et al. 2003; dive et al. 2004; corradi et al. 2006 corradi et al. , 2007 kröger et al. 2009; anthony et al. 2010) . for example, the substitution of tyr369 in the n-domain for phe391 in the c-domain is crucial for the c-domain selectivity of rxpa380, with phe391 forming an aromatic interaction with the phenyl moiety at the p 2 position of rxpa380 that tyr369 is unable to replicate (gordon et al. 2003; corradi et al. 2007 ). both the anoace5 and 6 models have a phenylalanine at the position of c-domain phe391, suggesting that they could form an interaction with an aromatic group at p 2 . anoace5 and 6 also have a conserved negative charge at the position of glu403 of the c-domain, which is replaced by arg381 in the n-domain fig. 7a . these two observations suggest that anoace5 and 6 and the c-domain of sace may accommodate substrates with similar functionalities at the p 2 position. in contrast, the anoace2-4 models have a tyrosine at the position of n-domain tyr369, indicating that they would not tolerate an aromatic group at p 2 , and may instead have a preference for a group to which they could form a hydrogen bond. unlike the n-domain anoace2-4 do not have a large positive side chain at the position of arg381. like the cdomain anoace2, 3a and 3b have a glutamate, whilst anoace4 has a serine fig. 7b . the models therefore suggest that anoace2-4 share some features of both the n-and cdomains of human sace at the s 2 subsite and this may well be reflected in their substrate and inhibitor binding properties. based on the models of anoace2-6, there appear to be some potentially significant differences in the s 1 subsite compared to the two domains of sace. for example, the n-and c-domain have a conserved phenylalanine reside (phe490 and phe512 respectively) which is replaced by tyrosine in anoace2-6 fig. 7c . this could have potentially significant consequences on inhibitor and substrate interaction, as the stacking interaction between the phenyl moiety at p 1 of both rxpa380 and rxp407 would not be maintained. interestingly, a tyrosine is conserved at this position in d. melanogaster ance. there are also interesting substitutions at the position equivalent to the c-domain glu143. the negative charge here is retained in ance and the anoace2 model. in the ndomain and anoace3a, 3b, 5 and 6 it is lost and replaced by serine (n-domain) or glutamine. most significantly though, in anoace4 lys133 occupies this position fig. 7c . the large positive lysine side chain is likely to have a significant effect on the type of groups that anoace4 can accommodate in the s 1 subsite. the anoace2-6 models also all have a conserved positive charge (either lysine or arginine) in the s 1 subsite, which, although conserved as lys62 in ance, is replaced by gln54 and leu81 in the n-and c-domains respectively fig. 7c . the introduction of these large positive side chains into what is in both domains of sace quite a large pocket of space is likely to influence the size and charge of functionalities that can be accommodated by anoace2-6 in the s 1 subsite. based on our models, anoace5 and 6 have an additional negative charge in their s 1 subsites compared to ance, the two domains of sace and anoace2-4. both have glu54, which takes the place of a small, non-polar side chain in the other subsites fig. 7c . overall, the models suggest that the s 1 subsites of anoace2-6 are quite polar environments compared to sace. the models presented here are not sufficient to determine the effect that this may have on binding properties, further work will be needed to investigate this. there is comparatively little variation in the s 1 ' and s 2 ' subsites of the anoace2-6 models compared to in the other subsites. a potentially significant difference is at the position of c-domain val380. this residue has been implicated in the selectivity of rxpa380 for the c-domain. it contributes to a hydrophobic environment for the tryptophan moiety of the inhibitor that is lost in the n-domain as val380 is replaced by thr358. interestingly; the valine at this position is conserved only in anoace2 and is replaced by a threonine in anoace3-6. this small difference, which has such a significant effect in determining the domain selectivity of rxpa380, highlights how subtle variations in the subsites of these enzymes can have significant effects on their biochemical properties. table 1 comparison of the amino acid residues comprising the binding pockets of the n-and c-domains of human sace, drosophila melanogaster ance and the models of the anopheles gambiae ace homologues; anoace2-7 c-n-ance ano2 ano3a ano3b ano4 ano5 ano6 ano7 y62 s35 e43 q59 t58 a47 a52 g50 g50 n302 a63 v36 t44 t60 t59 t48 s53 t51 t51 v303 n66 s39 t352 t358 n374 n373 n362 t368 t364 t365 t621 d377 q355 q361 e377 d376 d365 q371 q367 q368 d624 v380 t358 t364 v380 t379 t368 t374 t370 t371 t627 t282 s260 q266 t282 t281 t270 g275 d272 k273 anoace2-7 are clustered together on the same chromosome and thought to have arisen from a relatively recent gene duplication event. anoace1 is predicted to be considerably different to the other anoace homologues, belonging to a different functional grouping, and is likely to have acquired highly diverged functions (burnham et al. 2005; akif et al. 2012) . it is perhaps not surprising then that anoace1 was much more challenging to model and so an experimentally determined crystal structure will surely be needed to study the mechanism of action of this enzyme in more detail. it seems that this enzyme will make an exceedingly interesting case study and could reveal more unknown roles of ace homologues, aiding our understanding of this increasingly complex enzyme. the discovery of the anoace9 gene which appears to code for a protein with two homologous catalytic domains arranged in tandem provided the first evidence of a two domain ace homologue in insects. this now means that this domain arrangement, also seen in sace has been selected for on three separate occasions (burnham et al. 2005) . this organisation must surely then be in some way advantageous. studies of human sace have resulted in the suggestion that there may be some negative co-operativity between the two domains (georgiadis et al. 2003; binevski et al. 2003; andújar-sánchez et al. 2004; burnham et al. 2005; corradi et al. 2006 ) however more evidence is required before a conclusion can be reached. studying the structure and function of anoace9 could potentially yield a lot of information about the benefits of this organisation. angiotensin converting enzyme has been known of and studied for half a century; however it is only in the last decade that we have truly begun to understand the diverse range of physiological functions of this enzyme and its homologues. we now appreciate that ace and its homologues have extensive functions outside of blood pressure regulation. structural studies have greatly improved our understanding of the different biochemical properties of ace homologues. the elucidation of the human tace structure in 2003 was a huge breakthrough in the study of ace and was followed by the report of the n-domain structure in 2006. the structure of the d. melanogaster homologue ance is also frequently used as a model of human ace. the discovery and subsequent study of ace homologues from other organisms, most significantly d. melanogaster, has highlighted the diverse range of functions of this enzyme. ace homologues are conserved in insects, where, amongst other proposed roles, there is substantial evidence for them having an essential role in reproduction. studying insect ace homologues has already greatly improved our understanding of the mammalian enzyme, but these enzymes are also important to study in their own right. nowhere is this more evident than in the a. gambiae ace homologues. the a. gambiae genome codes for more ace homologues than has been seen in any other insect genome thus far and includes a two-domain enzyme comparable to sace. initial genomic studies have indicated that these enzymes may be involved in metabolism, the immune response and reproduction, however much more evidence is required to confirm this. this could potentially be achieved by treating the mosquitoes with ace inhibitors and observing the effect that this has. a similar approach has previously been taking with a. stephensi, which indicated that ace homologues were involved in reproduction . however this is unlikely to distinguish between the functions of the different anoace homologues, hence a better approach may be to use rnai to selectively knockdown the individual genes. liao et al. recently used this approach successfully in their work on acer (liao et al. 2014 ). fig. 7 features of the active sites of the anoace2-6 models compared to the n-and c-domains of human sace. in all images the n-domain selective inhibitor rxp407 is shown as sticks with green carbon atoms and the c-domain selective inhibitor rxpa380 as sticks with orange carbon atoms. the catalytic zinc ion is a grey sphere. (a) the s 2 subsite of anoace5 and 6. key residues of anoace5 and 6 are shown as grey sticks using anoace5 numbering. the c-domain is shown as cyan sticks and the n-domain magenta. anoace5 and 6 share a conserved phenylalanine (phe381) with the c-domain which is replaced by a tyrosine in the n-domain. this residue makes important contacts with the domain selective inhibitors and so suggests that anoace5 and 6 may share some binding preferences with the c-domain. there is also a conserved negative charge, asp393 which is replaced by positive arg381 in the n-domain, and a conserved valine, val508, replaced by thr496 in the n-domain. there is some similarity with the n-domain though, for example ser566 which is replaced by phe570 in the cdomain. (b) the s 2 subsite of anoace2-4. the s 2 subsites of the anoace2-4 models are very similar and so are represented here by anoace3a, shown as light orange sticks. there are some features shared with both the n-and c-domains of sace; tyr390 is conserved in the n-domain, but replaced by phe391 in the c-domain, whilst glu402 is conserved in the c-domain but replaced by the positive arg381 in the n-domain. these residues are likely to have significant effects on substrate and inhibitor binding properties. (c) the s 1 subsite of anoace2-6. common features of the anoace2-6 models are represented by anoace3a in light orange, with unique features of anoace4 and anoace5 and 6 shown as violet and grey sticks respectively. the anoace2-6 models share a conserved tyrosine at tyr511 which is replaced by phenylalanine in both the n-and cdomain. all of the models have a unique positively charged residue not found in the n-or c-domains, shown here by lys71 in anoace4 and arg77 in anoace3. anoace4 has a further positively charged residue, lys133, which is replaced by neutral or negatively charged residues in the n-and c-domains and the other models. in contract anoace5 and 6 have an additional negative charge introduced by glu54. these substitutions of charged residues are likely to have a significant effect on substrate and inhibitor binding properties as we have discussed, d. melanogaster ance has been successfully used as a model for inhibitor binding to human ace. it is not unreasonable to consider that it may be possible to use anoace9 in a similar way as a model for human sace in order to learn more about the rationale for the presence of two homologous domains arranged in tandem. here we have modelled the active sites of the a. gambiae ace homologues based on the structure of d. melanogaster ance and compared them to the human and d. melanogaster enzymes. whilst this is of course no substitute for experimentally determined crystal structures, it has indicated some key differences that could potentially be exploited in the design of insect specific ace inhibitors; perhaps targeting their role in reproduction. in the longer term such inhibitors could be designed for use in insecticides, which would be particularly useful when considering a. gambiae as a vector for the transmission of the malaria causing parasite p. falciparum. ace revisited: a new target for structure-based drug design high-resolution crystal structures of drosophila melanogaster angiotensin-converting 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oviposition and ecdysteroid levels in lepidoptera temperature-induced selective death of the c-domain within angiotensin-converting enzyme molecule the two homologous domains of human angiotensin i-converting enzyme interact differently with competitive inhibitors * the two homologous domains of human angiotensin i-converting enzyme are both catalytically active* cloning and characterisation of angiotensinconverting enzyme from the dipteran species, haematobia irritans exigua, and its expression in the maturing male reproductive system crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor drugs targeting the reninangiotensin-aldosterone system key: cord-355807-q3bngari authors: yepes-pérez, andres f.; herrera-calderon, oscar; quintero-saumeth, jorge title: uncaria tomentosa (cat’s claw): a promising herbal medicine against sars-cov-2/ace-2 junction and sars-cov-2 spike protein based on molecular modeling date: 2020-10-29 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1837676 sha: doc_id: 355807 cord_uid: q3bngari covid-19 is a novel severe acute respiratory syndrome coronavirus. currently, there is no effective treatment and vaccines seem to be the solution in the future. virtual screening of potential drugs against the s protein of severe acute respiratory syndrome corona virus 2 (sars-cov-2) has provided small molecular compounds with a high binding affinity. unfortunately, most of these drugs do not attach with the binding interface of the receptor-binding domain (rbd)–angiotensin-converting enzyme-2 (ace-2) complex in host cells. molecular modeling was carried out to evaluate the potential antiviral properties of the components of the medicinal herb uncaria tomentosa (cat’s claw) focusing on the binding interface of the rbd–ace-2 and the viral spike protein. the in silico approach starts with protein–ligand docking of 26 cat’s claw key components followed by molecular dynamics simulations and re-docked calculations. finally, we carried out drug-likeness calculations for the most qualified cat’s claw components. the structural bioinformatics approaches led to the identification of several bioactive compounds of u. tomentosa with potential therapeutic effect by dual strong interaction with interface of the rbd–ace-2 and the ace-2 binding site on sars-cov-2 rbd viral spike. in addition, in silico drug-likeness indices for these components were calculated and showed good predicted therapeutic profiles of these phytochemicals found in u. tomentosa (cat’s claw). our findings suggest the potential effectiveness of cat’s claw as complementary and/or alternative medicine for covid-19 treatment. communicated by ramaswamy h. sarma the severe acute respiratory syndrome corona virus 2 (sars-cov-2) is a part of coronavirus family (cov) and was initially identified in wuhan, china. covid-19 (coronavirus disease 2019) is highly contagious in humans, which has rapidly spread and caused an unprecedented pandemic, with a large number of deaths and economic crisis in the world (prajapat et al., 2020) . according to the latest report of the world health organization (who), over 32.7 million cases and 991 000 deaths of covid-19 were confirmed as of september 27, 2020 (world health organization, 2020 . in developing countries of latin america and the carribean, the public health has been the most affected because people do not have the opportunities to access a modern health system and medicines . phytotherapy based on natural products might be a proper alternative for treating viral diseases (akram et al., 2018) . according to who estimates, about 80% of the population in developing countries uses traditional medicine in primary health care, mainly medicinal plants (world health organization, 2018) . the selection of natural products for the study of their biological properties has been addressed through three fundamental methodologies: the selection of random natural sources, the selection based on chemotaxonomy (screening of similar compounds in organisms belonging to the same family or genus) and selection based on ethnomedicine (heinrich, 2002) . ethnomedicine has been considered the most effective therapy and consists of the study of natural products that have a long history of use in some communities for the treatment of certain diseases and are part of the phytotherapeutic arsenal of popular knowledge (wu & tan, 2019) . on the other hand, uncaria tomentosa (willd. ex schult.) dc. named cat's claw ('uña de gato' in spanish) is a woody vine indigenous to the peruvian amazon and other tropical areas of south and central america that belongs to rubiaceae family (sandoval et al., 2000) . currently, the raw material of u. tomentosa is dispensed in public hospitals of the social health insurance (essalud-peru) as complementary medicine service (cms) (gonzales et al., 2010) . traditionally, extracts prepared by roots and barks decoction are used against several diseases, such as allergies, arthritis, inflammations, rheumatism infections and cancer (araujo et al., 2018) . bioactive constituents of u. tomentosa extracts include proanthocyanidins [proanthocyanidin b2 (the main component), proanthocyanidin b4, proanthocyanidin c1, an epicatechin trimer, epiafzelechin-4b!8-epicatechin and an epicatechin tetramer] (batiha et al., 2020; navarro-hoyos et al., 2017) , oxindole alkaloids (isopteropodine, pteropodine, rhynchophylline, mytraphylline, speciophylline, uncarine f and uncarine e), indole alkaloidal glucosides (cadambine, 3dihydrocadambine and 3-isodihydrocadambine) (batiha et al., 2020; kura s et al., 2009; laus et al., 1997; lima-junior et al., 2013; lock et al., 2016; navarro et al., 2019 snow et al., 2019) , quinovic acid glycosides (pavei et al., 2012) , tannins (ostrakhovich et al., 1997) , polyphenols, catechins, beta sitosterol (aquino et al., 1997; navarro et al., 2019) and proteins (lenzi et al., 2013) , which individually or synergistically contribute to their therapeutic properties. in regards to the antiviral properties of u. tomentosa, the alkaloid fraction has been demonstrated to be the most effective on human monocytes infected with dengue virus-2 (denv) in vitro (reis et al., 2008) . another study revealed that only the alkaloidal fraction has inhibitory activity on dengue virus, and the negative effect was observed with the nonalkaloidal fraction (lima-junior et al., 2013) . in another study, the antiherpetic activity of u. tomentosa seems to be associated with polyphenols or with their synergistic effect with pentacyclic oxindole alkaloids or quinovic acid glycosides (caon et al., 2014) . u. tomentosa hydroethanolic extracts have demonstrated a significant in vitro inhibitory effect on the replication of herpes simplex virus type 1, and the inhibition of viral attachment in the host cells was characterized as the main mechanism of its antiviral activity (terlizzi et al., 2016) . sars-cov-2 contains four structural proteins, namely the spike (s), membrane (m), envelope (e) and nucleocapsid (n) proteins. the s protein is responsible for the host attachment and fusion of the viral and host-cell membranes (wu et al., 2020) . otherwise, the angiotensin-converting enzyme 2 receptor (ace-2r) is the host cellular receptor with a higher affinity to sars-cov-2 (jamwal et al., 2020) . this process is triggered when the s1 subunit of s protein binds to a host-cell receptor (han & kr al, 2020) . to engage a host-cell receptor, the receptor-binding domain (rbd) of s1 undergoes transient hinge-like conformational motions (receptoraccessible or receptor-inaccessible states). u. tomentosa's constituents could block the virus from binding to human cell receptors and disrupt the virus cycle helping to prevent the protein maturation of sars-cov-2 and limit its infection spread . several molecular targets have been identified as the main druggable key of sars-cov-2 for new antiviral discovery. moreover, its x-ray structure has been recently released, hence allowing possible computational analysis. in fact, several computational studies have already been undertaken on this system including a long 20 ls molecular dynamics (md) study and virtual screening of several databases (huang et al., 2020) . with neither drugs nor vaccines approved against covid-19 yet, finding strategies to diminish the impact of the pandemic is fundamental. medicinal herbs and, more particularly, those with demonstrated antiviral activities as u. tomentosa could slow down the spreading of the disease. particularly in developing countries, in which the accessibility to these plants is easier and more economically viable, adding these medicinal herbs to the general medical kit may be beneficial. here, our study stands on an in silico strategy reminiscent to those applied at the early stage of current state-of-the-art drug discovery pipelines and includes (1) protein-ligand docking of all bioactive compounds of u. tomentosa against focusing both on the binding interface of the rbd-ace-2 and inside sars-cov-2 rbd spike protein, (2) simulations of ligand pathway of the best predicted compounds from step 1 to evaluate convenient entrance mechanism of the compounds to the binding site, (3) md simulation to assess the stability of the best protein-ligand complexes from 1, (4) calculation of pharmacokinetics parameters for the most qualified compounds resulting from the previous parts of the docking protocol. this study demonstrates the antiviral potential of u. tomentosa-based products to be applied as a rapid phytotherapeutic option for covid-19. calculated binding affinity of the main constituents of the u. tomentosa (table 1) was explored for its ability to disrupt the sars-cov-2/ace-2 complex and inhibit sars-cov-2 spike protein of novel coronavirus findings, a facile therapeutic option for anti-coronavirus therapy. to this purpose, the crystal structures of sars-cov-2/ace-2 complex and sars-cov-2 spike protein were downloaded from the protein data bank (pdb entry code 6m17 and 6vyb, respectively) (yan et al., 2020) and all bounded ligands, ions and solvent molecules were manually removed using the ds visualizer 2.5 program. for docking studies, the structures of the selected proteins were parameterized using autodock tools (trott & olson, 2009) . to facilitate the formation of hydrogen bonds, polar hydrogens were added. ligands used in this study are major components of the u. tomentosa extracts and a sulfated heparin octasaccharide (taken from pdb 5ue2), a potent sars-cov-2 inhibitor in vitro reported in the literature (kwon et al., 2020) . the 2d structures of the 26 cat's claw constituents were obtained as mol.2 files from the zinc database (chemaxon, 2016) . the resultant compounds were submitted to marvinsketch 8.3 (morris et al., 1998) to correct the protonation states of the ligands at physiological ph 7.4. in addition, the geometry optimization of all ligands was carried out using the hf/6-31g ã level of theory. then, the structures were parameterized using autodocktools to add full hydrogens to the ligands, to assign rotatable bonds and saving the resulting structure in the required format for use with autodock. all possible flexible torsions of the ligand molecules were defined using autotors in pdb autodocktools (morris et al., 2009; walls et al., 2020) to promote the calculated binding with the target structure. our docking protocol was performed using autodock vina and default procedures to dock a flexible ligand to a rigid protein. docking simulation of ligands was carried out on the interface between the sars-cov-2 and ace-2 (pdb code: 6m17) (yan et al., 2020) , where both proteins residues are in proximity. next, we used the cryo-em structure of sars-cov-2 spike protein (pdb code: 6vyb) in their open state (lipinski et al., 2012) to explore the potential inhibition of components of the cat's claw, selecting ace-2-binding pocket to this study. once a potential binding site was identified, 26 compounds which are the major components of the cat's claw extracts were docked to this enzymes-site to determine the most probable and the most energetically favorable binding conformations. to accomplish rigorous docking simulations involving a grid box to the identified catalytic site, autodock vina 1.1.2 (trott & olson, 2009 ) was used. the exhaustiveness was 20 for each protein-ligand pair (number of internal independent runs). the active site was surrounded by a docking box of 40 â 40 â 40 å with a grid spacing of 1 å. affinity scores (in kcal mol -1 ) given by autodock vina for all compounds were obtained and ranked based on the free energy binding theory (more negative value means greater binding affinity). the resulting structures and the binding docking poses were graphically inspected to check the interactions using the ds visualizer 2.5 (http://3dsbiovia.com/ products/) or the pymol molecular graphics system 2.0 programs. molecular interaction stability of protein-ligand complexes obtained by docking simulations were verified through md simulations by using the gromacs program (abraham et al., 2015) considering the sars-cov-2/ace-2 interface, as well as the sars-cov-2 spike protein active site and the best docking pose for proanthocyanidin c1, qag-2, proanthocyanidin b2 and 3-dihydrocadambine, respectively. force field parameters for protein and ligands were derived independently. for the selected protein, the amber03 force field was selected and assigned using the pdb2gmx tool of the gromacs program packages, meanwhile ligand force field parameters were prepared with the generalized amber force field (gaff) using the molecular geometries previously optimized with the hf/6-31g ã level of theory in gas phase, (foresman et al., 1992; glendening et al., 2012; roothaan, 1951) with the gamess-us program (schmidt et al., 1993) . in addition, each ligand was verified as a minimum through a harmonic vibrational normal mode analysis. atomic charges were obtained with the merz-kollman scheme (singh & kollman, 1984) by fitting a restricted electrostatic potential (resp) model by the gamess-us program (bayly et al., 1993) , and the output file was used into the resp sub-program of the ambertools program package (cornell et al., 1995) . assignment of gaff force field parameters was carried out by the antechamber program (wang et al., 2006) and the required input files for molecular dynamics simulations were prepared using the acpype python interface. protein and protein-ligand complexes were solvated in a rectangular box of tip3p waters. the obtained system was neutralized adding seven-sodium counter ions to neutralize the net negative charge of the protein, and then physiological conditions (298 k, ph 7.4, 0.9% nacl solution) were established (hammad et al., 2020) . to remove spurious contact, molecular geometries were optimized with the steepest descent algorithm with 100,000 steps, protein backbones atoms were constrained with a force constant of 1000 kj mol à1 . then, the md simulations were allowed to run for 1000 ps in the npt ensemble. in addition, 50 ns in the npt ensemble were calculated for the production stage. all simulations were carried out under periodic boundary conditions. a cubic box with the size of 25 â 25 â 25 nm was used. a 12 å cutoff distance was used to calculate nonbonded interactions. electrostatic interactions were treated with the ewald particle mesh (pme) method (nishizawa & nishizawa, 2010) ; while van der waals interactions were introduced by using the cut-off scheme. finally, table 1 . best binding energy (kcal mol -1 ) based on autodock scoring of the main constituents of the u. tomentosa into the rbd/ace-2 interface and sars-cov-2 spike protein binding domain (rbd) (pdb id: 6vyb). best binding energy rbd/ace-2 interface (kcal mol -1 ) best binding energy sars-cov-2 rbd (kcal mol -1 ) spiroxindole alkaloids uncarine f -7.1 -6.1 speciophylline -6.8 -6.8 mitraphylline -6.8 -6.2 pteropodine -6.7 -6.2 isopteropodine -6.7 -6.3 isomitraphylline -6.6 -6.1 rynchophylline -6.0 -5.5 isorynchophyllin -6.0 -5.7 indole glycosides alkaloids 3-isodihydrocadambine -7.6 -6.4 cadambine -7.6 -6.1 3-dihydrocadambine -7.0 -7.1 polyhydroxylated triterpenes uncaric acid -7.0 -5.5 floridic acid -7.0 -5.5 pht-1 -6.9 -5.6 quinovic acid glycosides qag-2 -8.2 -6.8 qag-5 -8.0 -6.1 qag-4 -7.8 -6.8 qag-6 -7.8 -6.8 3) b a hepos: heparin octasaccharide taken from pdb 5ue2 was used as positive control. b estimated by kwon et al. (2020) . the v-rescale thermostat at 300 k with a coupling constant of 1.0 ps was used and the pressure was kept constant at 1 atm using the parinello-rahman barostat (parrinello & rahman, 1981) with a coupling constant of 2.0 ps and a compressibility factor of 4.5 â 10 à5 bar à1 . all covalent bonds were constrained using the lincs algorithm and the contact list was updated every 40 fs. drug-likeness prediction along with further adme properties presents a wide of opportunities for a rapid new antiviral drug discovery. the drug-like and adme properties for the most active components of the u. tomentosa extract (constituents having the highest binding affinity) were screened using openaccess cheminformatics platforms such as molinspiration (for molecular weight -mw, rotatable bonds and polar surface area -psa descriptors), alogps 2.1 (for log p o/w descriptor) and the pre-admet 2.0 to predict four pharmaceutical relevant properties such as intestinal permeability (app. caco-2), albumin-binding proteins (k hsa ), madin-darby canine kidney (mdck line) cells permeation and intestinal absorption (%hia). these parameters establish movement, permeability, absorption and action of potential drugs (ertl et al., 2000) . the interpretation of both mdck and caco-2 permeability using preadmet is as follows: (1) permeability lower than 25: low permeability; (2) permeability between 25 and 500: medium permeability; (3) permeability higher 500: high permeability. this study was performed to identify whether certain components of u. tomentosa extracts have potential therapeutic effects against covid-19. to this purpose, a database of 26 compounds that have shown prevalence on the herbal therapeutic activity has been generated ( figure 1 ) (aquino et al., 1990 (aquino et al., , 1997 batiha et al., 2020; keplinger et al., 1998; kitajima et al., 2000; lima-junior et al., 2013; lock et al., 2016; montoro et al., 2004; navarro et al., 2019 pavei et al., 2012; peñaloza et al., 2015; snow et al., 2019; vera-reyes et al., 2015) . our initial hypothesis is that cat's claw should contain molecules with highest therapeutic profiles against sars-cov-2, by disrupting sars-cov-2/ace-2 association or by inhibiting sars-cov-2 spike protein. during covid-19 host infection, sars-cov-2 enters human epithelial cells through a first molecular recognition of rbd to the ace-2 protein. when coronaviruses bind directly to the peptidase domain (pd) of ace-2, it results in the loss of their primary physiological role, which includes vasoconstriction and blood pressure regulation. in consequence, binding of sars-cov-2 rbd to the human ace-2 receptor is associated strongly with cardiovascular diseases, such as hypertension, heart attack and chronic nephropathies. blocking the binding of sars-cov-2 to the human ace-2 receptor may result in the most promising approach to prevent virus entry into human cells. recently, the cryo-em cocrystal structures of the rbd of sars-cov-2 with human ace-2 have been solved (yan et al., 2020) , which open the possibility to design better and more specific inhibitors for suppression of viral infection. thus, to study the effectiveness of cat's claw against sars-cov-2/ace-2 complex, docking approaches were carried out in the ace-2-rbd binding interface as the druggable site, to establish the interaction between the selected site and the main constituents of cat's claw. we also performed molecular docking studies to find a potential association of constituents of cat's claw to the sars-cov-2 spike protein. this approach also could conduce to block the sars-cov-2 spike protein interaction with human receptor ace-2. hence, in this article, the structure of spike glycoprotein (pdb id: 6vyb) is to be considered as an additional druggable target. in addition, we have also performed the docking of a sulfated heparin octasaccharide (hepos) as positive reference, which have been recently reported as an effective in vitro inhibitor of sars-cov-2 by its interaction against the spike protein rbd (kwon et al., 2020) . overall, docking approach revealed that the most components founded in cat's claw could block sars-cov-2/ace-2 association because they displayed significant binding affinity at interface of ace-2-rbd complex in the range between -6.0 to -8.6 kcal mol -1 (table 1) . on the other hand, when structures were docked against the sars-cov-2 spike protein, good dock scores were obtained (specially, proanthocyanidins series) ranging from -5.5 to -7.2 kcal mol -1 . thus, we performed a rigorous exploration of the docking solutions obtained from these compounds when docking occurred against sars-cov-2-related enzymes. hence, based on the analysis of these different results and visual inspection, a clear behavior appears along the molecular docking that could be summarized as follows in the following sections. the recognition of sars-cov-2 by human ace-2 can be divided into several contacts. from the ace-2 structure: a 1 helix domain comprises 12 critical aminoacids, such as gln24, thr27, asp30, lys31, his34, glu35, asp38, tyr41, gln42, glu37, phe390 and gln388; a 2 helix domain located around met82 and four residues (lys353, gly354, asp355 and arg357) between b 3 and b 4 sheets are needed to recognize sars-cov-2. from sars-cov-2 structure, ten residues are essential for ace-2 binding, such as lys417, gln493, tyr453, tyr505, tyr449, asn501, gln498, thr500, gln474 and phe486. therefore, these key binding domains were considered in this article to explore the ability of u. tomentosa to disrupt sars-cov-2 rbd interaction to human ace-2 (yan et al., 2020) . in general, all compounds as part of u. tomentosa show docked structures that fit well into the rbd/ace-2 interface, particularly along a 1 , a 2 , b 3 and b 4 domains of human ace-2 ( figure 2 ) and with good predicted docking scores that range from -6.0 to -8.6 kcal mol -1 (table 1) . calculations revealed that most of ligands were located between b 3 and b 4 sheets, and a reduced group very close to a 1 and a 2 helix of ace-2. since during viral infection a 1 , a 2 , b 3 and b 4 domains are responsible for the recognition of sars-cov-2 by human ace-2, our findings open the possibility to use u. tomentosa against sars-cov-2/ace-2 association. notably, most predicted complexes have the interaction fingerprint with those of critical residues as part of ace-2-rbd binding interface. protein-ligand interaction analysis of the 26 constituents to the rbd/ace-2 complex revealed that the most of constituents strongly bonded to ace-2 through his34, asp30, glu37, phe390, gln388, lys353, thr27, glu35 residues, meanwhile with sars-cov-2 rbd showed critical contacts with tyr505, tyr453, lys417, tyr449 residues. due to these compounds as part of cat's claw and interaction with key aminoacids inside ace-2-rbd binding interface, we are encouraged to believe that u. tomentosa could affect the interaction of sars-cov-2 spike protein with ace-2. note that this approach may be useful as a rapid therapeutic option to prevent or treat covid-19. a simple view showed that all ligands were able to interact with those critical aminoacids involved in the molecular recognition of rbd to the ace-2 protein, where at least one compound from each chemical series showed great ability to bind to the sars-cov-2-rbd complex along the interface. hence, we focused on five compounds (those with highest negative energy value obtained after docking on each series) such as proanthocyanidin c1 (-8.6 kcal mol à1 ), qag-2 (-8.2 kcal mol à1 ), 3-isodihydrocadambine (-7.6 kcal mol à1 ), uncarine f (-7.1 kcal mol -1 ) and uncaric acid (-7.0 kcal mol à1 ), which had higher or comparable affinity than positive reference hepos (-7.3 kcal mol à1 ) to the sars-cov-2-rbd complex (table 1) . at this point it is worth mentioning that the docking calculations involving positive reference give a value in very good agreement with experimental one (-7.3 kcal mol à1 ), hence providing with a certain amount of confidence regarding the autodock scoring function of this project. a rigorous view of 2d plots ligand interactions into ace-2-rbd interface generated from ds visualizer program revealed which interactions are involved by the most docking active ligands and how their structures affect them. thus, the most active docking ligand proanthocyanidin c1 (which had affinity of à8.6 kcal mol à1 ) was able to interact with those critical aminoacids for binding sars-cov-2 spike to the human ace-2 receptor. we found that proanthocyanidin c1 establishes three strong hydrogen bond interactions between the hydroxyl groups in flavone moiety with tyr505 (sars-cov-2) and asp30 (ace-2 receptor) residues, one p-alkyl interaction with lys417 residue and several hydrophobic interactions between the molecule and the glu37, gln388, lys353, phe390, thr37 and his34 residues. in addition to these critical aminoacids, proanthocyanidin c1 revealed notable binding interactions with human ace-2 receptor very close to interface through four hydrogen contact, such as ala386, thr92, lys26 and ala337 that could may also affect the interaction of rbd with ace-2 (figure 3(a) ). furthermore, a closer look at the best possible binding pose of qag-2 (which display a high docking score of à8.2 kcal mol à1 ), reveals strong interactions at the contact interface between the two proteins. thus, it was found that qag-2 forms two hydrophobic interactions with critical aminoacids lys353 and asp355 located between b 3 and b 4 sheets in the ace-2 protein. we also observed that qag-2 displays one interaction at interface with val407 (from spike protein) through strong hydrogen bond. in addition, various hydrophobic contacts were observed between molecule and ser375, phe377, val510, cys379, ile410, gly404, asp405, phe356, ala386, arg408 and gly502 at the sars-cov-2 rbd-ace-2 interface (figure 3(b) ). note that the deprotonated carboxylate moiety does not form any important interaction, therefore does not play any specific role in the qag-2 binding at rbd-ace-2 interface. this fact can be explained primarily because positively charged residues (lysine, arginine or histidine) do not surround this moiety at the interface. on the other side, 3-isodihydrocadambine (-7.6 kcal mol -1 ) has key contacts with residues in the rbd/ace-2 binding interface (figure 3(a) ). thus, molecule showed several interactions with critical residues of human ace-2, which are essential for sars-cov-2 spike binding, among them, one hydrogen bond contact and one p-alkyl interaction with glu37 residue. importantly, the protonated nitrogen atom in the b-carboline moiety forms one salt bridge interaction with the carboxyl in asp30 (from ace-2 protein) and three hydrophobic interactions with phe390, thr27 and gln388. furthermore, 3-isodihydrocadambine also had a crucial h-bond contact with tyr505 amino acid of sars-cov-2 spike protein, which is well-reported as an initial contact point during ace-2 recognition. 3-isodihydrocadambine also showed one p-cation interaction between fused aromatic ring in the b-carboline moiety and the key residue lys417 and eight van der waals contacts with spike protein residues, including glu406, asp405, gln409, phe456 leu455, asp405, tyr453 and tyr421. further interactions were also observed that might promote the disruption of the interactions between sars-cov-2-rbd and ace-2, among them two h-bonds formed per arginine residues 393 (from ace-2 protein) and 403 (from spike protein) interacting with the sugar moiety. interestingly, uncarine f binds at rbd/ace-2 interface with binding affinity of à7.1 kcal mol à1 through several interactions with those base residues for the recognition of sars-cov-2 to the human ace-2 (figure 3(b) ). besides two strong hydrogen contacts with his34 of human ace-2 and tyr505 from the sars-cov-2 spike protein, uncarine f showed several hydrophobic interactions with key aminoacids at the interface, such as lys353, asp30, phe390, glu37, tyr453 and gln388. finally, analysis of the 2d-interaction map for uncaric acid (which had docking score of à7.0 kcal mol à1 ) revealed crucial contacts with essential aminoacids residues for ace-2-rbd binding. thus, uncaric acid binds at asp30 residue of ace-2 through a hydrogen bonding with one of the hydroxyl groups. furthermore, hydrophobic interactions between molecule and five of those key residues at interface were displayed as follows: his34, tyr453, phe390, gln388 and tyr505. notably, uncaric acid had five p-alkyl interactions with other interface residues that probably could promote the ace-2-rbd binding cleavage, such as leu29 (d), val93 (d), lys26 (d), pro389 (d) and lys417 (f). in accordance to abovementioned, our docking studies shows that several components of u. tomentosa may have the ability to disrupt the association of sars-cov-2 spike protein with the human ace-2 receptor. among these components, proanthocyanidin c1 (-8.6 kcal mol à1 ), qag-2 (-8.2 kcal mol à1 ), 3-isodihydrocadambine (-7.6 kcal mol à1 ), uncarine f (-7.1 kcal mol à1 ) and uncaric acid (-7.0 kcal mol à1 ) had good predicted binding affinity for binding interface and they can naturally access it without noticeable energetic cost. these findings suggest that u. tomentosa may be a viable treatment option during initial stage of the covid-19 infection. recently, cryo-em structure of the sars-cov-2 spike glycoprotein was resolved in their closed and open states (pdb id: 6vxx and 6vyb, respectively) (walls et al., 2020) . sars-cov-2 spike protomer consists of five functional domains, namely as ntd, rbd, fp, hr1 and cd (figure 4 ). because rbd domain is responsible for the binding to the host cell receptor (ace-2), we focused our docking investigations inside the several residues in the sars-cov-2 rbd have been identified as essential in the association to the human ace-2 during coronavirus (covid-19) infection, including tyr449, tyr453, gly496, thr500, tyr505, lys417, gln493, asn501 and gln498 (lan et al., 2020) . therefore, to demonstrate the ability of constituents of u. tomentosa to block binding of the sars-cov-2 spike protein to human ace-2 receptor, we performed molecular docking studies around aforementioned critical amino acids, meaning this docking runs were carried out inside ace-2 binding surface of rbd. from a general perspective, promising docking scores were obtained when the major constituents from u. tomentosa bind to the rbd of sars-cov-2 (ranging from -5.5 to -7.2 kcal mol à1 ). thus, to compare the best binding pose of the most docking-active components of u. tomentosa and hepos (positive reference) inside sars-cov-2 rbd, figure 5 illustrates the most stable binding poses based on autodock scoring listed in table 1 for all constituents on rbd binding domain. the superposition of the positive reference (hepos) and the best conformation obtained theoretically for selected docked compounds showed that the major constituents in the ethanolic extract of u. tomentosa can accommodated themselves into stable conformations occupying this binding site during docking process. notably, all main constituents of u. tomentosa had at least two interactions with those key aminoacids for sars-cov-2 rbd binding to human ace-2, through h-bonds, p-contacts or hydrophobic contacts, making this herb a promising treatment that may be used in the early stages of the covid-19 infection. among them, four exhibited high potential to bind rbd: 3-dihydrocadambine (in brown), proanthocyanidin b4 (in purple blue), proanthocyanidin b2 (in light blue) and proanthocyanidin c1 (in hot pink) ( figure 5) , which had the highest docking score of -7.1, -7.2, -7.2 and -7.0 kcal mol à1 , respectively, that would be comparable to that reported for the potent inhibitor hepos of -7.3 kcal mol à1 . as such, in searching those critical contacts that could blocks rbd-ace-2 interaction, an exhaustive analysis has been undertaken to the docking results for those components as mentioned in figure 6 . as shown in figure 7 , our findings revealed that the most docking active molecules complexed with the sars-cov-2 rbd had an interaction fingerprint involving seven critical residues implied in the sars-cov-2 spike attachment to the human receptor ace-2: tyr453, gly496, lys417, asn501, tyr505, gln498 and asn501. thus, 3-dihydrocadambine displays two strong h-bond through binding between sugar and acetyl moiety and key tyr453 and gly446 residues, respectively. notably, tyr453 also established one p-p stacking interaction with the fused aromatic ring in the b-carboline moiety. in addition, critical residues lys417 and arg403 from rbd was involved in the binding event by forming three p-cation contacts with 3-dihydrocadambine, while hydrophobic interactions formed with asn501, tyr449, gln493, tyr505 residues clearly favored its affinity for sars-cov-2 spike protein. however, a visual inspection to the 2d-protein-ligand interaction plot of 3-dihydrocadambine shows that the protonated nitrogen formed two unfavorable positive-positive interactions (represented in red dotted lines) that could have great significance in the stability of protein-ligand complex. proanthocyanidin b2 was well-fitted into the functional domain rbd and its hydroxyl groups formed four hydrogen bonding and four hydrophobic contacts with critical residues for binding to ace-2, including gly496, asn501, gln493 and tyr505, tyr453, lys417, gln498, respectively. one further hbond with ser494 and one hydroxyl group were predicted by docking when proanthocyanidin b2 binds to sars-cov-2 rbd. finally, proanthocyanidin c1 was able to bind sars-cov-2 rbd through three strong h-bonds with those critical residues (gly496, lys417, asn501) crucial to association with human ace-2. also, further van der waals interactions were predicted to form between proanthocyanidin c1 and crucial gln498, tyr453, gln493, tyr505, gln498 residues that may be stabilizing the binding event to the rbd domain. with higher confidence on the viability of our docking predictions of the best compound with highest binding affinity, such as proanthocyanidin c1 (-8.6 kcal mol à1 ) and qag-2 (-8.2 kcal mol à1 ) into the rbd/ace-2 interface and 3-dihydrocadambine (-7.1 kcal mol à1 ) and proanthocyanidin b2 (-7.2 kcal mol à1 ) within sars-cov-2 rbd binding domain, we further evaluated the stability of the docked complexes throughout md simulations for 50.0 ns. to accomplish this aim, we first calculated the root mean square deviation (rmsd) for ligands for 50 ns of md simulation at real natural conditions into the selected binding pocket. the md simulation results (figure 8(a-d) ) showed that the rmsd of the systems reached equilibrium after around %5 ns of simulation time. in general, after equilibration, small fluctuations in the rmsd were observed, suggesting substantial stability for all complexes during the simulations, which fall within the ideal range around 2 å (smaller rmsd values indicate higher stability of the simulation) (gohlke et al., 2000; kramer et al., 1999) . a rigorous exploration of the rmsd values for ligand-sars-cov-2 rbd complexes shows that structures of 3-dihydrocadambine and proanthocyanidin b2 display good signals of stability during 50 ns of md simulation with rmsds values of 1.866 ± 0.300 and 2.233 ± 0.304 å, respectively (figure 8(a,b) ). importantly, similar behavior had the docked complex composed of proanthocyanidin c1 and qag-2 into the rbd/ace-2 interface, which showed remarkable stability throughout the simulation time period at rmsd values of 2.151 ± 0.237 and 1.775 ± 0.361, respectively (figure 8(c,d) ). a closer look at rmsd plot for qag-2 into the rbd/ace-2 interface revealed that ligand gradually stabilized after 5 ns, which is an indication of its higher conformational flexibility within the interface between rbd and ace-2 proteins. taken together, these findings suggest binding stability of ligands toward the active domains of the sars-cov-2 rbd and rbd/ ace-2 viral targets. the radius of gyration (r g ) represents the compactness of the protein structure and conformational stability of the whole systems (i.e. protein-ligand complexes). if the radius of gyration remained relatively constant, the complex was considered to be stably folded; otherwise, it was considered to be unfolded. in this scenario, radius of gyration values was calculated in order to observe the conformational alterations and dynamic stability of each viral protein within the 50 ns simulation time. figure 9 (a-d) illustrates r g values for the protein and ligand in the complexes, respectively. as shown in figure 9 (a-d), calculated r g values for ligands into the sars-cov-2 rbd protein for 3-dihydrocadambine (5.065 ± 0.144 å), figure 8 . backbone rmsd values of (a) 3-dihydrocadambine within sars-cov-2 rbd active site (red) and protein without ligand (blue). b: proanthocyanidin b2 within sars-cov-2 rbd binding site (red) and protein without ligand (blue). c: proanthocyanidin c1 at rbd/ace-2 interface and rbd/ace-2 complex without ligand (blue). d: qag-2 into rbd/ace-2 interface (red) and rbd/ace-2 complex without ligand (blue). proanthocyanidin b2 (4.356 ± 0.087 å) and inside rbd/ace-2 binding interface for proanthocyanidin c1 (5.542 ± 0.095 å) and qag-2 (6.818 ± 0.083 å) remained relatively constant during the simulation, therefore each protein-ligand complex could be considered to be stably folded. in addition, although rmsd values would show that the ligands undergo a significant shift within the active domain, estimated r g values suggest that overall shape of the protein was stable upon binding of the ligand during the 50-ns md simulation. finally, these observations are also supported by 2d-binding interactions plots and 3d representation of the selected ligands into the binding pocket, respectively (see supporting information figures s1-s4 ). in addition, to show the conformational changes of the ligands into the viral proteins active site along the first 50 ns window md simulation (figure 9 ), these plots revealed that after md simulations the key protein-ligand interactions initially shown by the docking results were maintained and the 3-dihydrocadambine and proanthocyanidin b2 within sars-cov-2 rbd, as well as proanthocyanidin c1 at rbd/ace-2 remained stable in the binding pocket compared to the initial docking pose. thus, crucial binding ligand interactions with lys417, arg403, tyr453, gln493, asn501, tyr505, ser494, tyr449, tyr495 phe497 and gly496 were maintained after 50-ns md simulation into the binding pocket of sars-cov-2 rbd. similarly, in comparison with the docking results, those key ligand interactions with amino acid residues identified as essential for maintaining rbd-ace-2 stability are also present after md simulations. besides, 3d representations of the selected ligands into each binding pocket were used with the aim to compare the best conformation poses from md simulation and docking, hence we plotted the superposition of the docked complex 3d-structures before and after 50-ns md simulation into the active cavity (see supporting information figures s5-s8 ). in general, there is no significant difference between the structures extracted after 50-ns md simulation and the docking pose of ligands, only slight translational and rotational motions were observed. the obtained md simulation results suggest (1) the conformation of the binding pocket and ligands were stable during the md simulations, (2) ligands do not leave the binding pocket while running md simulation and (3) active pocket in both selected viral targets favored ligands binding, suggesting not only the rationality and validity of our docking studies, but also proposes that many of these constituents of u. tomentosa could act as a dual inhibitors of the sars-cov-2 spike protein and rbd/ace-2 complex, which are mostly responsible for the attachment and internalization of the novel coronavirus in the human host. in the final stage, selected ligands were redocked into the sars-cov-2 rbd (3-dihydrocadambine and proanthocyanidin b2) or inside the rbd/ace-2 interface (proanthocyanidin c1 and qag-2) starting from the mean geometries of the last 3 ns md simulations trajectories, aiming to obtain the correct binding energies and poses. the vina re-docked results are summarized in table 2 and the best ligand bound receptor figure 9 . radius of gyration (r g ) graphs: (a) for 3-dihydrocadambine into the binding cavity (red) and sars-cov-2 spike protein without ligand (blue). b: for proanthocyanidin b2 onto active site (red) and sars-cov-2 spike protein without ligand (blue). c: for proanthocyanidin c1 within binding cleft and rbd/ace-2 complex without ligand (blue and violet). d: for qag-2 into the binding site (red) and rbd/ace-2 complex without ligand (blue and violet). conformations are shown in the supporting information figures s9-s12 . notably, after re-docked process 3-dihydrocadambine displays higher binding energy (-7.5 kcal.mol à1 ) than the initial predicted docking score (-7.1 kcal.mol à1 ). 2d and 3d diagrams of protein-ligand interactions from 3d coordinates showed that 3-dihydrocadambine had a significant conformational change in their binding modes in comparison with observed binding poses initially predicted by docking. as can be seen from table 2 , interactions profile into the sars-cov-2 rbd binding site was substantially altered, showing contacts with those critical residues, including asn501 (h-bond), gly496 (three h-bond), tyr453 (h-bond), tyr505 (p-cation with cationic nitrogen). on the other hand, despite proanthocyanidin b2 showed a re-docking score significantly lower than the initial docking. however, readers can observe that proanthocyanidin b2 is well-accommodated inside the rbd binding pocket highlighting key contacts with tyr453, ser494, lys417, gln493, arg403, tyr495, tyr505, glu406 and phe497 that are essential for sars-cov2 binding to human ace-2 receptors. in addition, predicted binding poses were compared in supporting information figure s10 , thus the best binding pose obtained from re-docking studies is considerably different to that retrieved from the initial docking, this finding could be strongly associated with a lower binding energy after re-docking. interestingly, when proanthocyanidin c1 was re-docked using the mean geometry of the last 3 ns md simulation trajectory, a higher binding score (-9.0 kcal mol à1 ) than the initial docking was obtained (-8.6 kcal mol à1 ). as shown in supporting information figure s11 , proanthocyanidin c1 fitwell into the rbd/ace-2 interface interacting with those critical residues at the junction of sars-cov-2 to human ace-2, such us glu37, his34, asp30, gln388, tyr505, phe390, lys353, glu354, lys417. a 3d-comparison between the best initial docking pose and the best re-docking pose into the active site clearly revealed that this binding pose significantly favors proanthocyanidin c1 binding. finally, re-docking calculations into rbd/ace-2 interface for qag-2 showed a very similar binding affinity (-8.1 kcal mol à1 ) to the initial docking prediction (-8.2 kcal mol à1 ). a close view to 2d and 3d ligand-protein diagrams plot revealed that qag-2 fit-well inside the binding pocket, and is located closed to b3 and b4 sheets in the ace-2 protein. re-docked process confirms that qag-2 is capable of binding to aminoacids residues that are critical for the recognition of the sars-cov-2 by full-length human ace-2. after the re-dock analysis, we identified that qag-2 formed two hydrogen bond interactions with key gly354 and tyr505 residues, respectively. similarly, ligand was able to bind through van der waals contacts with three critical residues asp355, gln388 and glu37, which could have an effect on stabilizing the binding event. as can be seen in supporting information figure s12 , 3d-plots comparison between starting pose docking and docking pose after re-docking for qag-2 revealed similar binding modes at rbd/ace-2 interface, only a slight shift was observed. taken together, these computational results clearly evidenced that constituents of u. tomentosa are capable of binding to sars-cov-2 spike protein through strong interactions with those key aminoacids crucial for the viral attachment to human ace-2 in stable complexes. our investigations could be a new strategy for inhibiting the recognition of the sars-cov-2 rbd by ace-2, and therefore might interfere with the entry of coronavirus to its host cells. computational modeling demonstrated that components of this herb may cause ace-2 and spike protein cleavage, because they would interact with key residues within rbd/ace-2 interface forming very stable complexes. hence, we firmly believe that ethanolic extract of cat's claw may be a novel herbal-based therapeutic option to treat covid-19 infection because its components may cause disruption of sars-cov-2 rbd/ace-2 complex or could block attachment of sars-cov-2 to the entry receptor ace-2. calculated drug-likeness profiles play a critical role in assessing the quality of novel antiviral candidates. early predictions of pharmacokinetic behavior of the promising antiviral compounds based on its structure could help find safer and effective leads for preclinical testing. here, we calculated and analyzed various drug-likeness indices for the most qualified components of u. tomentosa predicted by docking studies (table 3) . to this purpose, ten pharmacokinetics parameters were calculated as drug-likeness filter for speciophylline, uncarine f, uncaric acid, cadambine, 3-isodihydrocadambine, 3-dihydrocadambine, proanthocyanidin b2, proanthocyanidin c1, proanthocyanidin b4, epiafzelechin-4b-8, qag-1, qag-2, qag-4, qag-5 and qag-6, respectively. results obtained revealed the druggability of the selected components from ethanolic extract of cat's claw, demonstrating their potential as likely orally active antiviral. despite major components had more than two violations to the rule of 5 and druggability, predicted values for speciophylline, uncarine f, uncaric acid and cadambine displayed favorable physiochemical profiles compared to positive reference hepos, which clearly displayed important violations in rof-5. the discussion focused on these four active compounds showed that according to lipinski rule of five (rof-5) (no more than one violation is acceptable) (lipinski et al., 2012) , compounds could be used as orally dosed drugs in humans. the predicted human intestinal absorption (% hia) for speciophylline, uncarine f and uncaric acid revealed greater hia value in the range between 81% and 90%, while cadambine having relatively low values of 27% may be acceptable within 95% of marketed drugs. note that, greater hia values denote that these compounds could be absorbed throughout the intestinal segments upon oral administration. on the other hand, we calculated the most important physicochemical property to correlated passive molecular transport through membranes and drug-membrane interactions, such as polar surface area (psa) (ertl et al., 2000) . predicted psa showed favorable values for compounds ranging from 82 to 158 å 2 , indicating they would penetrate more efficiently through the infected host cells. in addition, the partition coefficient between n-octanol and water (log p o/w ) was calculated in order to explore the ability of constituents to pass through lipid bilayers (veber et al., 2002) . notably, speciophylline, uncarine f and uncaric acid presented values within ideal range for approved-drugs (ranging from 1.7 to 4.20). binding to serum albumin (expressed as log k hsa ) is the most important parameter for distribution and transport of antiviral drugs in the systemic circulation (colmenarejo, 2003; zhivkova, 2015) . early prediction of this parameter reduces the amount of wasted time and resources for drug development candidates in the antiviral therapy and management. we found that ligands fit well within the recommended values range (ranging from à1.5 to 1.5), showing log k hsa numbers between -0.592 and 0.58. finally, we also predicted the passive transmembrane permeation using caco-2 cell monolayers or mdck cells as models. currently, both models are used as a simplified in vitro model for intestinal absorption in drug development (broccatelli et al., 2016; pham-the et al., 2018; press & di grandi, 2008) . our results showed that in comparison with reference drugs, speciophylline, uncarine f and uncaric acid have 72-342 nm/s. from such observations, these compounds displayed great values of human intestinal absorption (% hia) above 81%, very similar to the reference drugs values (above 96%). given the aforementioned results, we believe at least four components of cat's claw here reported may provide favorable characteristics as the drug like, hence u. tomentosa may constitute itself a promissory option to fight against covid-19 infection. by integrating in silico approaches, this article evidenced that several components of u. tomentosa could act disrupting the association of sars-cov-2 spike protein with the human ace-2 receptor or by blocking the rbd-ace-2 interaction during covid-19 virulence. further, we identified that various constituents of cat's claw bears optimal pharmacokinetics properties to be used orally as a potential antiviral response. therefore, we believe that ethanolic extract of u. tomentosa should be taken into consideration as a rapid response to the covid-19 during the early stages of infection. covid-19 outbreak that emerged from wuhan, china has acquired pandemic status and severe acute respiratory <0.0 ----5 a molecular weight of the hybrid (150-500). b polar surface area (psa) (7.0-200 å 2 ). c n-on number of hydrogen bond acceptors <10. d n-ohnh number of hydrogens bonds donors 5. e octanol water partition coefficient (log p o/w ) (-2.0 to 5.0). f binding-serum albumin (k hsa ) (-1.5 to 1.5). g human intestinal permeation (<25 poor, >500 great). h madin-darby canine kidney (mdck) cells permeation. i human intestinal absorption (% hia) (>80% is high, <25% is poor). j heparin octasaccharide used as positive reference in this work. syndrome requires the attention of academics to discover the possible safe and effective drug to ameliorate its effects worldwide. in the present study, 26 constituents of u. tomentosa were docked on the binding interface of the rbd-ace-2 and inside sars-cov-2 rbd spike protein of novel corona virus. it was observed that the components of u. tomentosa such as proanthocyanidin c1, qag-2, 3-isodihydrocadambine, uncarine f and uncaric acid had a good predicted binding affinity for interface of the rbd-ace-2 as compared to the sulfated heparin octasaccharide (hepos). likewise, 3-dihydrocadambine, proanthocyanidin b4, proanthocyanidin b2 and proanthocyanidin c1 had the highest docking score on sars-cov-2 spike glycoprotein in their open state, whereas md simulations at 50 ns demonstrated both the feasibility of the binding free energy predicted by docking protocols and the stability of the docked protein-ligand complexes. virtual prediction adme revealed that speciophylline, uncarine f and uncaric acid presented values of druggability according to lipinski rule, demonstrating their potential bioavailability as likely orally active antiviral. based on our findings and its ancestral use in the traditional medicine from south american countries, u. tomentosa can be performed as an herbal supplement with the safety and efficacy parameters at both preclinical and clinical stages to evaluate its effectiveness in the treatment of novel coronavirus disease . furthermore, all components found in u. tomentosa could work in synergism by different mechanisms to combat the spread of sars-cov-2. no potential conflict of interest was reported by the author(s). andres f. yepes-p erez http://orcid.org/0000-0001-6975-5119 oscar herrera-calderon http://orcid.org/0000-0001-7264-0961 jorge quintero-saumeth http://orcid.org/0000-0002-6544-9429 gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. softwarex, 1-2, 19-25 antiviral potential of medicinal plants against hiv, hsv, influenza, hepatitis, and coxsackievirus: a systematic review new polyhydroxylated triterpenes from uncaria tomentosa triterpenes and quinovic acid glycosides from uncaria tomentosa uncaria tomentosa improves insulin sensitivity and inflammation in experimental nafld uncaria tomentosa a wellbehaved electrostatic potential based method using charge restraints for deriving atomic charges: the resp model a second generation force field for the simulation of proteins, nucleic acids, and organic molecules predicting passive permeability of drug-like molecules from chemical structure: where are we? antimutagenic and antiherpetic activities of different preparations from uncaria tomentosa (cat's claw) chemaxon -software solutions and services for chemistry and biology. marvinsketch, version in silico prediction of drug-binding strengths to human serum albumin fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties toward a systematic molecular orbital theory for excited states natural bond orbital methods. wires computational molecular science knowledge-based scoring function to predict protein-ligand interactions the world summit of harmonization on traditional, alternative and complementary medicine pharmacophore development, drug-likeness analysis, molecular docking, and molecular dynamics simulations for identification of new ck2 inhibitors computational design of ace2-based peptide inhibitors of sars-cov-2 ethnomedicine and drug discovery de novo design of protein peptides to block association of the sars-cov-2 spike protein with human ace2 an updated insight into the molecular pathogenesis, secondary complications and potential therapeutics of covid-19 pandemic uncaria tomentosa (willd.) dc. -ethnomedicinal use and new pharmacological, toxicological and botanical results two new 19-hydroxyursolic acid-type triterpenes from peruvian "una de gato" (uncaria tomentosa) evaluation of the flexx incremental construction algorithm for protein-ligand docking effect of alkaloid-free and alkaloid-rich preparations from uncaria tomentosa bark on mitotic activity and chromosome morphology evaluated by allium test sulfated polysaccharides effectively inhibit sars-cov-2 in vitro structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor alkaloids of peruvian uncaria tomentosa effects of aqueous fractions of uncaria tomentosa (willd.) d.c. on macrophage modulatory activities searching therapeutic strategy of new coronavirus pneumonia from angiotensin-converting enzyme 2: the target of covid-19 and sars-cov uncaria tomentosa alkaloidal fraction reduces aracellular permeability, il-8 and ns1 production on human microvascular endothelial cells infected with dengue virus experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings bioactive compounds from plants used in peruvian traditional medicine identification and quantification of components in extracts of uncaria tomentosa by hplc-es/ms autodock4 and autodocktools4: automated docking with selective receptor flexibility automated docking using a lamarckian genetic algorithm and an empirical binding free energy function fractioning of proanthocyanidins of uncaria tomentosa. composition and structure-bioactivity relationship proanthocyanidin characterization and bioactivity of extracts from different parts of uncaria tomentosa l. (cat's claw molecular dynamics simulation analyses of viral fusion peptides in membranes prone to phase transition: effects on membrane curvature, phase behavior and lipid-water interface destabilization antioxidant activity of the extract from uncaria tomentosa polymorphic transitions in single crystals: a new molecular dynamics method hplc-pda method for quinovic acid glycosides assay in cat's claw (uncaria tomentosa) associated with uplc/q-tof-ms analysis chemical composition variability in the uncaria tomentosa (cat's claw) wild population in silico assessment of adme properties: advances in caco-2 cell monolayer permeability modeling drug targets for corona virus: a systematic review permeability for intestinal absorption: caco-2 assay and related issues immunomodulating and antiviral activities of uncaria tomentosa on human monocytes infected with dengue virus-2 new developments in molecular orbital theory cat's claw inhibits tnfa production and scavenges free radicals: role in cytoprotection general atomic and molecular electronic structure system an approach to computing electrostatic charges for molecules the amazon rain forest plant uncaria tomentosa (cat's claw) and its specific proanthocyanidin constituents are potent inhibitors and reducers of both brain plaques and tangles inhibition of herpes simplex type 1 and type 2 infections by oximacrov r , a cranberry extract with a high content of a-type proanthocyanidins (pacs-a) autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading molecular properties that influence the oral bioavailability of drug candidates monoterpenoid indole alkaloids and phenols are required antioxidants in glutathione depleted uncaria tomentosa root cultures structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a novel coronavirus outbreak of global health concern automatic atom type and bond type perception in molecular mechanical calculations annex 1 who guidelines on good herbal processing practices for herbal medicines coronavirus disease (covid-19): weekly epidemiological update. world health organization severe acute respiratory syndrome coronavirus 2: from gene structure to pathogenic mechanisms and potential therapy interaction between gut microbiota and ethnomedicine constituents structural basis for the recognition of sars-cov-2 by full-length human ace2 studies on drug-human serum albumin binding: the current state of the matter key: cord-277766-rxmpi61o authors: guang, cuie; phillips, robert d.; jiang, bo; milani, franco title: three key proteases – angiotensin-i-converting enzyme (ace), ace2 and renin – within and beyond the renin-angiotensin system date: 2012-06-15 journal: arch cardiovasc dis doi: 10.1016/j.acvd.2012.02.010 sha: doc_id: 277766 cord_uid: rxmpi61o the discovery of angiotensin-i-converting enzyme 2 (ace2) and a (pro)renin receptor has renewed interest in the physiology of the renin-angiotensin system (ras). through the ace2/angiotensin-(1–7)/mas counter-regulatory axis, ace2 balances the vasoconstrictive, proliferative, fibrotic and proinflammatory effects of the ace/angiotensin ii/at1 axis. the (pro)renin receptor system shows an angiotensin-dependent function related to increased generation of angiotensin i, and an angiotensin-independent aspect related to intracellular signalling. activation of ace2 and inhibition of ace and renin have been at the core of the ras regulation. the aim of this review is to discuss the biochemistry and biological functions of ace, ace2 and renin within and beyond the ras, and thus provide a perspective for future bioactives from natural plant and/or food resources related to the three proteases. angiotensine ; inhibiteurs de l'enzyme de conversion ; inhibiteur de l'enzyme de conversion ii ; rénine ; récepteur de la prorénine résumé la découverte de l'enzyme de conversion de l'angiotensine 2 (ac2) et un récepteur à la prorénine est une avancée récente dans la compréhension de la physiologie du système rénine-angiotensine. au sein de l'axe inhibiteur de l'enzyme de conversion de l'angiotensine 2/angiotensine 1/mas, l'ac2 contrebalance l'effet vasoconstricteur, prolifératif, fibrosant et pro-inflammatoire de l'axe ace/angiotensine 2/at1. le récepteur à la prorénine a une fonction angiotensine dépendante, liée à l'augmentation de la production d'angiotensine 1, et un aspect indépendant de l'angiotensine, lié aux signaux intracellulaires. l'activation de l'ac2 et l'inhibition de l'ace de la rénine ont été considérées comme au centre de la régulation du système rénine-angiotensine. l'objet de cette revue générale est de discuter les fonctions biochimiques et biologiques de l'ace, de l'ac2 et de la rénine au sein et au-delà du système rénine-angiotensine et ainsi de proposer une perspective de développement d'agents actifs extraits de plantes naturelles ou d'alimentation, produits liés à ces trois protéases. © 2012 elsevier masson sas. tous droits réservés. the renin-angiotensin system (ras) is not only an endocrine but also a paracrine and an intracrine system [1] . in mammals, the intravascular ras plays a key role in maintaining blood pressure homeostasis and fluid and salt balance, and the tissue or local ras is involved in physiological and pathological processes, such as tissue growth and remodelling, development and inflammation [2] . in a classical ras, the substrate angiotensinogen (agt), which is released into the circulation from the liver, is degraded by the enzyme renin that originates in the kidney, generating the inactive angiotensin i (ang i). when this decapeptide comes into contact with angiotensin-i-converting enzyme (ace) at the endothelial surface of blood vessels, the c-terminal dipeptide is cleaved, giving rise to angiotensin ii (ang ii), the main effector molecule of the ras. through interactions with specific receptors, particularly its type 1 or at1 receptor, ang ii stimulates a wide variety of signalling pathways in the heart, blood vessels, kidneys, adipose tissue, pancreas and brain, initiating most of the physiological and pathophysiological effects that have been attributed to the ras [3] . due to the function of directly generating the main effector ang ii, ace -together with the classical axis ace/ang ii/at1 -has been at the core of ras studies since its discovery. in 2000, a homologue of ace, known as angiotensin-iconverting enzyme 2 (ace2), was cloned by two independent research groups [4, 5] . evidence indicates that ace2 negatively regulates the activated ras by degrading ang ii to the heptapeptide ang(1) (2) (3) (4) (5) (6) (7) . moreover, through the mas receptor (a g protein-coupled receptor), the resulting ang-(1-7) counterbalances the cardiovascular effects of ang ii by opposing many at1 receptor-mediated actions [6] . ace2 and the axis ace2/ang-(1-7)/mas are becoming the focus of intense research regarding the ras [1] . the discovery of a (pro)renin receptor ([p]rr) and the introduction of renin inhibitors have also brought (pro)renin back into the spotlight [7] . far from being a straightforward cascade containing one substrate (agt), two proteases (renin and ace), two peptides (ang i and ang ii) and one receptor (at1), the ras currently consists of several axes upstream and downstream of the classical cascade, which include more than two dozen peptidases, nearly a dozen ang fragments and at least six different receptors [8] . here, we review three critical proteases (ace, ace2 and renin) within and beyond the ras and thus intend to find new connections between natural plant and/or food resources and the ras. occurrence, gene encoding and structure of ace ace (ec 3.4.15.1) is a monomeric glycoprotein that is distributed in many tissues and biological fluids. there are two isoforms of ace in humans: somatic ace (sace) and germinal ace (gace). somatic ace is found in many types of endothelial and epithelial cells [9] . germinal ace or testicular ace is present exclusively in germinal cells in the male testis. although ace is a type i integral membrane protein, it can also be released as a soluble enzyme into extracellular fluids, such as plasma and seminal and cerebrospinal fluids, following post-translational proteolytic cleavage by a membrane protein sheddase or secretase [10] [11] [12] . somatic ace and gace are encoded by a single gene containing 26 exons. the promoter for sace is situated in the 5 flanking region of the first exon, whereas that for gace is within intron 12, which results in different lengths for the two isoforms. the longer sace (150-180 kda) is transcribed from exon 1 to exon 26, excluding exon 13, whereas the shorter gace (90-110 kda) is transcribed from exon 13 to exon 26. exon 13 encodes a unique sequence for the n-terminus of gace, whereas downstream exons encode a common sequence for both isozymes [13] . somatic ace and gace both consist of a 28-residue hydrophilic c-terminal cytoplasmic domain, a 22-residue hydrophobic transmembrane domain that anchors the protein in the membrane and an n-terminal ectodomain ( fig. 1 ) that is heavily glycosylated with mannose, galactose, fructose, n-acetylneuraminic acid and n-acetylglucosamine [14] . the ectodomain of sace is further divided into two similar domains (n domain and c domain) encoded by the homologous exons 4-11 and 17-24, respectively, and each domain contains an active his-glu-x-x-his (hexxh) sequence [9] . somatic ace is the only known metallopeptidase with two homologous active sites [15] , which implies that there has been a gene duplication event during evolution [16] . except for a unique sequence constituting its n-terminus, gace is identical to the c-terminal half of sace [17] . due to cleavage of the membrane-bound residues by ace secretase, soluble circulating ace lacks a transmembrane portion and a cytosolic domain [18] . the three-dimensional x-ray crystallographic structure of a deglycosylated truncated version of gace (c domain of sace), reveals a preponderance of ␣-helices with a zinc ion and two chloride ions incorporated. a deep narrow channel separates the molecule into two subdomains and the active site is located toward the bottom of this channel. an n-terminal 'lid' on the top of molecule appears to allow only small peptide substrates access to the active site cleft. in fact, the structure bears little similarity to that of carboxypeptidase a (m14 family) on which the initial drug development of ace inhibitors was based. instead, it resembles rat neurolysin (m3 family) and pyrococcus furiosus carboxypeptidase (m32 family), despite sharing little sequence similarity with these two proteins [17] . corradi et al. [18] reported the crystal structure of the n domain of sace. similarly, it has an ellipsoid shape with a central groove dividing it into two subdomains, one of which contains the n-terminal region that covers the central binding cavity. but the structure reveals differences in the active site and it contains only one chloride ion, equivalent to chloride ii of gace. the three-dimensional structures of c domains (based on gace) and n domains provide an opportunity to design domain-selective ace inhibitors that may exhibit new pharmacological profiles [17, 18] . according to the catalytic mechanism and the critical amino acid residue involved, peptidases are classified into four major types: serine, cysteine, aspartic and metallo [15] . ace is an m2 family metallopeptidase: ma(e), the gluzincins [19] . two histidine residues of the functional motif hexxh and a third distant glutamate positioned 23-24 residues further towards the c-terminus are the ligands for the zinc cofactor [20] . an activated water molecule complexed to zn 2+ serves as the nucleophile to attack the carbonyl group of the targeted peptide bond [15] . the activity of ace is also chloride dependent. chloride primarily activates the active sites of ace and enhances the binding of substrates [9] . each active domain of ace displays differences in sensitivity to chloride activation [21] . the activity of the c domain of sace depends highly on chloride ion concentration and is inactive in its absence, whereas the n domain can be completely activated at relatively low concentrations of this anion and is still active in the absence of chloride [22, 23] . germinal ace depends on chloride to a lesser extent compared with the c domain of sace [9] . cushman and cheung [24] reported an optimal in vitro ace activity of rabbit rung acetone extract in the presence of 300 mm nacl at ph 8.1-8.3. the two active domains of sace are also subtly different in substrate specificity. they hydrolyze bradykinin almost equally but the c domain active site can hydrolyze ang i, substrate p [23] and hippuryl-his-leu [25] more efficiently, while the n domain active site preferentially hydrolyzes ang-(1-7) [26] , luteinizing hormone-releasing hormone (lh-rh) [23] , the haemoregulatory peptide n-acetyl-ser-asp-lys-pro (acsdkp) [27] and alzheimer amyloid ␤-peptide (a␤) [28] . fuchs et al. [29] proved that the c-terminal catalytic domain was the main site of ang i cleavage in mice. the differentiation of catalytic specificity might be due to very subtle variation in substrate-specific amino acids [22] and chlorideinduced conformational alteration of active sites [23] . ace acts as an exopeptidase to cleave dipeptides from the free c-termini of two typical substrates, ang i and bradykinin. for certain substrates such as cholecystokinin [30] , substrate p [31] and lh-rh [32] , which have amidated c-termini, ace not only displays exopeptidase activity but also acts as an endopeptidase [33] . the most prominent example of endopeptidase activity is ace hydrolyzing the synthetic a␤-(1-40) peptide into four fragments: an a␤peptide and the others corresponding to products of a␤-(1-7) hydrolysis [34] . thus, ace might have a more general impact on the metabolism of biologically active peptides than previously recognized [16] . the two substrates used most often for measuring ace activity and inhibition in vitro -hippuryl-his-leu and n-[3-(2-furyl)acryloyl]-lphenylalanylglycylglycine (fapgg) -only have the n-termini blocked and substrates with two termini blocked have been developed [22] . ace was originally isolated in 1956 as a 'hypertensinconverting enzyme' [35] . in the ras, ace cleaves the decapeptide ang i-(1-10) (asp-arg-val-tyr-ile-his-pro-phe-his-leu) into the octapeptide ang ii-(1-8) by removing the c-terminal dipeptide his-leu. when the ras is overactive, ang ii exerts its harmful effects primarily via the at1 receptor, whereas the at2 receptor may oppose and counterbalance those effects mediated by at1 receptor to exert protective actions [1] . ang ii is a potent vasoconstrictor, stimulates the release of aldosterone and antidiuretic hormone or vasopressin and increases the retention of sodium and water. these effects act directly in concert to raise blood pressure. a nonapeptide derivative of ang i, des-asp1-ang i-(2-10), prevents infarction-related and non-infarction-related cardiac injuries and disorders. his-leu can be cleaved from the peptide by ace to produce ang iii-(2-8) [14] , which has 40% of the vasoconstriction activity of ang ii. ang iii exerts its effects, in principle, in a similar manner to ang ii, and may be equally or even more important in mediating the release of vasopressin [1] . ace also degrades ang-(1-9) to ang-(1-7) then further degrades this peptide to the inactive ang-(1-5) (fig. 2 ). in addition, ace (also termed kininase ii) inactivates the vasodilators bradykinin-(1-9) (arg-pro-pro-gly-phe-ser-pro-phe-arg) and kallidin (lys-bradykinin) in the kallikrein-kinin system, by cleaving the c-terminal dipeptide phe-arg. ace eventually cleaves its primary metabolite bradykinin-(1-7) into the shorter fragment bradykinin-(1-5) [36] . through ang ii and aldosterone, ace may also be implicated in the impairment of nitric oxide bioavailability and cell oxidative stress, augmenting the generation of reactive oxygen species and peroxynitrite [37, 38] . with the ability to hydrolyze neuropeptides such as enkephalin [39, 40] , substrate p, neurotensin [31] and lh-rh, ace may be involved in the functioning of the brain and nervous system. ace may affect the digestive system by hydrolyzing the peptide hormone cholecystokinin and gastrin [30] . the in vivo experiment conducted by azizi et al. [41] proved that acute ace inhibition could increase the level of the natural stem cell regulator acsdkp in plasma. acsdkp substantially inhibits cell cycle entry of normal haematopoietic stem cells and protects haemopoiesis against damage caused by cycleactive cytotoxic agents [42] . acsdkp can also inhibit the proliferation of hepatocytes [43] and lymphocytes [44] and stimulate angiogenesis [45] in vivo. the in vivo antifibrotic effect of ace inhibition is partially mediated by acsdkp [46] . ace may also affect susceptibility to alzheimer's disease by degrading a␤ and preventing the accumulation of amyloid plaques in vivo [47] . in the brains of amyloid precursor protein swedish mutation transgenic mice, ace converts a␤ 1-42 to a␤ 1-40 and degrades a␤, and chronic inhibition of ace with captopril enhances predominant a␤ 1-42 deposition [48] . however, through the inhibition of brain ace activity in the a␤ 25-35 -injected mice, perindopril ameliorates cognitive impairment and may therefore have a beneficial effect on alzheimer's disease as well as hypertension [49] . using the assumed mechanistic analogy to other zinc metallopeptidases, plus the knowledge that several snake-venom peptides potentiate the action of bradykinin by inhibiting ace, efforts were undertaken to develop orally-active peptide analogues for potential use in the treatment of hypertension [9] . the first such compound, captopril or d-3-mercapto-2-methylpropanoyl-l-proline, is an analogue of the ala-pro sequence, with sulphydryl as a strong chelating group for the zinc ion. its adverse effects, which were the same as those caused by mercapto-containing penicillamine, prompted the design of non-sulphydryl ace inhibitors [50] . the results were two active inhibitors: enalaprilat and lisinopril. they are both essentially tripeptide analogues with a zinc-co-ordinating carboxyl group and a phenylalanine that occupies the s1 groove in the enzyme. lisinopril is a lysine analogue of enalaprilat but it is hydrophilic, with greater affinity than enalaprilat. the later compounds are all variations of the first three inhibitors, with most of the differences residing in the functionalities that bind the active site zinc and the s2 pocket. in addition to phosphonates, ketones are also useful as chelators [51] . currently, there are more than 10 ace inhibitors marketed that are widely used as first-line therapy for cardiovascular diseases, including hypertension, heart failure, heart attack and left ventricular dysfunction. according to the functional moiety, they are divided into three types: thiol (captopril), carboxylate (benazepril, enalapril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril) or phosphate (fosinopril). some ace inhibitors are now administrated clinically as ethyl-ester prodrugs, which have good bioavailability but are inactive in their own right. they are then converted to the active diacid molecules in vivo by esterases. as a drug class, ace inhibitors are very effective, have a relatively low incidence of side effects and are well tolerated. a common side effect of ace inhibitors is a dry cough, which appears in 5-20% of patients and may result in the discontinuation of treatment. another serious problem is angioedema, which affects 0.1-0.5% of patients and can be life-threatening. the two side effects have generally been attributed to altered concentrations of bradykinin [51] . use of ace inhibitors during the second and third trimesters of pregnancy is contraindicated because of their association with an increased risk of foetopathy, a group of conditions that includes oligohydramnios, intrauterine growth retardation, hypocalvaria, renal dysplasia, anuria, renal failure and death. exposure to ace inhibitors during the first trimester of pregnancy may place the infant at increased risk for major congenital malformations [52] . the initial drug development of clinical ace inhibitors was based on the assumption of an active site related to that of carboxypeptidase a but organized to remove a dipeptide rather than a single amino acid from the cterminus of its substrate. it is now known that sace has two active sites, neither of which resembles that of carboxypeptidase a, and that these sites are not identical. clinical ace inhibitors, however, show little discrimination between these two active sites [9] . ang i is hydrolyzed predominantly by the c domain of sace in vivo [29] but bradykinin is hydrolyzed by both active sites [53] ; therefore a c domain-selective inhibitor would allow some degradation of bradykinin by the n domain and this degradation could be enough to prevent the accumulation of excess bradykinin that has been observed during attacks of angioedema. that is, the c domain-selective inhibition could possibly result in specialized control of blood pressure with fewer vasodilatorrelated adverse effects [51] . a structure-activity study has proved that the group substitution involving the phenyl ring and carbon chain at the sulphonyl and propionyl moieties of captopril is essential for better activity towards the c domain of ace [54] . there is increasing evidence that the n domain of sace is responsible for the in vivo degradation of the natural haemoregulatory hormone acsdkp [27, 41, 55] . so, n domain-selective inhibition might open up novel therapeutic areas. two phosphinic tetrapeptides, rxpa380 and rxp407, have been found to be highly selective inhibitors of the c and n domains of sace, respectively [51] . the availability of the three-dimensional structures of the c and n domains of sace makes the structure-based design of active site-specific inhibitors possible [17, 18] . ace2 (ec 3.4.17.-) is also a type i transmembrane glycoprotein and its expression has now been recognized as being ubiquitous. it appears to be susceptible to cleavage that releases the catalytical active ectodomain. a disintegrin and metalloprotease 17 (adam17; also known as tumour necrosis factor-alpha cleavage enzyme [tace]) is a major protease for soluble ace2 shedding, while phorbol ester, ionomycin, endotoxin and the proinflammatory cytokines interleukin-1␤ and tumour necrosis factor-alpha can also acutely induce ectodomain release [56] . calmodulin binding sites have been identified in the cytoplasmic tail of ace2 and calmodulin inhibitors are proved to increase ace2 shedding [57] . the cleavage site resides between amino acids 716 and 741 near the predicted transmembrane domain and residue lys 548 in the ectodomain plays an important role in dictating ace2 ectodomain shedding [58] . ace2 has 805 amino acids encoded from 18 exons and shares about 42% sequence identity with the n and c domains of sace [16] . ace2 also belongs to the m2 family of metalloproteases but consists of a single active site domain that, by sequence comparison, more closely resembles the n domain than the c domain of sace ( fig. 1) [11] . in addition to the conserved zinc metallopeptidase consensus sequence hexxh (amino acids 374-378), there is a conserved glutamate residue 24 residues c-terminal to the second histidine of the zinc motif, which serves as the third zinc ligand [16] . the threedimensional structure of a truncated extracellular region of human ace2 shows that the active site domain (residues 19-611) can be further divided into two subdomains i and ii, which form two sides of a long deep cleft and are connected only at the floor of the active site cleft by a prominent ␣helix. the deeply recessed and shielded proteolytic active site of ace2 is a common structural feature of proteases and can avoid hydrolysis of correctly folded and functional proteins. the zinc is co-ordinated by his 374 , his 378 , glu 402 and one water molecule in the subdomain i near the bottom, whereas a chloride ion is co-ordinated by arg 169 , trp 477 and lys 481 in the subdomain ii [58] . the ace2 transmembrane c-terminal domain shares 48% sequence identity with collectrin (fig. 1) , a non-catalytic protein shown to have a critical role in amino acid reabsorption in the kidney [59] , pancreatic beta cell proliferation and possibly insulin exocytosis [6] . unlike sace and gace, which are primarily dipeptidylcarboxypeptidases, ace2 functions predominantly as a monocarboxypeptidase, with a substrate preference for hydrolysis between proline and a hydrophobic or basic cterminal residue [6] . it is like carboxypeptidase a in its action model but is different in active structure because the latter has an hxxe zinc-binding motif with the third ligand (histidine) positioned 108-135 residues further towards the c-terminus [16] . ace2 efficiently cleaves a single residue phenylalanine from ang ii to generate ang(1-7) , with about 400-fold higher catalytic efficiency than the conversion of ang i to ang-(1-9) by removing the c-terminal leucine residue (fig. 2 ). other substrates with high catalytic efficiency are apelin-13, dynorphin a-(1-13) and des-arg9bradykinin [60] . arg 514 of ace2 has been identified as a residue critical to substrate selectivity [61] . ace2 activity is also regulated by chloride ions; it has been proposed that chloride binding induces subtle changes in the conformation of the active site, which either facilitate or hinder substrate binding [6] . for the substrate ang i, ace2 activity increases with increased [cl − ] and a plateau is reached at approximately 500 mm cl − . for the substrate ang ii, an increase in ace2 activity is observed as [cl − ] increases from 0-100 mm but any further increase in [cl − ] decreases ace2 activity until a plateau is reached at 500 mm cl − ; ace2 activity at 500 mm cl − is even lower than that in the absence of cl − (0 mm). consequently, an increase in [cl − ] above 100 mm, which is the physiological concentration in human plasma, increases ang i and decreases ang ii cleavage by ace2. this has an effect on the localized concentration of ang ii in the kidney, where ace2 has a high level of expression and extracellular chloride ion levels fluctuate. thus, in vivo cl − sensitivity may serve as a homeostatic regulatory mechanism [61] . ace2 activity is unaffected by inhibitors of ace (captopril, lisinopril and enalaprilat) or carboxypeptidase a (benzylsuccinate and potato carboxypeptidase inhibitor) [16] . the major function of ace2 is to counter-regulate ace activity by reducing ang ii bioavailability and increasing ang-(1-7) formation. as a result, ace2 plays a crucial role in maintaining the balance between the two axes ace2/ang-(1-7)/mas and ace/ang ii/at1 of the ras; a chronic and sustained imbalance may lead to pathophysiology of the cardiovascular, renal, pulmonary and central nervous systems [62] . studies have shown that ace2 overexpression and recombinant ace2 treatment can attenuate hypertension in animal models [63, 64] , while in humans, there is a strong association between ace2 polymorphisms and hypertension in han chinese [65] . in addition to the ang ii system, ace2 may regulate blood pressure through other peptide systems, such as bradykinin and/or apelin [6] . ace2 gene delivery [66] and ace-(1-7) infusion [67] also have beneficial effects on atherosclerosis, whereas ace2 deficiency accentuates vascular atherosclerosis and inflammation [67] in animal models. regarding heart function, ace2 null mice display impaired cardiac contractility [68] and the loss of ace2 in wild-type mice accelerates adverse ventricular remodelling by potentiation of ang ii effects by means of the at1 receptors [69] . ang-(1-7), through interaction with the mas receptor, can improve atrial tachyarrhythmias [70] , myocardial performance, cardiac modelling and survival [3, 6] in rodent heart failure models. in humans, ace2 gene variants might be involved in modulation of left ventricular mass in men [71] and soluble ace2 activity is increased in patients with heart failure and correlates with disease severity to exert cardioprotective actions [72] . deletion of the ace2 gene in mice leads to the development of glomerulosclerosis and increased albuminuria [73] , while treatment with the ace2 inhibitor mln4760 can worsen renal damage in streptozotocin-induced diabetic mice [74] . chronic treatment with ang-(1-7) improves renal endothelial dysfunction via the mas receptor in apolipoprotein e-deficient mice, by increasing levels of endogenous nitric oxide [75] , whereas genetic deletion of the mas receptor in mice leads to a reduction in urine volume, sodium retention, microalbuminuria and reduced renal blood flow, which are associated with upregulation of the at1 receptor and transforming growth factor-beta messenger ribonucleic acid [76] . this evidence indicates the protective role of ace2/ang-(1-7)/mas in renal function. ace2 has also been shown to regulate cardiovascular functions in brain regions. overexpression of ace2 in the rostral ventrolateral medulla reduces high blood pressure and heart rate in spontaneously hypertensive rats (shrs) [77] . in the nucleus tractus solitarius of shrs, ace2 gene transfer improves baroreceptor heart rate reflex [78] . in the mouse subfornical organ, ace2 overexpression inhibits at1 receptor expression and prevents ang ii-mediated pressor and drinking responses [79] . in addition, ace2/ang-(1-7)/mas can exert cerebroprotective functions in endothelin-1-induced ischaemic stroke in rodent models [80] . ace2 exerts a host of actions on the cardiopulmonary system, which include prevention of endothelial dysfunction, reduction in pulmonary oxidative stress, attenuation of vascular impairment, anti-inflammatory effects and anticardiac remodelling effects. all these properties are responsible for the protective role of ace2 against pulmonary arterial hypertension [81] . structure-based drug screening has identified two ace2 activators: a xanthenone (1-[(2-diethylamino)ethyl-amino]-4-(hydroxymethyl)-7-[(4-methylphenyl)sulphonyloxy]-9h-xanthene-9-one; xnt) and resorcinolnaphthalein. xnt hydrogen bonds with ace2 residues lys 94 , tyr 196 , gly 205 and his 195 , and resorcinolnaphthalein is involved in three hydrogen bonds with residues gln 98 , gln 101 and gly 205 . xnt and resorcinolnaphthalein modulate ace2 activity possibly by two mechanisms. logically, the closed conformation of the enzyme cannot allow the substrate into its active site. in the presence of compound, the free enzyme may be shifted to the open form, effectively increasing the activity coefficient of the enzyme. alternatively, product release is a rate-limiting step in ace2 turnover. ace2 activity may be enhanced in the presence of compound, as the enzyme-product complex empties more quickly and ace2 becomes available to start another cycle. xnt and resorcinolnaphthalein enhance in vitro ace2 activity in a dose-dependent manner and show no significant effects on ace activity. administration of xnt to shrs can result in a decrease in blood pressure, improvements in cardiac function and reversal of myocardial, perivascular and renal fibrosis [82] . the protective role of xnt against hypertension-induced cardiac fibrosis is associated with activation of ace2, increases in ang-(1-7) and inhibition of extracellular signal-regulated kinases [83] . furthermore, ace2 activation by xnt attenuates thrombus formation and reduces platelet attachment to vessels [84] . xnt also prevents pulmonary vascular remodelling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension [85] . development of xnt is being pursued to translate the vasoprotective concept of ace2 into an effective cardiovascular therapy [62] . diminazine aceturate, a small molecule antiprotozoal agent that shares structural similarity with xnt, has recently been demonstrated to be a potent activator of ace2 and to decrease mean arterial pressure and myocardial fibrosis in shrs [86] . intracerebroventricular infusion of diminazine aceturate, prior to and following endothelin-1-induced middle cerebral artery occlusion, significantly attenuates cerebral infarct size and neurological deficits in a rat model [80] . these data suggest that diminazine aceturate may serve as a lead compound for the discovery of the next generation of drugs for the treatment of cardiovascular disease and hypertension [86] . interestingly, peptidase-independent actions of ace2 have also been elucidated. ace2 has been identified as an essential receptor for the coronavirus (cov) that causes severe acute respiratory syndrome (sars). the spike protein of sars-cov attaches the virus to its cellular receptor ace2 with a binding domain on the spike protein mediating the interaction [87] . this ace2-spike interaction leads to endocytosis of virus particles through internalization with ace2, induces the fusion of virus with host cells and establishes sars-cov infection with the release of viral rnas into cytoplasm [6] . the spike protein of sars-cov also induces tace-dependent shedding of the ace2 ectodomain, with the involvement of the ace2 cytoplasmic domain. cellular tace and the ace2 cytoplasmic tail promote viral entry and infection. these observations indicate that ace2 shedding and its causative cellular signals may be attributable to sars-cov-induced tissue damage [88] . an ace2 inhibitor, n-(2-aminoethyl)-1 aziridine-ethanamine, has been identified as being effective in blocking sars-cov spike proteinmediated cell fusion [89] . the sars-cov receptor function of ace2 is independent of its catalytic activities for ang ii degradation but ace2-mediated ang ii degradation is still important for lung protection from sars pathogenesis [6, 90] . so, the consequence of long-term activation of ace2 must be determined and the effects of ace2 activators on the immune competence of animals and their vulnerability to sars-cov infection must be tested before these molecules are ready for preclinical trials [64] . ace2 is also the receptor for the human coronavirus nl63 that was discovered in 2004 in the netherlands and was shown to be globally distributed [91] . additionally, due to the high homology with collectrin at its transmembrane, ace2 binds to b 0 at1-family amino acid transporters and contributes to the absorption of neutral amino acids in animal intestines [6, 92] . thus, ace2 may be involved in multiple biological functions. renin (ec 3.4.23.15) is a key enzyme of the ras. since its discovery 100 years ago, an impressive quantity of information about renin has been compiled. it is generally accepted that the active renin found in the circulation of mammals almost exclusively originates from the kidney. in addition to systemic renin, there are a number of extrarenal tissues that express renin as part of the local or tissue-specific rass. within the kidney, renin is predominantly produced by the juxtaglomerular cells. (pro)renin gene transcription in these cells is controlled through several mechanisms, among which cyclic adenosine monophosphate (camp)/protein kinase a/camp response element binding protein (stimulatory) and calcium/protein kinase c (inhibitory) cascades are employed by physiological cues, whereas signal transducers, activators of transcription and nuclear factor b transcription factors (inhibitory) and members of the nuclear receptor superfamily probably become relevant in pathological situations. prorenin is stored in vesicles, activated to renin and then released upon demand. the release of renin is under the control of the intracellular camp (stimulatory), ca 2+ (inhibitory) and cyclic guanosine monophosphate signalling pathways. meanwhile, a great number of intrarenally generated or systemically acting factors have been identified that control renin secretion directly at the level of renin-producing cells, by activating the camp or ca 2+ signalling pathways [93] . renin is an aspartyl protease with a typical structure made of two lobes. the cleft between the lobes contains the active site, characterized by two catalytic aspartic residues. renin is a highly specific enzyme and has only one known substrate (agt) [94] . prorenin is the 'inactive' precursor of renin. in addition to the main organ, the kidney, other tissues, such as the brain, the adrenal gland, the submandibular gland, the glands of the reproductive system and adipose tissue, are also able to secrete prorenin in the surrounding milieu and in plasma. in contrast to renin, prorenin is released constitutively; renin and prorenin levels are usually well correlated. under some physiopathological circumstances, such as pregnancy and diabetes, prorenin levels exceed by far those of renin [94] . prorenin has a prosegment of 43 amino acid residues attached to the n-terminus of mature renin; the prosegment folds into an active site cleft of mature renin to prevent catalytic interaction with agt [95] . prorenin-renin conversion occurs in the kidney before renin is released from the juxtaglomerular cells. several enzymes, including proconvertase 1 and cathepsin b, have been proposed to be responsible for this irreversible cleavage of the prosegment from prorenin to form mature renin. a (reversible) non-proteolytic activation of prorenin may also occur. at acidic ph and/or low temperature, prorenin is capable of undergoing a conformational change, involving the unfolding of the prosegment from the enzymatic cleft. this non-proteolyticallyactivated prorenin is fully enzymatically active [96] . under physiological conditions, approximately 2% of plasma prorenin is in the open form and can also display enzymatic activity [94] . the traditional assumptions of renin being just an enzyme responsible for the cleavage of agt and prorenin being just an 'inactive' proenzyme were challenged by the cloning of a human (p)rr in 2002 [94] . the (p)rr is a single transmembrane-domain protein of 350 amino acids, with a large unglycosylated and highly hydrophobic n-terminal domain responsible for renin and prorenin binding and a short cytoplasmic tail of about 20 amino acids involved in intracellular signalling [97] . the (p)rr was first identified on cultured human mesangial cells and bound well to renin and prorenin with a k d in the nanomolar range [96] . binding of prorenin induced a conformational change in the molecule, increasing its enzymatic activity from virtually zero to values similar to those of active renin in solution without proteolytic removal of the prosegment [93, 98] . two regions in the human prorenin segment, namely t 7p fkr 10p (a gate that is not accessible by its specific antibodies until it is loosened from the active site cleft) and i 11p flkr 15p (a handle, a protruding pentameric segment), have been identified as being crucial for the non-proteolytic activation [99] . renin bound to the (p)rr displays a 3-to 5-fold increase in the catalytic efficiency of agt conversion to ang i compared with free renin [97, 100] . in addition to the ang-dependent function on the cell surface related to the increased catalytic activity of receptor-bound (pro)renin (renin activity of activated prorenin and increased activity of renin) that leads to the formation of ang i from agt, the (p)rr system also has ang-independent intracellular effects that are not necessarily related to the ras [7] . pathological conditions like high blood pressure upregulate the receptor, whereas elevated (pro)renin concentrations downregulate the receptor via translocation of the transcription factor promyelotic zinc finger protein to the nucleus [98] . in the ang-independent signalling pathways, prorenin-induced activation of mitogen-activated protein kinase p38 and subsequent phosphorylation of heat shock protein 27 result in actin polymerization, while (pro)renin-induced activation of the extracellular signal-regulated kinase 1/2 (p44/p42) signalling cascade leads to the intracellular expression of profibrotic genes, giving rise to the synthesis of transforming growth factor-␤, plasminogen activator inhibitor-1, collagen 1 and fibronectin. these effects potentially increase cardiac and renal hypertrophy and fibrosis [97, [101] [102] [103] . the discovery of the (p)rr has confirmed the hypothesis that renin is also a hormone. it has been suggested that blocking the (p)rr may be a new target for renal and cardiac end-organ protection [1] . additionally, the mannose-6-phosphate/insulin-like growth factor ii receptor can bind both renin and prorenin with high affinity and is believed to serve as a clearance receptor for renin/prorenin. an intracellular renin-binding protein has been also cloned and found to be an inhibitor of renin activity but its deletion affected neither blood pressure nor plasma renin [94] . after the discovery of the receptor, a (p)rr antagonist was designed, based on the idea that the prosegment contains a handle region that binds to the receptor, allowing prorenin to become catalytically active in a non-proteolytic manner [96] . the antagonist (also known as hand region peptide, hrp), consisting of 10 amino acids (nh 2 -rillkkmpsv-cooh), resembles the handle region (i 11p llkk 15p ) of rat prorenin prosegment and will thus competitively bind to the (p)rr as a decoy peptide and inhibit the receptor-mediated activation of prorenin [95] . treatment with the hrp in diabetic mice and rats decreased the renal content of ang i and ii and inhibited the development of nephropathy without affecting hyperglycaemia [104, 105] . in stroke-prone shrs, continuous subcutaneous administration of the hrp inhibited activation of the tissue ras without affecting the circulating ras or arterial pressure and significantly attenuated the development and progression of proteinuria, glomerulosclerosis and cardiac fibrosis [106, 107] . infusion of the hrp in human (p)rr transgenic rats significantly inhibited the development of glomerulosclerosis, proteinuria, mitogen-activated protein kinase activation and transforming growth factor-␤ expression in the kidneys [108] . future research should be able to determine to what degree the beneficial in vivo effects of hrp are due to prorenin blockade [98] and are related to interference with renin binding and the ras [4] . decoy effects of the hrp, however, were not confirmed by other research groups [109] [110] [111] [112] . ace inhibitors and at1 receptor blockers (arbs) are proven to be effective therapeutic agents in the treatment of cvd. however, both ace inhibitors and arbs lead to a substantial compensatory rise in circulating active renin and ang peptides that may eventually limit their therapeutic potential [113] . moreover, the increased ang i seen with ace inhibitors can subsequently be converted to ang ii by non-ace pathways, mediated by chymase and chymotrypsinlike ang-generating enzyme [114] . in addition to the side effects of ace inhibitors, such as cough and angioedema, a meta-analysis of randomized controlled trials in 2010 suggested that arbs are associated with a modestly increased risk of new cancer diagnosis, although conclusions about the exact risk of cancer associated with each particular drug have not been drawn [115] . based on this study, the united states food and drug administration is conducting a review to assess the possible link between the use of arbs and cancer. therefore, direct renin inhibition may be an alternative pharmacological approach to ras inhibition. renin is the rate-limiting step of the ras and the concept of blocking the ras at its origin by inhibiting renin has existed for at least 50 years [113] . the first-generation renin inhibitors were peptide analogues of agt and the secondgeneration compounds were peptidomimetic agents that are dipeptide transition-state analogue inhibitors of the active site. but these renin inhibitors had limited clinical use due to poor metabolic stability and oral bioavailability, short duration of action, weak antihypertensive activity and high cost of synthesis [114] . a combination of molecular modelling and x-ray crystallographic analysis of the active site of renin led to the development of aliskiren, a new non-peptide low-molecular-weight orally-active renin inhibitor. aliskiren has a high binding affinity for renin and appears to bind to both the hydrophobic s1-/s3-binding pocket and a large, distinct subpocket that extends from the s3-binding site toward the hydrophobic core of the enzyme. aliskiren is a potent competitive inhibitor of renin but very poorly inhibits related aspartic peptidases. it shows no effects on cytochrome p450 isoenzyme activities and is not bound extensively to blood proteins, therefore having a low potential for drug interactions [113] . compared with the earlier renin inhibitors, aliskiren is more resistant to intestinal degradation and possesses significantly improved oral bioavailability. the terminal half-life ranges from 23 to 40 hours, which makes it suitable for once-daily dosing. aliskiren monotherapy displays an antihypertensive efficacy similar if not superior to that of other first-line antihypertensive drugs, with a safety and tolerability profile similar to that of arbs. combined with various antihypertensive agents, aliskiren exhibits synergistic effects [114] . from 2007, aliskiren was approved by regulatory bodies in both europe and the united states, for use alone and with other agents in the treatment of hypertension [113] . recent studies showed that aliskiren treatment also markedly increased the plasma concentration of (pro)renin in patients and failed to inhibit the non-catalytic effects of (pro)renin [110, 116] . the pharmaceutical company novartis reported that the addition of aliskiren to standard therapy for patients recovering from a heart attack did not provide the anticipated effect of limiting adverse changes to the heart's left ventricle. in december 2011, novartis announced the early termination of the aliskiren trial in type 2 diabetes using cardio-renal endpoints (altitude) and advised that aliskiren should not be used in combination with ace inhibitors or arbs in patients with diabetes. the study was conducted in type 2 diabetic patients at high risk of fatal and non-fatal cardiovascular and renal events; aliskiren 300 mg was given in addition to an ace inhibitor or an arb. the overseeing data monitoring committee concluded that study patients were unlikely to benefit from aliskiren and that there was a higher incidence of adverse events related to non-fatal stroke, renal complications, hyperkalaemia and hypotension in this highrisk population after 18-24 months of combined therapy (www.novartis.com). aliskiren has been shown to be a very safe antihypertensive after more than 20,000 patient-years of data. however, as we have seen in ongoing telmisartan alone and in combination with ramipril global endpoint trial (ontarget) that the combination of telmisartan (an arb) and ramipril (an ace inhibitor) is associated with more adverse events without an increase in benefit in patients with vascular disease or high-risk diabetes without heart failure [117] , the combination of multiple safe drugs in this class may no longer be a promising strategy. over the last decade, a number of food-derived compounds have been shown to have in vitro ace inhibitory activity. some of them display significant antihypertensive activity in rats and humans. among these compounds, foodderived ace inhibitory peptides have been most widely studied [14, 118] . a quantitative structure-activity relationship study indicated that a potent ace inhibitory dipeptide should have a large and hydrophobic amino acid at the c-terminus and a non-polar amino acid or possibly a positively charged amino acid at the n-terminus. for tripeptides, the most favourable residues for the c-terminus are aromatic amino acids, while hydrophobic amino acids are preferred for the n-terminus and positively charged amino acids are preferred for the middle position [119] . the in vitro half-maximal inhibitory concentration (ic 50 ) values of food-derived ace inhibitory peptides are about 1000fold higher than that of synthetic captopril but they have higher in vivo activities than would be expected from their in vitro activities. it has been suggested that food-derived peptides might act via different antihypertensive mechanisms, possess higher affinities for tissues and be more slowly eliminated than synthetic captopril [118] . until now, only two lactotripeptides (vpp and ipp) have been successfully commercialized. data from van mierlo et al., however, did not support a blood pressure-lowering effect of the two tripeptides in caucasians [120] . given the discrepancy between in vivo and in vitro results, further investigation into the in vivo and clinical antihypertensive effects and mechanisms of food-derived ace inhibitors is necessary. three dipeptides (ir, kf and ef) in pea protein hydrolate have been identified as inhibiting renin activity in vitro [121] . oleic acid and linoleic acid isolated from rice and some cereals also have in vitro renin inhibitory activity [122, 123] . a series of studies have shown that saponins from different food/plant sources, primarily from soybean, can inhibit in vitro renin activity [124] [125] [126] [127] ; among them, orally administered soybean saponin can lead to a reduction of blood pressure in shrs but the direct evidence for saponin suppressing plasma renin activity in vivo is still lacking [124] . experiments are required to further investigate the biochemical and biological properties of these plant/food-derived non-peptides that are related to renin inhibition. significant conceptual progress made in the last few years leads us to conclude that ace2 could serve as a new direction for improved therapeutics for cardiovascular disease. compared with ace inhibitor therapy, ace2 is an endogenous regulator of the ras. targeting ace2 would not only produce the vasoprotective/antiproliferative peptide ang-(1-7) but would also influence the vasoconstrictive/proliferative effects of the ace/ang ii/at1 axis. as a part of the vasoprotective/antiproliferative axis, ace2 can effectively control fibrosis and structural remodelling and is extremely beneficial for pulmonary hypertension. ace2 activation may provide improved protection and reversal of ischaemia-induced neural damage. thus, direct activation of ace2 could result in better outcomes in cardiovascular disease. additionally, ace2 is a multifunctional enzyme with many biologically active substrates. the effects of ace2 on substrates other than ang i and ang ii may hold relevance for the treatment of cardiovascular disease [62] . xnt analogues exist in many natural plant resources and have shown various physiological functions. using these resources to search for in vitro ace2 activators would be a good starting point for developing plant food-derived ace2 activating agents. ongoing experiments would characterize these ace2 activators. compared with ras regulatory drugs, these plant/food-derived bioactives appear more natural and safer to the consumer. as part of a daily diet, bioactives from food sources may result in a much lower healthcare cost. plant/food-derived ras regulators could be applied in the prevention of hypertension and as initial treatment in mildly hypertensive individuals [118] . the authors declare that they have no conflicts of interest concerning this article. renin-angiotensin system revisited physiology and pharmacology of the (pro)renin receptor an ace in the hole alternative pathways of the renin angiotensin system and their potential role in cardiac remodeling a novel angiotensinconverting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a human homolog of angiotensin-converting enzyme. cloning and functional expression as a 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c-domain inhibitors of angiotensin-converting enzyme: angiotensin-(1-7) and keto-ace the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensinconverting enzyme the n-terminal active centre of human angiotensin-converting enzyme degrades alzheimer amyloid beta-peptide angiotensin-converting enzyme c-terminal catalytic domain is the main site of angiotensin i cleavage in vivo novel activity of angiotensin-converting enzyme. hydrolysis of cholecystokinin and gastrin analogues with release of the amidated c-terminal dipeptide hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase novel activity of human angiotensin i converting enzyme: release of the nh2-and cooh-terminal tripeptides from the luteinizing hormone-releasing hormone molecular basis of exopeptidase activity in the c-terminal domain of human angiotensin i-converting enzyme: insights into the origins of its 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inhibition increases the plasma level of the natural stem cell regulator n-acetyl-seryl-aspartyl-lysylproline the synthetic tetrapeptide acsdkp protects cells that reconstitute longterm bone marrow stromal cultures from the effects of mafosfamide (asta z 7654) in vivo effect of the tetrapeptide, n-acetyl-ser-asp-lys-pro, on the g1-s transition of rat hepatocytes the tetrapeptide acsdkp, a negative regulator of cell cycle entry, inhibits the proliferation of human and chicken lymphocytes the tetrapeptide acsdkp, an inhibitor of primitive hematopoietic cell proliferation, induces angiogenesis in vitro and in vivo prevention of myocardial fibrosis by n-acetyl-seryl-aspartyl-lysyl-proline in diabetic rats angiotensin-converting enzyme degrades alzheimer amyloid beta-peptide (a beta) a beta aggregation, deposition, fibril formation; and inhibits cytotoxicity angiotensin-converting enzyme converts amyloid beta-protein 1-42 (abeta(1-42)) to -40), and its inhibition enhances brain abeta deposition effect of a centrally active angiotensin-converting enzyme inhibitor, perindopril, on cognitive performance in a mouse model of alzheimer's disease a new class of angiotensin-converting enzyme inhibitors ace revisited: a new target for structure-based drug design major congenital malformations after first-trimester exposure to ace inhibitors roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin i and bradykinin: insights from selective inhibitors 3d-qsar studies on angiotensinconverting enzyme (ace) inhibitors: a molecular design in hypertensive agents rxp 407, a selective inhibitor of the n-domain of angiotensin i-converting enzyme, blocks in vivo the degradation of hemoregulatory peptide acetyl-ser-asp-lys-pro with no effect on angiotensin i hydrolysis ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia tumor necrosis factoralpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndromecoronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis kidney amino acid transport hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ace and ace2) identified by site-directed mutagenesis therapeutic implications of the vasoprotective axis of the reninangiotensin system in cardiovascular diseases transgenic angiotensinconverting enzyme 2 overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function targeting the degradation of angiotensin ii with recombinant angiotensin-converting enzyme 2: prevention of angiotensin ii-dependent hypertension correlation of angiotensin-converting enzyme 2 gene polymorphisms with stage 2 hypertension in han chinese angiotensin converting enzyme-2 confers endothelial protection and attenuates atherosclerosis genetic ace2 deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse candesartan ameliorates cardiac dysfunction observed in angiotensin-converting enzyme 2-deficient mice loss of angiotensin-converting enzyme 2 accelerates maladaptive left ventricular remodeling in response to myocardial infarction the angiotensin-(1-7)/mas receptor axis is expressed in sinoatrial node cells of rats association of angiotensinconverting enzyme 2 (ace2) gene polymorphisms with parameters of left ventricular hypertrophy in men. results of the monica augsburg echocardiographic substudy detection of soluble angiotensin-converting enzyme 2 in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system loss of angiotensinconverting enzyme-2 leads to the late development of angiotensin ii-dependent glomerulosclerosis ace2 inhibition worsens glomerular injury in association with increased ace expression in streptozotocin-induced diabetic mice chronic treatment with angiotensin-(1-7) improves renal endothelial dysfunction in apolipoproteine-deficient mice genetic deletion of the angiotensin-(1-7) receptor mas leads to glomerular hyperfiltration and microalbuminuria overexpression of angiotensin-converting enzyme 2 in the rostral ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats gene transfer of angiotensin-converting enzyme 2 in the nucleus tractus solitarius improves baroreceptor heart rate reflex in spontaneously hypertensive rats angiotensin-converting enzyme 2 overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type 1 receptor downregulation cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke ace2, a promising therapeutic target for pulmonary hypertension structure-based identification of small-molecule angiotensinconverting enzyme 2 activators as novel antihypertensive agents angiotensin-converting enzyme 2 activation protects against hypertension-induced cardiac fibrosis involving extracellular signal-regulated kinases ace2 activation promotes antithrombotic activity evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension diminazene aceturate is an ace2 activator and a novel antihypertensive drug structure of sars coronavirus spike receptor-binding domain complexed with receptor modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace2 induces tnf-alpha production and facilitates viral entry structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury differential downregulation of ace2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl63 tissue-specific amino acid transporter partners ace2 and collectrin differentially interact with hartnup mutations physiology of kidney renin prorenin and the putative (pro)renin receptor inhibition of diabetic nephropathy by a decoy peptide corresponding to the ''handle'' region for nonproteolytic activation of prorenin prorenin and the (pro)renin receptor-an update pivotal role of the renin/prorenin receptor in angiotensin ii production and cellular responses to renin the (pro)renin receptor: a new addition to the renin-angiotensin system? human prorenin has ''gate and handle'' regions for its non-proteolytic activation binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system renin-stimulated tgf-beta1 expression is regulated by a mitogen-activated protein kinase in mesangial cells renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin ii-independent mechanisms prorenin induces intracellular signaling in cardiomyocytes independently of angiotensin ii prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type 1a receptor-deficient mice regression of nephropathy developed in diabetes by (pro)renin receptor blockade contribution of nonproteolytically activated prorenin in glomeruli to hypertensive renal damage nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension slowly progressive, angiotensin ii-independent glomerulosclerosis in human (pro)renin receptor-transgenic rats prorenin is the endogenous agonist of the (pro)renin receptor. binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor prorenin and renininduced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide the putative (pro)renin receptor blocker hrp fails to prevent (pro)renin signaling pro)renin receptor peptide inhibitor ''handle-region'' peptide does not affect hypertensive nephrosclerosis in goldblatt rats aliskiren: a new renin inhibitor as antihypertensive new class of agents for treatment of hypertension: focus on direct renin inhibition angiotensinreceptor blockade and risk of cancer: meta-analysis of randomised controlled trials prorenin engages the (pro)renin receptor like renin and both ligand activities are unopposed by aliskiren telmisartan, ramipril, or both in patients at high risk for vascular events plant food-derived angiotensin i converting enzyme inhibitory peptides structural requirements of angiotensin i-converting enzyme inhibitory peptides: quantitative structure-activity relationship study of di-and tripeptides lactotripeptides do not lower ambulatory blood pressure in untreated whites: results from 2 controlled multicenter crossover studies identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate the occurrence of renin inhibitor in rice: isolation, identification, and structure-function relationship renin inhibitory activity in rice and cereals reduction of blood pressure by soybean saponins, renin inhibitors from soybean, in spontaneously hypertensive rats inhibition of human renin activity by saponins human renin inhibitory activity in legumes isolation of human renin inhibitor from soybean: soyasaponin i is the novel human renin inhibitor in soybean this work was supported partly by fundamental research funds for the central universities (jusrp211a06) and the self-determined research program of jiangnan university (no. 5815210232110800 & no. 5815210372100530). key: cord-352230-8mazd3eu authors: beeraka, narasimha m.; sadhu, surya p.; madhunapantula, subbarao v.; rao pragada, rajeswara; svistunov, andrey a.; nikolenko, vladimir n.; mikhaleva, liudmila m.; aliev, gjumrakch title: strategies for targeting sars cov-2: small molecule inhibitors—the current status date: 2020-09-18 journal: front immunol doi: 10.3389/fimmu.2020.552925 sha: doc_id: 352230 cord_uid: 8mazd3eu severe acute respiratory syndrome-corona virus-2 (sars-cov-2) induced coronavirus disease 19 (covid-19) cases have been increasing at an alarming rate (7.4 million positive cases as on june 11 2020), causing high mortality (4,17,956 deaths as on june 11 2020) and economic loss (a 3.2% shrink in global economy in 2020) across 212 countries globally. the clinical manifestations of this disease are pneumonia, lung injury, inflammation, and severe acute respiratory syndrome (sars). currently, there is no vaccine or effective pharmacological agents available for the prevention/treatment of sars-cov2 infections. moreover, development of a suitable vaccine is a challenging task due to antibody-dependent enhancement (ade) and th-2 immunopathology, which aggravates infection with sars-cov-2. furthermore, the emerging sars-cov-2 strain exhibits several distinct genomic and structural patterns compared to other coronavirus strains, making the development of a suitable vaccine even more difficult. therefore, the identification of novel small molecule inhibitors (nsmis) that can interfere with viral entry or viral propagation is of special interest and is vital in managing already infected cases. sars-cov-2 infection is mediated by the binding of viral spike proteins (s-protein) to human cells through a 2-step process, which involves angiotensin converting enzyme-2 (ace2) and transmembrane serine protease (tmprss)-2. therefore, the development of novel inhibitors of ace2/tmprss2 is likely to be beneficial in combating sars-cov-2 infections. however, the usage of ace-2 inhibitors to block the sars-cov-2 viral entry requires additional studies as there are conflicting findings and severe health complications reported for these inhibitors in patients. hence, the current interest is shifted toward the development of nsmis, which includes natural antiviral phytochemicals and nrf-2 activators to manage a sars-cov-2 infection. it is imperative to investigate the efficacy of existing antiviral phytochemicals and nrf-2 activators to mitigate the sars-cov-2-mediated oxidative stress. therefore, in this review, we have reviewed structural features of sars-cov-2 with special emphasis on key molecular targets and their known modulators that can be considered for the development of nsmis. covid-19 is a devastating disease caused by a coronavirus related to the one that caused outbreaks of severe acute respiratory syndrome (sars) in the year 2002 (1, 2) . middle east respiratory syndrome (mers)-related coronavirus is an infamous member of this cohort. covid-19, which is caused by the sars-cov-2 infection, was detected in wuhan, china in december 2019. the world health organization (who) declared this infection a pandemic on march 11 2020 due to its severity and rapid spread across the globe. as of june 11 2020, sars-cov-2 had infected 7.4 million individuals, and caused 4,17,956 deaths across 212 countries worldwide ( table 1) . coronaviruses (cov) belongs to a family of single-stranded rna viruses (+rna) that can infect a variety of mammals such as bats and humans (3) . sars-cov-2 contains rna of 29,891nucleotide length, which codes for 9,860 amino acids (4) . the rna has a 5' cap and 3' poly-a tail and produces a poly-protein 1a/1ab (pp1a/pp1ab) in the host (4) . sars-cov-2 belongs to beta cov category and appears in a crown shape with a size of ∼60-140 nm (figure 1) . gene sequencing data revealed that sars-cov-2 has 89 and 82% sequence similarity with bat sars-like-cov-zxc21 and human sars-cov, respectively (4, 5) . the spike (s) proteincoding gene mutation in the nsp2 and nsp3 regions results in the replacement of glycine (g) with serine (s) at 723 position (g723s), and an isoleucine (i) replaced with proline (p) at 1010 amino acid position (i1010p). due to these mutations, the invading potential of sars-cov-2 has increased significantly toward host tissues. this virus can also be transmitted through the respiratory droplets from coughs and sneezes of infected individuals (4) . this mode of aerosol transmission is possible, especially, when protracted exposure occurs in closed areas (4) . the incubation time of the virus varies significantly from individual to individual. in general it takes about 6 days from the day of infection to the first appearance of symptoms. however, in a few cases the symptoms may appear only after 2 weeks (6) . members of coronaviridae are known to induce respiratory complications in humans (7, 8) . at first, sars-cov, mers-cov, and sars-cov-2 varieties were transmitted from animals to humans which triggered severe respiratory diseases (9) (10) (11) . however, subsequent transmission occurred among humans primarily due to physical contact. hence, conventional preventive measures such as physical isolation were implemented to avoid propagation of early infection across the human population (1, 12) . similar to the sars-cov, the pathological manifestations of sars-cov-2 could induce lung malfunction in humans as indicated by the severe acute respiratory syndrome and pneumonia (12) . recent studies reported that sars-cov-2 infection can induce mild, moderate, and severe illness in infected patients (4) . clinical manifestations of this infection include chronic pneumonia, sepsis, septic shock, fever, and dry cough (4) . a progressive respiratory failure during this infection may lead to sudden death (4) . mild illness resulting from a sars-cov-2 infection is characterized by the presence of malaise, headache, low fever and dyspnea. in the case of moderate illness from sars-cov-2, the complication is manifested by the presence of cough and mild pneumonia. severe illness from sars-cov-2 is associated with chronic pneumonia, cough, sars, hypoxia, and tachypnea (in children) followed by respiratory, and cardiovascular system failure (4). the autopsy and biopsy reports of sars-cov-2 patients revealed severe edema with pulmonary tissue exudates, focal reactive hyperplasia, damage to pneumocytes as well as alveolar macrophages, and patchy cellular infiltration (13) . coronavirus-induced lung damage has been demonstrated experimentally by several investigators in animal models (14) . for instance, the sialodacryoadenitis virus and parker's rcov were shown to induce damage to alveolar type-i cells through the expression of pro-inflammatory cytokines, and chemokines such as cinc-2, cinc-3, lix, mip-3α, and fractalkines (15) (16) (17) (18) (19) (20) (21) . for example, fractalkine promotes the infiltration of cytotoxic lymphocytes in the alveolar epithelium thereby inducing a severe inflammatory response (15, 22) . similarly, mip-3α confers the chemotaxis of immune cells via il-1β and tnf-α inflammatory mediators (17, (22) (23) (24) (25) . therefore, these animal models could be used to develop effective pharmacological agents against sars-cov-2 infections. studies from several laboratories have demonstrated that the entry of sars-cov-2 into human cells is facilitated by ace-2 (26) . ace-2 is a member of the renin-angiotensin system (ras), which plays a vital role in cardiovascular and renal homeostasis. ace-2 and tmprss2 facilitates the entry of the virus into host cells during sars-cov-2 infection (7). in addition, there are other proteases such as aminopeptidase n (apn) which plays a prominent role for the entry of hcov-nl63 and hcov-229e into host cells (27) (28) (29) (30) . apn is a membrane-bound glycoprotein that mediates the zincdependent protease activity during the entry and or replication of coronavirus strains into host cells (29, 31, 32) . hence, the ace-2 receptor's down-modulation may prevent sars-cov-2 viral entry/replication (33) . the s-protein of sars-cov and other coronavirus strains are different in their structural and functional domains (3). s-protein can bind to the n-terminus of ace-2 receptors on the outer surface of host cells including respiratory epithelium of the lungs (34) (35) (36) . identifying the key amino acid residues in s-protein of the sars-cov-2 strain may benefit virologists and medical scientists to develop better therapeutic agents. however, to date these details are not known, hence, there is an immediate requirement to identify the amino acids involved in binding s-proteins to ace-2 receptors on host cell surfaces. furthermore, investigations should also focus on establishing the structural similarities of s-protein motifs that are interacting with the ace-2 receptors of other coronavirus strains (37) (38) (39) (40) (41) . these investigations might help in deciphering molecular strategies to target receptor binding sites of ace-2 proteins with sars-cov-2 using novel therapeutics and vaccines to avoid membrane fusion process and viral entry (7) . the tmprss2 protease can foster the entry of the sars-cov-2 virus by activating the s-protein for virus-host cell membrane fusion, consequently enhancing viral replication in the host cells (7, (42) (43) (44) (45) (46) . tmprss2 plays a vital role in generating inflammatory cytokines and chemokines in lung epithelial cells by cleaving s-protein during coronavirus infections including sars-cov-2. hence, tmprss2 is another potential therapeutic target to consider for the novel drug development against sars-cov-2 (46) (47) (48) . prevention and treatment of sars-cov-2 infections are achieved at different levels (49) . the primary approach involves physical isolation to prevent the spread of virus from individual to individual; the second approach involves inhibiting the entry of virus into human cells and the third method includes treating the infected individuals to minimize inflammatory reactions and blocks cathepsin l required for sars-cov processing note: yet to be examined against sars-cov-2 infection blocks sars-cov interaction with ace-2 note: yet to be examined against sars-cov-2 infection [n-(9,10-dioxo-9,10-dihydroanthracen-2yl)benzamide] blocks sars-cov fusion to host cell membrane note: yet to be examined against sars-cov-2 infection (67) nct numbers were obtained from https://clinicaltrials.gov/. pulmonary damage. although physical isolation is the ideal way of limiting the spread, in reality this approach is difficult to execute, hence, many pharmacological companies are actively involved in developing small molecule inhibitors to prevent the entry of the virus into human hosts (7, 49) . in this regard several nsmis have been investigated to treat sars-cov; but, significant breakthroughs are yet to come for treating sars-cov-2 (48) ( table 2) . adedeji et al. (49) reported the discovery and characterization of novel inhibitors to block sars-cov replication via different mechanisms. one mechanism uses screening of small molecule inhibitors using "hiv-1 pseudotyped with sars-cov surface glycoprotein s (sars-s)" (49, 68) . "ssaa09e2" is a novel small molecule inhibitor, which blocks the interaction of cov sars-s with ace-2 receptors, thus blocking the viral entry (49) . another nsmi is "ssaa09e1" reported to be involved in blocking the cathepsin l, which is required for cov-sars-s processing to mediate viral entry into the host cell (49) . ssaa09e3 is another nsmi, which can block the fusion of viral membranes with host cell surfaces (49) (figure 2 ). since the pathological aspects and genomic similarity of sars-cov-2 virus with sars-cov, the above strategies of inhibition may figure 2 | molecular pathogenesis of sars-cov-2 in human lung cells. binding of s-protein of sars-cov-2 to the ace-2 receptors triggers the processing of ace-2 through adam-17/tnf-α-converting enzyme and induces the "ace-2 shedding" into the extracellular space and facilitates uptake of sars-cov-2 followed by the development of sars. alternatively, the entry of sars-cov-2 by membrane tmprss2 serine protease'/hat (human airway trypsin-like protease)-mediated cleavage of ace2 can facilitate sars-cov s-glycoprotein-mediated virus entry. even though, several nsmis targeting these processes were described and their mode of action against coronavirus were delineated, their efficacy against sars-cov-2 is yet to be tested. be considered for developing potent pharmacological agents to prevent sars-cov-2 infections (49) . however, the prospective research should address the efficacy of these inhibitors against sars-cov-2 infections. cytokine storm was predominantly reported during sars-cov-2 infection. targeting cytokine-mediated inflammatory responses induced by sars-cov-2 is another viable approach for mitigating the complications of viral infection. in this regard, chang et al. (35) , documented the inflammatory cascades mediated through intracellular signaling pathways conferred by the sars-cov in both lung epithelial cells and fibroblasts. authors of this study have reported that s-protein of sars-cov efficiently mediate the il-8 release in the infected lung cells by activating mapkinases, and activator protein-1 (ap-1) without intervention of nf-kb cascade (35) . this study suggested a promising lead for novel rational drug design through the identification of a "specific sequence motif of sprotein functional domain, " which is responsible for inducing il-8-mediated inflammatory response in lungs (35) . baricitinib is a pharmacological agent, which was reported to block the sars-cov-2 viral entry and inflammation through the inhibition of ap2-associated protein kinase 1 (aak1), cyclin g-associated kinase, and janus kinase-1 and 2 (69) . chloroquine (cq) and hydroxychloroquine (hcq) were reported to be effective in mitigating the coronaviral load (70, 71) . cq and hcq not only inhibit the entry of sars-cov-2 but also change the ph of acidic intracellular organelles such as endosomes and lysosomes thereby preventing membrane fusion reactions. however, many contradictions and queries prevail pertaining to the use of hcq for the treatment of covid-19. at the time of the submission of this review, results of many clinical trials are yet to be announced, hence, the efficacy of hcq for inhibiting sars-cov-2 infection is still a possibility. prospective studies should focus on testing the fda approved inhibitors of "abl-1 kinases, " "pi3k/akt/mtor" signaling, and "mapkinase" pathways against sars cov-2. since these pathways are involved in cell survival, inflammatory cytokines production, and proliferation of cells, targeted downregulation of these pathways is likely to mitigate the exacerbations induced by coronavirus. in this direction, many of these inhibitors are currently being tested against sars-cov-2 ( table 3 ) (72) (73) (74) . for instance, sorafenib, which inhibits raf, is being experimented in preclinical models and early clinical trials (72) . likewise, the efficacy of il-1 receptor antagonists and tnf-α receptor antagonists for blocking the rat coronavirus-mediated chemokine production was already proven effective in animal models (15, 75, 76) . further studies testing the safety and efficacy are warranted before considering these inhibitory agents for treating individuals infected with sars-cov-2 (76). however, the concept of "one drug to treat all" should be followed to combat several devastating viral infections (77, 78) . for instance, the ebola, marburg, and sars-cov-2 are undoubtedly devastating viral pathogens, which can induce high mortality as they transmit rapidly via air and body fluids (78) . outbreaks of these viruses occur sporadically and currently there are no clinically approved nsmis available to combat these viruses. a recent report by taylor et al. (79) demonstrated the efficacy of a synthetic adenosine analog, bcx4430 in blocking a broad spectrum of viral species viz., "coronaviruses, paramyxoviruses, and bunyaviruses" as these viruses could induce sars, measles, and mumps. bcx4430 could efficiently block both ebola and marburg viral titers in non-human primate models by targeting viral rna polymerase (78, 79) . hence, this molecule should be tested for further studies against sars-cov2 infections in humans. targeting the membrane protease involved in viral s-protein processing and the viral entry into host cells is another approach in mitigating sars-cov-2. the host cellular proteases viz., "trypsin, ", "miniplasmin, " "human airway trypsin like protease, " "tryptase clara, " and "tmprss2" could cleave the ha glycoprotein located in influenza a virus and thereby promote viral entry into lung cells (80) . the usage of serine protease inhibitors such as camostat and aprotinin significantly blocked the replication of influenza virus in epithelial cells of lungs and bronchioles (81) . in addition, these nsmis could block the release of inflammatory mediators such as cytokines, il-6 and tnf-α, during this infection (81) . tmprss2 is a key protein involved in the pathogenesis of several seasonal viral infections including influenza, h1n1, h3n2, and h7n9 (82) (83) (84) (85) . tmprss2 cleaves the s-protein of coronavirus to produce unlocked, fusion-catalyzing viral forms and binds to the host cell surface thereby enhancing rapid viral entry (43, 44, (86) (87) (88) (89) (90) . both sars-cov and mers-cov could rapidly enter into the host cells as tmprss2 can facilitate viral binding to the cell surface (42, 43, 45, 87, 91, 92) . tmprss2 also plays a vital role in the immuno-pathology of coronavirus infections including sars-cov-2 across lungs by inducing lung fibrosis (46) . hence, the emerging research should promote the development of nsmis to target these proteases thereby hindering the entry of sars-cov-2 into host cells. a proof-of-concept study by iwata-yoshikawa et al. (46) reported that sars-cov failed to replicate in the bronchioles and lungs of tmprss2 knockout mice. authors of this study reported elevated expression of tlr3-mrna expression in the lungs of "sars-cov-inoculated tmprss2-deficient mice" and showed enhanced tlr-3 mediated localization of dsrna into endosomes (46) . in this study, tmprss2 knockout has resulted in downregulation of inflammatory cytokines and chemokine expression, which are involved in the bronchiolitis obliterans organizing pneumonia (boop), sars, and pulmonary fibrosis in sars-cov infection (46, 93, 94) . the intricate sars-cov-2 pathogenesis is similar to that of sars-cov. studies have reported the efficacy of ifns to block sars-cov in cell line models but not against sars-cov-2. among ifnα/ -β/ and -γ, the ifn-β was reported to be the most potent blocker of sars-cov growth (3, (95) (96) (97) (98) . furthermore, ifn-β and-γ have a synergistic effect in blocking sars-cov viral replication (62, 63) . however, the effect of this combination against sars-cov-2 is not yet reported. therefore, future studies should focus on determining the efficacy of ifnα/ -β/ and -γ against sars-cov-2 infections. unlike small molecule inhibitors, sirnas are specific and can be designed to mitigate sars-cov associated structural proteins by targeting orf4 (99, 100), orf5 (101, 102), orf9a (50, 103, 104) , and orf7a (50, (105) (106) (107) . for example, sirnas sisc2 and sisc5 have shown success in cultured cells as well as in preclinical mouse models in inhibiting the sars infection without causing toxicity (108) . several other reports have also recently demonstrated the efficacy of sirnas to inhibit the expression of sars-cov genes coding for 3cl protease in cell line models (108) (109) (110) (111) (112) (113) (114) . the activity of sars-cov 3cl protease is essential for viral replication as this protein is involved in the processing of viral proteins (114) . selective optimization and screening of hexa-chlorophene analogs can be "active 3cl protease inhibitors" during a sars-cov infection (114) . hence, the pharmacological agents/sirnas targeting these pathways may likely produce effective clinical outcomes in sars-cov-2 infections. however, clinical studies should test the utility of these agents/sirna in reducing the burden of infections caused by sars-cov-2 (111). the genome of coronaviruses is reported to be significantly involved in coding both structural proteins, and non-structural proteins (nsp's) for the effective viral replication (115) . the nsp's (nsp8c and nsp7) are required for novice cov viral particle formation through viral orf 1ab polyprotein processing (115) . several nsmis were reported to target these non-structural proteins in coronavirus infections to treat sars (115) . for instance, grl0617, a bendioxolane derivative, could target papain-like proteinases like nsp3 (51, 52, 116, 117) , whereas 5choloropyridinyl indolecarboxylate targets nsp5 (53, (118) (119) (120) and a "combination of zinc derivatives with pyrithione" targets nsp12 (121, 122) ; ranitidine bismuth citrate targets nsp13 (123) (124) (125) (126) (127) . monoclonal antibodies; cr3014 (128), mab-201 (129), mdef-201 (130) , ampligen (131), polyiclc (61, 132) , stinging nettle lectin (131) , and tapi-2 (a tace-inhibitor) (59) are anticoronaviral agents tested in vivo models of sars. for instance, a study showed that amiodarone (a known anti-arrhythmic agent) effectively targets coronaviral spreading in in vitro models (55) . working in a similar fashion, 2878/10 humanized antibodies can neutralize coronaviruses thereby reduce the complications caused by viral infections (54) . however, the above nsmis should be tested against sars-cov-2 viral associated proteins and against the activity of nsp's to derive an effective therapeutic intervention. prospective research must focus on the development of novel "helicase inhibitors, viral attachment inhibitors, and activity of rhesus θ -defensin" that block sars-cov-2 infection using in vitro, in vivo, and clinical studies (115) . hence, the development of nsmis to target the synthesis of nsp's in sars-cov-2 may deliver cellular antiviral responses by blocking their replication in host cells (115, 133, 134) . repurposing existing drugs is another strategy widely under consideration to target key proteins involved in the sars-cov-2 infection. in this regard, the existing nsmis viz., antivirals (umefenovir, remdesivir, nitazoxanide, favipiravir, ritonavir, lopinavir, ifns), anticytokines, antimalaria drugs (chloroquine, hydroxychloroquine), and passive antibody therapies are currently being evaluated to improve clinical outcomes in sars-cov-2 infected patients (3, 47, 64, 135, 136) . however, these agents require additional experimental and clinical validations before being tested in sars-cov-2 infections. for example, hydroxychloroquine (anti-malarial drug) and the tocilizumab (immunosuppressive drug) are preferred currently to mitigate viral entry and cytokine production in the sars-cov-2 infection. these drugs are being tested in ongoing trails in china and italy (135, 137) . priming the spike (s)-protein of coronavirus by host cells using membrane proteases is a necessary process for viral entry and replication, which further determines zoonotic potential of coronaviruses (138) . a recent report by markus hoffmann et al. (7) investigated the protease dependence of sars-cov-2 for its entry into cells. for example, sars-cov-2 uses the tmprss2 protease for its priming (7) . inhibition of tmprss2 using camostat mesylate retarded the viral entry into caco-2 cells (7). camostat mesylate could be recommended as an nsmi for human clinical trials to combat the sars-cov-2 virus (7). this report delineated the ability of neutralizing antibody responses against s-protein to block the sars-cov-2 entry into host cells (139) . the serum antibody responses raised to combat the "sars-s protein/ace-2 interface" during the sars-cov-2 infection indicates that the vaccination strategy may be an effective therapeutic modality against the covid-19 infection (7). ace-2 catalytic efficacy is significantly higher than ace for angiotensin-ii (140) . several compounds, such as mln-4760, were screened according to structure-based/substrate-based studies through virtual screening for inhibiting ace-2 activity (140) (141) (142) (143) . ace-2 is predominantly expressed in lungs, brain, heart, blood vessels, and renal organs (144, 145) . ace-2 is essential for cardiovascular homeostasis, and cns homeostasis as ace-2 confer redox homeostasis by mitigating ang-ii-induced oxidative stress (146) . however, in covid-19, ace-2 acts as receptor on human respiratory epithelial cells for sars-cov-2 binding (7). a recent report by markus hoffmann et al. (7) provided evidence that the sars-cov-2 strain use its spike (s)-protein to bind to ace-2. authors of this paper have also demonstrated the efficiency of tmprss2 in sars-cov-2 viral strain priming in host cells (7) . therefore, targeting ace-2 could be a viable strategy to prevent the entry of sars-cov-2 into the human system. however, a recent report by guan et al. (147) cautioned that the administration of ace inhibitors significantly induced adverse clinical outcomes in covid-19 patients due to severe hypertension, coronary artery disease, and chronic renal failure; hence, further use of ace inhibitors to treat covid-19 infections was halted (147) (148) (149) . in another report diaz (149) hypothesized that covid-19 patients receiving i.v. infusions of aceis and arbs (at1-receptor blockers) are at a higher risk of attaining severe disease pathogenesis. hence, they supported the development of nsmis such as "tmprss2 inhibitors to treat sars-cov-2 infections (7)". the failure of disease management and lack of selective therapies could be due to the intricate covid-19 pathogenesis induced by the sars-cov-2 infection. hence, the early recognition of disease is essential for effective management of covid-19 (48) . although, several reports delineated the efficacy of certain nsmis viz., ribavirin, promazine, and imp dehydrogenase inhibitors to inhibit in vivo models of sars-cov replication, later, they were proven ineffective (60, (150) (151) (152) . a report by reghunathan et al. (153) showed that the immune response produced against sars-cov may be different from other viral infections as indicated by the lack of upregulation in mhc-i genes, cytokines, and ifns or complementmediated cytolysis in peripheral blood mononuclear cells (pbmcs). the failure in the development of a vaccine is due to antibody-dependent enhancement and th-2 immunopathology (154) (155) (156) . pegylated ifn-α inhibits viral replication of sars-cov and offers protection against type i pneumocytes in lungs (2) . a significant reason for the failure or lack of selective therapies against sars-cov-2-induced sars is the intricate immune system mediated pathophysiology (4) . other reports by law et al., also detailed similar mechanisms (157, 158) . sars-cov can evade host ifn-mediated viral growth inhibition by activating ifn-regulatory factor 3 (157) . furthermore, sars-cov could induce apoptosis in lymphocytes in vitro using "orf 7a, orf 3a, and orf 3b, e protein, and n protein" (159) (160) (161) (162) . for instance, the sars-cov can evade immunity as indicated by the decline in cd4 and cd8 t cells (163) . therefore, it is necessary to uncover the complement-based cytolysis in human patients in response to the sars-cov-2 strain as this virus executes unusual mechanisms to evade the human immune system consequently inducing pathogenesis and mortality. the prospective research should focus on this viral-mediated immune signaling with respect to sars-cov for developing effective nsmis. intravenous (iv) hyperimmune globulin therapy is one of the immunotherapies known to downmodulate pro-inflammatory cytokines and mitigate the severity of infection in covid-19 patients. iv infusion of immunoglobulins composed of a high dose of antibodies, which can bind to a number of inhibitory receptors viz., fc gamma receptor iib (fcγriib) (164, 165) and fcγriic (166) and confer anti-inflammatory responses against sars-cov-2 (completed clinical trials: hyperimmune plasma nct04321421). oxidative stress is significantly induced by several viral infections inside the lungs through the downregulation of redox regulator nuclear factor-erythroid 2 related factor 2 (nrf-2) (167). nrf-2 is a leucine-zipper transcription factor (167) expressed predominantly in nasal epithelium, epithelial cells of lungs, and alveolar macrophages (168, 169) . disruption of nrf-2 and keap1 interaction triggers the activation of the anti-oxidant defense mechanism (169) . for instance, nrf-2 activation offers protection against inflammation and lung injury induced by influenza viral infections and respiratory syncytial virus (rsv) through the anti-oxidant defense pathway (170) . several viral proteins in the host cells can foster optimum levels of ros-mediated oxidative stress to facilitate viral metabolism and the viral replication cycle without killing host cells (168, (171) (172) (173) (174) . recent seminal studies described the active role of viruses in inhibiting the nrf-2 pathway (175) (176) (177) . for instance, the positive regulation of nrf-2 in modulating the thiol redox system and oxidative stress for the survival of infected astrocytes was observed in moloney murine leukemia virus and hiv virus (178) . the hcv virus could induce the downregulation of nrf-2 dependent nqo1, gclc, and gpx and modulate oxidative stress (179, 180) . an rsv infection mediates proteasomal degradation, deacetylation, and sumoylation of nrf-2 consequently causing the downregulation of nqo1, cat, and sod1 gene expression (181) . hence, nrf-2 activators are potential anti-viral agents, which can be tested against the sars-cov-2 infection (167) . future research is highly imperative in unraveling the underlying activity of nrf-2 for emerging sars-cov-2 survival by analyzing nrf2 target genes nqo1, gclc, and gpx. in addition, the sars-cov-2 mediated expression of serine and cysteine proteases in different cell lines should be investigated in relation to nrf-2 activation, which is a beneficial strategy to combating sars-cov-2 pathogenesis. however, in the case of certain viral infections, it is imperative to develop nrf-2 inhibitors to protect the host cells (182) . for instance, the marburg virus (a causative agent for lethal hemorrhagic fever) can modulate oxidative stress by activating nrf-2 dependent signaling through the blockade of "vp-24 viral protein" binding to keap1 (183) . therefore, vp-24 dependent nrf2 activation can mediate the upregulation of genes ho (heme oxygenase)-1, nqo1, and gclm (183) . in the case of dengue virus, the viral particles could induce er stress and activate nrf-2 signaling, which then lead to tnf-α secretion (184) . in this scenario, it is crucial to uncover any underlying mechanisms of emerging sars-cov-2 survival through the modulation of oxidative stress via nrf-2 signaling in different cells of different organs including lungs (183) . the prospective lindera erythrocarpa makino (195) celastrol (quinone methide triterpene) inhibits tat-induced hiv-1 infection tripterygium wilfordii (197) bakuchiol (phenolic isoprenoid) psoralea corylifolia l. (199) rupestonic acid (sesquiterpene) artemisia rupestris l. (continued) frontiers in immunology | www.frontiersin.org research studies should focus on the development of nrf-2 modulators against sars-cov-2. natural products were proven to offer protection against virusinduced oxidative stress by modulating anti-oxidant defense pathways (185, 186) . for instance, the administration of egcg has mitigated viral replication of "influenza a/bangkok/1/79 infection" by activating nrf-2 to attenuate virus-induced oxidative stress, inflammation, and apoptosis in lung cells (186) . similarly, the cytoprotective and antioxidant efficacy of nrf-2 was reported against pr8 influenza-a viral infection in at-i and at-ii cells (186) . prospective research should focus on testing the efficacy of several natural products to block sars-cov-2 viral replication by ascertaining nrf-2 mediated antioxidant responses. studies have also shown the activation of host cellular transmembrane proteases (for example, serine proteases, cysteine proteases), which can further foster a prompt viral entry and viral replication in host cells by reducing nrf-2 expression (4). decline in proteolysis of the above proteases can actuate the propagation of several human viruses viz., influenza, hiv, nipah, ebola, and coronaviruses (sars-cov, mers-cov, sars-cov-2) (42, 90, (187) (188) (189) . in this scenario, similar to influenza-a virus (190) , it is highly important to unravel the influence of nrf-2 expression on tmprss2, and human airway trypsin-like protease during sars-cov-2-mediated inflammatory conditions and oxidative stress in lungs. the downregulation of the nrf-2 gene is correlated to serine protease activity and consequent influenza viral entry (185) . recent studies have demonstrated the efficacy of natural nrf-2 activators viz., egcg and sulforaphane (sfn) for blocking viral entry/viral replication as well as promoting antiviral mediators rig-i, ifn-β, and mxa (185) . in this context, it is essential to demonstrate the effects of nutritional interventions like sfn and egcg against sars-cov-2 induced oxidative stress by modulating nrf-2 signaling. evidence has demonstrated the use of naturally occurring nrf2 activators for mitigating viral infections/post-viral infection induced complications. for example, α-luminol is a natural nrf-2 activator, which confers the protection of astrocytes against the momul virus (191) . egcg enhances nuclear nrf-2 levels during tat-induced hiv-1 infection and offers protection against virus induced oxidative stress (192) . tanshinone ii a can induce upregulation of nrf-2 expression and mitigates ros production during tat-induced hiv-1 infection via modulating ampk/nampt/sirt1 signaling in host cells (193) . sfn enhances the phagocytic function of "hiv-infected alveolar macrophages in lungs" by activating nrf-2 signaling, which further induces downstream antioxidant cascades (194) . lucidone is effective for the nrf-2 mediated blockade of dengue virus by inducing heme oxygenase-1 (195) ; rographolide could induce nrf-2 induced antioxidant defenses against influenza a in lung cells (196) ; celastrol can mediate nrf-2 induced antioxidant defenses against hiv-1 tat-induced inflammation (197) . broccoli sprouts containing sfn acts as a nrf-2 activator to reduce influenza-induced infection in lung cells (198) . bakuchiol and rupestonic acid are phytoconstituents that confer nrf-2 activation thereby promoting nqo1 gene expression and ho-1-mediated interferon activity to enhance antioxidant response against influenza virus in lung cells (199, 200) . curcumin is another significant compound that can modulate nrf-2 signaling and enhance the generation of ifn-β to offer protection against the influenza virus (201) . curcumin can mitigate this viral infection by modulating tlr2/4, p38/jnk mapk, and nf-κb pathways (201) . however, studies are required to decipher the activity of these phytochemicals against sars-cov-2 ( table 4) . a recent report by drȃgoi (209) hypothesized that the potent natural nrf-2 activators viz., resveratrol, sfn, curcumin, and asea redox should be evaluated in different combinations with conventional drugs against sars-cov-2 infection in both in vitro and in vivo models and to further deduce a correlation between nrf-2 activity and sars-cov-2 viral entry/replication. sars-cov-2 is progressively inducing a high mortality rate across the globe due to the lack of selective therapeutic interventions or vaccination. recent reports by lu et al. (210) and xu et al. (148) delineated that the s-protein of sars-cov-2 and sars co-v exhibit similar 3-d pharmacophore in the receptor binding domain (rbd) of ace-2 of human cells. covid-19 patients are characterized by the severe viral pathogenesis due to extensive cytokine storm viz., tnf-α, il-1β, il-10, ifnγ, and mcp-1 in infected lung tissues (48) . a report by chen and du (211) hypothesized that the phyto-constituents such as "baicalin, scutellarin, hesperetin, nicotianamine, and glycyrrhizin" may deliver anti-sars-cov-2 effects. hesperetin glycoside abundant in citrus fruits, which can inhibit the sars-cov 3clpro (212) . the activity of this molecule must be examined against serine/cysteine proteases, which support sars-cov-2 viral entry/replication. traditional citrus flavonoids were frontiers in immunology | www.frontiersin.org reported to have a potential to act against sars-cov-2 as studied by molecular docking studies. molecular docking simulations, lc-ms studies described the efficacy of citrus flavonoids (ex. naringenin) in binding to ace-2, and mitigating inflammationinduced lung injury by the sars-cov-2 virus (213) . further studies should evaluate these compounds in preclinical models to determine the safety and efficacy against the sars-cov-2 infection. natural products such as di/tri-terpenoids, lignoids were proven to inhibit the viral replication of coronaviruses in vitro (214) ; griffithsin could block coronaviral entry by binding to the sars-cov spike glycoprotein (215) . tsl-1 can block coronaviral entry/replication; leaf extracts of toona sinesis roem effectively blocked sars-cov replication (57) . betulinic acid, savinin can act as competitive inhibitors against sars-cov 3cl protease to block viral entry (214) . the research gap must be filled to develop nutritional therapeutic interventions by investigating the efficacy of these phytochemicals against sars-cov-2 viral entry. seeds of psorelia corylifolia exhibit inhibitory effects against the sars-cov papain-like protease required for coronavirus entry/replication. the efficacy of these molecules should be examined against sars-cov-2 (216) ( table 5 ). the active site pockets of main proteases such as 6lu7 and 2 gtb in sars-cov-2 are reported to be involved in conferring viral entry/fusion; hence, these sites should be considered as the potential drug targets against sars-cov-2 (66) . a molecular docking study by khaerunnisa et al. (65) reported the predicted efficacy of bioactive compounds against above sars-cov-2 main protease (mpro) sites viz., "nelfinavir, lopinavir, kaempferol, quercetin, luteolin-7glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin" ( table 5) . the life-threatening consequences of the covid-19 pandemic remain high due to lack of selective targeted therapies and vaccination strategies. this is primarily due to extreme genomic variability of rna viruses as well as variations in the host-cell invading mechanisms. hence, this review benefits virologists, medical scientists, and cell biologists to ascertain and develop nsmis, nrf-2 modulators, and clinically viable vaccines to combat this devastating sars-cov-2 strain. however, many more preclinical and clinical studies are required to uncover the therapeutic efficacy of potential phytochemicals, natural nrf2 modulators, and several nsmis against 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protein 10 by sars-cov infection, interferon alfacon 1 and interferon inducer in human bronchial epithelial calu-3 cells and balb/c mice increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons interferon-beta and interferongamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro potential inhibitor of covid-19 main protease (mpro) from several medicinal plant compounds by molecular docking study nelfinavir was predicted to be a potential inhibitor of 2019-ncov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation antiviral drugs specific for coronaviruses in preclinical development a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 baricitinib as potential treatment for 2019-ncov acute respiratory disease inhibition of human coronavirus 229e infection in human epithelial lung cells (l132) by chloroquine: involvement of p38 mapk and erk chloroquine and hydroxychloroquine as available weapons to fight covid-19 antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis repurposing of kinase inhibitors as broad-spectrum antiviral drugs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection biologic basis for interleukin-1 in disease differences between anti-tumor necrosis factor-alpha monoclonal antibodies and soluble tnf receptors in host defense impairment protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 designing defenses against deadly viruses bcx4430-a broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of ebola virus disease host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses the serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells tmprss2 is essential for influenza h1n1 virus pathogenesis in mice tmprss2 is a host factor that is essential for pneumotropism and pathogenicity of h7n9 influenza a virus in mice the host protease tmprss2 plays a major role in in vivo replication of emerging h7n9 and seasonal influenza viruses a mutant h3n2 influenza virus uses an alternative activation mechanism in tmprss2 knockout mice by loss of an oligosaccharide in the hemagglutinin stalk region influenza and sars-coronavirus activating proteases tmprss2 and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein wild-type human coronaviruses prefer cell-surface tmprss2 to endosomal cathepsins for cell entry a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research vegf and bfgf are highly expressed in intraluminal fibromyxoid lesions in bronchiolitis obliterans organizing pneumonia in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds treatment of sars with human interferons severe acute respiratory syndrome-related coronavirus is inhibited by interferon-α potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifn-α/β) but not by type ii interferon (ifn-γ) the molecular biology of sars coronavirus an overall picture of sars coronavirus (sars-cov) genome-encoded major proteins: structures, functions and drug development m and n proteins of sars coronavirus induce apoptosis in hpf cells the sars-coronavirus membrane protein induces apoptosis through modulating the akt survival pathway ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the n-terminal domain of n protein structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein sars coronavirus 7a protein blocks cell cycle progression at g0/g1 phase via the cyclin d3/prb pathway sars coronavirus protein 7a interacts with human ap 4 a-hydrolase using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque sirna targeting the leader sequence of sars-cov inhibits virus replication inhibition of genes expression of sars coronavirus by synthetic small interfering rnas inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells inhibition of sars-cov replication by sirna prophylactic and therapeutic effects of small interfering rna targeting sars coronavirus screening of drugs by fret analysis identifies inhibitors of sars-cov 3cl protease recent developments in anti-severe acute respiratory syndrome coronavirus chemotherapy deubiquitinating activity of the sars-cov papain-like protease selectivity in isg15 and ubiquitin recognition by the sars coronavirus papain-like protease drug design targeting the main protease, the achilles' heel of coronaviruses characterization and inhibition of sarscoronavirus main protease enzymatic activity of the sars coronavirus main proteinase dimer a second, non-canonical rna-dependent rna polymerase in sars coronavirus zn2+ inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins molecular cloning, expression, and purification of sars-cov nsp13 multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase inhibition of sars coronavirus helicase by bismuth complexes bismuth complexes inhibit the sars coronavirus human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets single-dose intranasal administration with mdef201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal sars-cov balb/c mouse model interferon alfacon 1 inhibits sars-cov infection in human bronchial epithelial calu-3 cells a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo toll like receptor-3 ligand poly-iclc promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine cns tumor models nonstructural proteins 7 and 8 of feline coronavirus form a 2: 1 heterotrimer that exhibits primer-independent rna polymerase activity new nsp8 isoform suggests mechanism for tuning viral rna synthesis breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies a trial of lopinavirritonavir in adults hospitalized with severe covid-19 the proximal origin of sars-cov-2 trypsin treatment unlocks barrier for zoonotic bat coronavirus infection twoyear prospective study of the humoral immune response of patients with severe acute respiratory syndrome hydrolysis of biological peptides by human angiotensinconverting enzyme-related carboxypeptidase evaluation of angiotensin-converting enzyme (ace), its homologue ace2 and neprilysin in angiotensin peptide metabolism ace2: from vasopeptidase to sars virus receptor structure-based pharmacophore design and virtual screening for novel angiotensin converting enzyme 2 inhibitors role of the ace2/angiotensin 1-7 axis of the renin-angiotensin system in heart failure angiotensin-converting enzyme 2 (ace2), but not ace, is preferentially localized to the apical surface of polarized kidney cells ace2-mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function clinical characteristics of coronavirus disease 2019 in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid-19 evaluation of interferon inducers, ribavirin and mouse hyperimmune serum in a pathogenesis/lethal mouse model using a mouse-adapted sars-cov enhancement of the infectivity of sars-cov in balb/c mice by imp dehydrogenase inhibitors, including ribavirin is the anti-psychotic, 10-(3-(dimethylamino) propyl) phenothiazine (promazine), a potential drug with which to treat sars infections?: lack of efficacy of promazine on sars-cov replication in a mouse model expression profile of immune response genes in patients with severe acute respiratory syndrome implications of antibody-dependent enhancement of infection for sars-cov-2 countermeasures pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 chemokine upregulation in sars-coronavirus-infected, monocyte-derived human dendritic cells the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors the 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in vero e6 cells overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway g0/g1 arrest and apoptosis induced by sars-cov 3b protein in transfected cells significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome selective blockade of the inhibitory fcγ receptor (fcγriib) in human dendritic cells and monocytes induces a type i interferon response program ligation of fc gamma receptor iib inhibits antibody-dependent enhancement of dengue virus infection immunomodulation by ivig and the role of fc-gamma receptors: classic mechanisms of action after all? front immunol therapeutic modulation of virus-induced oxidative stress via the nrf2-dependent antioxidative pathway oxidative stress induced by hiv-1 reverse transcriptase modulates the enzyme's performance in gene immunization thiol chemistry and specificity in redox signaling antiviral activity of nrf2 in a murine model of respiratory syncytial virus disease influenza a virus induction of oxidative stress and mmp-9 is associated with severe lung pathology in a mouse model mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis c virus core protein cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells the pathogenesis of influenza virus infections: the contributions of virus and host factors viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis respiratory syncytial virus induces nrf2 degradation through a promyelocytic leukemia protein-ring finger protein 4 dependent pathway hiv-1 decreases nrf2/are activity and phagocytic function in alveolar macrophages astrocytes survive chronic infection and cytopathic effects of the ts1 mutant of the retrovirus moloney murine leukemia virus by upregulation of antioxidant defenses hepatitis c virus impairs the induction of cytoprotective nrf2 target genes by delocalization of small maf proteins hepatitis c virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to dna damage and stat3 activation respiratory syncytial virus infection downregulates antioxidant enzyme expression by triggering deacetylationproteasomal degradation of nrf2 dimerization controls marburg virus vp24-dependent modulation of host antioxidative stress responses marburgvirus hijacks nrf2-dependent pathway by targeting nrf2-negative regulator keap1 activation of nrf2 by the dengue virus causes an increase in clec5a, which enhances tnf-α production by mononuclear phagocytes nrf2 expression modifies influenza a entry and replication in nasal epithelial cells nrf2 protects human alveolar epithelial cells against injury induced by influenza a virus endosomal proteolysis of the ebola virus glycoprotein is necessary for infection two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium retrovirusinduced oxidative stress with neuroimmunodegeneration is suppressed by antioxidant treatment with a refined monosodium α-luminol (galavit) egcg inhibits tat-induced ltr transactivation: role of nrf2, akt, ampk signaling pathway tanshinone ii a inhibits tat-induced hiv-1 transactivation through redox-regulated ampk/nampt pathway activating the nrf2-mediated antioxidant response element restores barrier function in the alveolar epithelium of hiv-1 transgenic rats lucidone suppresses dengue viral replication through the induction of heme oxygenase-1 activity of andrographolide and its derivatives against influenza virus in vivo and in vitro celastrol ameliorates hiv-1 tat-induced inflammatory responses via nf-kappab and ap-1 inhibition and heme oxygenase-1 induction in astrocytes effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study bakuchiol is a phenolic isoprenoid with novel enantiomer-selective antiinfluenza a virus activity involving nrf2 activation chinese herbal medicine compound yi-zhi-hao pellet inhibits replication of influenza virus infection through activation of heme oxygenase-1 inhibition of curcumin on influenza a virus infection and influenzal pneumonia via oxidative stress, tlr2/4, p38/jnk mapk and nf-κb pathways the spectral properties of (-)-epigallocatechin 3-o-gallate (egcg) fluorescence in different solvents: dependence on solvent polarity electrophilic tuning of the chemoprotective natural product sulforaphane pharmacokinetics and tissue distribution study of tanshinone iia after oral administration of bushen huoxue qubi granules to rats with blood stasis syndrome celastrol: a spectrum of treatment opportunities in chronic diseases the active compounds derived from psoralea corylifolia for photochemotherapy against psoriasis-like lesions: the relationship between structure and percutaneous absorption 3-triazolecontaining derivatives of rupestonic acid: click-chemical synthesis and antiviral activities against influenza viruses biological activities of curcuminoids, other biomolecules from turmeric and their derivatives-a review asea redox supplement" [ars]) should be clinically tested as adjuvants in all types of medium and severe cases of aggressive respiratory viral infections (including influenza a/b/c, sars, mers, covid-19, measles, avian influenza etc.) based on their extrapolated cytoprotective antioxidant effects (especially on vital organs), including the cytoprotection offered by ars on the cardiac muscle of dmd patients which can be extrapolated to the lungs -a very short medical communication genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plant-derived phenolic compounds citrus fruits are rich in flavonoids for immunoregulation and potential targeting ace2 specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia baicalin, a metabolite of baicalein with antiviral activity against dengue virus dual effect of glucuronidation of a pyrogallol-type phytophenol antioxidant: a comparison between scutellarein and scutellarin co-occurrence of nicotianamine and avenic acids in avena sativa and oryza sativa azetidine-2-carboxylic acid derivatives from seeds of fagus silvatica l. and a revised structure for nicotianamine betulin and betulinic acid: triterpenoids derivatives with a powerful biological potential the griffithsin dimer is required for high-potency inhibition of hiv-1: evidence for manipulation of the structure of gp120 as part of the griffithsin dimer mechanism research & consulting llc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 beeraka, sadhu, madhunapantula, rao pragada, svistunov, nikolenko, mikhaleva and aliev. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-282256-lqmixm7s authors: tsioufis, costas; dimitriadis, kyriakos; tousoulis, dimitrios title: the interplay of hypertension, ace-2 and sars-cov-2: emerging data as the “ariadne’s thread” for the “labyrinth” of covid-19 date: 2020-05-22 journal: hellenic j cardiol doi: 10.1016/j.hjc.2020.05.003 sha: doc_id: 282256 cord_uid: lqmixm7s nan dr. william h. stewart the us surgeon general during 1965-1969 is remembered primarily for his infamous statement: "it is time to close the book on infectious diseases…" five decades later, how erroneous this statement was! in 2020, humanity is facing one of the most devastating health urgencies in known modern history leading to health system collapse, economies "shutting down", unemployment surges and thousands of deaths worldwide due to covid-19 infection. 1 for the cardiovascular medical community there are important issues to be clarified and in this editorial our attempt is to provide the latest evidence-based directions for the intriguing labyrinth-like associations of hypertension, reninangiotensin system and covid-19. the hype of the hypertension link to covid-19 pandemic was fueled by the initial reports from wuhan, china, in which hypertension was present in 30% of cases and in 48% of non-survivors. 2 in 138 hospitalized patients with covid-19, hypertension was observed in 31% (58% in intensive units) 3 and the official data from the national health commission of china demonstrate that 35% of patients diagnosed with covid-19 have hypertension with the overall case fatality rate being 2.3% in the entire cohort but significantly higher (6%) in patients with hypertension. 4 the only available meta-analysis from wuhan of 46248 cases, supports that hypertension constitutes the most prevalent comorbidity in 17% of patients infected with the novel coronavirus. 5 the globality of the pandemic stresses the need for international data. in italy the most current analysis shows that of 69.1% of the deceased patients were hypertensives and 30% used angiotensin converting enzyme inhibitors (aceis) and 17% angiotensin receptor blockers (arbs). 6 despite the above observations, there are no robust data to suggest clearly an independent link of hypertension with increased covid-19 morbidity and mortality. 7 similarly, in other lower respiratory track infections, diabetes and heart failure are independently related to adverse outcome, while hypertension per se is not. 7 major pathophysiological pathways by which covid-19 causes cardiovascular and systemic complications is diffuse inflammatory response and ace-2 ( figure 1 ). the characteristic covid-19 "cytokine storm" is reflected by increased il-17, il-7, il-6, interferon-c, and tnfa. 7, 8 focusing on il-6 it is associated with the outcome from the viral infection and also plays a key regulator role of immuneinflammatory response in essential hypertension. 7 supportive to the latter, hypertension pathogenesis and progression of target organ damage is closely associated with low-grade inflammation 9 and this could partially explain the fact that hypertension is the most common comorbidity in covid-19 cohorts and related to worst course of the infection. 7 the confounding effect, however, of ageing and other comorbidities like diabetes and renal dysfunction in this inflammatory link should be taken in mind with future studies urgently needed in this setting. in humans, the entry of the new coronavirus into the target cells is facilitated by the spike protein that is anchored to the angiotensin converting enzyme-2 (ace-2) in parallel with cellular serine protease tmprss2 actions. 7 ace-2 converts angiotensin ii to angiotensin 1-7 ( figure 1 ). 10, 11 receptors of the ace-2 are expressed in the epithelial cells of lungs, intestine, kidneys, heart and vessels and their activation causes vascular dilatation and provide renal and cardiovascular protection. 7, 10, 11 in this sense promoters of ace-2 are studied as potential antihypertensive drugs. 10, 11 the novel coronavirus sars-cov2 by using the ace-2 "consumes" the enzyme and the protective actions of the latter are significantly attenuated. the non-counterbalanced actions of angiotensin ii are partially responsible along with inflammation and thrombotic milieu to the covid-19 related severe complications. at the level of the lungs the virus reduces the activity of the ace-2 and regionally stimulates the renin-angiotensin system with leucocytes infiltration facilitation further accelerating the progression of the inflammatory response. in a relative study, covid-19 patients were characterized by higher angiotensin ii levels that were closely related to the total viral load and the degree of lung injury. 12 recombinant ace-2 administration has been proposed as a means to reduce angiotensin ii levels and lung tissue lesions. 10, 11 concerning the heart and vascular component of covid-19 infection, dysregulation of the ace-2 related pathways are linked to cardiac muscle injury and potential hemodynamic collapse. 10, 11 given consideration of the above, the "million dollars question", is about the effects of renin angiotensin blockers on ace-2 expression/action and most importantly what is the clinical relevance of this modulation in humans in the context of covid-19. there are data from animal studies for a more consistent upregulation of ace-2 with the majority of arbs, while the effect of aceis was variable. 7 this knowledge led to the opinion of potential susceptibility of patients on aceis and arbs to covid-19 infection 13 but this is currently unjustified. firstly, the data in humans are scarce and only in the small intestine enterocytes upregulation of ace-2 is reported due to acei. 14 regarding the lungs there are zero evidence for humans and even if existed cannot directly lead to the conclusion that this upregulation can indeed facilitate viral cell entry. 7 if one wants to focus on pneumonia outside the covid-19 setting, the use of aceis and arbs reduces the risk of infection 15 and related mortality. 7 in the era of covid-19 "social distancing", the "scientific approaching" by rapid and free exchange of knowledge provides the scaffold for better therapeutic answers. we should be therefore thankful to the researchers for the important emerging data. [16] [17] [18] [19] firstly, zhang p et al conducted a retrospective, multicenter study including 1128 adult patients with hypertension diagnosed with covid-19 and admitted to hospitals in the hubei province. 16 they showed that the incidence of the 28-day all-cause death among hypertensives who had inpatient treatment with aceis/arbs is significant lower compared with acei/arb non-users (3.7% vs. 9.8%; p = 0.01). 16 even after matching and adjusting variables in-hospital use of aceis/arbs still exhibits remarkable association with reduced all-cause mortality. and even more recently, three simultaneous publications shed further light into the debate of renin-angiotensin therapies in the covid-19 setting. [17] [18] [19] the work by reynolds h et al showed that among 12594 patients who were tested for covid-19 there was no association of aceis/arbs with increased likelihood of a positive test or severe infection. 17 further insight is provided by mancia g et al focusing in 6272 cases with severe covid-19. 18 after adjustment for confounders there was no independent association for the use of aceis/arbs with susceptibility for infection or worse clinical outcome in contrast to loop diuretics that were linked to enhanced risk. 18 interestingly the publication by mehra m et al, included international data from 169 hospitals (11 countries in asia, europe, and north america) for 8910 patients with covid-19 for whom discharge status was available. 19 there was no harmful association of either aceis or arbs with mortality but also it was demonstrated that the use of statins and aceis was linked to less in-hospital deaths. 19 these findings clearly support recently published recommendations by many societies regarding continuation of aceis or arbs among patients with co-existing hypertension, cardiovascular disease and covid-19. 20,21 moreover, they provide further rationale for ongoing studies with drugs targeting the renin-angiotensin system for therapy of covid-19, like losartan for hospitalized (νct04312009) and non-hospitalized patients (nct043111177). 22 there is a trending notion that there is not only a "second" wave of covid-19 pandemic linked to the infection per se to be worried about but also a "third" and maybe a more important one! this "third wave" in the upcoming months and years is related to major events that would lead to a massive burden to the health systems because of covid-19 related postponed guideline care in major disease states among them the cardiovascular ones. for hypertensive patients this is highly relevant due to the lack of regular follow-up along with destabilization of blood pressure levels due to unjustified discontinuation or modification of dosage of aceis/arbs that can lead to heightened cardiovascular risk. 23 this outsidethe-norm approach to chronic diseases might be a reaction of the public, as well as of some health providers, to the media-induced frenzy for the novel coronavirus. attention should be paid to the latter phenomenon and the cardiovascular community has already played a key role on establishing guidance for treatment and continuity of care for high risk patients. 7, 20 interplay with the organization of the health systems and governments is needed to sufficiently address the "burden" of cardiovascular emergencies and routine cases throughout the covid-19 era. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan covid-19 and the cardiovascular system prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis hypertension, the renin-angiotensin system, and the risk of lower respiratory tract infections and lung injury: implications for covid-19 clinical features of patients infected with 2019 novel coronavirus in wuhan, china effects of oral paricalcitol therapy on arterial stiffness and osteopontin in hypertensive patients with chronic kidney disease and secondary hyperparathyroidism aldosterone system inhibitors in patients with covid-19 coronavirus disease 2019 (covid-19) and cardiovascular disease: a viewpoint on the potential influence of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers on onset and severity of severe acute respiratory syndrome coronavirus 2 infection clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? urinary angiotensin-converting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin ii receptor blocker risk of pneumonia associated with use of angiotensin converting enzyme inhibitors and angiotensin receptor blockers: systematic review and meta-analysis association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 renin-angiotensin-aldosterone system inhibitors and risk of covid-19 renin-angiotensin-aldosterone system blockers and the risk of covid-19 cardiovascular disease, drug therapy, and mortality in covid-19 the new guidelines for hypertension: navigating between scylla and charybdis of clinical practice controversies of renin-angiotensin system inhibition during the covid-19 pandemic effects of blood pressure lowering treatment in hypertension: 8. outcome reductions vs. discontinuations because of adverse drug events -meta-analyses of randomized trials with clinical strength, and scientific rigor, the "labyrinth" of unanswered questions will be solved and the covid-19 outbreak will be limited. concerning hypertension therapy, it seems at present to be "immune" to covid-19. let's be optimistic and let the emerging data be our "ariadne's thread". key: cord-326405-3446eyi3 authors: wysocki, jan; gonzález-pacheco, francisco r.; batlle, daniel title: angiotensin-converting enzyme 2: possible role in hypertension and kidney disease date: 2008-02-07 journal: curr hypertens rep doi: 10.1007/s11906-008-0014-1 sha: doc_id: 326405 cord_uid: 3446eyi3 the discovery of angiotensin-converting enzyme (ace) 2 adds a new level of complexity to the understanding of the renin-angiotensin system. the high catalytic efficiency of ace2 for the generation of angiotensin (ang)-1-7 from ang ii suggests an important role of ace2 in preventing ang ii accumulation, while at the same time enhancing ang-1-7 formation. ace and ace2 may have counterbalancing functions and a regulatory role in fine-tuning the rate at which ang peptides are formed and degraded. by counterregulating the actions of ace on ang ii formation, ace2 may play a role in maintaining a balanced status of the renninangiotensin system. this review focuses on the function of ace2 and its possible roles in kidney disease and hypertension. studies using models of ace2 ablation and the pharmacologic administration of an ace2 inhibitor suggest that decreased ace2 activity alone or in combination with increased ace activity may play a role in both diseases. the first human homologue of angiotensin-converting enzyme (ace), termed angiotensin-converting enzyme 2 (ace2) was identified in 2000 by two separate groups using genomics-based strategies [1, 2] . the gene encoding ace2 is located on the x chromosome. like ace, ace2 is a type 1 integral membrane protein; however, ace2 contains only a single active site domain and consists of 805 amino acids [1, 2] . ace2 acts as a carboxypeptidase, removing single amino acids from the c-terminus of its substrates, whereas ace acts predominantly as a peptidyl dipeptidase removing c-terminal dipeptides. the metalloprotease catalytic domains of ace2 and ace are 41% identical, and comparison of the genomic structures indicates that the two genes arose through duplication from a common ancestor [1, 2] . the subsequent elucidation of the three-dimensional structure of the extracellular domain of ace2 [3] revealed that the catalytic mechanism of ace2 closely resembles that of ace. however, the substrate-binding pockets differ significantly [3] , explaining the differences in substrate specificity between the two enzymes and the failure of ace inhibitors to bind to and inhibit ace2 [4] . ace2 is the only known enzymatically active homologue of ace in the human genome. the carboxyl end of ace2 is homologous to collectrin [5] . collectrin, unlike ace and ace2, lacks carboxypeptidase catalytic properties and was initially localized in the collecting tubule [5] . more recently, however, it has been localized to the proximal tubule, where it is involved in regulating amino acid uptake [6•,7] . collectrin also has been suggested to have a role in cystogenesis by interacting with ciliaspecific membrane integral proteins [8•] . another gene has been identified in the genomes of several mammalian species that encodes a novel, single-domain ace-like protein that was named ace3. in several species ace3 seems to lack catalytic activity as a zinc metalloprotease [9] . moreover, in humans, no evidence could be found that the ace3 gene is expressed, and the presence of deletions and insertions in the sequence suggests that in humans ace3 is a pseudogene [9] . ace2 was initially found in heart, kidney, and testis with lesser amounts in colon, small intestine, and ovary [10] . ace2 also has been found in the lungs where it plays an important role in angiotensin (ang) ii metabolism [11, 12] . ace2 protects mice from severe lung injury induced by acid aspiration, sepsis, and severe acute respiratory syndrome (sars) virus infection [11, 12] . we first suggested that ace2 could be renoprotective, particularly in combination with low levels of ace [13] . recent work by our laboratory is consistent with this hypothesis [14••,15••,16•] . in this review we discuss recent publications dealing with ace2 as a pathway for ang ii metabolism and its possible role in hypertension and diabetic kidney disease. the discovery of ace2 adds a new level of complexity to the understanding of the renin-angiotensin system (ras). ace2 is a carboxypeptidase that facilitates the conversion of ang ii to ang-(1-7) and the conversion of ang i to ang-(1-9) [1,2,17] (fig. 1 ). ang-(1-9) has no known effects on blood vessels but can be converted by ace to a shorter peptide, ang-(1-7), which is a blood-vessel dilator (fig. 1 ). ang i (with 10 amino acids) is an intermediate peptide without known biologic effects. ang i is converted to ang-(1-9) (with nine amino acids) by ace2, but is converted to the eight-amino acid ang ii by ace (fig. 1) . the actions of ace2 should act to prevent the accumulation of ang ii, a vasoconstrictor, and lead to formation of ang-(1-7), a vasodilator. ace and ace2 may have counterbalancing functions and a regulatory role in fine-tuning the rate at which ang peptides are formed and degraded. ace2 has a high catalytic efficiency for the generation of ang-(1-7) from ang ii [17] . this suggests an important role of ace2 in preventing ang ii accumulation, while at the same time enhancing ang-(1-7) formation. recently, it was shown that ang 1-7 inhibits ang ii-stimulated mitogen-activated protein kinase (mapk) phosphorylation in proximal tubular cells [18•] . generation of ang-(1-7) by proximal tubular ace2 could therefore also have a role in counteracting the effects of locally generated ang ii [18•] . ace2-mediated degradation of ang ii to ang-(1-7) has been documented in studies using kidney cortex preparations or isolated proximal tubules [19,20•,21•]. ace2 affinity for ang ii conversion has been generally found higher than for ang i to ang(1) (2) (3) (4) (5) (6) (7) (8) (9) [19,20•,21•]. in freshly dissected rat proximal tubular segments, however, li et al. [22••] observed ace2-dependent formation of ang-(1-7) from ang i, which depended on conversion of ang i to ang-(1-9), followed by ace-mediated cleavage to ang-(1-7). in another study in cardiac tissue, investigators suggested that ace2 can act through ang-(1-9) instead of ang-(1-7), as a counterregulator of ace [23] . the knowledge of ace2 biologic peptide substrates is useful in providing an initial understanding of the substrate specificity of the protease and the physiologic role of ace2. if ace2 is a "converting enzyme," as ace is, then its peptidase activity either produces or degrades (or both) a peptide with biologic activity [17] . therefore, an understanding of the biologic activity of ace2 substrates and products suggests putative physiologic roles for ace2 [17] . biologically active substrate peptides that are cleaved by ace2 are shown in table 1 . although the biologic peptides ang i and ang ii are the main known ace2 substrates, ace2 can hydrolyze several other target peptides. ace2 does not cleave bradykinin but inactivates both des-arg 9bradykinin and lys-des-arg 9 -bradykinin [17] . ace2 can also remove the c-terminal residue from apelin and other vasoactive peptides such as neurotensin, kinetensin (a neurotensin-related peptide), and des-arg bradykinin [10] . ace2 has high catalytic efficiency to hydrolyze apelin-13 and apelin-36 peptides [10] . apelin also induces an increase in myocardial contractility and a reduction of vasomotor tone [10] . two opioid peptides, dynorphin a and -casamorphin, are also cleaved by ace2 [10, 17] . these two ace2 substrate peptides stimulate and opioid g-protein-coupled receptors, and may have negative effects on cardiomyocyte contractility [10] . angiotensin i angiotensin 1-9 angiotensin ii angiotensin 1-7 a non figure 1 . the vertical arrows show the classic angiotensin (ang) ii-forming pathway driven by angiotensin-converting enzyme (ace) and non-ace mechanisms. horizontal arrows show the actions of the more recently discovered ace2. through the action of ace2, less ang i substrate is available for ace to generate ang ii because ang i is derived to ang-(1-9) (upper horizontal arrow). ang ii is converted to ang-(1-7) by ace2 (lower horizontal arrow). the net effect of ace2 is less ang ii accumulation and more ang-(1-7) formation. [16•,23,25-27] . in these assays, the substrate peptide contains a fluorescent 7-methoxycoumarin group (mca), which is quenched by energy transfer to a 2,4-dinitrophenyl moiety (dnp). this reaction is based on the cleavage of an amide bond between the fluorescent and quencher groups, resulting in an increase in fluorescence [28] . it can be used to measure the activity of ace2 and of other peptidases [28] . the fluorescence signal of mca-yvadapk(dnp) is partially quenched by specific inhibitors of both ace and ace2, but not by an inhibitor of another metalloprotease, carboxypeptidase a [16•]. we took advantage of this dual action to develop an assay to measure ace and ace2 activity concurrently [16•] . the combination of specific inhibitors for ace and ace2 quenched the signal almost completely and to the same degree as ethylenediaminetetraacetic acid (edta), a metal ion chelator, indicating that both metalloenzymes, ace and ace2, are involved in the degradation of the substrate. as noted previously, the dual cleavage of this substrate by ace and ace2 makes it possible to measure activity of these two carboxypeptidases concurrently in tissue samples [16•] . this is particularly important because ace2 and ace colocalize in numerous tissues where both enzymes have been shown to be enzymatically active [29] . the assessment of ace2 activity, the only active homologue of ace, in combination with ace activity is useful in evaluating the ras in the pathophysiology of different disease states [14••] . ace2 activity has also been measured using a different fluorogenic substrate, mca-apk(dnp) [25] [26] [27] . this approach detected ace2 activity in serum samples from mice overexpressing ace2 (but not in wild-type mice) [26] , and in rat heart tissue [25] and rat testes [27] . using this substrate in mouse tissues, however, we could not quench the fluorescence with mln-4760. moreover, the mca-apk(dnp) substrate is highly selective for ace2 [26] . accordingly, it cannot be used for dual measurements of ace and ace2 activity. ferrario et al. [24••] used an hplc-based method to measure ace2 activity in cardiac membranes; this method was later extended to measurements in other tissues and body fluids [21• ]. the assay is performed in two steps: 1) radiolabeled peptide precursors are incubated with ace2; and 2) hplc is used to separate the peptide products. the hplc method utilizes the ability of ace2 to hydrolyze ang ii to ang-(1-7) [17] . the conversion rate of the exogenous radioactively labeled ang ii to ang(1) (2) (3) (4) (5) (6) (7) in the presence and absence of ace2 inhibitor is the equivalent of the ace2 enzymatic activity [24••] . because this method requires hplc measurements, the process involved is relatively time consuming, but it provides information on ang-(1-7) formation directly. another newly developed assay also uses the natural substrate of ace2, ang ii; this method utilizes surface-enhanced laser desorption/ionization time-of-flight (seldi-tof) ms with proteinchip array technology (ciphergen biosystems, fremont, ca) [20•] . in this assay, mouse kidney samples are mixed with ang ii and incubated for a specified time. ace2 converts ang ii to ang-(1-7), which both can be quantified by seldi-tof ms. based on the observation of a linear relationship between recombinant ace2 concentrations and the quotient of the formed ang (1-7) to the hydrolyzed ang ii, the ratio of ang (1-7)/ang ii was proposed as a measure of tissue ace2 activity [20• ]. the levels of ace2 activity vary widely within tissues. for instance, using our fluorogenic assay the levels of ace2 activity in mouse kidney cortex are about 10-to 20-fold higher than in the heart, whereas ace2 activity is barely detectable in serum [16•] . studies using other measurement methods found very low or undetectable ace2 activity in plasma from rodents [20•,26] . similarly, in healthy humans, the levels of circulating ace2 are undetectable or very low [30] . rice et al. [30] found that circulating ace2 was 100-fold lower than ace, and ace2 was detectable in less than 10% of the study population. low circulating ace2 levels could result from less shedding of ace2 as compared with ace from the plasma membrane of endothelial cells [30] . the zinc metalloprotease adam17 is responsible for the regulated shedding of ace2; there is little constitutive shedding of ace2 as compared with ace [31•]. ace2 activity and protein have been detected in human urine from healthy subjects [32•] , and ace2 activity has been measured in urine and serum from sheep [21• ]. thus, ace2, like ace, appears to be secreted as a soluble protein in vivo. it would be important to determine whether circulating or urinary ace2 increases in certain disease states, either due to increased expression, increased shedding from the membrane, or decreased clearance from the circulation [30] . the pattern of ace and ace2 expression in the glomerulus of db/db mice is just the opposite of what we find in cortical tubules [13] . consequently, the final urine may not reflect the site of the nephron where the alteration primarily resides. in kidney cortex, a good correlation was found between ace2 protein abundance and enzymatic activity [16• ]. this indicates that the level of functional activity is highly dependent on the level of protein expression. in contrast, we found a lack of positive correlation between renal ace2 activity and renal ace2 mrna levels in the db/db as well as streptozotocin (stz)-induced diabetic mice. these findings illustrate the importance of not relying solely on mrna levels when assessing whether ace2 is altered. measurements of ace2 mrna alone may not reflect existing differences occurring at a posttranscriptional level [16•] . investigators must keep this in mind when interpreting data from studies reporting ace2 gene polymorphisms in diseases such as hypertension or diabetes. this further underscores the importance of having reliable assays to measure ace2 activity targeting the sites of possible ace2 alterations in the kidney and other organs. immunohistochemical analysis of ace2 distribution within the kidney demonstrated that renal tubules clearly show the highest intensity of immunostaining [13,15••,33] . in both kidney sections and cultured polarized renal epithelial cells, ace2 localizes predominantly to apical surface [15••,32•] where it can undergo regulated proteolytic shedding [31•] . ace2 is also present in the glomerulus, although the level of expression in mouse kidneys is reduced as compared with renal tubules (see below). li et al. [22••] have systematically examined the relative distribution of ace2 in the rat kidney at the mrna level. in microdissected rat nephron segments, semiquantitative reverse transcriptase-polymerase chain reaction (rt-pcr) revealed that ace2 mrna was widely expressed, with relatively high levels in the proximal straight tubule. the high level of ace2 expression in the proximal tubule suggests that it may regulate the local levels of ang ii, and generate ang-(1-7). in rats, ace2 has been localized by immunohistochemistry to the proximal tubule [34] . interestingly, ace2 colocalizes with ang(1-7) , and the proximal tubular staining for ace2 and ang-(1-7) increases in normal pregnancy [34] . this correlated with increased urinary excretion of ang-(1-7); however, ace2 in urine was not measured [34] . lely et al. [35] reported that in humans ace2 is expressed in tubular and glomerular epithelium, as well as in vascular smooth muscle cells and the endothelium of interlobular arteries. in renal biopsies from patients with primary and secondary kidney diseases, these authors found neo-expression of ace2 in glomerular and peritubular capillary endothelium [35] . our laboratory recently showed that ace2 colocalizes with glomerular epithelial cell (podocyte) markers, and its presence in the podocyte/slit diaphragm complex was confirmed by immunogold labeling [15••] . the presence of ace2 was further demonstrated in immortalized podocytes grown in culture by western blot and immunofluorescence [36] . velez et al. [37] examined the processing of angiotensin substrates by cultured glomerular epithelial cells. they showed that podocytes express a functional intrinsic ras characterized by neprilysin, aminopeptidase a, ace2, and renin activities, which predominantly lead to ang-(1-7) and ang-(1-9) formation and ang ii degradation [37] . our laboratory has characterized the pattern of glomerular staining for ace2 and ace in control and diabetic mice [15••] . in diabetic kidneys, glomerular immunostaining for ace2 is attenuated in db/db mice of 8 weeks of age. in sharp contrast, ace expression in glomeruli from diabetic db/db mice is increased as compared with glomeruli from their respective age-matched nondiabetic controls [15••] . tikellis et al. [33] found the opposite pattern in rats made diabetic by stz. however, glomerular staining for ace2 in mice is weaker than in rats; thus, there may be a species difference. in humans, as in mice, glomerular staining is weaker than tubular staining [38] . what may be the significance of reduced ace2 and increased ace at the glomerular level? we reasoned that in diabetic mice such a combination could foster renal injury by resulting either in ang ii accumulation, decreased ang-(1-7) formation, or both [15••] . to test this hypothesis, we used a specific ace2 inhibitor, mln-4760, to reduce ace2 activity. the administration of mln-4760 for 16 weeks exacerbated albuminuria in the db/db mice [15••] . this was associated with increased glomerular expression of fibronectin. in a different model of diabetes (stz-treated mice), soler et al. [14••] found both glomerular mesangial expansion and increased albumin excretion after mln-4760 treatment. we further found that the glomerular expression of ace was increased in stz-treated mice and further increased after mln-4760 administration [14••] . as outlined in figure 1 , changes in ace and ace2 can work in concert to regulate the level of angiotensin peptides. the scheme outlined in figure 1 predicts that a pattern of low ace and high ace2 would lead to less ang ii and more ang-(1-7) formation locally. a combination of increased ace and decreased ace2 in the glomerulus is apt to favor increased glomerular ang ii accumulation and foster an increase in glomerular permeability manifested by albuminuria. this hypothesis does not necessarily conflict with prevailing views of enhanced formation of ang ii in the diabetic kidney. rather, both enhanced ang ii formation and decreased ang ii degradation may occur in glomeruli of diabetic mice, thereby contributing to produce more damage. studies in animals with ace2 genetic ablation have shown the development of renal lesions, particularly within the glomerulus, although glomerular injury was seen only in older animals. in male mice with genetic ace2 ablation, early accumulation of fibrillar collagen in glomerular mesangium was followed by the development of glomerulosclerosis by 12 months of age. female ace2 mutant (ace2-/-) mice were relatively protected. urinary albumin excretion was increased as compared with age-matched control mice [39••] . these structural and functional changes in the glomeruli of male ace2 mutant mice were prevented by treatment with the ang ii type 1 receptor antagonist irbesartan. the glomerular injury in male mice associated with the deletion of the ace2 gene were further accentuated by diabetes [40••] . in this more recent study, ace2 knockout mice were crossed with akita mice, a model of type 1 diabetes mellitus. diabetic ace2 knockout mice (ace2-/yins2 wt/c96y) exhibited a twofold increase in urinary albumin excretion compared with akita mice not depleted in the ace2 gene. increased mesangial matrix scores and glomerular basement membrane thicknesses in ace2-/yins2 wt/c96y mice were accompanied by increased fibronectin and -smooth muscle actin staining in the glomeruli. there were no differences in blood pressure or heart function to account for the exacerbation of kidney injury. although kidney levels of ang ii were not increased in the diabetic mice, treatment with an ang ii receptor blocker reduced urinary albumin excretion rate in ace2-/yins2 wt/c96y mice, suggesting that acceleration of glomerular injury in these mice is ang ii mediated [40••]. a seminal paper by crackower et al. [41] , primarily describing an ace2 knockout and its associated cardiac pathology, also reported that ace2 was reduced at the gene and protein level in kidneys from three separate rat models of spontaneous and diet-induced hypertension. in various recombinant rat models, several quantitative trace loci (qtl) for hypertension have been identified [41] . ace2 maps to the x chromosome in humans and a qtl has been mapped to the x chromosome in several rat models of hypertension [41] . the finding by crackower et al. [41] that the ace2 gene maps to a defined qtl on the x chromosome suggests ace2 as a candidate gene underlying the loci linked to hypertension. more recently, tikellis et al. [42•] showed that the developmental pattern of ace2 expression in the spontaneously hypertensive rat (shr) kidney was altered before the onset of hypertension. specifically, the expression and activity of ace2 were increased in tubules from the shr, before the onset of hypertension. with the increase in blood pressure at 6 weeks of age, the tubular expression of ace2 is reduced in shr compared to wistar-kyoto rats (wky). over the course of renal development, ace2 expression does not significantly change in the shr kidney, whereas ace2 expression increases in the wky kidney [42•] . this finding seems to be consistent with the concept that ace2 is downregulated in kidneys from hypertensive rats. in humans, few studies have been conducted to examine possible association of ace2 gene polymorphisms with hypertension. in three studies performed on chinese cohorts, an association was found between single-nucleotide polymorphisms in ace2 gene and blood pressure in women with the metabolic syndrome [43] and in women with essential hypertension [44, 45] . moreover, one of the ace2 alleles associated with high blood pressure seemed to confer a risk for reduced antihypertensive response to ace inhibitors as well [45] . in another study, no link was reported between ace2 gene polymorphisms and essential hypertension in australian individuals of white anglo-celtic origin [46] . collectively, the role of the ace2 gene in conferring the predisposition to hypertension is far from clear; whether the discrepancies in genetic linkage analyses could be related to differences in ethnic origin of the cohorts studied needs further investigation. polymorphisms in the ace2 gene were also sought in the general population regarding echocardiographically determined parameters of left ventricular mass, structure, or function [47] . this study provides some evidence that genetic variants in the ace2 gene may be associated with left ventricular mass and left ventricular hypertrophy in men, but no association with blood pressure was found in either men or women. in a study exploring human renal biopsy specimens, a significant correlation was found between the mrna levels of ace and ace2 in a variety of renal conditions [48] . the correlation was highly significant (p < 0.001) but relatively weak (r = 0.396). interestingly, the ace to ace2 ratio was significantly higher in subjects with hypertension than in subjects without hypertension. these findings are in keeping with the idea that ace2 might play a role in maintaining a balanced status of local renal ras by acting to counterregulate the actions of ace. evidence is also emerging that ace2 may be altered in pregnancy-related hypertension [49] . joyner et al. [49] examined the questions of whether ang-(1-7) and ace2 colocalize and whether they change in parallel during normal and hypertensive pregnancies. the authors found that during normal pregnancies, concurrent changes of ace2 and ang-(1-7) occur, suggesting that ace2 plays a role in regulating the renal levels of ang-(1-7) at mid-to late gestation [49] . in contrast, in hypertensive pregnant rats, the ace2 activity in cortex and medulla were unchanged, whereas ang-(1-7) levels were reduced. it was therefore concluded that the decrease in renal ang-(1-7) content in the absence of a concomitant decrease in ace2 implicates the participation of other ang-(1-7) forming or degrading enzymes during hypertensive pregnancy [49] . genetic ablation of ace2 may not result in spontaneous hypertension. two different ace2 knockouts have been described, and hypertension is not an overt feature of either phenotype [41,50••] . in a study by gurley et al. [50••] in mice on the c57bl/6 background, ace2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ace2 had no effect on baseline blood pressures in 129/svev mice. however, this does not mean that ace2 is not important in blood pressure regulation. after acute ang ii infusion, plasma concentrations of ang ii were almost threefold higher in ace2-deficient mice than in controls; moreover, blood pressures were substantially higher in the ace2-deficient mice than in wild-type mice [50••] . severe hypertension in ace2-deficient mice was associated with exaggerated accumulation of ang ii in the kidney, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [50••] . this study provided strong evidence that complete ace2 insufficiency can increase blood pressure under conditions of ang ii excess. based on this study, one cannot conclude whether relative ace2 deficiency or surplus affects blood pressure. to more directly assess the role of ace2 on blood pressure regulation, we recently used recombinant ace2 (race2) administered over a period of 3 days via osmotic minipumps, with or without ang ii infusion [51] . the increase in blood pressure produced by ang ii alone was prevented by the concomitant administration of ace2 [51] . moreover, plasma ang ii levels were markedly reduced by race2 administration, thereby demonstrating the important role of this enzyme in the degradation of ang ii [51] . this suggests that the administration of race2 may have a role in the treatment of ang ii-dependent hypertension, and opens the way for the development of therapies based on ace2 modulators capable of selectively increasing ace2 activity. ace2 is the only enzymatically active homologue of ace, and it plays a significant role in maintaining a balanced status of the ras. ace2 could act by either preventing ang ii accumulation or enhancing ang-(1-7) formation, or both. there is also increasing evidence that alterations in ace2 may be involved in disease states, such as experimental diabetic kidney disease and possibly hypertension. investigating the role of ace2 in those two prevalent diseases and whether its effects are mediated by ang ii or ang-(1-7) and other biologically active peptides, which are also substrates of ace2, opens the way for developing new therapeutic targets in hypertension. no potential conflict of interest relevant to this article was reported. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensin-coverting enzyme-related carboxypeptidase (ace2) converts antiogensin i to angiotensin 1-9 ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis angiotensinconverting enzyme-2 (ace2): comparative modeling of the active site, specificity requirements, and chloride dependence collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ace2 and is developmentally regulated in embryonic kidneys essential role for collectrin in renal amino acid transport revealed a novel function of the ace2 homologue, collectrin, as an important regulator of renal amino acid uptake and showed that its primary localization is the proximal tubule aminoaciduria and altered renal expression of luminal amino acid transporters in mice lacking novel gene collectrin the role for hnf-1beta-targeted collectrin in maintenance of primary cilia and cell polarity in collecting duct cells the ace2 homologue, collectrin, forms complexes with proteins related to vesicle transport and fusion, and regulates cell polarity. this study provided evidence that collectrin is decreased in polycystic kidneys and suggested its role in primary cilia formation in kidney epithelium identification and characterisation of the angiotensin converting enzyme-3 (ace3) gene: a novel mammalian homologue of ace angiotensin-converting enzyme ii in the heart and the kidney a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure increased ace2 and decreased ace protein in renal tubules from diabetic mice: a renoprotective combination? hypertension ace2 inhibition worsens glomerular injury in association with increased ace expression in streptozotocin-induced diabetic mice the first study to show accentuated histologic damage at the glomerular level after pharmacologic ace2 inhibition in stz model of diabetes. also, administration of ace2 inhibitor to diabetic mice was associated with increased glomerular ace expression, suggesting that both decreased ace2 activity and augmented ace expression may be responsible for exaggerated glomerular injury in mice receiving ace2 inhibitor glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensin-converting enzyme: implications for albuminuria in diabetes determined the localization of ace2 within mouse glomeruli, and specifically within podocytes. further showed that diabetic db/db mice have decreased glomerular expression of ace2. also, pharmacologic ace2 inhibition was associated with increased albuminuria, suggesting a role of glomerular ace2 in diabetic kidney injury ace and ace2 activity in diabetic mice describes the development of a microplate-based fluorometric method for the concurrent determination of ace and ace2 activity in mouse tissues, and shows that both ace2 protein and enzymatic activity are increased in two murine models of diabetes hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase angiotensin-(1-7) inhibits angiotensin ii-stimulated phosphorylation of map kinases in proximal tubular cells -7), which binds to the mas receptor, suggesting that ace2-mediated generation of ang-(1-7) may counterbalance the effects of locally formed ang ii. 19. ferrario cm new mass spectrometric assay for angiotensin-converting enzyme 2 activity describes ms-based assay to measure tissue ace2 activity angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ace2-dependent processing of angiotensin ii the role of angiotensin converting enzyme 2 in the generation of angiotensin 1-7 by rat proximal tubules this important study provides information on levels of ace2 expression throughout the rat nephron at the mrna level and demonstrates that ace2 in proximal straight tubules facilitates conversion of ang i to ang enalapril attenuates downregulation of angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 provides evidence that ace inhibition and at1 blockade result in increased cardiac ace2 expression. suggests that the antihypertensive action of at1 antagonists may be due to increased ang ii metabolism by ace2 myocardial infarction increases ace2 expression in rat and humans heart block, ventricular tachycardia, and sudden death in ace2 transgenic mice with downregulated connexins the novel angiotensin-converting enzyme (ace) homolog, ace2, is selectively expressed by adult leydig cells of the testis novel peptide inhibitors of angiotensin-converting enzyme 2 angiotensin-converting enzyme 2 (ace2) and ace activities display tissue-specific sensitivity to undernutrition-programmed hypertension in the adult rat circulating activities of angiotensin-converting enzyme, its homolog, angiotensinconverting enzyme 2, and neprilysin in a family study tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensinconverting enzyme-2 (ace2) this cell culture study provided new insight into the way ace2 secretion might occur in vivo angiotensin-converting enzyme 2 (ace2), but not ace, is preferentially localized to the apical surface of polarized kidney cells examined cellular localization and cell fate of ace2 in comparison with ace in nonpolarized chinese hamster ovary (cho) cells and polarized mdckii cells. the results suggest that ace and ace2 have a distinct secretion pattern from the cell surface of kidney epithelium and distinct cellular localization characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy enhanced renal immunocytochemical expression of ang-(1-7) and ace2 during pregnancy renal ace2 expression in human kidney disease? ace2 is critically important for angiotensin ii metabolism in podocytes characterization of renin-angiotensin system enzyme activities in cultured mouse podocytes the emerging role of ace2 in physiology and disease loss of angiotensin-converting enzyme-2 leads to the late development of angiotensin ii-dependent glomerulosclerosis examined effect of ace2 gene ablation on kidney function, histology, and alteration in signaling pathways in the mouse kidney. this important contribution demonstrated for the first time that lack of ace2 is associated with sex-dependent glomerular injury in aging animals loss of angiotensinconverting enzyme-2 (ace2) accelerates diabetic kidney injury the akita model of diabetes was used to determine the possible effect of ace2 gene ablation on development of diabetic nephropathy in mice. ace2 knockouts with diabetes had increased urinary albumin excretion and exaggerated glomerular pathology as compared with diabetic mice not depleted in ace2 gene. these alterations could be reversed by administration of ang ii receptor blockade. this was the first demonstration that genetic ace2 deficiency in diabetes is associated with ang iix-mediated acceleration of glomerular injury angiotensinconverting enzyme 2 is an essential regulator of heart function developmental expression of ace2 in the shr kidney: a role in hypertension? described the developmental pattern of ace2 expression in the kidney of hypertensive shr rats compared with normotensive wky rats. ace2 expression and activity are initially increased in the shr kidney at birth, but with the onset of hypertension, the tubular expression of ace2 falls in shr compared with wky association of angiotensinconverting enzyme 2 gene a/g polymorphism and elevated blood pressure in chinese patients with metabolic syndrome association study of angiotensin-converting enzyme 2 gene (ace2) polymorphisms and essential hypertension in northern han chinese polymorphisms of ace2 gene are associated with essential hypertension and antihypertensive effects of captopril in women no association of angiotensin-converting enzyme 2 gene (ace2) polymorphisms with essential hypertension association of angiotensinconverting enzyme 2 (ace2) gene polymorphisms with parameters of left ventricular hypertrophy in men. results of the monica augsburg echocardiographic substudy synergistic expression of angiotensin-converting enzyme (ace) and ace2 in human renal tissue and confounding effects of hypertension on the ace to ace2 ratio temporal-spatial expression of ang-(1-7) and angiotensin-converting enzyme 2 in the kidney of normal and hypertensive pregnant rats altered blood pressure responses and normal cardiac phenotype in ace2-null mice this outstanding study demonstrated that ace2 deficiency in mice challenged with exogenous ang ii is associated with impaired blood pressure regulation. it also suggested that impaired blood pressure response could be related to decreased ang ii disposal, as shown by elevated plasma and kidney levels of ang recombinant ace2 attenuates angiotensin ii induced hypertension key: cord-322966-o65fo853 authors: arnold, ruth h. title: covid-19 – does this disease kill due to imbalance of the renin angiotensin system (ras) caused by genetic and gender differences in the response to viral ace 2 attacks? date: 2020-05-25 journal: heart lung circ doi: 10.1016/j.hlc.2020.05.004 sha: doc_id: 322966 cord_uid: o65fo853 abstract debate continues in the medical literature on the role of the renin angiotensin system (ras) in coronavirus disease 2019 (covid-19) pathophysiology and the implications for the use of cardiovascular drugs acting on the ras. could these drugs – which include angiotensin converting enzyme inhibitors (aceis) and angiotensin receptors blockers (arbs) – be harmful or potential key therapeutic agents in covid-19? [abstract] debate continues in the medical literature on the role of the renin angiotensin system (ras) in coronavirus disease 2019 (covid19) pathophysiology and the implications for the use of cardiovascular drugs acting on the ras. could these drugs -which include angiotensin converting enzyme inhibitors (aceis) and angiotensin receptors blockers (arbs) -be harmful or potential key therapeutic agents in covid19? debate has arisen due to the finding that underlying cardiovascular disease and hypertension are associated with significantly increased risk of hospitalisation and death in covid-19 [1, 2] , in addition to the viral receptor being angiotensin-converting enzyme-2 (ace-2) [3 -5] . coronaviruses downregulate ace-2 anti-inflammatory actions leading to imbalance in ace:ace-2 [4] which may be fundamental in covid-19 pathophysiology. key gender and genetic differences in the regulation of ace:ace-2 balance [6] [7] [8] may explain differences observed in disease severity [2, 9, 10] . social media amplification of concern about continuation of ras medications during the current pandemic prompted cardiovascular societies to publish position statements strongly advising continued use, given a lack of evidence that ras drugs were unsafe and the substantial risk of hospitalisation when these drugs are withdrawn from patients with valid indications for treatment [11, 12] . three (3) large observational studies published in the new england journal of medicine (nejm] now support the view that there is no evidence for harm due to ras medications in covid-19 [10, 13, 14] . however, given the fundamental role of the (ras) in covid-19, a broader therapeutic indication for acei and arbs, over and above treatment of patients with established cardiovascular indications has been suggested [15, 16] but is yet to be tested in randomised trials. vitamin d status may also be linked to the ras impacts of covid-19, as vitamin d is a negative regulator of renin synthesis, and a small study has reported high rates of vitamin d deficiency in covid patients requiring intensive care [17] . both vitamin d and ace have a role in the immune system, which may add a complex dimension to the issue of ras drugs and vitamin d status in covid-19 [18, 19] . covid-19 has a more severe course in patients with pre-existing cardiovascular disease [1, 2, 10] and causes a significant rate of cardiovascular events [20] . further research is required to define the pathological mechanisms and the basis for the observed gender, age, and racial differences in severity. ras medications can be used with greater confidence in covid-19 patients with valid indications for their use, but a broader role for manipulation of the ras in covid-19 will require randomised trial data. coronavirus-2 (sars-cov-19) is believed to gain cell entry via viral spike protein binding to the ace-2 receptor [3, 4] . virology research for other similar coronaviruses indicates that viral cell infection is followed by rapid downregulation of ace-2 [4, 5] . the ace-2 system is the yang of the yin-yang balance of the ras hypertension system. ace-2 is a zinc metalloprotease, discovered in 2000 [21] , which mainly breaks down angiotensin ii (ang ii), the main effector of the ras system, to ang(1-7), resulting in a decrease in blood pressure, vasoconstriction and decreased inflammation. ace-2 is usually membrane bound but is shed in covid infection, in acute lung injury and myocardial infarction [16] . whilst the role of circulating ace-2 is unclear, conditions such as advanced heart failure have higher plasma ace-2 proportional to worsening clinical status [22, 23] . serum ace-2 levels are also significantly higher in hypertensive patients [23] and gender differences are described with up to 50% greater levels in male patients [23] . there is debate as to whether the observed higher levels in male, hypertensive, and diabetic patients are related to increased ace-2 expression as a compensatory mechanism, or whether it is due to increased ace-2 shedding, limiting tissue ace-2 activity and causing excess tissue ang ii. the role of circulating, versus tissue, ace-2 levels in susceptibility to covid infection is unclear [24] . in viral downregulation of ace-2, unopposed ace generates high ang ii causing increased bp, vasoconstriction, inflammation and cell damage. to exacerbate matters, the active metabolite generated by ace-2, ang(1-7), can be broken down by ace itself, accentuating the ras imbalance caused by covid-19 [21] (figure 1 ). covid-19 is highly contagious spreading from one province, in december 2019, to the whole of china in 30 days, despite extreme shutdown measures [2] . by may 2020, the disease has infected 3,392,718 people with 239,178 deaths worldwide [9] and is causing enormous global economic and social impacts. epidemiology for 72,314 confirmed, suspected and asymptomatic cases in china suggested mild disease in 81%, with 2.3% fatality overall [2] . early mortality figures in different countries have shown striking differences, with greater mortality in europe, compared to asian countries [9] , which may relate to fundamental differences in racial ace and ace-2 polymorphisms, impacting both susceptibility to the disease and subsequent pathological severity. population demographics clearly play a role with elderly patients at greatest risk of severe disease and death. key differences in the speed of government actions to implement extensive public health measures, as recommended by the world health organization (who), have also impacted mortality rates as hospitals in some countries have been overwhelmed by a rapid rise in cases. why do 81% of cases have a mild disease and yet 19% have a more severe disease? the chinese center for disease control and prevention (ccdc) epidemiology showed that there was an overall mortality of 2.3% [2] . the death rate increased with age and with co-morbidities, particularly cardiovascular disease (13.2%), diabetes (9.2%) and hypertension (8.4%), all the risk factors typically seen in those with "metabolic syndrome". whilst it is clear that co-morbidities and age increase mortality, what is not clear is whether this excess is due to the impact of the co-morbid conditions on host inflammatory response alone, or to the impact of a treatment some of this patient group is taking [25] . since the ras system is fundamentally involved in covid-19 pathogenesis, there was great speculation that drugs widely used in hypertension and heart failure treatment, acting on the ras, may impact disease severity, with some suggesting a protective effect [15, 16] and others concerned about potential harm [24] . these drugs include arbs, aceis and the combination drug valsartan/sacubitril (entresto) which combines an arb with a neprilysin inhibitor (also known as an angiotensin receptor-neprilysin inhibitor [arni]). whilst there is evidence that acei and arb treatment may increase expression of ace-2 in some tissues in animal models, data supporting or quantifying this effect in human subjects at clinical doses of ras medications is lacking [24] . hence the relevance of possible medication-induced alteration in ace-2 levels, over and above the welldescribed significant differences in circulating levels of ace-2 in advanced heart failure, hypertension, males and diabetics, and the implications for covid-19 severity, is unclear [22, 23, 25] . increased ace-2 was speculated to increase susceptibility to covid-19 by allowing more virus into cells which, in turn, may exacerbate disease severity. the counter argument is that, once infected, having more ace-2 could be protective against viral attack and downregulation of the ace-2 antiinflammatory system [26, 27] . three (3) large observational studies reported in the nejm have now examined this issue and found no evidence for increased risk of adverse outcomes in covid-19 patients taking acei or arbs [10, 13, 14] adding weight to previously reported smaller retrospective clinical studies [28] [29] [30] which also showed no excess mortality for acei/arbs. mehra et al. [10] reported data for 8,910 patients admitted with covid-19 in 169 hospitals in europe, asia and north america, and confirmed that age, coronary disease, heart failure, cardiac arrhythmia, chronic obstructive pulmonary disease and current smoking were all associated with increased in-hospital mortality but there was no increased risk of mortality associated with risk of acei or arbs. they did find that use of acei and statins was more common in survivors, however this was not randomised data. a multicentre chinese study of 1,128 hospitalised patients with covid-19 and pre-existing hypertension showed a death rate of 3.7% in those on acei or arb versus 9.8% in non-users (p=0.03). the data was observational and not randomised, but even after adjustment for confounding variables a lower mortality, with a hazard ratio of 0.37 (p=0.03) persisted for those receiving acei/arbs, with a hr of 0.3 (p=0.03) compared to use of other antihypertensive agents [28] . the study did not separate treatment with arbs from acei [29] . the answer to observed differences in covid-19 death rates could lie in key differences in the ace:ace-2 system balance related to age, gender and racial variation in genetic ace and ace-2 polymorphisms and environmental factors influencing ace-2 expression [6, 31) . there are ace gene polymorphisms that cause insertion (i) or deletion (d) of a sequence of the gene, and ace activity levels in dd carriers are approximately twice that found in ii genotypes [7] . the possible impact of ace genotype in the covid-19 disease state, when the ability to breakdown ang ii is impaired, could be hypothesised to cause a greater quantity of circulating ang ii and a more marked imbalance of the ras and more severe disease. genetic and environmental factors influencing ace-2 expression could also result in differences in the risk of becoming infected in the first place by affecting the affinity of the ace-2 binding domain for the viral spike protein and by causing differences in the density of ace-2 receptors present in tissues such as the lungs. ace-2 polymorphisms may then contribute to significant differences in disease severity by determining the extent of the ace:ace2 imbalance, particularly in the endothelium of the lungs, where the virus attacks at the same time as disabling the ace2-mediated repair systems. chen and co-workers (2020) [8] have analysed 30 tissues in thousands of individuals and found significantly higher tissue ace-2 in asian females compared to males and other ethnic groups. they have described an age-dependent decrease in ace-2 expression and a highly significant decrease in type ii diabetic patients. the loci for higher ace-2 expression is almost 100% in east asians and >30% higher than other ethnic groups. ace-2 is suppressed by inflammatory cytokines, by diabetes and induced by oestrogen and androgen, both of which are decreased in the elderly. the work by chen and co-workers suggests a negative correlation between covid-19 mortality and tissue ace-2 levels, at both a population and molecular level [8] . circulating ace-2, however, has been described to increase in pathological states such as hypertension, diabetes and heart failure and perhaps differences in circulating, shed ace-2 and tissue-bound ace-2 are key in virus susceptibility and subsequent disease severity. gender differences in ace-2 ace-2 is on the x chromosome, hence men have only one allele and women have two [6, 31] . further to this, oestrogen is believed to upregulate ace-2 expression [32] , giving pre-menopausal women the advantage of two alleles and oestrogen upregulation, making a deficiency of ace-2 and its ability to rally less likely in the event of a viral attack. in support of this hypothesis, the chinese cdcc reported overall female mortality of 1.7% including suspected cases as well as serologically confirmed cases versus 2.8% in males. this difference was more marked in serologically confirmed cases, 2.8% female versus 4.7% male mortality. the overall ratio was 1.06 males to 1.0 females believed infected [2] . hence the men were not significantly less likely to become infected, just more likely to have a more severe pathophysiological expression of the disease. whilst the who suggested [9] that these gender differences may be driven by substantially greater rates of smoking in the chinese male population, the rates of smoking in the covid-19 cases were not provided in the epidemiological study. the x-linked nature of the ace2 gene, with females having a greater range of phenotypes, may have a greater role than has been yet been fully defined in coronavirus pathogenesis gender differences. the range of ace-2 phenotypes and their ability to upregulate compensatory mechanisms in the face of viral ace-2 downregulation may play a role. marked gender differences in disease have been confirmed in other large observational studies with marcia et al reporting 37% women in 6,232 patients with covid and mehra et al. reported 40% female patients in 8,910 covid-19 patients requiring hospital admission and furthermore reported improved survival in female patients, independent of older age [10] . serum ace-2 levels are reported to be significantly lower in females [23] and there are fundamental differences in immune responses to viral infections based on gender with stronger humoral and cellular immune responses described in females [33] . smoking has been linked to upregulation of ace and downregulation of ace-2 [34] , which could be hypothesised to make smokers less likely than the general population to become infected by virtue of having less ace-2 required for viral entry. however, if a smoker becomes infected, a pre-existing ace:ace-2 imbalance may result in more severe disease due to greater inflammation, higher risk of ards and multi-factorial issues including poor baseline lung function and bacterial superinfection. evidence supporting this picture is seen in data from wuhan. although smokers are said to comprise 52.1% of the male population in china [9] , the rate of current smokers reported in covid-19 series seems well below this; 7% by huang et al. [ due to the extreme complexity of the ras system, another component may be relevant in causing greater mortality in smokers with covid-19. neprilysin is a metalloprotease that has a number of functions but can act to breakdown ang ii to ang(1-7) 4 , the role usually performed by ace-2. it is highly expressed in airways, pulmonary interstitium and alveolar cells. a study examining circulating neprilysin levels in 1,536 community-based subjects >45 years, found that the lowest tertile group had the highest rates of smokers (p<0.001) and higher rates of hypertension (p=0.04) [38] . if smokers have low lung neprilysin activity, they may lack an important safety mechanism that could overcome virally mediated loss of the ace-2. this might be relevant in patients taking entresto (sacubitril/valsartan), a medication with proven benefit in the treatment of patients with heart failure. neprilysin inhibition by the sacubitril component of entresto may block the alternate method of generating the anti-inflammatory mediator (1-7) when ace-2, under viral attack, is less able to do so. clearly this is a complex question, impossible to answer without more data and even recently reported observational studies did not have sufficient patients taking entresto to report on the safety of this drug in covid-19 [10, 14, 22] . reports from china showed that patients with severe disease, leading to intensive care unit (icu) admission and death, had features consistent with unopposed ras activity, such as higher blood pressure (bp) on admission to hospital and higher bp on admission to icu in non-survivors [1, 35] , with a mean systolic bp of 145 mmhg in those requiring icu admission versus 122 mmhg in milder cases (p=0.018) [35] . lower serum potassium levels, combined with high urinary potassium has also been reported in more severe patients [39, 40] , suggesting ang ii-mediated activation of aldosterone. the presence of significant hypertension, instead of hypotension, in a group of patients admitted to icu versus those not requiring icu, is a particularly interesting clinical finding with implications for the pathogenesis of covid-19. excess ras activity without ace-2 balance to break down ang ii and block adverse effects, such as cellular damage and fibrosis, may play a key role in determining disease severity, similar to the ras pro-inflammatory effects well known to contribute to heart failure ( figure 2 ). autopsy data published for four covid-19 patients [41] has described platelet rich clot formation occluding small vessels and a microangiopathic picture, consistent with virally mediated vascular damage. the ace:ace-2 imbalance caused by viral ace-2 downregulation understanding the ace:ace-2 imbalance in covid-19, its role in age, gender and racial differences in disease severity, and the difference between tissue bound and circulating ace-2, could all prove key in treating this disease. in support of the fundamental ace:ace-2 imbalance having clinical relevance, it has been reported that serum ang ii levels are indeed significantly elevated in covid-19 and correlate with both viral load and lung injury [42, 43] . whilst ace formation of ang ii can be blocked by ace inhibitors, up to 40% of ang ii is formed via non-ace pathways such as chymase [4] , making ace inhibitors potentially less effective than arb blockers in diminishing the problem of the excess ang ii, since the acei may only block 60% formation of the ang ii. on the other hand, the arbs are highly selective blockers of the main receptor for the ang ii, the angiotensin receptor type 1 (at 1 ) receptor. arbs do not decrease the amount of ang ii produced in the first place, but by blocking the at 1 receptors they activate a key counter regulatory anti-inflammatory cascade to restore the ace:ace-2 balance. the excessive circulating ang ii, unable to bind blocked at 1 receptors, binds at2 receptors which trigger counter-regulatory anti-inflammatory actions and upregulates ace-2 expression [15] to remove excessive circulating ang ii. a protective role of increased tissue ace-2 expression seems plausible since groups with higher levels such as younger people, premenopausal women and possibly asian populations [8] do seem to have lower disease severity and mortality [2, 9] compared to european populations with an older population and high rates of co-morbidities such as diabetes, hypertension and cardiovascular disease, all of which have been described as having higher circulating ace-2 levels, due to increased ace-2 shedding. it has been pointed out that in another rna virus, human immunodeficiency virus (hiv), higher expression of binding sites such as cd4 actually protected from, rather than increased, virulence [15] . males have only one x-linked ace-2 allele and could be more vulnerable to phenotypes causing impaired ace-2 regulation. patients with pre-existing hypertension may have a greater frequency of the ace polymorphisms that result in excessive ang ii levels, exacerbated by viral impairment of ang ii clearance. covid-19 is a disease where a major battlefront is at the endothelium, fought by the tissue ras. there have already been cases reported in the media of families tragically affected by very high rates of severe disease and mortality, in line with genetic factors playing a significant role in covid-19 pathophysiology [44] . coronaviruses directly impact cellular systems, other than the ras, which are important in inflammatory pathways. rna viruses remodel host membranes and lipid metabolism creating a suitable environment for their replication [45] . lipid metabolism pathways are impacted in corona virus infection, with glycerophospholipids and fatty acids significantly elevated in sars cov-229e infected cells. up-regulated phosphatidylinositol (pi) may promote coronavirus entry [46] . covid-19 is associated with cardiac events and significant serum troponin level elevation in a clinical setting [36] and elevated lysophospholipids, including lp1, have been reported in acute coronary syndromes from other causes, undergoing coronary angiography [47] . hcov-229e infection up-regulates fatty acids, believed to promote efficient coronavirus replication. however, yan and co-workers [46] reported that exogenous addition of fatty acids may interfere with viral replication by upsetting the delicate balance of fatty acids, causing reversion of lysophospholipids into phospholipids, limiting viral replication. it is possible that the manipulation of fatty acids in a clinical setting may upset the balance needed for viral replication. use of drugs such as statins may also impact viral ability to harness cellular lipid pathways. clearly more data is needed to investigate the role of fatty acid and cholesterol manipulation. the finding that statins decrease in-hospital mortality, even in a non-randomised study [10] , is of interest. long term sequelae of sars, caused by a related coronavirus (sars co-v), have been reported in follow-up studies in survivors out to 15 years. these include sleep disturbance, characterised by increased stage 2 sleep percentage, diffuse myalgia, chronic fatigue, depression, lung damage and avascular femoral head necrosis [48] [49] [50] . deranged lipid metabolism has also been reported in sars survivors. lipid metabolites, including phosphatidylinositol (pi) and lysophosphatidylinositol (lpi), are associated with cellular entry and/or egress of respiratory viruses. lpis and pis were markedly upregulated in recovered sars patients. lpis are thought to have a critical role in glucose homeostasis. a large proportion of the recovered sars patients reported glucose metabolic disorders, including hyperinsulinaemia, insulin resistance, hyperglycaemia, and type 1 or 2 diabetes [49] . patients who died of sars were found to have extensive lung damage as well as features of systemic vasculitis. autoantibodies against human epithelial and endothelial cells can develop after sars-cov infection and this could explain the severe deterioration in some patients in phase ii of the disease and may lead to post-infectious cellular injury and sars-induced immunopathology in survivors [51] . this long-term immunopathology could also be a risk in covid-19, a related virus which also acts to inflame the endothelium and, due to gender differences in immune function, female survivors may be at greater risk to autoimmune mediated sequelae [33] . immune system role of vitamin d and ace vitamin d deficiency can be related to immune system dysfunction [18] and inflammation, and can cause a pro-thrombotic state; it is common in europe in winter. vitamin d is a potent negative endocrine regulator of the ras and works predominantly by suppressing renin synthesis [52] . vitamin d deficiency and genetic and geographical variation in its function may be another factor contributing to greater imbalance of the ace:ace-2 system leading to differences in disease severity in covid19 . it has been suggested that vitamin d supplementation could reduce the risk of covid-19 infection and death [53] . lau et al. [17] reported a small study of vitamin d levels in 20 patients which is yet to be peer reviewed. they described vitamin d deficiency in 84.6% of covid-19 patients in icu and 57.1% of patients admitted to a ward bed. they found that 100% of patients under age 75 admitted to icu had vitamin d deficiency (n=11). ace is best known for its role in blood pressure regulation by converting angiotensin i to ang ii, but in fact it cleaves many peptides and has a role in innate and adaptive immune responses, with ace expressed on both neutrophils and macrophages. the role of ace in immune responses and inflammation is complex and includes defence against intracellular pathogens, mediated via actions that are not dependent on ang ii, as reviewed by bernstein and co-workers [18] . these complex interactions mean that acei and arbs may not have the same impacts in covid-19, a disease impacting both haemodynamic and inflammatory pathways. many agents are under investigation or in clinical trial for the treatment of covid-19 but so far no anti-viral drugs or vaccine has been officially approved for covid-19 treatment. hundreds of trials are listed in the who covid-19 trial data base (www.who.int international clinical trials registry). a small trial using hydroxychloroquine and azithromycin showed some promise in decreasing viral carriage to only 3 to 6 days [54] , an improvement on 20 days reported from china [1] . this combination could, however, carry a risk of arrhythmia due to long qt, in a group of patients where abnormal troponins and endothelial dysfunction can be present, with some arrythmias reported in china [1] . longer trials will be needed. preliminary clinical data for the investigational rna antiviral agent, remdesivir, did not show a survival benefit in a study of 237 patients in china but did show a trend to earlier recovery [55] , and the adaptive covid-19 treatment trial (actt) a study of 1,063 patients with lung involvement in the usa has not yet been peer reviewed but a press release from the national institutes of health (nih) [56] reports a 31% faster recovery time (p<0.001) of 11 versus 15 days and possibly a trend to a survival benefit with 8% versus 11.6% mortality (p=0.059). a variety of therapeutic approaches, in addition to a vaccine, may contribute to covid-19 treatment and prevention in the medium and long term. however, in the current more urgent situation there are 2,075,668 active cases and 51,332 (2%) are serious or critical, with many countries still in the exponential rise phase of new cases. treatment that can be distributed in large quantities is needed if the economic impact of this pandemic is to be addressed. a meta-analysis of cardiac troponin results in covid patients reported that higher levels correlated with more severe disease [20] . this is consistent with either myocarditis or an acute coronary syndrome, conditions for which manipulation of the ras with commonly available drugs, already have an indication, based on treating endothelial and myocardial inflammation. use of these drugs in covid-19 patients meeting the definition for an acute coronary syndrome or for another valid cardiovascular indication can now be given with more confidence in light of recent studies showing no detriment for these drugs in covid-19 [10, 14, 22] .this approach may be one that is directed at fundamental viral vascular pathogenesis. a broader therapeutic role for ras inhibition in covid-19 is being investigated in two randomised trials examining the effects of the arb losartan on the severity and prognosis in covid-19, in hospitalised covid-19 patients (nct 04312009) and in non-hospitalised patients (nct04311177). in the current global covid-19 pandemic, many countries are in lockdown with social and economic paralysis. the impact of covid-19 has been described as a warzone with many patients dying a lonely death, isolated from loved ones in overcrowded and hastily expanded hospitals. currently, only supportive care is available. whilst many therapeutic agents are under investigation [57, 58] , none have yet been approved for use in covid-19, nor demonstrated a significant survival benefit. even if some agents are proven to be effective, it will take time for trials and for production on a large scale. thinking of covid-19 as a fundamentally cardiovascular disease that attacks the endothelium could provide momentum to use safe, established and widely available medications acting on the ras, where they are already indicated for acute coronary syndrome and cardiac dysfunction, present in many patients with covid-19. whilst more data on the role of vitamin d in covid pathogenesis is needed, replacing an essential vitamin when it is deficient, seems a safe, cost effective and easily achieved therapeutic strategy. any successful shift to milder disease severity will ease the burden on health care systems. switching on endogenous anti-inflammatory pathways may also play a key role in decreasing the risk of longterm vascular and autoimmune sequelae in covid-19 survivors, which could pose a further economic burden once the pandemic is over. although there may be more questions than answers and hot debate in the medical literature about the role of the ras and drugs acting on this system in covid-19, the consensus opinion is they are safe to use in covid-19 [11, 12] , a view now supported by retrospective clinical data [110, 14, 22, [28] [29] [30] . in managing a global pandemic with a high death rate, treatments aimed at the fundamental pathology, that can meet the scale of the problem, are an appealing option. data from randomised clinical trials examining a broader role for ras agents in covid-19, beyond existing indications, is awaited and will further define the role of these medications. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study novel coronavirus pneumonia emergency response 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family host lipids in positivestrand rna virus genome replication characterization of lipidomic profile of human coronavirus-infected cells: implications for lipid metabolism remodelling upon coronavirus replication lysophosphatidylinositol signalling and metabolic diseases chronic widespread musculoskeletal pain, fatigue, depression and disordered sleep in chronic post-sars syndrome; a case-controlled study altered lipid metabolism in recovered sars patients twelve years after infection long term bone and lung consequences associated with hospital -acquired severe acute respiratory syndrome: a 15 year follow-up from a prospective cohort study autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (sars)-associated coronavirus infection are vitamin d receptor agonists like angiotensin-converting enzyme inhibitors without side effects? evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths hydroxychloroquine and azithromycin as a treatment of covid-19; results of an open label non-randomised trial remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial nih clinical trial shows remdesivir accelerates recovery from advanced covid-19 updated approaches against sars-cov-2 world health organization. overview of the types/classes of candidate key: cord-298515-5n7hxhbg authors: oarhe, chinyere i.; dang, vinh; dang, mytrang; nguyen, hang; gopallawa, indiwari; gewolb, ira h.; uhal, bruce d. title: hyperoxia downregulates angiotensin-converting enzyme-2 in human fetal lung fibroblasts date: 2015-02-09 journal: pediatr res doi: 10.1038/pr.2015.27 sha: doc_id: 298515 cord_uid: 5n7hxhbg background: angiotensin (ang) ii is involved in experimental hyperoxia-induced lung fibrosis. angiotensin-converting enzyme-2 (ace-2) degrades ang ii and is thus protective, but is downregulated in adult human and experimental lung fibrosis. hyperoxia is a known cause of chronic fibrotic lung disease in neonates, but the role of ace-2 in neonatal lung fibrosis is unknown. we hypothesized that ace-2 in human fetal lung cells might be downregulated by hyperoxic gas. methods: fetal human lung fibroblast imr90 cells were exposed to hyperoxic (95% o(2)/5% co(2)) or normoxic (21% o(2)/5% co(2)) gas in vitro. cells and culture media were recovered separately for assays of ace-2 enzymatic activity, mrna, and immunoreactive protein. results: hyperoxia decreased ace-2 immunoreactive protein and enzyme activity in imr90 cells (both p < 0.01), but did not change ace-2 mrna. ace-2 protein was increased in the cell supernatant, suggesting protease-mediated ectodomain shedding. tapi-2, an inhibitor of tnf-α−converting enzyme (tace/adam17), prevented both the decrease in cellular ace-2 and the increase in soluble ace-2 (both p < 0.05). conclusion: these data show that ace-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas. they also suggest that hyperoxia decreases ace-2 through a shedding mechanism mediated by adam17/tace. supplementary information: the online version of this article (doi:10.1038/pr.2015.27) contains supplementary material, which is available to authorized users. s upplemental oxygen, which is frequently used in the treatment of pulmonary insufficiency in premature infants, has been implicated in the development of bronchopulmonary dysplasia (bpd). bpd remains a major cause of morbidity and mortality during the first year of life, and some of these infants have significant respiratory problems throughout childhood, including increased airway reactivity and the development of restrictive airway disease (1) . hyperoxia induces lung injury and may contribute to bpd. a comprehensive review of the literature reveals a strong association between exposure to hyperoxia and subsequent expression of comorbidities. hyperoxia can directly cause lung injury by generation of reactive oxygen radicals. prolonged exposure of neonatal mice to hyperoxia results in impaired alveolarization and capillary development and in increased lung fibrosis that is similar to human bpd (2) . among the mechanisms believed to contribute to the pathogenesis of lung fibrosis, ace-2 is of great importance, since the renin-angiotensin system (ras) is expressed by and is functional in human lung myofibroblasts, the cells believed to be most important in synthesizing the collagens that accumulate in fibrotic lungs (3) . in studies of tissue fibrogenesis, the tissue ras is believed to play a key role in initiation and progression of fibrous tissue accumulation in a variety of organs. both ace inhibitors and angiotensin receptor antagonists have been shown to prevent fibrogenesis of the heart (4), liver (5) , kidney (6) , and lungs (7, 8) . ang ii, produced by ace, is the main effector molecule in the ras. ang ii is a potential pulmonary profibrotic mediator which activates ang ii type i receptor and stimulates collagen synthesis. studies done in vivo showed that the angiotensin system was upregulated in oxygen-exposed fetal lung fibroblasts (9) . the main function of ace-2 is to convert the octapeptide angiotensin ii to a heptapeptide ang 1-7. this limits the accumulation of ang ii and protects against experimental lung fibrosis. imai et al (10) . demonstrated in three different models of acute respiratory distress syndrome (ards) which all used ace 2 knock-out mice, that these mice showed very severe disease compared with wild-type mice. previous work from this laboratory demonstrated that ace-2 protects against fibrosis, but is downregulated in both human and experimental lung fibrosis models (11) , and that inhibition of ace-2 is associated with increased lung tissue injury/fibrosis. earlier, uhal et al (3) . demonstrated the existence of an angiotensin/ tgf-β1 "autocrine loop" in human lung myofibroblasts. more important in the context of the neonatal lung, jiang et al (12) . showed the activation of the angiotensin system in hyperoxiainduced lung fibrosis in neonatal rats. chou et al (13) . showed that blocking ang ii activity through the at1r (angiotensin type i receptor) blocker losartan prevented lung fibrosis in response to hyperoxia. the role of ace-2 in these models, however, has not yet been evaluated. the function of ace-2 as the cellular receptor for sars-coronavirus was demonstrated by li et al (14) .. haga et al (15) . articles demonstrated the role of tnf-α-converting enzyme (tace), which can cleave the ectodomain of ace-2, in viral entry into the lungs. these authors also showed that tace-dependent "shedding" of ace-2 ectodomain is completely dependent on the binding of sars-s protein to ace-2 and that tace was required for viral entry (16) . further, the shedding was blocked by tace antagonists, tapi-0, and tapi-2 (15) . tace (also known as adam17), originally identified as a metalloprotease required for processing the membrane form of tnfα and its release as a soluble factor (17) , is now known to be involved in the cleavage and release of the ace-2 ectodomain in several tissues (18) . given the known involvement of other ras components in lung fibroblast function (3) and the profibrotic action of hyperoxic gas, we hypothesized that ace-2 might be expressed by fetal lung fibroblasts and further, might be downregulated by hyperoxia. we further theorized that the downregulation of ace-2 by hyperoxia might be prevented by inhibiting its shedding by an inhibitor of tace, the enzyme that causes shedding. we report here the findings that ace-2 protein and enzyme activity are severely decreased by hyperoxia in human fetal lung fibroblast cultures. we also report that tapi-2 prevents this decrease. table 1 shows the number of cells with disrupted plasma membrane integrity, as measured by trypan blue and expressed as a percentage of total cells. cell cultures exposed to hyperoxia showed a modest (less than 1%) but statistically significant increase in dead cells compared to cells exposed to room air, if the exposure was followed by a 24-h period of recovery (p < 0.01). figure 1 shows western blotting for caspase 9 as a marker of apoptosis which, when normalized to β-actin, showed no significant changes by densitometry. together, these data suggest a very slight decrease in the viability of hyperoxia-treated cells following a recovery period, but only in a very minor fraction (<1%) of the cells. figure 2 shows the effect of hyperoxia on ace-2 immunoreactive protein on western blotting. both ace-2 immunoreactivity and enzyme activity were significantly decreased in cells treated with hyperoxia when compared with cells treated with normoxia. this reduction (74.6%, p < 0.01 by densitometry) only occurred when cells were allowed to recover by incubating in normoxic serum free media for 24 h. in figure 3 , the enzymatic activity measured by the ace-2 synthetic substrate was completely inhibited by the synthetic peptide dx 600 (p < 0.01), a competitive inhibitor of ace-2 (11) . figure 3b showed no significant reduction of ace-2 in fetal lung fibroblasts exposed to hyperoxic or normoxic gas. imr 90 cells in confluent culture were exposed for 72 h to 95% oxygen with 5% co 2 . control cells were incubated in 21% o 2 with 5% co 2 for 72 h. at the end of 72 h, cells were either harvested or incubated for an additional "recovery" phase in serum free media for 24 h. western blotting for caspase 9 was then performed as described in materials and methods. despite the slight decrease in band intensity in hyperoxia-treated cells, quantitation by densitometry demonstrated no statistically significant difference in the caspase 9/β-actin ratio (not shown). hyperoxia with normoxic recovery downregulates angiotensinconverting enzyme-2 (ace-2) in human fetal lung fibroblasts. imr90 cells were exposed to hyperoxic or normoxic gas as described in figure 1 . (a) western blotting for ace-2 protein was then performed and normalized to β-actin. (b) there was no change in ace-2 without recovery but with recovery hyperoxia caused a significant decrease in ace-2 immunoreactive protein (74.6%, *p < 0.01 vs. 21% o 2 by student's t-test). figure 4 shows that the addition of tapi-2 during exposure to hyperoxia prevented the reduction of immunoreactive ace-2 that otherwise would occur in hyperoxia-treated cells (p < 0.05) next, ace-2 immunoreactivity was analyzed in the culture media collected from hyperoxia-exposed cells. figure 5 shows that treatment of fetal lung fibroblasts with hyperoxia increased the release of soluble ace-2 into the culture supernatant (p < 0.01) and that tapi-2 prevented the increase (p < 0.01). figure 6 shows that the treatment of hyperoxia followed by normoxic recovery significantly increased adam17/tace immunoreactive protein by threefold in the imr90 cells (p < 0.05). the hyperoxic gas alone (without recovery) did not increase adam17/tace (data not shown). as expected, the inclusion of the tace inhibitor tapi-2 (an active site blocker) had no ability to reduce the increase in tace protein induced by hyperoxia with normoxic recovery (data not shown). figure 6 , panel c shows that hyperoxia with normoxic recovery also caused a significant increase in adam17/tace mrna (2.2-fold, *p < 0.05). methods of preventing and/or reducing the effect of oxygen injury remain elusive. identification of molecular mechanisms of oxygen injury and endogenous protective factors . hyperoxia with normoxic recovery reduces angiotensin-converting enzyme-2 (ace-2) enzymatic activity but not ace-2 mrna. imr90 cells were exposed to hyperoxic or normoxic gas as described in figure 1 (with recovery only), but cells were harvested for assay of ace-2 enzyme activity (11) with and without inclusion of the ace-2 competitive inhibitor dx600 at 1 µmol/l in the enzyme assay vessel. (a) ace-2 enzymatic activity showed a statistically significant reduction by hyperoxia (with recovery) and complete blockage by dx 600 (*p < 0.01 vs. 21% o 2 by anova and dunnett's test). the (b) ace-2 mrna quantification by qrtpcr showed no significant change. see methods for details. . hyperoxia with normoxic recovery reduces cellular angiotensinconverting enzyme-2 (ace-2) by a tapi-2-sensitive mechanism. imr90 cells were exposed to hyperoxic or normoxic gas as described in figure 1 (with recovery), but in the presence or absence of the adam17/tace inhibitor tapi-2. cell monolayers were then harvested for ace-2 western blotting (upper panel). densitometry (lower panel) demonstrated a significant reduction in cell monolayer-associated ace-2 in hyperoxia treated cells compared to the control (*p < 0.05 vs. ctl by anova and tukey's multiple comparison test). addition of tapi-2 prevented the decrease. figure 5 . hyperoxia with normoxic recovery increases soluble angiotensin-converting enzyme-2 (ace-2) by a tapi-2-sensitive mechanism. imr90 cells were exposed to hyperoxic or normoxic gas as described in figure 1 (with recovery), but in the presence or absence of the adam17/ tace inhibitor tapi-2. cell-free culture supernatants were then harvested separately from the cell monolayer and were concentrated and subjected to western blotting for ace-2 (upper panel articles might provide much needed targets for future design of interventions. lang et al (9) . recently showed that angiotensin ii (angii) mediates the profibrotic effect of hyperoxia on cultured human lung fibroblasts, but these authors did not study the potential contributions of ace-2, which degrades angii, to the angiotensin-induced increase in collagen production stimulated by hyperoxia. moreover, previous work showed that ace-2, which protects against lung fibrosis in animal models by degrading angii, is downregulated in the lungs of patients with idiopathic pulmonary fibrosis through mechanisms yet to be fully identified (11) . together, these issues led to our hypotheses that: (i) hyperoxia might downregulate ace-2 and (ii) modulation of ace-2 might be useful in the management and prevention of hyperoxia-induced lung injury. for example, maintaining high levels of tissue ace-2 by prevention of its shedding, and/or replacement of the protein through administration of biologically active enzyme, may hold potential as therapeutic strategies for treating hyperoxic lung injury. in support of this theory, a pharmaceutical preparation of recombinant human ace-2 (rhace-2) is currently in clinical trials for acute lung injury (glaxosmithkline, pipeline drug gsk2586881). ace-2, a carboxypeptidase which degrades angiotensin ii, was shown to be protective but downregulated in human interstitial pulmonary fibrosis and in experimentally-induced lung fibrosis by li et al (11) .. a clear role for ace-2 in modulating acute lung injury was shown by the demonstrations of imai et al (10) . who showed that the absence of ace-2 in ace-2 knockout mice exacerbates experimental acute lung injury. to date, the potential role of ace-2 in hyperoxic lung injury has not been explored. in recent years, clinicians are seeing less of the classical "old bpd", which is characterized by pulmonary fibrosis alternating with cystic emphysema on chest x-ray with evidence of collagen deposition and fibrosis (19) . more recently, the milder form termed "new bpd" has been described, characterized by abnormalities in alveolar septation, pulmonary edema, and less overt fibrosis (19, 20) . in adult animal models of acute lung injury (10,21), ace-2 was shown to inhibit lung edema formation and inflammation as well as fibrogenesis. thus, although little is known about the role of ace-2 in alveolar septation, figure 7 . proposed role of angiotensin-converting enzyme-2 (ace-2) in hyperoxia-induced lung fibrosis. angiotensinogen (agt) produced by either alveolar epithelial cells or myofibroblasts (3) can be cleaved by a variety of pulmonary aspartyl proteases (3) to yield angiotensin (ang) i, which in turn is cleaved by various lung enzymes to yield the profibrotic angii. angii is degraded by ace-2 to yield the antifibrotic peptide ang1-7, which acts through its receptor mas. hyperoxia causes, by mechanisms yet unknown, the release of ace-2 from the cell through ectodomain cleavage mediated by increased adam-17/tace. through this mechanism, hyperoxia is theorized to promote fibrosis through both (a) promoting accumulation of angii (8, 9) and (b) reducing production of the antifibrotic peptide ang1-7 (21 figure 6 . hyperoxia with normoxic recovery increases adam17/tace in fetal human lung fibroblasts. imr90 cells were exposed to hyperoxic or normoxic gas as described in figure 1 (with recovery), then were recovered for western blotting of adam17/tace as described in materials and methods. (a) western blotting for adam17/tace protein was then performed and normalized to β-actin. (b) by densitometry, hyperoxia with normoxic recovery caused a significant increase in adam17/tace (3.1-fold, *p < 0.05 by student's t-test). (c) replicate wells were harvested for recovery of total rna followed by qrtpcr as described in methods; hyperoxia with normoxic recovery also caused a significant increase in adam17/tace mrna (2.2-fold, *p < 0.05 by student's t-test). articles oarhe et al. ace-2 might be expected to protect against the "new bpd" as well as the more fibrotic "old" form. regardless, a small percentage of children with bpd today present with classic findings of the "old bpd", and it may be that a continuum exists between the milder forms and the more severe presentations. therefore, the study of ace-2 and other components of the ras in hyperoxic lung injury and the possibility of ameliorating the most severe, and possibly the milder, forms of bpd associated with hyperoxic injury remains critical. ace-2 has also been shown to be involved in virus-induced lung disease. ace-2 is the cellular receptor the sars-coronavirus, is required for coronaviral entry, and inhibition of its shedding by the tace inhibitor is related to the severity of the sars-cov infection (14) (15) (16) . liu et al (22) . showed that ace2 protein levels were significantly downregulated after infection with h1n1 viruses, but was dispensable for viral replication. they also found that ace-2 cleavage could be regulated by influenza neuraminidase. zou et al (23) . showed that in experimental mouse models, infection with highly pathogenic avian influenza a h5n1 virus results in downregulation of ace-2 expression in the lung and increased serum ang ii levels. genetic inactivation of ace-2 causes severe lung injury in h5n1-challenged mice, confirming a protective role of ace-2 in h5n1-induced lung pathologies. the same authors also demonstrated that the administration of recombinant human ace-2 ameliorates avian influenza h5n1 virusinduced lung injury in mice. thus, the potential influence of ace-2 downregulation by hyperoxic gas on viral infection will be an interesting topic for future investigation. growing evidence in the study of the role(s) of ace-2 in normal pulmonary physiology or pulmonary pathophysiology has demonstrated that alveolar epithelial cells are the primary site of ace-2 expression in mice (24) , but relatively less attention has been directed toward understanding the role of fibroblasts in the ace-2 mediated pulmonary physiology or pulmonary pathophysiology. to our knowledge, the present study is the first to demonstrate that ace-2 is expressed by human lung fibroblasts and is downregulated by hyperoxia. it is also the first to document that this downregulation could be prevented by tace antagonist in hyperoxia-treated cells. given the known function of ace-2 in preventing lung collagen deposition in experimental lung fibrosis (11) , its expression by the fibroblastic cells most responsible for collagen synthesis is not surprising. indeed, other authors have shown that lung collagen accumulation in neonatal rats exposed to hyperoxic gas could be prevented by blockers of angiotensin receptor at1 (25) . the well-studied pulmonary toxicity of hyperoxic gas is known to involve apoptotic cell death of alveolar epithelial cells (26) , but exposure of the cell line imr90 to a high oxygen concentration that is often administered to premature neonates (95% o2) did not increase a marker of apoptotic cell death in these fetal human lung fibroblasts (caspase 9, figure 1 ). although the 95% oxygen did cause a very minor increase in necrotic cells (<1% see table 1 ), it seems unlikely that this small but statistically significant effect would have physiologic significance. the hyperoxic gas did, however, cause a significant decrease in ace-2 ( figure 2) ; whether or not subphysiologic oxygen concentrations reduce ace-2 will be an interesting topic for further research. in the light of all published evidence, the downregulation of ace-2 would be expected to lead to accumulation of ang ii and its downstream profibrotic pathways (see figure 7) . indeed, hyperoxic gas was previously shown to increase angiimediated collagen synthesis by human lung fibroblasts (9), and either inhibition or gene knockdown of ace-2 increased autocrine angii generation by human lung cells in culture (11) . furthermore, the heptapeptide ang1-7, the product of ace-2, was shown to be antifibrotic in lung tissue (21, 27) and inhibition of ace-2 reduces its production (see figure 7) . accordingly, the reduction of cellular ace-2 induced by hyperoxia would be expected to promote fibrogenesis through both increasing angii and simultaneously decreasing ang1-7. the mechanism by which hyperoxia decreases ace-2 appears to be at the protein level, as no significant change in the mrna occurred and the decrease was prevented by tapi-2, an inhibitor of the adam17/tace enzyme that cleaves the ace-2 ectodomain in response to sars coronavirus (16) . as was demonstrated by haga et al (15) ., the present study also showed that the effect of hyperoxia on ace-2 appears to involve the upregulation of adam17/tace mrna and protein (see figure 6) , which in turn mediates enzymatic shedding of the ace-2 ectodomain and its release into the extracellular space. determination of the signaling mechanism(s) by which hyperoxia upregulates adam17/tace will be an interesting and compelling topic for future investigations. it is notable that the decrease in ace-2 occurred only if the period of hyperoxic exposure was followed by normoxic recovery for 24 h (figures 2-6) . this sequence of redox poise variation is reminiscent of ischemia-reperfusion injury, in which many key signaling pathways are induced not during the ischemic period, but during the return to normoxia (28) . although unknown at present, the mechanisms induced by hyperoxic/ normoxic cycling may be speculated to involve mitochondrial or other redox sensing mechanisms known to be influenced by reactive oxygen or nitrogen species (29) . the elucidation of these pathways will also be a very interesting topic for future inquiry. in summary, this study showed that ace-2 is expressed not only by lung epithelial cells (24) , but also by fetal human lung fibroblasts. ace-2 protein and enzyme activity, but not mrna, were decreased by hyperoxia in cultured fetal lung fibroblasts, but ace-2 protein was increased in the cell culture supernatants. inhibition of these effects by tapi-2 suggests a mechanism mediated by adam-17/tace-induced shedding of ace-2 from the cell surface. since it is known that lung injury and fibrosis can be abrogated by ace-2, the demonstration of a direct effect of hyperoxia on ace-2 in human lung cells has significant implications for the pathogenesis of hyperoxic lung injury, fibrogenesis and viral infection of the lungs. the peptide dx 600 , a competitive inhibitor of ace-2, was purchased from phoenix pharmaceuticals, burlingame, ca. antibodies against ace-2 were obtained from santa cruz biotechnology. tapi-2 was cell lines cultured to confluence were treated as described above, with control cells at 21% oxygen and hyperoxia-exposed cells at 95% oxygen (both at 5% co 2 ). after 72 h exposure in 5% fetal bovine serum, cells were allowed to recover in serum-free media in normoxic gas (21% o 2 + 5% co 2 ) for 24 h. then, cells were treated with trypan blue and viewed under the microscope, and blue stained cells as a marker for dead cells were counted. the cells were then treated with propidium iodide for dna count as a marker for total cells. a total of at least 400 cells per microscopic field were counted over at least three microscope fields per culture well. the counts from each field in a given culture well were then averaged to obtain the data used to calculate the numbers reported as n = 3 (three culture wells of 1,200 cells each per condition). the total number of necrotic cells as a percentage of total cells was then calculated. to assess for possible apoptosis, caspase 9 was detected by western blotting of cell lysates collected at the end of the 72-h exposure to hyperoxia or at the end of the 24-h recovery in normoxic gas. blotting methods are described below. cells were harvested with protease inhibitor (protease inhibitor cocktail p840, sigma, st louis, mo). soluble protein lysate (45 µg) were loaded and run on 10% tris.hcl polyacrylamide gels, separated by sds-page, in 10× tris/glycine/sds buffer (biorad, berkeley, ca). gels were transferred to immun-blot polyvinylidene fluoride blotting membrane. blotting membrane was blocked by 5% nonfat dry milk in 0.1% tween 20 in tris-buffered saline. western blot analysis of ace-2 was performed with anti-ace-2 polyclonal antibody (1:4,000: abcam biotechnology, cambridge, ma). antibodies for caspase 9 and adam17/tace were obtained from cell signaling, danvers, ma. to ensure equal loading of proteins, membranes were probed with an antibody against β-actin (cell signaling technology). bands were visualized by hrp-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution, santa cruz biotechnology, santa cruz, ca) using enhanced chemiluminescence detection by standard techniques. imr-90s raised and treated in six-well collagen i-coated plates (bd biosciences, bedford, ma) were harvested in ice-cold complete tris-hcl buffer (tris-hcl (ph 6.5), 1× complete protease inhibitor cocktail edta-free (roche, indianapolis, in), and lisinopril (50 μg/l; sigma-aldrich). lisinopril was added to the buffer as a means to block the activity of ace. in a half-area black 96-well microtiter plate, the fluorogenic peptide substrate for ace-2, mca-yvadapk(dnp)-oh (r&d systems, minneapolis, mn) was added at a final concentration of 10 μmol/l to 30 μl cell lysate (in a total volume of 50 μl using complete tris-hcl buffer) on ice. dx 600 (at a final concentration of 10 µmol/l; phoenix pharmaceuticals, burlingame, ca), a competitive inhibitor of ace-2, was also added to half of the wells to compare enzymatic ace-2 activity inhibition. the plate was warmed to room temperature and the fluorescence was read on a plate reader (310/20 nm excitation and 420/50 nm emission) in a biotek fl600 microplate fluorescence reader (biotek, burlington vt) for 30 min. kinetic readings were normalized to their respective dna concentrations. in a clear 96-well microtiter plate, 4 µl of cell lysate and 46 µl of complete np-40 lysis buffer (np-40, 1× complete protease inhibitor cocktail ethylenediaminetetraacetic acid-free (roche)) were added. total volume was brought up to 100 µl with the addition of 50 µl hoescht dye (final concentration of 5 µmol/l). the microtiter plate was then covered and rocked for 30 s on medium speed. the plate was then placed in a 37 °c incubator for 15 min. fluorescence was read on a plate reader (360/40 nm excitation and 460/40 emission) (fl600 microplate fluorescence reader). imr 90 cells after treatment were extracted for total rna by trizol reagent (invitrogen, carlsbad, ca) following the manufacturer's protocol. first-strand cdna was synthesized from total rna using superscript ii reverse transcriptase and oligo(dt) [12] [13] [14] [15] [16] [17] [18] (invitrogen). real-time pcr was carried out with cdna synthesized from 50 ng of total rna, sybr green pcr core reagents (applied biosystems, foster city, ca) according to the manufacturer's instructions, and 0.2 μmol/l specific primers for human ace-2 (sense 5′-cattggagcaagtgttggatctt-3′, and antisense 5′-gagcta-atgcatgcca-ttctca-3′), adam17/ tace (sense 5′-ttggtggtagcagatcatcg-3′ and antisense 5′-ctgggagagccaacataagc-3′) and β-actin (sense 5′-aggccaaccgcg-agaagatgacc-3′ and antisense 5′-gaagtccagggcgacgtagc-3′). the thermal profile for ace-2 and β-actin started with 10 min activation at 95 °c followed by 40 cycles of denaturation at 95 °c for 60 s, annealing at 55 °c for 60 s, extension at 72 °c for 60 s, and ending with dissociation curve analysis of the pcr products. the thermal profile for adam17/ tace started with 10 min activation at 95 °c followed by 40 cycles of denaturation at 95 °c for 15 s, annealing at 55 °c for 15 s, extension at 72 °c for 30 s. reactions were performed in a steponeplus realtime pcr system instrument (applied biosystems). threshold cycle (c t ) data were collected using stepone software version 2.1 (applied biosystems). the relative ace-2 expression was normalized to β-actin and calculated with the comparative ct method of 2−δδct, where δct = ctace-2 − ctβ-actin and δδct = δct treatment − δct control . media from cells plated in a total volume of 1 ml were collected in a 15 ml conical tube containing 300 µl of 10× complete protease inhibitor edta-free cocktail (roche). cell debris from the sample was removed through centrifugation. samples were prepped for dialysis using spectra/por biotech cellulose ester dialysis membranes with a mwco 10 kda (spectrum laboratories, rancho dominguez, ca) according to manufacturer's protocol. membranes containing the samples were then kept at 4 °c in a 2 l beaker of deionized water with gentle stirring until the media sample became clear (~2 d). at the completion of dialysis, samples were transferred to a new 15 ml canonical tube and frozen at −80 °c. completely frozen samples were lyophilized using the labconco freeze dryer/freezone 4.5 system according the manufacturer's protocol. lyophilized samples were diluted in 100 µl of sterile deionized water. western blot for ace-2 and β-actin was done by standard protocols. very low birth weight outcomes of the national institute of child health and human development neonatal research network hypoxic stress exacerbates hyperoxia-induced lung injury in a neonatal mouse model of bronchopulmonary dysplasia angiotensin-tgf-beta 1 crosstalk in human idiopathic pulmonary fibrosis: autocrine mechanisms in myofibroblasts and macrophages recruitable ace and tissue repair in the infarcted heart angiotensin-ii type 1 receptor interaction is a major regulator for liver fibrosis development in rats apoptosis of lung epithelial cells in response to tnf-alpha requires angiotensin ii generation de novo abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor angiotensin ii and the fibroproliferative response to acute lung injury the renin-angiotensin system mediates hyperoxia-induced collagen production in human lung fibroblasts angiotensin-converting enzyme 2 protects from severe acute lung failure angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis activation of the renin-angiotensin system in hyperoxia-induced lung fibrosis in neonatal rats angiotensin ii type 1 receptor antagonist attenuates lung fibrosis in hyperoxia-exposed newborn rats angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus tace antagonists blocking ace2 shedding caused by the spike protein of sars-cov are candidate antiviral compounds modulation of tnfalpha-converting enzyme by the spike protein of sars-cov and ace2 induces tnf-alpha production and facilitates viral entry a metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) bpd: old and new problems serum levels of seven cytokines in premature ventilated newborns: correlations with old and new forms of bronchopulmonary dysplasia the angiotensin-converting enzyme 2/ angiogenesis-(1-7)/mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension downregulation of angiotensin-converting enzyme 2 by the neuraminidase protein of influenza a (h1n1) virus angiotensin-converting enzyme 2 protects from lethal avian influenza a h5n1 infections angiotensin converting enzyme 2 is primarily epithelial and is developmentally regulated in the mouse lung angiotensin ii type 1 receptor antagonist attenuates lung fibrosis in hyperoxia-exposed newborn rats synergetic effect of α-lipoic acid with keratinocyte growth factor on protecting alveolar epithelial type ii cells of rat fetus from hyperoxia -induced injury regulation of alveolar epithelial cell survival by the ace-2/angiotensin 1-7/mas axis oxygen, the lead actor in the pathophysiologic drama: enactment of the trinity of normoxia, hypoxia, and hyperoxia in disease and therapy mitochondrial targets for volatile anesthetics against cardiac ischemia-reperfusion injury portions of the work conducted by some authors (m.d., h.n., and i.g.) were conducted in partial fulfillment of the requirements for the degree doctor of philosophy from michigan state university. articles oarhe et al. key: cord-264828-6w13xo2a authors: albini, adriana; di guardo, giovanni; noonan, douglas mcclain; lombardo, michele title: the sars-cov-2 receptor, ace-2, is expressed on many different cell types: implications for ace-inhibitorand angiotensin ii receptor blocker-based cardiovascular therapies date: 2020-05-19 journal: intern emerg med doi: 10.1007/s11739-020-02364-6 sha: doc_id: 264828 cord_uid: 6w13xo2a sars-cov-2 is characterized by a spike protein allowing viral binding to the angiotensin-converting enzyme (ace)-2, which acts as a viral receptor and is expressed on the surface of several pulmonary and extra-pulmonary cell types, including cardiac, renal, intestinal and endothelial cells. there is evidence that also endothelial cells are infected by sars-cov-2, with subsequent occurrence of systemic vasculitis, thromboembolism and disseminated intravascular coagulation. those effects, together with the “cytokine storm” are involved in a worse prognosis. in clinical practice, angiotensin-converting enzyme inhibitors (ace-is) and angiotensin ii receptor blockers (arbs) are extensively used for the treatment of hypertension and other cardiovascular diseases. in in vivo studies, ace-is and arbs seem to paradoxically increase ace-2 expression, which could favour sars-cov-2 infection of host’s cells and tissues. by contrast, in patients treated with ace-is and arbs, ace-2 shows a downregulation at the mrna and protein levels in kidney and cardiac tissues. yet, it has been claimed that both arbs and ace-is could result potentially useful in the clinical course of sars-cov-2-infected patients. as detected in china and as the italian epidemiological situation confirms, the most prevalent comorbidities in deceased patients with covid-19 are hypertension, diabetes and cardiovascular diseases. older covid-19-affected patients with cardiovascular comorbidities exhibit a more severe clinical course and a worse prognosis, with many of them being also treated with arbs or ace-is. another confounding factor is cigarette smoking, which has been reported to increase ace-2 expression in both experimental models and humans. sex also plays a role, with chromosome x harbouring the gene coding for ace-2, which is one of the possible explanations of why mortality in female patients is lower. viral entry also depends on tmprss2 protease activity, an androgen dependent enzyme. despite the relevance of experimental animal studies, to comprehensively address the question of the potential hazards or benefits of ace-is and arbs on the clinical course of covid-19-affected patients treated by these anti-hypertensive drugs, we will need randomized human studies. we claim the need of adequately powered, prospective studies aimed at answering the following questions of paramount importance for cardiovascular, internal and emergency medicine: do ace-is and arbs exert similar or different effects on infection or disease course? are such effects dangerous, neutral or even useful in older, covid-19-affected patients? do they act on multiple cell types? since ace-is and arbs have different molecular targets, the clinical course of sars-cov-2 infection could be also different in patients treated by one or the other of these two drug classes. at present, insufficient detailed data from trials have been made available. also in other tissues like gut, heart, kidney as well as arterial and venous endothelial cells in all organs studied [5] ( fig. 1 ). similar to human immunodeficiency virus (hiv), the receptors and co-receptors are expressed also on cells of haematopoietic origin [6] , sars-cov-2 is able to infect and cause damage to t lymphocytes, despite their very low expression levels of ace-2, which argues in favour of an alternate receptor allowing viral entry into these cells, where the virus is not able to replicate [7] . among sars-cov-2-infected patients in italy who died until april 23 and with a median age of 80 years, 69% of them were hypertensive, 31% had type 2 diabetes, 27% suffered from ischemic heart disease, 21% from atrial fibrillation, and 16% from heart failure, with 82% of them showing 2 or more comorbidities [8] . this could contribute to explain the apparently much higher (around 13%) covid-19-related case-fatality rates in italy, as compared to those reported [9] in cohorts of china patients (around 5%). in china the lethal cases of sars-cov-2 infection have occurred in patients with a median age of 63 years [10] , with their most prevalent comorbidities being represented, albeit to a lower extent, by hypertension (24%), diabetes (16%) and cardiovascular conditions (9%) [11, 12] . among 201 sars-cov-2-infected patients, (average age 51 years), 42% developed severe acute respiratory fig. 1 ace-2 is expressed on lung, gut, kidney epithelial cells, cardiac, endothelial cells and in testis and to lesser extent in the breast, skin and other organs distress syndrome, 27% had hypertension, and 19% were diabetic [13] . in one retrospective study on 416 covid-19-affected patients, those with myocardial injury (transient increase of hs-t-troponin values) had significantly higher mortality than the subgroup without myocardial injury (51% vs 4.4%, respectively; p < 0.001) [14] . in another report on 187 covid-19-affected patients, 23% of whom died, the mortality rate during hospitalization was, respectively, 7.6% among the patients without cardiovascular disease and no elevation of hs-t-troponin levels, 13.3% among those with an underlying cardiovascular disease but normal troponin levels, 37% for those without previous cardiovascular disease but elevated troponin levels, and 69% among those showing both cardiovascular disease and an increased hs-t-troponin profile [15] . a high frequency of thrombosis and thromboembolism has been additionally reported in covid-19-affected patients [16] [17] [18] (fig. 1 ). ace-2 expression has been demonstrated in endothelial cells from arterial and venous vessels [5] and there is clear-cut evidence that endothelial cells are prone to acquire sars-cov-2 infection [19] , with subsequent development of endotheliitis, endothelial cell damage, systemic vasculitis and disseminated intravascular coagulation (dic). covid-19-affected patients with acute respiratory failure present a severe hypercoagulability rather than consumptive coagulopathy [20] , with a concurrent massive elevation of von willebrand factor [21] towards massive endothelial stimulation and damage with release of von willebrand factor from weibel-palade bodies. in chinese, italian and french cohorts of covid-19-affected patients, a substantial increase in d-dimer and fibrin degradation products, along with a longer prothrombin time and activated partial thromboplastin time has additionally been documented as compared to uninfected patients [18, 20, 22] . significantly higher levels of d-dimer and fibrin degradation products, along with longer prothrombin and activated thromboplastin times were observed in sars-cov-2-infected subjects who did not survive, with the 71.4% frequency of dic in non-survivors and 0.6% in survivors highlighting the critical role played by such condition in covid-19 severity and associated deaths [23] . all the above pathogenetic mechanisms, in fact, are increasingly known to make a significant contribution to the occurrence of severe disease patterns and covid-19-associated mortality [24] . the aforementioned findings raise the following concerns: 1. covid-19-affected patients with cardiovascular morbidity, hypertension and diabetes show a worse prognosis, particularly in advanced age. 2. many of these patients, in italy, are currently treated with angiotensin ii receptor blockers (arbs) or aceinhibitors (ace-is) [25] . 3. sars-cov-2 is known to damage the cardiovascular system, thereby causing myocarditis [26] , myocardial injury, coronary plaque instability, and type 1 myocardial infarction [27] , along with oxygen supply and demand imbalance, systemic vasculitis, disseminated intravascular coagulation (dic) [14, 22, 23] , and heart failure [14, 28] . 4. renin-angiotensin-aldosterone system (raas)-interfering drugs are likely to affect ace-2 receptor-sars-cov-2 interaction dynamics within lung, heart, vascular, kidney and gut tissues [5, 19] , while it is still not completely elucidated how such interactions are relevant to the clinical course of cardiovascular comorbidities in patients with covid-19 [29] . are these two drug classes (arbs, ace-is or other antihypertensive drugs) acting differently from each other in the pathogenetic evolution of sars-cov-2 infection? do they affect infection of different cell types? it has been demonstrated that human ace-2 enzyme is a negative regulator of raas [30, 31] , thus providing a crucial link between immunity, inflammation, increased coagulopathy, and cardiovascular disease, thereby serving as a protective mechanism against heart failure, myocardial infarction, lung disease, hypertension, vascular permeability, and diabetes [24, 32] (fig. 2) . ace-2 function, following sars-cov binding, is reduced due to endocytosis and proteolytic cleavage [33] and there are high levels of ang ii in the blood of patients with covid-19 [34, 35] . consequently, the up-regulation of human ace-2 induced by raas-antagonists in sars-cov-2-infected patients could be clinically useful, due to the cardiovascular protection elicited by the increased activity of angiotensin(1-7), thereby attenuating angiotensin ii effects on vasoconstriction and sodium retention [31, 34] . in vivo ace-2 deficiency results in augmented vascular inflammation and plaque instability [36] , while its overexpression has been also shown to reduce left ventricular damage during myocardial infarction [37] . it has been additionally suggested that ace-2 plays a key role in the reduction of left ventricular remodelling as well as in preserving ventricular function in humans [38] . also sex seems to play a role in the pathogenesis of sars-cov-2 infection, with most of covid-19-associated deaths having been recorded among male individuals both in china (70-73%) [11, 39] and in italy (64%) [8] . the fact that the ace-2-encoding gene is located on chromosome x [40] could be a relevant factor in this regard. the sars-cov-2 uses the transmembrane protease serine 2 (tmprss2), an androgen-regulated gene, for viral spike protein priming [1] . ace-2 receptor polymorphisms have, in silico, different affinities for the spike proteins sars-cov-2 [41] [42] [43] . another confounding factor is smoke, with cigarette smoking increasing ace-2 expression not only in experimental models [44] but also in humans [45] notwithstanding the well-established immunomodulatory effects of nicotine. respiratory tract inflammation can expand ace-2 receptor availability for respiratory cell infection by sars-cov-2. this point is also controversial [46] , with the percentage of covid-19-affected patients admitted to intensive care units in china being almost double in current smokers than in non-smokers [10] . these findings provide important insights into the molecular bases of the broad cross-talk between raas (fig. 2) and sars-cov-2 cellular recognition and infection [1] [2] [3] . experimental studies on lewis rats have documented a 4.7-fold increased expression of cardiac ace-2 gene after treatment with the ace-inhibitor lisinopril, and by 2.8-fold after treatment with the angiotensin ii (at)1 receptor blocker losartan; however, ace-2 activity remained paradoxically unchanged with lisinopril, being increased after losartan administration [47] . in spontaneously hypertensive rats treated with losartan, ace-2 increased in renal but not in cardiac tissues [48] . in contrast, patients treated with ace-is and arbs show an ace-2 down-regulation at the mrna and protein levels in kidney and cardiac tissues [49] . angiotensin ii is also able to up-regulate ace and down-regulate ace-2 in human kidney tubular cells, which were blocked by an at1 receptor antagonist (losartan), but not by an at2 receptor blockade [49] . these experimental observations have raised concerns that ongoing treatments with ace-is or arbs could worsen the prognosis of covid-19-affected patients suffering from simultaneously occurring cardiovascular disease conditions. in fact, it has been hypothesized that the ace-2 overexpression induced by these two drug classes, although elicited by different mechanisms, could favour sars-cov-2 human lung tissue colonization [50] . by contrast, it has been additionally claimed that both arbs [51] and ace-is [29] could result potentially useful in the clinical course of sars-cov-2-infected patients [34] (fig. 2) . due to the huge number of hypertensive, cardiovascular disease-affected and diabetic people currently treated with ace-is or arbs, many of whom are now requesting a strong reassurance by their cardiologists, we think it would be necessary to clarify the contents of such a relevant debate from a practical/clinical viewpoint. the recent hfsa/acc/aha statement [52] has claimed the need of continuation of ongoing treatments with raas antagonists in patients taking these drugs for various cardiovascular conditions, diabetic nephropathy or hypertension, despite theoretical concerns that their use might worsen the covid-19 clinico-pathological evolution, should these subjects become infected by sars-cov-2. also the position statement of the council on hypertension of the european society of cardiology [53] strongly suggests that, since sars-cov-2 binds to ace-2 enzyme to infect host's cells, and although ace-2 levels are increased following treatment with ace-is and arbs, there is currently lack of evidence supporting in humans the harmful effects of ace-is and arbs in the context of the covid-19 pandemic, thereby recommending that physicians and patients should continue ongoing treatments with their usual anti-hypertensive therapies. among the covid-19-affected, deceased italian patients, 24% of those followed were under treatment with ace-is and 16% with arbs [8] , with almost 70% of them also showing pre-existing hypertension. with the aim of reinforcing the above-reported statements, some very recent studies further contribute to clarify the debate [54] [55] [56] . in a retrospective study on 1128 hospitalized covid-19-affected patients with hypertension, including patients taking ace-is (17%) or arbs (83%) with a median age of 64-66 years (about 52% of whom males) and an additional 940 covid-19-affected patients treated by other anti-hypertensive drugs with a median age of 64 years (about 53% of whom males), the unadjusted casefatality rate was either significantly lower in the former group versus the non-ace-is/arb group (3.7% vs. 9.8%, p = 0.01) [55, 56] , or both groups were equal in terms of severe disease and deaths [54] . nevertheless, the chinese cohorts consisted of younger patients and had less comorbidities than the italian covid-19-affected patients who succumbed to the disease. interestingly, the platelet count was significantly lower [55] , similarly to the prothrombin time [54] and the d-dimer [56] levels, in the ace-is-or arb-treated, covid-19-affected patients. after multivariate analysis for age, sex, comorbidities, and in-hospital medications [56] , the adjusted hr for the ace-is/arb group was 0.42 (95% ci 0.19-0.92; p = 0.03). the cautious conclusion of the authors, due to the intrinsic limitations of a retrospective study, was that "it is unlikely that in-hospital use of ace-inhibitors/arbs was associated with an increased mortality risk" [56] . four very recent retrospective studies did not find any influence of the two raas-inhibitors on the clinical course [57] , the rate of inhospital death [58] or propensity of developing covid-19 infection [59, 60] . as a conclusive remark, we can summarize "the current state-of-the-art" by underscoring and expressing endorsement on the statements independently expressed by hfsa/ acc/aha and the council on hypertension of the european society of cardiology with just one sentence: "further data are needed in order to draw definitive conclusions". conclusions: due to the large number of older patients treated all over the world with raas-antagonists for hypertension, diabetic nephropathy and severe cardiovascular disease conditions, although both authoritative statements and some preliminary contributions seem to dispel the fear that raas-antagonists could worsen the clinical course of covid-19-affected patients, we claim the need for adequately powered, prospective studies aimed at providing an "ad hoc" answer to the following relevant questions: 1. do ace-is and arbs display similar or quantitatively divergent effects in covid-19 patients? 2. could ace-is or arbs eventually be resulting in neutral, useful, or even dangerous in terms of cardiovascular protection in the clinical course of sars-cov-2 frail, comorbid infected patients? 3. how they affect infection of the numerous different type of ace2 positive cells in the organism throughout the human body? at present, insufficient detailed data from trials have been made available. conflict of interest the authors declare that this "point of view" article was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. this article does not contain any studies with human participants or animals performed by any of the authors. informed consent informed consent was not required for this type of study which represents an opinion (point of view) paper. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the 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cardiovascular disease, drug therapy, and mortality in covid-19 association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease 2019 (covid-19) renin-angiotensin-aldosterone system inhibitors and risk of covid-19 acknowledgements we thank andrea sonaglioni (cardiology unit, san giuseppe hospital-multimedica, milan, italy) and mario milco d'elios (department of experimental and clinical medicine, university of florence, florence, italy) for helpful discussion and revision of the manuscript. this work has been supported by italian ministry of health ricerca corrente-irccs multimedica. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-024076-q9fw7ch1 authors: manga, pravin title: should ace inhibitors and angiotensin receptor blockers be withdrawn in the current setting of covid-19 infection? date: 2020-04-17 journal: nan doi: 10.18772/26180197.2020.v2nsia4 sha: doc_id: 24076 cord_uid: q9fw7ch1 nan the reports urging caution in the use of ace inhibitors and arbs for hypertension, diabetes and cardiovascular disease are based on laboratory data which found that sars-cov and covid-19 virus binds to ace 2 receptor which is found in the epithelial cells of the lung, kidney, intestine and blood vessels. (4, 5) the ace 2 is an integral protein and contributes to a host of physiologic functions and is highly expressed in alveolar cells of the lungs, providing the main entry site for the virus into human hosts. (5) once the covid-19 virus binds to ace 2 and gains entry, it subsequently downregulates ace 2 expression. it is postulated that this may lead to unopposed angiotensin ii activity and renin-angiotensin-aldosterone-system (raas) activation, leading to neutrophil infiltration into the lungs and lung injury. (6) however, ace 2 also has potential protective effects in that it counters the effects of angiotensin ii via angiotensin 1 receptor activation, by promoting the conversion of angiotensin ii to angiotensin 1-7, which in turn modestly lowers blood pressure through vasodilatation and increases renal excretion of sodium and water. ace 2 may also attenuate inflammation through the activation of nitric oxide pathways. in some experimental studies with animal models, it has been shown that there is increased expression and activity of ace 2 in the heart and brain after treatment with ace inhibitors and arbs.(7) furthermore, the upregulation of ace 2 in humans is supported by recent evidence showing an increased secretion of ace 2 in the urine of hypertensive patients treated with olmesartan, an arb. (8) although ace 2 receptor upregulation may be induced by ace inhibitors and arb treatment and hence a theoretical risk of increased susceptibility to covid-19 infection, currently there is no data that has demonstrated a definitive causal relationship between ace 2 activity and covid-19 mortality. interestingly, ace 2 and angiotensin 1-7 may in fact have a salutatory role in the lungs since it has been found to be protective in a number of lung injury models. in a mouse model with acid lung injury, ace 2 downregulation induced by sars-cov, the virus responsible for the sars virus outbreak in 2003, worsened lung injury, but this lung injury was improved by therapy with an arb.(9) these findings suggest that sars-cov may induce lung injury, but the injury can be ameliorated by arb administration.(9) these preclinical findings suggest a possible protective role of arb in sars-covassociated lung injury and lend credence to the hypothesis that primary activation of the raas in cardiovascular patients, rather than its inhibition, renders them more prone to a deleterious outcome. however, currently there is no clinical evidence that has proven that ace inhibitors or arb-induced ace 2 activity is an effective therapy for covid-19-induced lung injury. in addition, ace 2 activity may not correlate with the degree of severity of infection with covid-19 infection. although ace 2 is presumed to be an important mediator for sars-cov infection, the absence of sars-cov has been found in some cell types expressing ace 2.(10) on the other hand, infection was present in cells apparently lacking ace 2, suggesting that additional co-factors may be needed for adequate cellular infection. (10) because of the connection of the ace 2 pathway with covid-19 infection, there has been widespread concern amongst physicians and patients, whether raas antagonists such as ace inhibitors and arbs confer an increased risk of covid-19 infection. many patients and their physicians, including those in south africa, have contemplated a cessation of ace inhibitors or arb medications. in the wuhan study, (2) there was no information of how many of the patients with severe covid-19 infection were on ace inhibitors or arbs. furthermore, there are no published studies to date showing that diabetes and hypertension are independent predictors of mortality with covid-19 infection. thus, a clear causal relationship between those with cardiovascular disease, hypertension, heart failure with reduced ejection fraction (hfref), and diabetes with chronic kidney disease (dckd) on ace inhibitors or arb treatment and an increased risk of covid-19 does not exist. there are alternative agents that can be used for the management of hypertension, but dckd and hfref are compelling indications for these drugs. patients, influenced by the media, may request an alternative anti-hypertensive agent, and that may be appropriate for them, but it is inappropriate to recommend discontinuation of ace inhibitors or arb treatment in patients with hfref and dckd based on a hypothetical adverse outcome with covid-19 infection. importantly, the indication for which the drugs were originally prescribed may have changed since the drugs were initiated, and a patient with hypertension may not be aware of that they have developed lv systolic dysfunction or that a diabetic has developed proteinuria. a recent report suggested a reverse causality relationship by the fact that patients who are receiving ace inhibitors or arb may be more susceptible to viral infections and more likely to have a higher risk of dying because they are older and hence would have a higher prevalence of hypertension, renal disease and diabetes. (11) it is important to recognize that pathophysiological mechanisms that lead to cardiovascular disease are known to overlap with pathways that regulate immunological functions. thus, age is one of the strongest risk predictors for cardiovascular disease and the effect of ageing on immune function may be equally important for covid-19 susceptibility and severity. (12) a dysregulated immunologic state corresponds with an elevated risk of incident cardiovascular disease and thus other traditional cardiovascular disease risk factors such as diabetes and hyperlipidaemia impact immune function. (13) thus, the presence of cardiovascular disease may be a marker of accelerated immunologic ageing/dysregulation and relate indirectly to covid-19 prognosis.(12) in south africa, hypertension, hfref and diabetes are common non-communicable diseases, and a significant proportion of patients are being treated with generic versions of ace inhibitors or arbs. there is extremely strong scientific evidence for the benefit of raas inhibition in patients with cardiovascular disease. in patients with hfref, raas inhibition is a foundation of therapy for these patients. discontinuation of raas inhibition in patients with heart failure can precipitate clinical deterioration and may be associated with increased mortality. (14) furthermore, ace inhibitors and arbs are common therapies for hypertension and after myocardial infarction. (15, 16) there is significant mortality benefit with all three classes of these agents' post myocardial infarction. based on the solid scientific foundation of these three agents in cardiovascular disease, diabetes and renal disease, there is a potential for significant adverse outcomes in discontinuing these agents. covid-19 appears to be particularly severe in patients with cardiovascular disease and may cause myocarditis, myocardial stress and cardiomyopathy.(12) thus, discontinuing raas inhibition in these high-risk patients can potentially lead to higher mortality. change of therapy for patients with hypertension is less risky. however, they are associated with other risks such as medication errors, rebound increase in blood pressure, frequent monitoring to assess adequate blood pressure control and management of side effects of newly prescribed medications. it has been shown that even short periods of loss of control of blood pressure may be associated with increased cardiovascular risk. (17) in response to the reports of a hypothetical risk of ace inhibitors and arbs, many societies have issued statements strongly recommending the continuation of ace inhibitors and arb therapies. (18, 19) patients are strongly discouraged from making autonomous decisions about their cardiovascular therapy and must be guided by their informed treating physician. moving forward it is clear that more research is needed to clarify and understand the relationship between the ace 2 protein, ace inhibitors and arb use in cardiovascular disease and covid-19 prognosis. in this regard an ongoing randomized trial evaluating recombinant ace 2 in the setting of covid-19 may help one to provide mechanistic information in patients infected with this virus (clinicaltrials.gov identifier: nct04287686). (20) this therapy has the possible potential to both decrease viral load and ameliorate the harmful effects of angiotensin ii. until more robust evidence is available, it is prudent to advise that ace inhibitors or arb therapy should be continued in patients who are at risk for covid-19 infection or who have covid-19 infection. even in patients with current covid-19 infection, ace inhibitors or arb should be initiated in guideline-indicated conditions such as in patients with heart failure or myocardial infarction. we must not draw inappropriate conclusions from observational studies. in conclusion, in the current covid-19 pandemic in south africa, both practitioners and patients need to be advised that ace inhibitors and arb therapy should be continued in patients with cardiovascular disease and in associated conditions such as diabetes and renal disease. ongoing research efforts need to concentrate on assessing the role of ace 2 in covid-19 infection and the effect on mortality of known therapies for cardiovascular disease such as ace inhibitors and arbs. it is hoped that with the large numbers of patients currently infected worldwide with covid-19, this information will be elucidated in the near future. preventing a covid-19 pandemic: ace inhibitors as a potential risk factor for fatal covid-19 are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? clinical characteristics of coronavirus disease 2019 in china angiotensin-converting enzyme 2 is a functional receptor for coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury effect of angiotensin-converting-enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 urinary angiotensin-converting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin ii receptor blocker a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury pathology and pathogenesis of severe acute respiratory syndrome sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? cardiovascular considerations for patients, health care workers, and health systems during the coronavirus disease 2019 (covid-19) pandemic inflammation in atherosclerosis: from pathophysiology to practice withdrawal of pharmacological treatment for heart failure in patients with recovered dilated cardiomyopathy (tred-hf): an open-label, pilot, randomised trial esc/esh guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the european society of cardiology (esc) and the european society of hypertension (esh) esc guidelines for the management of acute myocardial infarction in patients presenting with st-segment elevation: the task force for the management of acute myocardial infarction in patients presenting with st-segment elevation of the european society of cardiology outcomes in hypertensive patients at high cardiovascular risk treated with regimens based on valsartan or amlodipine: the value randomised trial position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target key: cord-259933-ggx4v0bz authors: dalan, rinkoo; bornstein, stefan r.; el-armouche, ali; rodionov, roman n; markov, alexander; wielockx, ben; beuschlein, felix; boehm, bernhard o. title: the ace-2 in covid-19: foe or friend? date: 2020-04-27 journal: horm metab res doi: 10.1055/a-1155-0501 sha: doc_id: 259933 cord_uid: ggx4v0bz covid-19 is a rapidly spreading outbreak globally. emerging evidence demonstrates that older individuals and people with underlying metabolic conditions of diabetes mellitus, hypertension, and hyperlipidemia are at higher risk of morbidity and mortality. the sars-cov-2 infects humans through the angiotensin converting enzyme (ace-2) receptor. the ace-2 receptor is a part of the dual system renin-angiotensin-system (ras) consisting of ace-ang-ii-at (1) r axis and ace-2-ang-(1–7)-mas axis. in metabolic disorders and with increased age, it is known that there is an upregulation of ace-ang-ii-at (1) r axis with a downregulation of ace-2-ang-(1–7)-mas axis. the activated ace-ang-ii-at1r axis leads to pro-inflammatory and pro-fibrotic effects in respiratory system, vascular dysfunction, myocardial fibrosis, nephropathy, and insulin secretory defects with increased insulin resistance. on the other hand, the ace-2-ang-(1–7)-mas axis has anti-inflammatory and antifibrotic effects on the respiratory system and anti-inflammatory, antioxidative stress, and protective effects on vascular function, protects against myocardial fibrosis, nephropathy, pancreatitis, and insulin resistance. in effect, the balance between these two axes may determine the prognosis. the already strained ace-2-ang-(1–7)-mas in metabolic disorders is further stressed due to the use of the ace-2 by the virus for entry, which affects the prognosis in terms of respiratory compromise. further evidence needs to be gathered on whether modulation of the renin angiotensin system would be advantageous due to upregulation of mas activation or harmful due to the concomitant ace-2 receptor upregulation in the acute management of covid-19. coronaviruses (cov) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as middle east respiratory syndrome (mers-cov) and severe acute respiratory syndrome (sars)-cov. the sars-cov-2, has caused a rapidly spreading outbreak (covid19) with over 300 000 infected cases and more than 13 000 deaths globally [1] [2] [3] [4] [5] [6] [7] [8] , (https://coronavirus.jhu.edu/map.html). the sars-cov-2, a positive strand rna virus, has been seen to infect humans through the angiotensin converting enzyme -2 (ace-2) receptor [9] . in covid-19 infections, emerging evidence demonstrates that older individuals and people with underlying metabolic conditions of diabetes mellitus, hypertension, and hyperlipidemia are at higher risk of morbidity and mortality [1] [2] [3] [4] [5] [6] [7] [8] . in individuals with hypertension, diabetes, and other cardiovascular disorders with vascular complications, the renin angiotensin system (ras) is known to be activated with an increase in ace activity and a downregulation of ace-2. modulation of this system by ace-inhibitors or at 1 -receptor blockers is now considered as the first-line therapy as well as for prevention and management of vascular complications. in this regard, the questions arise if (i) differences in ace-2 may explain the exacerbated course of disease in patients with metabolic diseases and (ii) if ace modulation in covid-19 patients is neutral, beneficial, or harmful. the latter question may have immediate therapeutic consequences for millions of patients. moreover, ace-2-based therapy has been proposed as a potential therapeutic approach in covid-19 pneumonia [10] . the angiotensin converting enzyme 2 (ace-2), a single pass type 1 membrane monocarboxypeptidase, discovered 2 decades ago [11] consists of an n-terminal peptidase domain and c-terminal collectrin like domain [9] . it is the peptidase domain that is responsible for the main functions of the renin angiotensin system (ras) [9] . the ace-2 shares 40 % homology with the n-terminal catalytic domain of ace, and a hydrophobic region near the c-terminus likely to serve as a membrane anchor [9, 11] . the ace-2 protein is encoded by the ace-2 gene located on chromosome xp22. these ace-2 proteins are more abundantly expressed on the apical surface of the well-differentiated and mostly ciliated airway epithelium of the lungs (alveolar type-2 cells), and enterocytes of the small intestine [12] . furthermore, ace-2 protein is expressed in arterial and venous endothelial cells and arterial smooth muscle cells, in the heart, kidneys, adrenal glands, pancreas, skeletal muscle, and adipose tissues [11] . the coronavirus sars-cov-2, a single stranded rna virus, has been seen to infect humans through their envelope spike glycoprotein (s-protein), which is responsible for cov cell entry and hostto-host transmission. during viral infection, this s-protein cleaves into s1 and s2 [9] . the furin cleavage site in the sars-cov-2s protein may provide a priming mechanism [13] . the ectodomain s1 binds to the peptidase domain of the ace-2 enzyme, while the s2 is cleaved further by the host cell serine protease tmprss2 [14] resulting in membrane fusion. both these steps are essential for the viral entry into the cells. an in vivo study shows that the infection of human airway epithelia by sars coronavirus correlated with the state of cell differentiation and ace-2 expression and localization [15] . the infection tends to occur more readily through well differentiated ciliated cells with higher ace-2 expression [15] . it has been observed that ace-2 membrane expression and plasma levels are reduced after infection with sars coronavirus [16] . further, sars cov spike protein has been found to reduce ace-2 expression and to augment pulmonary injury [16] . however, treatment with blockers of the renin-angiotensin system reduced the pulmonary injury by activating the ace-2-ang-(1-7)-mas axis. in the classical endocrine model of the ras, renin cleaves its substrate, angiotensinogen (agt), to produce the inactive peptide, angiotensin i (ang i), which is then converted to angiotensin ii (ang ii) by endothelial angiotensin-converting enzyme (ace). the catalytic activity of ace to activate ang ii occurs most extensively in the lung. ang ii mediates vasoconstriction as well as aldosterone release from the adrenal gland, resulting in sodium retention and an increase in blood pressure through the angiotensin 1 receptor (at 1 r). however, recent evidence suggests that ras also includes local systems with autocrine (cell-to-same cell) and paracrine (cell-to-different cell) effects in addition to the classical circulating ras with its well-known classical endocrine effects. in particular, ang ii generation at the tissue level by the tissue specific ras appears to have physiologic effects that are as important as circulating/systemic ang ii and, under some circumstances, more important than circulating ang ii. therefore, the ras system is not only involved in controlling blood volume and blood pressure but with the tissue specific local systems it directs tissue remodeling, endothelial dysfunction, and fibrosis [17] [18] [19] [20] . simplistically, it can be seen as a dual function system, which acts through two apparently opposite arms: the one responsible for the main actions of this system is constituted by the ace ang ii-at 1 receptor axis and the other, a counterregulatory arm, is formed by the ace-2-ang-(1-7)-mas axis. the catalytic activity of ace results in increased ang ii levels and increased catabolism of ang-(1-7) whereas the catalytic activity of ace-2 is predominantly on ang i and ang ii and leads to formation of ang-(1-7). ang-(1-7) acts on the g-protein coupled receptor mas and is known to have counterregulatory actions on ang ii resulting in vasoconstriction and growth inhibitory effects [17] [18] [19] [20] (see ▶ fig. 1 ). a common mechanism by which diabetes, hypertension, and other metabolic disorders cause vascular complications is endothelial dysfunction, inflammation, and atherosclerosis. in these disorders, the ace-ang ii-at 1 receptor axis is activated with a downregulation of ace-2-ang-(1-7)-mas axis [19] [20] [21] , which leads to increased local and circulatory ang ii with a decrease in ang-(1-7) and subsequent increased oxidative stress, activation of the endothelium, smooth muscle cell migration, growth, proliferation and thrombosis [19] [20] [21] . deficiency in ace-2 leads to increased formation of atherosclerotic plaques and blocking of ace-2 results in prevention of atherosclerosis [20] (▶ fig. 1) . ace-2 is present in multiple organs and has various functions. in the heart, ace-2 prevents progressive cardiac fibrosis associated with aging and/or cardiac pressure overload and is beneficial in heart failure [19, 20] . in most forms of chronic kidney disease including diabetic and hypertensive nephropathy, expression of ace-2 has been reported to be reduced in kidney tubules with an increased expression in the glomerula [20] . this imbalance has been postulated as a potential cause of diabetic nephropathy. in the adrenal glands, the local secretory ras stimulates aldosterone production and serves as an amplification system for circulating ang ii. importantly the regulation of the secretory adrenal ras is independent of the circulatory ras and the activity of the adrenal ras correlates with aldosterone production and regulation of potassium serum concentrations [22] . severe diabetes has been associated with hyporeninaemic hyperactivity of the adrenal gland [23] . in the pancreas, the ace-2-ang-(1-7)-mas axis has been described to protect the function of insulin-producing beta cells by improving the function of islet microvascular endothelial cells. further, activation of endothelial nitric oxide synthase and no signaling pathways via the ace-2-ang-(1-7)-mas axis may have anti-inflammatory beneficial effects in pancreatitis [23, 24] . in the skeletal muscle, the ace-2-ang-(1-7)-mas axis has been described to decrease insulin resistance [25] . ace-2 may exert potential anti-obesity effects via stimulating brown adipose tissue formation and induction of browning in white adipose tissue [26] . a study done in the survivors of the sars infections, the organ involvement correlated with the organ expression of ace-2 with significantly higher immunostaining in the lung, kidney, heart, and islets of pancreas [27] . in this series, 20 of the 39 patients had diabetes during hospitalization, which resolved subsequently suggestive of acute islet cell damage and diabetes, probably due to the use of the ace-2 receptor domain for viral entry [27] . the respiratory system is a major site of ace-activity and source of systemic ang ii synthesis. locally produced ang ii may trigger increasing vascular permeability facilitating pulmonary edema. the ace-2-ang-(1-7)-mas axis that is highly expressed in the lungs, may potentially induce pulmonary vasoconstriction in response to hypoxia, which is an important process in preventing shunting in patients with pneumonia or lung injury [19] . in acute respiratory distress syndrome (ards) mouse models, ace-2 knockout mice displayed more severe ards symptoms compared with wildtype mice, while overexpression of ace-2 appeared to be protective [19] . ▶fig. 1 schematic representation to show the renin angiotensin system in diabetes and the interaction of the sars-cov 2 with the ace-2. 1. the sars-cov 2 interacts with the ace-2 through the spike proteins after priming by tissue serene proteases. it uses the ace-2 protein to enter the alveolar cells in the lungs. 2. the renin angiotensin system consists of renin which catalyzes the conversion of angiotensinogen to angiotensin 1 (ang 1). the subsequent axis depends on the balance between the angiotensin converting enzyme (ace) and ace-2. ace converts ang 1 to ang ii and this acts in the angiotensin receptor (at 1 r), whereas ace-2 converts it to ang-(1-7), which acts on the mas receptor. 3. in the respiratory system activation of ace leads to a proinflammatory, pro-fibrotic , pro-hyperresponsiveness response in the respiratory system, whereas ace-2-ang-(1-7)-mas induces a protective mechanism of anti-inflammatory, anti-fibrotic and anti-hyperresponsiveness. a lower ace-2 will put these individuals at higher risk of respiratory distress. 4. in hypertension, diabetes, and cvd, the ace related pathway is activated with downregulation of the ace-2 pathway. these results in the multi-organ complications seen in metabolic diseases with endothelial dysfunction promoting atherosclerosis, increased cardiac fibrosis and lv remodeling, diabetic nephropathy, hyperactivity of adrenal gland, and it decreases insulin release and increases insulin resistance. 5. infection with covid-19 may exacerbate the ace-2 deficiency in these patients in all organs and maybe responsible for the multiorgan failure. thus, stimulation of the ace-2-ang-(1-7)-mas axis may have anti-inflammatory and antifibrotic effects in the pulmonary system, and these actions could potentially be favorable in the recovery of patients with pulmonary inflammation. it is possible that an ace/ ace-2 imbalance is one of the potential mechanisms explaining why patients with cardio-metabolic problems are at higher risk for respiratory failure [20] . in an animal model study, it has been reported that infusion of sars cov spike protein reduced ace-2 expression and augmented pulmonary injury. however, treatment with blockers of the renin-angiotensin system reduced the pulmonary injury by activating the ace-2-ang-(1-7)-mas axis [16] . therefore, it may be assumed that the inherent downregulation of the ace-2-ang-(1-7)-mas axis (as seen in metabolic conditions) is exacerbated in the covid-19 state because (i) the virus uses the peptidase domain of the enzyme for entry into the cells and (ii) there is a decrease in ace-2 with an increase in ace [9] . in covid-19 infections, chest radiography typically shows patchy or diffuse asymmetric airspace opacities bilaterally more in the peripheral zones and in later stages ground glass opacities. ct scan typically shows bilateral involvement, which is different from sars and mers which starts unilaterally. moreover, it shows bilateral multifocal ground-glass opacities with a peripheral lung involvement in milder cases with more widespread consolidation in patients who go to the intensive care unit. pleural effusion, cavitation, pulmonary nodules, and lymphadenopathy have not been reported in patients with covid-19. an important clinical observation of covid-19 infection in china was that some individuals with early infection exhibited no symptoms. interestingly, they even had negative swab results (due to absence of infection in the upper respiratory tract) and normal chest x-rays, however, showed significant changes in the ct scan of the lungs [28] . histopathological examination of early covid-19 infected lungs, which were serendipitously detected in specimens operated for lung cancer, revealed exudative and proliferative phases of acute lung injury, edema and congestion of alveolar septa with inflammatory infiltrates (macrophages), type ii pneumocytes hyperplasia with viral inclusions, organization of inflammatory exudates and interstitial fibrosis [29] . these pathological findings corroborated with the ct findings of patchy ground glass opacities. intriguingly, these pathological findings of inflammatory and fibrotic changes are known to be exacerbated in animal models of ace-2 knockout mice [19] . histopathological examination from autopsies of individuals who have succumbed from covid-19 infections showed degenerated and necrotized cells in the myocardium and blood vessels with interstitial inflammatory infiltrates and focal necrosis. in the kidneys, proteinaceous exudates in the bowman's capsule surrounding the glomerulus, degeneration and shedding of the renal tubular epithelial cells and hyaline casts, microthrombi and fibrotic foci have been reported [30, 31] . some of these changes of myocardial fibrosis and renal changes are already seen in the chronic phase of metabolic disorders due to ace-2 downregulation and these may get exacerbated [19, 20] . the vast majority of covid-19 patients have mild symptoms. however, a significant percentage is critically ill with admission to an intensive care unit (icu, approx. 10 %) with 4 % case fatality rate globally (john hopkins coronavirus resource center: https://coronavirus.jhu.edu/). mortality is higher in patients with underlying hypertension, type-2 diabetes mellitus, or cardiovascular disease [1] [2] [3] . to address this further, we performed a cumulative analysis of published data from china and south korea, which showed that a disproportionately higher percentage of patients with a higher morbidity (requiring icu care, ards) had hypertension (28 %), type-2 diabetes (17 %), cvd (8 %) as an underlying disease. moreover, patients admitted to the icu had a higher systolic bp when compared to asymptomatic cases [4] [5] [6] [7] and in a recent series reported none of the nonsurvivors had hypotension [3] (▶table 1). a limitation of the data summarized in ▶ table 1 is the posibility that some cases were reported twice in different series. interestingly, however, in a recent case report on a mild case extensively followed in australia, a low level of the monocyte chemoattractant protein-1 (mcp-1), a key biomarker of type-2 diabetes related vascular complications, was found to be associated with minor disease pathology [32] . in a recent preprint study, hypokalemia due to renal potassium wasting has been reported with 93 % of the critically ill patients having hypokalemia independent of gastrointestinal symptoms [33] . the presence of higher expression of ace-2 receptors or its upregulation although initially thought to be a risk factor for infection is unlikely the case. in a recent preprint study, wherein they integrated public genomics, epigenomics, and transcriptomics data, they found that the expression of ace-2 is relatively high in asian females and young people while it is lower in males, and further decreases with age and with the progression of type-2 diabetes [34] . this contradicts the hypothesis that higher expression is directly related to increased susceptibility as we know that sars-cov-2 infections are more common in males and in elderly patients with metabolic disorders. taken together, these observations suggest that patients with underlying type-2 diabetes, hypertension and/or cardiovascular diseases are at higher risk for requiring critical care and ventilation. there is evidence that although they are critically ill, they tend to demonstrate normotension to hypertension and hypokalemia, which are classical features of an activated ace related ras with a downregulation of the ace-2 pathway. as per current data, it does not appear that higher ace-2 expression is associated with an immediate predilection as individuals at risk will tend to have lower ace-2 expression. since ace-2 is the entry receptor for cellular infection by sars-cov-2, blocking entry using ace-2-related therapy could be feasible to prevent the spreading of infection in the lungs and the whole body. convalescent sera containing neutralizing antibodies against sars-s protein offer protection against sars-cov-2 infection [35] . therefore, it was suggested to use recombinant human ace-2 protein to saturate the viral s-protein and thus prevent cellular entry of sars-cov-2 [10] . this is especially of interest, since ace-2 has already been de-veloped as an experimental drug (apn01, gsk2586881) for adult respiratory distress syndrome (ards) and is therefore available for clinical trials. it was shown that ace-2 could successfully prevent lung injury by the original sars-cov-2 [16] . for ards, it had favorable effects in mice and piglets, but the results from a recent study in humans has been disappointing [35] [36] [37] [38] [39] . another crucial step in cellular infection involves the serine protease tmprss2 that cleaves the viral s-protein. the tmprss2 inhibitor camostat mesylate has been approved in japan for pancre-atic inflammation and has been shown to prevent cellular infection by sars-cov-2 [14] . consequently, it could prove being useful in covid-19. both ace inhibitors and at 1 receptor blockers have been used in diabetes and cardiovascular disorders to modulate this system in preference for vasodilation and maintenance of endothelial integrity. furthermore, in the diabetic kidney, reduced ace-2 protein expression could be prevented by ace inhibitor therapy suggestive of an upregulation with ace inhibitor [19] . it is worthwhile men-▶table 1 mortality (death) and morbidity (icu stay) associated with cardiometabolic disease based on reported cases in the literature. tioning that ace-2 receptor upregulation due to angiotensin receptor blockers that are commonly used in hypertension could be considered to be risk factor for increased transmission of sars-cov-2 . however, it has been seen that the at 1 receptors tends to associate with ace-2 in these cases, which prevent the internalization of the ace-2 receptor and hence may not be amenable to viral entry [40] . moreover, as mentioned earlier, higher expression of ace-2 was not associated with higher susceptibility to infection with sars-cov. according to the scientific data detailed above, it is tempting to speculate that blocking the ace-ang ii-at 1 receptor axis with at 1 blockers commonly used as antihypertensives may tilt the balance in favor of the ace-2-ang-(1-7)-mas axis, which may help to accelerate respiratory recovery in patients suffering from covid-19 [41] . however, it is intriguing that the patients most likely to be treated with ace-i or at 1 blockers are the ones with the poorest prognosis in covid-19 [1] [2] [3] [4] [5] [6] [7] . moreover, ace-2 upregulation concomitant with ras inhibitions may ease viral infection. consequently, it is also possible, that ras blockade may have a detrimental effect in covid-19. currently, there is no clinical data to support one or the other direction. thus, the heart failure society of america, the american college of cardiology, and the american heart association have all issued statement to suggest that there is no definitive evidence of harm or benefit with the use of ace-inhibitors or at 1 receptor blockers and that patients should continue to take their medications as usual [42] . it is of the utmost priority to evaluate the growing stock of clinical data from china and worldwide to determine if ras inhibitors are beneficial or deleterious in covid-19. only then, we can know how to counsel our patients taking ras inhibitors . in fact some researchers are planning a clinical trial to study losartan in covid-19 patients [43]. individuals with underlying hypertension, type 2 diabetes, or cardiovascular disease are at higher risk for respiratory failure and mortality in covid-19. one of possible mechanisms for this predilection may be the imbalance in the ace pathways and therapeutics targeting viral entry through the ace-2 receptor or the ace-ang ii-at 1 receptor axis as well as stimulating the ace-2-ang-(1-7)-mas axis may be beneficial in these individuals. further evidence needs to be gathered on whether modulation of the renin angiotensin system would be advantageous due to upregulation of mas activation or harmful due to the concomitant ace-2 receptor upregulation in the acute management of covid-19. special expert group for control of the epidemic of novel coronavirus pneumonia of the chinese preventive medicine association korean society for antimicrobial therapy, korean society for healthcare-associated infection control and prevention clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china for the china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease endocrine and metabolic link to coronavirus infection structural basis for the recognition of the sars-cov-2 by full length human ace-2 angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov-2 receptor ace2 and tmprss2 are predominantly expressed in a transient secretory cell type in subsegmental bronchial branches (preprint); biorxiv sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia a crucial role of angiotensin converting enzyme 2(ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 and new insights into the renin-angiotensin system the renin-angiotensin system and diabetes: an update angiotensin-converting enzyme (ace2) is a key modulator of the renin angiotensin system in health and disease ace2, angiotensin-(1-7) and mas receptor axis in inflammation and fibrosis upregulation of angiotensin converting enzyme 2 by shear stress reduced inflammation and proliferation in vascular endothelial cells local renin-angiotensin systems in the adrenal gland adrenal renin-angiotensin-aldosterone system in streptozotocin-diabetic rats local renin-angiotensin system regulates the differentiation of mesenchymal stem cells into insulin -producing cells through angiotensin type 2 receptor the role of the renin-angiotensin system in the development of insulin resistance in skeletal muscle ace2 exerts anti-obesity effect via stimulating brown adipose tissue and induction of browning in white adipose tissue binding of sars coronavirus to its receptor damages islets and causes acute diabetes radiology perspective of coronavirus disease 2019 (covid-19): lessons from severe acute respiratory syndrome and middle east respiratory syndrome pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer chinese clinical guidance for covid-19 pneumonia diagnosis and treatment pathological findings of covid-19 associated with acute respiratory distress syndrome breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 hypokalemia and clinical implications in patients with coronavirus disease 2019 (covid-19) individual variation of the sars-cov2 receptor ace2 gene expression and regulation the convalescent sera option for containing covid-19 ace-2 protects from severe acute lung failure mechanical stress and the induction of lung fibrosis via the midkine signalling pathway acute respiratory distress syndrome leads to reduced ratio of ace/ace-2 activities and is prevented by ang-(1-7) or an ang ii receptor antagonists a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome angiotensin ii mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin ii receptor type 1 receptor-dependent mechanism hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 the authors declare that they have no conflict of interest. key: cord-286638-bqxyb61p authors: singh, awadhesh kumar; gupta, ritesh; ghosh, amerta; misra, anoop title: diabetes in covid-19: prevalence, pathophysiology, prognosis and practical considerations date: 2020-04-09 journal: diabetes metab syndr doi: 10.1016/j.dsx.2020.04.004 sha: doc_id: 286638 cord_uid: bqxyb61p background and aims: high prevalence of diabetes makes it an important comorbidity in patients with covid-19. we sought to review and analyze the data regarding the association between diabetes and covid-19, pathophysiology of the disease in diabetes and management of patients with diabetes who develop covid-19 infection. methods: pubmed database and google scholar were searched using the key terms ‘covid-19’, ‘sars-cov-2’, ‘diabetes’, ‘antidiabetic therapy’ up to april 2, 2020. full texts of the retrieved articles were accessed. results: there is evidence of increased incidence and severity of covid-19 in patients with diabetes. covid-19 could have effect on the pathophysiology of diabetes. blood glucose control is important not only for patients who are infected with covid-19, but also for those without the disease. innovations like telemedicine are useful to treat patients with diabetes in today’s times. the disease burden of coronavirus infectious disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus-2 (sars cov-2) has been increasing continuously with more than a million confirmed patients and more than 45 thousand deaths globally [1] . with a high prevalence of diabetes, it is important to understand the special aspects of covid-19 infection in people with diabetes. this becomes even more important, as most parts of the world are seeing restrictions on mobility of patients in order to contain the pandemic. recently our group has published an article highlighting special considerations in the management of diabetes in today's times with covid-19 pandemic [2] . much more data from various parts of world has accumulated since then about the association between diabetes and covid-19, management of diabetes in those with covid-19 infection, and innovative strategies for medical consultation in view of limited access to healthcare facilities for patients with chronic diseases. this review aims to collate currently available data about diabetes and covid-19 infection. it specifically looks at the relation between diabetes and covid-19 in terms of epidemiology, pathophysiology and therapeutics. the review is updated till the time of writing; however, the data is evolving, and the conclusions made here might change later. we searched pubmed database and google scholar using the key terms 'covid-19', 'sars-cov-2, 'diabetes', 'antidiabetic therapy' up to april 2, 2020 . full texts of the retrieved articles were accessed. diabetes and associated complications can increase the risk of morbidity and mortality during acute infections due to suppressed innate and humoral immune functions. the levels of glycated hemoglobin (hba1c) > 9% have been linked to a 60% increased risk of hospitalization and pneumonia-related severity during bacterial infection [3] . past viral pandemics have witnessed the association of diabetes to increased morbidity and mortality. diabetes was considered as independent risk factor for complications and death during 2002e2003 outbreak of severe acute respiratory syndrome (sars-cov-1) [4] . similarly, the presence of diabetes tripled the risk of hospitalization and quadrupled the risk of intensive care unit (icu) admission during influenza a (h1n1) infection outbreak in 2009 [5] . during the 2012 outbreak of middle east respiratory syndrome coronavirus (mers-cov), diabetes was prevalent in nearly 50% of population and the odds ratio (or) for severe or critical mers-cov ranged from 7.2 to 15.7 in diabetic cohort [6] as compared to overall population. mortality rate in patients with mers who had diabetes was 35% [7, 8] . emerging data suggests that covid-19 is common in patients with diabetes, hypertension, and cardiovascular disease (cvd), although the prevalence rate varied in different studies as well in country-wise data. in the pooled data from the 10 chinese studies (n ¼ 2209) on characteristics of comorbidities in patients with covid-19, singh et al. [9] have reported a prevalence of hypertension, diabetes and cvd in 21%, 11%, and 7% patients, respectively. similarly, in a meta-analysis of 8 trials that included 46,248 covid-19 patients, yang et al. [10] reported a prevalence of 17%, 8%, and 5% for hypertension, diabetes and cvd respectively, in patients with covid-19. epidemiology working group of chinese center for disease control and prevention that investigated 20, 982 patients of covid-19 have shown that hypertension, diabetes and cvd were associated in nearly 13%, 5% and 4% of patients respectively [11] . in contrast, an italian study by onder et al. found diabetes in nearly 36%, while cvd was associated in nearly 43% of 355 patients admitted with covid-19 [12] . similarly, in a small study of 24 patients from united states, bhatraju et al. [13] reported diabetes to be associated with 58.0% patient with covid-19. while the study from the covid-19 surveillance group of italy (n ¼ 481) has shown that 34% patients with covid-19 had diabetes who died, the covid-19 response team from centers for disease control and prevention (cdc), usa reported a prevalence of 11% from the data of 7162 covid-19 patients [14, 15] . table 1 summarizes the prevalence of these comorbidities in all available studies to-date in patients with covid-19 [11e27] . it should be noted here that these findings could be a mere reflection of the high prevalence of diabetes across the globe including china (being the diabetes capital of the world), and thus causality cannot be inferred from the observed elevated proportions. evolving data also suggest that patients of covid-19 with diabetes are more often associated with severe or critical disease varying from 14 to 32% in different studies [15e18, 20, 22, 24] . wang et al. [20] in a study of 138 patients reported that 72% patients of covid-19 with comorbidities including diabetes required admission in icu, compared to 37% of patients without comorbidities. in an analysis of 201 patients with covid-19, wu et al. [24] table 2 summarizes the prevalence of non-severe (mild to moderate) to severe or critical disease in patients with diabetes with covid-19. fig. 1 illustrates the graphical representation of non-severe and severe covid-19 in patients with diabetes. interestingly, the prevalence of non-survivors was also higher in diabetic subjects with covid-19 and it varied from 22 to 31% in different studies [17, 21, 23, 24, 27] . table 3 summarizes the prevalence of survivors and non-survivors among patients with diabetes and covid-19. fig. 2 depicts the graphical representation of these data. in a univariate analysis of 191 patients with covid-19, zhou et al. [21] found diabetes to have an or of 2.85 (95% ci, 1.35 to 6.05; p < 0.001) for in-hospital mortality. however, this association of . however, the cfr was as high as 10.5% in patients with cvd, 7.3% in diabetes and 6.0% in hypertension [29] . based on these findings and acknowledging the higher morbidities and mortality associated with comorbidities, researchers have recently proposed that the course of treatment and prognosis of covid-19 should be stratified based on the absence or presence of co-morbidities in to type a, b and c. while type a represents covid-19 patients with pneumonia with no comorbidities, type b denotes covid-19 pneumonia with comorbidities; and type c denotes covid-19 pneumonia with multi-organ dysfunction [30] . 7. special aspects of pathophysiology of diabetes and relationship of anti-diabetic drugs in the context of covid-19 sars cov-2, like sars cov-1 utilises ace-2 as receptor for entry into cell [31] . ace-2 is expressed not only in the type i and ii alveolar epithelial cells in the lungs and upper respiratory tract, but also several other locations like heart, endothelium, renal tubular epithelium, intestinal epithelium, and pancreas. s-glycoprotein on the surface of sars cov-2 binds to ace-2 and causes a conformational change in the s-glycoprotein. this allows proteolytic digestion by host cell proteases (tmprss2 and furin) ultimately leading to internalization of the virion [32] . cellular entry of the virus triggers inflammatory response with recruitment of t helper cells which produce interferon g. this leads of recruitment of other inflammatory cells leading to a 'cytokine storm' which could lead to organ damage and multi-organ failure seen in severe disease. as discussed, diabetes is associated with poorer outcomes in covid-19. a study in 161 patients with covid-19 in wuhan found increased time for viral clearance in patients with diabetes [33] . apart from the usual mechanisms (impaired neutrophil chemotaxis and phagocytosis) by which diabetes predisposes to infections in general, there are several specific factors responsible for increased risk and severity of infection with sars cov2 in diabetes. a) increased ace-2 expression: diabetic mice have been found to have increased expression of ace-2 in renal cortex, liver and pancreas, but not in lungs [34] . recently, a phenome-wide mendelian randomization study found diabetes to be causally related to ace-2 expression [35] . though the significance of these observations is not clear at present, increased ace-2 expression might predispose people with diabetes to infection with sars cov2. b) increased furin: diabetes is associated with an increase in furin, which is a type-1 membrane-bound protease, belonging to the proprotein convertase subtilisin/kexin family (pcsk). it is involved in the entry of coronaviruses into the cell and increased furin has been reported in diabetes, which might facilitate viral replication [36] . c) impaired t-cell function: alterations in cd4 lymphocytes have been reported in animal models with mers [37] . lymphocytopenia has been observed in patients with covid-19 and correlated with prognosis [17] . d) increased interleukin-6 (il-6): several cytokines are increased in covid-19 infection. amongst these, il-6 is increased in diabetes and may play a more deleterious role in covid-19 infection [38] . monoclonal antibody against il-6 receptor (tocilizumab) is being tested in a trial in covid-19 [39] . ace-2 receptors are expressed in pancreatic islets and infection with sars cov-1 has been seen to cause hyperglycaemia in people without pre-existing diabetes. hyperglycaemia was seen to persist for 3 years after recovery from sars indicating a transient damage to beta cells [40] . though the similar effect has not been reported in covid-19, it may be important to monitor blood glucose levels in acute stage and during follow up. there is no data on the differential effects of oral antidiabetic drugs on the disease course in covid-19. metformin has antiproliferative and immunomodulatory effects by virtue of inhibition of amp activated protein kinase and has shown protective role in pneumonia in mouse models [41] . in one study in patients with tuberculosis, patients treated with metformin had better survival than those who did not receive metformin [42] . in a median 6.2 years of follow up of 5266 patients with diabetes, mendy et al. [43] showed that metformin was significantly associated with a decreased risk of mortality in patients with chronic lower respiratory diseases (hr: 0.30, 95% ci, 0.10 to 0.93), even after the adjustment for multiple confounding factors. in a study of 4321 patients with a follow up of 2-year period, ho et al. [44] showed metformin users had a significantly lower risk of death (hr, 0.46; 95% ci, 0.23 to 0.92), compared with non-metformin users, in patients with coexistent chronic obstructive pulmonary disease and diabetes. thiazolidinediones (tzd) seen to increase the risk of pneumonia in a study when compared to sulfonylureas [45] . experimental studies suggest that pioglitazone reduces steatohepatitis by increasing the ace-2 expression in liver tissues [46] . this purported increase in ace-2 expression and its relation to covid-19 has led some researchers to propose avoiding tzd in patients with diabetes and covid-19. experimental studies also suggest that liraglutide, a glp-1 receptor agonist increases the ace-2 expression in lungs in type 1 diabetic rat and improves right ventricular hypertrophy [47] . implications of these findings in the current context of covid-19 and its relation to anti-diabetic drugs is not yet fully clear. dipeptidyl peptidase-4 (dpp4) are tissue oligopeptides involved in multiple biological processes that include control of the activity of growth factors, chemokines and bioactive peptides and t-cell activation beside regulating glucose metabolism [48] . the relationship of coronavirus to this cellular type-ii transmembrane protein dpp4 (cd26) has generated a great interest recently. dpp4 serves as the receptor for mers-cov, in the same way as ace-2 is the receptor for sars cov and sars cov2 [49, 50] . experimental studies have suggested that certain polymorphisms of dpp-4 are associated with reduced chance of mers-cov infection [51] . this finding might explain the perplexing absence of mers-cov cases in africa, despite the presence of virus in camels, presumably because of frequent presence of protective polymorphisms of dpp-4 in africans [51] . moreover, this has generated an immense interest whether use of dpp4 inhibitors (dpp4i) can reduce the viral entry of mers-cov. in one in vitro study, sitagliptin, vildagliptin and saxagliptin could not block the coronavirus viral entry into cells [52] . though ace-2 is recognized as the main receptor for sars cov-2, a recent modeling study did not rule out its interaction with cd26 or dpp4 [53] . moreover, a possible interaction of dpp4 and renin-angiotensin system (ras) pathways seems to be plausible, although not completely studied. interestingly, dipeptidyl aminopeptidase i-iii cleaves the angiotensin ii (1e8) to angiotensin iii (2e8) and iv (3e8) which has cascading favorable effect through angiotensin-4 (at-4) receptors. similarly, various endo-and oligopeptidase cleaves angiotensin i (1-10) directly to angiotensin (1e7) which has a very favorable cascading effect. this suggest that a plausible interaction of non-specific dpp-4i with ace-2 is theoretically possible, and therefore, this area needs a future research. in this regard, some of the studies found that co-administration of angiotensin-converting enzyme inhibitors (ace-i) with dpp4 inhibitors led to an increased sympathetic tone and a consequent adverse hemodynamic effect [54e56]. there has been an interaction observed between ace-i and vildagliptin where a 4-to 5-folds increased risk of angioedema was noted, possibly due to the diminished degradation of bradykinin or substance p [57] . in contrast, in the experimental study, sitagliptin was shown to inhibit ace which could partially explain the purported beneficial cv effects [58] . dpp4 inhibitors have been associated with an increased risk of upper respiratory infections, however these agents have not been shown to lead to increased risk of pneumonia [45] . at present, there is insufficient evidence either for or against the use of dpp-4i in patients with diabetes and covid-19 [59] . glycemic control is important in any patient who has covid-19. though there is limited data about the association of blood glucose levels with disease course in covid-19 at present, data from other infections like sars and influenza h1n1 has shown that patients with poor glycemic control have increased risk of complications and death [60, 61] . most patients with mild infection and with normal oral intake can continue the usual antihyperglycemic medications. however, it is advisable to discontinue sglt-2 inhibitors because of the risk of dehydration and euglycemic ketosis. metformin may also need to be stopped if there is vomiting or poor oral intake. doses of other antihyperglycemic drugs like sulfonylureas and insulin may have to be altered depending upon the blood glucose levels. most hospitalised patient with covid-19, especially those with respiratory distress, would require insulin. ideally, patients with very poor oral intake or those on mechanical ventilation would require intravenous insulin infusion with frequent monitoring of blood glucose (every hour or every 2 h, see next section). however, the adjustment of infusion rates would necessitate visit to the patient and increase exposure to the medical personnel. there is a need to explore alternate insulin administration strategies. one of these is use of subcutaneous short acting insulin analogues, an approach which has been used successfully in mild to moderate diabetic ketoacidosis; however, its safety in critically ill patients is less clear [62, 63] . secondly, single dose of basal insulin has been attempted in critically ill patients as in one study from thailand. this could be an attractive option as it would reduce contact with the patient considerably [64] but needs more research especially in critically sick patients. finally, insulin pump or continuous subcutaneous insulin infusion (csii) could be an option and some models have the advantage of altering the insulin rates remotely via bluetooth [65] . fully automated closed-loop glucose control has been tried in critical illness and if feasible, could be useful in treating patients with covid-19 [66] . blood glucose monitoring poses a special challenge as it necessitates frequent visits to patient's bedside, especially if the patient is critically ill and receiving intravenous insulin. however, attempts could be made to minimize exposure. if the patient is not critically ill, he/she may be given a glucose testing device and selfmonitoring may be taught. blood glucose readings can then be communicated on phone and necessary action taken. continuous glucose monitoring (cgm) could be of help, especially the systems where the data can be remotely accessed without visiting the patient. though, there is some evidence of cgm interference with commonly prescribed medications like acetaminophen, atenolol and lisinopril, it has been shown to be useful and reliable in critically ill patients [67, 68] . treatment with ace inhibitors and arb has the potential to cause up regulation of ace-2 [69] . mice with coronavirus induced lung injury showed improvement when treated with an angiotensin receptor blocker, losartan [70] . a retrospective analysis showed reduced rates of death and endotracheal intubation in patients with viral pneumonia who were continued on ace inhibitors [71] . however, a contrary view is that increased expression of ace-2 could theoretically increase the risk of infection with sars cov-2. this could be a concern in people with diabetes who are at already elevated risk of infections because of many other factors. however, there is no evidence to support this hypothesis currently. in a retrospective analysis of 112 covid-19 hospitalised patients with cardiovascular disease in wuhan, there was no significant difference in the proportion of acei/arb medication between nonsurvivors and survivors [72] . in view of lack of robust evidence for either benefit or harm, it is reasonable for patients to continue using ace inhibitors and arb, as recommended by european society of cardiology council on hypertension, european society of hypertension and american heart association [73e75]. there are several studies about the protective effect of statins in pneumonia [76] . statins are known to increase ace-2 levels and may protect against viral entry of sars cov-2 [77] . however, this increase in ace-2 could be counterintuitive in the current context. nevertheless, statins are known to inhibit nuclear factor kappa b (nfkb) activation and might help in blunting the cytokine storm [78] . calcium channel blockers (ccb) have been shown to reduce severity of disease and mortality in patients with pneumonia, presumably by inhibiting calcium influx into the cell [79] . the precise role of these agents in covid-19 has not been studied, however it seems safe to continue these drugs for control of blood pressure in hypertensive patients. since ccb has no effect on ace2 expression, some researchers have proposed its preferable use in patients with covid-19 and hypertension [80] . though aspirin has anti-inflammatory properties, it may not be advisable to continue it in patients with sepsis and disseminated intravascular coagulation. however, in patients with underlying coronary artery disease, it needs to be continued as anticoagulant unless otherwise contraindicated. treatment of diabetes poses challenge in the current times when the world is going through an unprecedented pandemic. there are 'lockdowns" in most places with people confined at home. opportunities for exercise are limited and regular walks and visits to gyms or swimming pools are not possible. there is also considerable mental stress because of the unpredictability of the disease as well as social immobility. alterations in the daily routine affect the dietary intake as well. stress could lead to inappropriate eating. access to fresh fruits and vegetables could be limited and there may be a tendency to eat packaged foods high in calories, saturated fat and trans-fat. patients may find it difficult to procure medicines, insulin, needles and glucose strips etc. because of partial or complete lockdowns. the problem becomes more pronounced with elderly who are living alone. all these factors could cause glucose dysregulation and could predispose the patients to complications like infections, hyperosmolar coma, ketoacidosis and even acute cardiac events. care should be taken not to vary the calorie intake markedly. healthy balanced diet with good amount of protein, fiber and limitation of saturated fats is important to maintain a good glycemic control. 2. exercise should be continued. home based exercise like cycling, treadmill, stationary jogging and resistance exercise with small weights are beneficial. 3. regular intake of antidiabetic drugs and insulin is important and should be emphasized. 4. telemedicine can be very helpful in these times. patients can consult their physician via telemedicine and appropriate advice about treatment can be given [81] . an article dealing with telemedicine is under publication in the special issue. 5. care of feet should be emphasized in order to avoid foot related complications. there are telemedicine temperature mats which can screen for inflammation without having to visit the clinic. the patients who show inflammation can then be called to the clinic [82] . 6 . patients need to be educated about the need to visit the hospital urgently in emergency situations like vomiting, drowsiness, shortness of breath, chest pain, weakness of limbs, altered sensorium etc. diabetes is associated with increased incidence and severity of covid-19. there is experimental evidence of the effect of diabetes on viral entry into cell and inflammatory response to the infection. it is important to control blood glucose in patients who are infected with covid-19. treating diabetes at present with restrictions on movement is challenging; however, innovations like telemedicine can be useful in these trying times. we hereby declare that we have no conflict of interest related to this article. world health organization clinical considerations for patients with diabetes in times of covid-19 epidemic bacterial 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position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers esh statement on covid-19 hfsa/acc/aha statement addresses concerns re: using raas antagonists in covid-19 bin abdulhak aa. the use of statins and risk of community-acquired pneumonia hiding in plain sight: an approach to treating patients with severe covid-19 infection statins may decrease the fatality rate of middle east respiratory syndrome infection preadmission use of calcium channel blockers and outcomes after hospitalization with pneumonia: a retrospective propensity-matched cohort study are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? virtually perfect? telemedicine for covid-19 all feet on deck-the role of podiatry during the covid-19 pandemic: preventing hospitalizations in an overburdened healthcare system, reducing amputation and death in people with diabetes key: cord-349445-yh6ndtgm authors: mohammed el tabaa, manar; mohammed el tabaa, maram title: targeting neprilysin (nep) pathways: a potential new hope to defeat covid-19 ghost date: 2020-05-27 journal: biochem pharmacol doi: 10.1016/j.bcp.2020.114057 sha: doc_id: 349445 cord_uid: yh6ndtgm covid-19 is an ongoing viral pandemic disease that is caused by sars-cov2, inducing severe pneumonia in humans. however, several classes of repurposed drugs have been recommended, no specific vaccines or effective therapeutic interventions for covid-19 are developed till now. viral dependence on ace-2, as entry receptors, drove the researchers into ras impact on covid-19 pathogenesis. several evidences have pointed at neprilysin (nep) as one of pulmonary ras components. considering the protective effect of nep against pulmonary inflammatory reactions and fibrosis, it is suggested to direct the future efforts towards its potential role in covid-19 pathophysiology. thus, the review aimed to shed light on the potential beneficial effects of nep pathways as a novel target for covid-19 therapy by summarizing its possible molecular mechanisms. additional experimental and clinical studies explaining more the relationships between nep and covid-19 will greatly benefit in designing the future treatment approaches. coronavirus disease 2019 (covid19) is an infectious viral disease that is caused by a newly discovered coronavirus, namely severe acute respiratory syndrome coronavirus-2 (sars-cov-2) [1] . it is a virus belonging to order nidovirales of family coronaviridae, which are singlestranded rna viruses [2] and broadly distributed in both humans and other mammals [3] . majority of the people infected with 2019 novel coronavirus (2019-ncov) may recover without requiring special treatment [4] , except the elderly people and those with some medical problems such as hypertension, diabetes [5] and chronic respiratory disease [6] . such patients are more liable to be infected with covid-19 and might develop a fatal pneumonia with clinical presentation greatly resembling that caused by the previous beta-coronavirus sars-cov appeared in 2003; severe acute respiratory syndrome (sars) [7] . in such cohort, patients firstly complain of fever, dry cough, and at late stage, may suffer from dyspnea that makes them in a desperate need of intensive care unit (icu) admission and oxygen therapy [8] . however, the time between icu admission and ards development is recorded to be as short as 2 days [9] , being the reason for the high mortality rate associated with covid-19 at this stage [10] . till present, no covid-19-specific vaccines or medications are available, although some national medical authorities recommend testing the efficacy of antiviral medication in especially sever clinical trials [11] . in addition, several medical researchers suggest starting the supportive and symptomatic treatment till the new medications are developed [12, 13] . early evidences have shown that the pathogenicity for covid-19 may be enhanced with increased the body susceptibility to induce a phenomenon called cytokine storm, in which the immune system gears up by overreacting and producing more inflammatory mediators to fight the infection [14] . as a result, a severe inflammatory reaction is produced accompanied with significant damage, including organ failure [15, 16] . receptors [27] ; disrupting, of course, the normal physiological function of ace-2/ang (1-7)/masr axis of the renin-angiotensin system (ras) pathway [28] . therefore, researchers suggested that the use of angiotensin converting enzyme inhibitors (aceis) and/or angiotensin receptor blockers (arbs), may show a positive trend towards the severe inflammatory reactions and endothelial dysfunction caused by stimulating the function of ace/ang ii/at-1 axis and thereby, towards the bad pulmonary effects associated with the covid-19 infection [29, 30] . however, no clinical trial, till this time, has been proved its efficacy in preventing or even reducing the covid-19 severity [31, 32] . pursuing clinical trials with the absence of whole data describing the structure of covid-19 virus and its related mechanisms may lead to unsatisfied outcomes. as well, understanding such data will greatly help in determining the most appropriate effective treatment protocols. consequently, the first step to prevent and slow down the disease progression should involve the deduction of proper pathophysiology of that novel viral disease. identifying ace-2 as a viral entry receptor will emphasize on the important role of classical ras pathway in covid-19 pathophysiology, however, neither the link between ace-2 and other ras components nor their exact roles in the covid-19 pathogenesis have been neatly studied till now. one ras component, namely neprilysin (nep), has been established to potentiate the physiological beneficial role ascribed to the ace-2/ang (1-7)/masr axis; by an alternative pathway [33] . nowadays, nep has emerged as a pharmaceutical target for many drugs; especially those used in treating cardiovascular diseases and alzheimer's disease (ad) [34, 35] . considering the respiratory system, nep was reported to play a vital role in protecting lungs from inflammation and fibrosis [36] [37] [38] . a long time ago, mentioned that rats infected with common respiratory tract pathogens, such as parainfluenza virus type-1, rat coronavirus, and mycoplasma pulmonis showed low nep activity; resulting in an increase in their susceptibility to inflammatory responses [39] . however, the precise nep's role against life-threatening lung injuries has not been investigated yet. consequently, this current review aims to examine the cellular signaling pathways involving nep in covid-19 pathogenesis and to summarize the evidences supporting the potential beneficial effects of nep as a novel target for therapy. all previous and recent studies agreed with the fact that the surface of all human pathogenic coronaviruses is covered with crown like projections called spike (s) glycoproteins; giving the viruses their name [40] . in addition to s protein, another three main structural proteins have been recognized in sars-cov-2, namely envelope (e), nucleocapsid (n) and membrane (m) proteins [41] , figure (1) . specifically, s proteins are very important for sars-cov-2 infection to get started [42] . they possess two functional subunits by which the virus can enter into its target host cells involving; s1 subunit, which enables it to bind with receptors on the host cell surface and s2 subunit, that allows the virus to fuse into the cellular membrane [43] . unlike other coronaviruses, several experimental analyses proved that sars-cov-2 does not use the common known viral entry receptors, such as aminopeptidase n (apn) [41] and dipeptidyl peptidase 4 (dpp4) [44] , but, instead, can utilize ace-2 receptor [27] . additionally, it was revealed that transmembrane protease serine 2 (tmprss2) is very critical for the host cell entry of sars-cov-2 [45] and its plasma membrane fusion [46] resembling sars-cov [47] . firstly, the virus starts its destructive journey with binding of s1 subunit to ace-2 enzyme receptor on the cell membrane surface, which in turn activates tmprss2. activation of tmprss2 is followed by cleavage of both virus's s1 subunits from its s2 subunits and so of the ace-2 receptors. secondly, activated tmprss2 can also act on the s2 subunit, activating it through prompting an irreversible conformational change that will facilitate the virus-cell fusion [48, 49] . within this view, understanding the precise involvement ace-2, as a member of pulmonary ras pathway, in covid-19 pathophysiology may open new therapeutic possibilities for management. for years, ras was depicted as a hormonal circulating system involved in fluid and electrolyte balance, systemic vascular resistance and blood pressure regulation [50, 51] . however, a wealth of data showed that lung epithelial cells, fibroblasts and alveolar macrophages could also express the major constituents of the ras [52, 53] supporting the existence of a "local" pulmonary ras that can be differentiated from the "systemic" circulating ras [54] . as regards "local" ras in the lung, it has been reported to involve multiple cellular mechanisms affecting the vascular permeability, fibroblast activity and alveolar epithelial cells [55, 56] . by the time, it is well recognized that activating pulmonary ras can influence the pathogenesis of lung injury in different lung diseases such as pulmonary hypertension, acute respiratory distress syndrome (ards), asthma and pulmonary fibrosis [54] . as a consequence, pulmonary ras has been described to participate in disease process or play a protective role against tissue injury [57] and thus, modulating ras in the lung can successfully play a good role in the treatment of inflammatory lung disease [52, 58] . the functional steps of ras was firstly initiated by hepatic synthesis of a plasma globulin called renin substrate (or angiotensinogen) which could be enzymatically converted through renin secreted by juxtaglomerular cells of the kidney, into the biologically inactive decapeptide, namely angiotensin (ang) i [59] . subsequently, ras exhibits two main axes based on two distinct enzymes responsible for cleavage of ang i into angiotensin (ang) ii or ang 1-7 [51] , first axis involves generation of ang ii as the main effector via the angiotensin converting enzyme (ace) and named the classical vasopressor axis ace/ ang ii/ ang ii type 1 receptor (at1) [60] . for the second axis, angiotensin converting enzyme ii (ace-2) was identified as a depressor for ras activation through producing ang 1-7 [61] . henceforth, this axis ace-2/ ang 1-7/ mas-1 has become an important area of scientific research interest [62] . the ace/ang ii/at-1 axis has been well documented to drive a set of deleterious reactions in the lung through generating ang ii, the principal effector of this classical axis, involved in ras mediated vasoconstriction, sodium retention, fluid overload, inflammation and fibrosis [63, 64] . in addition, ace has been reported to evoke bradykinin and substance p degradation, two local pro-inflammatory and protussive peptides which can stimulate cough reflex and nitric oxide (no) release [65, 66] . specifically, pulmonary vascular inflammation contributes to a phenomenon called ace "shedding," in which endothelial surface-bound ace is released into the interstitium resulting in significant increase in the level of ang ii [67] , which explains the detection of higher ang ii level in covid-19 patients than normal [68] . several studies declared that ang ii could act through two distinct g protein-coupled receptors (gpcr) subtypes, angiotensin ii receptor type 1 (at1r) and type 2 (at2r), that were found to be expressed in human lung tissue; proving the local generation of ang ii in lung [65, 69] . the majority of actions evolved by ang ii could be mediated by at1 receptor via enhancing many complex intracellular signaling pathways including mapk/erk, plcb/ip3/diacylglycerol, tyrosine kinases, and nf-kb [70] . activating at1 receptor had been shown to further stimulate monocytes, macrophages and vascular smooth muscle cells to produce the proinflammatory cytokines such as tnf-α and il-6 [71] [72] [73] . on the contrary, at2 receptor was documented to have a number of counterregulatory interactions against lung tissue injury via inhibiting inflammation, and fibrosis [74, 75] . in addition to the known ang ii effects on promoting vasoconstriction, and pro-inflammatory cytokine release, there is also an increasing evidence that ang ii could induce vascular endothelial dysfunction [76, 77] , promoting the activation and aggregation of platelets and thereby, pro-thrombotic milieu [78] . ang ii could be associated with noticeable rise in the plasma level of endothelin-1 (et-1) [79] , which is a peptide secreted mainly from vascular endothelial cells (ecs), and basically works through the endothelin type a receptor (eta) in vascular smooth muscle cells and the endothelin type b receptor (etb) in ecs [80] . consequently, et-1 is described as the most potent bronchoconstrictor [81] and vasoconstrictor in the airways [82] . practically, et-1 acts as a key player of angiotensin ii-induced endothelial dysfunction and platelets activation via inducing il-6 release [83, 84] , which was documented to be correlated directly with the extent of endothelial dysfunction [85] . endothelial dysfunction is generally characterized by an imbalance between endotheliumdependent relaxing and contracting factors, attributed to reduced production/availability of nitric oxide (no); namely endothelium-derived relaxing factor (edrf) [86, 87] . no acts as a potent endogenous vasodilator that could prevent platelet aggregation and leukocyte adhesion to ecs [88, 89] . since il-6 would inactivate endothelial nitric oxide synthase (enos), it could disrupt no production [90] , decreasing its level and inducing a state of oxidative stress that may lead to ang ii-induced impairment in endothelial responses [91] postulating impaired endothelium functions as a principal factor in the pathogenesis of heart failure, hypertension and diabetes, it will be expected to classify the patients of such diseases as high risk groups for covid-19 development [92] [93] [94] . furthermore, platelets activation and aggregation may be noticed as a result of inflammatory reactions aggravated by endothelial dysfunction, leading to imbalance between coagulation and fibrinolysis [87] . it is clearly evident that continuing release of il-6 can enhance hepatic thrombopoietin (tpo) mrna expression resulting in thrombopoiesis stimulation and increase in circulatory platelets' numbers, known as (inflammatory thrombocytosis). within this context, et-1 can also mediate the synthesis of platelet activating factor (paf), a potent phospholipid mediator of platelet activation and aggregation, that may activate platelets to stick together and aggregate. as a result, a platelet plug is formed as an initiator for blood clotting and intravascular thrombus formation [88, 89] , which is considered as a starting point for developing stroke [95] . even though the high incidence of inflammatory thrombocytosis in covid-19 patients, the laboratory results of severe cases showed the opposite; suffering from thrombocytopenia [23] .the possible explanation is the depletion of both platelets and megakaryocytes as a result of multiple blood clots formed at the injured site, leading to less platelet production with more consumption as the disease severity increases [96] . interestingly, endothelial dysfunction and platelet activation can successively together worsen the severity of covid-19 infection. as known, both et-1 and activated platelets, associated with endothelial dysfunction, [97] could promote leukocytes rolling, adherence to endothelium, activation and migration into the inflammatory sites, sharing in enhancement of leukocytes recruitment [98, 99] . in addition, endothelial dysfunction can drive the fibrotic consequences following sars-cov-2 infection, developing pulmonary fibrosis as a result of releasing transforming growth factor-β1 (tgf-β1), the main fibrogenic cytokines implicated in pulmonary fibrosis, which could be induced by et-1 action [100, 101] . taken into consideration the numerous harmful effects possibly induced by ang ii during covid-19 pathogenesis, we found that most novel studies aim to use the anti-hypertensive drugs which act either by inhibiting the ace activity or by blocking at1 receptor, suggesting that action may mitigate the disease severity in covid-19 patients. on the other side, it has been proposed that enhancing the counter regulatory axis composed by ace-2/ang (1-7)/masr axis may be the most helpful. ace-2/ang (1-7)/masr axis, the depressor arm of ras, was identified to mitigate the deleterious actions mediated by ace/ ang ii/ at1 [51] . within this axis, ace-2 competes with ace by hydrolyzing ang i into the nonapeptide angiotensin (ang 1-9) which is further cleaved by the action of ace into heptapeptide angiotensin (ang 1-7), thus decreasing the amount of ang i available for ang ii generation by ace [102, 103] . to the same extent, ace-2 could also hydrolyze ang ii converting it into ang (1-7) [104, 105] . until the year 2000, ace has emerged as the key enzyme in the pulmonary ras, but this was challenged by the discovery of its homologue; ace-2 [106] which was found out to be broadly expressed in almost all types of lung cells, within the vascular endothelial and smooth muscle cells, types i and ii alveolar epithelial cells and bronchial epithelial cells [107, 108] . ace-2 could negatively regulate the ras in the lung through reducing ang ii/at1 receptor signaling and activating the counterregulatory ang (1-7)/ mas receptor pathway [54] . that finding was compatible with the animal studies showed that the use of ras inhibitors could effectively relieve the symptoms of acute severe pneumonia and respiratory failure [109] . consequently, the increased ace-2 was addressed as a target for protection from predisposition to inflammatory lung diseases such as, acute respiratory distress syndrome (ards) [108, 110] . concerning covid-19 pathogenesis, binding of sars-cov-2 to ace-2 resulted in exhaustion of ace-2, breaking the balance of the ras system within the lung and then, exacerbation of pulmonary inflammatory reactions [109, 111] . being ace-2 receptors widely expressed on vascular endothelial cells (ec) within the lungs [112] , sars-cov-2 can exploit them to induce and diffuse inflammatory cascades within endothelial cells disrupting their function; evidenced by the existence of viral elements and inflammatory cells within endothelial cells [113] . thus, distributing ace-2, as the main functional receptor for sars-cov-2, within human tissues will be the determinator for spreading of viral infection within the lung and other organs [108, 114] . both alveolar and bronchial membranes were reported to highly express ace-2 , which may explain the higher tendency of sars-cov-2 virus to harmfully affect lower airways than the upper ones [110, 115] . however, recent reports indicate that few covid-19 patients may also develop some signs and symptoms of upper respiratory tract infection (e.g. rhinorrhea, sneezing, or sore throat) [7, 17, 116] . furthermore, it was reported that ace-2 is also expressed in many epithelial cells of other organs than lung including kidney, blood vessels, intestine [110] and brain [117] . the fact which may explain the existence of some extra-pulmonary co-morbidities as myocardial dysfunction [30] and acute kidney injury (aki) [118] , gastrointestinal manifestations (diarrhea, vomiting or abdominal pain) [119] , neurologic manifestations (altered mental status or seizures) [120, 121] . therefore, increased ace-2 may be useless by promoting viral entry into lung cells, potentiating its devastating effect and enhancing mortality [122] . consistent with these findings, suggesting ace-2 activators such as, diminazene aceturate (dize) [65] , 4-[2-(4carbamimidoyl phenyl) imino hydrazinyl] benzene carboximidamide and xanthenone (xnt) [54] for counteracting covid-19 pathogenesis will be in vain. as a consequence, in order to diminish sars-cov-2 entry and its subsequent lung injury, pulmonary ace-2 activity should be reduced. however, at the same time, reduced ace-2 activity may contribute to worsening of the lung inflammation, by unopposed angiotensin ii accumulation [31] and forcing the ras to continuously increase the expression of cytokines [123] . simultaneously, a previous data indicated that the decrease in the ace-2 activity in a rat model of ards was paralleled by low amounts of ang-(1-7) [124] , which has been reported to play a beneficial role against pulmonary inflammation and fibrosis [125, 126] . in this context, another study stated that treatment with protease-resistant, a cyclic form of ang (1-7) (cang 1-7), could restore the ace-2 activity; attenuated the inflammatory response; decreased lung injury and improved lung function [124, 127] . that confirms the positive relationship between ang 1-7 and preserving the depressor role of ace-2/ang (1-7)/masr axis in pulmonary ras. hence, these data will attract the attention to the pivotal role of ang (1-7) in covid-19 pathophysiology and therapy. ang (1-7) is a biologically active metabolite of the ras that had become a peptide of interest in the last decade, because of its effective role in activating a number of crucial events for the homeostasis of normal physiological functions [128] . it was reported that ang (1-7) could exert various effects, which are greatly in opposition to those of at-1 receptor activation such as vasodilator, anti-inflammatory, anti-hypertrophy, anti-proliferative, anti-fibrosis and antioxidant effects [54, 129] . clinical and epidemiological studies have revealed the existence of a powerful link between the vasodilator effect of ang 1-7 and its higher plasma levels in females; making them less liability to hypertension than males [130, 131] . several mechanisms have been attributed to ang 1-7 in lowering blood pressure which can be explained as follows (i) triggering enos to stimulate the release of no, which could play a critical role in promoting the relaxation of blood vessels and inhibiting the platelet aggregation [132, 133] (ii) inducing natriuresis/diuresis [134] , and (iii) activating peroxisome proliferator activator receptors (ppars), which in turn supports the availability of no [135] . interestingly, ang 1-7 was documented to directly blunt the activation of pro-inflammatory signaling pathways induced by the ang ii-associated phosphorylation of mapks and nf-kb signaling [136, 137] , suggesting also the anti-hypertrophic effects of ang 1-7 through normalization of mapks activity [130] . moreover, ang (1-7), by counteracting the ang ii effects, could also preserve the endothelial function through increasing nitric oxide bioavailability and inhibiting oxidative stress [138] . taken together, ang 1-7 could also reduce lung injury by suppressing the expression of fibrogenic molecules such as tgf-β1 [139] , which acts as a key mediator involved in pulmonary fibrosis [140] . moreover, several researches have pointed out the antioxidant role of ang 1-7 [54, 130] that might be established by; (i) limiting the activation of nadph oxidase, which is a membranebound enzyme complex involved in triggering ros production through generating superoxide radicals [141] , and/or (ii) normalizing the expression of antioxidant enzymes such as catalase and heme oxygenase-1 (ho-1) [130] , as well as the nuclear factor erythroid 2-related factor 2 (nrf2), that acts as an emerging regulator of cellular resistance to oxidants [142] . other observation and experimental evidence suggested that endogenous aceis effects of ang (1-7) can be mediated by its unique membrane bound g protein-coupled receptor known as mas (masr) [143] , which was revealed to be found in the thin areas of the bronchial epithelium and smooth muscle [144] . masr could direct the ang 1-7 biological responses via the masr/camp/protein kinase a (pka) signaling [145] , which was previously validated by administrating ave 0991 (a nonpeptide mimetic of ang 1-7 as a specific ligand (agonist) for masr [130] . subsequently, attention should be drawn to specifically trigger the ang (1-7) as a potential therapeutic agent able to mitigate the lung injury in patients with covid-19 infection, without increasing ace-2 activity. there are different formulations of ang 1-7 are being developed e.g. ave-0991 [146] , hpβcd/ang 1-7 [147] , cgen-856 [148] , norleu3-a [149] and cyclic ang 1-7 [150] and used to demonstrate its therapeutic potential in numerous animal models of human diseases, including hypertension, heart failure, stroke, diabetes mellitus, atherosclerosis, renal disease and pulmonary arterial hypertension [130, 145, 151] . however, therapeutic attempts and clinical trials are still underway because of ang (1-7) rapid in vivo degradation by ace [130] . on the other hand, previous studies emphasized that ang (1-7) could induce vasodilatation in rats of mas-deficient vessels [152] and in rats pretreated with a779 (masr blocker) [153] . the former observed data suggested that ang (1-7) might also interact with an additional specific receptor other than masr to elicit vasodilatation [154] . indeed, ang (1-7) have been shown to stimulate also the bradykinin (bks) pathway via preventing the bk hydrolysis [155] . bks are one of the formed kinins that can play significant roles in regulating tissue injury, inflammatory responses and vascular permeability [156] . they have a little direct impact on the activation and recruitment of inflammatory cells, but they could work indirectly through stimulating the airway epithelial cells and lung fibroblasts through producing a wide array of cytokines, including il-6, il-1, il-8, granulocyte colony stimulating factor (g-csf), gm-csf and macrophage chemoattractant protein-1 (mcp-1) [157] [158] [159] [160] . bk action was mediated by g-coupled receptors, namely bk receptor (br) [156] that amazingly found to be interacted with the mas receptors in order to regulate the vasodilator effect of the ang (1-7) [161] . at the same time, it was found that the reduction in ace-2 mrna expression within the lungs of stz diabetic rats was linked with an increase in circulating ang ii, but without any significant change in the production of ang (1-7) [162, 163] , ensuring that pulmonary ace-2 was not the only enzyme responsible for ang (1-7) production, but there might be another enzyme contributing to its synthesis. by the time, the classical view of ras has been further expanded and become well established than previously conceived. as a consequence, it was discovered that there was another alternative pathway by which ang (1-7) is produced, instead of that based on ace-2. that way was disclosed to degrade ang i directly into ang (1-7) by another ras member, called nep. nep (neutral endopeptidase or neprilysin, previously known as cd10) is a member of transmembrane zink-metalloendopeptidase that particularly highly expressed in both kidney and lung [164] [165] [166] . nep was also found in a number of other tissues, as epithelia of breast, prostate, stomach and in the central nervous system [167] [168] [169] . nep had been also shown to be present in a soluble circulating form (cnep) within several body fluids including urine, cerebrospinal fluid and plasma [170] . although nep can discriminately hydrolyze a broad spectrum of physiologically relevant substrates, it was found to possess an obvious substrate specificity. classically, nep exhibits a size-related specificity that enables it to hydrolyze only peptides with a small molecular weight generally at or below 3,000 dalton [171] . as well, nep has been also described to show a distinction between substrates being cleaved 'in vitro' and that being cleaved 'in vivo'. initially, nep was reported to specifically cleave more than 7 peptides 'in vivo' including natriuretic peptides (nps) (atrial natriuretic peptide (anp), c-type natriuretic peptide (cnp), and b-type natriuretic peptide (bnp)), bks, neuropeptides (substance p, enkephalins), gastrin, chemotactic peptide formyl-met-leu-phe (fmlp), versus 26 peptides 'in vitro' such as il-lβ, oxytocin, gastrin-releasing peptide (grp), et-1, ang i and ii……etc [172] [173] [174] . by time, more substrates had been proposed with varying levels of 'in vitro' and/or 'in vivo' proof of cleavage. among that, grp, et-1, ang i which have been proved to be additionally metabolized 'in vivo' by nep [162, 175, 176] , emerging the evidence that nep could play an important role in many physiological and pathological conditions [177] , table 2 for cvs patients, nps were known to be of therapeutic importance in lowering blood volume by inducing natriuresis, in which excess sodium can be excreted in urine with accompanying water by the renal tubules [35] . previous clinitcal trials proved that administering synthetic nps might be associated with some limitations especially on the long run [178, 179] , that pushed them to depend on using neprilysin inhibitors (nepi) such as (thiorphan or candoxatrilat) to prolong and potentiate the beneficial effects of vasoactive/nps via inhibiting endogenous nps degradation [180] . nepi have been used for decades to treat acute diarrhea [181] . however, they are now developed and emerged as a pharmaceutical target for different cvs diseases. nepi have been used mainly in people with congestive heart failure and a reduced left ventricular ejection fraction (lvef) [182] . however, using nepi (e.g. candoxatril) solely for hypertension treatment was ineffective, because nepi could inhibit ang ii degradation, increasing its associated simultaneous detrimental effects. therefore, combining nepi either with acei (e.g. omapatrilat) or arbs (e.g. lcz696) become a necessity to overcome this limitation [183, 184] . another critical aspect for the pharmacological role of nepi was achieved in treating diabetes via inhibiting the breakdown of some substrates known to modulate glucose metabolism, such as incretin glucagon-like peptide-1 (glp-1), nps and bks [185] . within the brain, nep had also proved to hold a great beneficial role in the neurological disorders such as alzheimer's disease (ad) on both in vitro and in vivo studies [186] [187] [188] . multiple lines of evidence highlighted the presence of many amyloid plaques in the form of βamyloid (aβ) peptide in the brains of ad patients with dementia [189] . it was worthy mentioned that nep was one of the major aβ peptide-degrading enzymes in the brain, whose expression was documented to be lower in the brain of ad patients [190] supporting the role that nep can play in the prevention and treatment of ad. in addition to the above mentioned, nep seems to play a protective role in the pathogenesis of lung injury since significant decrease in the nep enzymatic activity in the lung of mice with ali was associated with inactivation of the tachykinins degradation pathway and consequently, reducing uncontrolled inflammation in ali/ards and in other neurogenic respiratory diseases [37, 191, 192] . of both respiratory bronchioles and alveoli [193, 194] , resulting in excessive production of grp [195] . grp was known to be one of the bombesin-like peptides that can be expressed and released by pnecs into the surrounding airway parenchyma in response to various stimuli like hypoxia or irritation [196, 197] to regulate the neutrophil chemotaxis and macrophage infiltration within the lung tissue [198, 199] . grp could act via stimulating gastrin-releasing peptide receptor (grpr) at the surface of macrophages, which in turn, would enhance the release of early inflammatory mediators contributing to the recruitment of neutrophils [200, 201] . more neutrophil infiltration within the lung is usually associated with high tendency for lung tissue damage [202, 203] , since they would be involved in breakdown of basement membrane integrity within the bronchiolar/alveolar architecture and thereby, diminution of pulmonary function. moreover, during the deleterious inflammatory reactions, as in pneumonia, neutrophil lifespan is prolonged to generate more superoxide radicals resulting in damage of the surrounding normal tissue [204] . interestingly, nep can play a vital role during lung inflammation through its catabolic effect on (grp) [205] in addition to its presence on the plasma membrane of neutrophils modulating their chemotactic responsiveness via cleaving the chemotactic peptide formyl-met-leu-phe (fmlp) which resembles an effective chemotactic agonist in response to grp [206, 207] . thus, breaking both grb and fmlp peptide by nep will cut the way for recruiting more neutrophils into site of injury and consequently, grab the reins. on the other hand, once neutrophils being activated at inflammatory sites, they could secrete high concentration of several serine proteases into the extracellular environment to degrade host pathogens, recruit more cytokines and stimulate further tissue damage [208, 209] . cathepsin g was reported to be one of the neutrophil-derived serine proteases that is abundantly found in the azurophil granules and known to degrade both angiotensinogen and ang i into ang ii [209, 210] . so far, nep can additionally take a part in decreasing the pro-inflammatory, oxidative and profibrotic effects of ang ii by minimizing the release of cathepsin g and consequently, its action on ang i [211] [212] [213] . as nep was reported to have more catalytic activity than ace-2 in cleaving ang i into ang (1-7), it could also effectively enhance the protective activities associated with ang (1-7) in the lung [214] . as well, nep could not affect lung ang (1-7) metabolism because it was involved in the metabolism of ang (1-7) within tissues other than pulmonary tissues, as renal cortex. on the other hand, ace was recorded to be the major enzyme responsible for ang (1) (2) (3) (4) (5) (6) (7) metabolism in the pulmonary membranes by hydrolyzing it to ang (1-5), and then, into ang (3) (4) (5) by the action of aminopeptidases [215] . besides, nep shows higher activity than ace towards bks degradation, resulting in inhibition of the bradykinin-induced inflammatory cells influx [125, 216] . despite multiple data confirming the expected role of nep in relieving the pulmonary inflammatory response, its effect on reducing exacerbation of acute severe pneumonia in covid 19 patients has not been highlighted yet. hence, the question now, may the reviewed actions of nep pathways be sufficient to impose a new effective strategy for covid-19 management as compared to the suggested repurposed drugs? guidelines only provide supportive care. however, many drugs repurposed based on host response in order to defeat covid-19, table 1 [217]. recently, there is an empirical use of anti-inflammation therapy in critical patients of covid-19 presented with severe complications in order to prevent further injury and suppress cytokine storm manifestations as ards and other organs damage till even death. main antiinflammatory medications include, non steroidal anti-inflammatory drugs (nsaids), corticosteroids, chloroquine and statins [22] . numerous observational data support a strong association between use of nsaids in management of lower respiratory tract infections and higher incidence of complications including fulminating pneumonia, pleural effusions and dissemination of infection [218] . nsaids may also induce nephrotoxicity among susceptible covid-19 patient groups and is exacerbated by fever and dehydration [219] . consequently, some studies recommend avoiding use of nsaids e.g. ibuprofen and diclofenac as the first choice for fever control and pain symptoms in covid-19 infection and using paracetamol instead [220, 221] . previous study demonstrated that corticosteroids-treated asthmatic patients showed enhanced nep expression in their airway epithelium as compared to nonsteroid-treated ones. this fits the hypothesis that the anti-inflammatory effect of corticosteroids within the airways may be partially mediated by upregulating nep [222] . despite of possible benefits gained from the anti-inflammatory effect of corticosteroids, they will be associated with serious impairment in the immune system of severe covid-19 cases [22] . corticosteroids may delay the viral elimination and increase susceptibility for the secondary infection resulting in deterioration of the disease especially with immune system impairment [223] . other side effects associated with corticosteroid treatment include hyperglycemia, central obesity and hypertension which represent an obstacle against their use in people at higher risk for covid-19 especially diabetic, cardiac and hypertensive patients [217] . auyeung et al., 2005 reported that there is no survival benefit for treatment of sars patients with corticosteroids [224] . another study on corticosteroid therapy of mers patients has revealed no mortality difference with delayed clearance of mers-cov from lower respiratory tract [225] . moreover, there is no evidence from any clinical trials supporting its administration for covid-19 [22] . in order to overcome the immune suppression induced by corticosteroids, a variety of other therapies have been developed to act directly as specific anti-cytokines (e.g. anakinra or tocilizumab) [10] or anti-inflammatory cytokines (e.g. il-37 or il-38) [17] without targeting the immune system and have proven to be effective in treating several syndromes that triggered by cytokine storm [123] . according to previous studies, chloroquine (cq) and its less toxic metabolite, hydroxychloroquine (hcq) possess anti-inflammatory and immunomodulatory benefits by affecting cell signaling in viral infections. cq/hcq also exhibit a wide variety of antiviral reactions against several viruses including members of the flaviviruses, retroviruses, and coronaviruses [25] . cq and hcq can prevent the attachment of viral particles to their cell surface receptor, modulate ph in order to inhibit ph-dependent steps of viral replication or interfere with posttranslational modifications (ptms) of viral proteins [217, 226] . an enough pre-clinical evidence considering cq effectiveness for treatment of covid-19 showed reduction in the pneumonia exacerbation, improving lung imaging findings and high rate of virus nucleic acid test negativity. accordingly, cq phosphate in guidelines (version 6) for treatment of covid-19 has been recommended with oral administration twice daily at a dose of 500 mg (300 mg for chloroquine) for adults and no more than 10 days [22] . however, there is a narrow margin between cq/hcq therapeutic and toxic dose. its poisoning has been favored and life-threatening in patients with cardiac disorders [227] . it is also contraindicated for people with retinopathy, elevated liver enzymes, heart rhythm disorders (as qt prolongation) or allergy to cq/hcq [228] . further, efficiency and safety of cq/hcq for covid-19 is still unclear and needs a confirmation depending on more preclinical and clinical trials [229] . statins are the widely used cholesterol lowering drugs, that were also reported to improve endothelial functions via lipid-independent mechanisms; mediated by their anti-inflammatory and anti-oxidant properties as well as their ability to restore vascular no bioavailability [230] . because of their immunomodulatory effect, they have also proven to be beneficial as adjuvant therapy in patients with different auto-immune inflammatory conditions (e.g. systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis) [231] . as a consequence, some hospitals included statins in the treatment protocol of covid-19. although statin therapy is usually well tolerated, their use in covid-19 patients may increase the incidence and severity of myopathies and acute kidney injury [232] . moreover, statin drugs may increase il-18 levels which can promote severe pneumonia, deteriorating sars-cov-2-induced ards and mortality, especially in elderly patients who are more likely to use these drugs [233] . sex differences in health outcomes following covid-19 may be attributed to sex-dependent production of steroid hormones. estrogen has been reported to attenuate inflammation which might protect women compared to men [234] . estrogen signaling are also known to downregulate mcp-1 expression and promote adaptive t cell response by stimulating neutrophil recruitment [235] . it has been also reported that serms, like toremifene, exhibits potential effects in blocking various viral infections as sarscov and merscov virus [236] through interacting with and destabilizing the virus membrane glycoprotein resulting in inhibition of its replication [237] . wang et al., 2020 suggests that treatment with estrogens and estrogen-related compounds as estradiol (e2) could suppress the expression of tmprss2 in the lung resulting in decreased mortality to sars-cov infection [235] . an estrogen receptors/ras interaction has been demonstrated in several studies. e2 was detected to drive ras to increase ang-(1-7) production through estrogen receptor (er α) mediated stimulation of ace1 and ace2 mrna expression and activity. a sex difference has been revealed in the expression of ras components [238] . there is a controversy about the role of ras blockers including, ace inhibitors and/or angiotensin ii type 1 receptor blockers (arbs) in treating covid-19 and their exact roles still remain unclear [28, 32, 239] . their use has been suggested to be of a value through increasing ace-2 that may have a protective effect against virus-induced lung injury by preserving ace-2 in competition with sars-cov-2 entry into the cells [28] . another mechanism by which ace-2 can prevent lung damage and attenuate the pulmonary fibrosis, will be via enhancing the ace-2/ang-(1-7)/mas axis to increase the production of ang (1-7), which in turn can counteract the activity of the ace/angii/at1r axis [137] . on the other hand, it was known that ace inhibitors could block the breakdown of bradykinin increasing its level and then, promoting its associated inflammatory reactions resulting in more deterioration in the health state [240] . additionally, it has been expected that patients receiving arbs may show upregulation in the membrane bound ace-2 facilitating the coronavirus entry and worsen then its course [241] . the suggested explanation may be attributed to the increase in angiotensin ii level that probably pushes it to act as an increased substrate loaded on the ace enzyme, resulting in shifting a part of ang ii to be converted by the action of ace2 into ang (1-7) , that may be associated with ace-2 upregulation [32] . other agents acting on the ras, such as beta-blockers and direct renin inhibitors (dri) to lower angi formation and thereby, angii and ang (1-7). however, till now, no one discussed their impact on the severity and prognosis of covid-19 [28] . on the contrary, lacking ang (1-7) will be of negative effect on lung health. so, there is an urgent need to suppose a pathway that can ensure the increase of ang (1-7) level without upregulating ace-2. that effect may be attained by keeping ace activity to enhance the pulmonary metabolism of ang (1-7) and at the same time, shifting the ras system away from ace/ang ii/at-1 axis to avoid its associated inflammatory and oxidative activities. we suggest that nep may achieve this complicated equation. based on previous literature that addressed several beneficial and protective effects displayed by nep during lung injury, we postulate that increasing nep activity may mitigate covid-19 pathogenesis. lung of covid-19 patients showed pneumocyte hyperplasia with inflammatory cellular infiltration [242] , confirming the release of excessive grp into the surrounding airway parenchyma as a result of pnecs hyperplasia [193, 194, 196, 197] . considering the documented links between high grp level and both neutrophil chemotaxis and infiltration as well as reduction in food and water intake [243] , it is not surprising to detect high neutrophil count [244] and anorexia in severe covid-19 patients [245] . thus, we expect that grp is the first spark in initiating neutrophils recruitment as well as cytokine storm, which are the main pillars in covid-19 pathophysiology. our suggested hypothesis herein depends on two main aspects, figure (3 nep may abrogate grp-induced neutrophil chemotaxis via cleaving grp and degrading fmlp peptide that can modulate the chemotactic responsiveness of neutrophils. on the other aspect, nep may withstand the potent cytokine storm, which was prescribed to be one of causes for lung damage progression and death in covid-19 patients. we suggest that nep can diminish the release of inflammatory cytokines that may increase sensitivity of target cells for further stimulation by sara-co-2 virus [246, 247] . thus, nep can improve lung histopathology and enhance tissue survival through two mechanisms: firstly, interfering with ang ii formation via preventing the proteolytic cleavage of angiotensinogen and ang i into ang ii by neutrophil-derived cathepsin g [209] [210] [211] that is expected to be released continuously in response to uncontrolled neutrophil activation associated with covid-19 patients and via regenerating the synthesis of endogenous ang (1-7), expected to be minimized because of ace-2 exhaustion by sara-co-2 virus [109] . ang (1-7) by itself may protect against pulmonary fibrosis through reducing tgf-β1 expression [139] . secondly, breaking bks and thereby, inhibiting its role in activation and recruitment of the inflammatory cells. however, recorded findings suggest that lung damage caused by covid-19 is induced by an alternative mechanism rather than hyperinfammatory injury. it likely seems that endothelial activation and associated pulmonary intravascular coagulopathy are the contributing factors in covid-19 pathogenesis [18] . within this context, nep may exert a critical role in suppressing et-1 that mediates angiotensin ii-induced endothelial and platelets dysfunction. yet, nep may minimize the chance for ang ii formation, which was reported to be a potent stimulator of et-1 in endothelial cells [248] . even, nep can additionally degrades et-1, preventing its associated inflammatory injury and eventual fibrotic cascade in the lung [81, 175] . furthermore, we speculate that nep may be helpful in dealing with individuals at high risk groups for covid-19 that exhibit many obstacles in their management. nep may regulate blood pressure in cardiovascular and hypertensive patients indirectly via decreasing both blood and tissue levels of ang ii by: (i) increasing ang i substrate availability a result of inhibiting cathepsin g-mediated neutrophil release, and (ii) augmenting the rate of ang i conversion into ang (1) (2) (3) (4) (5) (6) (7) , that previously reported to exert a stimulatory effect on anp secretion via masr [249] . for diabetic patients, we demonstrate that nep may regulate pancreatic ras flux to improve glycemic conditions. in response to nep-mediated degradation 'in vitro', nep may evoke the insulin secretory ability of ang (1-7) via hydrolyzing it into the biologically active ang (1) (2) dipeptide in pancreatic islets [33, 250] . numerous 'in vivo' and 'in vitro' experiments have been made to increase nep expression. one study speculated that dexamethasone could enhance nep expression in airway epithelial cells via promoting its transcription and synthesis [251] . another prior finding demonstrated that valproic acid could reduce plaque formation and improve learning deficits via up-regulating nep in app/ps1 transgenic mice [252] . recently, serotonin and its derivatives were explored to ameliorate symptoms of ad induced in mouse via enhancing nep up-regulation [253] . several lines of evidence identified that hormones such as androgens [254] and estrogen [255] could also positively regulate nep expression, suggesting that decline in levels of androgen or estrogen associated with aging would accompany by decrease in nep synthesis, which may be an important factor for increasing the risk of covid-19 infection among elderly. on the other hand, several findings investigated the up-regulating effect of some natural products on nep expression such as apigenin, luteolin, and curcumin, (-)-epigallocatechin-3-gallate as well as resveratrol [256] [257] [258] . in china, naoerkang (nek), a traditional chinese herbal medicine, improved the ability of learning and memory in rats model of ad by increasing nep expression in their hippocampal tissues [259] . however, some scientists aimed to produce recombinant nep (r nep) instead, as park et al., 2013 who prepared recombinant soluble nep from insect cells to be intracerebrally injected into ad mice [238] . therefore, we finally suggest that therapeutic strategies aimed to increase nep expression and/or activity may be of great benefit for prevention and treatment of covid-19. despite the high widespread of covid-19 contagion worldwide, there is no specific efficient treatment that has been proved till now. several pharmacological interventions for covid-19 have been suggested targeting the host's immune response. following sars-cov-2 exposure, there is an overproduction of grp within lung tissue resulting in increased release of inflammatory cytokines such as il-1β, il-6, tnf-α, vegf, gm-csf and mcp-1. such cytokines are widely known to enhance neutrophil infiltration which, in turn, induce lung inflammation and respiratory distress as reported in covid-19 infected patients. as well, cytokines release is also presumed to develop sars-cov-2 -associated pulmonary fibrosis. novel findings define covid-19 as one of the pulmonary diseases associated with endothelial and platelets dysfunction. now, it is definitely known that viral invasion can be mediated by one of the ras signaling system components, namely ace-2. hence, covid-19 occurrence and progression can be attributed to imbalance in the pulmonary ras signaling resulting from sars-cov-2-induced ace-2 drain. interestingly, the decrease in ace-2 activity will be accompanied with a decrease in the generation of ang1-7 which was known to be the light side of ras. regarding the data emphasized on the protective role of ang (1-7) in lung injury, it may be recommended as a covid-19 therapy, but because of its short half-life, ang (1-7) exhibits a limitation for its use. since nep is a more potent alternative way than ace-2 for producing ang1-7, it is suggested to assess the possible beneficial role of nep in covidinduced lung injury. few studies have discussed nep-mediated protective pathways in experimental models of lung injury and fibrosis, however its actual role as a lung protective therapy has not been yet recognized. nep has been involved in degradation of many peptides that may be incorporated in covid-19 pathophysiology. so, we expect that nep can effectively interfere with the chemotactic responsiveness and recruitment of neutrophils by degrading both fmlp peptide and gpr, respectively. furthermore, we suggest that nep can minimize cytokine storm associated with sars-cov-2 invasion through inhibiting ang ii formation by neutrophil-derived cathepsin g and directing ang i for generating ang (1-7) which can in turn suppress tgf-β1 expression and its fibrogenic action, protecting against fibrosis. degrading both bks and et-1 by nep may be associated with low il-6 levels, which will be beneficial for stabilizing endothelium and restoring its function. in addition to its catabolic properties, we postulate that nep may possess an advantage for covid-19 high risk patients through modulating blood pressure and glucose homeostasis. practically, numerous invivo and in-vitro experimental manipulations were made to upregulate nep expression either by using drugs (dexamethasone and valproic acid), hormones (androgens and estrogen) or natural substances (apigenin, luteolin, curcumin and (-)-epigallocatechin-3-gallate). however, others directed their efforts towards preparing the recombinant nep (r nep). because most pre-clinical and clinical studies within the medical field are interested in studying nep inhibitors, there is a little data concerning use of nep as a therapeutic agent. consequently, its associated adverse effects have not yet been studied well. finally, we hope our hypothesis will be somewhat enough to direct a future work towards the therapeutic role of nep in modulating covid-19 pandemic and to target the subsequent therapies for enhancing nep activity in covid-19 patients. the authors declare no conflict of interest. the authors and their institutions are the only responsible for the financial support and the content of this work in the submitted manuscript. all other authors have no conflict of interests to disclose. 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that initiate blood clot formation ace-2 = angiotensin-converting enzyme 2 ang (1-7) = angiotensin at-1 = angiotensin ii type 1 receptor et-1 = endothelin-1; fmlp = formyl methionyl-leucyl-proline grpr = gastrin-releasing peptide receptor pnecs = pulmonary neuroendocrine cells; sars-cov-2 = severe acute respiratory syndrome coronavirus-2 key: cord-005386-p37rw8dh authors: szolnoki, zoltán; maasz, anita; magyari, lili; horvatovich, katalin; farago, bernadett; somogyvari, ferenc; kondacs, andras; szabo, mihaly; fodor, lajos; bodor, anita; hadarits, ferenc; melegh, bela title: coexistence of angiotensin ii type-1 receptor a1166c and angiotensin-converting enzyme d/d polymorphism suggests susceptibility for small-vessel-associated ischemic stroke date: 2006 journal: neuromolecular med doi: 10.1385/nmm:8:3:353 sha: doc_id: 5386 cord_uid: p37rw8dh the renin-angiotensin system plays an important role in the maintenance of blood pressure homeostasis. the angiotensin-converting enzyme (ace) converts angiotensin i into angiotensin ii. angiotensin ii, which binds the angiotensin ii type-1 receptor (at1r), is a potent vasoconstrictor. on a pathophysiological basis, both ace i/d and at1r a1166c polymorphism lead to an enhanced activity of the angiotensin ii-at1r axis, thereby possibly contributing to circulatory disturbances. a mutually facilitatory effect may be presumed between the two polymorphisms. we examined whether this synergistic effect is involved in the evolution of different types of ischemic stroke. genetic and clinical data on 308 consecutive patients with acutely developing ischemic stroke were analyzed. a total of 272 stroke and neuroimaging alteration-free subjects served as a control group. univariate and logistic regression statistical approaches were used. the ace d allele combined with the at1r 1166c allele did not yield a risk of ischemic stroke. however, the co-occurrence of the homozygous ace d/d and at least one at1r 1166c allele was more frequent in the ischemic stroke group than in the control group (22.4 vs 11%, p<0.005, or, 2.33; 95% ci, 1.46–3.7). after specific subgroup analysis, this synergistic association was even stronger for small-vessel ischemic stroke (or, 3.44; 95% ci, 1.9–6.24; p<0.0005). multivariate logistic regression analysis of the data confirmed this association (adjusted or, 3.54, 95% ci, 1.88–7.16; p<0.0005). our results demonstrate that ace d/d and at1r 1166c polymorphism were associated with the development of small-vessel ischemic stroke through a mutually facilitatory interplay between them. genetic interactions might contribute to the altered functional network in renin-angiotensin system in vascular disorders. the renin-angiotensin system (ras) plays an important role in the maintenance of blood pressure homeostasis (agachan, 2003; henderson, 2004; miller, 2004) . the angiotensin-converting enzyme (ace) converts angiotensin i into angiotensin ii. angiotensin ii, which binds the angiotensin ii type-1 receptor (at1r), is a potent vasoconstrictor and a stimulator of cardiac growth nicholls, 2005) . both ace and at1r display polymorphisms that can alter their functions. the ace d/d polymorphism, associated with an elevated angiotensin ii level, has been demonstrated to play an important role in the development of ischemic stroke and leukoaraiosis (szolnoki, 2002 (szolnoki, , 2003 (szolnoki, , 2004 . the at1r a1166c polymorphism, which leads to an enhanced responsiveness of the at1r, has been suggested as a mild susceptibility factor for ischemic stroke (rubattu, 2004; brenner, 2005) . as both polymorphisms are associated with an enhanced activity of the angiotensin ii-at1r axis, we postulated at a clinical level, a synergistic effect between them as regards the evolution of ischemic stroke. we also analyzed to what extent the different subtypes of ischemic stroke are related to the gene-gene interaction the data on 308 consecutive patients with acutely developing ischemic stroke who had never suffered a previous stroke event were analyzed. these subjects had been admitted to our department of neurology and neurophysiology between january 1998 and 2004 after being examined by an internist in the local emergency unit or by a family physician at their homes. all 308 subjects underwent a detailed clinical scrutiny, including the medical history, the family history, an evaluation of vascular risk factors, general physical and neurological examinations, urine analysis, extensive laboratory examinations, electrocardiography, extracranial and transcranial doppler sonography of the brain-supplying arteries, transthoracic and/or transoesophageal echocardiography where appropriate, and magnetic resonance imaging (mri) examinations within 2 d after the onset of the symptoms. all scans were read by an experienced investigator without knowledge of the clinical and laboratory data. the patients were enrolled immediately after the clinical neurological and mri examinations. subjects on whom mris could not be recorded or for whom the examined clinical parameters and risk factors could not be obtained with certainty in consequence of some technical cause or death were excluded from the study groups. patients with atrial fibrillation and cardio-embolic source were also excluded in order to make the study groups more homogeneous. following evaluation of the clinical and radiological features, the patients were enrolled into one or other of three subgroups. group 1 corresponded to large-vessel infarction (cortical or cerebellar lesions and/or brainstem infarcts or subcortical hemispheric infarcts more than 1.5 cm in diameter on the mris, with a cerebral cortical impairment or a brainstem or cerebellar dysfunction); group 2 corresponded to small-vessel occlusion (one or more subcortical hemispheric or brainstem infarcts with a diameter of less than 1.5 cm on the mris, with one of the features of the traditional clinical lacunar syndrome and without a cerebral cortical dysfunction); and group 3 corresponded to a mixed vascular pathology (one or more lacunar and large-vessel infarcts on the mris, with lacunar syndrome or cortical/cerebellar/brainstem dysfunction). this classification based on the clinical and radiological features was considered to be the most exact and quantifiable method with regard to the requirement that the subgroups reflect the main well-defined vascular pathologies and their overlapping, which may possibly be affected by the examined mutations. polymorphism were associated with the development of small-vessel ischemic stroke through a mutually facilitatory interplay between them. genetic interactions might contribute to the altered functional network in renin-angiotensin system in vascular disorders. doi: 10.1385/nmm:8:3:353 index entries:ace i/d; at1r a1166c polymorphism; angiotensin; network; receptor; stroke. as a control group, 272 stroke and neuroimaging alteration-free caucasian hungarian subjects were examined. the controls were randomly selected by using a sex-matched technique from general practice registers from the same locality as the stroke cases, with the requirement that they had negative brain mri or computed tomography (ct) findings in order to avoid silent brain infarctions. they were healthy and believed to be free of cerebrovascular disease. subjects with any previous clinical data suggesting a cerebrovascular or cardiovascular event (such as transient schemic attack or angina pectoris) were excluded from the control group. both the controls and the patients gave their informed consent to the clinical work-up and the dna analysis. the study was approved by the local ethics committee. the smoking and drinking habits and the presence of hypertension or diabetes mellitus were recorded in all groups. the serum cholesterol level, the serum triglyceride level, the platelet count, and the hematocrit were also measured and analyzed as important clinical parameters. hypertension was diagnosed when the blood pressure repeatedly exceeded 140 mmhg systolic and/or 85 mmhg diastolic or when the patient was taking antihypertensive medication. diabetes mellitus was diagnosed when the glucose level was at least 7.78 mm in a fasting state and/or at least 11.11 mm 2 h after a meal or 75 g oral glucose loading, according to the world health organization criteria 12 (who, 1985) . ischemic heart disease was diagnosed when a history of angina pectoris or acute myocardial infarction was present or if there was electrocardiogram evidence of coronary heart disease. patients were classified as smokers if they had ever smoked more than five cigarettes per day for at least a year. patients were considered to be moderately heavy drinkers if they drank 40 g of alcohol or more per day. the body mass index was calculated as the weight in kilograms divided by the square of the height in meters. genomic dna was extracted from 200 µl of peripheral blood anticoagulated with edta by the desalting method (miller, 1988) . all blood samples were stored at -20°c until dna isolation. for at1r (genbank accession no. nt 005612) genotyping, a new primer set was constructed. the forward primer was 5´ aaaagccaaatcccact-caa 3´, and the reverse primer was 5´cagga-caaaagcaggctagg 3´. pcr was performed with an mj research ptc-200 thermal cycler. the reaction volume was 50 µl, containing 1 µlof dna(40-80 ng), 0.2 µm of each of the primers, 5 µl of reaction buffer (100 mm, tris-hcl; ph, 9.0; containing 500 mm, kcl; and 15 mm, mgcl 2 ), 200 µm of dntp, and two units of taq polymerase. the polymerase chain reaction (pcr) conditions were as follows: initial denaturation at 96°c for 120 s, followed by 35 cycles of denaturation (96°c for 30 s), annealing (53°c for 30 s), and extension (72°c for 60 s). pcr products were digested with 1.5 u of dde i (new england biolabs, kvalitex scientific, technological, trading ltd, budapest, hungary) at 37°c overnight. the restriction fragments were separated by electrophoresis on 3% agarose gels containing ethidium bromide and visualized by ultraviolet illumination. the at1r 1166aallele results in 58 and 374 bp fragments, whereas the at1r 1166c allele results in 58, 143, and 231 bp fragments. the ace polymorphism was examined by a pcr method developed in order to decrease the examination time and provide a better detection of heterozygotes (somogyvari, 2001) . this method consisted of fluorescent probe melting point analysis performed with fluorescently labeled oligonucleotide hybridization probes on the lightcycler™ instrument (roche diagnostics, roche kft, budapest hungary). the genetic examinations were carried out without a knowledge of the results of the clinical work-up. the clinical data were expressed as means ± sd where appropriate. the differences between the clinical parameters in the stroke group and the controls were assessed by using the χ 2 test or the mann-whitney test where appropriate. the stroke groups were tested against the control group for the frequencies of the different genotypes and their combinations by the χ 2 test. logistic regression models were evolved to evaluate the importance of the co-occurrences of the ace d and at1r 1166c alleles in ischemic stroke. the main genotype categories examined were the homozygous at1r 1166c/c and ace d/d polymorphisms (with a score of 1 for the homozygous state, and of 0 for the 356 szolnoki et al. neuromolecular medicine volume 8, 2006 heterozygous state and lack of the given allele); and the presence of at least one of the ace d and at1r alleles (with a score of 1 for both the homozygous and heterozygous states, and of 0 for lack of the given allele). for all the ors, the 95% confidence intervals (95% ci) were calculated. logistic regression analyses were performed with the statistical package systat 10 (chicago) for windows. as a consequence of the great number of statistical comparisons, there arose a chance of statistically significant associations being registered between ischemic stroke and a given combination pair of the genotypes by accident, without valid biological associations. in order to decrease this possibility, we randomly divided our groups into two identical-sized samples and carried out statistical calculations independently for them. we considered an association between ischemic stroke and the occurrence of a given genotype biologically valid if that association was calculated to be statistically significant (p < 0.05) in both independent samples (data not shown). this methodical approach could resolve the statistical problems that stem from exploratory multiple testing. the clinical data are listed in table 1 . the at1r 1166c allele did not occur significantly more frequently in the stroke subgroups (large-vessel, 51.3%; small-vessel, 51.7%; mixed type, 50.7%; overall, 51.3%) than in the controls (52.9%) ( table 2 ). the ace d/d genotype meant a minor risk of smallvessel ischemic stroke (adjusted or, 1.81; 95% ci, 1.1-3.12; p < 0.05) ( table 3 ). this result was not confirmed in the divided stroke groups (data not shown). co-occurrence of the at1r 1166c and ace d alleles did not consist of a significant risk of ischemic stroke (table 3) . a synergistic effect was found between the at1r 1166c allele and the homozygous ace d/d genotype in the small-vessel stroke subgroup (adjusted or, 3.54; 95% ci, 1.88-7.16; p < 0.0005), the mixed vascular type (adjusted or, 2.41; 95% ci, 1.2-5.1; p < 0.05), and the overall ischemic stroke group (adjusted or, 2.42; 95% ci, 1.51-3.82; p < 0.005) ( table 3 ). the synergistic effect as shown earlier was not confirmed in the divided stroke groups for the mixed vascular type stroke group (data not shown). because of the low number of the homozygous at1r 1166cc genotype, its dose-dependent effect could not be evaluated statistically in combination with the ace d allele and aced/d genotype. in our population, we did not reveal a direct association between the at1r a1166c polymorphism and ischemic stroke. the ace d/d genotype was an independent minor risk factor for small-vessel infarcts. because of the low significance level, this result of our present study would need to be confirm. however, this association was consistent with earlier findings (szolnoki, 2002) . the co-occurrence of the at1r 1166c and ace d alleles did not enhance the risk of ischemic stroke in any subtype as compared with the control group. the combination of the homozygous ace d/d genotype and at least one at1r 1166c allele increased the risk of ischemic stroke. after specific subgroup analysis, this synergistic association was even stronger for small-vessel-associated ischemic stroke. although there was a tendency to an increase in this genotype combination in large-vessel pathology-associated cerebral infarction, a significant interplay was not observed in this subtype. a similar synergistic association has been described in ischemic heart diseases (fukazawa, 2004) . there is no exact explanation for this additive effect. on a pathophysiological basis, however, 358 szolnoki et al. neuromolecular medicine volume 8, 2006 both unfavorable polymorphisms result in an enhanced activity of the angiotensin ii-at1r axis. this enhanced activity can lead to an endothelial dysfunction and vasoregulation disturbances (de ciuceis, 2005; watanabe, 2005) . the local vasoregulation of the cerebral blood flow takes place at a small-vessel level. a normal endothelial function is essential for this process. accordingly, an endothelial dysfunction leads primarily to small-vessel circulatory disturbances. this association explains why the synergistic interaction we observed led to small-vessel infarction. the presence of the ace d/d genotype produces an elevated ace level, and thereby yields an increased level of the angiotensin ii (malik, 1997) . there are data showing that the at1r 1166c allele is associated with an increased angiotensin ii responsiveness (van geel, 2000; jones, 2003) . both the elevated level of the angiotensin ii and the enhanced responsiveness of its target might comprise the molecular basis of the synergistic effect seen at the clinical level. this interplay might have practical implications for everyday clinical practice. increasing attention has recently been paid to primary stroke prevention, this centering on endothelial protection (larose, 2004) . modern antihypertensive drugs such as ace inhibitors and at1r blockers protect against an endothelial dysfunction, and also lower blood pressure (de gennaro, 2005; higashi, 2005; saavedra, 2005; schmieder, 2005; zhou, 2005) . on a pathophysiological basis, they might directly counterbalance the unfavorable effects of both ace d/d and at1r 1166c polymorphisms (de gennaro, 2005; a p < 0.05. b p < 0.005. c p < 0.0005. d adjusted ors of the at1r 1166c allele and clinical risk factors from the logistic regression models after adjustment for differences in body mass index, serum cholesterol, serum triglycerides, diabetes mellitus, smoking, drinking habits, and ischemic heart disease. higashi, 2005; saavedra, 2005; schmieder, 2005; zhou, 2005) . identification of the population at a higher risk of ischemic stroke in consequence of the combination of ace d/d and at1r 1166c polymorphisms may help in the selection of patients for whom ace inhibitors and/or at1r blockers, either alone or in combination, should be chosen as specific preventive tools against ischemic stroke or an endothelial dysfunction. the association between the risky haplotype of ras and drug responsibility necessitates further studies. angiotensin converting enzyme i/d, angiotensinogen t174m-m235t and angiotensin ii type 1 receptor a1166c gene polymorphisms in turkish hypertensive patients reninangiotensin-aldosterone system in brain infarction and vascular death reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin ii-infused macrophage colony-stimulating factor-deficient mice: evidence for a role in inflammation in angiotensin-induced vascular injury angiotensin-converting enzyme inhibition and angiotensin at1-receptor antagonism equally improve endothelial vasodilator function in l-name-induced hypertensive rats possible synergic effect of angiotensin-i converting enzyme gene insertion/deletion polymorphism and angiotensin-ii type-1 receptor 1166a/c gene polymorphism on ischemic heart disease in patients with kawasaki disease multiple polymorphisms in the renin-angiotensinaldosterone system (ace, cyp11b2, agtr1) and their contribution to hypertension in african americans and latinos in the multiethnic cohort angiotensin ii type i receptor blocker and endothelial function in humans: role of nitric oxide and oxidative stress angiotensin-converting enzyme 2 protects from severe acute lung failure genetic variants of angiotensin ii receptors and cardiovascular risk in hypertension a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury statins and endothelial dysfunction renin-angiotensin system: genes to bedside the impact of renin-angiotensin system polymorphisms on physiological and pathophysiological processes in humans a simple salting out procedure for extracting dna from human nucleated cells good ace, bad ace do battle in lung injury gene polymorphisms of the renin-angiotensinaldosterone system and the risk of ischemic stroke: a role of the a1166c/at1 gene variant brain angiotensin ii: new developments, unanswered questions and therapeutic opportunities mechanisms for the clinical benefits of angiotensin ii receptor blockers real-time pcr assay with fluorescent hybridization probes for exact and rapid genotyping of the angiotensin-converting enzyme gene insertion/ deletion polymorphism evaluation of the interactions of common genetic mutations in stroke subtypes evaluation of the modifying effects of unfavourable genotypes on classical clinical risk factors for ischemic stroke specific apo e genotypes in combination with the ace d/d or mthfr 677tt mutation yield an independent genetic risk of leukoaraiosis angiotenin ii type 1 receptor a1166c gene polymorphism is associated with an increased response to angiotensin ii in human arteries inhibition of the renin-angiotensin system prevents free fatty acid-induced acute endothelial dysfunction in humans angiotensin ii at1 receptor blockade abolishes brain microvascular inflammation and heat shock protein responses in hypertensive rats this work was supported in part by grant ett 237/2003. key: cord-267519-a0bcmjkn authors: bravi, francesca; flacco, maria elena; carradori, tiziano; volta, carlo alberto; cosenza, giuseppe; de togni, aldo; acuti martellucci, cecilia; parruti, giustino; mantovani, lorenzo; manzoli, lamberto title: predictors of severe or lethal covid-19, including angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers, in a sample of infected italian citizens date: 2020-06-24 journal: plos one doi: 10.1371/journal.pone.0235248 sha: doc_id: 267519 cord_uid: a0bcmjkn aims: this retrospective case-control study was aimed at identifying potential independent predictors of severe/lethal covid-19, including the treatment with angiotensin-converting enzyme inhibitors (acei) and/or angiotensin ii receptor blockers (arbs). methods and results: all adults with sars-cov-2 infection in two italian provinces were followed for a median of 24 days. arbs and/or acei treatments, and hypertension, diabetes, cancer, copd, renal and major cardiovascular diseases (cvd) were extracted from clinical charts and electronic health records, up to two years before infection. the sample consisted of 1603 subjects (mean age 58.0y; 47.3% males): 454 (28.3%) had severe symptoms, 192 (12.0%) very severe or lethal disease (154 deaths; mean age 79.3 years; 70.8% hypertensive, 42.2% with cvd). the youngest deceased person aged 44 years. among hypertensive subjects (n = 543), the proportion of those treated with arbs or acei were 88.4%, 78.7% and 80.6% among patients with mild, severe and very severe/lethal disease, respectively. at multivariate analysis, no association was observed between therapy and disease severity (adjusted or for very severe/lethal covid-19: 0.87; 95% ci: 0.50–1.49). significant predictors of severe disease were older age (with aors largely increasing after 70 years of age), male gender (aor: 1.76; 1.40–2.23), diabetes (aor: 1.52; 1.05–2.18), cvd (aor: 1.88; 1.32–2.70) and copd (aor: 1.88; 1.11–3.20). only gender, age and diabetes also predicted very severe/lethal disease. conclusion: no association was found between covid-19 severity and treatment with arbs and/or acei, supporting the recommendation to continue medication for all patients unless otherwise advised by their physicians. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 novel coronavirus disease (covid-19) is spreading worldwide, and has caused over 250,000 deaths so far [1] . the mortality rate varies widely by age and across individuals, ranging from 0.2% among healthy, young-adults, to >10% among older persons with pre-existing conditions [1] . although the pharmacological treatment was not assessed, the first observational studies on patients with severe disease reported a high prevalence of comorbidities that are often treated with angiotensin converting enzyme (ace) inhibitors, such as cerebrovascular diseases, coronary heart disease, hypertension and diabetes [2] [3] [4] . observing that human pathogenic coronaviruses bind their target cells through angiotensin-converting enzyme 2 (ace2) [5] [6] [7] [8] , and that a few studies reported an increase in ace2 expression mediated by angiotensin ii type-i receptor blockers (arbs) and ace inhibitors (more consistently on animals than in humans) [9] [10] [11] [12] [13] [14] [15] [16] , some hypothesized that the increased expression of ace2 would facilitate infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), thus the hypertension treatment with ace2-stimulating drugs, as well as ace2 polymorphisms, might increase the risk of developing severe covid-19 [17] [18] [19] . consequently, this would lead to a serious conflict regarding treatment, because ace2 reduces inflammation and has been suggested as a potential new therapy for inflammatory lung diseases, cancer, diabetes, and hypertension [17, [20] [21] [22] [23] . in the wake of two preliminary cohort studies reporting a lower [24] or similar [25] covid-19 mortality among inpatients hypertensive subjects treated with arbs and ace inhibitors, the potential predictors of covid-19 and of disease severity, including anti-hypertensive medications, were recently analyzed by a few observational studies [26] [27] [28] . with one exception [27] , no increased risk emerged from the use of arbs or ace inhibitors; however, the role of other potentially linked predictors, including age and cardiovascular comorbidities [17, 29, 30] , differed across the population analyzed, and still requires confirmation. we have performed a case-control study on all sars-cov-2 infected subjects diagnosed in two italian provinces, retrieving admission and pharmacological data up to two years before infection, in order to confirm the potential independent predictors of severe/lethal covid-19, including treatment with ace inhibitors and/or arbs. this case-control, retrospective study compared the proportion of subjects treated with arbs and/or ace inhibitors among three groups of subjects with sars-cov-2 infection: a. asymptomatic infection or mild disease, defined as fever or malaise plus at least one of the followings: sore throat, muscle pain, shortness of breath, dry cough, headache, conjunctivitis, and diarrhea [31] , with no hospital admission; b. severe disease, requiring hospital admission, not in an intensive care unit; c. very severe or lethal disease, requiring admission in an intensive care unit and/or causing death. the sample includes all subjects with diagnosis of infection made in the province of ferrara, up to april 2, and the province of pescara, italy, up to april 24, 2020, by the central laboratory of the university hospital of ferrara or the central laboratory of the pescara hospital (and confirmed by the national institute of health). all diagnoses were made using (real time) reverse transcription polymerase chain reaction (rrt-pcr) on oropharingeal specimens. the assays were those originally proposed by the charité-universitätsmedizin berlin institute of virology [32] , and then endorsed by the who [33] . the data on background pharmacological treatment up to the previous two years (january 1, 2018) were obtained from the national database of drug prescription, and integrated with clinical chart information for hospitalized subjects. data have been collected on the following drugs: ace inhibitors (atc classes: c09a and c09b), arbs (c09c and c09d), and insulin or other anti-diabetic drugs (a10). information on age, gender, and pre-existing conditions of all subjects were obtained through data-linkage with hospital discharge abstracts (italian sdo), which have been queried from the day of the diagnosis until january 1st, 2015. all admission data have been revised manually by two physicians (lm and mef) and the following conditions have been included in the analyses: malignant tumors, major cardiovascular diseases (heart failure, myocardial infarction and stroke-cvd), type ii diabetes, renal disease and chronic obstructive pulmonary diseases (copd, bronchitis, pneumonia, asthma, and emphysema). the study complies with the declaration of helsinki, the research protocol was approved by the ethics committee of the emilia-romagna region (code 287, approved on march 24, 2020), and the requirement for informed consent was waived because of the retrospective and pseudo-anonymized nature of the data. first, the differences across groups with mild, severe or very severe/lethal disease were evaluated using two-way anova with post-hoc tukey hsd test for continuous variables, and mantel-haenzsel chi-squared test for categorical ones. a sample restricted to hypertensive subjects was used to compare the subjects treated and untreated with ace inhibitors or arbs. multivariate logistic regression was used to investigate the potential independent predictors of severe or very severe/lethal covid-19. four models were built, two were restricted to hypertensive subjects (a and b), and two included the total sample (models c and d) and. models a and c were fit to predict severe or very severe disease (grouped together), while models b and d to predict very severe/lethal disease only (and repeated to predict death, with similar results, which have not been shown to avoid redundancy). all recorded variables (age, gender, cvd, diabetes, renal disease, cancer and copd) were included a priori in all models, with the exception of treatments with ace inhibitors and arbs, that were excluded from models c and d because of multicollinearity with hypertension. standard diagnostic procedures were adopted to check all models validity: influential observation analysis (dbeta, change in pearson chi-square and similar), multicollinearity, interaction terms, hosmer-lemeshow test for the goodness of fit and c statistic (area under the receiving operator curve). statistical significance was defined as a two-sided p-value<0.05, and all analyses were carried out using stata, version 13.1 (stata corp., college station, tx, 2014). our sample of 129 hypertensive subjects with severe/lethal covid-19, and 414 hypertensive subjects with mild disease or asymptomatic infection, had 80% statistical power to detect a difference of 20% or higher (corresponding to a relative risk �1.20) in the risk of severe/lethal disease between users of arbs or ace inhibitors (exposed group), and non users (controls). the sample consisted of 1603 subjects (mean age 58.0y; 47.3% males): 454 (28.3%) had severe symptoms, 192 (12.0%) very severe or lethal disease ( table 1 ). the sample of infected subjects, compared with the available estimates of the general population of the two provinces [34] , showed higher prevalence of diabetes (12.1% in the sample versus ffi5% in the general population); hypertension (33.9% versus ffi17%), copd (6.0% versus ffi3%) and cvd (16.1% versus ffi3%). 154 subjects deceased (mean age 79.3 years; 54.6% males); of them, 70.8% were hypertensive, 42.2% were diagnosed a cvd; 27.9% diabetics. twenty subjects with very severe disease were younger than 60 years; the youngest being a male aged 33 years. of those deceased, eight were younger than 60 years, and the youngest was a female aged 44 years. at univariate analysis, as compared to the subjects with mild disease, those with severe or very severe/lethal disease were significantly more likely to be older, diabetics, hypertensive, diagnosed with copd, cvd, and renal disease, and treated with arbs and/or ace inhibitors (all p<0.05). among hypertensive subjects (n = 543), however, the proportion of those treated with arbs or ace inhibitors (whose medication was not discontinued during the follow-up) 11-3.20) , and older age, which showed an exponential increase after 70 years: compared with the subjects younger than 50 years, the aors of those aged 70-79 and �80 years were 5.72 (3.81-8.58) and 9.06 (6.04-13.6), respectively (model c; table 3 ). only male gender, older age and diabetes also predicted very severe/lethal disease (model d; table 3 ). the main findings of this retrospective, observational study, are the following: first, it is confirmed that, among hypertensive subjects, the use of ace inhibitors or arbs up to two years preceding sars-cov-2 infection did not affect the severity of covid-19. second, older age, male gender, diabetes, and the presence of copd or cvd were independent predictors of severe disease, with a sharp increase of risk among subjects older than 70 years. third, only older age, male gender and diabetes were associated with a higher likelihood of very severe/ lethal disease. several hypotheses have been made on the association between covid-19 progression and treatment with ace inhibitors and arbs [20-22, 35, 36] . on one side, some asked whether the therapy should be discontinued during sars-cov-2 pandemic [17, 37] because covid-19 was strongly associated with hypertension, which is frequently treated with arbs and ace inhibitors [2] [3] [4] . it was indeed hypothesized that: (a) ace2 up-regulation mediated by arbs (and, to a lesser extent, by ace inhibitors) might increase patients' susceptibility to sars--cov-2 entry into host cells and further viral propagation [18, 19] , (b) virus binding to ace2 might reduce its activity, thus leading to increased levels of angiotensin ii and consequent pulmonary vasoconstriction, inflammation and oxidative organ damage, and increased risk of acute lung injury [20] . on the opposite side, other scientists suggested that, other than being harmful, arbs and ace inhibitors use in patients with cardiovascular risk factors and known or suspected covid-19 may even exert a beneficial effect, as ace2 up-regulation could increase the conversion of angiotensin ii to angiotensin-(1-7), a peptide with potentially protective anti-inflammatory properties [35, 36] . recently, a few large observational studies based upon in-and outpatient electronic health records [26] [27] [28] examined the association between antihypertensive medications and the risk of covid-19 and/or a severe/lethal disease: our results are in line with most of the previous findings on an absence of risk with ace inhibitors and/or arbs use. in brief, the studies by mancia et al [26] , and reynolds et al [28] reported no difference in covid-19 severity or death between treated and untreated subjects, for both arbs and ace inhibitors. instead, the results by mehta et al [27] were partially discordant: the study found no association between treatment with arbs and death, but those treated with ace inhibitors showed a significantly higher risk of icu admission. in our sample, in order to further investigate the potential beneficial effects of ace inhibitors, the impact of which might be larger in patients with diabetes, copd, or cardiovascular diseases, we performed additional analyses, stratified by comorbidities. we found however no significant differences in the risk of severe/lethal covid-19 among treated and untreated patients with either cvd, or diabetes and copd (data not shown). besides sample size and provenance, there were few differences between this and previous studies: all studies included all infected subjects, hospitalized or not, evaluated disease progression beyond mortality, and retrieved medications and admissions from electronic health records (with the exception of mehta et al, who assessed the medications exclusively at the time of testing for sars-cov-2 [27] ). overall, the present and previous findings confirm those from preliminary chinese cohorts [24, 31] , and although confirmation from randomized studies is required [38] , they strongly support the statements of several experts [39, 40] and scientific societies, including the european medicines agency [41] , the european society of cardiology [42] , and the american heart association [43] , who recommend continuation of arbs or ace inhibitors medication for all patients, unless otherwise advised by their physicians. with regard to the role of the other risk factors that have been suggested for severe covid-19, including age, male gender, hypertension, diabetes, copd, and major cardiovascular diseases, it has been correctly argued that, so far, available data were unadjusted, thus the relative importance of underlying health conditions was unclear [21, 29] . in this study, we found support for a potential role of gender, diabetes, copd and cvd, beyond age, in covid-19 progression to a severe disease, whereas only gender and diabetes significantly increased the risk of a lethal or very severe outcome. thus, the present study confirms prior findings on the independent relationship of older age and male gender with death [44] , and of copd with progression towards severe disease [26] . instead, at least two issues may have influenced the conflicting results on the role of cvd and copd in predicting very severe/ lethal disease (an association showed in some prior populations [44] -but lacking in the present as well as in other recent findings [26] ): first, the relatively small sample of the present study; second, a marked difference in the population here enrolled, as compared to previous studies which included randomly selected sars-cov2 negative subjects as controls [26] . given the present scenario, further population-based cohort studies, with longer follow-up are clearly needed [29] to clarify these findings. in addition to a relatively small sample, a limitation of the present study is the lack of tobacco smoking and body mass index among the variables that have been recorded, because we could not extract such information for half of the deceased subjects, as well as for many of those that were not hospitalized. other limitations are the lack of an evaluation of the severity of the underlying cardiovascular diseases, and the absence of data on other antihypertensive medications. however, their potential role in altering the relationship between arbs or ace inhibitors and risk of severe/lethal covid-19 remains unclear: none of the previous studies on the topic assessed cardiovascular diseases severity [26] [27] [28] , and the two studies that included the use of other antihypertensive drugs into multivariable analyses did not find substantial differences between the adjusted and unadjusted relative risks of death [26, 28] . in conclusions, the present study did not find any association between covid-19 severity and treatment with arbs, ace inhibitors, or both, and confirms previous findings in supporting the recommendation of several scientific societies to continue arbs or ace inhibitors medication for all patients, unless otherwise advised by their physicians, who should thus be reassured. the adjusted analyses substantially confirm prior reports, indicating that the risk of severe or lethal covid-19 largely and significantly increases among the elderly, males, diabetics, and those with copd or major cardiovascular diseases. covid-19 coronavirus outbreak clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of coronavirus disease 2019 in china clinical characteristics of 140 patients infected with sars-cov-2 in wuhan sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a crucial role of 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angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors. hypertension localization of ace2 in the renal vasculature: amplification by angiotensin ii type 1 receptor blockade using telmisartan are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? preventing a covid-19 pandemic: ace inhibitors as a potential risk factor for fatal covid-19 coronavirus disease 2019 (covid-19) infection and renin angiotensin system blockers covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: what is the evidence? drugs and the renin-angiotensin system in covid-19 renin-angiotensin-aldosterone system inhibitors in patients with covid-19 association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan renin-angiotensin-aldosterone system blockers and the risk of covid-19 association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease renin-angiotensin-aldosterone system inhibitors and risk of covid-19 covid-19: risk factors for severe disease and death case-fatality rate and characteristics of patients dying in relation to covid-19 in italy evaluation and treatment coronavirus detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr world health organization. laboratory testing for coronavirus disease (covid-19) in suspected human cases-interim guidance aspects of daily life-health status-regions and cities [aspetti della vita quotidiana: stato di salute-regioni e tipo di comune renin-angiotensin-aldosterone system inhibitors in patients with covid-19 renin-angiotensin system blockers and the covid-19 pandemic: at present there is no evidence to abandon renin-angiotensin system blockers covid-19 and the cardiovascular system inhibitors of the renin-angiotensin-aldosterone system and covid-19 switching antihypertensive therapy in times of covid-19: why we should wait for the evidence sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? european heart journal ema advises continued use of medicines for hypertension, heart or kidney disease during covid-19 european society of c. position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers patients taking ace-i and arbs who contract covid-19 should continue treatment, unless otherwise advised by their physician2020 opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients key: cord-269151-r426u5dz authors: turner, jeffrey m.; kodali, ravi title: should angiotensin-converting enzyme inhibitors ever be used for the management of hypertension? date: 2020-07-09 journal: curr cardiol rep doi: 10.1007/s11886-020-01352-8 sha: doc_id: 269151 cord_uid: r426u5dz purpose of review: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are commonly used anti-hypertensive medications in a number of clinical settings. they are often used interchangeably, but we pose the provocative question as to whether they should be. we review the literature to evaluate for any differences in efficacy between the two classes in order to determine if the greater side effects associated with angiotensin-converting enzyme inhibitors are offset by any advantageous effects on outcomes to warrant their use over angiotensin receptor blockers. recent findings: in many clinical scenarios, the data supports similar efficacy between ace inhibitors and arbs, while in a minority of others, there are murky signals from previous trials that suggest ace inhibitors may be better. however, when reviewing the literature in its entirety, and taking into account recently published pooled analysis and head to head trials, it is reasonable to conclude that ace inhibitors and arbs have similar efficacy. this is in contrast to data on adverse effects, which consistently favors the use of arbs. summary: from the available data, it is reasonable to conclude that ace inhibitors and arbs have equal efficacy yet unequal adverse effects. it is in this context that we take the provocative stance that ace inhibitors should not be used to treat hypertension. renin angiotensin aldosterone system (raas) plays a significant role in systemic blood pressure control. several studies have shown its importance since renin was first discovered in 1898 by tigerstedt and bergman [1] . subsequently, goldblatt et al. [2] first demonstrated the development of hypertension in dogs when renal artery stenosis was experimentally induced. the compounds responsible for this were consequently isolated, initially named hypertensin, but eventually named angiotensin [3] . eventually, through concerted efforts of various scientist groups, the whole cascade was progressively discovered. the first of the raas inhibitors to be introduced for widespread human use for hypertension were angiotensinconverting enzyme (ace) inhibitors. the first ace inhibitor approved by the fda was captopril in 1981 [4] . several ace inhibitors have since been synthesized and are in clinical use currently. angiotensin receptor blockers (arbs) were discovered more than a decade later with the first arb losartan being approved by the u.s. food and drug administration in 1995 [5] . both these classes of medications are used as antihypertensive agents in a variety of clinical conditions including essential hypertension, coronary artery disease, congestive heart failure, and chronic kidney disease. over the past 40 years, a wealth of data has been produced from a number of well-designed trials regarding their impact on disease outcomes as well as their side effect profiles. regarding side effects, the results disfavor ace inhibitor use, because although the overall incidence is rare, angioedema-related this article is part of the topical collection on hypertension deaths from these agents are significantly more frequent as compared to arbs. it is in this context that if we conclude the benefits on outcomes are equivalent between ace inhibitors and arbs, then agents from these two classes should definitely not be considered interchangeable. the question therefore being posed is, should ace inhibitors ever be used to treat hypertension? the therapeutic benefit of ace inhibitors and arbs is derived from their ability to disrupt raas, albeit the mechanism by which this occurs differs by class. the cascade of raas initiates the production of renin from the juxtaglomerular apparatus of the kidneys (fig. 1) . renin then converts angiotensinogen produced in the liver to angiotensin i. angiotensin i is subsequently cleaved by angiotensinconverting enzyme to angiotensin ii. angiotensin ii acts via the at1 receptor to directly promote vasoconstriction as well as sodium reabsorption in the proximal tubule of the kidney and via the at2 receptor to stimulate aldosterone release from the adrenal cortex. aldosterone independently acts in the distal nephron to regulate sodium and potassium homeostasis; in addition, aldosterone promotes end-organ fibrosis in the kidneys, heart, and vasculature, among other organ systems [6] . ace inhibitors block the activity of angiotensin-converting enzyme, thereby preventing the conversion of angiotensin i to angiotensin ii. inhibition of this enzyme also disrupts the breakdown of bradykinin, which in turn promotes vasodilation through the kinin-kallikrein-bradykinin system. elevated bradykinin is implicated in the pathogenesis of ace inhibitorinduced cough and angioedema. alternatively, arbs directly inhibit the binding of angiotensin ii to at1 receptors. unlike ace inhibitors, arbs do not significantly affect bradykinin levels, and therefore, cough and angioedema are much less common. drugs that inhibit raas were originally designed for the purpose of lowering blood pressure. given the mechanisms by which ace inhibitors and arbs reduce blood pressure are unique for each class, significant research efforts have focused on understanding their differences. the ontarget trial directly compared the blood pressure lowering efficacy of ramipril at a dose of 10 mg daily with that of telmisartan at a dose of 80 mg daily [7] . the mean blood pressure reduction at 6 weeks was − 7.5/− 5.0 mmhg in the group receiving telmisartan and − 6.4/− 4.3 mmhg in the group receiving ramipril, demonstrating that telmisartan was non-inferior to ramipril for lowering blood pressure in patients with vascular disease or diabetes. in another head to head study, fixed dose olmesartan 40 mg in combination with amlodipine 10 mg demonstrated superior central systolic blood pressure lowering and 24 h ambulatory blood pressure lowering effects when compared to fixed dose perindopril 8 mg in combination with amlodipine 10 mg [8] . additionally, the newer arb agent azilsartan was shown to be superior to ramipril for blood pressure lowering efficacy [9] . in elderly individuals (65-89 years fig. 1 mechanism of action of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. acei, angiotensin-converting enzyme inhibitor; arb, angiotensin receptor blocker; at1, angiotensin ii receptor type 1; at2, angiotensin ii receptor type 2; ace2, angiotensin-converting enzyme 2 old), olmesartan had a greater effect on lowering 24 h ambulatory blood pressure than did ramipril [10] . finally, in a systematic review of 47 head to head studies that reported comparative blood pressure effects between ace inhibitors and arbs, 37 demonstrated no difference, 2 favored ace inhibitors, and 8 favored arbs [11] . there was no overall significant difference between ace inhibitors and arbs in the pooled analysis. together, these data overwhelmingly conclude that the anti-hypertensive effects of arbs are not inferior to ace inhibitors. the arb myocardial infarction paradox theorizes that not only are arbs inferior to ace inhibitors for reducing cardiovascular events but arbs may actually increase these risks [12] . this theory originated in 2004 following the release of results from the value trial, which compared reductions in cardiovascular morbidity and mortality between valsartan and amlodipine [13] . the group treated with valsartan had a 19% relative increase in myocardial infarctions as compared to those treated with amlodipine (p = 0.02). similar findings were reflected in previous placebo-controlled arb trials at that time. the charm-alternative trial reported a 36% relative increase in myocardial infarctions with candesartan versus placebo in congestive heart failure (chf) population, and the scope trial demonstrated a non-significant trend toward higher myocardial infarctions in elderly subjects with hypertension who received candesartan versus placebo [14, 15] . these results were in stark contrast to previous placebocontrolled trials with ace inhibitors that demonstrated consistent cardioprotective benefits in various populations. more recently, a number of indirect comparisons with pooled analysis of several randomized placebo control trials have been done to further elucidate whether ace inhibitors and arbs have differing effects on the risk of myocardial infarction [16] [17] [18] [19] . these data also suggest a superior benefit of ace inhibitors over arbs for the reduction of myocardial infarction and/or cardiovascular death. however, while the theory of the arb myocardial infarction paradox has persisted, it is important to recognize that the abovementioned data are only part of the story, and despite it, there is still good reason to believe that ace inhibitors and arbs are in fact similar in their impact on myocardial infarction. first, it is important to recognize that a generational gap exists between many of the ace inhibitor placebo-controlled trials versus the arb placebo-controlled trials. most of the ace inhibitor trials occurred prior to the year 2000, and most of the arb trials occurred after the year 2000. this results in important differences between the standard clinical practices in place for primary and secondary prevention of cardiovascular events, namely statins and effective blood pressure management. due to this, event rates in a study population from the 1980s and 1990s are likely to be vastly different from those in the 2000s and 2010s. this was demonstrated in the meta-analysis by bangalore, which reported placebocontrolled trials conducted before the year 2000 (which were most of the ace inhibitor trials) had higher event rates in the control group as compared to those conducted after the year 2000 (which were most of the arb trials) (event rate 10.5% pre-2000 versus event rate 5.0% post-2000) [16] . the authors conducted a sensitivity analysis restricting the comparison of placebo-controlled ace inhibitor and placebo-controlled arb studies only to those conducted after the year 2000, and this showed similar cardiovascular event outcomes between the two anti-hypertensive classes. more importantly, we now have well-designed head to head randomized trials comparing the effects of myocardial infarction and cardiovascular death from ace inhibitors and arbs. the largest of these is the ontarget trial where more than 17,000 subjects were randomized to receive either telmisartan or ramipril as monotherapy [7] . in this study, there was no difference in the incidence of myocardial infarction or cardiovascular death observed between the two agents. finally, in the previously mentioned bangalore paper, a separate meta-analysis of the only head to head trials between ace inhibitors and arbs also showed no difference in the risk for myocardial infarction or cardiovascular death between the two classes [16] . while debate still exists regarding the differences of ace inhibitors versus arbs on the reduction of myocardial infarction and cardiovascular death, we feel the complete story of these data concludes there is truly no difference between the two classes. the role of arbs as first-line therapy in the treatment of heart failure with left ventricular dysfunction is also controversial. both european and american guidelines state that arb therapy in heart failure with reduced ejection fraction (hfref) should be reserved for those who are either intolerant of ace inhibitors or who remain symptomatic despite ace inhibitor therapy, and in the latter scenario, subjects with hfref should be treated with an arb in the form of an angiotensin receptor neprilysin inhibitor [20, 21] . these guidelines stem from differences in the robustness of available data on the allcause mortality benefit from treatment with ace inhibitors versus treatment with arbs in heart failure trials. in patients with left ventricular dysfunction, ace inhibitors have consistently shown significant reductions in both cardiovascular morbidity and mortality as well as all-cause mortality in placebo-controlled trials [22] [23] [24] [25] . because of these known mortality benefits of ace inhibitor treatment in hfref, the subsequently undertaken heart failure studies using arbs could not ethically be designed as placebo control trials in which ace inhibitor therapy was excluded outright in the placebo group. for this reason, there is much less available data assessing the all-cause mortality benefit of arbs as compared to placebo in subjects not taking an ace inhibitor. however, the limited data that does exist argues that the benefit is equal. the charm-alternative trial, which compared candesartan to placebo in patients with hfref who were intolerant to ace inhibitors, reported a significant reduction in cardiovascular mortality, chf hospitalizations, and all-cause mortality similar to those seen with previous ace inhibitor trials [15] . additionally, in randomized trials designed to compare ace inhibitors and arbs head to head in patients with hfref, the impact on all-cause mortality, as well as cardiovascular mortality and morbidity, was similar [26, 27] . finally, in a recently published network meta-analysis of 57 randomized control trials conducted over the past 30 years, a similar impact on all-cause mortality for ace inhibitor and arb therapies was reported [28] . therefore, despite the limited availability of data for all-cause mortality reduction with arbs in hfref, there still exists enough credible evidence to conclude that arb agents provide a similar benefit as compared to ace inhibitors for all-cause mortality, as well as other outcomes, in hfref. it is therefore reasonable to conclude these agents could be used interchangeably as first-line therapy solely based on their efficacy to impact outcomes. robust data support the benefit of using either ace inhibitors or arbs on improving renal outcomes in patients with chronic kidney disease with proteinuria. in those with diabetes and either microalbuminuria with preserved glomerular filtration rate or diabetes with chronic kidney disease (ckd), ace inhibitors have demonstrated benefit in reducing proteinuria, slowing progression of the disease, and delaying the time until reaching end-stage kidney disease in multiple studies [29] [30] [31] [32] [33] . angiotensin receptor blockers have shown similar benefits on renal outcomes in diabetics, albeit, the majority of these data are in type 2 diabetics [34] [35] [36] . in non-diabetics with ckd and proteinuria, improvement in renal outcomes has been demonstrated with ace inhibitors; however, studies with arbs in this population are notably lacking [37, 38] . in head to head studies, ace inhibitors and arbs had a similar benefit on renal outcomes in those with ckd [39, 40] . overall, there is no compelling evidence to suggest that there is superiority in renal outcomes in ace inhibitors compared to arbs. for those subjects who have previously had an ischemic stroke or transient ischemic attack, blood pressure reduction with anti-hypertensive medications is critical for secondary prevention. data from the hope study showed that ramipril is beneficial for secondary prevention of stroke based on subgroup analysis [41] . the progress study showed the combination of the ace inhibitor perindopril plus the thiazide diuretic indapamide to be more effective at blood pressure lowering and prevention of recurrent stroke than perindopril alone [42] . meanwhile, data from the ontarget study showed no difference in primary stroke prevention between telmisartan and ramipril [7] . there is a dearth of data available to directly compare the impact of ace inhibitors versus arbs on secondary stroke prevention, but overall, the consensus based on extrapolation from previous trials is that ace inhibitors and arbs are equal in this regard. this sentiment is also reflected in the 2017 acc/aha/hfsa hypertension guidelines [43] . the most common side effect in patients using ace inhibitors is cough. although not conclusive, it is thought to be induced by high levels of bradykinins due to inhibition of its degradation to inactive peptides. the reported incidence of cough with ace inhibitors varies widely in different studies but is generally accepted to be around 5-20% [44] . the incidence is higher among asian populations. a meta-analysis by matchar et al. showed that among randomized controlled trials in the analysis, the rate of cough in ace inhibitors group was 9.9% while arbs group had a rate of 3.2% (absolute risk difference of 6.7%) [11] . angioedema is another complication of raas blockers. although it occurs with much less frequency, it is a much more serious complication. in a metaanalysis by makani et al., the incidence of angioedema with ace inhibitors was 0.3% (95% ci 0.28-0.32), while the incidence with arbs was 0.13% (95% ci 0.08-0.19) [45] . the risk of angioedema was twice as high in ace inhibitors as compared to arbs (rr 2.2, 95% ci 1.5-3.34, p < 0.0001). there was no statistically significant difference between arbs and placebo. other reported side effects with raas blockers include hyperkalemia and acute kidney injury (aki); while these are rare in patients with normal renal function, the incidence progressively increases in patients with advancing stages of kidney disease. the burden of these adverse events is equal between ace inhibitors and arbs. one of the main reasons cited in favor of acei use in the past was the cost difference that existed. in the 1990s, acei had already become generic while arbs were just introduced and were much more expensive. as previously mentioned, losartan was the first arb to be approved by the fda in 1995. it is also the first arb to be made generic in 2010. since then, the majority of arbs have been made available as generic formulations as well and the prices have significantly dropped. although the prices vary quite significantly even within ace inhibitors and arbs, and whether they are generic or branded, the most used generic preparations are comparable in price between these groups and are equally affordable. hence, currently, when considering only the price of these medications in the generic market, there is no clear winner. in general, the availability of ace inhibitors and arbs is similar in the usa and other developed countries. however, it is worth mentioning that in 2018 and 2019, a series of recalls involving three arb agents, valsartan, losartan, and irbesartan, did have a temporary impact on the supply of these drugs [46] . the basis of these recalls involved the presence of probable carcinogens that were introduced through manufacturing processes from two factories in china and india. production of valsartan, losartan, and irbesartan from other manufacturers did not contain the identified carcinogens, and therefore, the recall did not remove the availability of these drugs from the market in their entirety. in total, about one-sixth of drug companies producing arbs in the usa were impacted. it is now well established that angiotensin-converting enzyme 2 (ace2) acts a receptor for sars-cov2, the virus responsible for covid-19, and enables its entry into cells [47] . ace2 is a homologous enzyme to ace and converts angiotensin ii into angiotensin 1-7 which has vasodilatory properties. several observational studies during the pandemic have noted that patients with hypertension are more susceptible to covid-19 infections and its complications including severe respiratory illness, icu stay, and death [48, 49] . given what is known about viral entry into cells, an indirect conclusion was drawn that raas inhibitors can increase susceptibility to covid-19 infection by increasing ace2 levels. however, many studies involving ace inhibitors and arbs have shown inconsistent results with ace2 mrna and enzyme level expression [50, 51] . direct evidence on the infectivity of the virus and its complications in patients taking raas blockers is sparse. on the contrary, based on data from a non-covid-related lung injury, it is postulated that elevated ace2 levels during covid-19 infection could be protective of the lung parenchyma, thus preventing more serious illness [52] [53] [54] . this has led various organizations including aha to recommend against discontinuation of ace inhibitors or arbs when diagnosed with covid-19. angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have resulted in significant improvements in outcomes related to congestive heart failure, cardiovascular disease, diabetes, stroke, and kidney disease. it goes without saying that the widespread use of these therapies has saved a countless number of lives. however, as we look toward the future, it is important to ask how we can continue to maximize the benefits and minimize the risk of therapies related to raas inhibition. it appears we may now be at a critical point in which enough data has amassed to allow us to conclude that the impact on clinical outcomes between ace inhibitors and arbs are equivalent, but the risk associated with these agents clearly favors using arbs. the loudest arguments against equivalency in outcomes between ace inhibitors and arbs have surrounded concerns regarding higher myocardial infarction risks with arbs, the so-called arb myocardial paradox. however, when the data is critically reviewed in the context of the different event rates that existed due to the generational gap between placebo control ace inhibitor and arb trials, in addition to looking at more contemporary data from head to head studies, these arguments do not hold. regarding outcomes in hfref, there are similar gaps between trials conducted with ace inhibitors versus arbs. as a result, there is less robust data regarding the improvement in all-cause mortality with arbs in hfref, despite there being similarities in reductions of cardiovascular death and heart failure hospitalizations. however, when closely examining the few trials that directly address this, including head to head studies and contemporary meta-analysis, the data concludes equal efficacy in all-cause mortality between the two classes in subjects with hfref. on the other hand, the literature consistently demonstrates a small but statistically significant worse side effect profile with ace inhibitors compared to arbs. while the overall risk of death from angioedema with an ace inhibitor is not high, it is high enough that it should still discourage their use over arbs. we therefore conclude that the use of arbs for raas inhibition is a superior therapeutic strategy over the use of ace inhibitors, and clinical practice patterns should shift in this direction. conflict of interest jeffrey m. turner reports personal fees from tricida. ravi kodali declares no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. niere und kreislauf1 studies on experimental hypertension : i. the production of persistent elevation of systolic blood pressure by means of renal ischemia synthesis of a biologically active octapeptide similar to natural isoleucine angiotonin octapep-tide1 angiotensin ii receptor antagonists contribution of aldosterone to cardiovascular and renal inflammation and fibrosis telmisartan, ramipril, or both in patients at high risk for vascular events the fixed-dose combination of olmesartan/ amlodipine was superior in central aortic blood pressure reduction compared with perindopril/amlodipine: a 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clinically proven protease inhibitor risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study differential regulation of renal angiotensinconverting enzyme (ace) and ace2 during ace inhibition and dietary sodium restriction in healthy rats localization of ace2 in the renal vasculature: amplification by angiotensin ii type 1 receptor blockade using telmisartan a pilot clinical trial of recombinant human angiotensinconverting enzyme 2 in acute respiratory distress syndrome angiotensinconverting enzyme 2 inhibits lung injury induced by respiratory syncytial virus angiotensin-converting enzyme 2 protects from severe acute lung failure publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-005927-a9sj00y8 authors: kondoh, gen; tojo, hiromasa; nakatani, yuka; komazawa, nobuyasu; murata, chie; yamagata, kazuo; maeda, yusuke; kinoshita, taroh; okabe, masaru; taguchi, ryo; takeda, junji title: angiotensin-converting enzyme is a gpi-anchored protein releasing factor crucial for fertilization date: 2005-01-23 journal: nat med doi: 10.1038/nm1179 sha: doc_id: 5927 cord_uid: a9sj00y8 the angiotensin-converting enzyme (ace) is a key regulator of blood pressure. it is known to cleave small peptides, such as angiotensin i and bradykinin and changes their biological activities, leading to upregulation of blood pressure. here we describe a new activity for ace: a glycosylphosphatidylinositol (gpi)-anchored protein releasing activity (gpiase activity). unlike its peptidase activity, gpiase activity is weakly inhibited by the tightly binding ace inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for gpiase differ from those for peptidase activity. ace shed various gpi-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. the released products carried portions of the gpi anchor, indicating cleavage within the gpi moiety. further analysis by high-performance liquid chromatography–mass spectrometry predicted the cleavage site at the mannose-mannose linkage. gpi-anchored proteins such as tesp5 and ph-20 were released from the sperm membrane of wild-type mice but not in ace knockout sperm in vivo. moreover, peptidase-inactivated e414d mutant ace and also pi-plc rescued the egg-binding deficiency of ace knockout sperms, implying that ace plays a crucial role in fertilization through this activity. supplementary information: the online version of this article (doi:10.1038/nm1179) contains supplementary material, which is available to authorized users. in mammals, more than 200 cell surface proteins with various functions, such as hydroxylation, cellular adhesion and receptor, are anchored to the membrane by a covalently attached gpi moiety 1, 2 . gpi deficiency causes developmental abnormalities, failure of skin barrier formation and female infertility in mice, indicating that a gpi anchor is essential for cell integrity [3] [4] [5] . patients with the acquired hematopoietic disorder paroxysmal nocturnal hemoglobinuria have defective biosynthesis of gpi in hematopoietic stem cells 1, 2 . the discovery of the piga gene and its mutations in paroxysmal nocturnal hemoglobinuria has opened the avenue for investigating the mechanisms involved in gpi biosynthesis, although the information on the metabolic system of gpi-anchored proteins is still limited 6, 7 . prions are infectious pathogens that cause fatal neurodegenerative diseases in both human and cattle through the modification of prion protein 8, 9 . because prion protein is a gpi-anchored protein and its release is strictly linked to its pathogenic role 10, 11 , studies on the general mechanisms and biological meaning of released gpi-anchored protein are necessary to establish new strategies for defeating these incurable neurodegenerative diseases. to investigate the fate of gpi in vivo, we previously developed gpianchored enhanced green fluorescent protein (egfp-gpi) transgenic mice and found a considerable amount of egfp-gpi in the extracellular fluid 12 . this phenotype prompted us to identify gpi-anchored protein releasing factors, in order to identify new mechanisms of gpi-anchored protein turnover other than the recycling between plasma membrane and endosomes 13, 14 . the angiotensin-converting enzyme (ace) is a well-characterized zinc peptidase that regulates bioactivities of circulating peptides such as angiotensin i and bradykinin as a dipeptidyl carboxypeptidase 15, 16 , leading to upregulation of blood pressure. two isoforms of ace, the somatic and testicular forms, have been characterized and both contain the zinc binding motif hexxh in the center of the active site 17 . its peptidase activity is completely abolished by either chelating the zinc ion or exchanging the conserved amino acid residues 18 . furthermore, ace inhibitors, which are widely used as antihypertensive agents, specifically bind and compete with substrate peptides at this active site, indicating that the active site of ace is indispensable for the peptidase activity 19 . because the molecular size of ace is rather large (150-180 kda and 100-110 kda for somatic and testicular isoforms, respectively), it is conceivable that the enzyme has other undiscovered functions. in this regard, a homolog of ace, the ace-2 (ref. 20) , which is also a zinc peptidase and acts as an antagonist for ace peptidase function, was recently found to act as a receptor for the virus that causes severe acute respiratory syndrome (sars) 21 . in this report, we describe a new function for ace; it has a gpi-anchored protein releasing activity and that it has a crucial role in fertilization through this activity. first, we established an assay system to monitor the gpiase activity, taking advantage of the temperature-dependent phase separation of the aqueous tritonx-114 solution, which can separate water-soluble released proteins (products) from detergent-soluble gpi-anchored proteins (substrates) at 37 °c. when gpi-anchored proteins are deprived of their lipid moiety, they shift from the detergent-soluble phase to the water-soluble phase. this assay system was used for monitoring gpiase activity through purification. we examined this activity in various organs using both egfp-gpi and the placental alkaline phosphatase (plap) as probes for gpi-anchored proteins and found a substantial activity in testicular germ cells (see supplementary fig. 1 online) . we also examined the expression of gpld1, the gene that encodes a gpi anchor-cleaving enzyme in mammals 22, 23 , in various tissues. because testicular germ cells do not express gpld1, this activity may be compensated by other factors (supplementary fig. 2 online) . therefore, we decided to identify the molecular entity of the enzyme responsible for gpiase activity in these cells. using serial liquid chromatography that started from detergent-soluble fraction of mouse testicular germ cells, we purified a 100-kda protein and subsequently identified it as ace by proteomics analysis (fig. 1a , table 1 and supplementary fig. 3 online). to confirm that this gpiase activity involved ace, we performed a similar assay using either a recombinant product of testicular isoform ace (ace-t; supplementary fig. 4 online) or a commercially available rabbit ace (ace-s) and purified plap as substrate ( supplementary fig. 5 online). the products released by this activity had the same size as those of bacterial phosphatidylinositol-specific phospholipase (pi-plc), a commonly used gpi anchor-cleaving enzyme, implying that this activity cleaves gpi-anchored protein located in proximity to the cleavage site of pi-plc (fig. 1b) . similar activities were shown by both molecules in a dose-dependent manner (fig. 1c , data not shown), confirming that ace was responsible for gpiase activity and did not require other factors for the action. to examine whether the ace gpiase activity is identical to the pi-plc activity, we used several pi-plc inhibitors, such as myo-inositol, inositol monophosphate and antibody specific for pi-plc, as well as plc inhibitors such as a23187, u-73122 and c48/80 on ace gpiase assay. none of these compounds had any inhibitory effects, even when used at a high dose (100 mm each), indicating that ace gpiase activity is not an endogenous pi-plc-like activity (data not shown). we also assessed the effect of ace-specific inhibitors, such as captopril and lisinopril, which bind to the catalytic center with ligation of its thiol to the zinc ion and completely inhibit the peptidase activity, but found only a minor inhibitory effect on the gpiase assay ( fig. 1c , 1 × 10 -3 m captopril produced 40% inhibition and data not shown). moreover, we assessed the effect of chloride ion, which activates the peptidase activity of ace 15, 16 , but found no effects on the gpiase activity (data not shown). we then assessed the metal requirement for gpiase activity. first, ethylenediaminetetraacetic acid (edta), which can remove various metal ions from proteins, was added at different doses to the plap conversion assay. because edta has some inhibitory effects on plap enzyme action 24 , immunoblotting was performed using antibody specific for plap to check for plap release. gpiase activity was inhibited by 1 mm of edta but not by an amount (10% less) at which peptidase activity was considerably inhibited (fig. 2a) . we also assessed the effects of other metal chelating reagents, such as trans-1,2-diaminocyclohexane-n, n, n´, n´-tetraacetic acid (cydta) and ethylene glycol bis (beta-aminoethyl ether)-n, n, n´, n´-tetraacetic acid (egta). the membrane-rich fraction of germ cells from mouse testis was solubilized in a buffer containing 1% triton x-100, centrifuged and the supernatant was collected and subjected to chromatographic fractionation. the plap conversion assay was performed on the eluted fractions and the maximum values are used here. all reactions were performed using pi-plc (1.0 iu/ml), the value of which was defined as maximum reaction. the peptidase activity was vigorously inhibited by 1 mm of each of these reagents, but found no inhibition of gpiase activity. to confirm the difference in gpiase and peptidase activities, we replaced glu414 with aspartate or replaced both his413 and his417 with lysine in acet ( supplementary fig. 4 online), which activates a water nucleophile as a general base or captures a zinc ion, thus becoming essential for peptidase activity 18 . these mutants showed only a trace peptidase activity but retained gpiase activity at a level comparable with that of wildtype ace-t (fig. 2b) . the gpiase activity of h413k-h417k mutant, which cannot capture the zinc ion at the peptidase active site, was also inhibited by high-dose edta but not by cydta or egta (fig. 2c) , suggesting that edta-induced inhibition of gpiase activity is mediated in a non-metal-chelating manner 25, 26 and that the gpiase action of ace does not require zinc ion. considered together, these results suggest that the active site element for gpiase activity differ from that for peptidase activity. to examine whether ace gpiase activity catalyzes gpi-anchored proteins in intact cells, we developed the f9 cell clone, which stably expressed egfp-gpi on the cell surface. although ace exerted little effect on egfp-gpi expression, pretreatment of cells with filipin, a commonly used reagent for lipid raft disruption 27 , allowed ace to shed egfp-gpi from the cell surface ( fig. 3a,b) . most of the gpi-anchored proteins are localized and packed in the lipid raft [28] [29] [30] . furthermore, increased accessibility of gpi-anchored proteins to shedding enzyme was observed upon disruption of lipid raft 31 . because filipin has no pharmacological effects on ace gpiase activity, as assessed by plap conversion assay (data not shown), exogenous ace seems to be prevented from accessing the substrate molecules by this membrane microstructure. in contrast, both ace and pi-plc had no effect on the transmembrane protein e-cadherin, implying that the activity of ace is unique for gpi-anchored protein shedding. moreover, ace displayed both time-and dose-dependent enzymatic characteristics (data not shown). we examined the effect of this activity on various endogenous gpianchored proteins including sca-1 and thy-1 in f9 cells; cd59 and the decay-accelerating factor (daf) in hela cells; and prion protein (prp) and cd59 in hek293 cells (fig. 3c) . all proteins, except transmembrane protein e-cadherin, were efficiently shed but at various degrees. in contrast to f9 cell molecules, gpi-anchored proteins on human cells were readily released from the cell surface without filipin treatment. to determine the ace cleavage profile on the substrate, we performed the following studies. first, we used radiolabeling to clarify whether the released molecules contained gpi components. we metabolically labeled the f9 cells expressing egfp-gpi with radioactive phosphate or ethanolamine, both of which could be incorporated into the gpi anchor moiety but not in the egfp protein. we treated cells with ace, pi-plc or mouse glandular kallikrein (mgk), which digests egfp protein near the carboxy termini (data not shown) and trapped the released products from the supernatants using antibody specific for gfp. following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transfer onto nitrocellulose membrane, we performed autoradiography followed by immunoblotting to detect egfp-gpi protein. the ace-released egfp-gpi was labeled with [ 32 p]-phosphate or [ 3 h]-ethanolamine, but not mgk-treated products, indicating the presence of a portion of gpi anchor structure in the ace-released molecules (fig. 4a) . the radioactivity of released products was about one-third when it was labeled on the phosphate and one-half on the ethanolamine compared with those of pi-plc-released molecules, suggesting that ace cleaves gpi anchor between mannoses or at the linkage between phosphate and the third mannose. we then examined the difference in the cleavage mechanism between ace and pi-plc. k562 human erythroleukemia cell line was treated with either enzyme to test the shedding of the surface daf. because an additional fatty acylation on the inositol hydroxyl is frequently found on mature gpi anchor 2 fig. 6 online) . we also attempted to identify the structure of the carboxyterminal peptides by high-performance liquid chromatography-mass spectrometry with trypsin treatment of the released egfp-gpi. under high-peptide coverage, we found a single charged fragment at massto-charge ratio (m/3) 660.5, corresponding to the carboxy-terminal peptide len, carrying an ethanolamine-phosphate-mannose structure, suggesting that ace cleaves gpi anchor between the first and second mannose of the gpi moiety (fig. 4b) . this conclusion was supported by the fact that its tandem mass spectra showed intense dehydrated ions of (m + h) + at m/z 642 and less intense ions at m/z 498 that underwent neutral loss of mannose-h 2 o. these results suggest that ace cleaves gpi anchor as an endo-mannosidase, different from pi-plc cleavage, which is more proximate to the protein. the most prominent phenotype of ace knockout mouse is male infertility. although the physiological parameters of ace knockout sperm, such as number, shape, viability, mobility and rates of capacitation and acrosomal reaction, were not different from those of wild-type sperm, they showed defective sperm-egg binding at the zona pellucida 33, 34 . to show a functional ace gpiase activity in vivo and assess its role in fertilization, we first checked the state of gpi-anchored proteins in the sperm. we collected epididymal sperm from both wild-type and ace knockout mice 33 and compared the distribution of gpi-anchored proteins in water-soluble and detergent-soluble fractions. in the sperm, the water-soluble fraction contains soluble ingredients of the acrosome, whereas the detergent-soluble fraction consists of membrane components. two gpi-anchored proteins, tesp5 and ph-20, were examined, because both proteins are released from the sperm during fertilization 35, 36 . immunoblotting showed both tesp5 and ph-20 in the water-soluble fraction of wild-type sperm but not in the ace knockout sperm (fig. 5a) , implying that ace participates in converting gpianchored proteins from the membrane-bound form to a soluble form. finally, we examined the effect of ace on sperm-egg binding. we prepared epididymal sperm of both genotypes and treated them with either wild-type or peptidase-inactivated ace (ace-e414d) or pi-plc and then incubated them with unfertilized eggs from c57bl/6 mice. these treatments had no effect on wild-type sperm-zona pellucida binding (data not shown). in contrast, treatment with wild-type or ace-e414d vigorously restored sperm-zona pellucida binding of the ace knockout mice (fig. 5b) . moreover, pi-plc treatment apparently restored the egg-binding ability of ace knockout sperm to a level comparable with both ace treatments, confirmed by inhibition with inositol monophosphate, a pi-plc-specific inhibitor (fig. 5c) . then, we transplanted the wild-type oocytes fertilized with ace-pretreated knockout sperm into pseudopregnant females to assess their developmental potential. normal ace +/pups were born (supplementary fig. 7 online) . these findings indicate that ace gpiase activity is crucial for the egg-binding ability of sperm. the major finding of the present study was the discovery that ace is a gpi-anchored protein releasing factor. furthermore, this activity of ace might also be different from that of gpi-pld, the only enzyme known so far to cleave gpi anchor in mammals 22, 23 . it has been reported that gpi-pld releases gpi-anchored protein only when it is expressed intracellularly in culture cells 23 . in contrast, ace efficiently released gpi-anchored protein from the cell surface. because gpi-pld cleaves gpi anchor at the phosphorus-oxygen bond of the phosphatidylinositol backbone 22 , inositol acylation may also prevent completion of gpi-anchored protein release by gpi-pld. indeed, gpi-pld could not release daf from the intact erythrocyte 37 . in contrast, a recent crystallographic study has shown that positioning of glu414 is consistent with its function and that ace has a single fissure-like groove with zinc buried in the center 19 . two histidines in the well-characterized core sequences of the catalytic site, hemgh and downstream glutamate 17 , are ligated with zinc. ace-specific inhibitors also bind here, implying that this structure is indispensable for the peptidase activity. on the other hand, the results of amino acid replacements and metal chelating experiments suggest that the microstructure necessary for the gpiase activity is different from that required for peptidase activity. high-performance liquid chromatography-mass spectrometry predicted that ace cleaves the mannose linkage in the gpi moiety. we searched for consensus motifs of glycosidase or lectin on the ace amino acid sequences in silico but found no significant similarities. because ace does not seem to cleave conventional sugar chain and specifically cleaves the mannose linkage of the gpi anchor, there might be some unique motifs for this activity. serial mutagenesis of ace could be used to determine such motifs. shedding of gpi-anchored proteins on the cell surface was accelerated by filipin treatment, suggesting that gpi-anchored proteins, the majority of which are packed in the lipid raft, were inherently protected from ace attack, whereas spontaneous disruption of lipid raft was suggested on sperm capacitation, which might be caused by cholesterol depletion 38 . we believe that the sperm capacitation process includes a similar reaction caused by filipin treatment of culture cells. in this regard, gpi-anchored proteins were not released in sperms of ace knockout mice (fig. 5a) . considered together, our results indicate that ace participates in the release of gpi-anchored proteins in vivo. both the peptidase-inactivated mutant of ace and pi-plc efficiently cured the phenotype of ace knockout sperm, indicating that the release of gpi-anchored protein is crucial for the sperm binding ability. in this regard, male mice lacking angiotensinogen and kallikrein were fertile, excluding the involvement of ace substrates such as angiotensin i and bradykinin in fertilization 39, 40 . based on these findings, our results suggest two scenarios: (i) functional activation of some gpi-anchored proteins on the sperm surface upon their release; (ii) exposure of a zona pellucida-binding factor after the shedding of some gpi-anchored proteins. by using a cell-surface biotinylation technique, we found that ace released several proteins from the membrane fraction of germ cells (g.k., unpublished data). these proteins might also contain gpi-anchored proteins that do not directly contribute to the sperm-egg binding, although how such proteins influence sperm-egg binding remains to be investigated. our results also showed shedding of cell surface prp by ace (fig. 3c) . with regard to the pathogenesis of prion-related diseases, shedding of surface molecules seems to reduce the chance of pathogenic conversion 10, 11 . moreover, a soluble form of prp also prevented the pathogenic conversion and prolonged the lifespan of scrapie-form prp (prp sc )-transferred mice 41 . based on this scheme, we propose that ace might be a promising compound that could prevent the production of pathogenic prion proteins. the peptidase-inactivated ace mutant, which reduced the side effect of peptidase activity, might be potentially useful for the treatment of prion disease. purification of gpi-anchored protein releasing activity. germ cells prepared from 500 testes of adult icr mice were crushed in a buffer containing 3 mm tris ph 7.4, 2 mm mgcl 2 , 1 mm edta, 0.25 m sucrose and complete protease inhibitor (boehringer mannheim), and homogenates were centrifuged at 100,000g. we solubilized the pellet in a buffer containing 20 mm tris, ph 8.0, 1% tritonx-100 and complete protease-inhibitor. we ultracentrifuged lysates (100,000g), and collected the supernatant. this sample was applied to serial liquid chromatography: (i) deae-cellulose (seikagaku); (ii) phenyl-sepharose cl-4b (amersham bioscience); (iii) cona-sepharose 4b (amersham bioscience); and (iv) tsk gel 3000sw (tosoh). to identify the 100-kda protein, the peak fraction of tsk gel chromatogram was separated by sds-page and stained with gelcode blue stain reagent (pierce). we excised the cognate band and digested in gel with 1.5 µg/ml trypsin (sigma) in 50 mm ammonium bicarbonate and 5 mm cacl 2 for 16 h at 37 °c. after eluting peptides with 50% acetonitrile/5% formic acid several times, we applied them to the capillary high-performance liquid chromatography (magic, michrom)/lcq ion-trap mass spectrometry (thermoelectron). a perfluorinated polymer-coated electrospray tip (fortis tip) was used 42 .the tandem mass spectra were subjected to database search using sequest and mascot engines. we performed two independent experiments with similar results. we prepared the sample for investigating the structure of released egfp-gpi as follows: we treated 1 × 10 9 f9 egfp-gpi-expressing cells with 10 µg/ml filipin/ phosphate-buffered saline (pbs; sigma) for 1 h at 0 °c and then with 1.0 µm of ace-s for 1 h at 37 °c. the released egfp-gpi molecules were trapped with an anti-gfp antibody column, eluted with 0.1 m glycine, ph 2.8, and subjected to sds-page. we stained the gels with copper stain and destain kit (bio-rad) and excised the cognate band. in our experiments, the amount of released egfp was about 30 µg. we treated all samples with trypsin and applied eluted peptides to the capillary high-performance liquid chromatography, eluted with acetonitrile gradient from 5% to 90% vol/vol and analyzed using the lcq mass spectrometer. the tandem mass spectra were subjected to database search. plap conversion assay on tritonx-114 partition. we prepared plap by expressing cdna in cos7 cells, extracted it using buffer containing 20 mm tris, ph 8.0, 150 mm nacl, 1% tritonx-114, complete protease inhibitor and collected the detergent-soluble phase after partitioning at 37 °c. plap was purified by deae-cellulose and anti-plap antibody columns. we measured plap activity with an alkaline phosphatase detection kit (nacalai tesque). the conversion reaction was performed in 100 mm tris, ph 7.5, 5 mm cacl 2 , 150 mm nacl and 0.3 iu/ml of plap for 90 min at 37 °c. we stopped the reaction by adding tritonx-114 at a final concentration of 2% and 1 mm edta, followed by microcentrifugation at 25 °c. the water-soluble phase was collected and plap activity was measured by the alkaline phosphatase detection kit. the gpiase activity represented plap activity released in the water phase using the extinction coefficient of the product (32.4 cm -1 mm -1 ) at 595 nm. to examine the metal requirement of gpiase activity, we added edta (nacalai tesque), cydta (dojindo) or egta (nacalai tesque) to the assay. after stopping the reaction, we subjected the water phase to immunoblotting using a polyclonal anti-plap antibody (biomeda) to detect released plap. immunoblotting. tissue extracts were prepared as described previously 12 . the protein-transferred membranes were probed with a rabbit polyclonal antibody against gfp (medical & biological laboratories), plap (biomeda), tesp5 (ref. 35) , ph-20 (ref. 36) , acrosin or mouse monoclonal antibody against fertilin-β and detected using the ecl system (amersham bioscience). ace samples. ace cdna was obtained by reverse transcription-polymerase chain reaction (rt-pcr) using mouse testis cdna as a template and primer pairs, 5´-tgaattccaccatgggccaaggttgggctactccagg-3´ and 5´-gaattcgtcacttatcatcatcatccttataatcctgctgtggctccag gtacaggc-3´. this encodes a flag-tagged version of the soluble testicular isoform. peptidase-inactivated mutants with amino acid glu414 replaced by aspartate or his413 and his417 replaced by lysine were synthesized by sitedirected mutagenesis using 5´-cttggtgatagcgcaccacgatatgggcc acatccagtatttcatgca-3´ and 5´-catggaggacttggtgatagcgc acaaggaaatgggcaagatccagt atttcatgc-3´ as mutation primers, respectively. the culture supernatants of transfected cos7 cells were collected and recombinant ace was purified by anti-flag m2-agarose affinity column (sigma) and tsk gel 3000sw (tosoh). in this study, we also used the somatic isoform of ace (ace-s) from rabbit lung (sigma a-6778). the ace peptidase activity was measured as described previously 43 . we measured both gpiase and peptidase activities of recombinant proteins and ace-s three times independently and obtained similar results. in the sperm of wild-type and ace knockout mice detected by immunoblotting. the acrosin and fertilin-β are indicated for water-soluble and detergent-soluble fraction controls, respectively. +/+, wild-type; -/-, ace knockout. ds, detergentsoluble fraction; ws, water-soluble fraction. (b) binding of ace-knockout sperm to the zona pellucida after various treatments. the amount of ace used for treatment was equivalent to the endogenous ace activity. (c) number of sperm bound to the egg. values are mean ± s.e.m. *p < 0.001, **p < 0.005, compared with buffer control; p < 0.3, comparison of wild-type ace with ace-e414d; p < 0.5, comparison of wild-type ace with pi-plc; p < 0.05, comparison of pi-plc with pi-plc + inositol-p (student's t-test). a b c gpi-anchor synthesis in mammalian cells: genes, their products, and a deficiency glycosylphosphatidylinositol (gpi)-anchored proteins developmental abnormalities of glycosylphosphatidylinositol-anchordeficient embryos revealed by cre/loxp system tissue-specific knockout of the mouse pig-a gene reveals important roles for gpi-anchored proteins in skin development infertility in female mice with an oocyte-specific knock of gpianchored proteins the cloning of pig-a, a component in the early step of gpi-anchor biosynthesis deficiency of the gpi anchor caused by a somatic mutation of the pig-a gene in paroxysmal nocturnal hemoglobinuria proc. natl. acad. sci. usa 95 mammalian prion biology: one century of evolving concepts filipin prevents pathological prion protein accumulation by reducing endocytosis and inducing cellular prp release phorbol ester-regulated cleavage of normal prion protein in hek293 human cells and murine neurons tissue-inherent fate of gpi revealed by gpi-anchored gfp transgenesis cholesterol-dependent retention of gpianchored proteins in endosomes gpi-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway angiotensin converting enzyme: implications from molecular biology for its physiological functions the angiotensin-converting enzyme gene family: genomics and pharmacology families of zinc metalloproteases the two homologous domains of human angiotensin i-converting enzyme are both catalytically active crystal structure of the human angiotensin-converting enzyme-lisinopril complex angiotensin-converting enzyme 2 is an essential regulator of heart function angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus primary structure and functional activity of a phosphatidylinositolglycan-specific phospholipase d posttranslational modification of glycosylphosphatidylinositol (gpi)-specific phospholipase d and its activity in cleavage of gpi anchors mutations of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase non-chelating inhibition of the h10n variant of human liver arginase by edta that zincing feeling: the effect of edta on the behaviour of zinc-binding transcriptional regulators rapid cycling of lipid raft markers between the cell surface and golgi complex functional rafts in cell membranes functions of lipid rafts in biological membranes gpi-anchored proteins are organized in submicron domains at the cell surface sphingolipid deficiency induces hypersensitivity of cd14, a glycosylphosphatidylinositol-anchored protein, to phosphatidylinositol-specific phospholipase c mammalian glycosylphosphatidylinositol anchor transfer to proteins and posttransfer deacylation male-female differences in fertility and blood pressure in ace-deficient mice angiotensin-converting enzyme and male fertility a mouse serine protease tesp5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein a hyaluronidase activity of the sperm plasma membrane protein ph-20 enables sperm to penetrate the cumulus cell layer surrounding the egg lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase c molecules involved in mammalian sperm-egg interaction genetic manipulation of the rennin-angiotensin system decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice soluble dimeric prion protein binds prp sc in vivo and antagonizes prion disease properties of an electrospray emitter coated with material of low surface energy colorimetry of angiotensin-i converting enzyme activity in serum distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria the mean fluorescence value of pi-plc-treated population was defined as the maximum shedding and that of ace(-) as no shedding. we performed three independent experiments and obtained similar results.radiolabeling analysis. we metabolically labeled f9 cells expressing egfp-gpi with 0.2 mci/ml of [ 32 p]-orthophosphoric acid (amersham bioscience) or 0.1 mci/ml of [ 3 h]-ethanolamine (amersham bioscience) for 16 h. filipin-pretreated cells were incubated with 1.0 µm of ace-s, 1.0 iu/ml of pi-plc or 10% lysate of mouse submaxillary gland (containing mgk) for 1 h at 37 °c. released egfp was immunoprecipitated with antibody specific for gfp, subjected to sds-page and transferred onto nitrocellulose membrane. we evaluated the quantity of egfp-gpi protein by measuring the density of bands detected on egfp immunoblotting by using a densitometer (molecular device) and determined the radioactivity of the cognate band using a liquid scintillation counter (aloka). three independent experiments were performed with similar results.sperm-egg binding assay. all gametes were handled as described previously 35 we thank k. ohishi and y. tashima for technical assistance, t. baba for providing anti-tesp5 antibody and p. primakoff for providing anti-ph-20 antibody. we are also grateful to v.w. keng and d.g. myles for the critical reading of the manuscript. this work was supported by grants from the osaka medical research foundation for incurable diseases and the ministry of education, science, sports, and culture of japan. the authors declare that they have no competing financial interests. key: cord-005931-iggkxbbf authors: phillips, m. ian; de oliveira, edilamar menezes title: brain renin angiotensin in disease date: 2008-04-02 journal: j mol med (berl) doi: 10.1007/s00109-008-0331-5 sha: doc_id: 5931 cord_uid: iggkxbbf a brain renin angiotensin system (ras) and its role in cardiovascular control and fluid homeostasis was at first controversial. this was because a circulating kidney-derived renin angiotensin system was so similar and well established. but, the pursuit of brain ras has proven to be correct. in the course of accepting brain ras, high standards of proof attracted state of the art techniques in all the new developments of biolo1gy. consequently, brain ras is a robust concept that has enlightened neuroscience as well as cardiovascular physiology and is a model neuropeptide system. molecular biology confirmed the components of brain ras and their location in the brain. transgenic mice and rats bearing renin and extra copies of angiotensinogen genes revealed the importance of brain ras. cre-lox delivery in vectors has enabled pinpoint gene deletion of brain ras in discrete brain nuclei. the new concept of brain ras includes ace-2, ang1–7, and prorenin and mas receptors. angiotensin ii (ang ii) generated in the brain by brain renin has many neural effects. it activates behavioral effects by selective activation of ang ii receptor subtypes in different locations. it regulates sympathetic activity and baroreflexes and contributes to neurogenic hypertension. new findings implicate brain ras in a much wider range of neural effects. we review brain ras involvement in alzheimer’s disease, stroke memory, and learning alcoholism stress depression. there is growing evidence to consider developing treatment strategies for a variety of neurological disease states based on brain ras. of drinking obviously requires neural circuits in the brain. it was reasoned that the effects of ang ii in the brain were isolated from circulating ras by the blood-brain barrier (bbb). however, circumventricular organs (cvos) lack a bbb and the circulating ras and the endogenous brain ras appear to have a complex interface in the cvos [5, 6] . to prove a brain ras existed, neurophysiological techniques were required. felix and phillips [7] showed that ang ii activated neurons in the brain and in the subformical organ (sfo), and a peptide antagonist of ang ii, saralasin, inhibited ang ii-induced activity in those neurons. this suggested that ang ii is a nonclassical neurotransmitter. taking the concept a step further, it was reasoned that if ang ii, formed by brain renin, was involved in hypertension, then inhibiting brain ras would lower blood pressure. simply injecting saralasin alone into the brain of stroke prone hypertensive rats (spshr) temporarily reduced high blood pressure [8] . this occurred even in the absence of kidney renin when the spshr rats were bilaterally nephrectomized [8] . the finding was later confirmed by molecular inhibition of ang ii synthesis in the brain [9] . it meant that brain ras plays a role in neurogenic hypertension. immunocytochemistry with renin antibodies showed staining in neurons [10, 11] . renin-like activity was evident in the hypothalamus, pituitary, and pineal glands. antibodies to ang ii revealed a clear distribution of ang iilike staining in the hypothalamus and cvos and in higher concentration in shr [12] . what was needed were actual measurements of the renin and ang ii proteins. this was the achieved with high-performance liquid chromatography [13, 14] and direct protein assay [15] . brain levels of ang ii were higher than circulating levels of ang ii, suggesting independence of the two systems. having shown that ang ii was in the brain, the next level of proof required demonstrating that it had been synthesized in the brain. molecular biology was used to measure and locate mrna for agt and renin in the brain [16, 17] . stornetta et al. [18] identified glial cells as the site of synthesis of agt using in situ hybridization mrna. mendelsohn et al. [19] , using autoradiography with pseudocolor imaging, were able to map the entire distribution ang ii receptor binding sites in the brain. this proved that there were sites within the brain that had evolved to be activated by ang ii made in the brain. minute injections of ang ii into key brain nuclei showed that ang ii could differentially produce pressor effects, elicit drinking, release vasopressin, inhibit the baroreflex, and increase sympathetic nervous system activity [20, 21] . the confirmation that there was a separate brain ras, independent of the circulating ras, gave rise to the more generalized concept of local or tissue angiotensins in most other tissues including heart, blood vessels, and, ironically, the kidney [21] whitebread et al. [22] and chiu et al. [23] reported that there were two subtypes of the ang ii receptor, at 1 and at 2 . from this has sprung not only insight into genes involved in brain function but also the highly successful angiotensin receptor blockade class of antihypertensive drugs. the development of the transgenic mice [24] was relevant to brain ras with transgenic mice overexpressing agt by a metallothionein promoter [25] . transgenic mice, overexpressing agt in brain and liver, led to high blood pressure [26, 27] . but, what about renin? renin exists in only one form in humans, ren-1, but in two forms in mice, ren-1 and ren-2. after inserting a mouse ren-2 gene into a transgenic rat [tgr(mren-2)27], mullins et al. [28] reported fulminant hypertension in transgenic rats, even though circulating ang ii levels were low. the homogenous offspring developed malignant hypertension and had high mortality. the heterozygous offspring had end organ damage in the heart and kidney with extreme hypertension. hypertension disappeared when the brains of these mice was anesthetized. curt sigmund's group [29] developed transgenic mice that overexpressed human renin and human agt. with either a glial fibrillary acidic protein promoter or a synapsin-1 promoter, to stimulate gene expression in neurons or glial cells, hypertension and increased drinking resulted. transgenic animals must undergo embryonic development. if the inserted gene or the deleted gene of the transgenic animal is not embryonically lethal, it may be compensated for by other genes duplicating its role. this may account for the surprisingly benign effects of at 2 knockout transgenic mouse [30, 31] . to overcome this possibility, we developed gene suppression methods in adult animals using in vivo rna-based inhibition. antisense inhibition of agt mrna or at1r mrna oligonucleotides delivered to the brain reduces hypertension in shr [32] . antisense inhibition of at1r specifically diminishes at1 receptors in the paraventricular nucleus and organum vasculosum of the lamina terminalis [33] . functionally, at1 antisense inhibited the effects of ang ii in the brain including the pressor response, vasopressin release, and drinking [34] . in the double transgenic mice developed by sigmund and colleagues [35] , to overexpress both human renin and human agt so that they constantly have high ang ii levels, antisense to at1r mrna dramatically reduced the blood pressure. [phillips et al., unpublished] . in an elegant study, davisson et al. [36] , using a combination of at 1a -and at 1b -deficient mice, showed that at 1a receptors are mainly involved in central blood pressure (bp) control, while at 1b receptors mediate the drinking response. the question of which genes and exactly where in the brain cardiovascular effects are regulated may now be answered with greater precision. davisson and colleagues, who previously demonstrated uptake of vectors into specific location of the brain, reported an approach to gene deletion in mice with the cre-lox system [37] . cre, a bacteriophage p1-derived dna recombinase, serves to recombine precisely defined dna sequences that have been "floxed" by loxp sequences prior to embryonic development. because cre recombines the loxp sequences, the floxed segment is deleted and rendered nonfunctional. sinnayah et al. [37] microinjected cre with a promoter into selective targets, such as the sfo and supraoptic nucleus, delivered by adenovirus vectors or feline immunodeficiency virus. with this technique, the sigmund group have demonstrated the presence of brain ras and its physiological and blood pressure effects [38, 39] . the classical brain ras components must now include ang iii, ang iv, ang (1-7), ace2, prorenin, and mas receptors (fig. 1) . ang iii is a metabolite of ang ii found in brain and cerebrospinal fluid. it appears to act on the at1 receptor. ang iv is an endogenous agonist that has been identified in the brain in areas distinct from those where at1 and at2 are located and probably acts on an at4 receptor. an action of brain ras is implied in memory and learning by the finding that stimulation of at4 receptors potentiates the release of acetylcholine from hippocampal brain slices [40] . ace 2 generates ang (1-9) from ang i [41, 42] ; ace 2 is a human ace homolog that differs greatly from ace in substrate specificity and its activity is not modified by ace inhibitors [41] . ace 2 is widely expressed in a variety of tissues including brain [41] [42] [43] [44] . mice lacking the ace 2 gene showed elevation in plasma ang ii level [45] and enhanced pressor response to ang i. ang (1-7) is formed from ang ii via the action of several tissue-specific endopeptidases or from ang i via ace 2. ang (1-7) can be metabolized by ace to ang (1) (2) (3) (4) (5) ; it is a peptide with no known biological activity. ang (1-7) produces its biological effects such as vasodilatation, diuresis, natriuresis, and antitrophic via the mas receptor [46] . mas receptor stimulation also induces to production of nitric oxide (no) and prostacyclin. this new [47, 48] . this points to a complex interface between the brain ras and the systemic ras in controlling vital cardiovascular functions. prorenin receptors were first cloned by nguyen et al. [48] . they bind prorenin with higher affinity than renin [49] . tissue prorenin [50, 51] is active and renin per se has cellular effects independent of ang ii generation [48, 52] . prorenin receptors may help to explain the presence of tissues ras, where the generation of ang ii is independent of systemic ras. there are two forms of renin expressed in the brain of rodents and humans: secreted prorenin and nonsecreted renin. this suggests that renin is constitutively active [53] . using transgenic mice that express nonsecreted active renin or secreted prorenin in the brain, bp and drinking volume were increase similarly in both models. bp was normalized by an intracerebral ventricular injection of losartan in both models, whereas the same dose given systemically had no effect. these data support the concept of an intracellular form of renin in the brain, which may promote functional changes in fluid homeostasis and bp regulation [53] . ace 2, ang (1-7), and mas receptors ace 2 is a carboxypeptidase that counterregulates the vasoconstrictors effects of ang ii to degrade it to the vasodilator peptide ang (1-7) [54] . the first functional evidence of the ang (1-7) in the brain showed that the peptide stimulates the release of vasopressin, equivalent to that obtained with ang ii [55] . in the central nervous system, ang (1-7) acts as neuromodulator, especially in areas related to tonic and reflex control of arterial pressure. its effects are blocked by a mas receptor antagonist a-779 [56] . becker et al. [57] showed mas receptor in cardiovascular and hydroelectrolytic control areas of the rat brain. ang-(1-7) appears to act through the mas receptor. the distribution of ace 2 is widespread in the mouse brain, predominantly in neurons. ace 2 mrna is found in rat medulla oblongata, and ace 2 overlaps with compo-nents of the ras in the same areas [43, 58] . ace-2, ang (1-7), and mas receptors are all localized in brain areas related to the control of cardiovascular function. the physiological effects of central ang ii are mainly mediated by at1r, including vasoconstriction effects, vasopressin release, retention of salt and water, cell growth, and stimulation of aldosterone from the adrenal gland [21] . however, via at2, the ang ii mediates a wide variety of actions, including vasodilatation, inhibition of cellular growth, cell differentiation, and apoptosis. in addition to the brain ras role in neurogenic hypertension discussed above, evidence is accumulating that brain ras is involved in alzheimer's disease, stroke, memory and learning, alcoholism, depression and, emotional stress. alzheimer's disease (ad) is an age-related, neurodegenerative disease with a prevalence of 5-10% in individuals over 65 years [59] . the dementia presents as primary indicator with memory loss following progressive cognitive impairment and personality changes [69] . two hypotheses have been proposed to explain the pathogenesis of ad [60] . the amyloid plaque (ap) hypothesis postulates that ap is generated by proteolytic activity of the βand γ-secretase processing the amyloid precursor protein (app). however, although ap was observed in transgenic mice with mutations of app and presenilin-1 (ps1) genes, the mice did not exhibit neurodegeneration or memory loss observed in ad patients. supporting the presenilin hypothesis, it postulates the neurodegenerative and behavioral effects independent of ap. an increase in the ace activity and alteration in others components of brain ras has been demonstrated in ad [61] [62] [63] [64] . the beneficial effects of ace inhibitors and at1 blockers have been shown in animals and humans suggesting reducing brain ras activity is important. there are many effects of decreased ang ii activity including reduced blood pressure, less acetylcholine release, and increase of substance p-a substrate of ace which is reported to increase neprilysin activity, a recognized amyloid β degrading enzyme [59] . treatment with ace inhibitors and at1 antagonists has been shown to prevent stroke in spshr and in salt-loaded dahl salt-sensitive rats [65] . some clinical trails (syst-eur, systolic hypertension in europe; progress, peridopril protection against recurrent stroke) and randomized studies using antihypertensive treatments have been performed using different combinations of ace inhibitors: at1 receptor antagonists, β-blockers, and diuretics treatments [61] [62] [63] [64] . at1 receptor antagonists normalized the expression of cerebrovascular nitric oxide synthase, cerebrovascular compliance, and protected cerebrovascular flow [66] . similarly, cerebral blood flow was increased by ang (1-7) and reduced by a selective ang (1-7) antagonist a-779 [67] . these data suggest that ang (1-7) could have protective effects in ischemia. in general, the results of treatment show a reduction of bp, stroke, vascular dementia, and cognitive decline [64] . zhou et al. [68] showed evidence of an active local ras in brain microvessels influenced by circulating prorenin and ang ii with a predominant localization to the microvessel endothelium. kagiyama et al. [69] induced stroke with middle cerebral artery occlusion in rats and found that local angii levels and at2 receptor concentrations increased. they concluded that brain ras and at2 receptors increase apoptosis in cerebral ischemia. several studies have indicated brain ras in memory function. at1 antagonist treatment reduced anxiety and improve learning, spatial working memory, and motor performance in the aged rat [70] [71] [72] [73] . denobel et al. [70] reported that intracerebroventricular infusion of renin disrupts passive avoidance learning. coadministration of ace inhibitor (captopril) or at1 antagonist attenuates this deficit induced by renin. at2 receptor antagonist was not effective. ace inhibitors have been shown to enhance memory learned by fear or habituation [71] . recently, bonini et al. [72] showed that the intrahippocampal infusion of ang ii in rats induces amnesia. the effect was completely reversible and did not alter locomotion activity, exploratory behavior, or anxiety state. this effect of ang ii was blocked by the at2 antagonist but not by losartan. long-term potentiation (ltp) is a correlation of learning and memory processes. ang iv and ang (1-7) increase the excitability and ltp of neurons of the hippocampus [73] . furthermore, mas receptor knockout mice do not show alteration of hippocampal ltp [73] . a picture emerges of inhibitory influence by ang ii acting at the at1 receptor, and a facilitatory role by ang iv acting at the at4 to increase excitability, ltp, associative and spatial learning, and memory. although, the ang ii participation in memory consolidation is controversial, results suggest that ang iv and ang 1-7 via mas receptor in the brain are involved in cognitive behavior. alcohol intake ang ii induces drinking, but does it induce alcohol drinking? losartan, the at1 receptor blocker, has been reported to block toxic effects of low doses of ethanol [74] . maul et al. [75] showed that in at1 knockout mice, ang ii affected alcohol consumption, but at2 receptor and bradykinin receptors are not involved [75] . furthermore, ethanol-induced inhibition of hippocampal ltp is mediated by at1 receptors [76] . recently, sommer et al. [77] , using rats treated with spirapril-a blood-brain-penetrating inhibitor of ace-or transgenic rats [tgr(asraogen)680] with reduced central agt expression, showed that increased ethanol consumption was associated with an upregulation of agt transcript levels and a downregulation of local at1 mrna. taken together, these data suggest that reducing brain ras could be a strategy for treating alcoholism. the etiology of depression involves several neurotransmitter systems. the first evidence that brain ras may be important in depression was observed in hypertensive patients undergoing captopril treatment. depressed patients in the group noted reduced depression [78] . in this context, ras polymorphisms have been investigated for their association with depression, including the agt t allele, ace d allele, and at1r c allele, which are associated with markedly higher plasma agt and ace activity and with greater responsiveness to ang ii at lower concentrations. the ace d allele was more prevalent in depressed patients of japanese population [79] . agt m allele was associated with increased susceptibility for bipolar affective disorder in brazilian patients [80] . recently, saab et al. [81] observed that at1 c allele was prevalent in a depressed lebanese population. ras polymorphisms associated with depression are, in some cases, associated with suicidal behavior [82] . among males, the risk for suicide in i/i homozygotes was about 80% higher than in subjects with other genotypes. ace i/d polymorphism may also increase the vulnerability to suicide [82] . however, not all studies have found associations of these polymorphisms with depression, so the jury is still out. brain ras distribution of at1 receptors follows the hypothalamic-pituitary-adrenal axis. during stress, both the peripheral and the central ang ii systems are stimulated, with increases in at1 receptors expression and ang ii levels. different types of stress have been associated with peripheral sympathetic nerve stimulation, increased plasma renin activity, and higher circulating ang ii [83] . at1r are increased in brain by isolation stress induced by short periods of isolation and to 24-h isolation stress. at1 receptor blocker prevented this response and the ulcerations of the gastric mucosa produced by isolation stress [66] . peng and phillips [84, 85] demonstrated that in cold-induced hypertension, agt and ang ii in the brain predominate and in different brain areas, at1r are upregulated and at2r downregulated. at1 antisense treatment reduced the cold-stress-induced high blood pressure [84] . experimental studies also showed that pretreatment with losartan provide protection from stress induced by immobility or forced swimming [86] . ang ii is an important stress hormone, and blockade of at1 receptors might be considered for stress-induced disorders. to inhibit central at1 receptors may be impractical, but the hypothesis of paton suggests inhibition of at1r in brain capillaries could be an alternative if no is involved [47, 87] . detlev ganten's bold hypothesis in 1971 has proven correct. the brain ras is a model for neuropeptide processing and action in the brain. brain ras has neuronal effects far beyond cardiovascular and fluid homeostasis and may be developed for therapies for neurological dysfunctions the discovery of renin 100 years ago angiotensin-forming enzyme in brain tissue a micromethod for the measurement of renin in brain nuclei: its application in spontaneously hypertensive rats the cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin subfornical organ: a dipsogenic site of action of angiotensin ii angiotensin ii in central nervous system physiology specific angiotensin ii receptive neurons in the cat subfornical organ lowering of hypertension by central saralasin in the absence of plasma renin antisense inhibition of at1 receptor mrna and angiotensinogen mrna in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin wide distribution of immunoreactive renin in nerve cells of human brain renin-like immunocytochemical activity in the rat and mouse brain angiotensin-like immunoreactivity in the brain of the spontaneously hypertensive rat angiotensin synthesis in the brain and increased turnover in hypertensive rats angiotensin ii in rat brain comigrates with authentic angiotensin ii in blood pressure liquid chromatography presence of renin in primary neuronal and glial cells from rat brain localization of angiotensinogen messenger rna sequences in the rat brain identification of renin and angiotensinogen messenger rna sequences in rat brain astrocytes synthesize angiotensinogen in brain autoradiographic localization of angiotensin ii receptors in rat brain visualization of specific angiotensin ii binding sites in the brain by fluorescent microscopy functions of angiotensin in the central nervous system preliminary biochemical characterization of two angiotensin ii receptor subtypes identification of angiotensin ii receptor subtypes germ-line transformation of mice generation of transgenic mice with elevated blood pressure by introduction of the rat renin and angiotensinogen genes studies on the regulation of renin genes using transgenic mice high blood pressure in transgenic mice carrying the rat angiotensinogen gene fulminant hypertension in transgenic rats harbouring the mouse ren-2 gene the brain reninangiotensin system in transgenic mice carrying a highly regulated human renin transgene behavioural and cardiovascular effects of disrupting the angiotensin ii type-2 receptor in mice effects on blood pressure and exploratory behaviour of mice lacking angiotensin ii type-2 receptor antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate genes a decrease in angiotensin receptor binding in rat brain nuclei by antisense oligonucleotides to the angiotensin at1 receptor antisense oligonucleotide to at1 receptor mrna inhibits central angiotensin induced thirst and vasopressin the brain renin-angiotensin system contributes to the hypertension in mice containing both the human renin and human angiotensinogen transgenes divergent functions of angiotensin ii receptor isoforms in the brain targeted viral delivery of the cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain localization of renin expressing cells in the brain using a ren-egfp transgenic model efficient liver-specific deletion of the floxed human angiotensinogen transgene by adenoviral delivery of the cre recombinase in vivo potentiation of cholinergic transmission in the rat hippocampus by angiotensin iv and lvv-hemorphin-7 a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 distinct roles for ang ii and ang-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes new mass spectrometric assay for angiotensin-converting enzyme 2 activity angiotensin-converting enzyme 2 is an essential regulator of heart function angiotensin-(1-7) is an endogenous ligand for the g protein-coupled receptor mas detection of angiotensin ii mediated nitric oxide release within the nucleus of the solitary tract using electron-paramagnetic resonance (epr) spectroscopy pivotal role of the renin/prorenin receptor in angiotensin ii production and cellular responses to renin binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system tissue activity of circulating prorenin in vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen evidence supporting a functional role for intracellular renin in the brain effects of reninangiotensin system blockade on renal angiotensin-(1-7) forming enzymes and receptors release of vasopressin from the rat hypothalamoneurohypophysial system by angiotensin-(1-7) heptapeptide cardiovascular effects of angiotensin-(1-7) injected into the dorsal medulla of rats immunofluorescence localization of the receptor mas in cardiovascular-related areas of the rat brain differential expression of neuronal ace2 in transgenic mice with overexpression of the brain reninangiotensin system is inhibition of the reninangiotensin system a new treatment option for alzheimer's disease transcriptional regulation of the presenilin-1 gene: implication in alzheimer's disease effects of brain-penetrating ace inhibitors on alzheimer disease progression angiotensin-converting enzyme inhibitors and incidence of alzheimer's disease in japan prevention of dementia: lessons from syst-eur and progress prevention of dementia in randomised double-blind placebo-controlled systolic hypertension in europe (syst-eur) trial stroke prevention by losartan in stroke-prone spontaneously hypertensive rats brain angiotensin ii: new developments, unanswered questions and therapeutic opportunities systemic and regional. hemodynamic effects of angiotensin-(1-7) in rats at1 receptor blockade regulates the local angiotensin ii system in cerebral microvessels from spontaneously hypertensive rats expression of angiotensin type 1 and type 2 receptors in brain after transient middle cerebral artery occlusion in rats non-peptide angiotensin ii receptor antagonist and angiotensin-converting enzyme inhibitor: effect on a renin-induced deficit of a passive avoidance response in rats angiotensin ii blocks memory consolidation through an at2 receptor-dependent mechanism angiotensin ii disrupts inhibitory avoidance memory retrieval angiotensin-(1-7) enhances ltp in the hippocampus through the gprotein-coupled receptor mas losartan reduces ethanol intoxication in the rat central angiotensin ii controls alcohol consumption via its at1 receptor ethanol and diazepam inhibition of hippocampal ltp is mediated by angiotensin ii and at1 receptors plasticity and impact of the central renin-angiotensin system during development of ethanol dependence captopril treatment of major depression with serial measurements of blood cortisol concentrations frequency of the fragile x syndrome in japanese mentally retarded males angiotensinogen and angiotensin converting enzyme gene polymorphisms and the risk of bipolar affective disorder in humans renin-angiotensin-system gene polymorphisms and depression association between a functional polymorphism in the renin-angiotensin system and completed suicide angiotensin ii-an important stress hormone reduction of cold-induced hypertension by antisense oligodeoxynucleotides to angiotensinogen mrna and at1-receptor mrna in brain and blood the predominant role of brain angiotensinogen and angiotensin in environmentally induced hypertension brain and peripheral angiotensin ii play a major role in stress differential baroreflex control of sympathetic drive by angiotensin ii in the nucleus tractus solitarii acknowledgements em oliveira is recipient of a brazil-cnpq fellowship. mip was supported by nih merit award r37-hl027334 and nih r01, r01-hl077602. key: cord-001773-mqk0sx5n authors: lo, chao-sheng; shi, yixuan; chang, shiao-ying; abdo, shaaban; chenier, isabelle; filep, janos g.; ingelfinger, julie r.; zhang, shao-ling; chan, john s. d. title: overexpression of heterogeneous nuclear ribonucleoprotein f stimulates renal ace-2 gene expression and prevents tgf-β1-induced kidney injury in a mouse model of diabetes date: 2015-08-01 journal: diabetologia doi: 10.1007/s00125-015-3700-y sha: doc_id: 1773 cord_uid: mqk0sx5n aims/hypothesis: we investigated whether heterogeneous nuclear ribonucleoprotein f (hnrnp f) stimulates renal ace-2 expression and prevents tgf-β1 signalling, tgf-β1 inhibition of ace-2 gene expression and induction of tubulo-fibrosis in an akita mouse model of type 1 diabetes. methods: adult male akita transgenic (tg) mice overexpressing specifically hnrnp f in their renal proximal tubular cells (rptcs) were studied. non-akita littermates and akita mice served as controls. immortalised rat rptcs stably transfected with plasmid containing either rat hnrnpf cdna or rat ace-2 gene promoter were also studied. results: overexpression of hnrnp f attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (agt) and angiotensin (ang) ii levels, renal fibrosis and profibrotic gene (agt, tgf-β1, tgf-β receptor ii [tgf-βrii]) expression, stimulated anti-profibrotic gene (ace-2 and ang 1–7 receptor [masr]) expression, and normalised urinary ang 1–7 level in akita hnrnpf-tg mice as compared with akita mice. in vitro, hnrnp f overexpression stimulated ace-2 gene promoter activity, mrna and protein expression, and attenuated agt, tgf-β1 and tgf-βrii gene expression. furthermore, hnrnp f overexpression prevented tgf-β1 signalling and tgf-β1 inhibition of ace-2 gene expression. conclusions/interpretation: these data demonstrate that hnrnp f stimulates ace-2 gene transcription, prevents tgf-β1 inhibition of ace-2 gene transcription and induction of kidney injury in diabetes. hnrnp f may be a potential target for treating hypertension and renal fibrosis in diabetes. electronic supplementary material: the online version of this article (doi:10.1007/s00125-015-3700-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users. diabetic nephropathy (dn), a leading cause of endstage renal disease (esrd), accounts for ∼50% of all esrd cases [1, 2] . while glomerulopathy is a hallmark of early renal injury in dn [3] , tubulointerstitial fibrosis and tubular atrophy are major features of late-stage dn and are closely associated with loss of renal function [4] [5] [6] [7] . the mechanisms underlying tubulointerstitial fibrosis, however, are incompletely understood. tgf-β1 is considered to be the most potent inducer of fibrogenesis [8] . indeed, patients and animal models with type 1 or 2 diabetes have significantly elevated serum and urinary tgf-β1 levels [9] [10] [11] as well as heightened tgf-β1 mrna and protein expression in glomeruli and the tubulointerstitium [12] [13] [14] [15] [16] . we previously reported that high glucose milieu enhances expression of angiotensinogen (agt, the sole precursor of all angiotensins) through generation of reactive oxygen species (ros) in cultured rat renal proximal tubular cells (rptcs) [17, 18] . rat agt overexpression in rptcs leads to hypertension, albuminuria and rptc hypertrophy, and enhances tgf-β1 expression in diabetic agt-transgenic (tg) mice [19, 20] . conversely, rptc-selective overexpression of catalase or pharmacological blockade of the renin-angiotensin system (ras) attenuates hypertension, ros generation, kidney injury and normalised rptc ace-2 expression in mouse models of diabetes [21] [22] [23] [24] . taken together, these observations indicate that oxidative stress-induced upregulation of agt expression and downregulation of ace-2 expression in rptcs, resulting in higher angiotensin (ang)ii/ang 1-7 ratio, may be key determinants of development of hypertension and nephropathy in diabetes. we reported that insulin inhibits high glucose stimulation of rat renal agt gene expression via two nuclear proteins-heterogeneous nuclear ribonucleoproteins f and k (hnrnp f, hnrnp k)-that interact with the insulin-responsive element (ire) in the agt gene promoter [25] [26] [27] [28] , and that hnrnp f overexpression in rptcs inhibits agt gene expression and kidney hypertrophy in akita hnrnpf-tg mice [29] . here, we report that overexpression of hnrnp f stimulates ace-2 gene transcription and suppresses profibrotic gene (tgf-β1, tgf-βrii) expression in rptcs of akita hnrnpf-tg mice. we have confirmed these changes by in vitro studies in rat rptcs. we also show that hnrnp f overexpression prevents tgf-β1 signalling and inhibition of ace-2 gene expression in rptcs. finally, we identified the putative dna response elements (res) in the ace-2 gene promoter that are responsive to hnrnp f and tgf-β1. chemicals and constructs active human recombinant tgf-β1 was obtained from r&d systems (minneapolis, mn, usa). sb431542 (a tgf-β receptor i [ri] inhibitor) and other chemicals were purchased from sigma-aldrich (oakville, on, canada). the antibodies used in the present study are listed in electronic supplementary material (esm) table 1. the pkap2 plasmid containing the kidney-specific androgen-regulated protein (kap) promoter was a gift from c. d. sigmund (university of iowa, iowa city, ia, usa) [30] . full-length rat hnrnpf cdna fused with ha tag (encoding amino acid residues 98-106 [ypydvpdya] of human influenza virus hemagglutinin) was inserted into pkap2 plasmid at the noti site at both 5′ and 3′ termini [25, 29] . pgl4.20 vector containing luciferase reporter was obtained from promega (sunnyvale, ca, usa). rat ace-2 gene promoter (n-1,091/+83) was cloned from rat genomic dna with specific primers (esm table 2 ), as described by milsted et al [31] and then inserted into pgl4.20 plasmid at hindiii and kpni restriction sites. scrambled silencer negative control no. 1 small interfering rna (sirna) and hnrnpf sirna were bought from ambion (austin, tx, usa). quickchange ii site-directed mutagenesis kit and lightshift chemiluminescent electrophoretic mobility shift assay (emsa) kit were procured from agilent technologies (santa clara, ca, usa) and thermo scientific (life technologies, burlington, on, canada), respectively. the primer biotinlabelling kit was purchased from integrated dnatechnologies (coralville, ia, usa). physiological studies adult male heterozygous akita mice (mus musculus) with a mutated ins2 gene (c57bl6-ins2 akita /j) were purchased from jackson laboratories (bar harbor, me, usa: http://jaxmice.jax.org). akita tg mice (c57bl/6 background) overexpressing rat hnrnp f-ha in rptcs (line 937) were created in our laboratory (by j. s. d. chan) [29] . male adult non-tg and non-akita littermates served as wild-type (wt) controls, and were tested along with hnrnpf-tg, akita and akita hnrnpf-tg mice. all animals were housed individually in metabolic cages for 24 h before euthanasia at age 20 weeks. all animals were fed standard mouse chow and water ad libitum. animal care and procedures were approved by the crchum animal care committee and followed the principles of laboratory animal care (nih publication no. 85-23, revised 1985: http:// grants1.nih.gov/grants/olaw/references/phspol.htm). blood glucose levels, following 4-5 h fasting, were determined with an accu-chek performa system (roche diagnostics, laval, qc, canada). body weight (bw) was recorded. urine was collected and assayed for albumin/creatinine ratio (acr) by enzyme-linked immunosorbent assays (albuwell and creatinine companion, exocell, philadelphia, pa, usa). gfr was measured as described by qi et al [32] as recommended by the animal models of diabetic complications consortium (www.diacomp.org) with fluorescein isothiocyanate inulin [23, 28, 33] . kidneys were removed immediately after gfr measurement, decapsulated and weighed. the left kidneys were processed for histology and immunostaining, and right renal cortices were harvested for renal proximal tubules (rpts) isolation by percoll gradient centrifugation [23, 24, 28, 29] . aliquots of freshly isolated rpts from individual mice were immediately processed for total rna and protein isolation. immunohistochemical staining immunohistochemical staining was performed by the standard avidin-biotin-peroxidase complex method in four to five sections (4 μm thick) per kidney and three mouse kidneys per group (abc staining system; santa cruz biotechnology [santa cruz, ca, usa]) [23, 24, 28, 29] . staining was analysed under light microscopy by two independent, blinded observers. the collected images were assessed by national institutes of health image j software (http://rsb.info.nih.gov/ij/) [23, 24, 28, 29] . urinary agt, ang ii and ang 1-7 measurement mouse urinary agt, ang ii and ang 1-7 levels were analysed by elisa (immuno-biological laboratories, ibl america, minneapolis, mn, usa) and normalised by urinary creatinine levels as described [23, 24, 28, 29, 34] . cell culture immortalised rat rptcs (passages 12-18) [35] were cultured in 5 mmol/l d-glucose dmem containing 5% fbs until they reached 60-70% confluence. the media were then changed to serum-free dmem, ensuring that endogenously secreted tgf-β1 would not interfere in the assay. after 45 min preincubation, active human recombinant tgf-β1 [36] (0 to 10 ng/ml) was added (considered as time 0 h) and incubated for various time periods up to 24 h. in separate experiments, rptcs were incubated for 24 h in serum-free medium in the presence or absence of tgf-β1± various concentrations of sb431542. real-time quantitative pcr hnrnpf, ace, ace-2, masr, tgf-β1, tgf-βri, tgf-βrii, collagen type iv, collagen type i, fibronectin 1 and β-actin mrna expression levels in rpts were quantified by real-time quantitative pcr (rt-qpcr) with forward and reverse primers (esm table 2 ) [23, 24, 28, 29] . western blotting western blotting (wb) was performed as described previously [23, 24, 28, 29] . the relative densities of hnrnp f, ace, ace-2, ang 1-7 receptor (masr), tgf-β1, tgf-β ri, tgf-β rii, fibronectin 1, p-smad2/3, smad2/3 akita compared with wt mice and hnrnpf-tg mice. overexpression of hnrnp f had no effect on blood glucose levels in akita hnrnpf-tg mice. systolic bp (sbp), kidney weight (kw)/bw and kw/tibial length (tl) ratios, gfr and acr were all elevated in akita mice, compared with both wt controls and hnrnpf-tg mice. hnrnp f overexpression in rptcs markedly attenuated these changes in diabetic akita hnrnpf-tg mice. furthermore, akita mice exhibited elevated urinary agt and ang ii levels, parallel with decreased ang 1-7 levels, compared with wt mice. hnrnp f overexpression partially reduced urinary agt and ang ii levels, whereas it completely normalised urinary ang 1-7 levels-a novel finding. effect of hnrnp f overexpression on agt, ace, ace-2 and masr expression in akita hnrnpf-tg mouse kidneys immunostaining revealed that hnrnp f (fig. 1a) was overexpressed in rptcs of hnrnpf-tg and akita hnrnpf-tg mice compared with wt and akita mice, respectively. ace-2 ( fig. 1b) and masr (fig. 1c ) expression was decreased in akita mice compared with wt controls and normalised in akita hnrnpf-tg mice. rptc ace (fig. 1d ) expression did not differ between wt and hnrnpf-tg mice, whereas ace expression was significantly higher in akita mice than in wt controls and was not normalised in akita hnrnpf-tg mice. wb and rt-qpcr for hnrnp f, ace-2, masr and ace protein and their mrna levels ( fig. 1e -l, respectively) confirmed these observations. effect of hnrnp f overexpression on tgf-β1, tgf-β rii and tgf-β ri expression in akita hnrnpf-tg mouse kidneys immunostaining of tgf-β1 (fig. 2a) and tgf-β rii (fig. 2b) , wb of tgf-β1 (fig. 2d) and tgf-β rii expression values are means+sem corrected to β-actin, n=6. *p<0.05; **p<0.01 (fig. 2e) , and rt-qpcr of tgf-β1 (fig. 2g) and tgf-βrii (fig. 2h ) mrna expression showed significantly higher tgf-β1 and tgf-β rii expression in rptcs of akita mice than in wt controls and hnrnpf-tg mice, and they were attenuated in akita hnrnpf-tg mice. in contrast, tgf-β ri expression was similar in all groups studied (fig. 2c,f,i) . hnrnp f overexpression suppresses renal fibrosis in akita hnrnpf-tg mice akita mice developed renal structural damage compared with wt and hnrnpf-tg mice (esm fig. 1a , pas staining), including tubular luminal dilatation with accumulation of cell debris, increased extracellular matrix proteins in glomeruli and tubules, and proximal tubule cell atrophy. hnrnp f overexpression markedly reversed but never completely resolved these abnormalities in akita mice. we detected significant increases in masson's trichrome staining (fig. 3a) and immunostaining for collagen type iv (fig. 3b) , fibronectin 1 expression (fig. 3c ) and collagen type i (fig. 3d) in glomerulotubular areas in akita mice compared with wt controls and hnrnpf-tg mice. these changes were reduced in akita hnrnpf-tg mice. quantification of masson's trichrome-stained (esm fig. 1b) , immunostaining of collagen iv (fig. 3e) , fibronectin 1 (fig. 3f) and collagen i (fig. 3g) , and rt-qpcr quantification of mrna levels (fig. 3h-j) confirmed their expression. hnrnp f overexpression enhances ace-2 and suppresses agt, tgf-β1 and tgf-βrii gene expression and protein levels in rat rptcs in vitro rptcs stably transfected with pcdna 3.1/hnrnpf (rptc-pcdna 3.1/hnrnpf) exhibited considerably higher levels of hnrnp f (fig. 4a,b) , lower amounts of agt (fig. 4a,c) and a higher amount of ace-2 (fig. 4a,d) than non-transfected rptcs or rptcs stably transfected with pcdna 3.1 (rptc-pcdna 3.1). in contrast, tgf-β1 and tgf-β rii protein levels were significantly decreased in rptc-pcdna 3.1/hnrnpf compared with non-transfected rptcs or rptc-pcdna 3.1 (p<0.01) (fig. 4e,f,g, respectively) . tgf-β ri protein level was similar in non-transfected rptcs, rptc-pcdna 3.1 or rptc-pcdna 3.1/hnrnpf (fig. 4h) . rt-qpcr of hnrnpf, agt, ace-2, tgf-β1, tgf-βrii and tgf-βri mrna levels confirmed these findings (esm fig. 2a-f ). tgf-β1 signalling and inhibition of ace-2 gene expression in rat rptcs tgf-β1 inhibited rat ace-2 gene promoter activity (fig. 5a) , rat ace-2 mrna expression (fig. 5b) and rat ace-2 protein level (fig. 5c) in a concentration-dependent manner, which was reversed by sb431542 (a tgf-β ri inhibitor) (fig. 5d-f, respectively) . furthermore, tgf-β1 stimulated smad 2/3 phosphorylation in a concentration-and timedependent manner (fig. 5g) and reversed by sb431542 (fig. 5h) . these data demonstrate that tgf-β1 inhibition of ace-2 gene transcription is mediated, at least in part, via smad2/3 signalling. hnrnp f overexpression prevents tgf-β signalling, and tgf-β inhibition of ace-2 and induction of fibrotic gene expression in rptcs tgf-β1 had no detectable effect on hnrnp f protein levels (fig. 6a,b) . intriguingly, hnrnp f overexpression prevented tgf-β1 stimulation of smad 2/3 phosphorylation (fig. 6a,c) , tgf-β rii expression (fig. 6a , d) and fibronectin 1 expression (fig. 6a,e) . hnrnp f overexpression also prevented tgf-β1-induced downregulation of masr (fig. 6a ,f) content in rptcs. addition of tgf-β1 did not affect tgf-β ri expression in rptcs (fig. 6a,g) . furthermore, overexpression of hnrnp f prevented the inhibitory effect of tgf-β1 on ace-2 protein (fig. 6a,h) and ace-2 mrna (fig. 6i ) expression in rptc-pcdna 3.1/ hnrnpf. localisation of hnrnpf-and tgf-β1 (or smad)-re in rat ace-2 gene promoter to localise the putative dna-re(s) that mediate(s) the action of hnrnp f or tgf-β1 on ace-2 gene promoter activity, plasmids containing various lengths of the rat ace-2 gene promoter were transiently transfected into rptc-pcdna 3.1 or rptc-pcdna 3.1/ hnrnpf. the activity of pgl4.20-ace-2 promoter (n-1,091/+ 83) and pgl 4.20-ace-2 promoter (n-499/+83) exhibited respective fivefold and 12-fold increase as compared with the control plasmid, pgl 4.20 in rptc-pcdna 3.1 (fig. 7a) . further deletion of nucleotides n-499 to n-241 (pgl 4.20-ace-2 promoter [n-240/+83]) significantly reduced the rat ace-2 promoter activity. moreover, the activity of pgl4.20-ace-2 promoter (n-1,091/+83) and pgl4.20-ace-2 promoter (n-499/+83) was further increased by 1.5-2.0-fold, whereas the activity of pgl4.20-ace-2 promoter (n-240/+83) did not increase in rptc-pcdna 3.1/hnrnpf as compared with rptc-pcdna 3.1 (fig. 7a) . interestingly, addition of tgf-β1 inhibited the promoter activity of pgl 4.20-ace-2 promoter (n-1,091/+83) and did not affect the activity of pgl 4.20-ace-2 promoter (n-499/+83) and pgl 4.20-ace-2 promoter (n-240/+83) in rptc-pcdna 3.1 (fig. 7b) . however, tgf-β1 had no inhibitory effect on the promoter activity of these constructs in rptc-pcdna 3.1/hnrnpf (fig. 7c) . in contrast, transfection of hnrnpf sirna significantly inhibited the promoter activity of pgl 4.20-ace-2 promoter (n-1,091/+83) and pgl 4.20-ace-2 promoter (n-499/+83) without affecting the activity of pgl 4.20-ace-2 promoter (n-240/+83) in rptc-pcdna 3.1 (fig. 7d) . deletion of the nucleotides n-401 to n-393 (5′-ggggagagg-3′) in the ace-2 gene promoter markedly attenuated the promoter activity of pgl 4.20-ace-2 promoter (n-1,091/+83) and pgl 4.20-ace-2 promoter (n-499/+83) in rptc-pcdna 3.1/hnrnpf (fig. 7e) . interestingly, deletion of the putative proximal smad-re (nucleotides n-511 to n-504 [5′-cagagaca-3′]) or distal putative smad-re2 (nucleotides n-789 to n-784 [5′-gagaca-3′]) in the ace-2 gene promoter partially attenuated whereas deletion of both res (nucleotides n-511 to n-504 and nucleotides n-789 to n-784) completely abolished the inhibitory action of tgf-β1 on pgl 4.20-ace-2 promoter (n-1,091/+83) activity in rptc-pcdna 3.1 (fig. 7f) . furthermore, emsa showed that the double strand dna fragments, nucleotides n-405 to n-387 (putative hnrnpf-re), nucleotides n-518 to n-497 (putative proximal smad-re1) and nucleotides n-797 to n-776 (putative distal smad-re2) bind to the nuclear proteins from rptcs and they could be displaced by the respective wt dna fragments, but not by mutated dna fragments (fig. 7g,h, respectively) . importantly, addition of anti-hnrnp f and anti-smad 2/3 antibody induced a supershift of the respective hnrnpf-re and smad-res with the nuclear proteins, respectively (fig. 7g,h) . the present report identifies a novel mechanism by which hnrnp f prevents hypertension and kidney injury in diabetic akita mice, i.e. hnrnp f stimulation of renal ace-2 gene transcription and mitigation of the inhibitory effect of tgf-β1 on ace-2 gene transcription. we reported previously that overexpression of hnrnp f prevents systemic hypertension, and inhibits renal agt gene expression and rptc hypertrophy in diabetic akita hnrnpf-tg mice [29] . the present paper provides new in vivo and in vitro evidence that hnrnp f stimulates ace-2 gene transcription via binding to the dna-re of the ace-2 gene promoter, which is critical for the formation of renal ang 1-7 and subsequent expression of its antihypertensive and renoprotective actions in akita mice [37] . hnrnp f, a member of the pre-mrna-binding protein family [38] regulates gene expression at both the transcriptional and post-transcriptional levels. indeed, hnrnp f engages in alternative splicing of various genes [39] [40] [41] and associates with tata-binding protein, rna polymerase ii, nuclear cap-binding protein complex and various transcriptional factors. [42, 43] the akita mouse is an autosomal-dominant model of spontaneous type 1 diabetes in which the ins2 gene is mutated. akita mice develop hyperglycaemia and systemic hypertension, leading to cardiac hypertrophy, left ventricular diastolic dysfunction, glomerulosclerosis and enhanced oxidative stress in rpts, closely resembling those observed in patients with type 1 diabetes [44, 45] . our study provides evidence for a novel mechanism for hnrnp f lowering of sbp: inhibition of intrarenal agt gene expression and ras activation, concomitant with upregulation of the ace-2/ang 1-7/masr axis. indeed, our results show that hnrnp f overexpression inhibited renal agt and agt mrna expression (esm fig. 1 c-e), lowered urinary agt and ang ii levels and normalised urinary ang 1-7 levels. we consistently observed decreased renal ace-2 expression in akita mice as previously reported [23, 24] . decreased ace-2 expression also has been reported in male streptozotocin (stz)-induced diabetic mice [46] , stzinduced diabetic rats [47, 48] and human type 2 diabetic kidneys [49, 50] . the precise mechanism by which hnrnp f overexpression leads to upregulation of renal ace-2 and masr gene expression in diabetes remains unclear. one possibility is that hnrnp f binds to putative hnrnpf-re(s) in the ace-2 and masr gene promoters, subsequently enhancing ace-2 and masr gene transcription. this possibility is supported by our findings that hnrnp f considerably augments the activity of an ace-2 gene promoter and that the hnrnpf sirna and deletion of the putative hnrnpf-re markedly reduced the rat ace-2 gene promoter activity in rptcs. furthermore, the biotinylated-labelled hnrnpf-re specifically bound to rptc nuclear proteins and the addition of anti-hnrnp f antibody yielded a supershift of biotinylated-labelled hnrnpf-re binding with nuclear proteins in emsa. these data demonstrate that hnrnp f binds to the putative hnrnpf-re and stimulates ace-2 gene transcription. of note, hnrnp f is not specific for ---+ + + + + + + + + + + -bsa + + + + + + + + + ace-2 gene expression but also affects the expression of agt [25] and other genes [51, 52] . currently, little is known about the mechanisms by which tgf-β1 downregulates renal ace-2 gene expression in diabetes. chou et al [53] reported that sb431542 inhibited high glucose and tgf-β1 inhibition of ace-2 mrna expression in cultured nrk-52 cells. our findings confirm these observations. our present studies also demonstrate that tgf-β1 inhibits the activity of pgl 4.20-rat ace-2 promoter (n-1, 091/+83) and that deletion of putative smad-res in the ace-2 gene promoter mitigates the inhibitory effect of tgf-β1 on the ace-2 gene promoter activity. furthermore, biotinylated-labelled smad-res bound to rptc nuclear proteins and the addition of anti-smad2/3 antibody yielded a supershift of labelled dna with nuclear proteins. these data demonstrate that the inhibitory effect of tgf-β1 on ace-2 gene transcription is mediated, at least in part, via the smad-res in the ace-2 gene promoter. intriguingly, hnrnp f overexpression prevented tgf-β1 signalling on smad2/3 phosphorylation and on tgf-β1 inhibition of ace-2 gene promoter activity in rptcs. at present, the underlying molecular mechanism of how hnrnp f prevents tgf-β1 inhibition of ace-2 gene transcription is not yet defined. one possibility might be that hnrnp f directly inhibits tgf-β1rii gene expression as shown in our studies. the second possibility is that hnrnp f might interfere or prevent the interaction of smad2/3 with other transcriptional factor(s) to inhibit ace-2 gene transcription. clearly, more studies are needed to define the molecular interaction of hnrnp f with smad2/3 on ace-2 gene transcription. in summary, the present study suggests a major role for hnrnp f in attenuating systemic hypertension and renal fibrosis in experimental diabetes and possibly in diabetic human kidneys. our observations raise the possibility that selective targeting of this antihypertensive and anti-fibrotic protein may represent a novel approach for preventing or reversing the pathological manifestations of dn, particularly tubular fibrosis. identification of hnrnpf-re and smad-re in the ace-2 gene promoter. (a) luciferase activity of the plasmid containing various lengths of ace-2 gene promoter in rptc-pcdna 3.1 (white bars) and in rptc-pcdna 3.1/hnrnpf (black bars) ng/ml htgf-β1); and (c) in rptc-pcdna3.1/hnrnpf±tgf-β1 (white bars, without htgf-β1; black bars, with 2 ng/ml htgf-β1); (d) in rptc-pcdna 3.1±hnrnpf sirna (white bars, treated with 50 nmol/l scrambled sirna; black bars, treated with 50 nmol/l hnrnpf sirna), cultured in normal glucose media for 24 h. (e) promoter activity of the ace-2 gene ±hnrnpf black bars) or (f)±smad-res in rptc-pcdna 3.1 in the absence or presence of tgf-β1 (white bars, without htgf-β1; black bars, with 2 ng/ml htgf-β1). values are mean+sem, n=6. the experiments were repeated twice res (h) with rptc nuclear proteins±excess unlabelled wt hnrnpf-re or mutated hnrnpf-res (m1 to m4 are mutants of hnrnpf-re with nucleotides mutated or deleted in the binding motif as shown in esm table 2) or wt smad-re or mutant smad-res (smad-re1 [m1 and m2] and smad-re2 [m1 and m2] are mutants of respective smad-re1 and smad-re2 with nucleotides mutated in the binding motif as shown in esm table 2). rabbit igg or rabbit anti-hnrnp f or anti-smad2/3 antiserum was added to the reaction mixture and incubated for 30 min on ice before incubation with the biotinylated probe. results are representative of three independent experiments endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat the early natural history of nephropathy in type 1 diabetes: ii. early renal structural changes in type 1 diabetes mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure the tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? significance of tubulointerstitial changes in the renal cortex for the excretory function and concentration ability of the kidney: a morphometric contribution tubulointerstitial damage leads to atubular glomeruli: significance and possible role in progression transforming growth factorbeta regulates tubular epithelial-myofibroblast transdifferentiation in vitro accurate measurement and clinical significance of urinary transforming growth factor-beta1 utility of serum and urinary transforming growth factor-beta levels as markers of diabetic nephropathy increased excretion of urinary transforming growth factor beta 1 in patients with diabetic nephropathy evidence that tgf-beta should be a therapeutic target in diabetic nephropathy increased renal production of transforming growth factor-beta1 in patients with type ii diabetes expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy the renal expression of transforming growth factor-beta isoforms and their receptors in acute and chronic experimental diabetes in rats increased glomerular and tubular expression of transforming growth factor-beta1, its type ii receptor, and activation of the smad signaling pathway in the db/db mouse high glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells high glucose stimulates angiotensinogen gene expression and cell hypertrophy via activation of the hexosamine biosynthesis pathway in rat kidney proximal tubular cells ras blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney overexpression of angiotensinogen increases tubular apoptosis in diabetes catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice attenuation of interstitial fibrosis and tubular apoptosis in db/db transgenic mice overexpressing catalase in renal proximal tubular cells overexpression of catalase prevents hypertension and tubulointerstitial fibrosis and normalization of renal angiotensin-converting enzyme-2 expression in akita mice dual ras blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in akita angiotensinogen-transgenic mice heterogenous nuclear ribonucleoprotein f modulates angiotensinogen gene expression in rat kidney proximal tubular cells characterization of a putative insulin-responsive element and its binding protein(s) in rat angiotensinogen gene promoter: regulation by glucose and insulin heterogeneous nuclear ribonucleoprotein k modulates angiotensinogen gene expression in kidney cells heterogeneous nuclear ribonucleoproteins f and k mediate insulin inhibition of renal angiotensinogen gene expression and prevention of hypertension and kidney injury in diabetic mice heterogeneous nuclear ribonucleoprotein f suppresses angiotensinogen gene expression and attenuates hypertension and kidney injury in diabetic mice the kidney androgenregulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice regulation of multiple renin-angiotensin system genes by sry characterization of susceptibility of inbred mouse strains to diabetic nephropathy angiotensin ii type ii receptor deficiency accelerates the development of nephropathy in type i diabetes via oxidative stress and ace2 catalase overexpression prevents hypertension and tubular apoptosis in angiotensinogen transgenic mice rat proximal tubule cell line transformed with origin-defective sv40 dna: autocrine ang ii feedback transforming growth factor-beta 1 stimulates angiotensinogen gene expression in kidney proximal tubular cells angiotensin-(1-7) prevents systemic hypertension, attenuates oxidative stress and tubulointerstitial fibrosis, and normalizes renal angiotensinconverting enzyme 2 and mas receptor expression in diabetic mice functional diversity of the hnrnps: past, present and perspectives the generally expressed hnrnp f is involved in a neural-specific pre-mrna splicing event heterogeneous nuclear ribonucleoprotein f/h proteins modulate the alternative splicing of the apoptotic mediator bcl-x essential role for the interaction between hnrnp h/f and a g quadruplex in maintaining p53 pre-mrna 3′-end processing and function during dna damage association of the rat heterogeneous nuclear rna-ribonucleoprotein f with tata-binding protein interaction between the human nuclear cap-binding protein complex and hnrnp f a novel locus, mody4, distal to d7mit189 on chromosome 7 determines early-onset niddm in nonobese c57bl/6 (akita) mutant mice complications of iga nephropathy in a non-insulin-dependent diabetes model, the akita mouse ace2 deficiency modifies renoprotection afforded by ace inhibition in experimental diabetes characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy glomerular renin angiotensin system in streptozotocin diabetic and zucker diabetic fatty rats decreased glomerular and tubular expression of ace2 in patients with type 2 diabetes and kidney disease expression of ace and ace2 in individuals with diabetic kidney disease and healthy controls proteomic identification of proteins associated with the osmoregulatory transcription factor tonebp/orebp: functional effects of hsp90 and parp-1 global profiling of alternative splicing events and gene expression regulated by hnrnph/f interaction between tgf-beta and ace2-ang-(1-7)-mas pathway in high glucosecultured nrk-52e cells duality of interest the authors declare that there is no duality of interest associated with this manuscript.contribution statement jsdc is the principal investigator and was responsible for the study conception and design. csl drafted the manuscript and contributed to the discussion. csl, ys, syc, sa, ic and slz contributed to the in vivo and in vitro experiments and collection of data. jgf and jri contributed to the discussion and reviewed/edited the manuscript. all authors were involved in analysis and interpretation of data, and contributed to the critical revision of the manuscript. all authors provided final approval of the version to be published. jsdc is guarantor of this work and, as such, had full access to all study data, taking responsibility for data integrity and the accuracy of data analysis.open access this article is distributed under the terms of the creative commons attribution 4.0 international license (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord-006082-x1kankxd authors: romero, cesar a.; orias, marcelo; weir, matthew r. title: novel raas agonists and antagonists: clinical applications and controversies date: 2015-02-10 journal: nat rev endocrinol doi: 10.1038/nrendo.2015.6 sha: doc_id: 6082 cord_uid: x1kankxd the renin–angiotensin–aldosterone system (raas) regulates blood pressure homeostasis and vascular injury and repair responses. the raas was originally thought to be an endocrine system critically important in regulating blood pressure homeostasis. yet, important local forms of the raas have been described in many tissues, which are mostly independent of the systemic raas. these systems have been associated with diverse physiological functions, but also with inflammation, fibrosis and target-organ damage. pharmacological modulation of the raas has brought about important advances in preventing morbidity and mortality associated with cardiovascular disease. yet, traditional raas blockers such as angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers (arbs) only reduce the risk of disease progression in patients with established cardiovascular or renal disease by ∼20% compared with other therapies. as more components of the raas are described, other potential therapeutic targets emerge, which could provide improved cardiovascular and renal protection beyond that provided by an ace inhibitor or arb. this review summarizes the present and future pharmacological manipulation of this important system. supplementary information: the online version of this article (doi:10.1038/nrendo.2015.6) contains supplementary material, which is available to authorized users. more than seven decades ago, angiotensin, a poly peptide that is the product of the enzymatic activity of renin, was found in venous samples of ischaemic dog kidneys. 1 in the 1950s and 1960s, two forms of angiotensin were reported: angiotensin-1 (ang i), which is 10 amino acids long, and angiotensin-2 (ang ii), with 8 amino acids. 2 the latter form results from the metabolism of ang i by a dipeptidyl carboxypeptidase named angiotensinconverting enzyme (ace). the substrate of renin was found to be angiotensinogen, a serum globulin produced by the liver. 3 also at this time, the role of ang ii in the regulation of aldosterone production by the adrenal cortex emerged. 4 in the 1970s, the main catalytic cascade of the reninangiotensin-aldosterone system (raas) was described ( figure 1 ). 5 plasma angiotensinogen is cleaved by renal renin, producing ang i, which is then converted to ang ii by endothelial ace, a process that occurs most extensively in lung tissue. ang ii was considered the most important raas mediator, with increased levels of ang ii being associated with vasoconstriction and increased blood pressure. ang ii binds to the type-1 ang ii receptor (at1) in a variety of tissues, including vascular smooth muscle and the adrenal grand, to mediate many mechanisms that lead to raised blood pressure. the stimulation of aldosterone production via the at1 receptor in the adrenal gland facilitates sodium retention by the kidney when aldosterone binds to the mineralocorticoid receptor. on the basis of the first experimental studies of ang ii, in which supraphysiological doses of this peptide were tested in dogs, the raas was related to the pathophysiology of hypertension mainly through the effects of this system on vascular constriction. 1 however, more recent studies have helped clarify that blood pressure control by ang ii at physiological concentrations is more related to sodium and water handling than to arterial constriction. 6 ang ii shifts the natriuresis-blood pressure curve, with increased levels of ang ii requiring increased blood pressure to eliminate the same quantity of sodium. part of this effect occurs via direct mediation by ang ii, which activates sodium transporters in the proximal tubules of the kidney. [7] [8] [9] furthermore, ang ii, through binding to the at1 receptors, is the most potent regulator of aldosterone secretion in the adrenal cortex. 10, 11 aldosterone binding to the mineralocorticoid receptor induces nongenomic (rapid) and genomic effects in many tissues, but mainly in the kidney, where it increases the epithelial expression of epithelial sodium channel (enac, also known as amiloride-sensitive sodium channel) at the level of the distal tubules, which favours the retention of sodium and water. 12 blockade of the raas was a major breakthrough in the treatment of cardiovascular disease, lowering mortality and improving quality of life in patients with hyper tension, chronic kidney disease (ckd), myocardial infarction and heart failure. [13] [14] [15] [16] [17] the classic raas antagonists target the angiotensinogen-angiotensin-at1-aldosterone axis. however, as knowledge about the raas expands, the number of potential therapeutic targets in this system is increasing. in this review we the components of the raas are schematically represented in figure 1 . renin is the rate-limiting step in ang ii production and is released by the juxta glomerular apparatus in the kidney in response to decreased renal perfusion pressure, low tubular salt load or sympathetic activation. renin is synthesized from prorenin, a pre-p roenzyme that is constitutively released, with plasma levels 10-fold higher than those of active renin. 18 prorenin can be proteolytically activated in the kidney by neuroendocrine convertase 1 (also known as prohormone convertase 1 and proprotein convertase 1) or cathepsin b and non-proteolytically (reversibly) in many tissues by the renin receptor (also known as renin/p rorenin receptor). these two types of activation require the propeptide to expose its active site, a cleft between two similar domains. 19 the renin receptor also binds to mature renin, increasing its catalytic activity by 4-5 fold. 19 the renin receptor can initiate ang ii-independent signal ling pathways, such as the mapk/erk pathway and pathways involving the zinc finger protein plzf. 20 the effects of these signalling pathways are not clear and the renin receptor has not been shown to have a role in cardiovascular disease when the effects of ang ii are blocked. 21 the secretion of renin is controlled by short-term and long-term negative feedback loops. short-term regulation is mediated directly by ang ii through binding to at1, which induces inhibition of renin gene expression. 22 the long-term negative feedback loop is an indirect consequence of the effects of increased ang ii levels: increased blood pressure, sympathetic inhibition of baroreceptor mechanisms and increased intratubular sodium levels. the classic effects of ang ii are mediated by the at1 receptor, which is present in practically all tissues (box 1). blood pressure effects are modulated by vascular at1 receptors, whose activity produces vasoconstriction (rapid pressure effects) and by renal at1 receptors that mediate sodium and water reabsorption. many fibrotic and inflammatory effects of ang ii are also a consequence of at1 receptor activation. 23 however, ang ii can also activate the type-2 ang ii receptor (at2), which is more sparsely expressed in body tissues than the at1 receptor, but which is upregulated in many cases of injury (for example, to the brain, heart and kidney). at2 is part of the protective arm of the raas, and, when activated, has antifibrotic and anti-inflammatory effects (box 1). activation of the at2 receptor does not seem to have blood pressure effects. 24 other non-classic raas components have been described in the past few years. a novel enzyme similar to ace was identified and called angiotensin-converting enzyme 2 (encoded by ace2 and hereby referred to as ace2). 25 this carboxypeptidase has great ang ii affinity and can transform this peptide to angiotensin 1-7 (hereby referred to as ang 1-7). ace2 can also convert ang i to angiotensin 1-9 (ang 1-9), which is then converted to ang 1-7 by ace. similarly, plasmatic endopeptidases can convert ang i to ang 1-7. ang 1-7 binds to a g-protein-coupled receptor called proto-oncogene mas (encoded by mas1 and hereby referred to as mas1). 26 this ace2-ang 1-7-mas1 axis has been reported to function as a counterbalance to the classic ace-ang ii-at1 axis, with opposite effects than those mediated by the classic axis (box 1). 27 another non-classic axis in the raas is the pathway mediated by angiotensin-4 (ang iv) and its receptor, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase, insulinresponsive aminopeptidase or irap). 28, 29 this receptor, which was originally reported to colocalize with the glucose transporter glut-4, has a role in the degradation of vaso pressin and oxytocin. ang iv inhibits the enzymatic activity of irap and knockout models and studies of non-peptide inhibitors suggest a role of irap in c ardiovascular disease, mainly in target-organ damage. [30] [31] [32] the effects of irap inhibition are listed in box 1. beyond the original description of the endocrine roles of the raas, experimental studies have demonstrated that the system also has paracrine, autocrine and intracrine functions. different tissues (reproductive and digestive tissues, heart and brain) and cells (adipocytes and white blood cells) express raas components, which not only perform the classic raas function (fluid volume homeostasis), but are also involved in other physiological functions, such as ovulation, 33 secretion of insulin by the pancreas, 34 cognitive function, 35 inflammation 36 and cancer. 37, 38 in addition, activation of local raass is strongly related to target-organ damage, mostly in the kidney and heart. 39 four clinically established groups of drugs inhibit the raas: ace inhibitors, angiotensin receptor blockers (arbs, which block the at1 receptor), renin inhibitors and mineralocorticoid receptor blockers (box 2). despite the important improvements achieved with these agents in slowing the progression of established cardiorenal disease, the ace inhibitors and the arbs only provide a 20% reduction in the relative risk of key points ■ renin-angiotensin-aldosterone system (raas) blockade with an angiotensin converting enzyme inhibitor or an angiotensin receptor blocker provides a 20% relative risk reduction for the progression of established cardiorenal disease compared with other non-raas blocking therapies ■ the raas is an endocrine, paracrine and autocrine system that regulates blood pressure homeostasis through effects on a variety of target organs, as well as having a role in the responses to vascular injury and repair ■ the raas is a complex system with a variety of sites suitable for pharmacological intervention ■ novel molecules that alter the production of various raas peptides or that alter receptor density, function or responsiveness to these peptides could have an important influence on haemodynamics and vascular structure and function www.nature.com/nrendo progression of cardiovascular disease when compared with non-raas blocking therapy. 14, 16, 17, 40 moreover, no data have demonstrated beneficial effects of ace inhibitors or arbs in the primary prevention of either cardiac or kidney disease. therefore, much consideration has been given to how to improve upon raas inhibition with the classic, established drugs, by investigating new agonists and antagonists of the complex raas cascade. many theories about why traditional raas inhibition has not provided more clinical benefits have been proposed, but none of these theories has been validated clinically. in the early 1980s, early clinical studies of the first oral ace inhibitor, captopril, demonstrated this agent has antihypertensive properties and improves the clinical status of patients with heart failure. 41 ace inhibitors do not affect the levels of ace2. even though the main mechanism of action of ace inhibitors is to limit the formation of ang ii, other activities, such as reducing the catabolism of other vasodilator oligopeptides (such as bradykinin) by ace, might have a role in the pharmacological effects of ace inhibitors. another tetrapeptide, n-acetyl-seryl-aspartyl-lysil-proline (ac-sdkp), whose levels are elevated in patients receiving ace inhibitors, could also be responsible for many of the antifibrotic and immune-beneficial effects of these agents. 42 furthermore, ace inhibitors increase the levels of ang i, which shifts raas activity to the axis producing ang 1-7. 43, 44 some of the favourable pharmacological effects of ace inhibitors might be mediated though increased activity of the ace2-ang 1-7-mas1 axis. 19, 20 currently, ten ace inhibitors are available in the usa and several more are available globally. the clinical effects of the various ace inhibitors are not markedly different, except for trandolapril and quinapril having a longer half-life and broader tissue distribution than the other agents owing to their liposolubility. many years of use have confirmed very good safety and tolerability profiles for these drugs, with cough (6-20% of patients) and angioedema (1% of patients) being the most common adverse effects. this drug class is the first-line therapy for patients with hypertension, ckd and heart failure, [45] [46] [47] with ace inhibitors successfully controlling blood pressure and reducing left-ventricular hypertrophy 48 and all-cause mortality. 49 the first oral arb, losartan, was made available in the 1990s, and currently nine arbs exist for clinical use (box 2). despite different pharmacokinetic characteristics, 50 most arbs are used as once-daily drugs and have fewer adverse effects than ace inhibitors. 51 in general, the indications for using arbs are the same as for using ace inhibitors; arbs are efficacious in hypertension 52-54 and heart failure, 55 figure 1 | components of the renin-angiotensin-aldosterone system and main classes of pharmacological activators and inhibitors of the system. prorenin can be activated proteolytically in the kidneys (by neuroendocrine convertase 1 or cathepsin b) or nonproteolitically by the renin receptor in many tissues. circulating renin can also bind to the renin receptor, which increases its enzymatic activity. renin converts angiotensinogen to ang i, which can then enter three main pathways. these three axes, ace-ang ii-at1-aldosterone, ace2-ang 1-7-mas1 and ang iv-irap, are highlighted. activation of the at1 receptor in the adrenal gland results in production of aldosterone, which can then bind to the mineralocorticoid receptor. abbreviations: ace, angiotensin-converting enzyme; ace2, angiotensin-converting enzyme 2; ang, angiotensin; arb, angiotensin receptor blocker; arn, angiotensin receptor-neprilysin; at1, type-1 ang ii receptor; at2, type-2 ang ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas1, proto-oncogene mas; rh, recombinant human. protection. 57 as with ace inhibitors, use of arbs increases the levels of ang ii as a substrate of ace2 and could have clinical benefits resulting from stimulation of the ace2-ang 1-7-mas1 axis. 19, 20 in theory, combining ace inhibitors with arbs or with a renin inhibitor could have pharmacological benefits. however, no evidence of clinical benefits beyond those achieved with single-agent approaches was seen when combination therapy was used in patients with hypertension, coronary artery disease or ckd, and c ombination therapy could potentially be harmful. 58, 59 mineralocorticoid receptor antagonists the mineralocorticoid receptor antagonist spironolactone was first used in the 1960s as a potassium-sparing diuretic. since then, the systemic effects of aldosterone in hypertension, heart failure and target-organ damage became better known. randomized clinical trials confirmed the effectiveness of spironolactone to treat hypertension and showed that the drug has antifibrotic and anti-inflammatory properties. 48 spironolactone binds to other receptors in addition to the mineralocorticoid receptor, such as androgen and progesterone receptors, which can have adverse effects such as gynaecomastia and decreased libido. in 2002, a second mineralocorticoid receptor antagonist, eplerenone, was introduced. eplerenone has less affinity for sex hormone receptors than spironolactone; the drug has a nearly similar antihypertensive effectiveness to that of spironolactone, yet is less potent and has fewer adverse effects. 60,61 a third antagonist, canrenone, is available in some countries but scant clinical data on this drug exist. 62 in addition to their use in primary hyperaldosteronism, mineralocorticoid receptor blockers are commonly used to treat ascites, 63 heart failure 64 and resistant hypertension. 65, 66 direct renin inhibitors in 2007, and after many attempts, the first oral direct renin inhibitor (dri), aliskiren, became available, replacing first-generation inhibitors of low bioavailability. aliskiren is an effective antihypertensive agent and dual therapy with aliskiren and an arb was tested in the altitude trial. 58 however, in this trial, the combination of aliskiren and losartan did not show any benefits on cardiovascular or renal outcomes versus losartan alone, and dual therapy was associated with an increased rate of adverse events. 58 improving the benefits of raas blockade beyond the effects of ace inhibitors or arbs will be challenging, but research in this area is abundant. several new nonpeptide drugs have been developed that modify raas activity (box 2); however, most of them are at preclinical or early clinical stages. angiotensin-receptor-neprilysin inhibitors natriuretic peptides have an important role in sodium and water homeostasis, inducing natriuresis, inhibiting the hypothalamic-pituitary-adrenal system at all regulatory levels 67, 68 and promoting vasodilatation; they also possess antiproliferative properties. 69, 70 neprilysin is a vasopeptidase that metabolizes natriuretic peptides and other peptides, such as bradykinin. neprilysin inhibition increases the plasma levels of natriuretic peptides, but has modest effects in reducing blood pressure. 71 however, combining neprilysin with a raas blocker is a powerful tool to increase natriuresis and vasodilation. the first therapeutic agent in this class was omapatrilat, a vasopeptidase that inhibits both neprilysin and ace. the cardiovascular effects of omapatrilat were encouraging, but a high rate of angioedema stopped clinical trials of this drug. 72 the next generation of drugs in this group combines a natriuretic peptide inhibitor with an arb. lcz696, the first agent of the angiotensinreceptor-neprilysin (arn) inhibitor class, combines a neprilysin inhibitor moiety with a valsartan moiety. this dual therapy decreases blood pressure in animals and healthy humans with low rates of adverse effects. 73 this type of drug intervention is promising in the fields of h ypertension and heart failure. abbreviations: at1, type-1 angiotensin ii receptor; at2, type-2 angiotensin ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas1, proto-oncogene mas; raas, renin-angiotensin-aldosterone system. a randomized clinical trial (rct) evaluated the effect of lcz696 in the treatment of hypertension. 71 patients with mild to moderate hypertension (n = 1,212) received a single dose of 100, 200 or 400 mg of lcz696, or 80, 160 or 320 mg of valsartan in a 12-week placebo-controlled trial. lcz696 showed a good safety profile and was s uperior to placebo and valsartan in reducing office blood pressure and pulse pressure. more than 70% of patients had their blood pressure controlled with the 200 mg dose of lcz696, whereas this proportion was only 56% in the group receiving 160 mg of valsartan. in approximately 50 patients per arm, ambulatory blood pressure monitoring was utilized. mean diastolic 24 h blood pressure was not different between the groups receiving the two therapies, but mean systolic blood pressure was significantly reduced in the lcz696 group because of larger nocturnal decreases in blood pressure than those observed in the valsartan or placebo groups. the greater blood pressure reduction seen in patients receiving lcz696 when compared with patients in the other groups was not related to increased levels of the second messenger (cgmp) that mediates the actions of natriuretic peptides. a second rct in an asian population also confirmed the efficacy of lcz696 in treating hypertension. 74 the parameter study 75 is an ongoing study in elderly patients with wide pulse pressure, which evaluates the vascular effects of arn inhibition, including changes in arterial stiffness and central haemodynamics, and efficacy in reducing blood pressure. the trial will be finished in 2015. the paramount study 76 evaluated the effects of 200 mg of lcz696 compared with 160 mg of valsartan in almost 300 patients with heart failure and preserved ejection fraction. even though the levels of n-terminal probnp (also known as b-type natriuretic peptide) were decreased and functional new york class of heart failure was improved at 36 weeks of follow-up, no differences in echocardiographic parameters were observed. no changes were observed in the levels of plasma inflammation markers. surprisingly, proteinuria increased in the lcz696 group, an increase that was not observed in patients treated with lcz696 in the hypertension trials. this increase could be related to increased intraglomerular pressure related to increased levels of natriuretic peptides. 77 the most important observations in this study were a 9 mmhg decrease in systolic blood pressure and a 4 mmhg decrease in diastolic blood pressure, which both persisted at 36 weeks of follow-up in the lcz696 group, whereas the decreases in blood pressure seen in the valsartan group were 1.5 mmhg and 0.3 mmhg, respectively, over the same period. the paradigm-hf trial, in which therapy with 200 mg of lcz696 was compared with therapy with 10 mg of enalapril for the treatment of heart failure (with decreased ejection fraction), was stopped according to prespecified rules because lcz696 reduced the frequency of the primary outcome, a composite of death from cardiovascular causes or hospitalization for heart failure (hr 0.80, p <0.001). 78, 79 blood pressure was signifi cantly lower in the lcz969 group than in the enalapril group. the lcz696 group had higher proportions of patients with hypertension and nonserious angioedema, but fewer patients with renal impairment and hyperkalaemia than the enalapril group. urinary levels of cgmp in the lcz969 group did not change during the trial. some post-hoc analyses have noted that the benefits of lcz969 in the treatment of heart failure do not correlate with the antihypertensive effects of the drug. 80 arn inhibition could be a breakthrough in the hypertension field. the observed reductions in systolic nocturnal blood pressure and in pulse pressure might be an indication of a substantial effect of lcz969 on vascular function or on the central nervous system rather than an effect on natriuresis. nonsteroidal mineralocorticoid receptor antagonists (third-generation and fourth-generation compounds) have been developed with the aim of achieving cardiovascular benefits with fewer renal adverse effects than those of classic mineralocorticoid receptor antagonists (spironolactone, eplerenone and canrenone). abbreviations: ace, angiotensin-converting enzyme; ace2, angiotensin-converting enzyme 2; ang, angiotensin; arb, angiotensin receptor blocker; arn, angiotensin receptorneprilysin; at1, type-1 angiotensin ii receptor; at2, type-2 angiotensin ii receptor; irap, leucyl-cystinyl aminopeptidase (also known as insulin-regulated membrane aminopeptidase or insulin-responsive aminopeptidase); mas1, proto-oncogene mas; raas, renin-angiotensin-aldosterone system. finerenone, previously called bay 94-8862, 81 is a potent and promising mineralocorticoid receptor antag onist that conferred extensive cardiovascular and renal protection in rat models of hypertension and heart failure when compared with eplerenone. 82 the arts trial assessed the efficacy and safety of this drug in patients with systolic heart failure and mild ckd. 83 this phase ii study consisted of two parts. part a, which involved 65 patients, evaluated the safety and tolerability of 2.5, 5 or 10 mg daily doses of finerenone versus placebo. part b assessed the safety and efficacy of the drug in 392 patients randomized in a 1:1:1:1:1:1 design to receive finerenone orally at doses of 2.5, 5, or 10 mg daily, or 5 mg twice daily; placebo; or open-label oral spironolactone, which was given at an initial dose of 25 mg daily and uptitra ted to 50 mg daily if serum potassium concentrations allowed. finerenone decreased the levels of bnp and n-terminal probnp. the drug decreased albuminuria by a similar degree to spironolactone and was associated with lower incidence of hyperkalaemia and acute renal failure. more data are needed to assess the clinical effects of finerenone. the arts hf trial, which has been completed but has not been published yet, assessed the efficacy and safety of this drug when compared with eplerenone in patients with diabetes mellitus but without ckd or chronic left-ventricular dysfunction. 84 another nonsteroidal drug, br-4628, has high potency and selectivity for the mineralocorticoid receptor but also blocks the l-type calcium channel, which might help lowering blood pressure. after binding to the mineralo corticoid receptor, br-4628 triggers degradation of this receptor. 85 no clinical trials of br-4628 have been reported. pf-3882845 is a potent and selective nonsteroidal inhibitor of the mineralocorticoid receptor that has been tested in healthy humans 86,87 and patients with diabetic nephropathy, 88 but was discontinued owing to adverse effects. sm-368229 is another agent of this class that has potent activity as an antagonist of the mineralocorticoid receptor and, in hypertensive rats, diminishes blood pressure and reduces the urinary levels of inflammatory markers. 89 sm-368229 has not been tested in humans. 90 aldosterone synthase inhibitors a different approach to avoid the deleterious effects of aldosterone is by inhibition of the expression of cyp11b2, the gene than encodes aldosterone synthase. inhibition at this level of the raas would block the effects that are mediated through the mineralocorticoid receptor and also those that are independent of this receptor. an additional potential benefit of this approach is the prevention of a reactive increase in aldosterone levels, which occurs when the receptor is blocked. the potential downside of this strategy is that aldosterone synthase inhibitors might also suppress cortisol release by inhibiting cytochrome p450 11b1, mitochondrial (also known as steroid 11β-hydroxylase, encoded by the cyp11b1 gene), because the two enzymes have a high sequence homology. fad 286a is an isomer of fadrozole, an aromatase inhibitor. this drug decreased plasma and urinary aldosterone concentrations and improved cardiac and renal target-organ damage in several animal models. [91] [92] [93] fad 286a has only a mild antihypertensive effect and, therefore, might be more clinically important in the treatment of heart failure than other approaches that have a more potent effect in lowering blood pressure. lci699 is an orally active and nonselective aldosterone synthase inhibitor that has been evaluated in human studies. in phase i studies, a 0.5 mg daily dose of lci699 was well tolerated and lowered plasma and urinary aldosterone levels without affecting cortisol secretion. 94 at higher doses, however, cortisol secretion was diminished. phase ii studies demon strated that plasma and urinary aldosterone levels are decreased with lci699 therapy, but the raas is upregulated and slight hyponatraemia and increased serum potassium levels were reported. 95 in a large study, 524 patients were randomized to receive either lci699 0.25 mg once daily (n = 92), 0.5 mg once daily (n = 88), 0.5 mg twice daily (n = 97) or 1.0 mg once daily (n = 86); eplerenone 50 mg twice daily (n = 84); or placebo (n = 77) for 8 weeks. 96 lci699 intake was associated with a modest reduction in blood pressure that was lower than that achieved with eplerenone.when aldosterone synthase is inhibited by lci699, cortisol levels remain normal, but 11-deoxycorticosterone levels increase, which means that the adrenocorticotropichormone-cortisol axis is stimulated by the inhibition of expression of the cyp11b1 gene. 97 the stimulation of this axis might be one of the reasons why the decrease in blood pressure is only modest even at high lci699 doses. lack of aldosterone synthase selectivity, a short halflife and lack of adequate antihypertensive efficacy in primary aldosteronism and resistant hypertension hinder the usefulness of lci699 in these areas. this agent, however, might be of value in the treatment of cushing disease. 98 therefore, research is now set to develop second-generation aldosterone synthase inhibitors with better selectivity than lci699, which would be associated with improved effects on reduction of blood pressure. n-(pyridin-3-yl) benzamides are potential agents for future development in this drug class. 99, 100 post-transcriptional regulation of cyp11b1 and cyp11b2 expression by dicer-dependent micrornas (mirnas) can affect secretion of aldosterone and cortisol in adrenocortical cells. 101 therefore, adrenal mirnas might also be potential therapeutic targets. the rationale behind complete raas inhibition through blockade of the rate-limiting enzyme renin, and the expectation that this approach would more effectively prevent ang ii production than approaches targeted at other components of the raas 102 is theoretically attractive. however, aliskiren is the only dri available. as add-on therapy to arbs, aliskiren did not improve renal or cardio vascular outcomes and was associated with more adverse effects than arb therapy alone. 58 aliskiren has not been evaluated as initial or solo therapy, because other drugs have proven benefits when administered in this way. in the usa, the package insert of aliskiren contains a formal recommendation by the fda that the drug should not be used as dual therapy in association with arbs or ace inhibitors in patients with diabetes mellitus and renal disease. in 2013, a new dri, act-077825, was shown to have an adequate safety profile in humans when administered once daily. 103 act-077825 inhibits plasma renin activity, while the levels of immunoreactive renin increase, a pattern typical of dris. 102 however, the decrease in plasma renin activity is transient and is not dose-dependent at 7 days. 103 the mechanism under lying these observations is not clear, because the plasma levels of act-077825 remain constant. 103 the effects of this dri on blood pressure are controversial, as only one study showed a decrease in blood pressure when act-077825 was used, and this effect was seen in the supine position, 104 whereas other studies showed no differ ences between the effects of act-077825 and placebo on blood pressure in healthy individuals and patients with hypertension. 103, 105 more studies are necessary to clarify this inconsistency. the same pharmaceutical company that developed act-077825 is developing another dri, act-178882. in early phase i and phase ii studies, act-178882 has shown a good safety profile. 106 not much information is yet a vailable about the antihypertensive efficacy of act-178882. for 20 years, different agonist and antagonist peptides were used to study the function of the at2 receptor. the reported protective effects of this receptor were related to its antifibrotic and anti-inflammatory properties 24 (box 1). in 2004, a selective non-peptide at2 agonist, compound 21, was introduced for research. most of the known effects of this agent have been described in different animal models. in rats with spontaneous hypertension, the drug reduces the amount of collagen in the extra cellular matrix and in vascular tissue in a bloodpressure-independent manner. 107 compound 21 has a similar effect in animal models of myocardial infarction, reducing scar formation and improving cardiac function. 108 compound 21 reduced proteinuria in the kidney of rat models of hypertension and kidney disease, and also decreased the levels of inflammatory markers in these models. 109, 110 in the central nervous system, at2 receptor activation has neuroprotective effects and induces neuro regeneration. compound 21 reproduces these effects, but has to be administered centrally to bypass the blood-brain barrier. 111 however, systemic administration of compound 21 might still have effects on neuroregeneration. 112 compound 21 does not have a net effect on blood pressure, which is similar to the situation seen with other at2 agonists. 24 however, when the effects of the at1 receptor on vasoconstriction are blocked, the vasodilator properties of compound 21 are revealed, and blood pressure decreases. 8, 9 an at2 antagonist can reverse this effect. a study published in 2014 demonstrated that the increased urinary sodium excretion that is induced by compound 21 is mainly due to effects on proximal tubular cells of the kidney, namely internalization of sodium/ hydrogen exchanger 3 (nhe-3) and na + /k + atpase. 113 continuous compound 21 infusion recruited more at2 receptors to the proximal tubule brush border than an acute infusion. acute compound 21 infusion did not change blood pressure, but in a 7-day experiment in which concomitant intrarenal ang ii infusion was administered, compound 21 blocked the blood pressure effects of ang ii, increasing natriuresis. more studies are needed to clarify whether infusion time, time to achieve at2 receptor upregulation, or administration route can influence blood pressure responses to compound 21. even though no clinical trials are published yet, available evidence suggests that compound 21 could modulate fluid retention, h ypertension and target-organ damage. the benefits of ace2 in vascular disease might be brought about by two different mechanisms. first, by participating in ang ii degradation, ace2 decreases the interaction of ang ii with at1 receptors, and second, by increasing ang 1-7 synthesis, ace2 induces vasodilation. the main function of ace2 has been proposed to be the counter balancing of the effects of the ace-ang ii-at1-aldosterone axis. 114 decreases in ace2 levels or increases in ace and ang ii levels induced damage in animal models of diabetic nephropathy or hyper tension. 115, 116 in animal models, the human recombinant form of ace2 (rhace2) reduced progression of diabetic nephro pathy, decreased blood pressure (in models of hyper tension), and decreased cardiac fibrosis. 117, 118 in 2013, the pharmaco kinetic and pharmacodynamic characteristics of rhace2 were described in healthy humans. 119 daily rhace2 intake decreased the levels of ang ii in a dose-dependent manner over 24 h, with a transient increase in ang 1-7 levels, and more sustained increases in ang 1-7 degradation products, such as ang 1-5. the authors of this study suggest that rhace2 is likely to reach the extravascular space, a hypothesis that is based on the large spread of rhace2 distribution they observed. this enzyme has a good safety profile and did not induce immunogenicity, even when repeated doses were administered. similarly to results reported in healthy animals, rhace2 did not affect blood pressure or heart rate in humans. thus, rhace2 is a promising drug for treatment of patients with intolerance to other drugs that target raas components, and also perhaps in acute illnesses, such as myocardial infarction or decompensated heart failure, in which the balance between ace and ace2 is disturbed. this drug is attractive in the renal field, because rhace2 might quickly reduce proteinuria, even in associ ation with an ace inhibitor. these two classes of drugs used together might have important protective effects mediated through substantial increases in ang 1-7 levels. we look forward to more human clinical trials in this area. aside from cardio vascular effects, some evidence exists that ace2 might facilitate recovery from acute lung injury, as rhace2 has been reported to ameliorate virus-mediated lung injury in mouse models. 120 thus, rhace2 therapy in virus-mediated lung injury, especially influenza, in which ang ii levels are correlated with the degree of lung injury and with mortali ty, 121 is potentially promising. similarly to ace2 administration, ang 1-7 binding to the mas1 receptor has shown beneficial effects in animal models; these effects are related to decreased fibrosis and inflammation and to blood pressure reduction by vaso dilation and enhancement of baroreflex sensitivity. 44 more than 10 years ago, the non-peptide compound ave 0991 showed similar effects to those of ang 1-7 in endo thelial cells. 122 subsequent data demonstrated that ave 0991 and ang 1-7 compete for the same mas1 receptor in the kidney. 123 this non-peptide mas1 receptor agonist has been studied in animals, in which it demon strated a capa city to decrease blood pressure, alone or in combination with renin inhibitors. 124 ave 0991 was also demonstrated to attenuate acute and chronic renal injury in animal models. 125, 126 in 2014, ave 0991 was shown to improve penile erection through nitric oxide release in rats. 127 ave 0991 has also been shown to ameliorate kidney inflammation, 125 and, more interestingly, improve cell infiltration, cytokine release and histology in two rodent models of arthritis. 128 unfortunately, no clinical trials have yet been performed to test this promising therapeutic approach in humans. the development of compounds that protect ang 1-7 from enzymatic degradation has also been the focus of interest. the combination of ang 1-7 with hydroxylpropyl-β-cyclodextrin protects ang 1-7 from degradation and acts as a long-lasting release system, enabling uptake of ang 1-7 in the colon. this drug combination has been tested in animal models of hypertension, in which it showed antihypertensive effects, 129 and also had beneficial effects in models of diabetes mellitus and atherosclerotic inflammation. 130, 131 phase i clinical trials are currently testing the safety profile of this combination therapy in humans (r. a. santos, personal communication). many oligopeptides result from ang ii degradation by endopeptidases. the hexapeptide ang 3-8, known as ang iv, has been studied because it might have a role in protecting cognitive function. 29 irap, the ang iv receptor, which is inhibited by ang iv, was identified in 2001. 132 irap colocalizes with the glucose transporter glut-4 and was originally thought to contribute to basal intracellular retention of the transporter. 133 irap is linked to vasopressin and oxytocin degradation. a group of nonpeptide inhibitors of irap has been developed. hfi-419 is an irap-selective pyridinyl compound that enhances memory in rats. 134 an australian group is working on the cardiovascular-protective effects of irap inhibition with hfi-419 in mice. preliminary data demonstrate that hfi-419 prevents cardiac and endothelial damage induced by ang ii, independent of blood pressure. 32 hfi-419 also has anti-inflammatory properties. 32 the association of irap with glut-4 is intriguing, and whether irap inhibitors could be used to improve insulin sensitivity remains an interesting opportunity. more studies are n ecessary to explore therapeutic uses for irap inhibitors. vaccines against the raas the high prevalence of hypertension in the world and the related cost of this burden have made the development of vaccines against the raas an attractive strategy, with the aim of achieving increased treatment compliance and public access to therapy, and a decrease in public-health costs. many attempts were made to generate vaccines against renin, ang i, ang ii and at1. 135 however, most of these attempts have been harmful or have not had clinical efficacy. an rct in which an anti-ang ii vaccine (cyt006-angqb) was tested passed phase iia without safety problems, and showed a promising 24 h reduction in blood pressure of 9.0 mmhg (systolic) and 4.0 mmhg (diastolic). 136 however, subsequent studies could not confirm these results. novel strategies are currently in development, such as the association of ang ii as an epitope to a hepatitis a virus-like particle to generate an improved immune response. 137 future studies are needed to explore the efficacy and safety of an i mmunological, chronic and irreversible blockade of the raas. although to improve upon the cardiovascular and renal protection offered by ace inhibitors and arbs will be diffi cult, novel drugs are being developed to shift the activity of the raas towards the protective cardiovascular arm (at2 and mas1 receptors). a role for raas blockade in other, non-cardiovascular pathologies (such as lung injury and cognitive disorders) is emerging. the 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erection anti-inflammatory effects of the activation of the angiotensin-(1-7) receptor, mas, in experimental models of arthritis an orally active angiotensin-(1-7) inclusion compound and exercise training produce similar cardiovascular effects in spontaneously hypertensive rats treatment with angiotensin-(1-7) reduces inflammation in carotid atherosclerotic plaques oral administration of angiotensin-(1-7) ameliorates type 2 diabetes in rats evidence that the angiotensin iv (at(4)) receptor is the enzyme insulin-regulated aminopeptidase insulin-regulated aminopeptidase is a key regulator of glut4 trafficking by controlling the sorting of glut4 from endosomes to specialized insulin-regulated vesicles identification and characterization of a new cognitive enhancer based on inhibition of insulin-regulated aminopeptidase vaccine for hypertension: modulating the renin-angiotensin system effect of immunisation against angiotensin ii with cyt006-angqb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase iia study construction, expression and immunogenicity of a novel anti-hypertension angiotensin ii vaccine based on hepatitis a virus-like particle author contributions c.a.r. researched data for the article. c.a.r. and m.r.w. wrote the article. all authors contributed substantially to discussion of content and reviewing and/or editing the manuscript before submission. key: cord-002468-onpmkjaz authors: roca-ho, heleia; riera, marta; palau, vanesa; pascual, julio; soler, maria jose title: characterization of ace and ace2 expression within different organs of the nod mouse date: 2017-03-05 journal: int j mol sci doi: 10.3390/ijms18030563 sha: doc_id: 2468 cord_uid: onpmkjaz renin angiotensin system (ras) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. in this sense, since local ras has been described, the aim of this study is to characterize angiotensin converting enzyme (ace) and ace2 activities, as well as protein expression, in several tissues of the non-obese diabetic (nod) mice model. after 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ace and ace2 enzyme activities and protein expression. ace and ace2 enzyme activities were detected in different tissues. their expressions vary depending on the studied tissue. thus, whereas ace activity was highly expressed in lungs, ace2 activity was highly expressed in pancreas among the studied tissues. interestingly, we also observed that diabetes up-regulates ace mainly in serum, lung, heart, and liver, and ace2 mainly in serum, liver, and pancreas. in conclusion, we found a marked serum and pulmonary alteration in ace activity of diabetic mice, suggesting a common regulation. the increase of ace2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of ras. angiotensin converting enzyme (ace)2 is an enzymatically active ace homologue, which shares 42% of its amino acidic sequence identity in its catalytic domain; however, ace and ace2 show several differences. whereas ace is a dipeptidylcarboxypeptidase, presenting both n-and c-terminus catalytic domains with two zinc-binding motifs (hexxh, where x is any amino acid); ace2 is a monocarboxypeptidase with only one zinc-binding motif at its n-terminal domain [1] [2] [3] . through this catalytic domain, ace2 hydrolyzes angii (angiotensin ii) to generate ang1-7, a peptide that binds to the mas receptor (ang1-7 receptor) and activates vasodilation, anti-fibrosis, anti-proliferation, and anti-inflammatory effects as well as counterbalances the ace-angii-atr1 axis actions [4] [5] [6] . renin angiotensin system (ras) was classically described as a regulation system responsible for blood pressure regulation, electrolyte, and fluid homeostasis, exerting most of its actions through kidneys [7] . later on, its elements were also found in extrarenal tissues, indicating the presence of a local paracrine system that coexists with the circulating ras [8] . thus, ace expression was mainly found in the surface of endothelial cells of lungs, renal brush border membranes, intestines, choroid plexus, placenta [9] [10] [11] , and, to a lesser extent, in cardiac, hepatic, pancreatic, and adrenal tissues [12] . in contrast, ace2 protein expression was initially reported to be more tissue restricted and higher than ace, being described preferentially in kidneys (renal tubules and glomeruli), heart, and testis [13] . subsequently, ace2 expression was also found widespread in many other organs such as lungs, pancreas [14, 15] , bladder, stomach, ileum, adipocytes, and liver [16, 17] . ras activation is known to play a key role in several diseases, namely diabetes, and renal and cardiovascular pathologies [18, 19] . ace2 and ace enzymes have found to be altered in the kidney from different diabetic experimental models, the db/db (type 2) and streptozotozin (stz) (type 1) diabetic mice [20] [21] [22] . however, ace2 and ace enzymes have not been extensively studied in other tissues from diabetic mice. the non-obese diabetic (nod) mice is a strain that spontaneously develops autoimmune diabetes that mimics type 1 diabetes in humans [23, 24] . we hypothesized that ace2 and ace enzymes are present in different tissues other than kidney and differentially expressed. in addition, the administration of insulin can alter ace2 and ace expression within these tissues from nod mice. for this purpose, we studied both ace and ace2 activities as well as their protein expression in several tissues from nod diabetic mice. we also analyzed the effect of diabetes on ace2 and ace modifications in tissues from nod diabetic mice as compared to non-obese resistant (nor) mice. in addition, we assessed the effect of insulin administration on ace2 and ace expression in the studied tissues. ace enzymatic determination technique was set up using two buffers. for this study, human recombinant ace (hrace) was used. phosphate buffer (pb) and borate buffer (bb) were incubated with increasing amounts of hrace ( figure 1a ). we observed that ace activity at lower hrace amounts was only detected by the use of bb. in addition, the hrace activity measurement was found to be linear with bb as compared to pb. pb needed higher amounts of hrace for its detection, suggesting a lower sensitivity when this buffer was used. after the above-mentioned experiments, we decided to use bb for our ace activity assays. the next step was to test the optimal amounts of tissue protein for ace activity measurements (figure 1b,c) . increasing amounts of protein for each tissue of both control (cont, dashed lines) and diabetic (db, continuous lines) mice were used. pulmonary tissue presented a linear enzymatic activity in both cont and db mice due to its high enzymatic activity ( figure 2b ). in contrast, no linearity in increasing amounts of heart (triangle), pancreas (circle), and liver (cross) samples due to the low ace activity detection was observed ( figure 1c ). furthermore, it is worth noting that ace activity in db was higher as compared to cont, and lung presented the highest levels of ace activity. ace2 enzymatic activity was already previously established and validated in our laboratory by using a specific ace2 quenched fluorogenic substrate [25] [26] [27] . the optimal amount of each studied tissue was determined by reactions with increasing amounts of protein in pulmonary, cardiac, hepatic, and pancreatic tissues of both cont and db samples ( figure 2 ). higher ace2 activity was observed in lung, heart, liver, and pancreas from cont mice, with increased amounts of protein. interestingly, increased levels of enzymatic activities were found in tissue samples from db mice as compared to controls. among all studied tissues, pancreas presented higher levels of ace2 activity followed by heart, lung, and liver. figure 1 . implementation of angiotensin converting enzyme (ace) activity assays with different buffers and in different tissues. (a) ace activity with borate (bb) and phosphate buffer (pb) after increasing amounts (ng) of human recombinant ace (hrace). ace activity at lower concentrations of hrace was only detected by the use of bb; (b) ace activity in lung measured with bb (black rhombus), and (c) ace activity in heart (blue triangle), pancreas (green circle), and liver (red cross) from control (dashed lines) and diabetic (continuous lines) mice. increasing amounts of micrograms of total protein extracts were tested. ace2 activity assays in different tissues and with increasing protein amounts. ace2 activity was measured in lung (black rhombus), heart (blue triangle), pancreas (green circle), and liver (red cross) from control animals (cont) (left panel) and diabetic animals (db) (right panel) mice. increasing amounts of micrograms of total protein extracts from each studied tissue were tested (1, 3, 5, 7.5, and 10 μg of protein). increasing amounts of micrograms of total protein extracts from each studied tissue were tested (1, 3, 5, 7.5, and 10 μg of protein). ace2 activity assays in different tissues and with increasing protein amounts. ace2 activity was measured in lung (black rhombus), heart (blue triangle), pancreas (green circle), and liver (red cross) from control animals (cont) (left panel) and diabetic animals (db) (right panel) mice. increasing amounts of micrograms of total protein extracts from each studied tissue were tested (1, 3, 5, 7.5, and 10 µg of protein). serum, lung, heart, liver, and pancreas from diabetic, insulin-treated, and control mice in serum samples, ace activity was significantly increased in db at early and late stages of diabetes as compared to cont mice (p = 0.04 in 21-day follow up and p = 0.0003 in 40-day follow up). insulin administration slightly decreased circulating ace activity in db mice at both early and late stages (p = ns, not significant) (figure 3a ). ace activity was significantly increased in lungs from db mice at early and late stages of diabetes as compared to cont mice (p = 0.045 and p = 0.026, respectively). insulin administration did not modify ace activity in lungs from db as compared to cont mice (p = ns) (figure 3b ). regarding heart samples, no statistically significant differences were observed in ace activity in db as compared to their respective cont mice (p = ns). furthermore, no significant effect was observed in cardiac tissue after insulin treatment in db as compared to cont mice (p = ns) ( figure 3c) . ace activity was also tested in liver. as observed in heart, no differences in ace activity levels between db and cont mice were found in liver tissue (p = ns). no changes were observed after insulin administration (p = ns) ( figure 3c ). no significant differences in ace activity levels were found in pancreas from db and cont mice (p = ns) ( figure 3c ). in serum samples, ace activity was significantly increased in db at early and late stages of diabetes as compared to cont mice (p = 0.04 in 21-day follow up and p = 0.0003 in 40-day follow up). insulin administration slightly decreased circulating ace activity in db mice at both early and late stages (p = ns, not significant) (figure 3a ). ace activity was significantly increased in lungs from db mice at early and late stages of diabetes as compared to cont mice (p = 0.045 and p = 0.026, respectively). insulin administration did not modify ace activity in lungs from db as compared to cont mice (p = ns) (figure 3b ). regarding heart samples, no statistically significant differences were observed in ace activity in db as compared to their respective cont mice (p = ns). furthermore, no significant effect was observed in cardiac tissue after insulin treatment in db as compared to cont mice (p = ns) ( figure 3c) . ace activity was also tested in liver. as observed in heart, no differences in ace activity levels between db and cont mice were found in liver tissue (p = ns). no changes were observed after insulin administration (p = ns) ( figure 3c ). no significant differences in ace activity levels were found in pancreas from db and cont mice (p = ns) ( figure 3c ). in serum samples, there was a significant increase of ace2 activity in db mice at early and late stages of diabetes as compared to cont (p = 0.0003 and p = 0.0003, respectively) and insulin administration significantly decreased ace2 activity in db mice at early and late stages (p = 0.001 and p = 0.001, respectively) ( figure 4a ). in lung homogenates, no changes were observed between db and cont mice (p = ns). interestingly, insulin administration significantly increased ace2 activity in db mice at late stage as compared to db mice (p = 0.05) (figure 4b ). at cardiac level, there was a in serum samples, there was a significant increase of ace2 activity in db mice at early and late stages of diabetes as compared to cont (p = 0.0003 and p = 0.0003, respectively) and insulin administration significantly decreased ace2 activity in db mice at early and late stages (p = 0.001 and p = 0.001, respectively) ( figure 4a ). in lung homogenates, no changes were observed between db and cont mice (p = ns). interestingly, insulin administration significantly increased ace2 activity in db mice at late stage as compared to db mice (p = 0.05) (figure 4b ). at cardiac level, there was a significant increase of ace2 activity in db mice in both early and late stages of db as compared to cont mice (p = 0.011 and p = 0.029, respectively), however, insulin administration did not modify this pattern (p = ns) (figure 4b) . in liver samples, no changes were observed between the studied groups ( figure 4b ). pancreatic ace2 activity was increased in db mice as compared to cont mice (p = 0.014 in 21-day follow up and p = 0.0003 in 40-day follow up of study). in addition, insulin administration did not change ace2 activity in pancreas from db mice (p = ns) (figure 4b ). int. j. mol. sci. 2017, 18, 563 5 of 13 significant increase of ace2 activity in db mice in both early and late stages of db as compared to cont mice (p = 0.011 and p = 0.029, respectively), however, insulin administration did not modify this pattern (p = ns) (figure 4b ). in liver samples, no changes were observed between the studied groups ( figure 4b ). pancreatic ace2 activity was increased in db mice as compared to cont mice (p = 0.014 in 21-day follow up and p = 0.0003 in 40-day follow up of study). in addition, insulin administration did not change ace2 activity in pancreas from db mice (p = ns) (figure 4b ). after ace and ace2 activities were measured, ace2/ace activity ratios were calculated for each studied tissue to infer the status of ras in different tissues as an ace-ace2 balance. in serum samples, ace2/ace activity ratio in db mice at early and late stages was higher as compared to cont (p = 0.011 and p = 0.0003, respectively). insulin administration significantly decreased circulating ace2/ace activity ratio in db mice (p = 0.001) (figure 5a ). in lung, there were no differences in ace2/ace activity ratio between the db and cont groups at early stage. interestingly, ace2/ace activity ratio was significantly decreased in db at late stage as compared to cont mice (p = 0.001). insulin administration significantly increased ace2/ace activity ratio in db mice (p = 0.014) (figure 5b) . at cardiac level, ace2/ace activity ratio was significantly increased in db mice in both early and late stages as compared to cont mice (p = 0.0003 and p = 0.008, respectively), but insulin administration had no effect on ace2/ace activity in db (figure 5c ). after ace and ace2 activities were measured, ace2/ace activity ratios were calculated for each studied tissue to infer the status of ras in different tissues as an ace-ace2 balance. in serum samples, ace2/ace activity ratio in db mice at early and late stages was higher as compared to cont (p = 0.011 and p = 0.0003, respectively). insulin administration significantly decreased circulating ace2/ace activity ratio in db mice (p = 0.001) (figure 5a ). in lung, there were no differences in ace2/ace activity ratio between the db and cont groups at early stage. interestingly, ace2/ace activity ratio was significantly decreased in db at late stage as compared to cont mice (p = 0.001). insulin int. j. mol. sci. 2017, 18, 563 6 of 13 administration significantly increased ace2/ace activity ratio in db mice (p = 0.014) (figure 5b) . at cardiac level, ace2/ace activity ratio was significantly increased in db mice in both early and late stages as compared to cont mice (p = 0.0003 and p = 0.008, respectively), but insulin administration had no effect on ace2/ace activity in db (figure 5c ). in pancreas, ace2/ace activity ratio was significantly increased in db mice in both early and late stages as compared to cont mice (p = 0.05 and p = 0.001, respectively), but insulin administration had no effect on ace2/ace activity in db ( figure 5, panel c) . in liver, there were no differences in ace2/ace activity ratio in db as compared to their respective cont (p = ns). insulin administration did not modify ace2/ace activity in liver (figure 5c ). in pancreas, ace2/ace activity ratio was significantly increased in db mice in both early and late stages as compared to cont mice (p = 0.05 and p = 0.001, respectively), but insulin administration had no effect on ace2/ace activity in db ( figure 5, panel c) . in liver, there were no differences in ace2/ace activity ratio in db as compared to their respective cont (p = ns). insulin administration did not modify ace2/ace activity in liver (figure 5c ). to assess ace protein expression, immunoblotting techniques were performed. in lungs, ace protein expression was significantly increased in db mice at early and late stages as compared to cont (p = 0.008 and p = 0.012, respectively). in addition, insulin administration significantly decreased ace protein expression in db mice (p = 0.05 and p = 0.024, respectively) ( figure 6a ). in heart, ace protein expression was significantly increased in db mice at late stage as compared to cont (p = 0.03) (figure 6b ). insulin administration did not modify ace protein expression. in liver and pancreas, there were no differences between db and cont mice (figure 6c) . in lungs, ace2 protein expression was significantly increased in db mice at early stage as to assess ace protein expression, immunoblotting techniques were performed. in lungs, ace protein expression was significantly increased in db mice at early and late stages as compared to cont (p = 0.008 and p = 0.012, respectively). in addition, insulin administration significantly decreased ace protein expression in db mice (p = 0.05 and p = 0.024, respectively) ( figure 6a ). in heart, ace protein expression was significantly increased in db mice at late stage as compared to cont (p = 0.03) (figure 6b ). insulin administration did not modify ace protein expression. in liver and pancreas, there were no differences between db and cont mice (figure 6c ). protein expression in db mice (p = 0.05) (figure 6a ). in heart, ace2 protein expression was significantly increased in db mice at early stage as compared to cont mice (p = 0.022 and p = 0.035, respectively). insulin administration did not modify ace2 protein expression in db mice (p = ns) (figure 6b ). in liver, ace2 protein expression was significantly increased in db mice at late stage as compared to cont mice (p = 0.008) and insulin administration did not modify ace2 protein expression (figure 6c ). in pancreas, there were no changes observed when ace2 protein expression was studied (figure 6d ). mean blood glucose levels in control animals were 160.25 ± 9.47 mg/dl at the end of the study. in diabetic animals, blood glucose levels were significantly increased to 550 ± 33.08 mg/dl in animals followed for 21 days and 555 ± 3.47 mg/dl in the animals followed for 40 days. insulin pellets in lungs, ace2 protein expression was significantly increased in db mice at early stage as compared to cont mice (p = 0.008). in addition, insulin administration significantly decreased ace2 protein expression in db mice (p = 0.05) (figure 6a ). in heart, ace2 protein expression was significantly increased in db mice at early stage as compared to cont mice (p = 0.022 and p = 0.035, respectively). insulin administration did not modify ace2 protein expression in db mice (p = ns) (figure 6b ). in liver, ace2 protein expression was significantly increased in db mice at late stage as compared to cont mice (p = 0.008) and insulin administration did not modify ace2 protein expression (figure 6c ). in pancreas, there were no changes observed when ace2 protein expression was studied (figure 6d ). mean blood glucose levels in control animals were 160.25 ± 9.47 mg/dl at the end of the study. in diabetic animals, blood glucose levels were significantly increased to 550 ± 33.08 mg/dl in animals followed for 21 days and 555 ± 3.47 mg/dl in the animals followed for 40 days. insulin pellets significantly reduced blood glucose levels at 110.65 ± 11 mg/dl and 97.67 ±9.48 mg/dl for 21 and 40 days of diabetes, respectively. blood glucose levels positively correlated with ace2/ace ratio in serum (r = 0.455, p = 0.003) and negatively correlated with ace2/ace ratio in lung (r = −0.546, p = 0.0005). several works have been focused on the study of ace and ace2 enzymes within the diabetic kidney [21, 28] . ace2 has been shown to be increased in the kidney from different models of diabetic nephropathy, the stz-diabetic model and the non-obese diabetic mice (nod), among others [20, 25] . however, in other tissues from diabetic animals, such as liver and heart, ace and ace2 enzymes have not been widely studied. in the present study, we demonstrated that ace and ace2 activities are present in different tissues. interestingly their expression is different depending on the tissue: ace is highly expressed in lung, whereas ace2 is highly expressed in pancreas among the studied tissues. we also observed that diabetes up-regulated ace and ace2 activity and protein expression in the majority of the studied tissues. a large number of ace activity detection techniques have been described, such as radioassays, high-performance liquid chromatography, and colorimetric-based assays [29] [30] [31] . of note that, ace activity measurement based on fluorimetric quantification is the most widely used technique because of its sensitivity, simplicity, speed, and high reproducibility [32] . in 1971, cushman and cheung developed an ace activity assay using synthetic ace-specific substrates, including one of the substrates most commonly cited in literature, hippuryl-l-histidyl-l-leucine (hhl) [33] . as an indirect determination, this technique is based on the hydrolysis of hhl and the measurement of fluorescence through the o-phthalaldialdehyde adducts formation with hhl [34] . in this work, ace activity was indirectly measured using hhl as the substrate for ace, as previously described. previous studies have shown that ace activity from the same samples differs depending on the homogenization buffer used [32, 35] . we now show that borate buffer is the most suitable to detect lower levels of ace activity, showing better linearity, conferring less variability, and offering a more reliable assay. our results showed that ace activity in lungs was higher as compared to other studied tissues. moreover, a significant increase was observed in serum and lung from diabetic mice at early and late stages as compared to control mice. the same pattern was observed when ace protein expression was studied. these observations are consistent with previous studies from huang and co-authors showing increased ace activity in plasma and mrna levels in lung from stz-induced diabetic c57bl/6 mice [36] . thus, there is a coupled (serum and pulmonary) alteration in ace activity, suggesting that pulmonary and circulating ras may exert a common regulation. since ras has a key role in cardiovascular diseases, recent studies have associated ras with liver fibrosis, cirrhosis, and portal pressure regulation [37] . interestingly, pancreas is found to express local ras. the role of ras components in diabetes have been previously studied [15, 25, 38] . in addition, ace was also increased in liver from db mice at late stage of follow-up. we found that ace activity was decreased in heart from db mice, but only at early stage. however, in previous studies by colucci and co-authors, there were no differences between control and diabetic mice [12] . in all studied tissues, the insulin-treated group presented no significant differences compared to the non-treated diabetic group. ace2 enzyme, a novel ace homologue, was discovered in the last decade [1, 2] . for years, the detection of endogenous ace2 activity in mouse tissues was difficult and ace2 activity assay is a relatively recent reliable technique [39, 40] . ace2 activity was performed in serum, lung, heart, liver, pancreas, and kidney tissues by measuring the hydrolysis of mca-apk(dnp), a quenched-fluorescent specific ace2-substrate. our findings showed that ace2 activity levels were higher in pancreas as compared to other studied tissues. interestingly, significant increases in serum, pancreas, and heart in ace2 activity from nod diabetic mice as compared to nor mice at 21 and at 40 days after the onset of diabetes were observed. in addition, ace2 activity was also increased in liver at 40 days of diabetes in nod mice. with regard to protein expression, ace2 expression levels were increased in lungs and heart at early stage of diabetes and in pancreas at late stage of diabetes. previous studies postulated that the differences observed in ace2 activities are related to adam17 sheddase activity [41] . however, no differences were found when studying ace2 and adam17 activities and gene expression in pancreas islet from db/db mice as compared to the respective db/m controls [42] . it is of note that insulin administration mainly restored ace and ace2, and ace2/ace ratio activities in serum samples at longer time of follow-up. however, these results were not consistent in other tissues. these findings may be ascribed to the direct correlation observed between glucose levels and ace2/ace ratio. thus, the results observed may indicate in part a protective effect of insulin on normalizing circulating ras activities. in conclusion, we assessed ace and ace2 activities in different tissues of nod mice and demonstrated that ace activity is highly detected in lungs, whereas ace2 activity is highly detected in pancreas. in diabetic mice, there is a coupled (serum and pulmonary) alteration in ace activity that suggests that pulmonary and circulating ras may exert a common regulation. the increase of ace2 activity within the circulation in diabetic mice may be related to a compensatory ras mechanism. nod/shiltj and nor/ltj female mice (from the jackson laboratory, bar harbor, me, usa) were housed in cages under 12 h light/dark cycle in a specific pathogen germ free (spf) environment. female mice only were used because the development of diabetes is more predictable in female than in male nod mice [43] . mice were fed with a chow diet and were provided access to tap water ad libitum. the ethical committee of animal experimentation of the barcelona biomedical research park (ceea-prbb) (mso-08-1106) and the catalan government (dmah: 4097) approved this study. mice had their blood glucose levels determined every two weeks starting at 10 weeks of age. fasting blood samples from tail vein were obtained and used for glucose level determination with the accu-chek compact ® (roche, st. cugat, spain). female nod mice were considered diabetic when glucose blood level higher than 250 mg/dl was first detected. nod diabetic mice were randomly assigned to two groups, without (db) or with insulin treatment (db + ins). for blood glucose levels control, insulin pellets (~0.1 u/24 hr/pellet, linbit, linshin canada inc, toronto, canada) were subcutaneously implanted under anesthesia with ketamine and medetomidine. after surgery, atipamezol was injected to revert the effects of medetomidine. diabetic animals were compared to the non-diabetic strain, nor (cont). studied animals were weekly controlled for body weight and glucose blood levels and were followed for 21 and 40 days after diabetes diagnosis, and then final surgery was performed. the total number of animals included in each study group were the following: eight animals in each cont group, seven animals in each db group, and five animals were studied for 21 days with insulin pellet and six animals for 40 days with insulin treatment. studies were performed in serum, heart, lungs, liver, and pancreas. animals were sacrificed under anesthesia, with pentobarbital. blood samples were obtained by intracardiac puncture and organs were next perfused with phosphate-buffered saline (pbs) solution by transcardiac puncture. serum was obtained after 10 min of centrifugation at 6000× g and stored at −80 • c. tissues were quickly removed and snap frozen in liquid nitrogen. they were then stored at −80 • c until use. ace activity was first set up using human recombinant ace (hrace) and, for this reason, two different buffers were used with increasing concentrations of recombinant. one buffer contained 0.5 m potassium phosphate (pb) ph 8.3 [34] and the other was a mix of 0.4 m borate buffer (bb) ph 7.2, 0.34 m sucrose, and 0.9 m nacl [32] . after that, increasing amounts of hrace were incubated with 27.3 m hip-his-leu at 37 • c for 25 min. reaction was stopped with 0.28 m naoh and then 20 mg/ml of o-phtaldialdehyde in methanol were added to generate an adduct formation. the reaction was incubated at room temperature (rt) in dark conditions for 10 min and then it was stopped using 3 n hcl. samples were clarified for 5 min at 800× g and his-leu fluorescent adduct was measured fluorometrically at 360-nm excitation and 485-nm emission using a fluorescence plate reader tecan infinite 200 (tecan instruments, männedorf, switzerland). for mouse samples, bb was used following the protocol previously described. for serum, 2 µl of sample were used and for tissues, between 5 and 10 µg of total protein were analyzed depending on the tissue. results were expressed as rfu (relative fluorescent units) per µl of serum or µg of protein (rfu/µl or rfu/µg). ace2 enzymatic activity assay was performed as previously published by our group [25, [44] [45] [46] and adapted to different tissues. briefly, 5 µl of serum or 5 µg of tissue samples that were previously homogenized were incubated using a 100 mm tris-hcl, 600 mm nacl, 10 µm zncl 2 , ph 7.5 buffer in the presence of protease inhibitors containing 100 µm captopril, 5 µm amastatin, 5 µm bestatin (all from sigma-aldrich, madrid, spain), and 10 µm z-pro-prolinal (enzo life sciences, grupo taper, madrid, spain). samples were incubated with 20 µm mca-ala-pro-lys(dnp)-oh (enzo life sciences), a specific ace2 quenched fluorogenic substrate, at 37 • c. enzymatic activity was determined after 4 hours of incubation in tissue, and 16 h of incubation in serum. the plates were read using a fluorescence plate reader tecan infinite 200 (tecan instruments) at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. results were expressed as rfu (relative fluorescent units) per µl of sample or µg of protein and per hour (rfu/µl/h or rfu/µg/h). protein expression was analyzed by western blotting techniques using tissue homogenates. briefly, 30 µg of protein were denatured by heat shock. samples were loaded into 8% acrylamide/bisacrylamide gel and transferred to hydrophobic pvdf (polyvinylidenedifluoride) membranes (amersham hybond-p, ge healthcare, madrid, spain) using trans-blot ® turbo™ transfer system (bio-rad laboratories, madrid, spain). membranes were blocked using 5% skimmed milk in tris-buffered saline (tbs) containing 0.1% tween-20 for 1 h at room temperature. membranes were then incubated using primary antibodies for ace (f940 1:500, bioworld, st. louis park, mn, usa) and ace2 (ab15347 1:2000, abcam, cambridge, uk), followed by incubation with hrp-conjugated secondary antibodies (dako, barcelona, spain). to control for protein loading, all membranes were probed with mouse monoclonal anti-β-actin (a1978 1:4000, sigma, madrid, spain) or mouse monoclonal anti-tubulin (t5168 1:10000, sigma). densitometric analyses of protein bands were performed using imagej software (1.47v, nih, usa) and corrected by control protein. values of each data are expressed as mean ± sem. comparisons between groups were assessed by kruskal-wallis for multiple comparisons and mann-whitney u-test for two group comparisons (spss version 18 for windows). statistical significance was considered when p ≤ 0.05. a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase angiotensin-converting enzyme-2 (ace2): comparative modeling of the active site, specificity requirements, and chloride dependence -7) in ischemic cardiomyopathy angiotensin ii at1-receptor blockade inhibits monocyte activation and adherence in transgenic (mren2)27 rats recent advances in the angiotensin-converting enzyme 2-angiotensin(1-7)-mas axis the preparation and function of the hypertensin-converting enzyme physiology of local renin-angiotensin systems tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis angiotensin-converting enzyme 2 and the kidney quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme renin-angiotensin system may trigger kidney damage in nod mice angiotensin-i-converting enzyme and its relatives angiotensin-converting enzyme 2 protects from severe acute lung failure angiotensin i-converting enzyme type 2 (ace2) gene therapy improves glycemic control in diabetic mice organ-specific distribution of ace2 mrna and correlating peptidase activity in rodents an update on the islet renin-angiotensin system the role of the renin-angiotensin system in the development of cardiovascular disease increased ace 2 and decreased ace protein in renal tubules from diabetic mice: a renoprotective combination? hypertension ace and ace2 activity in diabetic mice glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensin-converting enzyme: implications for albuminuria in diabetes ace2 inhibition worsens glomerular injury in association with increased ace expression in streptozotocin-induced diabetic mice autoimmune syndromes in major histocompatibility complex (mhc) congenic strains of nonobese diabetic (nod) mice. the nod mhc is dominant for insulitis and cyclophosphamide-induced diabetes cellular and humoral immune responses in the early stages of diabetic nephropathy in nod mice effect of insulin on ace2 activity and kidney function in the non-obese diabetic mouse circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous history of cardiovascular disease investigators from the nefrona study. circulating angiotensin converting enzyme 2 activity as a biomarker of silent atherosclerosis in patients with chronic kidney disease ace2 deficiency modifies renoprotection afforded by ace inhibition in experimental diabetes pulmonary angiotensin-converting enzyme antienzyme antibody colorimetry of angiotensin-i converting enzyme activity in serum sensitive method for quantitation of angiotensin-converting enzyme (ace) activity in tissue standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats. braz spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung a high-throughput fluorimetric assay for angiotensin i-converting enzyme an improved fluorometric assay of rat serum and plasma converting enzyme genetically increased angiotensin i-converting enzyme level and renal complications in the diabetic mouse update on new aspects of the renin-angiotensin system in liver disease: clinical implications and new therapeutic options angiotensin-converting enzyme 2: enhancing the degradation of angiotensin ii as a potential therapy for diabetic nephropathy hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ace2) and its implication for ace2 activity assays paricalcitol modulates ace2 shedding and renal adam17 in nod mice beyond proteinuria dynamics of adam17-mediated shedding of ace2 applied to pancreatic islets of male db/db mice sex steroids influence pancreatic islet hypertrophy and subsequent autoimmune infiltration in nonobese diabetic (nod) and nodscid mice albumin inhibits the insulin-mediated ace2 increase in cultured podocytes circulating angiotensin-converting enzyme 2 activity in kidney transplantation: a longitudinal pilot study role of circulating angiotensin converting enzyme 2 in left ventricular remodeling following myocardial infarction: a prospective controlled study we thank marta rebull for her technical assistance. this work has been supported by grants from fondo de investigacion sanitaria-instituto carlos iii-feder (isciii-feder fi13/00598); fondo de investigacion sanitaria-instituto carlos iii-feder (isciii-feder pi14/00557); and red de investigacion renal, fondo de investigacion sanitaria-instituto carlos iii, subprograma retics (rd16/0009/0013_redinren). the authors declare no conflict of interest. renin angiotensin system angii angiotensin ii ace angiotensin converting enzyme ace2angiotensin converting enzyme 2 nod non-obese diabetic nor non-obese resistant key: cord-015859-5kt59ose authors: esch, joep h.m. van; danser, a.h. jan title: local angiotensin generation and at(2) receptor activation date: 2007 journal: frontiers in research of the renin-angiotensin system on human disease doi: 10.1007/978-1-4020-6372-5_12 sha: doc_id: 15859 cord_uid: 5kt59ose nan the renin-angiotensin system (ras) plays an important role in the regulation of blood pressure and body fluid homeostasis. traditionally, the ras has been viewed as a circulating system ("circulating" ras). however, it is now well-established that angiotensin (ang) generation also occurs at tissue sites ("tissue" ras). the complexity of the system has increased even further now that we know that ang ii activates more than one receptor, that ang ii has metabolites which activate their own receptors, and that there may even be receptors for renin and prorenin. this review summarizes the latest insights on tissue angiotensin generation and focuses in particular on the activation of the ang ii type 2 (at 2 ) receptor by locally generated ang ii. renin belongs to the family of aspartyl proteases and has only one known substrate, angiotensinogen, the precursor of all angiotensin peptides. structure analysis revealed that renin consists of 2 homologous lobes which form a cleft containing the active site. renin has an inactive precursor, prorenin, in which the active site is covered by the prosegment. the renin gene was cloned in the 1980s in human, rat and mouse. most species have one renin gene (ren-1 c ), although some mouse strains have two renin genes, ren-1 d and a submandibular variant, designated as ren-2. the ren-2 gene is encoding for a nonglycosylated prorenin, as opposed to the ren-1 gene which can be glycosylated at three asparagine residues. the renin gene is located on chromosome 1 in human and mouse, whereas it is localized on chromosome 13 in rat. the renin gene encodes for pre-prorenin consisting of a presegment of 23 amino acids, a prosegment of 43 amino acids and the actual renin protein of 340 amino acids (morris 1992) . the presegment functions as a signal peptide directing prorenin to the secretory pathway. recently, a splice-variant of the renin gene was discovered which lacks the signal peptide and part of the prosegment. this truncated prorenin displays enzymatic activity because the truncated prosegment only partially covers the enzymatic cleft. it is thought to remain intracellular (clausmeyer et al 2000) , although truncated prorenin has also been demonstrated extracellularly (shinagawa et al 1992) . mice lacking the ren-1 d gene are characterized by sexually dimorphic hypotension (leading to a significant reduction of blood pressure in female mice), absence of dense secretory/storage granule formation in juxta-glomerular cells, altered morphology of the kidney, and a significant increase of plasma prorenin levels (clark et al 1997) . deletion of the ren-2 gene resulted in increased renin and decreased prorenin levels (sharp et al 1996) , but no changes in blood pressure, nor morphological changes occurred. transgenic mice overexpressing human renin did not develop hypertension whereas transgenic mice expressing both human renin and human angiotensinogen showed a significantly increased blood pressure (fukamizu et al 1993) . the plasma concentrations of ang i and ang ii were 3-5-fold increased in double transgenic mice as compared to either control mice or transgenic mice overexpressing human renin. these results demonstrate that human renin does not crossreact with mouse angiotensinogen, thereby illustrating the unique species specifity of the ras. prorenin can be activated through cleavage of the prosegment (proteolytic activation) or via a conformational change induced by low ph or low temperature (non-proteolytic activation) (danser and deinum 2005) (fig. 1) . proteolytic activation is an irreversible process in which the prosegment is cleaved, e.g., by kallikrein, trypsin or plasmin. in vivo, proteolytic activation is probably mediated by a proconvertase in the renin-producing cells of the juxta-glomerular apparatus of the kidney. non-proteolytic activation of prorenin is a reversible process in which prorenin is converted from the 'closed ' (inactive) to the 'open' (active) conformation by unfolding of the prosegment from the enzymatic cleft . acid activation leads to complete activation of prorenin whereas exposure to cold ('cryoactivation') only leads to partial activation (∼15%). kinetic studies have shown that an equilibrium exists between the closed and open conformations of prorenin, and that under physiological conditions (ph 7.4, 37 o c) <2% of prorenin is in the open conformation (danser and deinum 2005) . the kidneys are the main source of circulating (pro)renin. however, following a bilateral nephrectomy, prorenin, in contrast with renin, remains detectable. this suggests that prorenin is also produced outside the kidney. potential extrarenal prorenin-producing tissues are the eye, adrenal, ovary and testis (sealey et al 1988; danser et al 1989; itskovitz et al 1992; clausmeyer et al 2000) . normally, the concentration of prorenin in human plasma is 10 times higher than that of renin. the reasons for this excess are unknown, as prorenin does not seem to be activated (lenz et al 1990) . one possibility is that prorenin has functions unrelated to angiotensin generation. in this regard, it is of interest to note that it has recently been suggested that prorenin binds to a '(pro)renin receptor', thereby activating second messenger pathways in a manner that is independent of ang ii (nguyen et al 2002; saris et al 2006) . (pro)renin receptors may also mediate the uptake of renin and/or prorenin into tissues that do not synthesize renin and prorenin themselves, like the heart and the vessel wall. to date, two (pro)renin-binding receptors have been identified: the mannose-6phosphate (m6p) receptor (saris et al 2001) and the above-mentioned (pro)renin receptor. the m6p receptor is identical to the insulin-like growth factor ii (igfii) receptor and binds igfii, m6p-containing proteins such as prorenin and renin, and retinoic acid at distinct sites (kornfeld 1992; kang et al 1997) . prorenin and renin are both rapidly internalized after binding to this receptor, and internalized prorenin is proteolytically converted to renin. however, binding to this receptor did not result in angiotensin generation, either intra-or extracellularly. this, in combination with the fact that intracellularly generated renin was found to be degraded within a few hours, suggests that m6p/igfii receptors function as clearance receptors for (pro)renin. alternatively, since binding of m6p-containing proteins to m6p/igfii receptors results in the activation of second messenger pathways involving g-proteins (di bacco and gill 2003) , (pro)renin may act as an m6p/igfii receptor agonist. the (pro)renin receptor was cloned by nguyen and co-workers (nguyen et al 2002) . prorenin and renin bind equally well to this receptor, without being internalized or degraded. interestingly, the catalytic activity of bound renin was increased 5-fold, and receptor-bound prorenin became fully active in a non-proteolytic manner. thus, apparently, this receptor allows prorenin to generate angiotensins at tissue sites. importantly, binding of (pro)renin to the (pro)renin receptor in human mesangial cells also induced ang ii-independent effects, such as an increase in dna synthesis, activation of the mitogen-activated protein kinases (mapks) extracellular signal-regulated kinase (erk)1 (p44)/erk2 (p42), and plasminogenactivator inhibitor-1 release. furthermore, in cardiomyocytes, prorenin activated the p38 mapk/heat shock protein 27 pathway, resulting in changes of actin filament dynamics (saris et al 2006) . these non-angiotensin-mediated effects may underlie the blood pressure-independent cardiac hypertrophy in rats with a hepatic prorenin overexpression (véniant et al 1996) . finally, peters and co-workers demonstrated ren-2 prorenin internalization in cardiomyocytes of transgenic rats expressing the mouse ren-2 gene in the liver (peters et al 2002) . since ren-2 prorenin is nonglycosylated, this phenomenon cannot be mediated by m6p/igfii receptors. the internalization contrasts with the observations on the recently cloned (pro)renin receptor. thus, there may be a third (pro)renin receptor, the identity of which is currently unclear. angiotensinogen, the precursor of all angiotensin metabolites, is the only known substrate for renin. the angiotensinogen gene encodes for a glycoprotein of 453 amino acids with a molecular weight of ∼60 kda. the gene is located as a single copy on, respectively, chromosome 19 in rats, chromosome 8 in mice and chromosome 1 in humans. in 1983, doolittle reported a significant sequence homology of angiotensinogen to 1 -antitrypsin (23%), ovalbumin (21%) and antithrombin iii (18%) (doolittle 1983 ). these proteins are members of the serine proteinase inhibitor family and are closely associated with acute inflammation reactions. acute inflammation induces gene expression via acute-response which increases the angiotensinogen concentration in plasma (kageyama et al 1985) . the similarity between the structural organization of the angiotensinogen and 1antitrypsin genes suggests that both genes have evolved from a common ancestor (kitamura et al 1987) . although angiotensinogen mrna has been detected in brain, adipocytes, heart and the reproductive system, its main source is the liver (paul et al 2006) . hepatocytes constitutively secrete angiotensinogen into the extracellular fluid, without intracellular storage. blood plasma/extracellular fluid functions as the major reservoir for angiotensinogen. angiotensinogen plasma concentrations (∼1 μm) approximate the michaelis-menten constant of the renin reaction, which makes ras activity sensitive to small changes in angiotensinogen concentration. deletion of the angiotensinogen gene in mice leads to hypotension, low body weight gain after birth, and an abnormal morphology of kidney and heart . in turn, overexpression of angiotensinogen led to the development of hypertension (kimura et al 1992) . two isoforms of ace exist: somatic ace and testis (germinal) ace. somatic ace is abundantly expressed throughout the body, whereas testis ace is exclusively expressed in the testis. cloning of the ace gene provided a better understanding of the relationship between somatic and testis ace. both forms are transcribed from the same gene by using different promoters (hubert et al 1991) . in humans the ace gene is located on chromosome 17. somatic ace has 2 homologous domains which share 60% sequence homology. both domains contain a catalytically active site (wei et al 1991a) and are situated at the n-and c-terminal side of ace. according to their position they are designated as n-and c-domain. the majority of somatic ace is membrane-bound on endothelial cells. circulating ace is derived from ace-expressing cells by proteolytic cleavage at the juxta-membrane stalk region (wei et al 1991b) . testis ace possesses only one catalytic domain which is identical to the c-domain of somatic ace. studies selectively blocking the cand n-domain of somatic ace revealed that conversion of ang i to ang ii by membrane-bound ace depends on the c-domain, whereas both domains contribute to this conversion in soluble ace (van esch et al 2005) . degradation of bradykinin at tissue sites also required both domains (tom et al 2001) . deletion of both somatic and testis ace (ace −/− ) in mice led to hypotension, male infertility and changes in kidney morphology (esther et al 1996) . vascular expression of germinal ace in ace null mice restored renal morphology but did not normalize blood pressure, thus demonstrating that germinal ace cannot functionally substitute for somatic ace (kessler et al 2007) . recently, a homologue of somatic ace called ace2 was discovered (donoghue et al 2000) . ace2 shares 42% homology with the c-and n-terminal domains of somatic ace. the gene encoding ace2 is located on the x chromosome and ace2 is mainly expressed in endothelial cells of heart, kidney and testis. like somatic ace, ace2 can be released into the circulation after proteolytic cleavage (turner and hooper 2002) . unlike somatic ace, ace2 has only one catalytically active site which can convert ang i and ang ii to ang (1-9) and ang (1-7), respectively (donoghue et al 2000; vickers et al 2002) . these data suggest a potential role of ace2 in the counterregulation of high blood pressure by inactivation of ang ii. indeed, in a model of ang ii-dependent hypertension, blood pressures were substantially higher in ace2-deficient mice than in wildtype controls (gurley et al 2006) . mice lacking the ace2 gene were originally described to develop an abnormal heart function with severely impaired contractility (crackower et al 2002) , but this was not confirmed in a follow-up study (gurley et al 2006) . remarkably, ace2 also functions as a receptor for the virus causing severe acute respiratory syndrome, thereby stressing the importance of ace2 in a manner unrelated to the ras (li et al 2003) . initially, it was thought that the responses to ang ii were mediated by a single ang ii receptor. at the end of the 1980s, the discovery of specific ang ii receptor ligands such as losartan, pd12377, pd123319 and cgp42112 made it possible to identify several ang ii receptor subtypes. we now know that the biological actions of ang ii in man are mediated by at least two types of ang ii receptors: ang ii type 1 (at 1 ) and at 2 receptors (fig. 3 ). at 1 receptors mediate virtually all the known physiological actions of ang ii, such as vasoconstriction, inotropy, chronotropy, aldosterone release, noradrenaline release and growth stimulation. the at 1 receptor gene encodes for a protein of 359 amino acids with a molecular weight of 41 kda. the gene was first cloned in 1991 from rat vascular smooth muscle cells (murphy et al 1991) and bovine adrenal gland (sasaki et al 1991) . cloning and genetic analysis of the human at 1 receptor gene revealed that the human at 1 receptor gene is located on chromosome 3 and can produce two isoforms by alternative splicing. both isoforms have similar binding -and functional properties. in rodents two subtypes of the at 1 receptor have been identified: at 1a and at 1b (elton et al 1992) . the origin of these subtypes lies in a gene duplication which occurred after the divergence of rodents from the human/artiodactyls group about 24 million years ago. at 1a and at 1b share 94% sequence homology and are located on chromosome 17 and 2 in rat and chromosome 13 and 3 in mice, respectively. not surprisingly, both subtypes have similar ligand binding affinities and signal transduction properties but varying expression levels in different tissues. the at 1a receptor predominates in heart, kidney, lung, liver and vascular smooth muscle, whereas the at 1b receptor is mainly expressed in the adrenal and pituitary gland (burson et al 1994) . to date, there are no pharmacological antagonists which clearly discriminate at 1a and at 1b receptors. studies in mice using targeted gene manipulation provided more insight in the functional role of both subtypes in vivo. deletion of the at 1a receptor gene significantly decreased resting blood pressure in both heterozygous at 1a +/− and homozygous at 1a −/− receptor mice (ito et al 1995) . ang ii infusions resulted in a diminished pressor response in at 1a +/− receptor mutants whereas this response was virtually abolished in at 1a −/− mutants. additionally, both the expression levels of renin mrna and plasma renin activity were markedly increased in at 1a receptor knockout mice (sugaya et al 1995) . deletion of the at 1b receptor gene did not affect resting blood pressure, nor altered the pressure response to ang ii (chen et al 1997) . taken together, these findings indicate the important role of the at 1a receptor in mediating the pressure response in mice. at 1a or at 1b receptor deficiency is not associated with an impaired development or survival, but double knockout mice lacking both receptors display a phenotype similar to that observed in angiotensinogen knockout mice (tsuchida et al 1998) . these observations, together with the fact that ang ii does cause a pressor response in at 1a knockout mice after enalapril pretreatment (oliverio et al 1997) , suggest a compensatory role for the at 1b receptor. additionally, in vitro studies demonstrated that the at 1b receptor is the most important regulator of ang ii contractile responses in the mouse aorta and femoral artery (zhou et al 2003) . the at 1 receptor belongs to the seven-transmembrane g-protein-coupled receptor superfamily, and couples to a wide variety of second messenger systems, including the phospholipase c/inositol-1,4,5-triphosphate/diacylglycerol/protein kinase c pathway, the phospholipase a 2 /arachidonic acid pathway, the phospholipase d/phosphatidylcholine/phosphatidic acid pathway, and tyrosine kinases such as the map kinases erk1/2, p38 and c-jun n-terminal kinase (mehta and griendling 2007) . at 1 receptor stimulation results in a rapid internalization of the ang ii-at 1 receptor complex, followed by either receptor degradation in lysosomes or receptor recycling to the cell surface (mehta and griendling 2007) . internalized ang ii has been proposed to activate cytoplasmic or nuclear receptors prior to its intracellular degradation (thomas et al 1996) . furthermore, zou and co-workers recently demonstrated that mechanical stretch resulted in at 1 receptor activation in a ligandindependent manner. interestingly, the consequences of such activation could be prevented by an at 1 receptor blocker (zou et al 2004) . several reports have described crosstalk between at 1 receptor and other receptors, e.g. the bradykinin type 2 (b 2 ) receptor, the at 2 receptor, and the 1adrenoceptor. at 1 and b 2 receptors form stable heterodimers with an enhanced g-protein activation and altered receptor sequestration (abdalla et al 2000) . at 1 receptor-1 -adrenoceptor crosstalk enhances the response to 1 -adrenoceptor agonists (purdy and weber 1988) . interestingly, although the postjunctional at 1 receptor interacting with the 1 -adrenoceptor is of the at 1a subtype, the prejunctional at 1 receptor which facilitates noradrenaline release from sympathetic nerve endings is of the at 1b subtype (guimaraes and pinheiro 2005) . in contrast to the well-characterized at 1 receptor, the function of the at 2 receptor is much less understood. in general, it is assumed that at 2 receptors counteract the responses mediated by the at 1 receptor (hein et al 1995; ichiki et al 1995; abdalla et al 2001; schuijt et al 2001; batenburg et al 2004) . at 2 receptors are involved in physiological processes like development and tissue remodeling (by inhibiting cell growth and by stimulating apoptosis), regulation of blood pressure (vasodilatation), natriuresis and neuronal activity. evidence for at 2 receptor mediated vasodilatation is largely based on two approaches: an indirect approach, showing an enhanced response to ang ii in the presence of at 2 receptor blockade or gene disruption (hein et al 1995; ichiki et al 1995; batenburg et al 2004; van esch et al 2006) , and a direct approach showing at 2 receptor-induced responses by applying either the (partial) at 2 receptor agonist cgp42112a or ang ii in the presence of an at 1 receptor blocker (widdop et al 2002; li and widdop 2004) . the at 2 receptor gene was first cloned in 1993 (mukoyama et al 1993) . the at 2 receptor gene shares 34% sequence homology with its at 1 receptor counterpart and encodes for a protein of 363 amino acids with a molecular mass of 41 kda. it is located on the x chromosome in both humans and rodents. in fetal tissues the at 2 receptor is the predominant subtype. this situation changes rapidly after birth, resulting in the at 1 receptor becoming the dominant subtype in most adult tissues (widdop et al 2003) . yet, in adults, at 2 receptors can still be detected in a variety of tissues, including uterus, ovary, adrenal medulla, heart, blood vessels and brain (bottari et al 1993) . here it is important to consider that the distribution of the at 2 receptor depends on age and species, but is also subject to changes in expression during pregnancy and pathological conditions such as hypertension, heart failure and vascular injury (see below) (bottari et al 1993; de gasparo et al 2000) . in 1995, two groups reported that deletion of the at 2 receptor in mice led to an increased pressor response to ang ii (hein et al 1995; ichiki et al 1995) . additionally, ichiki et al reported a significant increased blood pressure in hemizygous at −/y 2 receptor mice whereas blood pressure was not significantly increased in a similar model described by hein and co-workers. mutants lacking the at 2 receptor gene showed a lower body temperature and impaired exploratory behavior. remarkably, despite its wide expression in the fetus, the at 2 receptor does not seem to be required for embryonic development, as no morphological and developmental differences were found between homozygous at −/− 2 or hemizygous at −/y 2 receptor mice and their wildtype controls. possibly, at 2 receptor knockout mice display a delayed expression of calponin and h-caldesmon after birth (yamada et al 1999) . during pregnancy, ang ii levels are elevated. because the fetus is also exposed to these high ang ii levels, it has been postulated that the at 2 receptor plays a role in the regulation of ang ii responsiveness in order to prevent fetal hypertension (perlegas et al 2005) . like at 1 receptors, at 2 receptors belong to the g protein-coupled receptor superfamily. however, in contrast to the at 1 receptor, the at 2 receptor is not internalized upon binding of ang ii (widdop et al 2003) . two major pathways have been described for at 2 receptor signaling (nouet and nahmias 2000) : (a) activation of protein phosphatases causing protein dephosphorylation and (b) activation of the nitric oxide (no)/guanosine cyclic 3', 5'-monophosphate (cgmp) pathway. up to now, three specific phosphatases have been linked to at 2 receptor activation: mapk phosphatase 1, sh2-domain-containing phosphatase 1 and protein phosphatase 2a. growth factors, including ang ii via the at 1 receptor, mediate their growth promoting actions through tyrosine kinase receptors and several kinase-driven phosphorylation steps. activation of the at 2 receptor counteracts these growth-promoting actions by dephosphorylation through subsequent activation of phosphatases. in addition to the inhibitory effect on growth, dephosphorylation (e.g., of erk1/2) also seems to play an important role in the stimulation of apoptosis (horiuchi et al 1998) . several studies have shown that at 2 receptor-mediated vasodilation is an endothelium-dependent phenomenon involving b 2 receptors, no and cgmp (wiemer et al 1993; siragy and carey 1997) (fig. 2) . initially, in vitro studies using endothelial cells showed that the stimulatory effect of ang ii on cgmp production, figure 2 . at 2 receptor-mediated relaxation involves either intracellular activation of kininogenase and subsequent bradykinin type 2 (b 2 ) receptor activation, or a direct activation of no synthase (nos) a downstream signaling product of no production, was abolished by blocking both b 2 receptors and nitric oxide synthase (nos) (wiemer et al 1993) . subsequent in vivo studies confirmed that the at 2 receptor-induced rise in cgmp involves bradykinin and no (siragy and carey 1997) . in vitro studies in endothelial cells reported that intracellular acidosis, as a result of at 2 receptor activation, stimulates bradykinin formation by activating kininogenases (tsutsumi et al 1999) . katada and majima were able to show production of bradykinin after at 2 activation in rat mesenteric arteries, suggesting that the b 2 receptor mediates vasodilatation by endogenous bradykinin released upon at 2 receptor activation (katada and majima 2002) . in agreement with this concept, deletion of the b 2 receptor enhanced the ang ii-induced hypertensive response in vivo (cervenka et al 2001) . additional studies concluded that no production following at 2 receptor stimulation may also occur independently of b 2 receptors, through direct nos activation (abadir et al 2003) , possibly involving the calcineurin/nuclear factor of activated t cells pathway (ritter et al 2003) . as both at 2 and b 2 receptors are co-expressed in various tissues, the hypothesis was raised that both receptors form heterodimers which can interact through receptor crosstalk. recent studies in rat pheochromocytoma cells, applying fluorescence resonance energy transfer, confirmed this hypothesis (abadir et al 2006) . heterodimer formation appeared to be dependent on the receptor number that was expressed, but also required at 2 receptor stimulation. as a consequence of heterodimer formation, it is possible that at 2 receptor activation results in b 2 receptor activation without intermediate bradykinin synthesis (batenburg et al 2004) . in addition to its interaction with the b 2 receptor, at 2 receptors are also known to interact with their at 1 counterpart. transfection studies in fetal fibroblasts showed that at 1 and at 2 receptors form heterodimers in which the at 2 receptor functions as a specific at 1 receptor antagonist (abdalla et al 2001) . possibly, at 2 receptorinduced vasodilatation depends on simultaneous at 1 receptor activation, as no at 2 receptor-mediated responses were noted in the absence of at 1 receptors (van esch et al 2006) . furthermore, it is important to consider that data obtained in absence of the at 2 receptor are complex because at 2 receptors downregulate at 1 receptors in a ligand-independent manner (jin et al 2002) and at 2 receptor knockout mice display an increased at 1 receptor expression . in addition to its interaction with at 1 receptors, the at 2 receptor also downregulates renin biosynthesis, thereby inhibiting the formation of ang ii (siragy et al 2005) . ang i and ii are metabolized by a whole range of peptidases ('angiotensinases'). although initially it was thought that all metabolites other than ang ii were inactive, it is now clear that at least several of these metabolites have functions of their own, which are sometimes mediated via non-at 1 /at 2 receptors. the most important of these peptides are ang (1-7), ang (2-8) (ang iii) and ang (3-8) (ang iv) (fig. 3) . ang (1-7) can be formed from ang i by the action of neutral endopeptidase or prolyl endopeptidase but also from the ang i degradation products ang (1-9) and ang ii (vickers et al 2002) . ang (1-7) is generally believed to counteract the response of ang ii although there are reports of similar or distinct actions from ang ii (santos et al 2000) . ang (1-7) induces relaxation in several vascular beds. the fact that this relaxation could be blocked by the selective ang (1-7) antagonist a-779 [d-ala 7 -ang (1-7)] suggested the involvement of a specific ang (1-7) receptor (santos et al 2000) . indeed, in 2003 the mas proto-oncogene, a g protein-coupled receptor, was proposed to be the receptor for ang (1-7) (santos et al 2003) . ang (1-7) potentiates bradykinin-induced responses (tom et al 2001) and releases no (brosnihan et al 1996) via mas receptor stimulation. mas receptor mrna expression has been detected in heart, testis, kidney and brain (metzger et al 1995) . mice deficient for the mas-receptor lack the antidiuretic action of ang (1-7) after an acute water load, and their aortas no longer relax in response to ang (1-7) (santos et al 2003) . mas −/− mice are also characterized by an impaired heart function, indicating an important role of the mas receptor in the maintenance of the structure and function of the heart (santos et al 2006) . although the mas-receptor is now held responsible for most of the responses to ang (1-7) , there are several other pharmacological mechanisms and receptors that are affected by ang (1-7) . as a slow substrate for ace, ang (1-7) may also function as an ace inhibitor, resulting in decreased ang ii formation and potentiation of bradykinin-induced vasodilatation (tom et al 2001) . furthermore, ang (1-7) acts as an at 1 receptor antagonist at low concentrations (stegbauer et al 2003) , and exerts at 1 receptor agonistic effects at high concentrations (van rodijnen et al 2002) . a link between ang (1-7) and the at 2 receptor has recently been proposed, because infusion of ang (1-7) during at 1 receptor blockade unmasked a vasodepressor response in conscious shr rats that could be attenuated by blockade of at 2 receptors, b 2 receptors and nos . possibly, mas-at 1 and/or mas-at 2 receptor heterodimers exist (castro et al 2005; lemos et al 2005) . through the action of aminopeptidase a, ang ii is converted to ang iii, which in turn can be converted to ang iv by aminopeptidase n (ardaillou and chansel 1997) . ang iii mediates some of the classical responses of ang ii (such as stimulation of aldosterone secretion and vasoconstriction) and this most likely involves binding to at 1 and at 2 receptors. the affinity of ang iii for these receptors is somewhat lower than that of ang ii (wright and harding 1995) . the responses to ang iii are less efficacious than those of ang ii, possibly due to its accelerated metabolism in the circulation. the latter relates to the wide distribution of aminopeptidase n that initiates the hydrolysis of ang iii but not ang ii. it is thought that ang iii might be the final mediator of some of the actions of ang ii. for example, the central action of ang ii on vasopressin secretion in rats is dependent on ang iii, as this effect was absent after specific blockade of aminopeptidase a (zini et al 1996) . additionally, ang iii, and not ang ii, mediates the excretion of na + excretion through at 2 receptors in the presence of at 1 receptor blockade (padia et al 2006) . ang iv was initially believed to have no biological activity. this was based on two important findings: both at 1 and at 2 receptors display a poor affinity for ang iv, and ang iv does not elicit the characteristic ang ii responses like ang iii. the discovery of a specific ang iv binding site, designated as the at 4 receptor, changed this view (swanson et al 1992) . after purification, the receptor was identified as insulin-regulated aminopeptidase (albiston et al 2001) , a protein which is abundantly found in vesicles containing the insulin-sensitive glucose transporter (glut4) (keller et al 1995) . at 4 receptor expression occurs in brain, spinal cord, heart, kidney, colon, prostate, adrenal gland, bladder and vascular smooth muscle cells (wright and harding 1995; de gasparo et al 2000) . ang iv and the at 4 receptor appear to be involved in the facilitation of memory and learning (wright et al 1999) . ang iv infusions cause vasorelaxation in cerebral and renal vascular beds, possibly by increasing nos activity (patel et al 1998) . on the other hand, there are also studies showing that ang iv, because of its weak agonistic activity towards the at 1 receptor, induces vasoconstriction (van rodijnen et al 2002) . the close association of the at 4 receptor with glut4 suggests that ang iv might modulate glucose uptake. as soon as it was realized that angiotensin production at tissue sites is of greater importance than angiotensin generation in the circulation, many investigators started to unravel how and where such local angiotensin production might occur. initially, it was thought that all components required for local ang ii production (i.e., renin, angiotensinogen and ace) would be produced at tissue sites. infusions of radiolabeled angiotensins, allowing the quantification of uptake of blood-derived angiotensin in tissues, confirmed that the majority of tissue ang i and ii is produced at tissue sites, and not derived from blood (schuijt and danser 2002) . ace is well-known to be abundantly expressed in virtually every tissue of the body, its main site being the surface of endothelial cells. thus, its local synthesis is beyond doubt. although angiotensinogen mrna has been detected outside the liver, direct proof for actual angiotensinogen synthesis at important sites of local angiotensin production (e.g., heart and vessel wall) is lacking. for instance, the isolated perfused heart does not release angiotensinogen . therefore, the majority of tissue angiotensinogen is probably of hepatic origin. the fact that angiotensinogen is neither internalized, nor binds to membranes, combined with the observation that angiotensinogen-synthesizing cells release angiotensinogen to the extracellular space (klett et al 1993) , rather than storing it intracellularly, indicates that angiotensin generation must occurs extracellularly. thus, tissue angiotensin generation is restricted to the interstitial space and/or the cell surface (fig. 4) . following a bilateral nephrectomy, tissue renin and angiotensin levels drop to levels at or below the detection limit (campbell et al 1993; danser et al 1994; katz et al 1997) . this suggests that the majority of tissue renin is not locally produced, but kidney-derived, and that without renin, there is no angiotensin production. the presence of renin in cardiac membrane fractions (danser et al 1994) suggested that circulating renin, in addition to its diffusion into the interstitial space (katz et al 1997; van den eijnden et al 2002) , may bind to renin-binding proteins or receptors at tissue sites. the recent discovery of several of such receptors, as discussed above, supports this concept. an interesting additional observation is that these receptors also bind prorenin, and that prorenin, upon binding, becomes catalytically active. in view of the much higher prorenin than renin levels, an attractive concept is that prorenin rather than renin contributes to tissue angiotensin generation. studies with (pro)renin receptor blockers in diabetic rats confirmed this concept (ichihara et al 2004) . unexpectedly however, these blockers did not affect tissue angiotensin levels in control rats, although the prorenin levels of the latter rats were only ≈2-fold lower than those of the diabetic rats. moreover, despite the fact that prorenin is still present in circulating blood after a nephrectomy (danser et al 1994) , tissue angiotensin levels are close to zero. this suggests that, if prorenin contributes to tissue angiotensin production, this involves prorenin of renal rather than extrarenal origin. currently, the only known difference between renal and extrarenal prorenin relates to their degree of glycosylation. in vitro studies using the isolated perfused rat langendorff heart fully confirmed the idea of renin and angiotensinogen uptake underlying tissue angiotensin production. during buffer perfusion, no release of ras components could be demonstrated in the coronary effluent or interstitial fluid . after adding renin to the perfusion fluid, renin started to accumulate in the interstitial fluid, reaching steady-state levels in this compartment that were identical to its levels in the coronary circulation. findings on angiotensinogen were similar. stopping the exposure to renin revealed a biphasic washout curve, in agreement with the concept that renin is not only present in extracellular fluid but also binds to receptors. angiotensinogen washout was mono-phasic. angiotensin synthesis only occurred during simultaneous perfusion with renin and angiotensinogen. interestingly, in hearts of transgenic rats overexpressing angiotensinogen, angiotensin release continued after stopping the renin perfusion, i.e., when renin was no longer present in the coronary circulation (müller et al 1998) . this was due to the fact that receptor-bound renin continued to generate ang i. at steady state, the cardiac tissue levels of ang i were as high as expected assuming that ang i is restricted to the extracellular fluid (de lannoy et al 1998; schuijt et al 1999) . in contrast, the tissue ang ii levels were much higher. pretreatment with an at 1 receptor antagonist greatly reduced the cardiac tissue ang ii levels during renin + angiotensinogen perfusion. this suggests that locally generated ang ii accumulates at tissue sites through binding to at 1 receptors. subsequent subcellular fractionation studies confirmed that tissue ang ii, but not ang i, is located intracellularly (schuijt et al 1999; van kats et al 2001) . this is due to the fact that at 1 receptor-bound ang ii is rapidly internalized, after which intracellular degradation occurs. based on these observations, it is not surprising that the tissue ang ii content correlates directly with tissue at 1 receptor density . a wide range of in vitro studies has provided evidence for the existence of enzymes other than renin and ace generating ang i and ii, including cathepsin d, kallikrein, tonin and chymase (hackenthal et al 1978; urata et al 1990) . the in vivo importance of these alternative pathways is questionable. the fact that ang i and ii are virtually absent in plasma and tissue of nephrectomized animals (including humans) argue against a role of non-renin angiotensinogen-converting enzymes in vivo. a similar situation exists for chymase which is present in the cardiac interstitium, mast cells and endothelial cells. in vitro studies have provided evidence for an important role of chymase in the conversion of ang i to ang ii (urata et al 1990; tom et al 2003) , but in vivo evidence for chymase-dependent ang ii generation could not be obtained (saris et al 2000) . moreover, angiotensinogen and ace knockout mice have similar phenotypes (tanimoto et al 1994; krege et al 1995) , and ace deletion reduced the ang ii levels in both tissue and circulation by up to 99% (campbell et al 2004) . thus, at least in mice, ace is the main, if not only ang ii-generating enzyme in vivo. as discussed above, at 2 receptor expression is low or undetectable in adult tissues, in contrast with its high expression in fetal tissues. however, at 2 receptors reappear under pathophysiological conditions. for instance, in the kidney, at 2 receptor expression increases when inflammation, apoptosis, and proteinuria occur (ruiz-ortega et al 2003) . interestingly, transgenic at 2 receptor-overexpressing mice displayed less glomerular injury, proteinuria and transforming growth factor expression in a subtotal nephrectomy model (hashimoto et al 2004) . this suggests that the re-appearance of at 2 receptors under pathological conditions is part of a protective mechanism, for instance related to enhanced no production (hiyoshi et al 2005) . however, not all studies confirm the counterregulatory, protective actions of at 2 receptors in the kidney. duke and co-workers report that at 2 receptors mediate vasoconstriction in the renal medulla of 2-kidney, 1-clip rats, as opposed to the vasodilator effects mediated by at 1 receptors in this model (duke et al 2005) . in the heart, a wide range of animal studies revealed increased at 2 receptor expression under pathological conditions, e.g. during pressure overload, hypertension and ischemia, and post-myocardial infarction (wiemer et al 1993; wu et al 1994; schuijt et al 2001; yayama et al 2004) . studies in failing human hearts confirmed the animal data, and simultaneously showed a downregulation of at 1 receptors (asano et al 1997; wharton et al 1998) . from studies with at 1 receptor antagonists it is widely accepted that at 1 receptors play a major role in the post-myocardial remodeling process, mediating both fibrosis and hypertrophy (schieffer et al 1994) . since the beneficial effects of at 1 receptor blockade following myocardial infarction were diminished in at −/y receptor mice , it is reasonable to assume that the increased ang ii levels that will occur during at 1 receptor blockade (see below) exert beneficial effects via at 2 receptor stimulation. indeed, transgenic mice overexpressing at 2 receptors in the heart displayed improved cardiac hemodynamics post-myocardial infarction in an no-dependent manner bove et al 2004) . furthermore, treatment with either an at 2 receptor antagonist or a b 2 receptor antagonist reduced the beneficial effects of at 1 receptor blockade in wildtype mice following myocardial infarction . therefore, the beneficial effects of at 2 receptors in the heart involve the b 2 receptor/no/cgmp pathway. in contrast with these observations, a few studies have shown that at 2 receptors, like at 1 receptors, induce cardiac hypertrophy and fibrosis (senbonmatsu et al 2000; ichihara et al 2001) . to explain these discrepant data, it has been hypothesized that at 2 receptor upregulation is beneficial in the early pathological phase, by counteracting hypertrophy and fibrosis, but that chronic stimulation of the at 2 receptor (for instance by the high ang ii levels that will occur during at 1 receptor blockade) has deleterious effects on cardiac recovery (schneider and lorell 2001) . knowledge on the effects of at 2 receptors in the human heart comes from polymorphism studies, although the data are often contradictory. at 2 receptor gene variants have been linked to both cardiac hypertrophy and coronary ischemia (schmieder et al 2001; herrmann et al 2002; alfakih et al 2005) , without knowing however whether this is based on inceased or decreased at 2 receptor density. at 2 receptor-mediated vasodilation in isolated human coronary microarteries increases with age (batenburg et al 2004) . since endothelial function decreases with age, this could point to increased at 2 receptor expression in the face of decreased endothelial function, again in agreement with the concept that at 2 receptor density increases under pathological conditions. at 2 receptor expression also increased in peripheral resistance arteries of hypertensive diabetic patients during treatment with an at 1 receptor blocker, and this resulted in enhanced ang ii-induced vasodilation (savoia et al 2007) . recent studies have shown that at 2 receptors are also expressed in various carcinomas (deshayes and nahmias 2005) . assuming that at 1 receptors contribute to tumor growth and vascularization (fujita et al 2002) , one may predict that, here too, at 2 receptors will counteract the effects of the at 1 receptor stimulation, thus inhibiting growth and vascularization (silvestre et al 2002) . however, proangiogenic effects of at 2 receptors have also been described, occurring in conjunction with at 1 receptor activation . blocking the ras is possible at three levels: renin, ace and the at receptors. beta-adrenoceptor blockers, by antagonizing the renin-releasing 1 -adrenoceptors in the juxta-glomerular cells, were the first drugs to suppress the ras. these drugs will lower renin (campbell et al 2001) , ang i and ang ii, thereby reducing the degree of at 1 and at 2 receptor stimulation (table 1) . subsequently, the ace inhibitors were introduced. these drugs will lower ang ii. given the wide variety of available angiotensinases, this will not lead to substantial ang i accumulation, but rather result in metabolism of ang i through different (compensatory) pathways, e.g. by neutral endopeptidase. as a consequence, ang-(1-7) levels will rise during ace inhibition, thereby allowing ang-(1-7) to contribute to the beneficial effects of ace inhibitors (tom et al 2001) . simultaneously, due to the interference with ang ii generation, the negative feedback loop system regulating renin release is affected, and thus, the kidneys will release more renin. therefore, depending on the degree of ace inhibition, ang ii levels may rise again, sometimes to levels above baseline (campbell et al 1993; van kats et al 2000) . for instance, at 90% ace inhibition, a 10-fold rise in renin is sufficient to fully restore ang ii levels, whereas a 20-fold rise in renin would increase ang ii twofold above its baseline levels. in addition, prolonged ace inhibition is known to upregulate ace. given these compensatory mechanisms, it is not surprising that it has proven difficult to show that blood plasma and tissue ang ii levels remain suppressed during continuous ace inhibition (van kats et al 2000) . indeed, in pigs treated with captopril for 3 weeks post-myocardial infarction, cardiac ang ii levels were increased as compared to untreated control pigs (fig. 5) . although this ang ii may theoretically stimulate at 1 and at 2 receptors, it must be kept in mind that such receptor stimulation may occur less efficiently than normal. without ace inhibitor treatment, ace generates ang ii in a highly efficient manner, in close proximity of at receptors. during chronic ace inhibition, the increase in ang i generation will still allow ang ii generation, either by noninhibited ace or by non-ace converting enzymes like chymase (van kats et al 2005) . however, this type of ang ii generation is less efficient, because it does not result in a high level of regional at receptor stimulation. in particular, ang ii generated by chymase (which is localized in the adventitia) will be subject to rapid metabolism in the interstitial space on its way to at receptors (schuijt et al 1999; de lannoy et al 2001) and thus is less likely to result in a high regional at receptor occupancy. therefore, a low overall at receptor occupancy will occur, below the minimum per cell required to induce an effect. at 1 receptor blockers, available since the early 1990s, will also cause a rise in renin. ang i and ii in blood and tissues (as well as their metabolites) will increase in parallel with renin, and although this will not result in at 1 receptor stimulation, non-at 1 receptors (including at 2 receptors and mas) may now be stimulated excessively. as discussed above, it is feasible that, at least part of the beneficial effect of at 1 receptor blockers is due to such at 2 receptor stimulation (widdop et al 2002) . finally, renin inhibitors will soon be clinically available. these drugs lower both ang i and ii, and evidence for this, at least in blood plasma, is already available (nussberger et al 2002; azizi et al 2004) . whether renin inhibitors also decrease tissue ang i and ii levels is not yet known. this relates to the fact figure 5 . plasma and cardiac tissue angiotensin levels in pigs that were either untreated or treated with the ace inhibitor captopril or the at 1 receptor antagonist eprosartan for 3 weeks after a myocardial infarction. *p<0.05 vs. untreated. data are derived from (van kats et al 2000) that renin inhibitors primarily block human renin, and not (or to a much lesser degree) rat, mouse or porcine renin. thus, renin inhibitors cannot be tested easily in well-established animal models. theoretically, the decreased ang i and ii levels during renin inhibition will prevent at 1 and at 2 receptor stimulation, as well as the stimulation of any other receptor by angiotensin metabolites. although renin will rise during renin inhibitor treatment (like it does during any ras blocker treatment), this renin cannot be enzymatically active due to the presence of the renin inhibitor. thus, renin inhibitors may offer a more complete suppression of the ras, although this also implies that the putative beneficial effects mediated by at 2 or mas receptors will now no longer occur. so far, this does not appear to diminish the effects of renin inhibitors, at least on blood pressure (gradman et al 2005) . ang ii generated at tissue sites stimulates both at 1 and at 2 receptors. this local generation depends largely on angiotensinogen and renin and/or prorenin taken up from blood, the latter uptake possibly involving the recently discovered (pro)renin receptor. ace is generated locally, and appears to be the main, if not the only, ang ii-generating enzyme. ang ii has a whole range of metabolites, the most important of which are ang (1-7), ang iii and ang iv. the enzymes generating these metabolites, including ace2, have recently been characterized, as well as their putative (non-at 1 /at 2 ) receptors, like the mas and at 4 receptor. stimulation of at 2 receptors most likely contributes to the beneficial effect of ras blockers, in particular during at 1 receptor antagonism. these receptors are upregulated under pathophysiological conditions, and are generally believed 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infarction up-regulation of angiotensin ii type 2 receptor in rat thoracic aorta by pressure-overload at1b receptor predominantly mediates contractions in major mouse blood vessels identification of metabolic pathways of brain angiotensin ii and iii using specific aminopeptidase inhibitors: predominant role of angiotensin iii in the control of vasopressin release mechanical stress activates angiotensin ii type 1 receptor without the involvement of angiotensin ii key: cord-002307-gk84fnb9 authors: kehoe, patrick gavin; wong, steffenny; al mulhim, noura; palmer, laura elyse; miners, j. scott title: angiotensin-converting enzyme 2 is reduced in alzheimer’s disease in association with increasing amyloid-β and tau pathology date: 2016-11-25 journal: alzheimers res ther doi: 10.1186/s13195-016-0217-7 sha: doc_id: 2307 cord_uid: gk84fnb9 background: hyperactivity of the classical axis of the renin-angiotensin system (ras), mediated by angiotensin ii (ang ii) activation of the angiotensin ii type 1 receptor (at1r), is implicated in the pathogenesis of alzheimer’s disease (ad). angiotensin-converting enzyme-2 (ace-2) degrades ang ii to angiotensin 1–7 (ang ﻿﻿(1-7)) and counter-regulates the classical axis of ras. we have investigated the expression and distribution of ace-2 in post-mortem human brain tissue in relation to ad pathology and classical ras axis activity. methods: we measured ace-2 activity by fluorogenic peptide substrate assay in mid-frontal cortex (brodmann area 9) in a cohort of ad (n = 90) and age-matched non-demented controls (n = 59) for which we have previous data on ace-1 activity, amyloid β (aβ) level and tau pathology, as well as known ace1 (rs1799752) indel polymorphism, apolipoprotein e (apoe) genotype, and cerebral amyloid angiopathy severity scores. results: ace-2 activity was significantly reduced in ad compared with age-matched controls (p < 0.0001) and correlated inversely with levels of aβ (r = −0.267, p < 0.001) and phosphorylated tau (p-tau) pathology (r = −0.327, p < 0.01). ace-2 was reduced in individuals possessing an apoe ε4 allele (p < 0.05) and was associated with ace1 indel polymorphism (p < 0.05), with lower ace-2 activity in individuals homozygous for the ace1 insertion ad risk allele. ace-2 activity correlated inversely with ace-1 activity (r = −0.453, p < 0.0001), and the ratio of ace-1 to ace-2 was significantly elevated in ad (p < 0.0001). finally, we show that the ratio of ang ii to ang (1–7) (a proxy measure of ace-2 activity indicating conversion of ang ii to ang (1–7)) is reduced in ad. conclusions: together, our findings indicate that ace-2 activity is reduced in ad and is an important regulator of the central classical ace-1/ang ii/at1r axis of ras, and also that dysregulation of this pathway likely plays a significant role in the pathogenesis of ad. electronic supplementary material: the online version of this article (doi:10.1186/s13195-016-0217-7) contains supplementary material, which is available to authorized users. ad in human brain tissue [4, 5] . angiotensin ii type 1 receptor blockers (arbs) and angiotensin-converting enzyme inhibitors (aceis) reduce the amount of ad-like pathology and improve cognitive performance in most but not all mouse models of ad [6] [7] [8] [9] [10] [11] . translation of these treatments in ad is also supported in secondary outcomes of clinical trials of various arbs and aceis, as well as in epidemiological studies where the prevalence of ad was reduced [12] [13] [14] [15] [16] . last, the ace-1 indel polymorphism (rs1799752) is a genetic risk factor for sporadic ad [17] . this finding has previously been supported by several meta-analyses [18] [19] [20] [21] [22] but not by recent genome-wide association studies. ace-2 is a zinc metallopeptidase which shares 42% sequence homology within the ace-1 catalytic region [23, 24] . the ace-2 metalloprotease is expressed mostly as a transmembrane protein, but it also exists in an active soluble truncated form [24] . it is expressed predominantly in endothelial and arterial smooth muscle cells throughout the body [25] , but it is also expressed in non-vascular cells within the brain, including neuronal cell bodies [26] and astroglial cells [27] . upon its discovery, ace-2 was shown to generate angiotensin 1-7 (ang (1-7)) from ang ii, and, to a lesser extent, angiotensin 1-9 (ang (1-9)) from ang i [23, 24, 28] . emerging data suggest that ace-2-mediated conversion of ang ii to ang (1-7) and subsequent activation of the mas receptor by ang (1-7) (comprising the ace-2/ang (1-7) /mas axis) oppose the local actions of the classical ras pathway in both the periphery (reviewed in [29] ) and brain (reviewed in [30] [31] [32] [33] ). in experimental animal studies, ace-2 regulates blood pressure by counteracting the effects of the classical axis. a reduction in ace-2 expression has been implicated in cardiac and renal pathologies (reviewed in [30] ) associated with chronic hypertension. activation of brain ace-2 has been shown to be neuroprotective in animal models of ischaemic stroke [34, 35] . previous studies have suggested a link between reduced activity of the ace-2/ang (1-7)/mas axis and neurodegenerative conditions, including multiple sclerosis [36] . a recent study provided the first clues of an association with ad and reported reduced serum ace-2 activity in patients with ad compared with control subjects [37] . notably, this study also identified that ace-2 converts aβ 43 (an early deposited and highly amyloidogenic form of aβ that seeds plaque formation [38] ) to aβ 42 , which in turn is cleaved by ace-1 to less toxic aβ 40 and aβ 41 species [37] . ang (1-7) levels were also reduced in a mouse model of sporadic ad in association with hyperphosphorylation of tau [39] . in the present study, we investigated the expression and distribution of ace-2 in relation to ad pathology and the classical ras axis in human post-mortem brain tissue. we show, for the first time to our knowledge, that ace-2 activity is reduced in human post-mortem brain tissue in ad in relation to aβ and tau pathology, and also that ace-2 correlates inversely with ace-1 activity. we also show that the ratio of ang ii to ang (1-7) (a proxy measure of ace-2 activity) was increased in ad, indicating reduced conversion of ang ii to ang (1) (2) (3) (4) (5) (6) (7) . together, these data indicate that the ace-2/ ang (1-7)/mas axis is dysregulated in ad and that loss of function of this regulatory arm of ras may contribute, at least in part, to overactivation of the classical ras axis associated with ad pathogenesis. brain tissue was obtained from the south west dementia brain bank, university of bristol, uk, with local research ethics committee approval (national research ethics service 08/h0106/28 + 5). tissue was dissected from the mid-frontal cortex (brodmann area 9) in 90 cases of ad and 59 age-matched controls. brains had been subjected to detailed neuropathological assessment according to the national institute on aging-alzheimer's association guidelines [40] , and ad pathology was a sufficient explanation for the dementia in these cases. control brains were from people who had no history of dementia, had been extensively assessed neuropathologically, and had few or absent neuritic plaques, braak tangle stage iii or less, and no other neuropathological abnormalities. the demographic data for these cases are presented in table 1 , and the medical research council uk brain banks network (mrc uk-bbn) database identifiers are shown in additional file 1: table s1 . previous measurements of ace-1 activity, measured by fluorogenic activity assay, were available for all cases [4, 41] . total soluble (nonidet p-40-extracted) and insoluble (6 m guanidine hydrochloride-extracted) aβ levels were measured previously by sandwich enzyme-linked immunosorbent assay (elisa) [42] , and cerebral amyloid angiopathy (caa) severity, which was graded semi-quantitatively on a 4-point scale by a method adapted from that of olichney et al. [43] , had previously been reported [44] . phosphorylated tau (p-tau) load (area fraction of cerebral cortex immunopositive for p-tau) had been measured for all cases, as previously reported [45, 46] . ace1 genotype data for the alu 237-bp insertion(i)/deletion(d) (indel) polymorphism (rs1799752) in intron 16 of the ace1 gene were previously reported [5, 41] . last, all cases had previously been apolipoprotein e (apoe)-genotyped [44, 47] by a polymerase chain reaction method [48] . the right cerebral cortex had been fixed in 10% formalin for a minimum of 3 weeks before the tissue was processed and paraffin blocks were taken for pathological assessment. the left cerebral hemisphere had been sliced and frozen at −80°c until used for biochemical assessment. for each case, 200 mg of dissected frozen brain tissue was homogenised in a precellys homogeniser (stretton scientific, stretton, uk) as previously described [4, 5] . the samples were centrifuged at 13,000 rpm, and the clarified supernatants were aliquoted and stored at −80°c until required. total protein was measured using the total protein kit (sigma-aldrich, poole, uk) following the manufacturer's guidelines. all brain tissue was obtained within 72 h after death. ace-2 activity was measured in brain tissue using the sensolyte® 390 ace2 activity assay kit (catalogue number as-72086; anaspec, fremont, ca, usa). the assay was performed in black, flat-bottomed, non-binding, 96well nunc fluoronunc plates (fisher scientific, loughborough, uk) following the manufacturer's guidelines with minor modifications. brain tissue homogenates were prepared in assay buffer provided in the kit, to which 0.05% triton x-100 was added. samples were centrifuged at 13,000 rpm for 15 minutes at 4°c, and supernatants were removed and stored at −80°c until used. supernatants were diluted 1:100 in the proprietary ace-2 assay buffer and incubated for 10 minutes at room temperature prior to addition of the ace-2-specific fluorescence resonance energy transfer (fret) peptide and then incubated for 30 minutes in the dark. cleavage of the ace-2 fret peptide was measured using a bmg fluostar op-tima microplate reader (bmg labtech, aylesbury, uk) at an excitation/emission wavelength of 330/390 nm. ace-2 activity was interpolated from a serial dilution of 7-methoxycoumarin-4-yl-acetyl (mca) fluorescence reference standard, and measurements for each case were repeated in duplicate. to confirm the specificity of the commercial ace-2 assay kit, we measured ace-2 activity in a subset of samples (ten controls and ten ad) for which we had previously measured ace-2 activity as outlined above. the assay was performed in black, flat-bottomed, nonbinding, 96-well nunc fluoronunc plates. recombinant human ace-2 (440-6 ng/ml) (r&d systems, cambridge, uk) and brain tissue supernatants (diluted 1:20) were diluted in assay buffer (75 mm tris, 1 m nacl, ph 7.5) and pre-incubated with an ace-2 specific inhibitor, mln4760 (10 μm) (calbiochem, nottingham, uk) or assay buffer alone for 10 minutes at 37°c. an ace-2 fluorogenic peptide mca-apk(dnp) (enzo life sciences, exeter, uk) was then added, and the reaction was incubated at 37°c for 30 minutes in the dark. fluorescence was read at an excitation/emission wavelength of 330/ 405 nm using a bmg fluostar optima microplate reader. ace-2-specific activity was calculated after subtracting fluorescence in the presence of mln-4760 from the uninhibited sample. we observed a very strong correlation between the independent measurements of ace-2 in the presence of mln4760 (10 μm) and with the kit, indicating the specificity of the ace-2 assay kit (additional file 2: figure s1 ). ang ii levels were measured in brain tissue homogenates extracted in 1% sds lysis buffer (100 μm nacl, 10 mm tris, ph 6, 1 μm phenylmethylsulphonylfluoride, 1 μg/ml aprotinin [sigma-aldrich] and 1% sds in distilled water) using a commercially available sandwich elisa kit (abcam, cambridge, uk) following the manufacturer's guidelines. in brief, recombinant human ang ii or brain tissue supernatants (diluted 1:2 in pbs) were added in duplicate to wells that had been pre-coated with an ang ii-specific capture antibody and incubated for 2 h at room temperature. after a wash step, the wells were incubated for 2 h with biotinylated anti-ang ii antibody at room temperature. the plate was again washed, followed by a 30-minute incubation with streptavidin/hrp. after a final wash, 3,3′,5,5′-tetramethylbenzidine (tmb) substrate was added for 20 minutes, and the absorbance at 450 nm was read using a fluostar optima plate reader. the concentration of ang ii was interpolated from a serial dilution of recombinant ang ii (1000-62.5 pg/ml) and measured in duplicate for each case. ang (1-7) levels were measured in human brain tissue homogenates in 1% sds lysis buffer (see above) using an in-house direct elisa kit. recombinant human ang (1-7) (enzo life sciences) or human brain tissue homogenates (diluted 1:40 in pbs) were incubated for 2 h in a clear, high binding capacity nunc maxisorp plate (thermo fisher scientific, waltham, ma, usa) at 26°c with shaking. the wells were washed five times in pbs with 0.05% tween-20 and blocked for 1 h in pbs:1% bovine serum albumin (sigma-aldrich). after another five washes, the wells were incubated with biotinylated antihuman ang 1-7 (2 μg/ml in pbs) (cloud-clone, wuhan, china) for 2 h at 26°c with shaking, followed by a further wash step. streptavidin/hrp (1:200) in pbs/ 0.01% tween-20 was added to each well, which was incubated at room temperature for 20 minutes in the dark. tmb substrate (r&d systems) was added after a further wash and left to develop in the dark for 20 minutes. absorbance at 450 nm was read following the addition of 2 n sulphuric acid ('stop' solution) using a fluostar optima plate reader. ang (1-7) concentration was interpolated from a standard curve generated by serially diluting recombinant human ang (1-7) (5000-78.125 pg/ml). the assay showed minimal cross-reactivity with a number of closely related peptides, including ang i, ang ii and ang iii. formalin-fixed, paraffin-embedded tissue sections (7 μm) were cut and de-waxed prior to immunohistochemistry. sections were pre-treated in trisodium citrate buffer (9 mm), ph 6, and microwaved for 5 minutes, left to stand for 5 minutes, and boiled for a further 5 minutes before being left to stand for 15 minutes at room temperature. sections were then rinsed thoroughly and covered in horse serum blocking solution, rinsed again, and incubated overnight at room temperature with anti-ace-2 antibody (0.05 μg/ml, ab15348; abcam). bound antibody was visualised using a biotinylated universal antibody followed by vectastain elite abc avidin-biotin complex kit (vector laboratories, peterborough, uk) and a reaction with 0.01% h 2 o 2 . specificity of the antibody was assessed by preadsorption of the ace-2 antibody with a 250-fold molar excess of recombinant human ace-2 protein (r&d systems). unpaired two-tailed t tests or analysis of variance (anova) with bonferroni's post hoc analysis was used for comparisons between groups, and pearson's test was used to assess linear correlation with spss version 16 (spss, chicago, il, usa) and graphpad prism version 6 (graphpad software, la jolla, ca, usa) software. p values <0.05 were considered statistically significant. ace-2 enzyme activity is reduced in alzheimer's disease in association with increasing aβ load and tau pathology ace-2 activity was significantly reduced by approximately 50% in the mid-frontal cortex in ad compared with age-matched controls (p < 0.0001) (fig. 1a) . ace-2 varied according to disease severity when the controls and ad cases were grouped and stratified into the following braak tangle stage groups: 0-ii, iii-iv, and v-vi (p < 0.0001 by anova). post hoc analysis using the bonferroni correction for multiple comparisons revealed that ace-2 activity was significantly reduced in braak tangle stages v-vi compared with stages 0-ii (p < 0.0001) and stages iii-iv (p < 0.05) (fig. 1b) . no difference was observed between braak stages 0-ii and stages iii-iv. in a combined ad and control cohort, ace-2 activity correlated inversely with total insoluble aβ levels (r = −0.267, p < 0.01) (fig. 1c) but not with soluble aβ (data not shown). ace-2 correlated inversely with βsecretase activity (r = −0.277 p < 0.001) (additional file 3: figure s2 ). ace-2 correlated inversely with p-tau load (r = 0.327, p < 0.01) (fig. 1d) . ace-2 activity is reduced in relation to apoe and ace1 polymorphisms and caa severity ace-2 activity was significantly lower in individuals possessing an apoe ε4 allele, an established genetic risk factor for sporadic ad [49] , than in those without (p < 0.05) (fig. 2a) . ace-2 activity also differed significantly post hoc analysis revealed that ace-2 activity was reduced in braak tangle stages v-vi compared with stages 0-ii and iii-iv (p < 0.0001 and p < 0.05 respectively) and in braak tangle stages iii-iv compared with stages 0-ii, but the difference was not statistically different. the bars indicate the mean value and sem. c and d scatterplots showing that ace-2 activity was inversely correlated with insoluble amyloid-β (aβ) load (measured by enzyme-linked immunosorbent assay) (r = −0.267, p < 0.01) and phosphorylated tau (p-tau) load (measured by field fraction analysis) (r = −0.327, p < 0.001). the solid inner line indicates the best-fit linear regression and the outer lines the 95% confidence intervals. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. rfu relative fluorescence units between ace1 (rs1799752) indel genotypes (p < 0.05), with individuals who were homozygous ii for ace-1 (previously associated with increased risk for ad [17] ) having the lowest ace-2 activity, although post hoc analysis revealed that this did not reach statistical significance (fig. 2b) . we assessed ace-2 activity in relation to caa severity and found, as for ace-1 activity [4] , a tendency, although not significant, towards increased ace-2 activity in cases with moderate to severe caa compared with absent to mild caa (p = 0.08) (fig. 2c) . ace-2 is inversely correlated with ace-1, and the ratio of ace-1 to ace-2 is increased in alzheimer's disease ace-2 activity correlated inversely with ace-1 activity in a combined ad and control cohort (r = −0.453, p > 0.0001) (fig. 3a) . the same pattern was observed and remained statistically significant when the control (r = −0.390, p < 0.05) and ad (r = −0.257, p < 0.05) groups were analysed separately. previous reports have suggested the ratio of ace-1 to ace-2 is a good proxy measure for the activation status of classical and regulatory ras pathways [33] . with this in mind, we calculated the ace-1/ace-2 ratio for all cases and found that it was significantly increased in ad compared with controls (p > 0.0001) (fig. 3b) . the ace-1/ace-2 ratio also correlated positively with insoluble aβ level, approaching significance (r = 0.199, p = 0.059) (fig. 3c) , and significantly with p-tau (r = 0.252, p < 0.05) (fig. 3d) . the ace-1/ace-2 ratio was increased in individuals possessing an apoe ε4 allele, approaching significance (p = 0.093) (fig. 3e) , and differed significantly according to ace1 (rs1799752) indel polymorphism (p < 0.01). post hoc analysis revealed that the ratio was significantly higher in individuals with ace1 ii (ad risk factor) than in dd (p < 0.01) and in id than in dd (p < 0.05) (fig. 3f) . ang ii levels were significantly increased in mid-frontal cortex in ad compared with age-matched controls (p < 0.0001) (fig. 4a) , whereas ang (1-7) levels were unchanged (fig. 4b) . we calculated the ang ii/ang (1-7) ratio (as a proxy indicator of ace-2 activity) and found that the ang ii/ang (1-7) ratio was significantly increased in ad (p > 0.001) (fig. 4c) . these data indicate that the conversion of ang ii to ang (1-7) is likely to be reduced in ad because of lower ace-2 activity. ace-2 expression in human brain tissue ace-2 was localised primarily to capillaries but also had a perivascular distribution around larger arterioles (fig. 5a) . ace-2 labelled non-vascular cells that strongly resembled astrocytes ( fig. 5b and c) . labelling was not observed with pre-adsorption of the ace-2 antibody with recombinant human ace-2, demonstrating specificity of the antibody (fig. 5d ). in the present study, we show that ace-2 activity is reduced in post-mortem brain tissue in ad in association with increased aβ and tau pathology. the reduction in ace-2 was more pronounced in individuals carrying an apoe ε4 allele and in those who were homozygous ii for the ace1 (rs1799752) indel polymorphism (both of which are suggested genetic risk factors for ad [17] ). ace-2 activity correlated inversely with ace-1 activity (which we have previously shown to be increased in ad [4, 5] ), and the ace-1/ace-2 ratio was higher in ad. together, these data strongly suggest that reduced acefig. 2 angiotensin-converting enzyme 2 (ace-2) activity is reduced in association with apolipoprotein e (apoe) ε4 and ace1 (rs1799752) indel polymorphism and increased in cerebral amyloid angiopathy (caa). a bar chart showing reduced ace-2 activity in individuals with an apoe ε4 allele (p < 0.05). b bar chart showing that ace-2 activity varied according to ace1 indel polymorphism (p < 0.05), with a trend towards reduced ace-2 activity in ace-1 ii homozygotes. c bar chart showing elevated ace-2 activity in moderate to severe caa compared with absent to mild caa, approaching significance (p = 0.08). the bars indicate the mean value and sem. *p < 0.05. rfu relative fluorescence units fig. 3 angiotensin-converting enzyme 2 (ace-2) activity is inversely correlated with ace-1 activity, and the ace-1/ace-2 ratio is increased, in alzheimer's disease (ad). a scatterplot showing a strong inverse relationship between ace-1 and ace-2 activity in mid-frontal cortex (r = −.453, p < 0.0001). the inner solid line indicates the best-fit linear regression and the outer lines the 95% confidence intervals. each dot represents an individual brain. b bar chart showing elevated ace-1/ace-2 ratio in ad (p < 0.0001). c and d scatterplots showing positive correlation between the ace-1/ace-2 ratio and insoluble amyloid-β (aβ) load (r = 0.199, p = 0.059) and p-tau load (r = 0.252, p < 0.05). e bar chart showing a trend towards increased ace-1/ace-2 ratio in individuals who possessed an apolipoprotein e (apoe) ε4 allele. f bar chart showing lower ace:ace-2 ratio in individuals who were homozygous dd for the ace1 (rs1799752) indel polymorphism compared with ii (p < 0.01) and id (p < 0.05). the bars indicate the mean value and sem. *p < 0.05, **p < 0.01, ****p < 0.0001. rfu relative fluorescence units fig. 4 the ratio of angiotensin ii (ang ii) to angiotensin (1-7) (ang (1-7) ) (a proxy measure of ace-2 activity) is increased, indicating reduced conversion of ang ii to ang (1-7) in alzheimer's disease (ad). bar charts showing a elevated ang ii levels in ad and b unchanged ang (1-7) levels in ad compared with age-matched controls in mid-fontal cortex. c bar chart showing the ang ii/ang (1-7) ratio was significantly increased in ad (p < 0.001). the bars indicate the mean value and sem. ***p < 0.001, ****p < 0.0001 2 activity within the brain contributes to ad pathogenesis and is associated with increased activation of the central classical ras axis. the brain has its own intrinsic ras [50] [51] [52] , and we have shown in our previous studies that ace-1, the rate-limiting enzyme in the production of ang ii, is overactive in ad [4, 5] . it is widely accepted that ang ii-mediated signalling via at1r (commonly termed the classical axis) is overactive in ad and is associated with ad pathogenesis (reviewed in [1] ). this view has been supported in various animal studies in which infusion of ang ii resulted in elevated plaque and tau pathology and significant cognitive impairment [2, 3] . secondary observations in clinical trials and epidemiological studies have provided further evidence that ras-targeting drugs that either block the production of ang ii or prevent at1r-mediated signalling reduce the prevalence of ad [12] [13] [14] [15] [16] , while cognitive performance is improved and pathology reduced, in animal models of ad [6] [7] [8] [9] [10] [11] . until recently, the prevailing view of the ras in ad has been oversimplified because it has failed to consider the contribution of the other downstream ras regulatory pathways within the brain. in this study, we found reduced brain ace-2 activity in ad, which supports a recent study showing lower peripheral serum ace-2 levels in ad [37] . ace-2 activity correlated inversely with parenchymal aβ load and increased p-tau levels. we also observed a strong inverse relationship between ace-2 and β-secretase activity, suggesting that ace-2 may contribute in some way to regulating the amyloidogenic processing of app. there are several possible mechanisms that link reduced ace-2 activity to the pathogenesis of ad. firstly, lower ace-2 activity will, via a lower conversion of ang ii to ang (1-7), result in elevated ang ii levels (as we have shown in this study). an increase in ang ii/ang (1-7) ratio has commonly been reported in other chronic conditions associated with overactivation of the central axis [53] . secondly, ace-2 is primarily responsible for generating ang (1-7) from ang ii [24, 54, 55] , and subsequent ang (1-7) activation of the mas receptor counter-regulates the detrimental effects of the classical (ace-1/ang ii/at1r) axis [56] [57] [58] and has been linked with enhancing learning and memory processing [59, 60] . lastly, ace-2 has recently been shown to convert aβ 43 , a highly amyloidogenic form of aβ that seeds plaque formation [38] , to aβ 42 , which in turn is cleaved by ace-1 to aβ 40 or, to a lesser extent, aβ 41 , which have reduced toxicity [37] . lower ace-2 activity in ad may therefore promote the early deposition of aβ 43 and prevent downstream cleavage of aβ 42 by ace-1.together, these data suggest a putative protective role of the ace-2/ang (1-7)/mas pathway, not only against the development of pathology but also against the decline in cognitive function, that is lost in ad. our findings indicate that the balance between the classical (ace-1/ang ii/at1r) axis and regulatory (ace-2/ ang (1-7)/mas) axis of ras is disturbed in ad, as previously shown in various mouse models of cardiovascular disease [33] and diabetic nephropathy [53] . ace-2 activity is reduced in ad and is inversely correlated with increasing ace-1 activity, and the ace-1/ace-2 ratio is increased in ad in association with disease pathology. these findings support commonly observed traits in cardiac and renal pathologies showing that dysregulation of the ace-2/ang (1-7)/mas pathway, including reduced ace-2 activity, is associated with sustained hypertension mediated by overactivation of the classical axis (reviewed in [30, 61] ). despite the ratio of ang ii to ang (1-7) (a proxy measure of ace-2 activity) being increased in ad (i.e., reduced conversion of ang ii to ang (1-7)), we did not observe an overall reduction in total ang (1) (2) (3) (4) (5) (6) (7) in ad. this is inconsistent with a recent report showing reduced serum ang (1-7) levels, rather than reduced ace-2 activity, in senescence-accelerated mouse prone 8, a mouse model of sporadic ad (involving overexpression of app). the authors observed that ang (1-7) levels correlated inversely with ang ii and p-tau levels [39] . the reason for the discrepant findings between human and mouse brain tissue is unclear; however, both studies indicate that the ace-2/ang (1-7)/mas pathway is dysregulated in ad and that further work is required to determine the exact contribution of each component of the pathway in ad. activation of the ace-2/ang (1-7)/mas pathway, by inducing ace-2 activity, or infusion of ang (1-7) or a mas receptor agonist, is protective in various experimental animal models of cardiovascular disease and is associated with a reduction of the classical ras pathway (reviewed in [32, 61] ). neuronal overexpression of brain ace-2 is also neuroprotective in a chronic hypertension mouse model (transgenic for renin and angiotensinogen that overproduces ang ii) following experimental induction of ischaemic stroke [34, 35, 62] . these protective effects were partially reversed in the presence of a mas receptor antagonist, demonstrating the specificity of the ace-2/ang (1-7)/mas pathway, and they have been shown to be mediated by counter-regulating the effects of ang ii-mediated reactive oxygen species production [63] . in ad, there is growing recognition that re-positioning of brain-penetrating arbs and aceis may have clinical benefits in ad [64] . in addition to reducing the central pool of ang ii, arbs and aceis might also exert their protective effects by preventing at1r-mediated reduction in ace-2 activity [65] that can be reversed by arbs [27, [66] [67] [68] [69] . ace-2 activation is also associated with reduced ace-1 activity [70] and with down-regulation of ang ii levels and at1r expression [27, 65, [71] [72] [73] . these studies suggest that activation of ace-2 may exert protective effects in ad above and beyond dampening ras activation that the use of aceis and arbs currently allow. lastly, we explored the distribution of ace-2 within the mid-frontal and temporal cortices and found it to be localised predominantly within endothelial cells and smooth muscle cells of cerebral arteries, as previously reported [25] . interestingly, as for ace-1, we also observed extensive perivascular ace-2 expression and found that ace-2 activity was increased in individuals with moderate to severe caa, as has previously been shown for ace-1 [4] . we speculate that the sequential cleavage of aβ 43 , first by ace-2, and the subsequent cleavage of aβ 42 to aβ 40 (the predominant species in caa [74] ) by ace-1, provides a potential mechanistic link with caa. further studies are required to determine the relationship between ace-2 and caa severity. these data indicate that reduced activity of the ace-2/ ang (1-7)/mas axis is strongly linked to overactivity of the classical ras pathway and with ad-related pathology. additional file 1: table s1 . mrc identifiers for all cases. (doc 80 kb) additional file 2: figure s1 . scatterplot showing a strong positive correlation between two independent measures of ace-2 activity in brain tissue samples. ace-2 was measured using either a commercially available ace-2 activity assay kit (sensolyte® 390) or an ace-2 fluorogenic peptide substrate (mca-apk[dnp]) in the presence of a selective ace-2 inhibitor, mln4760 (10 μm). the solid inner line indicates the best-fit linear regression, and the outer lines the 95% confidence intervals. each point represents a separate brain. ****p < 0.0001. (tif 26 kb) additional file 3: figure s2 . scatterplot showing an inverse relationship between ace-2 activity and bace-1 activity in a combined alzheimer's disease and age-matched control cohort. ace-2 activity was measured using the sensolyte® 390 ace-2 activity assay kit, and bace-1 activity was measured using the β-secretase specific fluorogenic substrate angiotensins in alzheimer's disease -friend or foe? central angiotensin ii stimulation promotes β amyloid production in sprague dawley rats central angiotensin ii-induced alzheimer-like tau phosphorylation in normal rat brains angiotensin-converting enzyme (ace) levels and activity in alzheimer's disease, and relationship of perivascular ace-1 to cerebral amyloid angiopathy angiotensinconverting enzyme levels and activity in alzheimer's disease: differences in brain and csf ace and association with ace1 genotypes protective effects of intranasal losartan in the app/ps1 transgenic mouse model of alzheimer disease perindopril, a centrally active angiotensin-converting enzyme inhibitor, prevents cognitive impairment in mouse models of alzheimer's disease angiotensin ii type 1 receptor blocker losartan prevents and rescues cerebrovascular, neuropathological and cognitive deficits in an alzheimer's disease model cognitive deficit in amyloid-β-injected mice was improved by pretreatment with a low dose of telmisartan partly because of peroxisome proliferator-activated receptor-γ activation valsartan lowers brain β-amyloid protein levels and improves spatial learning in a mouse model of alzheimer disease effect of a centrally active angiotensin-converting enzyme inhibitor, perindopril, on cognitive performance in a mouse model of alzheimer's disease associations of antihypertensive treatments with alzheimer's disease, vascular dementia, and other dementias prevention of dementia in randomised double-blind placebo-controlled systolic hypertension in europe (syst-eur) trial use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis effects of blood pressure lowering with perindopril and indapamide therapy on dementia and cognitive decline in patients with cerebrovascular disease effects of telmisartan on cognition and regional cerebral blood flow in hypertensive patients with alzheimer's disease variation in dcp1, encoding ace, is associated with susceptibility to alzheimer disease systematic metaanalyses of alzheimer disease genetic association studies: the alzgene database alzheimer disease risk and genetic variation in ace: a meta-analysis haplotypes extending across ace are associated with alzheimer's disease large meta-analysis establishes the ace insertion-deletion polymorphism as a marker of alzheimer's disease candidate singlenucleotide polymorphisms from a genomewide association study of alzheimer disease a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 a human homolog of angiotensin-converting enzyme: cloning and functional expression as a captopril-insensitive carboxypeptidase tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis differential expression of neuronal ace2 in transgenic mice with overexpression of the brain renin-angiotensin system distinct roles for ang ii and ang-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes angiotensin-converting enzyme 2 is an essential regulator of heart function angiotensin-converting enzyme 2: central regulator for cardiovascular function angiotensin-converting enzyme 2 in the brain: properties and future directions ace2/ang-(1-7)/mas pathway in the brain: the axis of good ace2-ang-(1-7)-mas axis in brain: a potential target for prevention and treatment of ischemic stroke recent advances in the angiotensinconverting enzyme 2-angiotensin(1-7)-mas axis cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke suppressing inflammation by inhibiting the nf-κb pathway contributes to the neuroprotective effect of angiotensin-(1-7) in rats with permanent cerebral ischaemia angiotensin-converting enzyme (ace) and ace2 levels in the cerebrospinal fluid of patients with multiple sclerosis conversion of aβ43 to aβ40 by the successive action of angiotensin-converting enzyme 2 and angiotensin-converting enzyme aβ43 is the earliest-depositing aβ species in app transgenic mouse brain and is converted to aβ41 by two active domains of ace angiotensin-(1-7) is reduced and inversely correlates with tau hyperphosphorylation in animal models of alzheimer's disease national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach ace variants and association with brain aβ levels in alzheimer's disease higher soluble amyloid β concentration in frontal cortex of young adults than in normal elderly or alzheimer's disease the apolipoprotein e ε4 allele is associated with increased neuritic plaques and cerebral amyloid angiopathy in alzheimer's disease and lewy body variant apoe ε4 influences the pathological phenotype of alzheimer's disease by favouring cerebrovascular over parenchymal accumulation of a β protein cholinesterase inhibitors reduce cortical aβ in dementia with lewy bodies cholinesterase inhibitors may increase phosphorylated tau in alzheimer's disease apoe and cerebral amyloid angiopathy in the elderly apolipoprotein e genotyping by one-stage pcr apolipoprotein e ε4 and cerebral hemorrhage associated with amyloid angiopathy brain renin-angiotensin-a new look at an old system the brain renin-angiotensin system: a diversity of functions and implications for cns diseases brain renin angiotensin in disease angiotensin-(1-7): a novel peptide to treat hypertension and nephropathy in diabetes? brain angiotensin-converting enzymes: role of angiotensin-converting enzyme 2 in processing angiotensin ii in mice hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function angiotensin-(1-7): an update angiotensin-(1-7) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-(1-7) enhances ltp in the hippocampus through the g-protein-coupled receptor mas angiotensin-(1-7)/mas axis integrity is required for the expression of object recognition memory angiotensin-converting enzyme 2: a new target for neurogenic hypertension neuronal overexpression of ace2 protects brain from ischemia-induced damage activation of the ace2/ ang-(1-7)/mas pathway reduces oxygen-glucose deprivation-induced tissue swelling, ros production, and cell death in mouse brain with angiotensin ii overproduction current status of renin-aldosterone angiotensin system-targeting anti-hypertensive drugs as therapeutic options for alzheimer's disease angiotensin ii type 1 receptor-mediated reduction of angiotensin-converting enzyme 2 activity in the brain impairs baroreflex function in hypertensive mice ace and ace2: their role to balance the expression of angiotensin ii and angiotensin the ang-(1-7)/ace2/mas axis in the regulation of nephron function increased expression of angiotensin converting enzyme 2 in conjunction with reduction of neointima by angiotensin ii type 1 receptor blockade role of angiotensin-converting enzyme 2/angiotensin-(1-7)/mas axis in the hypotensive effect of azilsartan ace2 overexpression in the paraventricular nucleus attenuates angiotensin ii-induced hypertension exercise training normalizes ace and ace2 in the brain of rabbits with pacing-induced heart failure brain-selective overexpression of angiotensin-converting enzyme 2 attenuates sympathetic nerve activity and enhances baroreflex function in chronic heart failure angiotensin-converting enzyme 2 overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type 1 receptor downregulation amyloid β protein (aβ) in alzheimer's disease brain: biochemical and immunocytochemical analysis with antibodies specific for forms ending at aβ40 or aβ42(43) we acknowledge professor seth love, university of bristol, for his academic input and neuropathological assessment. all data within the article is linked to the mrc uk-bbn by a unique numeric mrc uk-bbn identifier (additional file 2: figure s1 ). there is no risk of disclosure of personal information, because all of the information held within the database has been anonymised.authors' contributions jsm carried out the angiotensin-ii measurements and validated the ace-2 activity measurements, performed the statistical analysis and was primarily responsible for drafting and finalizing the manuscript. sw carried out the ace-2 activity measurements, performed statistical analysis and helped to draft the manuscript. nam carried out the angiotensin (1-7) measurements, performed statistical analysis and helped draft the manuscript. lep carried out the ace-2 immunolabelling and analysis and revised the manuscript. pgk conceived and was responsible for overall planning and design of the study, and helped to revise and finalize the manuscript. all authors read and approved the final manuscript. all authors are members of the dementia research group, clinical neurosciences, school of clinical sciences, university of bristol, bristol, uk. the authors declare that they have no competing interests. the use of human brain tissue for this study was approved by the management committee of the south west dementia brain bank (human tissue authority licence number 12273) under the terms of bristol research ethics committee approval of the brain bank (reference 08/h0106/28 + 5). all participants provided consent to post-mortem removal of whole brain and csf and the retention of these for use in research. consent included access to the donor's medical records to collect information on past medical history relevant to the donation, but that in all publications this information would be anonymised. key: cord-317878-bqpj0ey0 authors: czick, maureen; shapter, christine; shapter, robert title: covid’s razor: ras imbalance, the common denominator across disparate, unexpected aspects of covid-19 date: 2020-09-11 journal: diabetes metab syndr obes doi: 10.2147/dmso.s265518 sha: doc_id: 317878 cord_uid: bqpj0ey0 a modern iteration of occam’s razor posits that “the simplest explanation is usually correct.” coronavirus disease 2019 involves widespread organ damage and uneven mortality demographics, deemed unexpected from what was originally thought to be “a straightforward respiratory virus.” the simplest explanation is that both the expected and unexpected aspects of covid-19 share a common mechanism. silent hypoxia, atypical acute respiratory distress syndrome (ards), stroke, olfactory loss, myocarditis, and increased mortality rates in the elderly, in men, in african-americans, and in patients with obesity, diabetes, and cancer—all bear the fingerprints of the renin-angiotensin system (ras) imbalance, suggesting that ras is the common culprit. this article examines what ras is and how it works, then from that baseline, the article presents the evidence suggesting ras involvement in the disparate manifestations of covid-19. understanding the deeper workings of ras helps one make sense of severe covid-19. in addition, recognizing the role of ras imbalance suggests potential routes to mitigate covid-19 severity. medieval english theologian william of occam is widely credited with the eponymous principle known as occam's razor: "entities should not be multiplied without necessity." or, in modern terms, "the simplest explanation is usually correct." in late winter/early spring of 2020, as novel coronavirus case numbers exploded in europe and parts of the united states, prominent newspapers and online newsmedia outlets reported with alarm a broad array of coronavirus disease 2019 (covid-19) demographic and clinical outcomes deemed to be surprising and unexpected, even by some of the physicians interviewed. as one washington post headline exclaimed: "once thought a relatively straightforward respiratory virus, covid-19 is proving to be much more frightening." 1 outcomes that initially jolted both the press and the medical community include cases of severe covid-19 in young, apparently healthy adults, 2 mortality rates among african-americans and latinos doubled compared to caucasians, 3, 4 similarly doubled mortality among men compared to women, 5 and a tripled death rate among certain cancer patients. 6 in the uk, government epidemiologic data suggested that blacks there are four times more likely to die of covid-19 compared to whites, with uk residents of indian/pakistani/bangladeshi ancestry also showing increased mortality rates. 7 journalists warned as covid-19 patients developed renal failure requiring dialysis, 8 and obesity was linked to higher mortality. 9 subsequent reports revealed occurrences of fatal covid-19 myocarditis, 10 strokes in young patients, 1, 11, 12 "silent hypoxia," 13 a mortality rate among ventilated covid-19 patients approaching 90%, 14 and a kawasaki-like syndrome in children. 15, 16 in the early days of the pandemic, the clear association of pre-existing hypertension with more severe covid-19 outcomes triggered consternation among physicians, 17 as did cases of the virus presenting with seemingly incongruent hyperglycemia, 18 and loss of taste and smell sensation. 8 the widespread organ involvement and demographic perplexities do at first seem to be a bewildering pathophysiologic maelstrom. but occam's razor posits that the most likely explanation is that a common underlying mechanism unites them all. when viewed through the lens of the renin-angiotensin system (ras), the covid-19 disarray slips into crystal clear focus. in addition to its better-known circulatory volume/pressure regulatory role, ras is also a cellular-level system, present in the tissues of practically every organ of the body, and mediating a broad array of inflammatory, metabolic, osmotic, growth, and repair functions. 19 the hallmarks of ras imbalance, either pre-existing or covid-19-mediated, are a common thread interwoven through wide-ranging covid-19 symptomatology and disparate sub-population outcomes. the commonality of ras dysregulation does not prove that ras is the sole mediator of covid-19 pathophysiology; there may be multiple factors that synergize to worsen covid-19 severity (table 1 ). yet the underlying presence of ras imbalance in so many aspects of covid-19 does suggest that ras may be the key to understanding and potentially mitigating covid-19. only after taking a deeper look at what ras is and how it works, does severe covid-19 itself begin to make sense. ras was first conceptualized as an exclusively endocrine effector system, activated in response to decreased arterial blood pressure, increased sympathetic nervous system tone, or decreased filtered solute delivery within the kidney tubules as, for instance, during hemorrhage or dehydration. [20] [21] [22] [23] ras was depicted as a linear sequence of enzymatic activations, with substrates circulating in the bloodstream to reach both their enzymatic activators and their target receptors. renin, released into circulation by renal juxtaglomerular (jg) cells, cleaves hepatically produced angiotensinogen to generate angiotensin i (ai), an intermediate and inactive compound, which circulates to encounter membrane-bound angiotensin converting enzyme (ace) on vascular endothelial cells, 24 primarily within the pulmonary circulation. 25 ace converts ai to angiotensin ii (aii), which transits to the adrenal cortex, triggering the release of aldosterone. aldosterone subsequently stimulates increasing renal reabsorption of sodium and water, thereby augmenting blood volume. 26 this sequence of ras activations has been dubbed the renin/ace/angiotensin/at1 axis or, more succinctly, the ace axis. 27 aii also binds to angiotensin type-1 receptors (at1) on vascular smooth muscle cells to mediate vasoconstriction and increase blood pressure. 26 at1 dispatches a g-protein to activate phospholipase c, which splits the membrane lipid phosphatidyl inositol bisphosphate (pip2) into two products, inositol tri-phosphate (itp) and diacylglycerol (dag), both of which increase intracellular calcium levels. 28 calcium-bound calmodulin next activates myosin light-chain kinase (mlck), which phosphorylates smooth muscle myosin, enabling actin binding, smooth muscle contraction, and vasoconstriction 28 (figure 1 ). circulating pressure/volume regulation within the vascular system is not the only function of ras. 29 after much debate, 30, 31 there is now widespread agreement that local tissue-level ras systems administer paracrine and autocrine effects in various structures including adipose tissue, the pancreas, vascular walls, and the heart, kidneys, and brain. 19, 32 evidence suggests that locally produced ras components affect physiology independently of blood pressure. 33 although this premise remains controversial, the original purpose of some ras components may not have involved blood pressure regulation and might even pre-date the evolution of blood vessels; 34 for example, the modern invertebrate fruit fly, which has an open circulatory system and therefore lacks discrete blood vessels, has an ace-like enzyme, 35 and a renin-like enzyme is found in the leech. 26 invertebrates demonstrate the value of a synergistic proinflammatory and procoagulant injury response system. they possess a sole cell-type having both platelet-like and macrophage-like functions which speeds injury response. 36 in open circulatory systems, blood directly contacts cells, so pathogens entering via a breach in the exoskeleton would rapidly begin cellular invasion. in the absence of blood vessels, there is no mechanism to restrict blood flow to the injury site, and this would be fatal without prompt coagulation. 36 with an open circulatory system, there is no time to lose. the evolution of vertebrate closed-circulatory systems interposed the vascular wall as a barrier to shield cells from blood-borne invaders. 36 coagulation and inflammation evolved along separate, specialized cell lines, 36 but the advantages of integration between coagulation and inflammation during injury response were preserved through crosstalk and mutual co-activation. [37] [38] [39] [40] [41] vasoconstriction to limit blood flow to injured areas is intertwined with inflammation and coagulation to establish a fully integrated injuryresponse mechanism. the vascular endothelium became the pivotal regulatory interface, with the ace axis mediating all three processes. ras is a critical survival mechanism, yet when dysregulated, it poses a significant danger. under normal conditions, the healthy vascular endothelium promotes vasodilation and inhibits coagulation and inflammation. 42, 43 however, when injury occurs, aii, together with proinflammatory cytokines from innate immune cells, transforms endothelium into an "activated" state that promotes vasoconstriction, inflammation, and coagulation. 37, 42, 43 endothelial activation is intended to be temporary; failure to recover a vasodilatory, anti-inflammatory, and anticoagulant baseline is termed endothelial dysfunction. 44 it results from excessive generation of reactive oxygen species (ros) by overactive aii, with consequent loss of nitric oxide (no) production capacity. 44 (figure 1 ) dysfunctional endothelium, due to ras dysregulation, promotes inflammatory and thrombotic damage in disease states such as diabetes, hypertension, and atherosclerosis. [45] [46] [47] the ace axis has direct growth-promoting effects on cells and cell matrix for post-injury repair. at1 activation of mitogen-activated protein kinase (map kinase) upregulates growth factors such as vascular endothelial growth factor (vegf), and inflammatory mediators including tumor necrosis factor (tnf) alpha, interleukin-1 (il1) beta, and nuclear factor kappa b (nf-κb). this upregulated signaling contributes not only to repair but also to inflammation, cancer, and second messenger pathways of the ace axis and bradykinin in vascular wall. to mediate vasoconstriction and increase blood pressure, aii binds to at1 receptors on vascular smooth muscle cells. 26 at1 activates plc, which splits the membrane lipid pip2 into two products, itp and dag, resulting in sr calcium release and increased intracellular calcium levels. 28 calcium binds to calmodulin; together they activate mlck, which phosphorylates smooth muscle myosin. phosphorylated myosin then binds actin, triggering smooth muscle contraction, and vasoconstriction. 28 bk initiates a pathway opposing the vasoconstrictive action of aii. bk binding to the b2 receptor 218 activates nos to produce no. 43 no diffuses from its site of production in vascular endothelial cells, to neighboring vascular smooth muscle, where it activates a vasodilatory second messenger cascade, culminating in activation of mlcp, which strips phosphate groups from smooth muscle myosin, such that it can no longer bind to actin. 43 nervous system function. 48 acting again via map kinase, at1 upregulates cytokines, such as transforming growth factor (tgf) beta, that drive fibrotic collagen deposition by myofibroblasts. 48, 49 fibrosis and hypertrophy can at first be reversible repair responses, but when dysregulated they progressively damage vascular smooth muscle, myocardium, and kidneys. 48, 50 in addition, some cellular damage may be too extensive for recovery, in which case the ace axis contributes to activating cell death pathways. 48, 51, 52 ros derived from at1 activation can inhibit anti-apoptotic bcl-2 and activate pro-apoptotic bax pathways, triggering programmed cell death. 52 aii can also impair mitochondrial function and increase protein misfolding, causing pathological protein aggregation. 48 and aii enhances autophagic catabolism of damaged organelles and protein aggregates, necessary for homeostasis, but capable of inciting cell death when dysregulated. 51 it is evident that a system as powerful and far-reaching as ras must be tightly regulated, to prevent potentially lethal misapplication of vasoconstriction, inflammation, and coagulation. the at2 receptor is generally described as a protective arm of ras, opposing the actions of the at1 receptor. 53, 54 the functional roles of at2 have been challenging to tease out, in part because of low adult at2 expression levels compared to at1, whose effects may overwhelm the more subtle alterations prompted by at2. 54 at2 has extensive expression during fetal development, but comparatively low-level expression in adults in select sites: heart, kidney, adrenal, brain, uterus, pancreas, retina, endothelium, and vascular smooth muscle. 55 aii also binds to at2, and in doing so, it counteracts at1 effects, promoting instead natriuresis, which can slightly lower blood pressure, as well as stimulating antiinflammatory, antithrombotic, and antifibrotic effects. 54 at2 activates superoxide dismutase (sod), which neutralizes ros and defends against oxidative stress created by at1. 52 at2 knockout mice have increased vascular hypertrophy, indicating that at2 has protective anti-hypertrophy functions, 55 congruent with at2 upregulation in the heart during disease states including heart failure, cardiac ischemia, and dilated cardiomyopathy. 55 some of the benefits of angiotensin receptor blockers (arbs), such as reducing vascular and cardiac hypertrophy, appear to be at2mediated, by shunting aii away from at1. 55 the principal at2 second messenger pathway involves production of nitric oxide (no), cyclic gmp (cgmp), and bradykinin, all of which promote vasodilation and oppose inflammation. 55 endothelial no upregulates at2 which then decreases ace activity. 54 cgmp also deactivates rhoa, an at1 second messenger, thereby antagonizing at1-mediated vasoconstriction. 55 ace has a counterbalancing "younger brother" enzyme known as ace2. 56,57 ace2 converts aii into angiotensin 1-7 (a1-7), so named because it possesses only seven of aii's original eight amino acids. a1-7 binds to mas receptors, yielding vasodilatory, anti-inflammatory, and antifibrotic effects. 27, 32, 33 in sum, ace2 depletes aii levels by converting it to a1-7, which binds to mas receptors, thereby counteracting at1 actions. [56] [57] [58] through a side-pathway, ace2 can also convert ai into angiotensin 1-9 (a1-9), 55 which possesses nine of ai's original ten amino acids. interestingly, ace then converts a1-9 into a1-7; therefore, ace plays a role in producing aii, but also in decreasing aii levels by shunting substrates toward a1-7 instead of aii. rather than being a linear cascade of enzymatic reactions, ras is now understood as a web of counterbalancing activations and deactivations 26, 55 (figure 2 ). the original sars coronavirus (sars-cov or sars), which caused severe acute respiratory syndrome, (also called sars) hitchhiked inside cells using cell-surface ace2. 56 the new, closely related coronavirus sars-cov-2 or sars2 (which is responsible for covid-19) uses the same method. 59 once inside the host cell, original sars virus dramatically reduces cellular expression of ace2. 60 sars2 virus evidently functions the same way. 61 because ace2 is critical for regulating the balance between the effects of aii and a1-7, 62 reduced ace2 expression leaves the "big brother" enzyme ace, unchecked, enabling unopposed oxidative, inflammatory and procoagulant effects 61 ( figure 3 ). in animal models and human cases of acute respiratory distress syndrome (ards), ace2 levels are severely decreased, 63 and ace/ace2 balance is lost. 57 overactive aii/at1 contributes to severe oxidative and inflammatory lung injury, 63 which in mouse models is blunted by ace2. 64 ace inhibitors (aceis) and arbs lessen pulmonary damage in mice injected with the original sars surface spike-protein, 65, 66 suggesting that unbalanced ace axis activity, after ace2 is downregulated, is the root of original sars lung pathophysiology. close assessment of covid-19 pathophysiology suggests the same mechanism. 61 as the following sections show, fingerprints of ras imbalance with ace/aii overactivation and diminished ace2/a1-7 are spread across the array of unexpected covid-19 organ system involvement. subpopulations manifesting higher rates of covid-19 mortality-including hypertensives, the elderly, the obese, diabetics, men, and african-americans-correlate with preexisting ras imbalance, with ace overactivity and/or ace2 underactivity priming these patients for more severe covid-19 outcomes. many hospitalized covid-19 patients reportedly display silent hypoxia: severe arterial hypoxemia without evident dyspnea, air hunger, or breathlessness. the neural reflex pathways mediating dyspnea have not been fully delineated, but likely involve integration among lung mechanosensory receptors, central and peripheral chemoreceptors, and emotion-regulation centers of the limbic system. 67, 68 the sensation of dyspnea has been reported to require intact chemosensory function. 67 the carotid bodies (neural crest-derived structures located at the carotid bifurcations), are the peripheral chemoreceptors, regulating the ventilatory response to hypoxia. 69 carotid body glomus cells scan their extensive blood supply for adequacy of oxygenation: 69, 70 if arterial partial pressure (pao 2 ) falls below approximately 60 mmhg, potassium background leak channels close, halting k + exit and depolarizing the glomus cells. 69 next, calcium influx through voltage-gated channels triggers the release of glomus cell neurotransmitters which initiate firing of action potentials by adjacent afferent terminals of the carotid sinus branch of cranial nerve ix. 69, 70 the action potentials travel first to the nucleus tractus solitarius (nts), and then to the prebotzinger complex in the medulla, augmenting respiratory drive as an attempt to correct the oxygen shortfall, 68, 69 ( figure 4 ) and simultaneously invoking the reflex pathways that mediate air hunger. the oxygen sensor which initiates glomus depolarization is still debated, but it may be ros-producing nadph oxidase, 69 which also happens to participate in at1 receptor signaling. whether or not at1 and nadph oxidase are involved in hypoxia sensing, ras is definitively activated during the hypoxic response. the carotid body is not only a driver of increased ventilatory effort; it is also a nexus for homeostasis regulation. 70 oxygen perturbations signify severe systemic stress with profound metabolic implications, requiring anti-stress responses including renal compensation to maintain ph, adjustment of cardiovascular functions, and autonomic and endocrine effectors, all of which ras integrates into the ventilatory response. 70 to mediate this integration, components of ras are expressed constitutively in glomus cells, including ace, ace2, at1, mas, at2. 71, 72 hypoxia increases expression of ace, angiotensinogen, at1 and at2. 72 hypoxia also increases sympathetic tone, which triggers renin release and elevates plasma aii levels. in turn, plasma aii has a stimulating effect on carotid body glomus cells, augmenting intracellular influx of calcium ions, and neurotransmitter release. 72 blood pressure and ventilatory responses crosstalk and reinforce each other. 72 sars2 coronavirus would be able to enter glomus cells utilizing membrane-bound ace2. the subsequent ace2 downregulation, amid hypoxemic pulmonary infection, would result in significant ace imbalance, 61 leading to carotid body hypoxic-signaling failure via two potential figure 2 the ras web. the ras system is now more appropriately conceptualized as a web of activations and counterbalancing deactivations, rather than its original depiction as a linear cascade. figure routes. first, aii overactivation could drive infected glomus cells into programmed cell death. this would be consistent with experimental aii/at1 apoptosis induction in rat and human alveolar epithelial cells. [73] [74] [75] alternatively, glomus failure could be akin to cellular dysfunction in heart failure, in which excessive aii triggers cell volume overload and impairment of cardiac electrical function. 76 although aii increases cell volume, a1-7 decreases it. 33 thus, with loss of both ace2 and a1-7, aii overactivity would be expected to volume overload glomus cells, impairing depolarization, and blocking neurotransmitter release. either scenario, or both acting synergistically, would result in failure of hypoxic signaling, with an absent dyspnea response despite severe hypoxemia in covid-19 patients. incidentally, those same two scenarios could also be at work in loss of olfaction and taste, both of these neural in contrast, ace2 expression is more restricted in the lungs, largely to type ii pneumocytes, 61 although there is evidence of some expression in pulmonary capillary endothelium. 240 ace2 deactivates aii, by converting it to a1-7, thereby blunting the pro-inflammatory, pro-fibrotic effects of the ace axis. in covid-19, novel coronavirus sars-cov-2 can enter type ii pneumocytes utilizing ace2 and then downregulate ace2 expression, 61 leaving the lungs vulnerable to damaging, unbalanced proinflammatory effects of ace/aii. 61 type ii pneumocytes have multiple protective functions, including producing surfactant, and serving as stem cells to replenish damaged type i pneumocytes (which function in gas exchange). 61 viral impairment of type ii pneumocytes, coupled with aii inflammatory damage to type i pneumocytes, would thus severely damage multiple aspects of alveolar function in covid-19, including oxygen exchange and capacity for repair. figure structures requiring depolarization and neurotransmitter release to mediate their sensory functions. both express tissue ras components. 77, 78 "atypical" ards: ras "mismatched" but why are covid-19 patients so severely hypoxemic in the first place? critical care anesthesiologists in the thick of the covid-19 crisis in italy suggested that severe covid-19 patients may not have classical ards. 79 unlike the situation in ards, lung compliance is preserved in covid-19. instead, covid-19 patients seemingly displayed catastrophic loss of hypoxic pulmonary vasoconstriction (hpv), with resulting ventilation-perfusion (v/q) mismatch hypoxemia. 79 in ards, lung compliance is poor because of decreased alveolar elasticity from noncardiogenic pulmonary edema. 80, 81 this decreased alveolar elasticity impairs gas entry into alveoli, lowering the alveolar partial pressure (pao 2 ), the top end of the gradient which drives the transfer of oxygen into pulmonary blood. oxygenation in ards patients often improves with ventilation using low tidal volumes and high positive end-expiratory pressure (peep) which increases pao 2 . 80,81 but severe covid-19 patients have not shown a similar benefit with this ventilation strategy. 82 even under normal physiologic conditions, pao 2 is not equal in all alveoli throughout the lungs. 83 because of postural differences in fluid hydrostatic pressures across the lung fields, alveolar expansion is not uniform and some alveoli inevitably have higher pao 2 than others. hpv is a protective physiologic mechanism that optimizes arterial oxygenation by regionally vasoconstricting within the pulmonary vascular bed, thereby diverting pulmonary blood flow away from alveoli with lower pao 2 , and vasodilating in other regions to increase blood flow to alveoli with higher pao 2 . 84 ( figure 5 ) optimal v/q figure 4 cellular mechanism of glomus cell hypoxic signaling. glomus cells, located within the carotid body, are neural crest-derived, and they exhibit voltage-dependent neurotransmitter release upon detection of hypoxemia. 69, 70 glomus cells detect hypoxemia by an as-yet undefined sensor, which is thought to close k+ channels, halting k+ exit and thereby depolarizing the glomus cell. 69, 70, 241, 242 depolarization leads to opening of voltage-gated ca2+ channels and neurotransmitter release, which triggers action potential firing in nearby cranial nerve ix. 69 matching yields a pao 2 of approximately 100 mm hg, when breathing air at atmospheric pressure. however, if the hpv response is impaired, blood flows through the lungs in an unregulated fashion, and poorly oxygenated blood with low pao 2 , enters the systemic circulation 85 ( figure 5 ). ras plays a role in hpv that is still not precisely defined but may involve hypoxic activation of nadph oxidase, generating ros that close potassium channels, depolarizing pulmonary vascular smooth muscle, which in turn opens voltage-gated l-type calcium channels, activating the smooth muscle contractile apparatus. 84, 86 mice lacking cytosolic nadph oxidase had a 25% decrease in initial phase hpv. 87 aii has been shown to activate pulmonary vasoconstriction in hypoxic humans, 88 and aceis and arbs have been shown to inhibit hpv. 89, 90 recombinant ace2 blunts pulmonary vasoconstriction in hypoxic pigs. 91 therefore, the loss of ace2 due to sars2 coronavirus together with the resultant ace imbalance 61 would be expected to dysregulate hpv and lead to hypoxemia. mortality rates in ards hover around 45%. 75 by contrast, a covid-19 case series of 5700 patients in and around new york city reported mortality rates of 88.1% in ventilated patients, essentially doubling the ards rate, 92 and providing further support that covid-19 is not traditional ards. 82 lung tissue locally expresses almost all components of ras. 75 ace is heavily expressed across the entire alveolar capillary network, and the lungs are the dominant site of circulating aii production. 75 in contrast, ace's antiinflammatory counterpart, ace2 is more restricted within the lungs, principally to alveolar type ii pneumocytes 75 ( figure 3 ). in addition, in healthy adults, pulmonary expression of anti-inflammatory at2 is low. 75 with such broad expression of the ace axis, and comparatively restricted expression of the protective arms of ras, the lung is poised for more severe ace imbalance in the event of ace2 downregulation by the sars2 virus. without ace2, ace actions would be essentially unchecked, 61 with no means to achieve appropriate v/q matching, amidst significant inflammatory destruction of lung tissue. 75 in animal models, mechanical ventilation has been shown to upregulate ace, promoting activation of aii. [93] [94] [95] it may be the case for many severe covid-19 patients that initiation of mechanical ventilation, and consequent further ace axis upregulation, becomes the final straw, especially when combined with anesthetic/sedating drugs that may further impair hpv. emerging data show a mixed picture of covid-19 effects on the heart and kidneys. preexisting cardiovascular risk factors are definitively associated with increased covid-19 severity. in an italian retrospective case series, 49% of 1591 patients admitted to the icu for covid-19 had preexisting hypertension, and 21% had cardiovascular disease. 96 among a retrospective cohort of 191 covid-19 cases hospitalized in wuhan, china, 30% had hypertension, and 8% had coronary heart disease. 97 hypertension and atherosclerosis have well-established associations with ace axis dysregulation. [98] [99] [100] [101] [102] moreover, ace2 levels in the heart are reported to be diminished in hypertension and in diabetes-associated cardiovascular disease. 103 thus, it makes sense that these comorbidities would be heavily represented among more severe covid-19 presentations. studies suggest that myocardial injury worsens covid-19 prognosis; 104 nevertheless, reported rates of cardiac as well as renal injury are lower than rates of pulmonary damage, suggesting that the heart and kidneys may possess some protection. for example, the wuhan cohort cited that among fatal cases, 93% had an ards diagnosis, but only 59% had a myocardial infarction (mi), and 50% had acute kidney injury (aki) prior to death. 97 among survivors, 7% had ards while only 1% had mi or aki. another study reported myocardial injury in 7% to 17% of hospitalized covid-19 patients, and 22% to 31% of icu cases. 105 in contrast, the italian icu cohort reported 88% requiring intubation and mechanical ventilation, indicative of dire pulmonary compromise. 96 kidneys locally express all ace axis components. 29, 106 additionally, the renal glomeruli filter most circulating ras proteins, which are then reabsorbed by the proximal tubule. 106 combined resorption and local production result in renal aii levels 100 to 1000 times higher than in plasma. 29, 106 dysregulated tissue ras, as occurs in hypertension, results in significant renal inflammatory damage, with progressive destruction of nephrons amid rasmediated remodeling and fibrosis. 49 similarly, the heart has dovepress significant local ras, which mediates inflammatory remodeling in conditions such as hypertension and infarction. 107 however, the counterbalancing anti-inflammatory at2 and ace2 arms of ras also have an important role in the heart and kidneys. ace2 is highly expressed in the heart, 62 in particular in the coronary arteries, 32 and in endothelial cells within the kidneys, 32 achieving significant production of a1-7. 27 therefore, with higher baseline ace2 levels, the heart and kidney may have a degree of shielding against the effects of ace2 loss perpetrated by the sars2 virus. in addition, cardiac and renal tissue ras often bypass ace, instead using chymase, trypsin, kallikrein, and cathepsins. 32 thus, local ras function in these organs could potentially be less imbalanced by the loss of ace2 due to the sars2 virus. additionally, the coronary arteries 52 and kidneys express comparatively higher levels of at2 during adult life. 55 the heart also has a demonstrated capacity to upregulate at2 during pathologic states such as ischemia. 55 therefore, the effects of covid-19-induced imbalance of ras may be blunted somewhat by aii binding to at2. there are also reports of covid-19-triggered myocarditis. 108 in one study, myocarditis was deemed likely causative in 7% of covid-19 deaths. 105 at1 has been shown to promote myocarditis in experimental models, secondary to actions on t-cell function. 109 t-cells have an intrinsic ras that regulates their proliferation and migration. 109 at1 fosters cytoskeletal changes that promote t-cell migration and trigger t-cell production of cytokines and chemokines, thereby increasing t-cell recruitment to regions of active inflammation. 109 by inhibiting at1, arbs decrease experimental myocarditis. 110 ras-driven t-cell-mediated effects in the heart, coupled with t-cell-mediated skin effects, which are also well described, 111, 112 could potentially explain the pediatric kawasaki-like condition linked to sars2. 16 besides ischemic and inflammatory tissue destruction, another aspect of cardiac pathology in covid-19 is the development of arrhythmias and sudden cardiac death. 113, 114 in addition to the arrhythmia-promoting effects of hypoxemia, arrhythmias in covid-19 patients bear hallmarks of ras dysregulation. cardiac remodeling by the ace axis is a known driver of arrhythmia events. in the atria, aii promotes fibrosis that facilitates reentry, 115 and aceis and arbs do provide some protection against the recurrence of atrial fibrillation after cardioversion in figure 5 hypoxic pulmonary vasoconstriction (hpv) and ventilation/perfusion (vq) matching. alveolar partial pressure of oxygen (pao 2 ) is the top end of an oxygen gradient, driving transfer of oxygen from the alveolar air spaces into the pulmonary blood. thick red arrows designate a sizable gradient driving oxygen transfer. thin blue arrows designate smaller oxygen gradients, with less potent driving force for oxygen transfer between alveolar air spaces and pulmonary blood. functional hpv regionally vasoconstricts (designated in the figure by small blood vessel tube) within the lungs, to divert pulmonary blood flow away from poorly oxygenated alveoli, which would have a more limited ability to transfer oxygen into the pulmonary blood. such appropriate vq matching ensures that adequately oxygenated blood, with normal arterial partial pressure of oxygen (pao2), is returned to the systemic circulation. when hpv fails, pulmonary flow is instead sent in excess to poorly oxygenated alveoli, such that larger volumes of poorly oxygenated blood, with low pao 2 , are returned to the heart, to then enter the systemic circulation. figure patients with left ventricular dysfunction. 48 aii promotes ventricular arrhythmias by prolonging qrs duration, and by promoting reentry through both fibrosis and slowed ventricular conductance. 48 these factors are likely playing out in covid-19 pathology, perhaps synergizing with potential arrhythmia-promoting effects of experimental drug treatments, including hydroxychloroquine, 114 which was studied earlier in the pandemic but the use of which has now been curtailed. during the initial united states covid-19 case escalation, stroke was reported in patients without traditional stroke risk factors. 12, 116 although unanticipated in the context of a viral respiratory tract infection, stroke risk is known to be elevated in conditions of ras overactivation such as diabetes and hypertension. 117,118 ras blockade with aceis and arbs has been shown to reduce stroke rates in high-risk populations. 55 with the downregulation of ace2, the coagulation-promoting effects of the ace axis would drive arterial thrombosis. healthy vascular endothelium promotes vasodilation and inhibits coagulation and inflammation, 42,43 through expression of natural anticoagulants, including tissue factor pathway inhibitor (tfpi), thrombomodulin (tm), tissue plasminogen activator (tpa), and the anti-inflammatory vasodilator no. 42 produced in endothelial cells by nitric oxide synthase (nos), 43 no diffuses to neighboring vascular smooth muscle to activate guanylate cyclase/cyclic gmp/protein kinase g-mediated vasodilation 43 (figure 1 ). no also has myriad anti-inflammatory and anti-coagulant functions, including inhibiting both platelet aggregation and monocyte adhesion. 119 during injury response, aii inhibits endotheliummediated vasodilation and anti-coagulation. 42 (figure 1 ). at1 also promotes increased expression of adhesion molecules such as vcam-1 and e-selectin that recruit inflammatory cells to the injury site, and inflammation synergistically promotes coagulation. 119,120 aii prompts endothelial cells to promote coagulation, by new expression of procoagulants-tissue factor (tf) to initiate the blood clotting cascade, 121, 122 von willebrand factor (vwf) to enhance platelet adhesion, 123 and plasminogen activator inhibitor (pai)-1 to blunt fibrinolytic degradation of blood clots. 37, 42, 43, 119 activation of these processes would then be the drivers of stroke in covid-19-induced ras imbalance, with overactive aii sabotaging the anti-coagulant actions of otherwise healthy endothelium. obesity and diabetes are reported to worsen covid-19 severity and mortality. in the case series of 5700 hospitalized covid-19 patients, 33.8% had diabetes as a preexisting comorbidity, and 41.7% were obese. 92 in a meta-analysis of 30 published studies that assessed the outcomes among more than 6400 patients, diabetes was found to more than double both the risk of severe covid-19 disease and the risk of mortality. 124 diabetes more than quadrupled the risk of severe ards-like pulmonary outcomes in covid-19. 124 a study in new york city reported that patients with body mass index (bmi) equal to or above 35 had more than triple the risk of in-hospital mortality from covid-19, compared to leaner patients [or: 3.78; 95% ci 1.45-9.83, p=0.006]. 125 multiple factors may be at work in these outcomes. immune function has been shown to be impaired by nutritional deficiencies in obesity, [126] [127] [128] and by poor glycemic control in diabetes. [129] [130] [131] obesity-compromised lung mechanics have been well described. [132] [133] [134] however, both obesity and diabetes also bear the hallmarks of ras dysregulation at baseline. in the developed world, nearly 90% of diabetes cases are the result of obesity, 135 and the two conditions are mechanistically intertwined via ras overactivation, 119,135-138 which primes for worse covid-19 outcomes. adipocytes locally produce a full set of ras components, including angiotensinogen, ace, aii, at1. 139 in obesity, this adipose ace axis is highly upregulated, 140 but with diminished counterbalancing ace2, 61 this ace imbalance negatively impacts insulin function. 140 the insulin receptor has two principal second messenger cascades. 135 the first, phosphatidyl inositol-3 kinase (pi3k) promotes cell membrane localization of glut transporters that facilitate glucose uptake. 139 pi3k also activates nos, which manufactures no to mediate the anti-inflammatory, anti-coagulant effects of insulin. 139 the second cascade, utilizing map kinase, is responsible for the growth-factor-like actions of insulin, including fibrosis, vascular smooth muscle proliferation, and cardiac myocyte hypertrophy. 136, 139 under normal circumstances, pi3k-produced no inhibits the map kinase arm. 139 in ras upregulation, however, superoxide produced by at1 eliminates no, thus shortcircuiting the pi3k function of the insulin receptor, so that glucose uptake fails, hence the connection between obesity and diabetes. 141 this mechanism would also explain the correlation between covid-19 and hyperglycemia. with ace axis overactivity, insulin receptor function is shunted to map kinase, 139 and as a result of this alteration, insulin receptors and at1 receptors-which also use map kinase as a second messenger-synergize to promote inflammatory and hypertrophic damage to the cardiovascular system which is a hallmark of diabetes. blocking ras overactivation with aceis or arbs helps restore normal insulin receptor function, improves insulin-mediated glucose uptake, and mitigates vascular damage. 139 as data accumulate showing elevated covid-19 mortality rates in african-americans, attention has focused on socioeconomic inequalities, including limited access to nutritious food, overcrowded housing conditions, and consequent health-related comorbidities, such as obesity, diabetes, and hypertension. these clearly evident factors undoubtedly play important roles, but less visible factors may also be at work. there is evidence of ras dysregulation that drives hypertension disparities in african-americans. this could also be contributing to the increased severity of covid-19 among this population. it has been firmly established that african-americans have higher prevalence, earlier onset, and more severe complications of hypertension than caucasians. 142 recent data from the us department of health and human services office of minority health attest that african-americans currently have higher rates of obesity than caucasians. 143 obesity greater than 20% above ideal body weight doublesto-triples hypertension risk, 144 and obesity significantly increases ace activity. 145 yet obesity alone cannot explain the unequal prevalence of hypertension. according to national health and nutrition examination survey (nhanes) data from 1988 to 1994, african-american and caucasian males had almost identical obesity rates during that time frame, but african-americans had 46% higher rates of hypertension. 146 research has identified critical physiologic distinctions among many african-americans that influence response to hypertension therapies. most african-american hypertensives have low-renin hypertension secondary to either a mutation of aldosterone synthetase (and consequent aldosterone overproduction) or of the epithelial sodium (enac) channels in the kidney, both of which lead to excessive renal sodium resorption. 147 although sodium retention inhibits renin release into the circulation, reduced activation of the circulating ras does not prevent hypertension. however, in mouse experiments, renin gene deletion solely within the kidneys did confer protection against developing aii-mediated hypertension, suggesting that tissue ras is more critical than circulating ras for blood pressure regulation-and for the development of hypertensive nephropathy. 106 in chronic kidney disease, renal tissue ras was found to be elevated, independent of circulating ras levels. 146 african-american hypertensives have increased incidence of ras-mediated organ damage, congruent with overactive tissue ras despite low circulating renin. 142 in keeping with this, african-americans taking aceis and arbs have comparatively poor blood pressure decrease, yet have significant protection against hypertensive end-organ damage. 146 several gene polymorphisms have been discovered to increase ras activity. for example, increased hypertension prevalence among african-americans has been linked to the −535t polymorphism of at1. 144 best studied, however, is the ace gene i/d polymorphism, distinguished by insertion (i) or deletion (d) of a nearly 300 base pair dna segment. 35 the dd genotype doubles ace activity, in both circulation and tissue, compared to the ii homozygous genotype; heterozygous ace id is intermediate between the two. 35 in a population-based study, the ace d polymorphism was slightly more common with african ancestry than with european or japanese ancestry. 148 african-american hypertensives in particular have been shown to have higher prevalence of the d allele. 149,150` it is important to stress, however, that although those of african descent may be more likely to carry the d allele, d is found among many ethnicities. for instance, it was found to occur at increased frequency among caucasian australians who suffered myocardial infarction compared to the general healthy population. 151 when ace d polymorphism occurs, it has often been linked to adverse medical conditions, including increased susceptibility to ards. 75 in children, the d allele was associated with worse outcomes in focal glomerulosclerosis 152 and single ventricle congenital heart disease. 153 ace d allele correlates with ventricular hypertrophy in endurance athletes. 35 additionally, the rotterdam study showed an association of d allele with early mortality. 35 despite current potentially negative consequences, ethnobiological studies have hypothesized an evolutionary survival benefit during ancient times for ras upregulation polymorphisms such as ace d allele. in tropical areas with sodium scarcity, these polymorphisms promoted sodium and water retention, and maintenance of blood pressure. 109, 154 however, because of the current abundance of dietary sodium, modern descendants face increased damaging consequences from those same polymorphisms. 109, 154 other ethnicities with reportedly increased covid-19 mortality, including latinos and south asians, are less well represented in the hypertension literature than african-americans. there is, nonetheless, some evidence for ras upregulation. low-renin hypertension is common in caribbean hispanics. 146 the ace-upregulating d allele has also been found with increased frequency among south indians with hypertension. 155 these observations suggest again that ras upregulation portends the worse covid-19 outcomes seen in some populations. in a retrospective case series of 1591 consecutive patients requiring icu level care for covid-19 in milan, italy, 82% were male. 96 researchers from beijing retrospectively analyzed mortality data from the earliest 500-plus reported cases of covid-19 in wuhan, china. 156 they reported that, while men and women had similar covid-19 prevalence, men were nearly 2.5 times more likely to die of covid-19. 156 simultaneous retrospective re-analysis of mortality data collected in 2003 showed that the original sars coronavirus also skewed toward higher mortality in men, 156 in keeping with prior published reports. 157 although gender-specific differences in immune response 158 may play a role in unequal outcomes of both sars and covid-19, there are definitive differences between the genders in the ras system and, in keeping with occam's razor, these differences likely contribute to worse male outcomes. sex hormones regulate the expression and function of ras components. 159 males generally have higher levels of ras than premenopausal females, 160 perhaps explaining why male hypertensive rats show a greater blood pressure decrease with aceis. 161 estrogen downregulates the expression of the at1 gene 162, 163 and suppresses both ros production in vascular smooth muscle and the enzymatic activity of ace. 159 estrogen also promotes the production of no, the counterweight to aii in the endothelium. 159 estrogen also positively impacts the protective arms of ras, including increasing ace2 levels in rats. 160 female mice display higher renal at2 expression than male counterparts, an effect that dissipates if ovaries are removed, 55 and estrogen upregulates at2 levels in the mouse kidney. 55 both the at2 and ace2 genes are found on the x chromosome, 164, 165 so it is reasonable to conclude that women would regulate and express these genes differently than men. in keeping with this, women have been reported to have higher levels of protective a1-7 than men, both in plasma 166 and in urine, 167 suggesting higher levels of ace2 activity both in the circulation and at the renal tissue level. these factors likely synergize to confer comparative covid-19 protection in females, especially pre-menopausally. when assessing the elevated death rates among cancer patients who develop covid-19, 168 immune impairment evoked by therapies and nutritional depletion cannot be ignored as contributory factors. however, ras dysregulation has also been shown to be associated with cancer. 169, 170 increased ace and at1 expression have been reported in patients with metastatic ovarian cancer and prostate cancer, compared to those with benign neoplasms. 171 at1 is also increased in squamous cell carcinoma, and in 20% of estrogen-receptor-positive breast cancers. 170 the cellular growth-promoting aspects of ras are brought into play by tumor cells to facilitate tumor expansion and invasion. aii promotes differentiation of tumorassociated macrophages (tams) from immature progenitors. 170 tams are abundant in the stroma surrounding the tumor, and they release factors that modulate tumor progression and metastatic migration. 170 tams also inhibit the body's anti-tumor immune mechanisms. 170 tams express at1, whose effect profile is proangiogenic, augmenting levels dovepress of vascular endothelial growth factor (vegf), and vegf type 2 receptor. 171 at1 facilitates epithelial-to-mesenchymal transition, which is crucial to metastatic progression. 170 knockout mice lacking the murine version of at1 have decreased tumor growth compared to normal counterparts. arbs blunt tumor size in animal models, 171 and they also decrease vegf levels, metastatic burden, and tumor vascularization. 170 similarly, aceis have been reported to inhibit tumor growth, angiogenesis, and metastasis. 170 among gastric cancer patients taking either aceis or arbs, prolonged survival has been noted. 169 also, fiveyear recurrence-free survival was significantly higher among patients with non-invasive bladder cancer who were receiving ras-blocking drugs. 169 a small retrospective study of advanced non-small-cell lung cancer estimated a 3-month prolongation in survival with ras blockers. 169 human studies of ras blockade to limit cancer progression may be confounded by the invisible effects of ras polymorphisms, which are only rarely assessed in trial subjects. 169 in the prospective rotterdam study, however, ace genotyping was performed and revealed that the high ace-activity dd genotype had the most significant benefit in cancer risk reduction with ras blockade. 169 the ace dd genotype has been associated with a higher risk of cancer, while the ace ii homozygote pools with a lower risk. 169, 170 these findings are congruent with ras overactivation portending poorer covid-19 prognosis. from the outset of the covid-19 epidemic, elderly patients have accounted heavily among fatalities. a retrospective comparison of covid-19 data from italy and china revealed a large increase in case fatality rates with increasing age in both population groups. 172 the italian case fatality rate was 1% in patients aged 50-59, 3.5% in those aged 60-69, 12.8% in those aged 70-79, and 20.2% in persons above 80 years of age. 172 among the chinese patients, case fatality rate was 1.3% in those 50-59, 3.6% in the 60-69 age group, 8% in those 70-79, and 14.8% above age 80. 173 elderly people often have nutritional deficiencies that can impair immune function. 174 increasing age is also associated with impairment of mucociliary clearance, which contributes to increased susceptibility to respiratory infections in the elderly, 175 because mucociliary clearance is the primary barrier defense mechanism of the lungs. 176 the elderly account for a higher percentage of patients having the various co-morbidities associated with poor covid-19 prognosis, such as hypertension, diabetes, cardiovascular disease, and cancer. however, even healthy aging also leads to ras imbalance, with elevated tissue ras components, and lower levels of counterbalancing protective arms of ras. 58 it is well described that circulating aii levels typically decline in the elderly. 55, 177 importantly, however, tissue levels of aii, ace, at1, and angiotensinogen increase with aging in the heart, kidney, and vasculature. 55, 177 data from african-american hypertensives, including those treated with aceis and arbs, revealed that tissue ras has a more significant deleterious effect on organ function than circulating ras. 142, 146 it is reasonable to expect that tissue ras levels, rather than circulating levels, would also be the more important variable in elderly individuals, coinciding with the higher incidence of the ras dysregulationassociated comorbidities in aged adults, such as hypertension and diabetes, which portend worse covid-19 outcomes. at the same time, there is evidence of lower levels of the protective counterbalancing arms of ras, 178 leaving the elderly comparatively more vulnerable to the damaging effects of the ace axis. animal experiments showed that the aortic vasodilatory response to a1-7 is lost in elderly female rats, but restored by exogenous estrogen replacement. 178 in rat lungs of both genders, ace2 levels have been found to decrease with aging 61, 179, 180 this loss of ace2 was more pronounced in elderly male rats than in similarly aged females, 61, 179, 180 which is in keeping with the worse prognosis reported for men compared to women with covid19. it has been posited that patients with baseline ace2 deficiency would be particularly vulnerable to severe outcomes because of unchecked ace activity with sars2 virus downregulating ace2 expression. 61 in elderly covid-19 patients, the higher basal tissue ace axis components and lower counterweight ace2 levels even at the start of infection would disadvantage these patients for poor outcome. the covid-19 outbreak in the west brought unexpectedly severe cases affecting younger adults. the explanation for this situation remains undefined and is probably multifactorial. lifestyle factors may impair immune defense against covid-19 in younger people. for instance, dehydration which may occur during strenuous exercise, impairs mucociliary clearance, 181 as does aerobic exercise in polluted urban environments. 182 rising obesity rates may play a role, as well as genetic factors, including high ras activity polymorphisms in the general population. [183] [184] [185] acquired, non-genetic ras dysregulation may also be playing a role here, due to the resurgence of inhaled nicotine and its effects upon ras expression. data reported on 191 patients with confirmed covid-19 from two hospitals in china showed that the odds ratio of dying among current smokers, compared to nonsmokers, was 2.23 (95% ci: 0.65). 97 the impact of e-cigarettes ("vaping") has not yet been reported in the context of covid-19. but experimental evidence has been accumulating, showing that e-cigarettes may be even more deleterious than traditional cigarettes, so it is reasonable to suppose that vaping will also influence covid-19 outcomes. smoking is known to inhibit mucociliary clearance, 186 an essential defense feature against airway pathogens. recent experimental evidence suggests that high nicotine levels in vaping may be even more harmful to this critical defense against inhaled pathogens. 181, 187 in a sheep model, high-nicotine-containing e-cigarettes caused a 50% reduction in experimentally modelled mucociliary clearance. 188 moreover, nicotine also adversely impacts ras levels, which would be deleterious during covid-19 infection. nicotine has been shown to decrease levels of mas receptor and a1-7, while increasing ace, aii, and at1. 189 additionally, nicotine decreased both ace2 and at2 expression in several cell types, including cardiac fibroblasts. 190 the rising use of e-cigarette products among young americans has alarmed the medical community and public health officials. cdc data showed that 3.7% of us adults -more than 9 million people-used e-cigarettes in 2014. 191 a 2018 gallup poll reported that vaping had increased to more than 9% of us adults; in addition, 20% of respondents reported smoking traditional cigarettes. 192 the national youth tobacco survey revealed that 5 million teenagers vaped in 2019, up from 3.6 million in 2018. 193 this constitutes a sizable portion of the younger us population who would be at increased risk during the current pandemic, given the evidence that baseline ace2 deficiency predisposes to poorer covid-19 outcomes. 61 with covid-19 deaths now surpassing 500,000 worldwide, and further increases becoming evident in some areas as economies reopen-let alone the possibility of a second wave of infections surging late in the yeardeveloping a therapeutic means to mitigate the severity of covid-19 has taken on utmost urgency. with the evidence of ace axis overactivation at the root of varied organ system pathology in covid-19, the most obvious recourse to attempt to blunt covid-19 progression and mortality would seem to be aceis and arbs. these drugs are widely available, inexpensive, and they target the very system that is unbalanced in covid-19. yet the use of these drugs in covid-19 is not clear cut. early in the covid-19 pandemic, concerns were raised that aceis and arbs might predispose to increased covid-19 severity. 194 in covid-19, the role of ace2 appears paradoxical. 61 ace2 facilitates entry of sars2 virus into cells, but at the same time, ace2 expression in alveolar cells protects against experimentally induced lung injury. 61, 64, 66 after viral entry into alveolar cells, the spike protein of the sars-cov-2 virus downregulates further expression of ace2, 61 thereby reducing the protective effect of ace2 on lung tissue. 60 in animal models, aceis and arbs elevate the expression of ace2 in the heart. 195, 196 during fetal kidney development, at1 exerts negative feedback to downregulate ace2 levels. 197 it has been suggested that aceis and arbs upregulate ace2 levels in cardiac and diabetic patients, 179, 198 and therefore could potentially accelerate sars2 entry. 61, 198 yet these drug classes have shown clear cardiovascular and renal protective effects, 199, 200 which might prove beneficial in blunting covid-19 organ damage. 201 in the midst of this "double-edged sword" debate, 194 the european society of cardiology and the american college of cardiology recommended that, without clear clinical evidence, already-prescribed aceis and arbs should not be discontinued. a case series of 1178 patients from wuhan, china found that aceis and arbs were not associated with increased covid-19 case severity or mortality. 202 an italian case-control study comparing records of 6272 covid-19 positive patients to more than 30,000 registered recipients of lombardy's regional health service came to the same conclusion. 203 an observational study of medicare advantage health records, conducted by yale university in partnership with united health group insurance corporation, announced that aceis-but not arbs-might even be protective against more severe covid-19 outcomes, since acei use correlated with a lower rate of covid-related hospitalization in persons over 65 years of age in this study. 204 multiple ongoing prospective trials are attempting to assess whether differences in covid-19 outcomes result from maintaining versus stopping aceis and arbs (eg, study nct04330300 in the who international clinical trials registry platform). readers may access the database of international clinical studies at http://www.who.int.ictrp). independent of a potential impact on virus entry into cells, these two drug classes have been shown in human studies and animal models to lower the risk and severity of numerous problems that covid-19 precipitates. among their many benefits, aceis are lung protective in ards, 75 and aceis and arbs reduce stroke rates. 55 arbs reduce cardiovascular oxidative stress, reverse endothelial dysfunction, and decrease inflammatory and oxidative renal damage in patients with diseases associated with ras dysregulation, such as hypertension and diabetes. 205, 206 arbs have mitigated the severity of experimental myocarditis 110 and have been shown to upregulate at2 in vascular endothelial cells. 52 moreover, at1 blockade may shunt existing aii to activate at2, yielding antiinflammatory effects. 205, 206 therefore, aceis and arbs might hold the potential to mitigate covid-19 organ damage. but clarity may come only once the drugs have been assessed as individual agents, rather than as aceis and arbs en masse. although there are definitive class effects of aceis and arbs, such as blood pressure lowering, 207 the drugs themselves each have unique pharmacological characteristics. evaluating them en masse may wash out otherwise important distinctions. for example, not all aceis have equivalent tissue penetration, due to differences in lipophilicity. 24 in a rat model, fosinopril had better cardiac entry than captopril, lisinopril, and ramipril. 24 similarly, quinapril had higher tissue penetration and better prevention of left ventricular hypertrophy than enalapril. 24 in spontaneously hypertensive rats, moexipril more potently inhibited lung, aortic, and cardiac ace than enalapril. 208 and among arbs, the unique structure of eprosartan seems to confer high renal affinity. 209 in addition, some aceis, such as captopril and perindopril, cross the blood brain barrier (bbb); others, such as enalapril and imidapril, do not. 109 this distinction could potentially hold importance, given that neurons express ace2, 210 and some severe covid-19 patients have manifested cns dysfunction, including encephalitis and a neurocognitive, dementia-like syndrome. 211 parenthetically, these reports are consistent with accumulating evidence that ras is also involved in the amyloid pathology of alzheimer's dementia. 212 among the arbs, only telmisartan has been shown to cross the bbb, 185 thereby reinforcing the importance of carefully evaluating arbs individually, rather than as whole drug classes, in covid-19 studies. there may be potential pitfalls deploying aceis and arbs against covid-19. arbs have two broad categories of action: irbesartan, candesartan, and telmisartan are noncompetitive inhibitors of at1, while losartan, valsartan, and eprosartan are competitive blockers. 209, 213 high concentrations of aii have been shown to overcome competitive blockade in vitro, 213 possibly negatively impacting the therapeutic potential of competitive at1 blockers in covid-19. additionally, many aceis are prodrugs, dependent on hepatic metabolism for activation, but there is evidence that, in some cases, the prodrugs may inhibit the function of the activated counterpart. 207 lisinopril, by contrast to other class members, is orally formulated as the active drug. 214 in the arb class, losartan and candesartan are prodrugs, but activated candesartan has 10 times higher affinity for at1 than activated losartan. 213 such pharmacological differences may impact the outcome of these therapies in covid-19. at least some aceis are dialyzable, but arbs are not, 209 and this may introduce additional difficulties using these agents in covid-19 patients with renal failure. because ras blockade inhibits hpv, 89,90 it is not known whether aceis and arbs would worsen hypoxia in covid-19 patients, or alternatively, push v/q matching back toward normal. because aceis, but not arbs, impact the metabolism of the vasodilator bradykinin (bk), the distinction between the two drug classes has the potential to differentially impact the course of treatment in covid-19 patients. bradykinin (bk) is the major vasoactive peptide in the kallikrein-kinin system, [215] [216] [217] interfacing with two principal receptors: 218,219 constitutively expressed b2, and inflammation-inducible b1. 218 in the lungs, bk receptors are situated on the endothelial cells of alveolar capillaries, with b1 upregulated in patients with covid-19. 220 to inactive fragments; thus, aceis may prolong the action of bk. [221] [222] [223] [224] [225] bk dysregulation can cause angioedema. [226] [227] [228] b2 receptors inhibit the function of adherens junctions between vascular endothelial cells, leading to plasma fluid extravasation into the interstitium 229 (figure 1) . clinical data indicate that this occurs as a side effect in about one or two patients per 1000 people taking an acei, 227,228 but the reason why only such a small percentage of patients are affected remains unknown. [230] [231] [232] the concept that angioedema-like changes occur in the lungs of patients with covid-19 is supported by both indirect (radiologic and clinical observation) evidence 233, 234 and by more direct evidence at autopsies. [234] [235] [236] 237 other than producing aii and inactivating bradykinin, ace also contributes to the production of a1-7, the counterbalance to aii. so, the interruption of these additional effects by aceis may potentially impact outcomes in covid-19 in ways that the arbs do not. as of this time, a small number of prospective clinical trials have begun enrolling patients to assess the potential of individual acei and arb drugs as a therapy to lessen covid-19 severity. among them, a study at the university of california san diego is evaluating the acei ramipril (nct04366050). trials at the university of kansas medical center (nct04335123) and the university of minnesota (nct04312009) are assessing the arb losartan, and telmisartan is being assessed at universidad de buenos aires (nct04355936). many other agents remain to be separately evaluated. aside from aceis and arbs to downregulate the ace axis, there are also potential agents to augment the activity of the protective arms of ras. recombinant ace2 has shown benefit in animal models of ards, 64 so it may prove beneficial in covid-19 treatment. apeiron biologics has announced that its recombinant ace2, called apn01, will enter phase ii trials in covid-19 patients. 238 in animal models, an at2 agonist called c21 has proven effective at countering damaging effects of ras. for example, c21 decreased collagen deposition fibrosis after mi and deceased arterial stiffness. 53 in addition, c21 blocked the rise in aii that accompanies a high sodium diet in obese zucker rats and blunted progression of glomerulosclerosis. 54 c21 also decreased renal at1 protein levels as measured by western blot. 54 a human trial of c21 in pulmonary fibrosis has been conducted in europe (the european union registry number of this trial is eudract 2017-004923-62). 75 this agent could potentially be studied in covid-19 patients. in animal studies, the vitamin d receptor inhibits ras activity, especially at the tissue level. 106 in human studies, vitamin d supplementation did not significantly lower blood pressure, although these studies were small and had few african-american participants, who are at elevated risk from covid-19 and who typically exhibit lower vitamin d levels than caucasians. 106 perhaps vitamin d could have a positive impact on covid-19 course if given to high-risk patients. no outcome in medicine is ever guaranteed, but the evidence of ras dysregulation in covid-19 supports attempting to mitigate covid-19 outcomes by modulating ras. such an approach would not be a shot in the dark, but rather would be guided by decades of experience modifying ras in other diseases. trials of rasmodulating therapies in covid-19 could be tailored to patients with high-ras profiles, and would likely need initiation of medication early in the symptomatic course, before the tipping point of severe organ damage has occurred. unfortunately, outpatient therapy opens doors to patient noncompliance, but enrollment of patients early in a hospitalization course may also be beneficial. despite potential pitfalls, the urgency to combat covid-19 requires tapping the full armamentarium of ras modifier agents for possible efficacy. a recent model predicts that even with continued social distancing, covid-19 fatalities could triple in the us alone by year's end. 239 human beings are now the ones with no time to lose. cha ae a mysterious blood-clotting complication is killing coronavirus patients belluck p 32 days on a ventilator: one covid patient's fight to breathe again virus is twice as deadly for black and latino people than whites in nyc reyes-velarde a younger blacks and latinos are dying of covid-19 at higher rates in california the coronavirus is killing men at twice the rate of women mcginley l patients with certain cancers are nearly three times as likely to die of covid-19, study says black people four times more likely to die of covid-19 in the uk compared to white people. cnn international but doctors 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peer-review system the authors would like to thank ms melinda judson and ms cassandra graybeal brown for assistance in digitizing the manuscript figures from the authors' hand-sketched originals. the authors report no conflicts of interest in this work. key: cord-286825-bu7j7kdr authors: macours, nathalie; poels, jeroen; hens, korneel; francis, carmen; huybrechts, roger title: structure, evolutionary conservation, and functions of angiotensinand endothelin-converting enzymes date: 2004-10-04 journal: int rev cytol doi: 10.1016/s0074-7696(04)39002-9 sha: doc_id: 286825 cord_uid: bu7j7kdr angiotensin-converting enzyme, a member of the m2 metalloprotease family, and endothelin-converting enzyme, a member of the m13 family, are key components in the regulation of blood pressure and electrolyte balance in mammals. from this point of view, they serve as important drug targets. recently, the involvement of these enzymes in the development of alzheimer's disease was discovered. the existence of homologs of these enzymes in invertebrates indicates that these enzyme systems are highly conserved during evolution. most invertebrates lack a closed circulatory system, which excludes the need for blood pressure regulators. therefore, these organisms represent excellent targets for gaining new insights and revealing additional physiological roles of these important enzymes. this chapter reviews the structural and functional aspects of ace and ece and will particularly focus on these enzyme homologues in invertebrates. of catalysis, classified into five major groups: serine proteases, cysteine proteases, aspartic proteases, metalloproteases, and proteases with an unknown mechanism (barret et al., 1998) . from these groups, the metalloproteases, with more than 30 families identified to date, are most diverse. in mammalian physiology, the high interest in metalloproteases originates from the fact that they are attractive targets for drug design. in insects, regulatory peptides are the major and most diverse class of signaling molecules. they act as transmitters and modulators in the nervous system and as hormones controlling key physiological processes, behavior, and development. peptidases are recognized as the key components of this peptidergic system, functioning in biosynthesis of active peptides from their precursors and in peptide inactivation. structural analysis of metalloproteases from invertebrate and vertebrate species indicates that these enzymes are very well conserved and arose early during evolution. the evolutionary conservation of universal peptidesignaling mechanisms has led to the general acceptance of the idea that invertebrates provide excellent models for the study of genetics and physiology. the information and knowledge gained by studying invertebrate peptidases will provide novel insights in the process of evolution and may reveal conserved functions of vertebrate peptidases that have remained unknown until now. angiotensin-converting enzyme (ace, dipeptidyl carboxypeptidase i [dcp i], peptidase p, carboxycathepsin, peptidyl-dipeptidase a, peptidyl-dipeptidase i, kininase ii, ec 3.4.15.1) was originally isolated in 1956 as a ''hypertensin-converting enzyme'' (skeggs et al., 1956) . classified as a member of the m2 gluzincin family, ace acts as a zinc-metalloprotease. the general reaction mechanism is identical for all zinc-metalloproteases. coordination of the zinc ion within the catalytic site of metalloproteases is typically avected by an hexxh motif, a third zinc ligand (e) located carboxyl to this motif, and a water molecule. the histidine residues within the active site motif are known to directly coordinate the active site zinc ion, whereas the hexxh glutamate has been shown to coordinate weakly to zinc via the activated molecule, thus facilitating the acid-base catalytic mechanism. the renin-angiotensin system (ras) plays a key role in the regulation of blood pressure and of fluid and electrolyte balance in mammals (inagami, 1994) . being part of the renin-angiotensin system, ace converts the decapeptide angiotensin i (angi) into the potent vasopressor angiotensin ii (angii). ace also inactivates the vasodilatory peptide bradykinin (bk) . hence, inhibition of ace results in lowering the blood pressure by preventing formation of the hypertensive angii and by inactivation of the hypotensive bk (fig. 1) . ace is one of the most studied mammalian peptidases, and in particular for medical applications. ace inhibitors are widely used in treatment of hypertension, diabetic nephropathy, and heart failure (dell'italia et al., 2002) . the production of captopril, the first clinically used ace inhibitor, represents a classical example of drug design (menard and patchett, 2001) . vertebrate ace is classified as a type i ecto-enzyme, with the major part of the protein, including the active site, located on the external surface of cells. ace is anchored in the plasma membrane by the c-terminal part of the protein. there is an n-terminal signal peptide present, required for the passage through the endoplasmatic reticulum. two distinct forms of ace are known: a somatic form (sace), which is found in many tissues, and a smaller iso-enzyme, germinal ace (gace, also called testicular ace [tace] ), which is found exclusively in testes, where it has a crucial role in fertility (fig. 2 ). the soluble, circulating form of the sace protein is produced by a ''secretase,'' probably a zinc-metalloprotease, which releases ace from the membrane by cleaving ov the extracellular part . the two forms diver in that the somatic isoform contains two active sites, whereas the germinal form contains only one. sace is composed of two very similar domains (c and n domain), which are both catalytically active (wei et al., 1991) . tace is, apart from its n-terminal sequence, identical to the c-domain of sace. the close similarity of the n and c domains of mammalian sace and the fact that exons 4-11 and 17-24 of the human ace gene, which code for the n and c domain, respectively, are very similar in size and have similar codon phases at exon-intron boundaries, strongly indicates that the coding gene arose from duplication of an ancestral ace gene coding for a single domain enzyme (cornell et al., 1995) . the ancestral copy is predicted to be the c-domain, equivalent to the germinal isoform (lattion et al., 1989 ). the fig. 2 schematic representation of human and drosophila ace protein and gene structure. exons are indicated by boxes. exons and corresponding amino acid regions in the protein are colored correspondingly. zinc-binding sites (hexxh) are indicated. human somatic and testicular promoters are indicated by arrows. one-kilobase intronic sequence was omitted at the // signs. two isoforms are transcribed from a single gene, under the control of two distinct promoters. sace is transcribed from a promoter region upstream of the duplication, and tace from a promoter within intron 12 (langford et al., 1991) . the two domains of sace have relatively broad substrate specificities and share some enzymatic features, but there are several biochemical properties that diverentiate the two active sites . for example, the hematopoietic stem cell regulator, n-acetyl-ser-asp-lys-pro (acsdkp), is an in vivo substrate for the n-domain, but not for the c domain of human ace (azizi et al., 1996) . the n and c domain can also be diverentiated by their response to chloride ions, with the c domain's specific activity being dependent on chloride ion concentration (wei et al., 1991) . their inhibitory aynity profiles also diver, illustrated by the existence of an n-domain-specific inhibitor (rxp 407) (dive et al., 1999) . ace acts as a peptidyl-dipeptidase, removing the c-terminal dipeptide from its substrate, but it can also act as an aminopeptidase and as an endopeptidase on peptides that are amidated at the c terminus. to date, only angiotensin i, bradykinin (yang, 1970) , and the haemoregulatory peptide ac-dskp (azizi et al., 2001) have been confirmed as in vivo substrates for mammalian ace. the conversion of angi into the vasoconstrictor angii and the degradation of the vasodilator bradykinin reflect the key role of ace as a component of the mammalian ras. in recent years, the ras turned out to be a far more complex regulatory system that involves more angiotensin-derived mediators (angi, angii, angiii, angiv, ang (1-9) ), ang (1-7) ) then initially recognized ( fig. 1) . to this day, it includes four identified receptors . in vivo models using ace-deficient mice have greatly advanced the knowledge of the ras and confirmed the role of ace in blood pressure regulation and fertility. knock-out mice all show reduced blood pressure, altered kidney morphology, impaired somatic growth, and male sterility (esther et al., 1996; eriksson et al., 2002) . these mice produce normal numbers of sperm that are indistinguishable from wild-type sperm in assays of viability, motility, capacitation, and induction of the acrosome reaction. absence of ace, however, does cause defects in sperm transport in the oviducts and in binding to zonae pellucidae (hagaman et al., 1998) . knockout of tace, leaving the sace intact, has proven that tace expression is both necessary and suycient for fulfilling the role of ace in male fertility (ramaraj et al., 1998) . recently, immunocytochemical studies have shown that tace is completely shed from the sperm membrane before ejaculation. this would imply that the defects in sperm transport in the oviducts and in binding to zonae pellucidae after tace knockout are not directly related with tace activity (metayer et al. 2002) , so the exact role of tace remains to be uncovered. the fact that to date no in vivo substrate has been found for tace is the main bottleneck in tace research. expression of sace, either in renal proximal tubes or in vasculature, is suycient for maintaining normal kidney functions. however, for maintaining blood pressure, sace must be expressed in vascular endothelial cells (kessler et al., 2003) . expression of tace alone is suycient to restore male fertility in mutant mice without curing other problems of ace à/à mice (ramaraj et al., 1998) . however, sace cannot substitute for tace, demonstrating that the two isozymes are not interchangeable for fertility functions (kessler et al., 2000) . yet tace expression in serum of aceà/à mice can substitute for sace in maintaining normal renal structure and functions without restoring blood pressure (kessler et al., 2002) . all this indicates that the two isoforms of ace are indispensable, as suggested by their high degree of evolutionary conservation. also, the specific physiological function of ace requires its expression in the correct tissue. the peptide acsdkp reversibly prevents the recruitment of pluripotent haematopoietic stem cells into the s phase of the cellular cycle by maintaining them into the g o phase. because this peptide has been found to be an in vivo substrate specific for the n domain of sace, it is suggested that ace is implicated in the process of hematopoietic stem cell regulation by permanently degrading this natural circulating inhibitor (azizi et al., 2001; rousseau et al., 1995) . however, in a very recent study, mutation of the n domain zinc-binding site in mice, rendering this n domain inactive indeed, resulted in accumulation of acdskp, but no physiological evect was seen (fuchs et al., 2004) . ace acts as an endopeptidase on the multifunctional neuropeptides substance p and cholecystokinin and may degrade the luteinizing hormone releasing hormone (lh-rh) (skidgel and erdos, 1985) . its biological significance in this context is not known, and the possibility of functional cross talk between the systems has not been excluded . the broad in vitro specificity (table i ) and the presence of ace in species that lack an identifiable ras shows that the relevance of ace to the animal must be more complex than just its role in the ras. up until now, only one other member of the m2 family other than ace has been identified. almost 50 years after the discovery of ace itself, two groups (donoghue et al., 2000; tipnis et al., 2000) introduced the first homolog of mammalian ace. this protein and its homolog were named ace2 and aceh, respectively, and were shown to be an essential regulator of heart function (crackower et al., 2002) . human ace2 is an 805-amino acid-long type i integral membrane protein, including a 17-amino acid n-terminal signal peptide and a putative c-terminal membrane anchor. the extracellular domain shares a 42% sequence identity with the catalytic domain of sace. it is 33% identical to human testicular (germinal) ace and also contains a single catalytic site. ace2 contains six putative n-linked glycosylation sites on the extracellular part (tipnis et al., 2000) . the c-terminal domain of ace2 shares a 48% sequence identity with collectrin (vickers et al., 2002; zhang et al., 2001) , a developmentally regulated renal transmembrane glycoprotein. the many similarities in the genomic sequence of ace2 and ace indicate that there is an evolutionary relationship between these two genes . ace2 transcripts are detected in heart, lung, kidney, the gastrointestinal tract, and testis (harmer et al., 2002; komatsu et al., 2002) . ace2 is located at the endothelium of coronary and intrarenal vessels and in the renal tubular epithelium. like ace, ace2 also appears to be susceptible to cleavage and secretion from the cell surface (donoghue et al., 2000) . however, the sequence identity of the juxtamembrane regions of ace and ace2 is diverent. the cleavage site present in ace (-ala-arg ser-glu-) is absent in ace2. hence, a diverent secretase may be involved in the shedding of ace2. ace2 does not have the same substrate specificity as ace, nor is it inhibited by the typical ace inhibitors such as enalapril, lisinopril, and captropril. ace2 hydrolyzes angi and angii but cleaves neither bradykinin nor the typical ace substrate hip-his-leu ( fig. 1) . to hydrolyze angi and angii, ace2 acts as a carboxypeptidase, removing one amino acid from the c terminus of the substrate. this is also the case for kinetensin, des-arg 9 bradykinin, and neurotensin (donoghue et al., 2000; . several other regulatory peptides, including the enkephalins, are not substrates of ace2. it seems that ace2 prefers to cleave a single c-terminal hydrophobic residue from its substrates (table i) and consequently does not hydrolyze bradykinin that contains an arginine at its c terminus. ace2 is the first mammalian carboxypeptidase identified that contains the hexxh active site. ace2 also acts on apelin-13 and apelin-36 peptides, of which the role is not yet fully understood but involves an evect on hypertension and vasoconstriction . two opioid peptides, dynorphin a(1-13) and b-casamorphin, are also ace2 substrates (eriksson et al., 2002; vickers et al., 2002) . these peptides activate g protein-coupled opioid receptors that regulate pain perception and are suggested to negatively avect cardiomyocyte contractility (pugsley, 2002; ventura et al., 1992; wenzlav et al., 1998) . the diverences in substrate specificity between ace and ace2 have important physiological consequences. whereas ace activity leads to the formation of the vasoconstrictor angii, ace2 is involved in the formation of vasodilator mediators (danilczyk et al., 2003) . the targeted disruption of ace2 in mice results in increased angii levels, impaired cardiac contractility, and up-regulation of hypoxia-induced genes in the heart (crackower et al., 2002) . renal ace is implicated in diabetic nephropathy. in the diabetic kidney, levels of ace2 transcription and protein expression are downregulated. it is suggested that this regulation occurs through the actions of ace2 in the ras (tikellis et al., 2003) . another, rather unexpected function of ace2 was recently discovered. it was found to act as a functional receptor for the severe acute respiratory syndrome (sars) coronavirus (cov) (dimitrov, 2003; li et al., 2003; xiao et al., 2003) . sars is the abbreviation for the cov-induced disease that causes sars, with a mortality rate of approximately 10% (ksiazek et al., 2003; kuiken et al., 2003) . the virus associates with cellular receptors to mediate infection of the target cells (gallagher and buchmeier, 2001; holmes, 2003) . ace2, acting as a cellular receptor, eyciently binds to the s1 domain of sars-cov, eyciently supporting the replication of sars-cov. adding a soluble form of ace2, but not that of ace, blocks the association of sars-cov and its target cell. replication of the virus in cells and the formation of syncitia can be blocked by antibodies against ace2, which indicates the importance of ace2 in the replication of sars-cov (li et al., 2003) . several attempts to reveal the three-dimensional structure of ace2 have been made very recently. this provides an insight into the structural variations underlying the diverences in substrate specificity between ace and ace2. it seems that these diverences occur in the ligand binding cavity, influencing the binding of the peptide carboxyterminus (guy et al., 2003) . the crystal structure of ace2, native and inhibitor bound, revealed a large inhibitordependent bending movement of the two enzyme domains to one another, positioning important catalytic residues (towler et al., 2004) . it has been suggested that ridges surrounding the cavity at the top of the molecule serve as a binding region for sars (prabakaran et al., 2004) . the discovery of this sars-related function of ace2, together with the knowledge of its structure, may contribute substantially to the possible treatment of sars patients. lamango and isaac (1993) reported the metabolization of [d-ala 2 , leu 5 ] enkephalin by head membranes of the housefly, musca domestica. this hydrolysis was only partially inhibited by phosphoramidon, an inhibitor of endopeptidase-24.11. the combination of phosphoramidon and captopril, however, fully inhibited the cleavage of enkephalin, suggesting the presence in this fly of a second enkephalin-metabolizing enzyme in addition to endopeptidase-24.11. it turned out to be zn 87-kda-enzyme that was activated by zncl 2 and inhibited by edta as well as the mammalian ace inhibitors captopril, fosinoprilat, and enalaprilat. this represented the discovery of the first invertebrate ace homolog . following the report on this ace-like activity, the first results of putative drosophila melanogaster ace cdna clones were published (cornell et al., 1993) . since then, ace homologs have been detected in many invertebrate species. cloning or purification of ace from the fruit fly d. melanogaster (cornell et al., 1995; tatei et al., 1995) , the cattle tick boophilus microplus (jarmey et al., 1995) , the leech theromyzon tessalutum (laurent and salzet, 1996) , the housefly m. domestica (lamango et al., 1996) , the buvalo fly haematobia irritans exigua (wijvels et al., 1996) , the mosquito anopheles stephensi , the silk moth bombyx mori , the grey fleshfly neobellieria bullata (vandingenen et al., 2002b) , and the locust locusta migratoria (macours et al., 2003a) have contributed to the molecular, biochemical, and evolutionary characterization of invertebrate aces. the first d. melanogaster ace homolog that was discovered is abbreviated as ance, because ace was already used for acetylcholine esterase in the fruit fly. of the invertebrate aces known today, the two d. melanogaster ace homolog (ance and acer) have been studied in most detail. ance is also known under the name race, as the enzyme was discovered, sequenced, and named by two groups simultaneously (cornell et al., 1995; tatei et al., 1995) . nowadays, the focus on ace research is beginning to shift from purification-characterization to a more physiological point of view. considering the fact that most invertebrates have an open circulatory system, there is probably no need for a renin-angiotensin system analogous to that found in vertebrates, at least not for the regulation of blood pressure. hence, understanding the biology of ace homologs in invertebrates may, in due time, provide substantial information on additional roles for ace. in contrast to the occurrence of both a double and a single domain ace in vertebrates, characterization of ace homologs from diverent invertebrates showed that the overall size of these proteins was comparable to the nonglycosylated testicular form of mammalian ace. cloning of the corresponding cdnas confirmed the existence of this single-domain form in insects. until now, there has been no evidence for a functional two-domain form of insect ace. in addition to the absence of a two-domain form, a structural diverence exists between insect ace and the mammalian enzyme, located in the n-and c-terminal ends. cloning of the d. melanogaster ace homologue (ance) revealed that the insect form of ace lacks both the heavily glycosylated n-terminal extension and the c-terminal transmembrane and cytoplasmic regions that are present in mammalian testicular ace. the enzyme contains an n-terminal secretory signal peptide, which indicates that the mature enzyme is being secreted. hence, ance was predicted to code for a soluble secreted protein (fig. 2) . confirmation of this prediction came when 90% of recombinantly expressed ance activity was found in the cell culture medium (cornell et al., 1995) . although insect ace activity was primarily detected in a membrane preparation from heads of the housefly, m. domestica , this ace protein was later found not to behave as an integral membrane protein. the membrane-associated activity was probably the result of a contamination of the membrane fraction with soluble ace (lamango et al., 1996) . therefore, the general form of insect ace seems to be a secreted single-domain protein with no recognizable c-terminal membrane anchor. the molecular identification of the ace enzyme in b. microplus, however (jarmey et al., 1995) , which does posses a c-terminal hydrophobic region, showed that the membrane-bound form of ace is not restricted to vertebrate organisms. also, in the cockroach leucophaea maderae and the noctoid moth lacanobia oleracea, a substantial amount of ace activity was detected in head membranes . the nature of this membrane-associated ace activity in these two organisms is, as yet, unidentified. sequence comparison between human testicular ace and several invertebrate ace homologs showed that the overall sequence identity is approximately 40% and exceeds 70% in the regions surrounding the active site. in human ace, 17 and 8 potential n-linked glycosylation sites (asn-xaa-ser/ thr motifs) are present in the somatic isoform and in the germinal isoform, respectively. this contrasts to the situation in invertebrates, where ace homologs have only three or fewer potential glycosylation sites. in d. melanogaster, it was shown that glycosylation of ace is not required for the enzymatic properties. expression of an unglycosylated mutant of ance resulted in the secretion of a catalytically active enzyme, with the same substrate and inhibitor aynity as the wild-type enzyme (williams et al., 1996) . kim et al. (2003) determined the crystal structure of the d. melanogaster ace homolog ance. ance is composed of 21 a-helices and three antiparallel b-strands. the substrate-binding channel exists as a large continuous internal angiotensin-and endothelin-converting enzymes path that comprises the entire protein molecule. the composition of this substrate-binding site is unusual in that it is composed of two unequal-sized ''chambers'' analogous to a peanut shell. the large chamber, referred to as the n chamber, contains the binding site of the n-terminal sequence of the substrate angiotensin i. the smaller chamber, called the c chamber, will bind the carboxy-dipeptide of the substrate. the size of these chambers is suyciently large for binding short peptide substrates. the bottleneck region connecting these two chambers encloses the zinc ion critical for catalysis and locates the inhibitor binding sites. the opening of these chambers to the exterior appears to be relatively small for the passage of peptide substrates, so flexibility and a so-called ''breathing motion'' are probably required for eycient catalysis. the presence of significant ace activity was reported in extracts of the horse anemone, actinia equine, a member of the cnidarians (coates et al., 2000b) . to this day, no ace peptidase activity has been detected in nematode tissues. however, there is one copy of an ace-like gene (cd42d8.5) with a recorded expressed sequence tag (est) present in the genome of the nematode caenorhabditis elegans. yet taking a closer look at this ace-like gene reveals that the amino acids crucial for enzyme activity are absent. the sequence lacks all three zinc coordinating his residues, which means that the c. elegans ace homolog does not have a functional zinc-binding domain. therefore, it cannot be classified as a metallopeptidase homolog. studies on this ace-like nonmetallopeptidase, termed acn-1, demonstrated that the protein plays an essential role in larval molting and adult morphogenesis. if acn-1 is indeed evolutionarily related to the ancestral ace, it means that during evolution it lost its peptidase activity but acquired sites for new molecular interactions that have a direct or indirect evect on the molting process in nematodes (brooks et al., 2003) . this phenomenon can be further expanded to other organisms and enzymes, as in the genome of d. melanogaster and anopheles gambiae, 15-25% of the protease-like genes lack one or more catalytic residues. some of these proteins have probably lost the peptidase activity of their ancestor while acquiring new functions. the analysis of the human ace gene at the structural level forwarded the view that the vertebrate somatic form was transcribed from a tandem duplication (lattion et al., 1989) . in all vertebrate species examined, from humans at the top down to the electric fish torpedo marmorata (turner et al., 1987) , the somatic ace enzyme exists as the two-domain form. the analysis of the ace gene of the housefly m. domestica revealed that this organism comprises a smaller form of the enzyme, indicating that this species does not contain the duplicated form of ace. the existence of this single-domain isoform was confirmed when the ance cdna of d. melanogaster was sequenced (cornell et al., 1995) . all other invertebrate ace enzymes that were discovered later on also consisted of the single-domain isoform. this leads to the view that invertebrate ace represents the ancestral copy of the vertebrate two-domain enzyme and that the duplication event occurred before vertebrate radiation. however, this course of ace evolution started to be questioned when the characterization of d. melanogaster ace homologs began. the high level of homology between ance and acer points toward the existence of an ancestral ace gene. in addition, studying the relationship of ance and acer to the n and c domains of human somatic ace showed that ance was more enzymatically related to the c domain than to the n domain of human ace (williams et al., 1996) . acer, however, has much less in common with this c domain. the active sites of the n domain of sace and acer share structural features that permit the binding of the rxp407 inhibitor, which does not bind the c domain of sace. an analysis of the genomic region around the ance gene in d. melanogaster has led to the identification of four additional ace-like genes (isaac et al., 2000) . from these four, ance 3, 4, and 5 seem to be transcribed, because ests were reported for these genes. with the information available today, no est seems to be present for ance 2. the predicted protein sequence corresponding the ance 3 gene includes a cterminal transmembrane anchor, as is seen in vertebrate ace. ance 2 codes for an ace-like protein with a putative signal peptide, but without a cterminal membrane region. both genes lack crucial active site residues. these two gene predictions are interesting because alternative splicing of the predicted transcripts would lead to a two-domain, membrane-bound ace-like product, as is seen in the vertebrate somatic form (coates et al., 2000a) . taken together, this information indicates that the duplication event that gave rise to the vertebrate tandem protein predates the divergence of the ecdysozoa and deuterostomes, and that the divering activities on the n and c domains have been maintained over a long period of time. insect ace is capable of hydrolyzing the known in vivo substrates of mammalian ace, angiotensin i, bradykinin, and the hemoregulatory peptide ac-dskp. however, there are no reports of insect homologs of these known in vivo substrates, and none of the many insect peptides isolated to date shows any structural resemblance to them. the search for angiotensin i-like peptides in the brain of several insect species could not prove otherwise (isaac et al., 1998b; schoofs et al., 1998) . in addition, a search in the d. melanogaster genome did not reveal any potential homologs of the precursor proteins of angiotensin i and bradykinin (isaac et al., 2000) . this is in contrast to the situation in leeches, where several reports on the presence of members of the renin-angiotensin system have been published (laurent and salzet, 1996; salzet and verger-bocquet, 2001) . cleavage experiments with ace from m. domestica showed that insect ace exhibits a broad in vitro substrate specificity. the in vitro substrates of mammalian ace, cholecystokinin, enkephalins, substance p, and lh-rh, are successfully cleaved by the endopeptidase activity of musca ace. this shows that insect ace can hydrolyze c-terminally amidated peptides in vitro, functioning as an endopeptidase (isaac et al., 1997a; lamango et al., 1997) . m. domestica ace is unable to hydrolyze either ccap (crustacean cardioactive peptide) or proctolin. apparently, the penultimate c-terminal prolyl residue of proctolin makes this peptide resistant to ace hydrolysis. the lack of hydrolysis of ccap is likely a result of the presence of a disulfide bridge, which results in a secondary structure that prevents access to the active site. a novel activity of insect ace, namely, prohormone processing, was suggested when it was found to be capable of successfully removing basic dipeptides from locustamyotropin oligopeptides possessing a c-terminal gly-lys-arg and gly-arg-arg extension (isaac et al., 1998b) . many insect peptides are synthesized as inactive prohormones and need posttranslational processing to acquire their biological activity. in several prohormones, the sequence -gly-arg/lys-arg-is an internal consensus recognition sequence for endoproteolysis by prohomone convertases, generating peptide intermediates with a c-terminal -gly-arg/lys-arg-extension. these are substrates for carboxypeptidase e (cpe) that sequentially removes the two basic amino acids from the c terminus, which is followed by amidation of the c terminus (isaac et al., 1997b) . the fact that insect ace is capable of hydrolyzing these partially processed prohormone-like peptides, together with the intracellular colocalization of the enzyme with locustamyotropin peptides, implies a role for ace in the biosynthesis of peptide hormones and neuropeptides . zhu et al. (2001) and coworkers demonstrated that the hexapeptide trypsin modulating oostatic factor from n. bullata (neb-tmof) (bylemans et al., 1994) is selectively hydrolyzed by ace that is present in substantial amounts in the fly's haemolymph . neb-tmof is capable of regulating vitellogenesis and is thought to be released by the ovaries and transported to the midgut, where it terminates the protein meal-induced trypsin biosynthesis. captopril feeding experiments suggest that neb-tmof represents a prime candidate for being an in vivo substrate for circulating insect ace (vandingenen et al., 2002a) . tmof was also detected in aedes aegypti, probably synthesized by the brain (borovsky and meola, 2004) . however, this aea-tmof, with six prolines at its c terminus, is not cleaved by neb-ace (k. hens, personal communication) . recently, new substrates were purified from the ovaries of n. bullata. their primary structures were identified as nklkpswqwisl (neb-odaif), nklkpsqwislsd (neb-odaif-1 1-13 ), nklkpsqwi (neb-odaif-1 1-9 ), nklkpsq (neb-odaif-1 1-7 ), slkpsnwltpse (neb-odaif-2), and leqiyhl. these peptides show significant homology to the n-terminal part of fly yolk proteins (hens et al., 2002; vandingenen et al., 2002b) . kinetic analysis of these peptides with insect ace and human ace shows that the n. bullata ace shares enzymatical properties with the c-domain sace, in addition to features that seem unique to invertebrate ace. characterization of the ance protein demonstrated that this enzyme eyciently hydrolyzes angiotensin i and bradykinin (cornell et al., 1995; williams et al., 1996) . ance can operate as an endopeptidase, for instance on tachykinins, substance p, and leucokinins. still, the nonamidated form of a peptide is always preferentially cleaved over the amidated form. furthermore, insect ace preferentially acts as a dipeptidase and shows reduced tripeptidase activity. of the insect peptides tested, locustatachykinin (lom tk-1) proved to be the best substrate for ance. however, the d. melanogaster tachykinins turned out to be poor ance substrates . acer is not capable of cleaving angiotensin i (houard et al., 1998) , and most peptides tested were hardly degraded by acer . to this day, there are no clues concerning the identity of the endogenous acer substrates. the structural and biochemical similarities between mammalian and invertebrate ace do not necessarily imply a functional conservation as well. although the presence of an invertebrate ras has been reported in a leech that feeds on ducks (laurent et al., 1995; salzet, 1995a,b, 1996) , no other components of the ras other than ace have been described in insects or other invertebrates. it is therefore highly improbable that this invertebrate enzyme would physiologically act as a converting enzyme for an angiotensin-like peptide. as already mentioned, functionality of mammalian ace is, however, not restricted to the ras. both its broad substrate specificity and widespread tissue distribution indicate its involvement in several other (but, unfortunately, still poorly described) physiological functions. to fully unravel the in vivo role of ace in insects, information about substrate specificity and tissue and cellular localization of the enzyme, as well as its putative colocalization with its substrates, has to be combined. these data, together with the studies of ace mutants and the evects of ace inhibitors on insect physiology, will eventually lead to the elucidation of the roles of insect ace. today's concept on insect ace functionality focuses on its possible role in reproduction, development, and defense. the discovery of ace in testes of several insect species, including h. irritans exigua (wijvels et al., 1996) , lymantria dispar (loeb et al., 1998) , n. bullata, the colorado potato beetle leptinotarsa decemlineata, and the locust l. migratoria schoofs et al., 1998) , implies that the function of tace may have been conserved during the course of evolution. in l. decemlineata, ace was detected in the germ cells, more particularly in developing spermatids and mature spermatozoa, whereas in l. migratoria, ace was found in peripheral somatic cell bodies of the apical compartment of the testicular follicles and not in the germ cells. in the haematophagus fly h. irritans, expression of testicular ace is induced after a blood meal, indicating a specific role in the maturation of spermatozoa (wijvels et al., 1996) . the possible importance of testicular ace in reproduction became apparent by the observation that male d. melanogaster transheterozygotes of two mutant ance alleles were found to be sterile (tatei et al., 1995) , with the infertility linked to the failure of the spermatocytes to develop into active mature spermatozoa. spermatogenesis proceeds normally in the mutants up to the spermatid stage, but these spermatids do not succeed in diverentiating into spermatozoa. this change is accompanied by nuclear scattering and an abnormal morphology of the spermatids. together with the observed expression pattern of ance, the enzyme is believed to be required for spermatid diverentiation through the processing of a regulatory peptide synthesized within the developing cyst (hurst et al., 2003) . the involvement of ace in the control of spermatogenesis was also demonstrated by studies of the evects of aii and bovine ace on the ability of the testes of l. dispar to synthesize and release ecdysteroids in response to testis ecdysiotropin (te). the evect of te on the initiation and up-regulation of ecdysteroid synthesis could be imitated by both aii and bovine ace, raising the possibility that ace is involved in the production of an aii-like peptide that modulates ecdysteroid synthesis in the testes of insects (loeb et al., 1998) . in female adults of the mosquito a. stephensi, ace activity increases by a factor 2.5 after a blood meal. it is not known where this induced ace is synthesized, but it accumulates in developing ovaries and in mosquito eggs . adding ace inhibitors (captopril and lisinopril) to the blood meal of the mosquito leads to a size reduction of the batch of eggs laid in a dose-dependent manner. the ace inhibitors reduce fecundity by interfering with the transfer of the oocytes along the oviducts (ekbote et al., 2003a) . in the reproductive tissue of the tomato moth lacanobia aleracea, almost all of the ace activity is concentrated in the accessory glands of the male and in the spermatheca and bursa copulatrix of the (virgin) female. these high activities may imply the importance of ace for peptide metabolism in the male and female reproductive tract. changes in the levels of ace activity in the reproductive tissues of males and females during mating, point toward a transfer of ace from male to female during time of copulation (ekbote et al., 2003b) . this male ace donated in the spermatophore might form a hitherto unknown male component of an energy-producing metabolic pathway, as is seen in the serine endopeptidase-dependent proteolytic cascade in the spermatophore of b. mori (aigaki et al., 1987 (aigaki et al., , 1988 . in the fleshfly n. bullata, feeding of captopril results in an increase of vitellogenin titers in the fly hemolymph. it has been suggested that this evect is mediated through the trypsin-modulating oostatic factor tmof. as already discussed, neb-tmof is an in vitro substrate of insect ace. however, if tmof were to be inactivated by ace, inhibition of ace would lead to an increase of tmof levels in the hemolymph, resulting in a reduced vitellogenin synthesis. therefore, an activating evect of ace on tmof is postulated . the discovery of ace interactive peptides in the ovaries of the fleshfly (vandingenen et al., 2002a) , and the discovery that these peptide substrates originate from yolk gene products, strengthen the idea that ace is a regulator of vitellogenic and developmental processes (hens et al., 2002; vandingenen et al., 2002b) . these physiological observations, in combination with the discovery of ace protein in the gonads of numerous insects, undoubtedly show that ace is a vital enzyme in insect reproduction. the further elucidation of the full role of ace in this process necessitates including the description of all endogenous substrates. the observation that some mutations in the d. melanogaster ance gene result in death during larval/pupal stages (tatei et al., 1995) indicates that insect ace is important for normal growth and development. many reports from diverent and independent laboratories have confirmed the role of insect ace in development and metamorphosis. along with the cloning of the first d. melanogaster ace homolog (race, ance), the gene coding for this protein was shown to be a target of the homeobox regulatory gene zerknullt (zen) and decapentaplegic (dpp) (tatei et al., 1995) . a 533-bp-long enhancer located in the ance promotor region covers three zen protein binding sites and was shown to mediate selective expression in the amnioserosa. during early development, ance is activated by zen and becomes associated with the diverentiating amnioserosa and with the anterior and posterior midgut, where it persists throughout embryogenesis. in late embryos, ance expression is detected in epithelial cells of the midgut and in the pericardial cells of the heart. ance expression is lost from the presumptive amnioserosa in zen à and dpp à mutants; the expression in the gut stays unavected. in d. melanogaster larvae, high expression of ance is found before and peaks during pupal development, indicating a physiological role for the enzyme during metamorphosis (wilson et al., 2002) . also, in the tomato moth, l. oleracea, ace activity increases approximately fourfold in the last larval instar and in the early pupal stage (ekbote et al., 2003b) . in insects, each larval molting is preceded by high ecdysteroid titers, and for pupal and adult development, multiple pulses of ecdysteroid hormones are essential. in wing discs of b. mori, the ace gene (bmacer) was found to be directly 30-hydroxyecdysone (20e) inducible in a dose-dependent manner. because this ecdysteroid-dependency was not found in other tissues examined, transcription of bmacer seems to be organ specific . during the transition from larva to pupa in d. melanogaster, the expression of ance in imaginal disc cells was found to be induced by ecdysteroids. these data point toward a fundamental role for ace during metamorphosis of holometabolous insects. several relationships between ace and metamorphosis are possible: the conversion of precursors into biologically active peptides necessary for metamorphosis, inactivation of signaling peptides, or recycling of amino acids from larval proteins for the synthesis of pupal and adult cuticle/tissues. in a. stephensi, ace expression is induced by a blood meal and ace accumulates in developing ovaries, from which it passes into the mosquito eggs . this indicates a role for ace-generating peptides that are essential for oocyte development and embryogenesis. the characterization of acn-1 in the nematode c. elegans shows that it is not necessarily the proteolytic function of ace that is involved in developmental processes. the hypodermal expression of acn-1 is under the control of nuclear hormone receptors that regulate molting in c. elegans. functional acn-1 knockout by rnai causes morphological defects in larvae and adults. more specifically, acn-1 was found to be required for larval molting, male tail development, and formation of adult alae (brooks et al., 2003) . prohormone processing? in n. bullata, l. migratoria, l. decemlineata, l. maderae, the walking stick insect carausius morosus, b. mori, and the caterpillar mamestra brassica, ace immunoreactivity is seen in neuropil regions of the brain. in l. migratoria, l. maderae, b. mori, and m. brassica, ace immunoreactivity is also present in neurosecretory cells of the brain . in l. migratoria, immunoreactive staining can also be traced in the controlateral nervus corporis cardiaca i (ncc i), corpora cardiaca (cc), and suboesophageal ganglion (sog), and in c. morosus, l. decemlineata, and l. maderae, ace immunoreactivity was also detected in the corpora cardiaca (veelaert et al., 1999) . the presence of ace in neurosecretory cells and neuropil regions of the insect brain points toward a dual role for ace in the insect nervous system . the localization of ace in the neuropil indicates the metabolic inactivation of peptidic neurotransmitters. the presence of peptides from the adipokinetic hormone (akh), kinin, and locustatachykinin family in the central nervous system (cns), as well as the knowledge that these peptides are hydrolyzed by ace (lamango et al., 1997) , indicates that they might be potential in vivo substrates for insect neuronal ace. in neurosecretory cells, ace is likely to be involved in the conversion of prohormones to active peptide hormones that are secreted into the hemolymph by the corpora cardiaca or corpora allata (isaac et al., 1998b) . because insect ace has been shown to exert in vitro prohormone processing activities against the locustamyotropins, and locustamyotropin-containing cells of the locust brain and suboesophageal ganglion also contain ace, this insect enzyme most probably plays a role in the biosynthesis of these hormones (veelaert et al., 1999) . in contrast to the situation in vertebrates, which have a dual system of immune reaction, namely, an innate and an acquired one, invertebrates only rely on their innate immune responses. this system comprises both humoral and cellular defense responses. well-known humoral responses include the synthesis of a broad spectrum of antimicrobial peptides/proteins (bulet et al., 1999) , as well as two proteolytic cascades: the blood coagulation system identified in the horseshoe crab (iwanaga, 1993 (iwanaga, , 2002 and the prophenoloxidase-activating system (pro-po-as) in insects and crustaceans (sö derhäll and cerenius, 1998) . cellular defenses cover hemocyte-mediated responses like phagocytosis, nodulation, and encapsulation. in insects, a large number of peptides is released into the hemolymph, where they are known to regulate a large diversity of physiological functions. the half-life of these peptides can be regulated by proteases and peptidases present in the hemolymph. the discovery of ace activity in the hemolymph of insects , together with the knowledge of putative prohormone converting activity of ace, indicated the possible involvement of ace in the proteolytic processing of insect antibacterial peptides from their precursors (isaac et al., 1998a) . supportive data concerning this hypothesis came from expression studies in larvae from the sheep blowfly lucilia cuprina and the old screwworm chrysomya bezziana, which are known to secrete or excrete chymotrypsins and trypsins in larval cultures and wound sites. the knowledge that an ace homolog is also secreted or excreted from these larvae indicated a dual role in wound formation. it may degrade chymotryptic and tryptic digestion products from host tissue proteins and may influence the host vascular and inflammatory response by processing or degrading host regulatory peptides (wijvels et al., 1997) . in the endoparasitic wasp, pimpla hypochondriaca, the female envenomates and oviposits into pupae of some lepidopteran species. this venom was shown to suppress the hemocyte-mediated immune responses of the host by impairing encapsulation and phagocytosis (parkinson et al., 2002; richards and parkinson, 2000) . the presence of ace in this venom, together with the presence of antibacterial activity, implies that ace could be one of the enzymes involved in the processing of antibacterial peptides from the venom sac (dani et al., 2003) . all the mentioned data indeed point toward an involvement of ace in the defense system, but they are still only hypothetical. the first physiological evidence for the existence of a connection between ace and defense came from studies in the locust l. migratoria. locusts that are subjected to an immune challenge through injection of lipopolysaccharides (lps) into the hemocoel showed an increase in ace mrna-expression. comparing the level of ace transcription from lps-treated animals with control animals revealed an approximately 10-fold increase in ace transcription 6 hours after the lps-injection (macours et al., 2003b) . the exact nature of the ace-defense connection remains to be clarified. in mammals, all blood cells derive from hematopoietic stem cells that diverentiate into diverent lineages. the involvement of ace here resides in the inactivation of ac-sdkp (n-acetyl-seryl-aspartyl-lysyl-proline), a peptide that prevents the recruitment of hematopoietic stem cells (azizi et al., 1996; baudin, 2002) . insects also continue to produce hemocytes via a division of stem cells or by continued division of hemocytes in circulation (jones, 1970; ratclive et al., 1985) . on the basis of this information, a possible connection between ace and the immune system can be suggested in the replenishment of the hemocytes that are involved in the insect immune response. the possibility of ace being involved in the processing of antimicrobial peptides in locust hemolymph cannot be excluded, but so far, except for lysozyme and coagulogen, no major antibacterial activity could be detected in locust hemolymph (van sambeek and weisner, 1999). the rise in ace expression that follows lps injection is not likely to result from an involvement of ace in the prophenoloxidase cascade, as this pathway is not activated by injection of lps. however, the immune system of the locust responds to lps injection through the formation of nodules (goldsworthy and chandrakant, 2002; hovman et al., 1974) . therefore ace may be required in the process of nodule formation. this speculation is in agreement with the recent findings of goldsworthy and colleagues (2003) , who investigated the evect of lps on the prophenoloxidase cascade and nodule formation in the locust. activation of prophenoloxidase by lps could only be achieved by coinjection with akh, not by the injection of lps alone. when captopril was coinjected, the activation of the ppo cascade remained unavected (with/without akh). however, captopril did inhibit the nodule formation induced by lps (goldsworthy et al., 2003) . considering these data, a possible role for ace in the immune system of insects may reside in the regulation of nodulation. to date, leeches are the only group of invertebrates in which the existence of a renin-angiotensin system has been reported. in theromyzon tessulatum, a parasite of ducks, an angiotensin ii (aii)-like peptide was isolated (salzet et al., 1993) . the full sequencing of such aii-like peptide was realized in another leech species, namely erpobdella octoculata . this leech aii peptide divers from the vertebrate aii by a c-terminal amidation. injection of this aii into the leech results in a decrease in mass, reflicting a diuretic evect. also in t. tessulatum, a renin-like peptide was isolated (laurent and salzet, 1995a) and partial peptidic sequences were identified. this enzyme was found capable of releasing ai from an ai-like precursor, angiotensinogen (laurent et al., 1995) , by cleavage of the leu 10 -leu 11 bond. the mass of this invertebrate renin (ca. 32 kda) was lower than that of vertebrate active renin (ca. 44 kda) but displayed a comparable activity. the leech renin is localized in the excretory and nervous system, which implies the involvement of the leech ras in osmoregulation. the angiotensin-converting enzyme of this leech-ras is a glycosylated membrane-bound enzyme of 120 kda that is released into the hemolymph in a hydrophilic form (100 kda) (laurent and salzet, 1996; vandenbulcke et al., 1997) . the presence of this ras in coelomocytes of leeches and the involvement of each part of this system in the immune response inhibition indicates the implication of the ras in host-parasite cross-talk (salzet and verger-bocquet, 2001) . endothelin-converting enzymes (eces) belong to the class of type ii integral membrane zinc metalloproteases named for their ability to hydrolyze big endothelins (big ets) into the smaller vasoactive endothelins. yanagisawa and colleagues (1988) were the first to predict the existence of eces based on the cloning of the prepro-endothelin gene. ece is classified into the neutral endopeptidase or m13 group of proteins, which contains type ii membrane glycoproteins with zinc peptidase catalytic activity. at present mammalian m13 family of zinc proteases consists of seven known members: neutral endopeptidase (nep); the endothelin-converting enzymes ece-1, ece-2, and ece-3; the kell blood group antigen (kell); the phosphate regulating gene (pex); x-converting enzyme (xce); and secreted endopeptidase (sep). each member of this family shows homology to the others, mainly in the c-terminal part of the sequence that is responsible for catalytic activity. in addition, there are structural similarities in that all m13 members have short intracellular domains. where identified, these enzymes have roles in the processing or metabolism of regulatory peptides and therefore represent potential therapeutic targets. ece-1 is a membrane-bound protein with a short n-terminal cytoplasmatic tail, a transmembrane hydrophobic domain, and a large extracellular domain, which shares high sequence homology to nep and other members of the metallopeptidase family. ten cysteine residues in the ece sequence are conserved among the other members of the family and are probably involved in disulfide bridges. the ece protein has 10 predicted sites for n glycosylation in the extracellular domain. this is consistent with the observation that ece is a highly glycosylated protein and explains the diverence between the predicted molecular mass and the mass shown on sds-page. purification and cloning studies of rat ece-1 have shown that it consists as a disulphidelinked dimer, in which cys 412 is solely responsible for the formation of the intermolecular disulphide bond (shimada et al., 1996) . the large extracellular domain contains the zinc-binding consensus sequence hexxh, in which the glutamic acid (e) is the most important residue for catalytic activity. the two flanking histidine (h) residues act as zinc coordinating amino acids. the third ligand for the zinc atom is e 651 , whereas h 716 is involved in the stabilization of the tetrahedral intermediate during the transition state (shimada et al., 1995) . four ece-1 isoforms have been identified in humans and were named ece-1a (758 amino acids), ece-1b (770 amino acids), ece-1c (754 amino acids) and ece-1d (767 amino acids) (jafri and ergul, 2003; valdenaire et al., 1995 valdenaire et al., , 1999a . the four isoforms are transcribed from a single gene and result from the use of alternate promoters located upstream of specific exons. they only diver in the first half (approximately) of their cytoplasmatic tail and are identical over their remaining sequence, including the enzymatic catalytic site. as a logical consequence, the isoforms display comparable converting activity. the specificity of the isoforms thus lies in their cytosolic tails, which results in a diverent subcellular distribution. whereas ece-1a and ece-1c are present in the plasma membrane, ece-1b and ece-1d are retained inside the cell. recently it was shown that ece-1 isoforms can heterodimerize. it was suggested that heterodimerization of ece-1 isoforms could constitute a means for regulating their subcellular distribution and, subsequently, their extracellular activity (muller et al., 2003) . considering the importance of the biosynthesis of both the intracellular and extracellular endothelins, the subcellular distribution of ece isoforms may play a central role in the regulation of the endothelin system. the endothelin system is composed of endothelin (et) peptides and their receptors (fig. 3a) , which together avect a wide variety of physiological functions and pathological conditions. the discovery of this system started in the mid-1980s, when an increasing awareness of the role of endothelial cells as active components of the vascular system led to the description of an endothelial cell-derived constricting factor (hickey et al., 1985) . in 1988, a 21-amino acid vasoconstricting factor termed ''endothelin'' was isolated from cultured porcine aortic endothelial cells (yanagisawa et al., 1988) . the expression of et is associated with many pathological processes and with tumor growth. to fulfill their wide spectrum of physiological functions, ets act through autocrine and paracrine mechanisms. their biosynthesis thus requires tight local control. endothelin-1 (et-1) is a 21-amino acid peptide with a hydrophobic c terminus. within 1 year of its discovery, two structurally related peptides were identified and termed et-2 and et-3, respectively. the three isoforms are encoded by three diverent genes and show highly conserved sequences in several species. all isoforms are composed of 21 amino acids with two intrachain disulfide bridges. et-2 exhibits the closest structural similarity to et-1, divering by only three amino acid residues, whereas et-3 divers by six amino acids. et isoforms are widely distributed among various cells and tissues. of the three isoforms, the most widely distributed, best studied, and most potent is et-1. et-1 shares great structural homology to sarafotoxin, a snake (atractapsis engaddensis) venom that induces myocardial infarction by exaggerated contraction of the cardiac vessels and interruption of the blood supply to the heart. ets are produced by a variety of organ and body tissues such as the lung and kidney as well as the brain, pituitary, peripheral endocrine tissues, and placenta. most abundantly, endothelial cells of the vascular endothelium produce et. et binds to two types of receptors (et a and et b ) (arai et al., 1990; sakurai et al., 1990) . both contain seven transmembrane domains and activate an overlapping set of g proteins, producing an array of diverent physiological responses (douglas and ohlstein, 1997) . although structurally highly similar, these receptors possess diverent aynities for the three isoforms of et. like many other regulatory peptides, the ets are maturated through proteolytic processing from larger precursor polypeptides, termed ''prepro-ets.'' this proteolytic cleavage generates an essentially inactive intermediate referred to as big et. the subsequent hydrolysis of big et into the final active product et, and a presumed inactive c-terminal fragment, is catalyzed by ece (fig. 3a) . in the case of big et-1 and et-2, proteolysis occurs at the trp21-val22 bond. et-3 is formed through cleavage of the trp21-ile22 bond of big et-3 (inoue et al., 1989) (fig. 3b) . the conversion of big et to et can occur in the extracellular medium and in the secretory pathway; cells thus secrete big ets either alone or together with endothelins . the physiological importance of the cleavage of big et is indicated by a 140-fold increase in vasoconstrictor potency on cleavage to et. this makes ece an important activating protease in the biosynthetic pathway of et. ece-1 null mice exhibit a phenotype similar to that of et-1-or et a -deficient mice, demonstrating the significance of ece-1 in generating bio-available et-1 (yanagisawa et al., 1998 ). the endothelin system plays a variety of roles in both normal physiology and pathological conditions in a number of tissues/organs of the body. in blood vessels, et maintains a basal level of vasoconstriction and is involved in the development of hypertension, atherosclerosis, and vasospasm after subarachnoid haemorrhage. in the heart, the endothelin system avects force and rate of contraction and mediates hypertrophy and remodeling in congestive heart failure. in lungs, et regulates the tone of both airways and blood vessels and is involved in the development of pulmonary hypertension. kidney-et regulates water and sodium excretion and acid-base balance, and it participates in the pathophysiology of acute and chronic renal failure. in the brain, the et system modulates cardiorespiratory centers and hormone release (kedzierski and yanagisawa, 2001) . in ovaries, et is shown to play a role in regulating the female reproductive cycle, where it functions as an important component of the luteolytic cascade (levy et al., 2003) . egidy and colleagues (2000) suggested that et, as a powerful vasoconstrictor and mitogenic peptide that is produced by many cell lines, might play a role in cancer progression of the colon. studies by johnson and colleagues (1999) revealed that ece-1 possesses a broad in vitro substrate specificity and is potentially involved in the metabolism of peptides distinct from et. they found that neurotensin, substance p, bradykinin, and the oxidized insulin b chain are in vitro substrates for ece-1. given this in vitro substrate specificity, it is likely that ece-1 is involved in the degradation and processing of many biologically active peptides, both at the cell surface and in the secretory pathway (johnson et al., 1999) . a second form of ece, ece-2, was cloned from bovine adrenal cortex and was reported to possess a 59% identity with ece-1. the main structural features of ece-1 are conserved in ece-2 (emoto and yanagisawa 1995) . both are type ii integral membrane proteins with the hexxh consensus sequence. the 10 cysteine residues conserved between ece-1, nep, and kell, are also conserved in ece-2, as is the cysteine residue responsible for dimerization of ece-1. ece-2 is also heavily glycosylated, with 10 possible glycosylation signals. although ece-2 shows specific activity to produce mature et-1 from big et-1, like ece-1, the striking diverence between ece-2 and ece-1 is the acidic ph optimum of 5.5 of ece-2, in contrast to the neutral ph optimum of ece-1. this indicates that ece-2 is involved in et synthesis in the intracellular compartments in which the ph is acidic (emoto and yanagisawa, 1995) . ece-2 has been reported to be expressed in various organs and tissues including cultured human vascular endothelial cells, uterus, ovary, heart, lung, and liver, indicating various functions for the enzyme. recent targeting studies with mouse ece-2 revealed that it plays crucial roles in the formation of cardiac outflow structures (yanagisawa et al., 2000) . four subisoforms of ece-2 that diver only in their n-terminal cytoplasmatic tails were termed ece-2a-1, ece-2a-2, ece-2b-1 and ece-2b-2, ece-2a-1 and ece-2a-2 are expressed in a variety of tissues including liver, kidney, adrenal gland, testis, and endothelial cells. ece-2b-1 and ece-2b-2 are abundantly expressed in the brain and adrenal gland (ikeda et al., 2002) . ece-3 was purified from bovine microsomes. this enzyme was found to be specific for the conversion of big et-3 to et-3 and was inhibited by phosphoramidon (hasegawa et al., 1998) . to the best of our knowledge, this is the only report on an ece-3 enzyme. neutral endopeptidase (neprilysin, nep), a 90-110-kda plasma membrane protein, is the prototype and best characterized member of the m13 zinc metallopeptidase family. nep is identical to the neutrophil and cluster-diverentiation antigen cd10 and is also known as the common acute lymphoblastic leukemia antigen (calla), which is mainly associated with the precursors of b lymphocytes (letarte et al., 1988) . purification and subsequent cloning of nep revealed it to be a type ii integral membrane protein of approximately 700 residues. neprylisin's extracellular domain, which includes the active site, also contains 12 cysteine residues, 10 of which are involved in highly conserved disulfide bridges that are important in the maintenance of structure and activity of the enzyme (oefner et al., 2000) . nep comprises three subisoforms that result from alternative splicing in the 5 0 untranslated region, indicating that these spliceforms do not exhibit functional diverences (ishimaru and shipp, 1995) . regarding its catalytic activity, nep shares some similarity with the bacterial zinc metalloproteinase thermolysin, and in particular in its substrate specificity (malfroy et al., 1988) . nep exists as an ectoenzyme that preferentially hydrolyzes extracellular oligopeptides (<5 kda) on the amino side of hydrophobic residues. nep is the only enzyme in this family of which the x-ray crystallographic structure has been determined. the structure revealed a restricted active site cleft preventing access of large peptides and proteins, explaining its oligopeptidase character (oefner et al., 2000) . nep is primarily expressed in the kidney, where it has the capacity to cleave and inactivate the natriuretic and vasodilatory peptide (anp), which controls arterial pressure (beaulieu et al., 1999) . the first physiological function of nep was found in the brain, where nep is located in neuronal cells. there, it controls the half-life of enkephalins and substance p (two neuropeptides involved in pain control) through inactivation (malfroy et al., 1978) . nep also plays a pivotal role in various cancers, such as prostate cancer and leukemia (letarte et al., 1988; papandreou et al., 1998) . a variety of other physiological substrates has subsequently been revealed for nep (turner and tanzawa, 1997) , although most attention has been focused on the vasodilator anp. the development of selective nep inhibitors, of which the best studied are thiorphan and phosphoramidon, has primarily been driven by their potential as novel cardiovascular agents (roques and beaumont, 1993) . the medicinal treatment of various azictions with nep inhibitors has become so prevalent that they now constitute a class of drugs used in both gastroenterology and cardiology (tanja et al., 2000) . there is, however, a potential downside to the clinical use of nep inhibitors, as downregulation of nep levels is seen in various cancers and in the lung, where it potentiates neurogenic inflammatory responses. recent reports that a nep-like enzyme can degrade the neurotoxic b-amyloid peptide involved in alzheimer's disease raises new concern about chronic blockade of nep-like activity in the cns . the kell blood group system is one of the major antigenic systems in human erythrocytes. over 23 diverent blood group antigens have been assigned to this system, and the molecular basis for this polymorphic variation has been shown to be the result of single-base mutations in the kell gene, resulting in the diverent kell phenotypes (lee, 1997) . the protein is of clinical importance because incompatibility involving kell antigens can cause severe hemolytic reactions to blood transfusions and leads to fetal and neonatal anemia. kell is a 93-kda type ii erythrocyte membrane glycoprotein that is attached to the cytoskeleton and that comprises a short n-terminal intracellular segment, a single transmembrane sequences and a large, 665-amino acid extracellular domain. in the c-terminal third of the molecule, kell shares a 33-36% amino acid identity with human nep, ece-1, pex, and xce (lee et al., 1991) . kell has 16 cysteine residues from which the 10 cysteine residues that are preserved in all members of the m13 family are conserved. one of the cysteine residues, cys 72 , forms a disulfide band with the xk protein, which spans the membrane 10 times (russo et al., 1998) . the absence of xk, which occurs in the mcleod patients, is correlated with acanthocytic red blood cells and a late-onset form of nerve and muscle disorder . studies using a recombinant, truncated form of kell, lacking the intracellular and transmembrane domain (skell), have shown that kell possesses converting enzyme activity for the substrate big et-3. conversion of big et-1 and big et-2 is much less eycient. the known substrates for nep are not hydrolyzed by skell. individuals lacking kell do have a normal et-3 biosynthesis, so the protein probably has other substrates than big et-3 and other biological functions . the ''phosphate-regulating gene with homology to endopeptidases on the x chromosome,'' pex, was identified from studies of patients with x-linked hypophosphataemic rickets (hyp consortium, 1995) . hyp is the most common inherited disorder of renal phosphate wasting characterized by hypophosphatemia and defective bone mineralization (scriver and tenenhouse, 1992) . it is postulated that the substrate of pex is an unidentified peptide hormone (termed ''phosphatin'') that modulates renal tubular phosphate handling. such an activity could involve either the processing of a phosphate-reabsorbing hormone precursor to its active form or the inactivation of a circulating phosphaturetic factor. in situ hybridization revealed the presence of pex in osteoblasts and odontoblasts, implicating the enzyme in the development of bones and teeth (ruchon et al., 1998) . in addition to these speculations, the physiological function of the pex protein in bone development and its relation to the mechanisms that lead to renal and skeletal abnormalities remain to be defined. cloning of the full-length pex cdna revealed it to be a type ii integral membrane protein with a 20-residue n-terminal cytoplasmatic tail and a c terminus of 700 amino acid residues in the extracellular compartment. recombinant pex functions as an endopeptidase by hydrolyzing parathyroid-hormone-derived peptides, although it is unlikely that these are physiological substrates, as they do not have the properties of the sought-after phosphatin (lipman et al., 1998) . a novel member of the metalloprotease family was isolated from mouse embryos in search for an alternative ece enzyme (ikeda et al., 1999) . this enzyme, called sep (soluble secreted endopeptidase) shares higher structural (55% identity, 74% similarity) and functional similarities with nep than with eces or other members of this family, indicating that sep and nep may constitute a subfamily within this group of metalloproteases. similar enzymes have subsequently been cloned and characterized from mouse testis (referred to as neprilysin-like 1; nl 1) (ghaddar et al., 2000) and rat brain (nep ii) (tanja et al., 2000) . in vitro alternative splicing of sep yields two isoforms, one membranebound isoform (sep á ) and another isoform that is secreted into the culture medium (sep). cellular expression of the cdna showed that most of the enzyme is secreted into the culture medium. after translation, both isoforms are inserted into the er as membrane type ii proteins. sep á then becomes an er resident, whereas sep is proteolytically cleaved and transported to the extracellular compartment (raharjo et al., 2001) . the presence of a furin cleavage site in the nl1 sequence indicated that a member of the substilinlike family of convertases is responsible for this secreted form (ghaddar et al., 2000) . in vitro enzymological analysis shows that sep can eyciently hydrolyze circulating vasoactive peptides, including et-1, angiotensin-i, anp, bradykinin, and substance p. the physiological relevance of sep mainly remains to be determined. its presence in testis and brain, however, indicates roles in fertility and neuropeptide inactivation, respectively, as it appears to localize in specific neuronal populations in the cns (tanja et al., 2000) . x-converting enzyme (xce) got its name from the fact that it is an orphan peptidase for which no substrate has as yet been identified. it was originally identified by homology cloning from a human brain cdna library. it is most abundant in the cns (valdenaire et al., 1999b) . the 775-amino acidlong xce is more homologous to ece-1 (42% identity in the last 500 residues) than to nep or any other member of this family. a rat neuronal membrane-bound endopeptidase termed dine (damage-induced neuronal endopeptidase), which is upregulated in response to neuronal injury and that was identified by diverential display pcr, appears to be the rat homolog of human xce (kiryu-seo et al., 2000) . therefore, it has been suggested that this enzyme may play a role in protecting injured neurons against neuronal death. although the conservation in the xce sequence clearly indicates a peptidase function for xce, its substrate remains to be identified. candidate substrates, for which no cleavage by recombinant xce could be detected, include et-1, big et-1, calcitonin, bradykinin, enkephalins, and galanin. inactivation of the xce gene in mice resulted in neonatal lethality caused by respiratory failure (schweizer et al., 1999) , indicating a role in respiration control. involvement of xce in defects in synaptic connections or transmission is a particularly attractive hypothesis because of the specific neuronal expression pattern of xce (in the medulla oblongata), together with its putative metaiopeptidase nature, indicating that one or more neuropeptide transmitters can be considered as potential candidate substrates for this enzyme. for many years, knowledge about the m1 family of metalloproteases was limited to vertebrates. nowadays, the number of research groups reporting on the presence of these enzymes in nonvertebrate organisms is increasing. this increase began with the notification of an endopeptidase-like activity in several invertebrates and has recently evolved into the direction of molecular identification. early reports described nep-like activity/homologs in invertebrates, but in recent years it has become clear that the presence of true ece activity/homologues in invertebrates also can no longer be denied. the distinction between a nep and ece homolog is not always easily made, as both enzymes share high sequence similarity and similar enzymatic properties. however, putative diverences between the two enzymes have come forward. in vertebrates, nep activity is discriminated from ece activity on the basis of their diverence in susceptibility to the inhibitors thiorphan and phosphoramidon. however, most of the data available on endopeptidase-activity in invertebrates reveal that these inhibitors are not always as eycient as in vertebrates. furthermore, the distinction between these inhibitory profiles does not always seem to be straightforward. because no expression studies with an ece-like protein in invertebrates have as yet been done, at present there is no clarity in this matter. on the molecular level, the distinction between nep and ece is made in the et-binding domain. this sequence, nayy in vertebrate ece, is replaced by a nafy sequence in nep. this knowledge came from mutational studies with vertebrate ece that have shown that the replacement of the first tyrosine by a phenylalanine in ece leads to an 18-fold reduction of the v max /k m for the conversion of big et-1 to et-1 (sansom et al., 1998) . to date, it is not clear whether this big et substrate of ece is present in invertebrates. the analysis of ets in nonvertebrates is mainly limited to immunocytochemical studies. a small number of studies have indicated the presence of endothelin in invertebrates, using antibodies to vertebrate endothelins (hasegawa and kobayashi, 1991; kohidai and csaba, 1995; montuenga et al., 1994) . neuropeptide-degrading endopeptidase activity was found in synaptic membranes of the locust, schistocerca gregaria. the locust adipokinetic hormone (akh-1) was used as a substrate. it was inactivated by cleavage at the asn-phe bond, resembling a cleavage pattern typical for mammalian endopeptidases. phosphoramidon partially inhibited this endopeptidase activity. this was the first time that the presence of an endopeptidase-like activity in insects was reported. the activity found in this insect species was less susceptible to inhibition by phosphoramidon and thiorpan compared to its mammalian counterparts. this may reflect a diverence in inhibitor binding (isaac, 1988) . the enzyme responsible for the inactivation of the neurohormone akh is located on the surfaces of tissues; this degradative activity could not be detected in hemolymph (o'shea and rayne, 1992) . in the mollusc aplysia californica, endopeptidase activity was detected with the use of the substrate leu-enkephalin. this activity could be inhibited by thiorphan and phosphoramidon for approximately 40% (bawab et al., 1993; zappulla et al., 1999) . the presence of an identical endopeptidase-like activity was confirmed by studies in d. melanogaster wilson et al., 2002) , m. domestica (lamango and isaac, 1993) , l. dispar (masler et al., 1996) , l. maderae, and l. migratoria . these researchers studied the cleavage of either akh or locustatachykinin (lomtk)-degradation. in d. melanogaster, the degradation of lomtk-1 by head membrane fractions yields two main fragments, fygvramide and gpsgfyg. the use of the nep inhibitor phosphoramidon led to 80% inhibition of the generation of the first fragment, whereas the formation of the second fragment was inhibited by only 50%. this points toward the presence of at least two distinct nep-like enzymes that are responsible for the degradation of lomtk-1, the first resembling nep and the second one resembling ece, which is less susceptible to phosphoramidon . surprisingly, the evect of the inhibitor thiorphan, which allows diverentiation between mammalian nep and ece, was not examined in this study on d. melanogaster endopeptidases. the first evidence of an ece-like activity in insects came from studies in neuronal membranes from the locust l. migratoria, where the conversion of big et-1 to mature et-1 was found to be less susceptible to inhibition with thiorphan when compared to phosphoramidon inhibition (macours et al., 2003c) . a second reason for not subscribing this activity to a typical nep enzyme comes from the fact that nep is known to rapidly degrade mature et-1 at multiple cleavage sites (vijayaraghavan et al., 1990) . also, the use of a fluorescent substrate highly selective for ece (luciani et al., 2001) confirmed the presence of ece-activity in the locust. this ece-like activity was found to be very abundant in the reproductive system (ovaries and testes) of the locust, indicating a role in reproduction and development. a lower but substantial amount of activity was found in midgut, brain, and fat body, so multiple functions of this enzyme in insects seem likely. the first invertebrate putative endothelin-converting enzyme homologue was cloned from the coelenterate hydra vulgaris. the coelentrates represent one of the oldest phyla of the animal kingdom. to the best of our knowledge, this is the only invertebrate organism, other than insects, from which an ece homolog has been cloned and reported. this sequence shows the highest percentage of identity (37%) to human ece-1 and a substantial amount of identity to the other members of the human m13 family. on the basis of this large sequence homology and the presence of the nayy motif, this molecule was classified as an ece homolog (fig. 4) . the cystein that is responsible for the dimerization of the protein in vertebrates is absent in this invertebrate ece homolog, indicating that early forms of this enzyme existed in monomeric form. expression patterns of h. vulgaris ece during head and foot regeneration point toward a role for this enzyme in development. homogenates from this organism also contained immunodetectable levels of big et-1 and et-1. human ets are able to induce contraction in h. vulgaris, indicating the involvement of a h. vulgaris endothelin-like system in contraction of the cytoskeleton (zhang et al., 2001) . however, human big et could not exert this evect on contraction, indicating that h. vulgaris ece does not properly convert human big ets or that h. vulgaris ece works intracellularly (sarras et al., 2002) . the unambiguous confirmation of the presence of a functional endothelin-like system in h. vulgaris will have to result from the molecular identification of h. vulgaris et and its precursor big et. sequencing of the entire genome of c. elegans in 1998 (the c. elegans sequencing consortium) revealed 22 possible nep/ece-like genes in this organism. intriguingly, one of the c. elegans genes codes for a two-domain product (cf. ace in vertebrates). the temporal and spatial expression of five c. elegans nep/ece-like genes has been studied, revealing all five genes to be expressed in a tissue specific manner, indicating that these genes control diverent physiological processes (turner et al., 2001) . simultaneously with the detection of ece-like activity in the locust, the first insect ece homolog was cloned (macours et al., 2003c) . the l. migratoria ece (lomece) sequence reveals a putative type ii transmembrane protein of 727 amino acid residues, with a calculated mass of 83 kda deduced from its longest open reading frame. the sequence contains a short n-terminal cytoplasmatic tail of nine amino acids, a hydrophobic stretch representing the putative membrane-spanning domain, and a large extracellular stretch of 695 amino acids containing the characterizing active site hexxh. hence, the overall structure of this insect ece resembles that of mammalian ece. ten potential glycosylation sites are present in lomece, indicating that like vertebrate ece, the insect form is also highly glycosylated. among these glycosylation signals, asn 589 and asn 608, which are known to be important for the bioactivity of mammalian ece-1 are conserved (nelboeck et al., 1998) . the putative zinc-coordinating residue glu 624, which is located 61 residues downstream from the active site, compares with a 61-amino acid separation in mammalian ece (shimada et al., 1995) in contrast to mammalian nep, which possesses a 64-residue separation (hooper et al., 1997 ). the c-terminal cevw sequence, critical for proper enzyme maturation and activity (macleod et al., 2001) , is present in lomece, as is the endothelinbinding site nayy (fig. 4) . the cysteine residue responsible for dimerization of the protein in mammalian organisms (shimada et al., 1996) is absent in lomece, supporting the hypothesis that early divergent forms of this enzyme may have existed in monomeric form (zhang et al., 2001) . the need for a dimeric form of ece for enzyme functionality was refuted by studies with solubilized recombinant ece-1 in which dimerization did not appear to be essential or preferential for full ece activity (korth et al., 1997) . studies with a monomeric and dimeric from of bovine ece demonstrated that these two forms posses a comparable biochemical profile. the major diverence between the two forms is that the dimeric form is found to be more eycient in catalyzing the conversion of big et-1 into et-1 (kulathila et al., 2002) , sustaining the possibility of other in vivo substrates for the invertebrate ece form. the fact that ece mrna is found in a variety of tissues in the locust is indicative of a major, and most probably diversified, biological role for the enzyme. in the d. melanogaster genome (adams et al., 2000; flybase consortium, 2003) , predictions of approximately 24 members of the m13 family can be made (coates et al., 2000b) . the identification and classification of these peptidase genes was performed with gene prediction strategies, sequence comparison with known genes, and searches for active site regions by use of the merops database (rawlings et al., 2002) . sequence comparison of these genes with members of the human metalloproteases family identified the cg9565 sequence to posses the highest homology with human ece-1 (fig. 4) . an m13 homolog that contained all the essential characteristic amino acids necessary for enzymatic activity was cloned from the cns of the slug aplysia californica. this molecule was assigned nep. the sequence contains the nayy sequence in combination with a 63-amino acid separation (64 in vertebrate nep) between the active site and the third zinc-binding ligand, thus combining both ece and nep characteristics (fig. 4) . the presence of nep in the central and peripheral nervous system may indicate that it could be a major player in the modulation of synaptic transmission in a. californica and in the metabolism of neuropeptides (zappulla et al., 1999) . in b. mori, an ecdysteroid-inducible neutral endopeptidase-like gene was cloned from the wing discs (bmnep). its sequence showed 36% identity with rat-nep. it contained the same zinc-binding motif (heith) as a mammalian nep and contained a 63-residue separation between the putative third zinc-coordinating ligand glu and the active site (fig. 4) . hence, it was classified as a nep homolog. the expression of bmnep is induced by 20hydroxyecdysone (20e) and is suggested to be a secondary response gene, following the expression of bmacer, which starts within 2 hours after 20e stimulation. from this point of view, it is suggested that this peptidase is involved in the activation or inactivation of peptides in wing discs, or peptide hormones secreted from diverent organs in the course of metamorphosis (zhao et al., 2001) . in the d. melanogaster genome, the cg5894 and cg9761 sequences display the highest homology to human nep. the m2 and m13 family of metallopeptidases contain both prokaryotic and eukaryotic members. in the genome of the bacteria, gloeobacter violaceus (accession bac91084), shewanella oneidensis (accession np_71808), and xanthomonas axonopodis (accession np_64155), the predicted protein sequences of ace homolog resemble the single-domain form of invertebrate ace, with a possible signal sequence and the absence of a transmembrane domain at the c terminus of the enzyme. an invertebrate nep/ece homolog was discovered in the gram-negative bacterium, porphyromonas gingivalis. this is an important pathogen in the manifestation of acute periodontitis. this chronic inflammatory disease is the major cause of tooth loss in adults (havajee and socransky, 1994; socransky and havajee, 1992) . the proteases of these bacteria, in addition to their primary function in providing peptides for growth, are thought to be directly involved in tissue invasion and modulation of the host immune system (lamont and jenkinson, 1998) . the porphyromonas gingivalis pepo sequence (pgpepo) shows 31% sequence identity with human ece-1 and 29.9% with human nep. instead of a membrane-bound domain, it contains a putative signal sequence of 25 amino acids at the n-terminal end of the sequence, indicating that the protease could be secreted across the plasma membrane, possibly with pathological consequences as a result. all activityessential amino acids are present in the pgpepo sequence, but the 10 cystein residues that are conserved between all members of the vertebrate nep/ece family are absent. because this also includes the absence of the cystein responsible for dimerization of the ece protein, the bacterial ece homolog exists in monomer form (awano et al., 1999) . the pgpepo protein displays a substrate specificity similar to mammalian ece. it converts big et-1, -2, and -3 into their respective mature endothelin peptides and cleaves substance p, angiotensin i and ii, and neurotensin at multiple sites. urotensin ii, angiotensin iii, and oxytocin are not hydrolyzed by pgpepo . the p. gingivalis bacteria were shown to succeed in invading and multiplication within aortic endothelial cells and are localized in atherosclerotic plaques (deshpande et al., 1998 (deshpande et al., -1999 . because several associations between periodontal and coronary artery diseases have been made (beck et al., 1996) , invasion of these bacteria into cells was suggested to cause a distortion of the finely balanced equilibrium involved in et production. this may provide a possible link between periodontitis and cardiovascular diseases . experiments using a mutant form of bacteria, showing pepo-gene deficiency, showed a decreased invasion of such bacteria in mammalian cells. this indicates that pepo gene is at least involved in the first step of the bacterial cycle; namely, the invasion and lysis of the mammalian cell membrane (ansai et al., 2003) . in the merops database for peptidases (rawlings et al., 2002) , a large number of bacteria can be found that all contain at least one m13 homolog in their genome. also in archeae, m13 homolog are reported, but the majority of the archeae do not contain an m13 homolog in their genome. the presence of homolog of these enzymes in prokaryotes could perhaps have resulted from a horizontal exchange of genes between prokaryotes and eukaryotes (froeliger et al., 1999) . however, because members of the m13 family are present in eubacteria and some archeabacteria, as well as in eukaryotes, the possibility that an ultimate ancestor of the m13 family may have been present in the organism that preceded both modern bacteria and eukaryotes cannot be excluded. although the exact evolutionary relationship between eukaryotic and prokaryotic metalloproteases cannot yet be determined, the high level of sequence conservation indicates that these enzymes are under strong evolutionary constraints, thereby protecting structural features essential for enzyme activity. the amyloidogenic pathway of abpp (amyloid beta precursor protein) processing leads to the production of the neurotoxic amyloid b-peptide (ab40 or ab42). the formation of the ab peptides occurs through the initial cleavage by a b secretase, followed by the action of a g secretase (fig. 5) . the pathological accumulation of ab in the brain (formation of plaques) is the primary cause of neuronal cell death and dementia in patients with alzheimer's disease (hardy and higgins, 1992) . the degree of ab accumulation in the brain is dependent not only on its production but also on its removal. the three zinc-metalloproteases nep, ece, and possibly ace have been put forward as ab degrading enzymes, thus limiting the amount of ab accumulation in the brain. in vitro assays revealed that ace is capable of preventing the aggregation of ab and prevents the deposition of ab onto a synthetic matrix. in addition, it inhibits ab fibril formation and ab-induced cytotoxicity (hu et al., 2001) . the catabolism of ab by ace occurs through a rather unusual cleavage, between the asp 7 -ser 8 bond of ab. however, infusion of ace inhibitors in the brain of abpp transgenic mice had no evect on the level of ab present in the brain. this raises some questions concerning the significance of ace in the ab catabolism in vivo. this is in contrast to the m13 metalloproteases, nep and ece, for which the in vivo involvement has been confirmed. in vitro models have characterized ece-1 as an ab-degrading enzyme that appears to act intracellularly, thereby limiting the amount of ab available for secretion. overexpression of ece-1 in cells that lack endogenous ece activity results in a pronounced decrease in ab accumulation in the conditioned media (eckman et al., 2001) . the physiological relevance of ece activity on ab catabolism was supported by the analysis of ab levels in the brain of mice deficient for ece-1. here, significant increases in the levels of ab were detected (eckman et al., 2003) . nep cleaves the ab peptide at five possible positions, with gly 9 -tyr 10 being the initial cleavage site (howell et al., 1995; shirotani et al., 2001) . chronic injection of thiorphan in the rat brain leads to an accumulation of ab (iwata et al., 2000) . in nep-deficient mice, increased levels of ab are detected because of a reduction in the level of exogenous ab catabolism. in addition, areas in the brain of patients with alzheimer's that are enriched in plaques display reduced levels of nep (yasojima et al., 2001a,b) . all this indicates that nep and ece are rate-limiting peptidases participating in ab catabolism (fig. 5) . future research may include the upregulation of these metalloproteases as a promising strategy for the treatment and prevention of alzheimer's disease. for many years, the study of m2 and m13 metalloproteases in invertebrates lagged behind that in mammals. the first reason was that it was not logical to start searching for peptidic regulators of blood pressure in animals with an open circulatory system. second, many invertebrates are small, and harvesting enough tissue for the purification of enzymes is diycult and time consuming. molecular studies in invertebrates have expanded rapidly with the development of microtechniques that make it much easier to work with these fig. 5 schematic representation of the synthesis of ab from its precursor. the shaded area represents the membrane-bound region. the cleavage of ab by nep, ece, and ace is shown by arrows. small quantities. many of the identified invertebrate genes are also known to be in vertebrate species, where they posses similar characteristics and functions. these observations support the fact that invertebrates provide excellent model systems for the study of genetics. by comparing the human genome with those of other organisms, regions of similarity and divergence can be identified, thereby providing critical clues about the structure and function of human genes. this information may be beneficial for developing better strategies for treating and preventing some human diseases. with each molecule that is sequenced, this approach becomes more powerful. the identification of ace and ece as key components in the regulation of the mammalian blood pressure system made them interesting targets for the development of eycient antihypertensive drugs. however, the large in vitro substrate specificity and the recent discovery of their involvement in alzheimer's disease asks for caution in the use of ace and ece inhibitors as therapeutic drugs. the large cellular and tissue distribution of ace and ece in the human body also points toward the possibility of as-yet-unknown physiological roles of these enzymes. the idea that invertebrate counterparts of these enzymes function as regulators of blood pressure is very unlikely. hence, these organisms can be useful in the clarification of these extra enzymatic functions. future research concerning the roles of these enzymes will have to involve the identification of new substrates and inhibitors. possible substrates can be tested on their in vivo characteristics by colocalization studies and knockout experiments. from the above listed data, it is clear that our knowledge about the occurrence and physiology of the m2 peptidase family in invertebrates is beginning to take shape, whereas research on the m13 peptidase family members is still in its infancy. available information on these enzyme families in invertebrates is restricted to ace, nep, and ece. however, the exploration of invertebrate genomes that have already been published indicates that a large number of m2 and m13 family members is present. further data mining of invertebrate genomes in combination with the recombinant expression of isolated sequences might lead to the identification of homologs to ace2, kell, sep, xce, ece-2, and ece-3 in invertebrates. the genome sequence of drosophila melanogaster a specific endopeptidase baee esterase, in the glandula prostatica of the male reproductive system of the silkworm, bombyx mori auginine carboxypeptidase activity in the male reproductive glands of the silkworm, bombyx mori construction of a pepo gene-deficient mutant of porphyromonas gingivalis: potential role of endopeptidase o in the invasion of host cells cloning and expression of a cdna encoding an endothelin a receptor sequencing, expression and biochemical characterization of the porphyromonas gingivalis pepo gene encoding a protein homologous to human endothelin-converting enzyme angiotensin i-converting enzyme and metabolism 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endopeptidase 24.11 loss in metastatic human prostate cancer contributes to androgen-independent progression analysis of venom constituents from the parasitoid wasp pimpla hypochondriaca and cloning of a cdna encoding a venom protein a live-cell assay for studying extracellular and intracellular endothelin-converting enzyme activity a model of the ace2 structure and function as a sars-cov receptor the diverse molecular mechanisms responsible for the actions of opioids on the cardiovascular system isolation and expression of the ecdysteroid-inducible angiotensin-converting enzyme-related gene in wing discs of bombyx mori alternative splicing regulates the endoplasmatic reticulum localization or secretion of soluble secreted endopeptidase invertebrate immunity: basic concepts and recent advances selective restoration of male fertility in mice lacking angiotensin-converting enzymes by sperm-specific expression of the testicular isozyme merops: the protease database kell, kx and the mcleod 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and catalysis the angiotensin-converting enzyme gene family: genomics and pharmacology mammalian membrane metallopeptidases purification and characterization of a peptidyl dipeptidase resembling angiotensin converting enzyme from the electric organ of torpedo marmorata the neprilysin (nep) family of zinc metalloendopeptidases: genomics and function aceh/ace2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors. can a fourth isoform of endothelin-converting enzyme (ece-1) is generated from an additional promoter molecular cloning and characterization xce, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in the cns organization of the gene encoding the human endothelin-converting enzyme (ece-1) biochemical identification and ganglionic localization of leech angiotensin-converting enzymes isolation and characterization of an angiotensin converting enzyme substrate from vittelogenic ovaries of neobellieria bullata presence of an angiotensin converting enzyme (ace) interactive factors in ovaries of the grey fleshfly neobellieria bullata captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfley successful parasitation of locusts by entomopathogenic nematodes is correlated with inhibition of insect phagocytes immonocytochemical distribution of angiotensin-i converting enzyme in the central nervous system of insects and speculations about its possible function kappa and delta opioid receptor stimulation avects cardiac myocyte function and ca2+ release from an intracellular pool in myocytes and neurons hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase the hydrolysis of endothelins by neutral endopeptidase 24 the two homologous domains of human angiotensin i-converting enzyme are both catalytically active diminution of contractile response by kappa-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive g protein cloning and characterisation of angiotensin-converting enzyme from the dipteran species, haematobia irritans exigua, and its expression in the maturing male reproductive system expression of angiotensin-converting enzyme-related carboxydipeptidases in the larvae of four species of fly drosophila melanogaster angiotensin i-converting enzyme expressed in pichia pastoris resembles the c domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity extracellular peptidases of imaginal discs of drosophila melanogaster the sars-cov s glycoprotein: expression and functional characterization disruption of ece-1 and ece-2 reveals a new role for endothelin-converting enzyme 2 in murine cardiac development dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption 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grey fleshfly, neobellieria bullata key: cord-017585-0llgr357 authors: chappell, mark c. title: role of ace, ace2 and neprilysin in the kidney date: 2007 journal: frontiers in research of the renin-angiotensin system on human disease doi: 10.1007/978-1-4020-6372-5_1 sha: doc_id: 17585 cord_uid: 0llgr357 nan from the initial description of renin activity over a century ago, ongoing study of the renin-angiotensin-aldosterone system (raas) continues to yield novel findings that redefine the functional nature of this system, as well as reveal the complexity of the interplay among the various raas components. indeed, the recent discoveries of angiotensin converting enzyme 2 (ace2) donoghue et al 2000; tipnis et al 2000) ; the renin receptor (nguyen et al 2002) and the angiotensin-(1-7) [ang-(1-7)] receptor (santos et al 2002) represent important examples of our evolving concepts of the raas and cardiovascular regulation. coupled with the emerging view that the raas is not defined as simply an endocrine system, these local or tissue systems may exhibit distinct functional and processing pathways (chappell et al 1989; chappell et al 2004; paul et al 2006) . the kidney is clearly an important target organ of the circulating raas, particularly the actions of ang ii and aldosterone to promote sodium and water reabsorption, as well as their influence on the progression of tissue injury and fibrosis (harris 1999) . the kidney also exhibits a local raas that expresses ang ii, ang-(1-7), and multiple ang receptor subtypes that mediate the distinct actions of these two peptides in both normal and pathophysiolgocial conditions such as hypertension or diabetes (burns 2000; carey & siragy 2003; navar et al 2000) . figure 1 illustrates one current view of the renal raas network that emphasizes the distinct synthetic pathways of ang ii and ang-(1-7), as well as functional actions mediated by the at 1 , at 2 and at 1−7 receptors. although the emergence of receptor subtypes distinguishes the distinct signaling pathways of ang ii and ang-(1-7), the post-renin enzymes that form and degrade these peptides must be considered in lieu of the overall regulation of the functional raas within the kidney. the inclusion of ang-(1-12) as a potential intermediate in ang ii formation via a renin-independent pathway reflects the recent 1 figure 1 . scheme that depicts the processing pathways involved in the formation and degradation of angiotensin ii (ang ii) and ang-(1-7) within the kidney. ang ii binds to either at 1 or at 2 receptor (r) subtypes, while ang-(1-7) recognizes an at 1−7 r. ace, angiotensin converting enzyme; eps, endopeptidases; nep, neprilysin demonstration of endogenous levels of the peptide in the kidney, circulation and other tissues (nagata et al 2006) . in this chapter, the roles of ace2, ace and the endopeptidase neprilysin in the functional expression of the intrarenal hormones of the raas are reviewed. ace may be considered the activation step in the catalytic cascade for the formation of ang ii from ang i (fig. 1) . although evidence of non-ace pathways for biosynthesis of ang ii is evident (sadjadi et al 2005a; tokuyama et al 2002) , ace likely represents the major, if not sole enzyme responsible for ang ii formation under normal physiological conditions in humans and other species. this is not to imply that ace has no other substrates than ang i (see below), but that a primary role for ace is the generation of ang ii. indeed, the identification of ace and the characterization of the enzymatic properties must be considered a pivotal achievement in our understanding of the raas and cardiovascular disease, as well as leading to the successful development of ace inhibitors in the treatment for hypertension and renal disease. ace is a metallopeptidase composed of a single monomeric protein. somatic ace contains two catalytic regions designated as the amino (n) and carboxy (c) domains. selective inhibitors against both catalytic domains of somatic ace are now available, however, the functional significance of the two domains is presently unknown (dive et al 1999; georgiadis et al 2003) . the enzyme cleaves two residues from the carboxy end of various peptides and, hence, its description as a dipeptidyl-carboxypepitdase. within the kidney, somatic ace is primarily a glycosylphosphatidylinositol-anchored membrane protein and the majority of the enzyme including both catalytic regions faces the extracellular space. ace is localized throughout the kidney with high concentrations in vascular endothelial cells, proximal tubules and interstitial cells. ace is also released from the apical surface of epithelial cells into the proximal tubular fluid and likely contributes to the urinary levels of the enzyme (hattori et al 2000) . indeed, the tubular fluid should be considered a distinct intrarenal compartment that contains raas processing enzymes and the peptide products may interact with ang receptors along the entire tubular area of the kidney. the release of ace from the cell membrane is a specific process as releasing enzymes or "sheddases" have been identified that recognize a unique motif on the stalk region of the enzyme (beldent et al 1993) . the conversion of membrane-bound ace to a soluble form does not appear to substantially alter the substrate preference or the catalytic properties of the enzyme. although the significance of this event is not currently understood, enzyme shedding may underlie an endocrine process to transport ace to more distal areas of the nephron that are deficient in this peptidase activity for the discrete production of ang ii. in this regard, casarini and colleagues have presented intriguing data that the urinary excretion of the n-terminal domain of ace may serve as a urinary marker in both humans and experimental hypertensive models (marques et al 2003) . extensive evidence suggests that intrarenal ace participates in the direct formation of ang ii from ang i. the renal administration of ace inhibitors reduces interstitial levels of ang ii and attenuates blood pressure. moreover, in an animal model of tissue-depleted ace that preserves circulating levels of the enzyme, renal ang ii is significantly reduced (modrall et al 2003) . interestingly, intrarenal levels of ang i were also markedly reduced in the tissue ace null mouse while renal ang-(1-7) concentrations were maintained (modrall et al 2003) . these data serve to emphasize that ace participates in the metabolism of other peptide hormones (skidgel & erdos 2004) . in the case of ang-(1-7), ace efficiently metabolizes the peptide to ang-(1-5), a product which is presently not known to exhibit functional activity deddish et al 1998; rice et al 2004) . we have postulated that the formation of ang-(1-7), particularly under prolonged activation of the raas, is considered to balance or attenuate the constrictor and proliferative actions of ang ii ferrario et al 2005c) . indeed, ang-(1-7) exhibits vasodilatory, natriuretic and anti-proliferative actions through the stimulation of nitric oxide and arachidonic acid metabolites (sampaio et al 2007) . ang-(1-7) abrogates the ang ii-dependent activation of map kinase in primary cultures of proximal tubule epithelial cells (su et al 2006) . moreover, the inhibitory actions of ang-(1-7) were blocked by the ang-(1-7) antagonist [d-ala 7 ]-ang-(1-7) suggesting a receptor mediated pathway distinct from either at 1 or at 2 receptor subtypes (su et al 2006) . similar effects of ang-(1-7) were originally demonstrated in non-renal cells (tallant et al 2005a) . in the circulation, ace inhibitors increase circulating levels of ang-(1-7) and augment the in vivo half life of the peptide by almost 6 fold yamada et al 1998) . the urinary excretion of ang-(1-7) increases in both human and experimental hypertensive models following acute administration of ace inhibitors yamada et al 1999) . the increased excretion of ang-(1-7) most likely reflects the reduced intrarenal metabolism of the peptide and the efficient shunting of the ang i pathway to formation of ang-(1-7). our recent studies in isolated sheep proximal tubules reveal that without prior inhibition of ace, ang-(1-7) derived from either ang i or ang ii was rapidly converted to ang-(1-5) (shaltout et al 2007) . blockade of ang-(1-7) partially reverses the beneficial actions of ace inhibitors on blood pressure in hypertensive rats as an ang-(1-7) monoclonal antibody or the [d-ala 7 ]-ang-(1-7) antagonist increase blood pressure (iyer et al 1997; iyer et al 2000) . moreover, studies by benter and colleagues find that the renoprotective effects of exogenous ang-(1-7) in lname-treated shr were not further improved with the ace inhibitor captopril (benter et al 2006a) . apart from ang ii and ang-(1-7), renal ace may also participate in the metabolism of other peptides including kinins, substance p and the hematopoietic fragment acetyl-ser-asp-lys-pro (ac-sdkp). bradykinin-(1-9) is very rapidly metabolized by ace in a two -step process to the inactive fragments bradykinin-(1-7) and bradykinin-(1-5). ace inhibition is associated with increased circulating and tissue levels of bradykinin-(1-9) and the renal content of kinin is higher in the tissue ace null mouse (campbell et al 2004) . in general, bradykinin is a potent vasodilator and inhibitor of cell growth through stimulation of nitric oxide, as well as exhibiting natriuretic actions within the kidney (scicli & carretero 1986) . interestingly, santos and colleagues have reported that the functional activity of ang-(1-7), under certain conditions, is dependent on the increased release of bradykinin (fernandes et al 2001) . moreover, the kinin b2 receptor antagonist hoe140 blocked nitric oxide release by the non-peptide ang-(1-7) agonist ave0991 (wiemer et al 2002) . similar to ang-(1-7), circulating levels of the ac-sdkp are markedly increased with ace inhibition and the enzyme cleaves the lys-pro bond of the tetrapeptide (azizi et al 1997; raousseau et al 1995) . although current evidence does not support a role for ac-sdkp in the regulation of blood pressure, the peptide does exhibit potent anti-fibrotic and anti-inflammatory actions (peng et al 2003) . indeed, exogenous administration of ac-sdkp attenuates proteinuria and improves renal function in several models of renal injury and hypertension (omata et al 2006) . interestingly, ang-(1-7) and ac-sdkp may be the only known endogenous substrates that are exclusively cleaved by the n-terminal catalytic domain of human ace (raousseau et al 1995; deddish et al 1998) . moreover, prolyl (oligo)endopeptidase, an enzyme that processes ang i or ang ii to ang-(1-7) in endothelial and neural cells (chappell et al 1990; santos et al 1992) , may also convert thymosin-2 to ac-sdkp in plasma and tissue (cavasin et al 2004) . the unusual specificity of the n-domain of ace for ang-(1-7) and ac-sdkp suggests an overlap of the activities of these two peptide systems within the kidney as well. although elucidation of the signaling mechanisms and receptors for ang-(1-7) and ac-sdkp is at an early stage, future studies should consider whether there is a basis for the functional similarities between these peptides. the role of raas enzymes including ace and renin has been primarily emphasized for their catalytic properties; however, compelling evidence now reveals receptor-like properties for these two enzymes. indeed, a renin receptor was recently cloned by nyguen and colleagues with significant concentrations of the protein in the glomerulus and vascular smooth muscle cells. (diez-freire et al 2006; nguyen et al 2002) . receptor-bound renin exhibits increase proteolyitc activity for ang i formation, but both pro-renin and renin also induce distinct signaling pathways following binding. in isolated mesangial cells, exogenous renin increased tgf-expression and other matrix proteins including plasminogen activator inhibitor (pai-1) and fibronectin that was apparently independent of ang ii synthesis (huang et al 2006) . ace inhibitors may also induce cell-specific signaling by inducing conformational changes in membrane-bound ace without alterations in ang ii or other peptides (benzing et al 1999) . two kinases, c-jun kinase and map kinase kinase 7 associate with the intracellular portion of ace. moreover, ace inhibitors increase the phosphorylation and nuclear trafficking of phosphorylated cjun kinase (kohlstedt et al 2002) . this aspect of ace-dependent activation of various kinases has been demonstrated in human endothelial cells and the question remains as to what extent this occurs in other cells or tissues. in addition, ace inhibitors or the angiotensin peptides ang-(1-9) and ang-(1-7) induce the association of ace and the bradykinin b2 receptor that prevents the rapid down-regulation of the ligand-receptor complex, thus potentiating the actions of bradykinin (burckle et al 2006; chen et al 2005) . almost 50 years following the discovery of ace, a new homolog of the enzyme termed ace2 was identified by two separate groups (donoghue et al 2000; tipnis et al 2000) . ace2 activity is not attenuated by ace inhibitors nor does the enzyme share the same catalytic properties. in this regard, ace2 contains a single zincdependent catalytic site that corresponds to the c-terminal domain of somatic ace. ace2 exhibits carboxypeptidase activity cleaving a single amino acid residue at the carboxyl terminus of various peptides. the original studies assessed ang i as the peptide substrate for ace2, given the similar homology to ace and the existing evidence for ace-independent pathways; however, ace2 converted ang i to the nonapeptide ang-(1-9) (donoghue et al 2000) . this product is currently not known to exhibit functional activity, but may serve as a substrate for the further processing to ang ii or ang-(1-7) . the subsequent kinetic studies of over 120 peptides found that the conversion of ang ii to ang-(1-7) was much preferred over that for ang i (vickers et al 2002) . indeed, ace2 exhibits an approximate 500-fold greater kcat/km for ang ii versus ang i and has the highest efficiency among the known ang-(1-7)-forming enzymes (fig. 2) . these studies also revealed that other peptides including apelin 13 and dynorphin are cleaved by ace2 at a similar or slightly greater efficiency than ang ii (vickers et al 2002) . at this time, the majority of studies have focused on the role of ace2 in the metabolism of angiotensins (see below), principally ang ii to ang-(1-7), and the role of ace2 in the processing of apelin or other peptides has not been sufficiently addressed. similar to ace, ace2 exists in both soluble and membrane-associated forms with high expression in the kidney, heart, brain, lung and testes (harmer et al 2002) . although there is significant circulating ace activity in various species, plasma levels of ace2 are quite low, but may vary among species (elased et al 2006; rice et al 2006) . recent studies in the sheep reveal appreciable ace2 in the plasma, albeit the activity was significantly lower than that for ace (fig. 3 , inset) (shaltout et al 2007) . for this assessment, we compared the enzyme activities using the endogenous substrates for both ace and ace2 (ang i and ang ii, respectively) data are n = 4-5, mean ± sem;* p < 0.05 vs. control at equimolar concentrations under identical incubation conditions. interestingly, as shown in fig. 3 , male sheep exhibited higher ace2 activity than females that likely contributes to the lower half-life (t 1/2 ) of serum ang ii in males . addition of the specific ace2 inhibitor abolished the conversion of ang ii to ang-(1-7) as measured by a hplc-125 i-detector and markedly increased the ang ii-[t 1/2 ] in both male (6 fold) and female (3 fold) sheep (fig. 3) . these ex vivo data in sheep serum demonstrate that circulating ace2 constitutes a major pathway in the metabolism of ang ii and support the increase in circulating ang ii levels in the ace2 null mouse . furthermore, we did not find that soluble ace2 in the serum contributed to the direct conversion of ang i to ang-(1-9) even in the presence of complete ace inhibition (shaltout et al 2007) . within the kidney, ace2 is primarily localized to the apical aspect of the proximal tubule epithelium. indeed, expression of ace2 in the renal mdck cell line revealed exclusive trafficking of the enzyme to the apical side, while the distribution of expressed ace was different, trafficking to the basolateral and luminal aspects of the cell (guy et al 2005) . consistent with the apical expression of ace2 in the renal epithelium, we found significant urinary ace2 activity that converted ang ii to ang-(1-7), but did not process ang i to ang-(1-9) (shaltout et al 2007) . the glycosylated form of ace2 is approximately 120,000 daltons and the filtration of the enzyme into the tubular fluid is highly unlikely (shaltout et al 2007) . in this regard, lambert and colleagues report that the metallopeptidase adam 17 may function as a secretase to release ace2 from extracellular side of the cell membrane . interestingly, adam 17 does not release ace suggesting that the regulation for the secretion for ace and ace2 is distinct. the localization of ace2 in the proximal tubule epithelium along with other elements of the ras (ace, angiotensinogen, ang receptors) supports a role for the enzyme in the processing of angiotensin peptides. in the rat kidney, burns and colleagues found no evidence that ace2 or other peptidases metabolize ang ii in proximal tubule preparations or in perfused proximal tubule segments isolated from male sprague dawley rats . however, ace2 activity was clearly evident in the rat tubules as the conversion of exogenous ang i to ang-(1-9) was sensitive to the ace2 peptide inhibitor dx-600 . ang-(1-9) was subsequently converted to ang-(1-7) by ace, a pathway similar to that reported for ang i metabolism in isolated cardiomyocytes (donoghue et al 2000) . in contrast to the rat, we found that ace2 was the predominant activity to convert ang ii to ang-(1-7) in sheep proximal tubules (shaltout et al 2007) . the addition of the nonpeptide ace2 inhibitor mln-4760 significantly attenuated the metabolism of ang ii at early time points. however, as shown in fig. 4 , the significant ace and neprilysin activities required prior inhibition to protect ang-(1-7) from rapid degradation in the proximal tubules. we could not demonstrate that ace2 participated in the direct metabolism of ang i, particularly under conditions where other enzymatic pathways were blocked (shaltout et al 2007) . indeed, ang i was directly converted to ang ii and ang-(1-7) via ace and neprilysin, respectively. the preferred conversion of ang ii to ang-(1-7) by ace2 in the sheep kidney is entirely consistent with kinetic studies on various peptide substrates by the human enzyme (rice et al 2004; vickers et al 2002) , as well studies in membrane fractions of mouse kidney and rat renal cortex that demonstrated ace2-dependent conversion of ang ii to ang-(1-7) ( elased et al 2006; ferrario et al 2005b) . an explanation for the discrepancy in the metabolism studies for angiotensin metabolism is not readily apparent; however, if the rat exhibits different kinetic properties for ang i and ang ii than sheep or human, then the role of ace2 is likely to be quite different among species. additionally, these studies have important figure 4 . ace2 inhibition blocks the conversion of ang ii to ang-(1-7) in isolated proximal tubules from female sheep. sequential addition of peptidase inhibitors on the formation of ang ii metabolites include: ap, amniopeptidase (amastatin, bestatin); chy, chymase, carboxypeptidase a (chymostatin, benzyl succinate); cy, cysteine protease (pcmb); nep, neprilysin (sch3977), ace, angiotensin converting enzyme (lisinopril); ace2 (mln-4760). data are n = 4, mean ± sem implications on the role of ace as well, particularly whether ace is involved in the formation or degradation of ang-(1-7) chappell et al 2000; yamada et al 1998) . although campbell and colleague demonstrate significant quantities of endogenous ang-(1-9) in the rat kidney (campbell et al 1991) , chronic ace inhibition or combined ace/at 1 blockade (chappell, unpublished observations) did not attenuate renal ang-(1-7) levels in the rat. in addition, ang-(1-7) levels within the kidney were maintained in tissue ace knockout mice (modrall et al 2003) . thus, these in vivo studies do not strongly support an ace2-ace cascade leading to the formation of ang-(1-9) and ang-(1-7) in the kidney. the molecular studies utilizing ace2 knockout mice provide additional evidence for the enzyme's role to balance the expression of ang ii and ang-(1-7). we originally showed that ace2 null mice exhibit higher circulating and tissue levels of ang ii . indeed, the increased ratio of renal ang ii to ang-(1-7) may contribute to the renal pathologies observed in older ace2 null mice (oudit et al 2006) . furthermore, the incidence of glomerulosclerosis and proteinuria in the male mice was markedly attenuated by at 1 receptor blockade. several hypertensive models including the spontaneously hypertensive rat (shr), stroke-prone shr and sabra salt sensitive rat exhibit lower mrna levels and protein expression for ace2 in the kidney than normotensive controls zhong et al 2004) , as well as human prehypertensives (keidar et al 2006) . tikellis and colleagues find that renal ace2 expression is actually higher in the shr than wky normotensive controls at day one following birth, similar at 42 days and then dramatically declines in adult shr by 80 days (tikellis et al 2006) . ace activity, however, was markedly lower in the shr kidney at all time points measured and declined in both strains at 80 days. apart from the interesting pattern of development for ace2 in the kidney, these data emphasize the need to at least consider alterations in both ace and ace2 in characterizing the functional output of the raas. moreover, parallel studies to document the changes in renal ang ii and ang-(1-7) during this developmental period are critical to establish the relevance to altered ace and ace2. it is clear that not all hypertensive models exhibit reduced ace2 in the kidney. our studies in the male mren2.lewis rat, a model of tissue renin expression with increased renal ang ii, found no difference in renal cortical ace2 activity as compared to the normotensive lewis strain, although cardiac activity was indeed lower in the hypertensive rats ferrario et al 2005b; pendergrass et al 2006) . chronic blockade with either an ace inhibitor or at 1 antagonist increased ace2 activity in the kidneys of both the mren2.lewis and lewis rats, but enzyme activity was significantly higher in the normotensive strain following treatment . this may reflect the situation where raas blockade does not completely reverse the extent of renal injury in the male mren2.lewis model. in this regard, the reduced ace2 and elevated renal ang ii in the injured kidney of albumin-loaded rats was associated with increased nf-b expression (takase et al 2005) . in contrast, ace2 and its product ang-(1-7) increase in the kidney of the rat during pregnancy . it is well known that the raas is activated during pregnancy, yet blood pressure is not altered in normal pregnancy, and it will be of interest to determine whether ace2 expression within the kidney is altered with pre-eclampsia. diabetic nephropathy is clearly dependent on an activated raas and both ace inhibitors and at 1 receptor antagonists are effective in attenuating the progression of injury. indeed, the renal expression of ace2 is reduced in the proximal tubules of the streptozotocin-induced model of type i diabetes (tikellis et al 2003; wysocki et al 2006) . moreover, the attenuation of renal injury in this model by ace inhibition is associated with increased ace2 expression. a protective role for renal ace2 is also evident from the findings that chronic ace2 inhibition in the diabetic db/db mice exacerbates the extent of albuminuria almost 3-fold . although angiotensin content was not measured, the db/db mice exhibited increased glomerular expression of ace and reduced ace2 as compared to the control db/dm mice. interestingly, the localization studies revealed distinct patterns of staining for ace2 and ace within the glomerulus -ace2 in podocytes and ace in the endothelial cells (liebau et al 2006) . ang-(1-7) or the nonpeptide agonist ave0991 attenuates proteinuria and improves renal vascular activity in the streptozoticin type 1 diabetic rat, but did not reverse the urinary excretion of lysozyme, a marker of tubulointerstitial damage (benter et al 2007) . moreover, the ratio of ang-(1-7) to ang ii formed from exogenous ang i was lower in glomeruli isolated from the kidneys of diabetic rats, however, the identity of the ang-(1-7)-forming activity was not determined in this study (singh et al 2005) . thus, in addition to the proximal tubule epithelium, the glomerulus may be a second key site within the kidney where ace2 may influence the local expression of angiotensin peptides and renal function. in the kidney, the endopeptidase neprilysin constitutes significant peptidase activity, particularly within the brush border region of the proximal tubules. similar to ace and ace2, neprilysin is a zinc-dependent metallopeptidase that is anchored to the apical or extracellular region of the membrane, but is apparently resistant to enzymatic shedding. although neprilysin was initially recognized for its enkephalindegrading activity and frequently referred to as "enkephalinase", studies now reveal that this enzyme contributes to the metabolism of various peptides with cardiovascular actions including adrenomedullin, angiotensins, kinins, endothelins, substance p and the natriuretic peptides (skidgel & erdos 2004) . indeed, the development of neprilysin inhibitors, and more recently, dual or mixed inhibitors that target ace as well remain potential therapies in cardiovascular disease (veelken & schmieder 2002) . in general, these dual inhibitors were either equally or more effective in lowering blood pressure and reducing renal injury as compared to monotherapy with an ace or neprilysin inhibitor (kubota et al 2003; tikkanen et al 2002) . however, two large clinical trials (octave, overture) with the mixed inhibitor omapatrilat revealed an increased incidence of angioedema. moreover, the drug was no more effective than an ace inhibitor alone (kostis et al 2004; packer et al 2002) . a subsequent experimental study has shown that omapatrilat inhibits amniopeptidase p and, although less potent than its actions against ace and neprilysin, this may further augment the local concentrations of kinins or substance p to exacerbate vascular permeability (sulpizio et al 2005) . in this aspect, the development of more selective inhibitors against ace and neprilysin may be of clinical benefit. the rationale for neprilysin inhibition primarily resides in preserving the "cardioprotective" peptides bradykinin and anp or bnp. however, neprilysin readily metabolizes ang ii to the inactive fragment ang-(1-4) which undergoes further hydrolysis to the dipeptides asp-arg and val-tyr. neprilysin also cleaves endothelin, although it is not clear to what extent reduced intrarenal levels of endothelin are beneficial given the functional diversity of the endothelin a and b receptor subtypes within the kidney (schiffrin 1999) . the additional ace inhibition would prevent the accumulation of ang ii and further contribute to the protection of kinins, as well as possibly reduce endothelin release. one possible caveat to this approach is that neprilysin is the major ang-(1-7)-forming activity from ang i or ang-(1-9) in the circulation (campbell et al 1998; yamamoto et al 1992) . indeed, acute administration of the potent neprilysin inhibitor sch3977 reduced circulating levels of ang-(1-7) and increased blood pressure in the shr chronically treated with the ace inhibitor lisinoropril (iyer et al 1997) . although plasma levels of neprilysin are low to non-detectable, the enzyme is appropriately localized to the ectocellular surface of endothelial and smooth muscle cells to contribute to the formation of ang-(1-7) within the vasculature (llorens-cortes et al 1992) . in the kidney, neprilysin may contribute to both the formation as well as the degradation of the ang-(1-7) (allred et al 2000) . neprilysin cleaves the pro 7 -phe 8 bond of ang i to ang-(1-7), but the very high levels of the enzyme in the kidney may continue to metabolize ang-(1-7) at the tyr 5 -ile 6 bond to form ang-(1-4) and ang-(5-7) (allred et al 2000; chappell et al 2001) . indeed, the mixed inhibitor omapatrilat augmented the urinary levels of ang-(1-7) in both human hypertensives and the shr model (ferrario et al 2002a; ferrario et al 2002b) . the clinical study revealed a strong correlation between the reduction in blood pressure and increased excretion of ang-(1-7) with the dual peptidase inhibitor (ferrario et al 2002a) . interestingly, chronic treatment of male shr with omapatrilat (2 weeks, 30 mg/kg daily) was also associated with the increased renal expression of ace2. as shown in fig. 5 , immunocytochemical studies demonstrate enhanced expression figure 5 . increased expression of ang-(1-7) and ace2 in the renal cortex of shr following treatment with omapatrilat. immunocytochemical staining for ang-(1-7) in control (a) and treated (b) shr; ace2 staining in control (c) and treated (d), group. ace2 staining in renal artery of treated shr; arrow indicates intimal layer (e). renal cortical ace2 mrna levels are significantly increased 2-fold following omapatrilat treatment (f); inset: ace2 and ef-1 bands in the presence of the specific rt primers (rt+). data are n = 7-8, mean ± sem of both ace2 and ang-(1-7) within the renal cortex of the treated-shr . omapatrilat treatment also revealed the renal vascular expression of ace2 with staining evident in the intimal, medial and adventitial regions of the renal artery (fig. 5e ); vascular staining for the enzyme was undetectable in the untreated shr group (fig. 5c ). cortical mrna of ace2 expressed as a ratio to ef-1 increased 2-fold suggesting that transcriptional regulation contributes to the enhanced expression of ace2 within the kidney (fig. 5f ). these studies are of interest as they reveal an additional mechanism of the vasopeptidase inhibitor that may result in the enhanced conversion of ang ii to ang-(1-7) by ace2, as well as protecting ang-(1-7) from both neprilysin-and ace-dependent degradation within in the kidney. furthermore, these data suggest an important ability of the dual peptidase inhibitor (as well as the administration of other raas inhibitors alone) to restore ace2 levels in the hypertensive kidney which may mitigate against the ang ii-at 1 receptor axis of the raas. indeed, raizada and colleagues show that lenti-viral expression of ace2 has amelioratory effects on blood pressure and cardiac fibrosis in the shr, although the renal effects of enhanced enzyme activity were not ascertained (diez-freire et al 2006) . their data clearly demonstrate that ace2 can markedly alter the balance of an activated raas pathway towards a normotensive phenotype. further study is required to determine the extent that the beneficial actions of increased ace2 reflect the greater inhibition of ang ii or the increased accumulation of ang-(1-7) in the kidney or other tissue. the positive influence of ace2 in the shr kidney following blockade of ace and neprilysin emphasizes the complex regulation of raas components within the kidney (see fig. 6 ). we have also shown that ace inhibition alone or at 1 receptor antagonism increases either renal ace2 mrna or activity (igase et al 2005; ferrario et al 2005b) . consistent with these data in the intact animal, gallagher figure 6 . a potential regulatory scheme for the stimulatory and inhibitory pathways of the reninangiotensin-aldosterone system within the kidney. ace, angiotensin converting enzymes; aogen, angiotensinogen; aldo, aldosterone; at 1 r, angiotensin type 1 receptor; jg, juxtaglomerular and colleagues demonstrate that ang ii directly down regulates ace2 through activation of the at 1 receptor . although ang-(1-7) alone did not influence the basal expression of ace2 in these cells, the peptide attenuated the inhibitory effects of ang ii on ace2 via a receptor-dependent mechanism . in contrast to the negative influence on ace2, ang ii increases ace expression within the kidney sadjadi et al 2005b) . ang ii also positively influences the expression of its precursor protein angiotensinogen (kobori et al 2001; zhang et al 2002) , and in selective areas of the kidney, either maintains or up regulates the at 1 receptor as well . this effect on the at 1 receptor may also lead to increased renal levels of ang ii via receptor mediated uptake and stable sequestration of the circulating peptide (ingert et al 2002) . in contrast, ang-(1-7) can down regulate the at 1 receptor through stimulation of a cylocoxygenase pathway (clark et al 2003; clark et al 2001) . there are few studies on the regulation of the ang-(1-7) receptor, although chronic ace or at 1 blockade reduced mas mrna expression in the renal cortex of the ren2 lewis congenic rat . consistent with the positive feedback concept, the current evidence suggests that aldosterone down regulates ace2 (keidar et al 2005; tallant et al 2005b) while the mineralocorticoid increases expression of ace, at 1 receptor, renin and intrarenal ang ii (bayorh et al 2006; klar et al 2004; schiffrin 2006) . thus, the renal raas appears to function in a positive regulatory manner on these components to promote or maintain ang ii content. in this regard, ace2 may serve as an important mechanism to break or reduce the positive gain of the system for ang ii production or enhanced signaling in the kidney. although ang ii potently reduces juxtaglomerular (jg)derived release and expression of renin, renin is not suppressed but increases in the collecting duct and distal tubules (prieto-carrasquero et al 2004) . the negative feedback by ang ii on jg renin may also be balanced by renin-independent pathways that contribute to the formation of ang i. alternatively, nagata and colleagues (nagata et al 2006) find significant concentrations of the novel peptide ang-(1-12) in the kidney and other tissues that may not require renin for the peptide's synthesis (see fig. 1 ). the infusion of ang-(1-12) produced an immediate increase in blood pressure that was abolished by either an ace inhibitor or an at 1 receptor antagonist (nagata et al 2006) . these data suggest that following formation of ang-(1-12), the peptide or its intermediate is converted to ang ii by ace. the elucidation of the enzyme(s) responsible for the formation of ang-(1-12) and the factors that influence its expression may greatly contribute to our understanding the regulation of the intrarenal raas, particularly under pathophysiological conditions. the majority of experimental studies on the raas and the regulation of blood pressure have utilized male animals. as there is overwhelming evidence for sex differences in the extent of hypertension and cardiovascular injury, the consideration of gender in the regulation of the renal raas enzyme cascade should be carefully considered (bachmann et al 1991; brosnihan et al 1999; reckelhoff et al 2000) . for example, the presence of renal damage in the male ace2 knockout mice was not evident in the estrogen replete female littermates and possibly there is greater expression of raas components in the males with the loss of ace2 (oudit et al 2006) . we and others have shown that estrogen depletion is associated with altered expression of renin, at 1 receptors, ace and nos isoforms, as well as exacerbates hypertension and salt-sensitive renal injury (bayorh et al 2001; brosnihan et al 1997; chappell et al 2003; chappell et al 2006; harrison-bernard et al 2003; roesch et al 2000; yamaleyeva et al 2007) . moreover, in lieu of the negative outcomes for estrogen or combined hormone replacement in older women (hers, whi), the influence of aging on the response of the intrarenal raas and other systems may be of equal importance. clearly, understanding the regulation and interplay of ace, ace2 and neprilysin within the kidney, as well as other areas including the heart, brain and vascular beds are critical to treating the burgeoning problem of cardiovascular disease. -7) metabolism in pulmonary and renal tissues high plasma level of n-acetyl-seryl-aspartyl-lysyl-proline: a new marker of chronic angiotensin-converting enzyme inhibition sexual dimorphism of blood pressure: possible role of the renin-angiotensin system effects of enalapril, tempol, and eplerenone on salt-induced hypertension in dahl salt-sensitive rats the role of gender in salt-induced hypertension proteolytic release of human angiotensinconverting enzyme: localization of the cleavage site angiotensin-(1-7) prevents development of severe hypertension and end-organ damage in spontaneously hypertensive rats treated with l-name angiotensin-(1-7) prevents diabetesinduced cardiovascular dysfunction angiotensin-converting enzyme inhibitor ramiprilat interferes with the sequestration of the b2 kinin receptor within the plasma membrane of native endothelial cells estrogen protects transgenic hypertensive rats by shifting the vasoconstrictor-vasodilator balance of ras bi-directional actions of estrogen on the renin-angiotensin system. braz enhanced renal immunocytochemical expression of ang-(1-7) and ace2 during pregnancy elevated blood pressure and heart rate in human renin receptor transgenic rats angiotensin ii and its receptors in the diabetic kidney effect of reduced angiotensin-converting enzyme gene expression and angiotensin-converting enzyme inhibition on angiotensin and bradykinin peptide levels in mice effects of neutral endopeptidase inhibition and combined angiotensin converting enzyme and neutral endopeptidase inhibition on angiotensin and bradykinin peptides in rats differential regulation of angiotensin peptide levels in plasma and kidney of the rat newly recognized components of the renin-angiotensin system: potential roles in cardiovascular and renal regulation prolyl oligopeptidase is involved in release of the antifibrotic peptide ac-sdkp angiotensin-(1-7) is an endogenous peptide in the rat brain: evidence for differential processing of angiotensin peptides processing of angiotensin peptides by ng108-15 neuroblastoma x glioma hybrid cell line metabolism of angiotensin-(1-7) by angiotensin converting enzyme angiotensin-(1-7) in hypertension release of angiotensin-(1-7) from the rat hindlimb: influence of angiotensin-converting enzyme inhibition pathways of angiotensin-(1-7) metabolism in the kidney omapatrilat treatment is associated with increased ace-2 and angiotensin-(1-7) in spontaneously hypertensive rats estrogen or the at1 antagonist olmesartan reverses the development of profound hypertension in the congenic mren2.lewis rat novel aspects of the renal renin-angiotensin system: angiotensin-(1-7), ace2 and blood pressure regulation estrogen and salt sensitivity in the female mren(2).lewis rat hydrolysis of angiotensin peptides by human angiotensin i-converting enzyme and the resensitization of b 2 kinin receptors angiotensin-(1-7) downregulates the angiotensin ii type 1 receptor in vascular smooth muscle cells angiotensin-(1-7) reduces renal angiotensin ii receptors through a cylocoxygenase dependent pathway angiotensin-converting enzyme 2 is an essential regulator of heart function n-domain specific substrate and c-domain inhibitors of angiotensin converting enzyme ace2 gene transfer attenuates hypertension-linked pathophysiological changes in the shr rxp 407, a phosphinic peptide, is a potent inhibitor of angiotensin i converting enzyme able to differentiate between its two active sites a novel angiotensin-converting enzymerelated carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 new mass spectrometric assay for angiotensin-converting enzyme 2 activity potentiation of bradykinin by angiotensin-(1-7) on arterioles of spontaneously hypertensive rats studied in vivo characterization of angiotensin-(1-7) in the urine of normal and essential hypertensive subjects effects of omapatrilat on the renin angiotensin system in salt sensitive hypertension vasopeptidase inhibition and angiotensin-(1-7) in the spontaneously hypertensive rat effect of angiotensin converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin converting enzyme 2 -7) forming enzymes and receptors advances in biochemical and functional roles of angiotensin converting enzyme 2 and angiotensin-(1-7) in the regulation of cardiovascular function distinct roles for ang ii and ang-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin i and bradykinin: insights from selective inhibitors membrane-associated zinc peptidase families: comparing ace and ace2 quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme potential mechanisms and physiologic actions of intracellular angiotensin ii intrarenal at 1 receptor and ace binding in ang ii-induced hypertensive rats postovariectomy hypertension is linked to increased renal at1 receptor and salt sensitivity angiotensin i-converting enzyme isoforms (high and low molecular weight) in urine of premature and full-term infants renin increases mesangial cell transforming growth factor-b1 matrix proteins through receptormediated, angiotensin ii-independent mechanisms angiotensin ii at1 receptors regulate ace2 and angiotensin-(1-7) expression in aorta of spontaneously hypertensive rats contribution of angiotensin ii internalization to intrarenal angiotensin ii levels in rats angiotensin-(1-7) contributes to the antihypertensive effects of blockade of the renin-angiotensin system evidence that prostaglandins mediate the antihypertensive actions of angiotensin-(1-7) during chronic blockade of the renin-angiotensin system effect of angiotensin ii blockade on a new congenic model of hypertension derived from transgenic ren-2 rats mineralocorticoid receptor blocker increases angiotensin-converting enzyme 2 activity in congestive heart failure patients ace2 activity is increased in monocytederived macrophages from prehypertensive subjects aldosterone enhances renin expression in juxtaglomerular cells expression of angiotensinogen mrna and protein in angiotensin ii-dependent hypertension ck2 phosphorylates the angiotensinconverting enzyme and regulates its retention in the endothelial cell plasma membrane omapatrilat and enalapril in patients with hypertension: the omapatrilat cardiovascular treatment vs. enalapril (octave) trial differential tissue and enzyme inhibitory effects of the vasopeptidase inhibitor omapatrilat in the rat tumor necrosis factor-a convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-co-v) receptor, angiotensin-converting enzyme-2 (ace2) the role of angiotensin converting enzyme 2 in the generation of angiotensin 1-7 by rat proximal tubules functional expression of the renin-angiotensin system in human podocytes identification and characterization of neutral endopeptidase in endothelial cells from venous or arterial origins n-domain angiotensin-i-converting enzyme with 80 kda as a possible genetic marker of hypertension depletion of tissue ace differentially influences the intrarenal and urinary expression of angiotensins isolation and identification of proangiotensin-12, a possible component of the renin-angiotensin system regulation of intrarenal angiotensin ii in hypertension pivotal role of the renin/prorenin receptor in angiotensin ii production and cellular responses to renin n-acetyl-seryl-aspartyl-lysyl-proline ameliorates the progression of renal dysfunction and fibrosis and wky rats with established anti-glomerular basement membrane nephritis loss of angiotensin-converting enzyme-2 leads to the late development of angiotensin ii-dependent glomerulosclerosis comparison of omapatrilat and enalapril in patients with chronic heart failure. the omapatrilat versus enalapril randomized trial of utility in reducing events (overture) physiology of local renin-angiotensin systems differential expression of nuclear at1 receptors and angiotensin ii within the kidney of the male congenic mren2.lewis rat ac-sdkp reverses cardiac fibrosis in rats with renovascular hypertension enhancement of collecting duct renin in angiotensin ii-dependent hypertensive rats the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensin-converting enzyme gender differences in development of hypertension in spontaneously hypertensive rats: role of the renin-angiotensin system circulating activities of angiotensin-converting enzyme, its homolog, angiotensin-converting enzyme 2, and neprilysin in a family study evaluation of angiotensin-converting enzyme (ace), its homologue ace2 and neprilysin in angiotensin peptide metabolism estradiol attenuates angiotensininduced aldosterone secretion in ovariectomized rats angiotensin converting enzyme-independent angiotensin ii production by chymase is up-regulated in the ischemic kidney in renovascular hypertension angiotensin ii exerts positive feedback on the intrarenal renin-angiotensin system by an angiotensin converting enzyme-dependent mechanism angiotensin-(1-7) through receptor mas mediates endothelial nitric oxide synthase activation via akt-dependent pathways angiotensin-(1-7) is an endogenous ligand for the g protein-coupled receptor mas production of angiotensin-(1-7) by human vascular endothelium role of endothelin-1 in hypertension effects of aldosterone on the vasculature renal kallikrein-kinin system angiotensin metabolism in renal proximal tubules, urine and serum of sheep: evidence for ace2-dependent processing of angiotensin ii a novel mechanism for angiotensin ii formation in streptozoticindiabetic rat glomeruli angiotensin converting enzyme (ace) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies angiotensin-(1-7) inhibits angiotensin ii-stimulated phosphorylation of map kinases in proximal tubular cells mechanism of vasopeptidase inhibitor-induced plasma extravasation: comparison of omapatrilat and the novel neutral endopeptidase 24.11/angiotensin-converting enzyme inhibitor gw796406 nf-kappab-dependent increase in intrarenal angiotensin ii induced by proteinuria angiotensin-(1-7) inhibits growth of cardiac myocytes through activation of the mas receptor differential regulation of cardiac angiotensin converting enzyme 2 (ace2) and ace by aldosterone characterization of renal angiotensin-converting enzyme 2 in diabetic nephropathy developmental expression of ace2 in the shr kidney: a role in hypertension? combined inhibition of neutral endopeptidase with angiotensin converting enzyme or endothelin converting enzyme in experimental diabetes a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase differential regulation of elevated renal angiotensin ii in chronic renal ischemia neutral endopeptidase inhibition: the potential of the new therapeutic approach in cardiovascular disease evolves hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase a comparison of the properties, and enzymatic activity of three angiotensin processing enzymes: angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11 distinct gender differences in the ace2-dependent metabolism of angiotensin ii in the serum of sheep ave 0991, a nonpeptide mimic of the effects of angiotensin-(1-7) on the endothelium ace and ace2 activity in diabetic mice converting enzyme determines the plasma clearance of angiotensin-(1-7) differential response of angiotensin peptides in the urine of hypertensive animals discoordinate regulation of renal nitric oxide synthase isoforms in ovariectomized mren2.lewis rats in vivo metabolism of angiotensin i by neutral endopeptidase (ec 3.4.24.11) in spontaneously hypertensive rats glomerular localization and expression of angiotensin-converting enzyme 2 and angiotensin-converting enzyme: implications for albuminemia in diabetes essential role(s) of the intrarenal renin-angiotensin system in transforming growth factor-beta1 gene expression and induction of hypertrophy of rat kidney proximal tubular cells in high glucose upregulation of angiotensinconverting enzyme 2 by all-trans retinoic acid in spontaneously hypertensive rats these studies were supported in part by grants from the national institute of health grants (hl-56973 and hl-51952) and the american heart association (aha-151521 and aha-355741). an unrestricted grant from the unifi corporation (greensboro, nc) and the farley-hudson foundation (jacksonville, nc) is also acknowledged. key: cord-018009-8j40876m authors: campbell, duncan j. john title: ace inhibition in heart failure and ischaemic heart disease date: 2007 journal: frontiers in research of the renin-angiotensin system on human disease doi: 10.1007/978-1-4020-6372-5_2 sha: doc_id: 18009 cord_uid: 8j40876m nan angiotensin converting enzyme (dipeptidyl carboxypeptidase i, kininase ii, ec 3.4.15.1, ace) plays a major role in the metabolism of many different peptides, including angiotensin (ang) i, bradykinin, kallidin, and n-acetyl-seryl-aspartyllysyl-proline (acsdkp). ace inhibitors are established therapy for heart failure and ischaemic heart disease, and alterations of ang ii, bradykinin, kallidin, and acsdkp peptide levels are implicated in the mechanisms of this therapy. this chapter briefly describes the renin angiotensin, kallikrein kinin, and acsdkp systems, and their role in cardiovascular physiology and disease. the role of ace inhibition in treatment and prevention of heart failure and ischaemic heart disease is summarised, and the possible mechanisms of the therapeutic benefits of ace inhibitors are described. this is not an exhaustive review, but focuses on those aspects most relevant to the clinical application of ace inhibitors. figure 1 shows an outline of the pathways of ang peptide formation and metabolism. in addition to the classical pathway involving renin and ace, alternative pathways have been proposed (campbell 2006) . there remain many questions concerning the mechanisms of ang peptide formation in discrete tissue compartments such as the heart. serine proteases, for example, may form ang ii by processes independent of renin at sites of inflammation or coagulation, where kallikrein and/or cathepsin g may be active. 21 figure 1 . pathways of ang peptide formation and metabolism. adapted from (campbell 2006) studies of nephrectomised animals show the main mechanism of ang peptide formation in the heart involves kidney-derived renin (campbell et al 1993; danser et al 1994) . renin messenger rna (mrna) levels in the heart are very low or undetectable (de mello et al 2000) . cardiac renin expression may, however, be induced by myocardial infarction and macrophages and myofibroblasts may express renin at the site of repair (sun et al 2001) . all ang peptides are derived from angiotensinogen. although angiotensinogen may be produced in low levels in the heart (dostal et al 1999; paul et al 2006) , plasma is the main source of angiotensinogen for ang peptide formation in the heart. ace is a membrane-bound zinc-containing metallopeptidase, some of which is cleaved from membranes and released as soluble ace found in plasma and other fluids (erdos 1990 ). ace has two catalytic domains with differential substrate specificities and susceptibility to ace inhibitors (wei et al 1991; wei et al 1992; jaspard et al 1993) . table 1 lists the many substrates of ace. those ace substrates most related to cardiac function are ang i, the bradykinin and kallidin peptides, and acsdkp. both catalytic domains of ace posses dipeptidyl carboxypeptidase and endopeptidase activities and can cleave ang i, bradykinin-(1-9), bradykinin-(1-7), and substance p. however, the n-terminal catalytic domain cleaves of luteinbradykinin-(1-9), bradykinin-(1-8), and bradykinin-(1-7) lys 0 -bradykinin-(1-9) (kallidin), lys 0 -bradykinin-(1-8), and lys 0 -bradykinin-(1-7) substance p n-acetyl-seryl-aspartyl-lysyl-proline (acsdkp) chemotactic peptide neurotensin luteinising hormone-releasing hormone (lh-rh) enkephalins cholecystokinin gastrin adapted from (ehlers et al 1990; erdos 1990; hooper 1991; rieger et al 1993) ising hormone-releasing hormone (lh-rh) and acsdkp more efficiently than the c-terminal domain (jaspard et al 1993; rousseau et al 1995) . the two catalytic domains of ace interact differently with ace inhibitors. captopril, enalapril, lisinopril, and trandolapril are all highly potent inhibitors of both domains. whereas trandolapril, lisinopril and enalapril show preference for the c-terminal catalytic domain, captopril shows preference for the n-terminal catalytic domain (wei et al 1992) . ace has a widespread tissue distribution, including vascular endothelium and smooth muscle cells, the brush border of proximal tubule cells of the kidney, and the brain (erdos 1990) . ace is expressed by the endothelium of the coronary vasculature, and by the endocardium and epicardium, but not by the valves in the human heart (dostal et al 1999) . ace is also expressed by cardiac fibroblasts, and fibroblast expression of ace is increased in the border zone of myocardial infarction (dostal et al 1999; burrell et al 2005) . cardiac ace expression is up-regulated in heart failure (hirsch et al 1991; studer et al 1994) . many different cell types express ang receptors in the heart. the type 1 ang (at 1 ) receptor is expressed by coronary smooth muscle and endothelial cells, cardiomyocytes, fibroblasts, nerves, and conduction tissue (regitz-zagrosek et al 1998) . at 2 receptors are expressed by fibroblasts and endothelial cells (regitz-zagrosek et al 1998) . in heart failure, cardiomyocyte at 1 receptor expression may be down-regulated, whereas fibroblast expression of both at 1 and at 2 receptors is increased (ohkubo et al 1997) . the at 1 receptor mediates most of the known actions of ang ii. there is continuing uncertainty about the role of the at 2 receptor, which may mediate actions of ang ii in the vasculature and heart that differ from those of the at 1 receptor (carey et al 2001; voros et al 2006) . the at 2 receptor is described further by danser in chapter 3 of this volume. human heart chymase was initially discovered in homogenates of human heart and proposed to be the major pathway of conversion of ang i to ang ii in the heart (urata et al 1990) . given that chymase is not inhibited by ace inhibitors, it represented a potential pathway of continued ang ii formation in patients taking ace inhibitor therapy (dell'italia et al 2002) , and thereby provided a rationale for a possible superiority of at 1 receptor blocker (arb) therapy over ace inhibitor therapy. however, studies of the effects of ace inhibition in rats, mice, and humans, and of ace gene knockout in mice, show ace is the dominant pathway of ang ii formation in the heart (campbell et al 1994; campbell et al 1999; zeitz et al 2003; campbell et al 2004a) . ace-related carboxypeptidase (ace2), like ace, is a membrane-associated and secreted metalloprotease expressed predominantly on endothelium (donoghue et al 2000; tipnis et al 2000; hamming et al 2004) . ace2 is expressed in all human tissues, with relatively high levels in renal and cardiovascular tissues, and also in the gut (harmer et al 2002) . in contrast to the dipeptidyl carboxypeptidase activity of ace, ace2 cleaves ang i to ang-(1-9) and also cleaves ang ii to ang-(1-7). ace2 is not inhibited by ace inhibitors. kinetic considerations make it unlikely that ace2 contributes to ang i metabolism in vivo (jaspard et al 1993; vickers et al 2002) . ace and ace2 have similar k m for ang i (16 and 6.9 μmol/l, respectively) but the k cat for ace (40 s −1 ) is approximately 1000-fold higher than that for ace2 (0.034 s −1 ), such that the k cat /k m ratio is approximately 500-fold higher for ace (2.5 x 10 6 l/mol per s) than for ace2 (4.9 x 10 3 l/mol per s). by contrast, the k m (2 μmol/l), k cat (3.5 s −1 ), and k cat /k m ratio (1.8 x 10 6 l/mol per s) of ace2 for ang ii (vickers et al 2002) make it more likely to participate in ang ii metabolism. initial genetic studies suggested an important role for ace2 in ang peptide metabolism in the heart. the ace2 gene knockout mouse was reported to have a cardiomyopathic phenotype associated with increased ang ii levels in plasma, heart, and kidney. additionally, the cardiomyopathic phenotype was ameliorated by concomitant ace gene knockout, suggesting that altered ang peptide metabolism contributed to the phenotype (crackower et al 2002) . in subsequent studies the ace2 gene knockout mouse had a normal cardiac phenotype, although it had an enhanced pressor response to ang ii administration (gurley et al 2006) . ace2 activity is reported to be increased in the hearts of patients with heart failure (zisman et al 2003) . however, measurement of ang peptides in coronary venous blood of patients with heart failure or ischaemic heart disease does not support an important role for ace2 in either ang i or ang ii metabolism in the human heart (campbell et al 2004b) . elucidation of the role of ace2 in ang ii metabolism must await the development of specific ace2 inhibitors. effects of the ras on the heart and vasculature both systemic and local actions of ang ii impact on the heart. systemic actions of ang ii include its vasoconstrictor action to increase blood pressure and the stimulation of aldosterone secretion. increased aldosterone levels may produce hypokalaemia and contribute to cardiac fibrosis (brilla et al 1993) . local cardiac actions of ang ii include inotropic and hypertrophic effects, and cardiac remodelling (paul et al 2006) . at 1 receptor stimulation induces both myocyte hypertrophy and collagen synthesis (regitz-zagrosek et al 1998) . moreover, ang ii may contribute to oxidative stress, inflammation, and thrombosis (dzau 2001; duprez 2006) . at 1 -mediated nadph oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis (li et al 2004; mehta et al 2007) . ang ii also activates gene transcription factors involved in vascular inflammation and remodelling (oettgen 2006) . ang ii and its metabolite ang iv may promote thrombosis by stimulating plasminogen activator inhibitor type 1 (pai-1) and pai-2 production by the vasculature (van leeuwen et al 1994; feener et al 1995; kerins et al 1995) . additionally, ang ii may promote thrombosis by activation of nuclear factor b-dependent proinflammatory genes and accelerating vascular expression of tissue factor (dielis et al 2005) . ang ii stimulates endothelin release (kohno et al 1992; moreau et al 1997) and endothelin blockade prevents some of the cardiovascular actions of ang ii (webb et al 1992; rajagopalan et al 1997; herizi et al 1998) . ang-(1-7) is a biologically active peptide (ferrario et al 1991) . the main pathway of ang-(1-7) formation is by cleavage of ang i by neutral endopeptidase (nep, endopeptidase 24.11) (yamamoto et al 1992; duncan et al 1999) (fig. 1 ). ang-(1-7) may also be formed by ace2 cleavage of ang ii, but the significance of this pathway remains to be established. many actions of ang-(1-7) are contrary to those of ang ii, and ang-(1-7) is proposed to function as a counter-regulatory hormone in blood pressure control, and in other cardiovascular actions of ang ii. ang-(1-7) reduces blood pressure and produces endothelium-dependent vasodilatation (benter et al 1993; pörsti et al 1994; benter et al 1995; nakamoto et al 1995; brosnihan et al 1996; le tran et al 1997) , actions that may be due in part to potentiation by ang-(1-7) of the hypotensive effects of kinins (paula et al 1995; lima et al 1997) and/or to stimulation of vascular prostaglandin production (benter et al 1993; paula et al 1995) . in support of a role for kinin-mediated nitric oxide production in its vasodilator effects, ang-(1-7) induced vasodilatation and hypotension were attenuated by nitric oxide synthase (nos) inhibition (pörsti et al 1994; gorelik et al 1998) , by the type 2 bradykinin (b 2 ) receptor antagonist icatibant (pörsti et al 1994; abbas et al 1997; lima et al 1997; gorelik et al 1998) , and also by at 2 receptor antagonism (lima et al 1997) . moreover, ang-(1-7) stimulation of nitric oxide release from coronary vessels was blocked by icatibant (brosnihan et al 1996) . high concentrations of ang-(1-7) inhibit ace, leading to the suggestion that ang-(1-7) potentiates the effects of bradykinin through ace inhibition (li et al 1997) . however, the ic 50 for ang-(1-7) inhibition of ace was 650 nmol/l and it is unlikely endogenous ang-(1-7) levels would be sufficient to produce this effect. ang-(1-7), like other ace inhibitors, may potentiate the actions of a b 2 receptor agonist by an indirect mechanism that is independent of bradykinin hydrolysis (deddish et al 1998) , possibly by sensitisation of the b 2 receptor (marcic et al 1999) . this mechanism of potentiation of kinin-induced hypotension by ang-(1-7) is unlikely to operate in vivo, however, because micromolar concentrations of ang-(1-7) were required to produce this effect (deddish et al 1998) . plasma ang-(1-7) levels are less than ang ii levels, except during ace inhibition when ang-(1-7) levels increase several-fold, in parallel with the increase in ang i levels (lawrence et al 1990; menard et al 1997) . tissue levels of ang-(1-7) are very low or undetectable, even with ace inhibition (campbell et al 1993; 1994) . there is, therefore, uncertainty whether ang-(1-7) levels are sufficient to play a role in cardiovascular physiology and disease states in humans. 3.1. figure 2 shows an outline of the pathways of kinin peptide formation. a proportion of kininogens is hydroxylated on pro 3 of the bradykinin sequence, leading to the formation of hydroxylated kinin peptides. the kininogens are the sole precursors of the kinin peptides and are coded by a single gene. differential splicing of the initial mrna transcript produces two different mrna coding for either high or low molecular weight kininogen. each is a glycoprotein that contains the kinin sequence in its mid portion. tissue kallikrein and plasma kallikrein are both serine proteases. whereas a single gene codes for plasma kallikrein there is a large family of tissue kallikrein genes, although klk1 is the only tissue kallikrein known to generate kinin peptides (yousef et al 2001) . kininogens and tissue kallikrein are expressed in many different tissues. plasma kallikrein is predominantly expressed in liver, although recent studies suggest expression of plasma kallikrein in the brain (takano et al 1999) . in humans, plasma kallikrein forms bradykinin from high molecular weight kininogen, whereas tissue kallikrein forms kallidin from high or low molecular weight kininogens (fig. 3) . by contrast, both plasma and tissue kallikrein generate . an outline of the formation of kallidin and bradykinin peptides in humans. a proportion of high molecular weight kininogen is hydroxylated on pro 3 of the bradykinin sequence, giving rise to both hydroxylated and non-hydroxylated peptides. adapted from (campbell 2003) bradykinin in rodents (bhoola et al 1992) . bradykinin may also be generated by aminopeptidase-mediated cleavage of kallidin. alternative pathways of kinin formation involving enzymes other than kallikreins may operate in disease states. although low molecular weight kininogen is a poor substrate for plasma kallikrein, it will form bradykinin in the presence of neutrophil elastase which, by cleaving a fragment from low molecular weight kininogen, renders it much more susceptible to cleavage by plasma kallikrein (sato et al 1988) . moreover, the combination of mast cell tryptase and neutrophil elastase releases bradykinin from oxidized kininogens that are resistant to cleavage by kallikreins (kozik et al 1998) . kinin production in vivo is controlled in part by endogenous inhibitors of the kallikrein enzymes. the main inhibitors of plasma kallikrein are c1 inhibitor, 2 -macroglobulin and antithrombin iii (bhoola et al 1992) . an important inhibitor of tissue kallikrein is kallistatin, although the function of kallistatin in vivo is uncertain (chao et al 1996) . all components of a functional kks are expressed in the heart . the heart and vasculature express tissue kallikrein (oza et al 1990; xiong et al 1990; nolly et al 1992; nolly et al 1994) . in addition, plasma kallikrein, a member of the contact system, generates bradykinin at the endothelial surface of blood vessels (campbell 2003) . . dose related effects of the ace inhibitor perindopril on ang ii, ang i, and bradykinin levels in the cardiac ventricles of control rats and rats with myocardial infarction. *, p < 0.05 compared to 0 mg/kg per day perindopril. data adapted from (campbell et al 1994; duncan et al 1996) kinins act via two types of kinin receptor, the b 1 and the b 2 receptors. the b 2 receptor normally predominates, whereas the b 1 receptor is induced by tissue injury. the kks generates 8 bioactive kinin peptides: bradykinin, hyp 3bradykinin, kallidin, and hyp 4 -kallidin act on the b 2 receptor, whereas their carboxypeptidase metabolites des-arg 9 -bradykinin, des-arg 9 -hyp 3 -bradykinin, des-arg 10 -kallidin, and des-arg 10 -hyp 4 -kallidin act on the b 1 receptor. hydroxylated kinins have similar biological activity to non-hydroxylated kinins. of particular interest is the recent report that the human b 2 receptor is activated by both plasma and tissue kallikrein (hecquet et al 2000) . cathepsin g and trypsin similarly activate the b 2 receptor and activation is blocked by icatibant. thus, the b 2 receptor may belong to a new group of serine-protease-activated receptors (hecquet et al 2000) . ace is one of many enzymes that metabolise kinin peptides (campbell 2003 ) and the efficiency of metabolism is an important determinant of their levels in blood and tissues. consequently, inhibition of any single enzyme that contributes to kinin metabolism causes only a modest increase in kinin levels. kinin peptides have a broad spectrum of activities and both systemic and local cardiac actions impact on the heart (bhoola et al 1992). kinin peptides act through many different second messenger systems, in particular nitric oxide and prostaglandins (bhoola et al 1992) . the b 2 receptor participates in an inhibitory interaction with endothelial nos (enos) that is reversed by bradykinin (ju et al 1998) . this interaction may recruit enos to the b 2 receptor and allow for effective coupling of bradykinin signalling to the nitric oxide pathway. kinins are potent vasodilators and promote diuresis and natriuresis. kinins in high concentration also participate in the cardinal features of inflammation, producing vascular permeability, neutrophil chemotaxis and pain (bhoola et al 1992) . cardiac bradykinin levels are increased during the acute phase of myocardial infarction in rats (duncan et al 1997) . by contrast, we found decreased kallidin levels in coronary sinus blood of subjects with heart failure, suggesting downregulation of the cardiac kks in heart failure (duncan et al 2000) . there is a large body of evidence demonstrating anti-hypertrophic and cardioprotective actions of the kks (griol-charhbili et al 2005; koch et al 2006; park et al 2006; spillmann et al 2006) . the cardioprotective effects of bradykinin included the reduction of arrhythmias, reduction of lactate, lactate dehydrogenase, and creatine kinase release, and increase in myocardial contractility and myocardial levels of glycogen, adenosine triphosphate and creatine phosphate during postischaemic reperfusion of the isolated working rat heart (linz et al 1992) . moreover, bradykinin suppressed endothelin release from the post-ischaemic rat heart (brunner et al 1996) . kinins protect against ischaemia-reperfusion injury by decreasing endothelial adherence of leukocytes, leading to attenuation of post-ischaemic leukocyte adherence, attenuation of disruption of the microvascular barrier and reduced tissue injury (shigematsu et al 1999) . many of the actions of kinins counteract those of ang ii, by causing endothelium-dependent vasodilatation through endothelial release of nitric oxide and prostacyclin (pelc et al 1991; lamontagne et al 1992; gallagher et al 1998) . kinins also counteract the hypertrophic actions of ang ii and reduce collagen formation (gallagher et al 1998; ritchie et al 1998) . administration of kinin receptor antagonists indicates a role for endogenous kinins in the regulation of the coronary vasculature and in the myocardial response to myocardial infarction. icatibant reduced flow-dependent vasodilatation of human coronary arteries, indicating a role for kinins in the regulation of coronary vasculature (groves et al 1995) . icatibant enhanced myocardial interstitial deposition of collagen following myocardial infarction in the rat, indicating a role for endogenous kinins in the modulation of collagen deposition; however, icatibant did not modify morphological and molecular markers of cardiomyocyte hypertrophy (wollert et al 1997) . kinins participate in the process of ischaemic preconditioning, and have also been shown to limit reperfusion injury (baxter et al 2002) . kinins may also protect against thrombosis by stimulating endothelial release of nitric oxide, prostacyclin, and tissue plasminogen activator (dielis et al 2005) . new properties of kinin peptides are being discovered. for example, b 1 receptors may have an important role in angiogenesis (emanueli et al 2002) . acsdkp is an inhibitor of pluripotent haemopoietic stem cell proliferation (lenfant et al 1989; bonnet et al 1993) , and is normally present in human plasma and mononuclear cells (pradelles et al 1990) . acsdkp is released from its precursor thymosin-ß 4 by prolyl oligopeptidase (cavasin et al 2004) and it is cleaved to an inactive form by the dipeptidyl carboxypeptidase activity of the n-terminal catalytic domain of ace (rousseau et al 1995) . acsdkp has a 4.5 min half-life in the circulation and is probably released continuously . the importance of ace in acsdkp metabolism is shown by the 5-fold increase in acsdkp plasma levels that accompany ace inhibition . acsdkp inhibits dna and collagen synthesis by cardiac fibroblasts (rhaleb et al 2001) , and both prevents and reverses myocardial inflammation and fibrosis in rats with heart failure after myocardial infarction . acsdkp and thymosin-ß 4 stimulate coronary vasculogenesis and angiogenesis (wang et al 2004; smart et al 2007) , and acsdkp increases myocardial capillary density in rats with myocardial infarction (wang et al 2004) . many clinical trials demonstrate the therapeutic benefit of ace inhibition in heart failure and ischaemic heart disease. it is of note, however, that the effects of ace inhibitors are dose related. large clinical trials, by necessity, use only one dose of any drug. the results of such trials are just as much a measure of the effect of the dose as they are a measure of the effect of the drug. use of a less than optimal dose may fail to reveal a drug's true therapeutic potential. this is of particular concern in a head-to-head comparison of two active drugs, where the result may be more due to choice of dose than to choice of drug. clinicians should strive to achieve drug doses that have proven to be of benefit in clinical trials. at present, a large proportion of patients receiving ace inhibitor therapy are receiving less than optimal doses (lenzen et al 2005) . measurement of plasma ang peptide levels is not feasible for the monitoring of ace inhibitor therapy, but measurement of plasma acsdkp levels may assist in this regard (struthers et al 1999) . heart failure is associated with neurohormonal activation that includes increased renin, ang ii, and aldosterone levels, and activation of the sympathetic nervous system (francis et al 1993) . increased ang ii, aldosterone, noradrenaline, and adrenaline levels predict increased mortality in heart failure patients (swedberg et al 1990) . therapies that counteract the effects of ras and sympathetic nervous system activation are the cornerstone of heart failure therapy (hunt et al 2001; swedberg et al 2005) . acute ace inhibition in heart failure patients promotes arterio-and venodilatation, with reduction in both afterload and preload, and an associated increase in cardiac output, stroke volume, and stroke work index, along with a decrease in pulmonary capillary wedge pressure, indicating improved left ventricular (lv) function (gavras et al 1978; ader et al 1980) . the cooperative north scandinavian enalapril survival study (consensus) demonstrated reduced mortality and improved symptoms with enalapril therapy in patients with severe heart failure (the consensus trial study group 1987). moreover, mortality was lower with enalapril therapy than with hydralazine-isosorbide dinitrate therapy in the second veterans administration cooperative vasodilator-heart failure trial (v-heft ii) (cohn et al 1991) . the studies of left ventricular dysfunction (solvd) confirmed the survival benefits of enalapril therapy in patients with reduced lv ejection fraction and heat failure (the solvd investigators 1991) and also demonstrated the prevention of heart failure in asymptomatic subjects with reduced lv ejection fraction (the solvd investigators 1992). ace inhibition improves survival, symptoms, and functional capacity, and reduces hospitalisation in patients with moderate and severe heart failure and lv systolic dysfunction (flather et al 2000; abdulla et al 2004) . ace inhibition is recommended as first-line therapy in patients with a reduced lv ejection fraction with or without symptoms, and should be up-titrated to the doses shown to be effective in clinical trials (hunt et al 2001; swedberg et al 2005) . although the patients recruited to the consensus, v-heft ii, and sovd studies had reduced lv ejection fraction due most often to ischaemic heart disease, they were enrolled several months or more after a myocardial infarction. studies in rats demonstrated survival advantage of ace inhibitor therapy commenced 14 days after myocardial infarction (pfeffer et al 1985b) . additionally, ace inhibition reduced arterial pressure and total peripheral resistance, attenuated lv remodelling, prevented deterioration in cardiac output and stroke volume index, and prevented the increase in lv volume, lv chamber stiffness and lv end diastolic pressure in rats with myocardial infarction (pfeffer et al 1985a) . these benefits of ace inhibition in rats with myocardial infarction were confirmed in patients. the survival and ventricular enlargement (save) trial showed reduced mortality with ace inhibitor therapy when commenced 3-16 days after myocardial infarction in patients with asymptomatic lv dysfunction (pfeffer et al 1992) . in addition, ace inhibitor therapy reduced the incidence of both fatal and nonfatal major cardiovascular events, including the development of severe heart failure and recurrent myocardial infarction. the benefits of ace inhibitor therapy after myocardial infarction were confirmed in the acute infarction ramipril efficacy (aire) and the trandolapril cardiac evaluation (trace) studies (the acute infarction ramipril efficacy (aire) study investigators 1993; kober et al 1995) . the aire study recruited patients 2-9 days after myocardial infarction who had shown clinical evidence of heart failure at any time. the trace study recruited patients 3-7 days after myocardial infarction who had a lv ejection fraction ≤35%. both the aire and trace studies showed survival advantage with ace inhibitor therapy and the trace study showed less development of severe heart failure. other large clinical trails confirmed the benefits of ace inhibition after myocardial infarction (gissi-3 gruppo 1994; isis-4 collaborative group 1995) . in addition to mortality benefit and reduction of severe heart failure, ace inhibition after myocardial infarction attenuates lv remodelling, lv enlargement and increase in lv mass, and improves lv ejection fraction after myocardial infarction (pfeffer et al 1988; sharpe et al 1991; sogaard et al 1993; johnson et al 1997) . by contrast, the consensus ii trial found the commencement of ace inhibitor therapy within 24 hours of myocardial infarction did not improve survival (swedberg et al 1992) . the failure of ace inhibition to improve outcomes in the consensus ii trial may have been due to its protocol. ace inhibitor treatment was started with intravenous infusion of 1 mg enalaprilat within 24 hours after the onset of chest pain, followed by administration of oral enalapril. intravascular administration of ace inhibitor had a negative inotropic effect in several human studies (foult et al 1988; haber et al 1994; zeitz et al 2003) , although not in another (friedrich et al 1994) . thus, the failure of ace inhibitor therapy to produce benefit in the consensus ii trial may have been due to the negative inotropic effect of intravenously administered enalaprilat, in addition to its administration within 24 hours of chest pain. current european society of cardiology guidelines recommend the initiation of ace inhibitors after the acute phase of myocardial infarction in patients with signs or symptoms of heart failure, even if transient, to improve survival and to reduce re-infarctions and hospitalisations for heart failure . the hope study was based on emerging evidence that ace inhibition reduced the risk of myocardial infarction in patients with low ejection fraction (pfeffer et al 1992; yusuf et al 1992; lonn et al 1994) . it examined the effects of addition of 10 mg ramipril to standard therapy in patients aged at least 55 years with a history of coronary artery disease, stroke, peripheral vascular disease, or diabetes, plus at least one other cardiovascular risk factor (hypertension, elevated total cholesterol level, low high-density lipoprotein cholesterol level, cigarette smoking, or microalbuminuria). patients were excluded if they had heart failure, were known to have a low ejection fraction, were taking an ace inhibitor or vitamin e, had uncontrolled hypertension or overt nephropathy, or had had a myocardial infarction or stroke within 4 weeks before the study began. during a mean follow-up of 5 years ramipril reduced the primary outcome (composite of myocardial infarction, stroke, or death from cardiovascular causes) from 17.8% to 14.0% (relative risk 0.78, 95% confidence interval 0.70 to 0.86; p < 0.001). treatment with ramipril reduced the rates of death from cardiovascular causes and all-cause mortality, myocardial infarction, revascularisation procedures, cardiac arrest, heart failure, and complications related to diabetes. the europa study examined the effects of addition of 8 mg perindopril to standard therapy in patients with previous myocardial infarction, angiographic evidence of coronary heart disease, coronary revascularization, or a positive stress test. past history of heart failure was recorded in 1.3% of subjects, but none had clinical signs of heart failure, with 10% in new york heart association class i and none in class ii or higher. during a mean follow-up of 4.2 years, perindopril reduced the primary outcome (composite of cardiovascular death, non-fatal myocardial infarction, cardiac arrest with successful resuscitation) from 9.9% to 8.0% (relative risk 0.80, 95% confidence interval 0.71 to 0.91; p<0.001). the main contributor to this reduction in the primary outcome was the reduction in non-fatal myocardial infarction. perindopril also reduced the incidence of heart failure requiring hospitalisation. by contrast, the prevention of events with angiotensin converting enzyme inhibition (peace) study failed to show an effect of ace inhibition on its primary endpoint (the peace trial investigators 2004). the peace study examined the effects of addition of 4 mg trandolapril to standard therapy on cardiovascular events in patients with stable coronary heart disease and preserved lv function. during a median follow-up of 4.8 years, trandolapril produced non-statistically significant reductions in the primary endpoint (composite of cardiovascular death, myocardial infarction, and coronary revascularization) from 22.5% to 21.9%, and in cardiovascular death and non-fatal myocardial infarction from 8.5% to 8.3%, although trandolapril reduced hospitalisation or death due to heart failure from 3.7% to 2.8%. participants in the peace study were at lower risk of cardiovascular events than those in the hope and europa studies. the baseline blood pressure of peace participants was less than that of patients in the hope and europa studies, and was similar to the level achieved with active therapy in the hope and europa studies. in addition, peace participants received more intensive management of risk factors than did those in the hope and europa studies, with 70% of peace participants receiving lipid lowering therapy (29% in hope, 56% in europa), and 72% had undergone coronary revascularization before enrollment (40% in hope, 54% in europa). thus, peace participants had an event rate similar to that of the general population (1.6% annualised rate of death), and the more aggressive management of their risk factors may have negated any potential benefit from ace inhibitor therapy. there has been debate about the reasons for the failure of the peace study to show an effect of trandolapril on the primary endpoint (pitt 2004; fox et al 2006a) . although the dose and type of ace inhibitor may be implicated, the most likely explanation is the low event rate in its relatively low risk population (necessitating the inclusion of revascularisation as part of the primary endpoint), such that the study did not have sufficient statistical power to achieve its aim. the ischemia management with accupril post bypass graft via inhibition of angiotensin converting enzyme (imagine) study similarly showed a lack of benefit from 40 mg quinapril in optimally treated low-risk patients after coronary artery bypass grafting (keuper et al 2005) . pooled analysis of the hope, europa, and peace trials showed ace inhibition reduced all cause and cardiovascular mortality, non-fatal myocardial infarction, stroke, heart failure, and coronary artery bypass surgery, leading to the recommendation that ace inhibitors be considered in all patients with atherosclerosis (dagenais et al 2006) . a meta-analysis of the hope, europa, peace, and other studies came to a similar conclusion (al-mallah et al 2006) . however, the number needed to treat for 4.4 years to prevent either one death, one nonfatal myocardial infarction, or one coronary revascularisation procedure was 100 (al-mallah et al 2006) . current european society of cardiology guidelines state: "ace inhibition is well established in the treatment of heart failure or lv dysfunction and in the treatment of diabetic patients. thus, it is appropriate to consider ace inhibitors for the treatment of patients with stable angina pectoris and co-existing hypertension, diabetes, heart failure, asymptomatic lv dysfunction and post-myocardial infarction. in angina patients without co-existing indications for ace inhibitor treatment the anticipated benefit of treatment (possible absolute risk reduction) should be weighed against costs and risks for side-effects, and the dose and agent used of proven efficacy for this indication" (fox et al 2006b) . et al 1990) . the systemic effects include the reduction of circulating ang ii and aldosterone levels and the increase in kinin and acsdkp levels. decreased ang ii and increased kinin levels contribute to the reduction of blood pressure by ace inhibition. there is ongoing debate about the extent to which the benefits of ace inhibition are related to blood reduction, as opposed to intrinsic benefits of ace inhibition (sever et al 2006) . a major contributor to the benefits of ace inhibition in heart failure and ischaemic heart disease may be the reduction in systemic blood pressure, and consequent reduction in heart work. ace inhibition may improve cardiac function by reducing coronary vascular resistance in patients with heart failure, thereby augmenting cardiac blood flow (dietz et al 1993) . ace inhibition reduces circulating and tissue levels of ang ii in both animals and humans (campbell et al 1994; duncan et al 1996; campbell et al 1999; zeitz et al 2003) . ace inhibition produced a modest reduction in ang ii levels in europa participants (ceconi et al 2007) . however, the effects of ace inhibition on ang ii levels can be variable, and depend on the responsiveness of renin secretion . in situations where renin shows little increase in response to ace inhibition, the levels of ang ii and its metabolites show a marked fall, with little change in the levels of ang i and its metabolites. by contrast, a large increase in renin levels in response to ace inhibition also increases the levels of ang i and its metabolites. the increased ang i levels promote ang ii formation by residual uninhibited ace and by serine protease pathways of ang i conversion, thereby buffering any fall in ang ii levels during ace inhibition . improved survival of heart failure patients with ace inhibitor therapy is associated with reduction in ang ii and aldosterone levels (swedberg et al 1990) . the role of renin in determining the response of ang ii levels to ace inhibition is most evident in heart failure, where many patients continue to have elevated ang ii levels despite ace inhibitor therapy (roig et al 2000; campbell et al 2001) . it is of note that maximally recommended doses of ace inhibitor do not completely prevent ace mediated formation of ang ii in heart failure (jorde et al 2000) . the beneficial therapeutic effects of concomitant ß-blocker therapy in heart failure may be due in part to the associated reduction in renin and ang ii levels (campbell et al 2001) . the effects of ace inhibitors on ang ii levels are dose dependent (fig. 3) . studies in rats showed tissue-specific differences in the dose-related effects of ace inhibition on ang ii levels (campbell 1996) . renal ang ii levels were reduced by lower doses of ace inhibitor than were required to reduce ang ii levels in other tissues such as the heart (fig. 3) . ang-(1-7) . this is due in part to the increase in ang i levels, with subsequent conversion to ang-(1-7). another mechanism for the increase in ang-(1-7) levels during ace inhibition is the inhibition of ang-(1-7) metabolism, given that ace is an important pathway of ang-(1-7) metabolism yamada et al 1998) . studies in rats led to the proposal that increased ang-(1-7) levels mediate in part the hypotensive effects of ace inhibition (iyer et al 1998a; iyer et al 1998b) . however, there is as yet no evidence that these mechanisms operate in patients receiving ace inhibitor therapy. there is ample evidence that kinin peptides contribute to the therapeutic effects of ace inhibitors (linz et al 1995; bönner 1997) . ace inhibitors increase circulating and tissue levels of bradykinin in animals (fig. 3) and humans (campbell et al 1994; duncan et al 1996; zeitz et al 2003) . the effect of ace inhibition on kinin peptide levels in any tissue compartment depends on the contribution of ace, relative to other kininases, to kinin peptide metabolism in that compartment. ace inhibitor therapy did not increase either bradykinin or kallidin peptide levels in cardiac atria of patients with ischaemic heart disease, despite the reduction in ang ii levels . the maintenance of low levels of kinin peptides by their efficient metabolism is relevant to the success of ace inhibitor therapy. ace inhibition has only a modest effect on kinin peptide levels because of the many other kininases that contribute to kinin metabolism. it is for this reason that ace inhibitors are generally free of the side effects, such as angioneurotic oedema, that one might expect from increased kinin peptide levels (nussberger et al 1998; nussberger et al 2002) . studies with kinin receptor antagonists indicate a role for kinins in the cardiovascular actions of ace inhibitors in animals and humans (linz et al 1995) . studies in humans indicate a role for the b 2 receptor in flow-dependent vasodilatation in normal volunteers (hornig et al 1997) and in the hypotensive effects in patients with hypertension (gainer et al 1998; squire et al 2000) . a role for the b 1 receptor is indicated in the systemic haemodynamic effects of ace inhibition in patients with heart failure (witherow et al 2001; cruden et al 2004) . cardioprotective effects of ace inhibition that were attenuated by icatibant included the reduction of arrhythmias, reduction of lactate, lactate dehydrogenase, and creatine kinase release, and increase in myocardial contractility and myocardial levels of glycogen, adenosine triphosphate and creatine phosphate during reperfusion of the ischaemic isolated working rat heart (linz et al 1992) . icatibant attenuated the ace inhibitor-induced increase in coronary flow and nitric oxide levels in dogs with myocardial ischaemia (kitakaze et al 2002) . icatibant also prevented the potentiation of ischaemic preconditioning by ace inhibition in human atria (morris et al 1997) . the post-ischaemic anti-arrhythmic effect of ace inhibition may be mediated by kinin-induced suppression of endothelin release (brunner et al 1996) . icatibant prevented the reduction in myocardial infarct size and the reduction in post-infarct remodelling by ace inhibition in animal models (linz et al 1992; hartman et al 1993; stauss et al 1994; mcdonald et al 1995; hu et al 1998) . however, a subsequent study in an in vivo canine model of myocardial ischaemic injury did not show an effect of ace inhibition on infarct size (black et al 1998) . moreover, icatibant did not modify the antihypertrophic effect of ace inhibition in rats with myocardial infarction, although it partially reversed the reduction in myocardial collagen deposition by ace inhibitor therapy in one study (wollert et al 1997) . possible mechanisms by which kinin peptides mediate the therapeutic benefits of ace inhibition include the promotion of endothelial production of nitric oxide and prostacylin, thereby contributing to the correction of endothelial dysfunction and reduced oxidative stress (linz et al 1995; bönner 1997; münzel et al 2001) . ace inhibition induced endothelial nos (enos) in vasculature of control rats, and attenuated the induction of inducible nos (inos) in rats administered bacterial lipopolysaccharide (bachetti et al 2001) . icatibant prevented the increase in nitric oxide formation in the heart and reduction in myocardial oxygen consumption that accompany ace inhibition in dogs (zhang et al 1997) . icatibant also prevented the antiproliferative effect of ace inhibition in neointima formation following endothelial injury to the rat carotid artery (linz et al 1992) , and the increase in capillary density induced by chronic ace inhibitor treatment in stroke-prone spontaneously hypertensive rats (gohlke et al 1997) . part of the benefits of ace inhibition may be due to the enhancement of insulin-mediated muscle glucose uptake, that is also attenuated by icatibant (henriksen et al 1996; henriksen et al 1999) . ace inhibition also affects the kks by mechanisms separate from prevention of kinin degradation. for example, chronic ace inhibition in mice and rats induced both renal and vascular b 1 receptor expression without modification of b 2 receptor expression (marin-castano et al 2002) . moreover, enalaprilat and other ace inhibitors in nanomolar concentrations were shown to directly activate the human b 1 receptor, in the absence of ace and b 1 receptor ligands (ignjatovic et al 2002) . several studies show ace inhibitors may potentiate the effects of bradykinin by a mechanism independent of prevention of kinin metabolism, that involves direct interaction between ace and the b 2 receptor (fleming 2006 ) and attenuation of the sequestration of the b 2 receptor (benzing et al 1999; chen et al 2006) . additionally, membrane ace appears to have its own signalling cascade that is activated by binding of ace inhibitors (fleming 2006) . one approach to differentiation of the respective roles of the ras and kks in mediating the therapeutic benefits of ace inhibition is the comparison of ace and arb therapy. comparison of ace inhibitor and arb therapy after myocardial infarction, or in patients with heart failure, did not show any difference in outcomes (pitt et al 2000; dickstein et al 2002; pfeffer et al 2003; mcmurray et al 2006) . these studies suggest ace inhibitor and arb therapy act through blockade of the ras, but a role for bradykinin cannot be excluded because losartan was shown to increase bradykinin levels in hypertensive humans (campbell et al 2005) . maximally recommended doses of ace inhibitors do not completely prevent ace mediated formation of ang ii in heart failure (jorde et al 2000) . combination of ace inhibitor and arb therapy produces more complete blockade of the ras that is dependent on the dose regimens of the individual therapies (menard et al 1997; azizi et al 2004) . this combination therapy improves outcomes in heart failure patients (cohn et al 2001; mcmurray et al 2003) , but not following myocardial infarction mcmurray et al 2006) . ace inhibition causes a several-fold increase in acsdkp levels that may contribute to decreased cardiac inflammation and fibrosis, and to increased myocardial capillary density after myocardial infarction (wang et al 2004; yang et al 2004) . elevated acsdkp levels during ace inhibitor therapy may also contribute to the anaemia experienced by heart failure patients receiving ace inhibitor therapy (van der meer et al 2005). heart failure patients have increased plasma aldosterone levels consequent to stimulation of aldosterone secretion by increased ang ii levels (weber 2001) . evidence that reduced aldosterone levels may contribute to the therapeutic benefits of ace inhibition is the reduced hypokalaemia in patients receiving ramipril therapy in the hope study (mann et al 2005) . in addition to promotion of sodium retention and oedema formation, aldosterone may promote cardiac fibrosis and deterioration in cardiac function (brilla et al 1993) . the possible clinical importance of this mechanism is shown by the benefits of aldosterone receptor antagonists in patients with heart failure, and in patients with lv dysfunction after myocardial infarction (pitt et al 1999; pitt et al 2003) . many authors have suggested the reduction in sympathetic activity that may accompany ace inhibition is due to a reduction in the stimulation of sympathetic activity by ang ii. however, although ace inhibitor therapy leads to reduction in sympathetic nervous system activity in heart failure, this is thought to be mainly secondary to the improvement of cardiovascular haemodynamics, rather than the specific consequence of reduced stimulation of the sympathetic nervous system by ang ii (esler et al 2001) . cardiac hypertrophy is well recognised as a risk factor for death and cardiovascular events (levy et al 1990) . ace inhibitors reduce cardiac hypertrophy in hypertensive patients (dahlof et al 1992) and also reduce progressive lv remodelling after myocardial infarction (ferrari 2006) . ventricular remodelling has a dominant role in the pathogenesis of heart failure, and the prevention of remodelling is considered to be an important mechanism of the benefit of ace inhibitor therapy in heart failure and after myocardial infarction (cohn 1995; abdulla et al 2007) . reduction of myocardial infarction and other ischaemic events by ace inhibition raises the possibility that these drugs inhibit atherosclerosis. ace inhibitors correct endothelial dysfunction in patients with heart failure and ischaemic heart disease mancini et al 1996; ceconi et al 2007) . these effects of ace inhibition may be due to the reduction of oxidative stress, vascular remodelling and inflammation by reduced ang ii levels and increased kinin levels. however, current evidence does not allow these data to be extrapolated to a reduction in atherogenesis by ace inhibition in humans. despite the prevention of atherosclerosis in animal models, ace inhibitor therapy was not able to reduce atherogenesis in patients. ace inhibition with cilazapril did not prevent restenosis after angioplasty (mercator), (mercator study group 1992; faxon 1995) . similarly, quinapril did not reduce restenosis after coronary stenting; in fact, late loss in minimum lumen diameter was significantly higher in the quinapril group than in controls (meurice et al 2001) . additionally, ace inhibition with enalapril failed to reduce progression of coronary atherosclerosis, as assessed by intravascular ultrasound, in patients with coronary artery disease (nissen et al 2004) . a meta-analysis of randomised controlled studies of the effect of antihypertensive therapies in progression of carotid intima-media thickness showed only a weak, non-significant reduction in progression of carotid intima-media thickness by ace inhibitor therapy, with significant heterogeneity between studies (wang et al 2006) . some studies showed a reduction in progression of intima-media thickness by ace inhibition and some did not. of note, calcium channel blockers were significantly more effective than ace inhibitors in their reduction of progression of intima-media thickness (wang et al 2006) . reduced rates of myocardial infarction with ace inhibitor therapy may also be due to an effect of this therapy on the mechanisms of thrombosis and fibrinolysis. ace inhibition reduced plasma levels of pai-1 antigen and activity in normal subjects on low salt diet and in subjects following myocardial infarction (wright et al 1994; moriyama et al 1997; oshima et al 1997; vaughan et al 1997; brown et al 1998; brown et al 1999) , although this effect of ace inhibition was not confirmed in other studies of patients with previous myocardial infarction (zehetgruber et al 1996; pedersen et al 1997) . ace inhibition also reduced pai-1 antigen, but not pai-1 activity, in subjects with congestive cardiac failure (goodfield et al 1999) . diabetes is well recognised to accelerate the processes of cardiovascular disease, and reduction of diabetes incidence may contribute to the therapeutic benefits of ace inhibition. many large clinical trials, including the hope, peace, and solvd studies, showed a reduced incidence of type 2 diabetes with ace inhibitor therapy (abuissa et al 2005) . however, the diabetes reduction assessment with ramipril and rosiglitazone medication (dream) study found ramipril did not reduce diabetes incidence among persons with impaired fasting glucose levels or impaired glucose tolerance, although it significantly increased regression to normoglycaemia (the dream trial investigators 2006) . this improvement in insulin resistance may be due in part to the enhancement of insulin-mediated muscle glucose uptake by ace inhibition (henriksen et al 1996; henriksen et al 1999) . aortic compliance is an important determinant of coronary blood flow (o'rourke et al 1999) . a recent meta-analysis showed ace inhibitors decrease arterial stiffness (mallareddy et al 2006) . ace inhibitors, by increasing aortic compliance, may reduce central systolic blood pressure and maintain diastolic blood pressure, thereby reducing heart work without compromising myocardial perfusion. decrease in arterial stiffness by ace inhibition may be due to reduced collagen deposition, as suggested by studies in spontaneously hypertensive rats (benetos et al 1997) . reduction of aortic collagen deposition by ace inhibition was not affected by icatibant, suggesting that this effect of ace inhibition was not mediated by kinins (benetos et al 1997) . atrial fibrillation is an important contributor to poor prognosis in heart failure (wang et al 2003) , and prevention of atrial fibrillation by ace inhibition may contribute to the therapeutic benefits of this therapy (vermes et al 2003). given that kinin peptides mediate in part the therapeutic benefits of ace inhibition, and that some of the actions of kinins are mediated by prostaglandins, the question arises whether a drug that inhibits prostaglandin synthesis may attenuate the effects of ace inhibition. this question was addressed in a systematic review of the interaction between aspirin and ace inhibitor therapy (teo et al 2002) . the solvd study found aspirin prevented the reduction of death by ace inhibition, but this interaction between aspirin and ace inhibitor therapy was not significant in the other trials examined. however, both solvd and the other trials showed aspirin attenuated the prevention of myocardial infarction or reinfarction by ace inhibition. by contrast, there was no evidence that aspirin attenuated the prevention of stroke, hospital admission for heart failure, or revascularisation by ace inhibitor therapy. when the composite of major vascular events including death, myocardial infarction or reinfarction, hospital admission for heart failure, stroke, and revascularisation was examined, aspirin did not significantly attenuate the benefits of ace inhibitor therapy. this analysis shows, therefore, that aspirin does interact with ace inhibitor 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a meta-analysis of randomized controlled trials temporal relations of atrial fibrillation and congestive heart failure and their joint influence on mortality: the framingham heart study the endothelin receptor antagonist, bq-123, inhibits angiotensin ii-induced contractions in rabbit aorta aldosterone in congestive heart failure the two homologous domains of human angiotensin i-converting enzyme are both catalytically active the two homologous domains of human angiotensin i-converting enzyme interact differently with competitive inhibitors bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure differential effects of kinins on cardiomyocyte hypertrophy and interstitial collagen matrix in the surviving myocardium after myocardial infarction in the rat effects of captopril therapy on endogenous fibrinolysis in men with recent, uncomplicated myocardial infarction identification, purification, and localization of tissue kallikrein in rat heart converting enzyme determines plasma clearance of angiotensin-(1-7) in vivo metabolism of angiotensin i by neutral endopeptidase (ec 3.4.24.11) in spontaneously hypertensive rats ac-sdkp reverses inflammation and fibrosis in rats with heart failure after myocardial infarction the new human tissue kallikrein gene family: structure, function, and association to disease effect of enalapril on myocardial infarction and unstable angina in patients with low ejection fractions the aceinhibitor lisinopril affects plasma insulin levels but not fibrinolytic parameters myocardial uptake and biochemical and hemodynamic effects of ace inhibitors in humans ace inhibitors promote nitric oxide accumulation to modulate myocardial oxygen consumption -7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace2 this work was supported by a senior research fellowship from the national health and medical research council of australia (id 395508), and by the national heart foundation of australia (id g 06m 2654). key: cord-253862-jl1zhg13 authors: khalaf, khalil; papp, natalia; chou, jadzia tin-tsen; hana, doris; mackiewicz, andrzej; kaczmarek, mariusz title: sars-cov-2: pathogenesis, and advancements in diagnostics and treatment date: 2020-10-06 journal: front immunol doi: 10.3389/fimmu.2020.570927 sha: doc_id: 253862 cord_uid: jl1zhg13 the emergence and rapid spread of sars-cov-2 in december 2019 has brought the world to a standstill. while less pathogenic than the 2002–2003 sars-cov, this novel betacoronavirus presents a global threat due to its high transmission rate, ability to invade multiple tissues, and ability to trigger immunological hyperactivation. the identification of the animal reservoir and intermediate host were important steps toward slowing the spread of disease, and its genetic similarity to sars-cov has helped to determine pathogenesis and direct treatment strategies. the exponential increase in cases has necessitated fast and reliable testing procedures. although rt-pcr remains the gold standard, it is a time-consuming procedure, paving the way for newer techniques such as serologic tests and enzyme immunoassays. various clinical trials using broad antiviral agents in addition to novel medications have produced controversial results; however, the advancement of immunotherapy, particularly monoclonal antibodies and immune modulators is showing great promise in clinical trials. non-orthodox medications such as anti-malarials have been tested in multiple institutions but definitive conclusions are yet to be made. adjuvant therapies have also proven to be effective in decreasing mortality in the disease course. while no formal guidelines have been established, the multitude of ongoing clinical trials as a result of unprecedented access to research data brings us closer to halting the sars-cov-2 pandemic. coronaviruses are widely known virulent pathogens affecting mammalian and avian species. previously, six globally distributed species of the virus have been identified to cause illness in humans. they are: human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), human coronavirus 229e (hcov-229e), human coronavirus nl63 (hcov-nl63), severe acute respiratory syndrome coronavirus (sars-cov), and middle east respiratory syndrome coronavirus (mers-cov) ( table 1) . sars-cov first emerged in [2002] [2003] in china, presenting as an atypical pneumonia with febrile state, headaches, and a marked cough that may rapidly deteriorate into respiratory failure. the sars-cov outbreak was the first to be declared a pandemic, and ultimately infected 8,000 persons in 29 countries, with a mortality rate of 10% (5). epidemiological analysis posited an infectious zoonotic agent, and further serological studies identified the intermediate host to be the palm civet (paguma larvata). spillover from animals to humans was hypothesized to be via the horseshoe bat (genus rhinolophus). an adaptation of the sars-cov virus was later proven to be due to interspecies dwelling (6) . a combination of ribavirin and pulse prednisolone showed positive results early in the outbreak and was adopted as the standard protocol. eventually, ribavirin was demonstrated to have increased cytotoxicity and lacked efficient antiviral action in vitro, and while pulse prednisolone showed efficacy in managing critically ill patients, its high dose administration was associated with disseminated fungal infections (7) . later, the combination of interferon-alpha1 (inf-α1) and corticosteroids was found to yield a better prognosis, but did not prove beneficial for patients in the late stage of the disease (8) . protease inhibitors produced an outcome similar to that of inf-α1 (9) . a novel coronavirus later named mers-cov emerged in saudi arabia in 2012. similar to sars, infected patients presented with a variety of clinical courses, from mild upper respiratory symptoms to fulminant pneumonia and multi-organ system failure. phylogenic and sequencing studies have proposed a bat origin, and surface protein modification was found to be derived from the intermediate hosts, dromedary camels. from 2012 to 2015, more than 2,000 confirmed cases were reported, mainly in middle eastern countries, with a mortality rate of 35% (10) . as with sars-cov, no definitive protocol was implemented, and most patients were treated with supportive therapy to preserve organ integrity (11) . in a cohort study conducted by omrani et al., a treatment protocol involving ifn-α2 and ribavirin was initiated, and while survival improved significantly at 14 days after treatment, this was not seen at day 28, necessitating further treatment options (12) . sporadic outbreaks occur to this day, with patients undergoing largely supportive therapy. the sars-cov-2 outbreak began in december 2019 in wuhan, china where clusters of atypical pneumonia yielded evidence of a novel strain of coronavirus. as of august 24, 2020, 16,036,308 cases had been reported worldwide, with a fatality rate of 5%. although this novel virus is less severe than the first sars-cov outbreak, human-to-human transmission remains very high and the number of cases continues to rise exponentially in major urban areas, highlighting the urgent need to develop new containment, diagnostic, and treatment protocols. as time is of the essence, the pathogenic mechanism cannot be studied with the rigor and comprehension otherwise expected, and the opportunities to compare therapeutic protocols are few. origin of the virus during the initial outbreak, liu et al. obtained samples of blood, bronchoalveolar lavage fluid, and anal and oral swabs from patients in wuhan, china suffering from a pneumonialike illness resembling the 2002-2003 sars-cov. a pan-cov pcr was performed on samples from five patients, and were positive for a pathogen from the coronaviridae family. this was compared with a full-length sequence of viral rna from a bat coronavirus (bat-covratg13), and demonstrated 96.2% similarity. thus, it is probable that the bat is the main reservoir of the novel coronavirus. identification of the intermediate host is an essential step in controlling the spread of disease, and became a priority for research teams. unfortunately, this was complicated by the many species of wild animals sold at the huanan seafood market, where the first cases were reported to have had contact. in 2019, a sars-cov-like pathogen known to be widely distributed in the malayan pangolin samples was discovered. the receptorbinding domain (rbd) present on the spike protein (s) is a crucial determinant in host range, as its interaction with the host receptor is responsible for the infection. rbd sequences from bat-covratg13, pangolin-sars-like cov and the novel sarslike pathogen were aligned. ninety three percentage similarity was demonstrated between the novel sars-like pathogen and the pangolin sars-like cov, and 89% similarity was demonstrated between the novel sars-like pathogen and the bat-covratg13. thus, on the basis of the rbd, the pangolin-sars-like cov is determined to be more likely than the bat-covratg13 to infect humans, making this the possible intermediate host (13) . xiao et al. conducted another study in which the pangolin-sars-like cov was isolated and amino acid sequence was compared to sars-cov-2. this yielded 100, 98.6, 97.8, and 90 .7% similarity with the s, m, e, and n proteins, respectively, of the novel sars-cov, strengthening the previous assumption that the pangolin was the intermediate host (14) . pathogenic classification is used to determine whether the pathogen is new or recurring in order to best implement safety and treatment protocols. while serological reactivity to viral proteins had been the mainstay of viral classification in the past, the process today now depends on replicated protein sequences. the international committee on taxonomy of viruses (ictv) maintains a study group for each viral family (15) . after analysis, the novel virus was assigned to the order nidovirales on the basis of the following domains: polyprotein protease (3clpro), catalytic domain of rna polymerase (rdrp), nidovirus-associated rdrp (niran), zinc binding domain (zbd), and helicase (hel1) (16) . subsequent next generation sequencing and phylogenic analysis placed the novel pathogen within the subgenus sarbecovirus of the genus betacoronavirus (17) ( table 2) . like other coronaviruses, the viral envelope is composed of a lipid bilayer derived from host cellular material, and the structural proteins are spike proteins composed of a trimeric glycoprotein projecting from the envelope like a crown. cryo-electron microscopy was used to compare the structural differences between the spike (s) proteins of sars-cov and sars-cov-2. unlike the previous sars-cov which possessed a single arginine targeted by a trypsin protease, sars-cov-2 possesses an s1/s2 protease cleavage site which is recognized by furin proteases. neuman et al. demonstrated the high level of protein organization and interaction within the envelope: the s protein was shown to be aligned with the npc, a crucial component of viral organization in protein-protein interactions that ensures high specificity and effectiveness for host invasion (19) . sars-cov-2 is an enveloped virus with a 29,904 bp positivesense non-segmented rna genome. the non-structural proteins (nsps) are described in table 3 . the remainder of the genome is responsible for accessory and structural proteins such as s, m, e and n. host cell recognition is the first and most essential step in viral pathogenesis. studies on the 2002-2003 sars-cov outbreak uncovered key interactions between the spike (s) protein, the rbd and angiotensin-converting enzyme 2 (ace-2). due to the previously mentioned similarities between sars-cov-2 and its predecessor, a viral infectivity study was performed using hela cells with and without the ace-2 receptor, which showed that only cells possessing the ace-2 receptor were infected (21) . the spike trimer is a class i fusion protein; upon infection the spike is cleaved by host proteases at the s1/s2 site for division of the two domains. the s1 domain possesses the rbd which recognizes and binds the ace-2 receptor in a prefusion state. structural rearrangement of the s protein subsequently exposes a furin cleavage site on the s2 domain which enables viral entry by means of fusion after the s1 domain is shed (22) . viral replication was hypothesized to occur via a process called autophagy: an evolutionary cellular process in which cytoplasmic proteins create isolation membranes surrounding materials destined for degradation (23) . evidence of coronavirus autophagy was first demonstrated by prentice et al., who showed that in coronavirus mouse hepatitis viruses, atg5-induced autophagy was required for the formation of double membrane vesicles (dmv) and for replication (23) . another study confirmed the use of atg5dependent autophagy of betacoronaviruses through nsp 6 (24). chen et al. previously showed that both sars-cov and mers-cov employ the use of papain-like proteases (plpro) to induce the formation of autophagosomes, but their fusion to lysosomes is inhibited which promotes the replication process (25) . however, other studies have demonstrated that key autophagy proteins atg5 or atg7 were not required for coronavirus mouse hepatitis or sars-cov viral replication (26) . reggiori et al. also yielded comparable results showing that the replication and release processes are not dependent on autophagy, but that the dmv's were coated with (lc3)-i (non-lipidated microtubule associated protein i light chain 3), thus showing the importance of this protein for viral replication (27) . a recent study by benveunto et al. has shown that sars-cov-2 induced mutations within the nsp6 protein, which in turn induced autophagosome formation by inhibiting phagosomelysosome fusion (28, 29) . sars-cov-2 may take advantage of this autophagy mechanism, as the acidic ph required for endosome maturation also functions to release the virion at the appropriate site. once all necessary proteins are translated and replication has taken place, virion assembly occurs in the golgi apparatus before release from the cell (30) . symptoms attributed to sars-cov-2 shereen et al. determined a mean incubation period of 5 days in the first 425 patients testing positive for sars-cov-2 (31). the time from infection to death varied from 6 to 41 days, with a mean of 14 days (32) . in january of 2020, huang et al. examined all patients exhibiting pneumonia-like symptoms in order to determine the clinical features of sars-cov-2; importantly, this study did not discriminate between previously healthy patients and patients suffering from chronic illnesses. data was collected from the 41 positive cases, and analysis yielded the following: fever was the first and most common primary symptom (98%), followed by dry cough (76%), lymphopenia (63%), dyspnea (55%), and fatigue/myalgia (44%). less common symptoms included sputum production (28%), headache (8%), hemoptysis (5%), and diarrhea (1%) (33) . a recent systematic review of 148 studies spanning nine countries disputes the above-mentioned data. in this analysis by grant et al., the most common symptoms were as follows: fever (78%), dry cough (57%), fatigue (31%), anosmia (25%), and difficulty breathing (23%) (34) . another study by clemency et al. assessed the likelihood that sars-cov-2 infection presents with symptoms. the positive likelihood ratio of having symptoms with disease from highest to lowest was as follows: anosmia and ageusia, followed by loss of appetite, fever, muscle pain, fatigue, dry cough, productive cough, diarrhea, difficulty breathing, and sore throat (35) . the stability of sars-cov-2 on various surfaces was compared to that of its predecessor sars-cov, and it was found that both viruses exhibit similar stability. the differences in epidemiological spread are therefore most likely to be dependent upon other factors such as high viral load and viral shedding in asymptomatic persons. the study is highly suggestive of an aerosolized pathogen as it remains viable and infectious in droplet form for up to 3 h (36). while resembling the previous sars-cov outbreak, this data confirms that sars-cov-2 is far more transmissible. kinetic studies employed to compare the two strains showed that sars-cov-2 binds to the ace-2 receptor with a 10-to 20-fold higher affinity than sars-cov, suggesting that this could be another main factor explaining the ease of transmission (37) . to further support this hypothesis, computational structural predictions were established to differentiate between the rate of association between the ace-2 receptor and the spike protein of both sars-cov and sars-cov-2. by incorporating the computer simulation into a mathematical model, it was shown that the novel pathogen has slower binding capacity than sars-cov, consistent with the varying life cycles of the two strains. in other words, the longer incubation period of sars-cov-2 was associated with the slower interaction between the spike protein and the ace-2 receptor. this allows for maintenance of an increased viral load in the body, thus contributing to increased human-to-human transmission (38) . the renin-angiotensin-aldosterone system (raas) is responsible for inducing a coordinated cascade regulating both cardiovascular and renal functions. angiotensin-converting enzyme (ace) is responsible for converting angiotensin i to angiotensin ii, with the latter functioning as a pro-sympathetic molecule throughout the body to regulate blood pressure. wakahara et al. measured ace and ace-2 expression under various conditions. in addition to the ace-2 role in local regulation of raas through the conversion of angiotensin ii into vasodilators and anti-trophic peptide, it acts and is expressed in synergy with ace concentrations and is believed to possess counter-regulatory effects to ace (39) . as the expression of the ace-2 receptor is vital for viral entry, it is essential to establish where this gene is expressed throughout the body to identify possible routes of infection and/or the extent of organ damage during infection. zou et al. employed the dataset systems of single-cell rna sequences from different body tissues to identify organs expressing the ace-2 gene in high concentrations. the symptoms reported to be associated with covid-19 (dyspnea, cardiac injury, kidney failure, diarrhea) were compared to ace-2 expression on target cells. it is on this basis that the organs at high risk of damage during viremia were recognized to be the lungs, heart, kidney, and upper respiratory tract (40) . a minority of patients is seen to present with gastrointestinal involvement, and a further study by wong et al. on 140 patients in wuhan demonstrated this complaint in 39% of their cohort. the virus was detected in stool by means of rt-pcr. staining of the n protein showed presence of the virus in the cytoplasm of duodenal, rectal and gastric epithelium thus indicating another possible mode of transmission and another sign of possible infection (41) . zhao et al. also compared the symptoms and disease characteristics of covid-19 patients with that of other pneumonias, indicating that liver damage was more prominent in covid-19 patients. however, it was not clear whether liver enzyme elevation was due to drug toxicity or viral shedding (42) . the ace-2-receptor mapping experiment, while important, provides little information on the broad expressions of the receptor, as ace-2 concentration may differ due to various clinico-pathologies such as hypertension, diabetes, heart failure, smoking and kidney injury (43, 44) . remuzzi and remuzzi in italy demonstrated that two-thirds of patients who died from sars-cov-2 were elderly, diabetic or suffered from cardiovascular disease (45) . their first line drug of choice was angiotensin receptor blockers (arbs). ferrario et al. demonstrated in 2005 that selective blockade of angiotensin ii synthesis or activity has an up-regulatory effect of ace-2 receptors in the kidneys and heart (46) . this ace-2 upregulation in the aforementioned comorbidities could therefore be responsible for the severity of infection in these patients. guo et al. demonstrated that in 187 sars-cov-2-positive patients, 28 .7% had an increase in troponin-t and crp levels that correlated with myocardial injury. sars-cov-2 patients who presented with an increase in cardiac markers exhibited a 59.6% mortality rate compared to those with normal cardiac markers (8.9% mortality rate). sars-cov-2 genomic material has also been isolated from cardiac biopsies. while the pathophysiologic development of myocardial injury is not yet fully understood, it is believed to be caused by either cytokine storm or direct myocardial damage through viral integration (47) . to further understand the organ damage and hemostatic changes, han et al. assessed the change in coagulation proteins in both covid-19 patients and healthy patients. they found that antithrombin levels were lower in covid-19 patients, whereas fibrinogen, fibrinogen/fibrin degradation products and d-dimer were high (48) . a recent cohort study by wichmann et al. reported a possible connection between covid-19 and incidence of thromboembolism. autopsies of 12 covid-19 patients revealed the incidence of deep vein thrombosis (dvt) in 58% of cases, and a third of patients suffered a fatal pulmonary embolism. further studies should be conducted to further elucidate the formation of thromboembolism in the course of covid-19 (49) . cd147, also known as emmprin (extracellular matrix metalloproteinase inducer) is a highly glycosylated transmembrane glycoprotein shown to be involved in facilitating sars-cov endocytosis. this presents an alternative route of viral invasion (50) . like the ace-receptor, cd147 is also present in various tissues such as lymphoid (51) , testes, liver, cerebral, and renal tissues, as well as cardiac and skeletal muscle (52) . in addition to enabling viral entry, cd147 has been linked to other pathologic conditions such as cancer (53, 54) , heart disease (55) and alzheimer's disease (56) . furthermore, the speed of invasion may be increased as cd147 has been shown to be upregulated upon t-cell activation (57) . wang et al. has since shown that the spike protein possesses high affinity for cd147, thus mediating viral invasion and facilitating viral replication. it acts as a negative regulator of the immune system which may contribute to the significant cd4+ decline during covid-19 (58) . under hypoxic conditions, this receptor is involved in the regulation of cell proliferation and apoptosis. the cd147 receptor, a marker of early-stage disease, can be useful for the assessment of pathological progression because it is often expressed on the surface of activated regulatory t cells (59) . lung epithelium is the largest area of the body in constant contact with the external environment, and is responsible for processing inhaled air containing large volumes of bacteria and viruses. the immune response to sars-cov was widely studied, and sars-cov-2 is believed to induce the same responses. innate immunity is the primary countermeasure against infection, and is maintained by a variety of cell types found in airway epithelium, including dendritic cells, innate lymphoid cells and alveolar macrophages. the protective signaling cascade begins as the pattern recognition receptors (prrs) of innate cells recognize pathogen-associated molecular patterns (pamps) of viruses. after recognition, type i and iii interferon (ifn) and other proinflammatory cytokines undergo transcription. autocrine and paracrine signals in both healthy and infected cells subsequently ensure the translation of these cytokines (60) . passive evasion refers to any mechanism of immune avoidance that does not directly interfere with the host immune system. many non-structural proteins (nsp) are translated in order to shield viral genomic material from the human immune system, as shown in table 3 . nsp 3, 4, and 6 are believed to function as modifiers of the intracellular membranes of organelles, where the virus may safely replicate. another passive mechanism is the conformational change of the viral mrna through either the addition of a 5'-cap (via formation of n-7-methyltransferase in nsp 14), or a 2'-o-methyl-transferase as with nsp 16, which marks the viral mrna to be of host origin. in another example, nsp 15 induces endoribonuclease activity against viral mrna at certain stages of its development to avoid detection (20) . nsp 1 was shown to inhibit host mrna translation through the activation of exonucleases. these induce degradation of the host genome and prevent the ifn signal transduction that is necessary to activate viral clearance. nsp 3 expresses plpro, which greatly resembles host cellular de-ubiquitinase action. this induces disruption of ubiquitin-mediated degradation and may also inhibit the innate immune response (61). as with the previous outbreak, immune clearance of sars-cov-2 relies mostly on the activity of th1 cells via the extensive secretion of il-2, il-10, ifn-γ, and tnf-α/β. this cytokine microenvironment activates macrophages and causes cytotoxic t-cell proliferation, initiating pathogen clearance. following degradation and the presentation of viral antigens on apcs, the humoral response acts to limit future infections through antibody neutralization (62) . in the lungs, alveolar macrophages are the first cells to make contact with microorganisms. their main function is to destroy any invaders without overstimulating the adaptive immune system, since foreign pathogens are ever-present. protective measures employed by alveolar macrophages include the surface expression of cd200 and tgf-β receptors, which function as negative regulators of dendritic cells and t cells (63, 64) . this inactivation is exploited by sars-cov, leading to an increase in viral load. additionally, law et al. demonstrated that macrophages and dendritic cells are targeted by sars-cov, resulting in a very low expression of antiviral cytokines. this inhibits apoptosis and hinders the bridging of the innate and adaptive immune systems, leading to lymphopenia (65) . in a cohort study by qin et al. of patients with confirmed sars-cov-2 infection, lymphopenia and an increase in neutrophil-to-lymphocyte ratio was routinely observed among infected patients and was especially evident in severe cases (66) . it has been shown that these two parameters are indicative of systemic inflammation, and were associated with the worst prognosis (67, 68) . because qin and colleagues did not observe any changes in either cd8+ or b cells, it was then hypothesized that sars-cov-2 plays a major role in indirectly damaging lymphocytes, with the release of proinflammatory cytokines and chemokines that results in the consumption of the lymphocytes necessary to prevent innate overactivation (69). qin et al. also noted that the increase in neutrophil-to-lymphocyte ratio was accompanied by an increase in procalcitonin, indicating bacterial co-infection (66). zhao et al. showed that mice depleted of alveolar macrophages and subsequently infected with a mouse-adapted sars-cov strain quickly developed activation of dendritic cells, which in turn activated cytotoxic t cells and initiated viral clearance (70) . while most viral infections end with eradication and development of immunological memory, adaptive immunity does on occasion fail to develop adequately. callow et al. demonstrated in 1990 that patients previously infected with human coronavirus 229e showed a decline in antibody concentration and were capable of being re-infected after 1 year (71) . other studies involving mers-cov have come to the same conclusion, determining that the concentration of virusneutralizing antibody was dependent upon disease severity (72, 73) . it is important to note that the failure of adaptive immunity could be due to either insufficient antibody response or decrease in t-cell durability (74, 75) . appropriate adaptive immunity requires early cd8+ and cd4+ responses. in the case of sars-cov-2, viral evasion of the innate immune system leads to an increase in cytokine production and late cd4+/cd8+ response, which then leads to pathogenic inflammation in patients with high viral loads. due to rapid and unopposed sars-cov-2 replication, cd4+ t lymphocytes are quickly activated to differentiate into th1 cells and are responsible for releasing pro-inflammatory cytokines il-6, gm-csf, and ifn-γ. gm-csf activates to further produce inflammatory monocytes (cd14+ and cd16+) which release more il-6. this disrupts the homeostasis of the immune system leading to cytokine storm (76) . sars-cov-2-related hyperinflammation involves very high levels of il-1-β, il-6, and tnf-α (77). sars-cov-2 is thought to bind to the toll-like receptor (tlr), activating inflammasomes and resulting in the cleavage of pro-il-1β to form il-1β, a mediator of inflammation, fever and lung injury (78) . the pathological immune response has a wide variety of clinical presentations, from mild symptoms to pulmonary failure and death. extensive lung damage is associated with neutrophil and macrophage infiltration while cd4+ and cd8+ count in peripheral blood are simultaneously lowered (79) . croft et al. performed serological analyses in both healthy and sars-cov-2-positive individuals. the cd4+ t cells in infected individuals demonstrated very high levels of cd69, cd44, and cd38, indicating a hyperactive state compared to the healthy group. further analysis showed high levels of the t-cell activation marker ox40 on cd4+ cells, which has been proven to be crucial for cell expansion, survival and cytokine production in both t cells and innate cells (80) . it is important to note that expression levels of ox40 also varied depending on the severity and progression of the disease, and may possibly be a marker of poorer prognosis. cd8+ t cells were also found in a hyperactive state, with higher expressions of cd69, cd44, and cd38. in various deteriorating icu patients, there was an increase in expressions of pd-1 and tim-3 in both cd4+ and cd8+ t cells, indicating immune exhaustion (76, 81) . aside from exhaustion antigens such as pd-1, bellesi et al. (82) revealed upregulated expression of the cd95 antigen on both cd4+ and cd8+ t cells. when cd95 apoptosis-related antigen is observed to be highly expressed, this is accompanied by a lower cd4+ and cd8+ count, which may partially explain the loss of lymphocytes in sars-cov-2-positive patients. the loss of naive cells appears to be particularly important in this context. complement activation can be initiated via the alternative pathway, classical pathway or lectin pathway. innate immunity recognizes pathogen-associated molecular patterns (pamps) in host cells and initiates a destructive response. the mannosebinding lectin (mbl) pathway has been shown to be the first line of defense against sars-cov (83). it is composed of pattern recognition molecules associated with serine protease from the mbl-associated serine proteases (masps), which circulate as zymogens and become active after recognition, binding to carbohydrate-based pamps (84) . to further understand the triggers of the excessive immune response to sars-cov-2, gao et al. showed that as with sars-cov, masp-2 contact with the n protein led to cleavage of c4 and c2 into c4a/c4b and c2a/c2b, respectively, and the mbl pathway proceeded as normal. none of the other pathways triggered complement activation (85) . due to the high infectivity and severity of the virus, the world health organization (who) has been prompted to develop new tools to ensure the fast and accurate diagnosis of the viral infection. many governments have given an unprecedented level of freedom to nominated laboratories to establish reliable diagnostic tests. the increased demand for rapid testing has fueled research in sequencing, characterizing, and understanding sars-cov-2, in order to definitively diagnose it in human samples. as of august 2020, the recommended sampling sites are as follows: the upper respiratory tract (nasopharynx, oropharynx, anterior nares), lower respiratory tract (sputum, bronchoalveolar lavage, pulmonary tissue biopsy) as well as urine, feces or whole blood. according to centers for disease control (cdc) guidelines, material from the upper and lower respiratory tracts are preferred, and should be sampled if possible. material should be collected by an experienced specialist to ensure high standards, and should be tested as soon as possible; exceptions are serum samples which can be stored for up to 2-4 weeks, to be referenced in future follow-up (86) . the overwhelming number of infected cases compounded with the shortage of healthcare personnel has led to the prioritization of testing of affected individuals. due to limited access and resources, the cdc has been compelled to define specific criteria for testing all 2019-ncov patients under investigation (pui) . in order to be tested, the pui needs to manifest with: -"fever and symptoms of lower respiratory illness (cough, difficulty breathing) after 14 days of travel to wuhan city or contact with a 2019-ncov pui within the last 14 days, " or -"fever or symptoms of lower respiratory illness (cough, difficulty breathing) after contact with a patient with a confirmed case of sars-cov-2 infection within 14 days" (87) . the introduction of restrictions not only guarantees good quality patient care and prioritizes acute and severe cases, but maintains the integrity of an already strained healthcare system. the high volume of cases demands fast and efficacious testing regimes for a variety of settings, including the hospital. depending on the type of technology and the personnel available, a few specialized diagnostic protocols have proven to be useful in the field. sars-cov-2 is an rna virus which sheds detectable genetic material in almost all excretions of an infected individual. this material can be detected by a simple nucleic acid test which is capable of identification and characterization of nucleotide sequences. in blood, sputum and other samples, the amount of genetic material is very sparse thus necessitating an additional amplification step in order to reach a particular detection threshold. this method is known as the nucleic acid amplification test (naat). another technique currently available is the polymerase chain reaction (pcr). real-time polymerase chain reaction (rrt-pcr) is the gold-standard molecular technique for detection of sars-cov-2 viral rna in all recommended samples. it is a primary diagnostic test that targets the following sequences that code for structural viral proteins: spike (s), membrane (m), envelope (e), nucleocapsid (n), and rnadependent rna polymerase (rdrp). the available literature suggests that the spike protein (s) may be the primary pivot in intracellular interactions with host cells, thus demonstrating high immunogenic character (88) . high infectivity of sars-cov-2 has compelled the cdc to publish rrt-pcr primers and probes together with all relevant literature for public access (87) . such advances in research were possible thanks to past experience with the previous betacoronavius epidemic. primer design was based on the nucleotide sequences that matched sars-cov and mers-cov with 80 to 90% accuracy (21) . the wide availability of protocols has accelerated progress in research and diagnostic measures. nevertheless, it should be noted that the high mutation rate and large genetic variability of the virus may negatively affect the performance of the assay, and may lead to an increasing number of false-negative results (89) . additionally, the difficulty of the assay, complexity of the logistic analysis, and protocol duration (45 min to a few hours) (90) confer some limitations to this diagnostic tool (62) . the full rna extraction protocol should be implemented in a biosafety cabinet at bsl-2 security level by trained and skilled personnel. it is recommended that none of the samples be heat-treated before rna extraction, which means that samples pose a high risk of infection to laboratory technicians (86) . false-positive results may also be obtained in cases where the amount of viral material in a collected sample is too low for detection (89) . based on a published summary report by the fda, the analytical sensitivity of rt-pcr is 80% with a limit of detection of 6.25 cp/ul. specificity showed no crossreactivity with the most common pathogens: bacterial (legionella pneumophilia, mycobacterium tuberculosis and m. pneumoniae, and streptococcus pneumoniae and s. pyogenes) and viral (adenovirus, parainfluenza, rhinovirus, rsv, etc.). the only detected cross-reactivity corresponded to the severe acute respiratory syndrome coronavirus (sars-cov), but this is not surprising since the n3 target gene in sars-cov-2 demonstrates more than 80% genetic similarity to other betacoronaviruses (91) . the diagnostic protocol formulated by the cdc clearly states the process of confirmation of sars-cov-2 infection, though the criteria depend on the area where the pui is being diagnosed. in areas where the concentration of sars-cov-2 virus is high, a single positive naat result is required to diagnose the patient. in areas with low viral circulation more than one of the following is required: -positive naat for at least two different targets on the sars-cov-2 genome (one specific for the virus) -1 positive naat for betacoronavirus (sars-cov, mers-cov, sars-cov2) with sars-cov-2 genome sequencing. it is indicated that the sequenced fragment must be longer or different from the fragment used in the naat assay (86) . in cases where all tested controls are positive, with all sars-cov-2 markers below growth thresholds, the naat result is negative. however, cases with high clinical suspicion may still produce a negative rt-pcr result. this may be due to a number of factors, such as poor quality and handling of specimens, specimen collection too early or late in the disease course, contamination, and/or technical errors (86) . in other words, the lack of a positive result does not exclude covid-19 disease. in such instances identification of the infected individual is based on clinical observation, patient history and epidemiological information. given the number of tests done to date, in the case of discordant opinions, re-sampling and use of different markers is advised (86) . the limitations of naat diagnostic techniques for sars-cov-2 have generated increasing demand for quicker and simpler tests based on serology. rt-pcr, while very reliable and precise, is only capable of identifying the presence of viral load, and cannot inform on the state or progress of the infection in a given patient. in contrast, serological testing and enzyme-based testing measure immunological responses to the virus, allowing for differentiation between exposed asymptomatic, acutely or mildly sick, and recovered cases. additionally, it is able to quantify the number of cases within a short period of time, and this is crucial for modeling the population-scale of infection, determining the level of prophylaxis, and has implications for the development of a vaccine against sars-cov-2. there are many immunoassay techniques currently approved or pending. their versatility and creativity augment the number of ways with which one can detect the pathogen in a studied sample. they can be divided into serologic tests and enzyme-based immunoassays. serologic tests exploit the natural responses of the human immune system, while enzyme-based immunoassays detect the antigen with specifically manufactured monoclonal antibodies. all tests in this category detect either the antigen shed by the virus or the antibody that is produced by b-cells in response to the pathogen. the rapid diagnostic test (rdt) is a serologic type of diagnostic tool that allows for detection of the target after 10-30 min (90) . it is a qualitative lateral flow type assay that resembles the standard rapid chromatographic test used for the detection of beta-hcg hormone in the urine of pregnant women. as a field-based test, it requires a drop of blood or other body fluid for identification of igg and/or igm antibodies, or the viral antigen itself (90) . antigen based-rdts target the structural viral proteins (s, n, etc.) mainly found in secretions of the upper respiratory tract, and react to the antibodies that are produced 10-30 days after the initial infection. sufficient time is required for the concentration of antibodies to reach the threshold of detection (92) . it is also advised to measure igg and igm titer baselines before or during the first few days after exposure (90) . the non-governmental organization find recognizes 10 cemarked rapid tests ready for use in the field (93, 94) . another report by john hopkins center for health and security lists a few rdts currently approved for diagnostic use worldwide, but the antibody-rdt lateral flow assay by cellex inc. is the only rdt approved in the united states. this test was developed in the usa and china, with a sensitivity and specificity equal to 93.8 and 95.6%, respectively (90) . the statistical significance was determined from studies in two chinese hospitals on 128 sars-cov-2-positive patients and 250 sars-cov-2-negative patients (90) . the test is currently available for purchase with a disclaimer from the fda that the test alone cannot be used for definitive diagnosis. in regards to the april 8 2020 who statement concerning immunoassays, the antibody-/antigen-detecting rdts are only to be used for research purposes (95) . according to recent data, these diagnostic tests do not produce results with appropriate reliability. bruning et al. (96) claims that immunodiagnostic testing for influenza infections in cases with a viral load comparable to sars-cov-2 showed test sensitivity that varied between 34 and 80% (96) . also, due to their abrupt development, the majority of available rapid tests appear not to have been properly validated by relevant institutions and may pose a serious risk to diagnostic efficacy. the simplicity of the rdt means it is neither capable of quantifying the number of antibodies and therefore the phase of infection, nor is it able to ascertain whether these antibodies are part of long-term immunity to the virus. as of august 2020, it is unknown whether exposure imparts immunity after recovery. a 10 day or more delay in diagnosis is a serious diagnostic limitation and may not be of increased benefit to acute-state patients. another type of serologic testing for sars-cov-2 is a neutralization assay. it is a valuable method that detects antibodies capable of clearing the infection. the procedure consists of the infection of a special type of cell (e.g., veroe6) with sars-cov-2, followed by incubation for 3-5 days at variable concentrations. during this time cells are titrated with human serum antibodies in order to detect the quantity of antibodies necessary to stop viral replication (90) . a study performed by zhou et al. demonstrated that 1:40-1:80 dilutions of igg-positive sample were capable of neutralizing 100tcid 50 (50% tissueculture-infective dose) in sars-cov-2 cultures (21) . other than this result, it remains a relatively novel area of research, and information regarding its usefulness in the diagnosis of covid-19 is sparse (90) . table 4 lists a number of exemplary testing kits currently available for diagnostic use. enzyme-linked immunosorbent assay (elisa) is currently one of the most popular commercially used enzyme based immunoassays. as indicated by the name, it is an assay using an enzymatic reaction to indicate a positive diagnostic result. plates covered with viral antigens, prepared by the manufacturer, are incubated with the patient's serum. in cases where the specific igg and igm antibodies are present, the antibody-antigen binding complex will be visualized through an enzymatic reaction (colorimetric change, fluorescence, etc.) the whole procedure may take up to 5 h and requires a large sample of blood, plasma, or serum. it is both a qualitative and quantitative type of assay, thus has great potential for future diagnoses of sars-cov-2 infections. in a study of sars-cov-2-specific antibody responses in covid-19 patients, nisreen et al. used elisa to demonstrate that severe viral infection resulted in higher antibody levels. additionally, they claim that igg seroconversion can be confirmed by applying the same technique in the second week of symptom onset (97) . another team proved that the accuracy of elisa is dependent on the timing of the infection. on days 0-10 after symptom onset, elisa showed <60% positive rate; the rate rapidly increased beyond that time frame for both igm and igg antibodies. liu et al. evaluated immunoassays for detection of antibodies against sars-cov-2, and demonstrated that viral protein s (spike)-based igm elisa had statistically higher sensitivity than n (nucleocapsid)based elisa (p < 0.05) in detecting igm antibodies. it is suspected that the level of immunogenicity of the s protein is the reason for the relatively higher specificity compared to the n protein (98) . based on this data, the antigen-based elisa will be a valuable diagnostic method for the fight against covid-19. currently, the ngo find lists 66 different immunoassay kits already commercialized, with 16 additional test kits in development (94) . with supplemental research, these have the potential to become a powerful rapid diagnostic tool for the hospital setting. however, due to their complexity they, unlike rdts, may not be used in the field. to reiterate, there is an insufficient amount of evidencebased research regarding elisa's specificity and sensitivity for use in diagnostic confirmation, to be comparable with rrt-pcr assays. a lesser known enzyme-based immunoassay that is nevertheless noteworthy is the chemiluminescent immunoassay (clia). very similar to elisa, it uses enzyme-labeled antibodies which activate an enzymatic reaction upon contact with their target. the photon of light emitted-luminescencecan then be quantified and directly corresponded to the volume of reagents. the benefit of this technique is its high sensitivity and the possibility of enhancing the reaction to allow for a larger threshold in samples with higher substrate concentration. clia can be used to detect versatile targets including igm, igg, and iga, and there seems to have been a recent increase in trust for this technique among clinicians. a systematic review by bastos et al. compared lfia, elisa, and clia tests, and the results suggest that clia exhibits the highest sensitivity and specificity for igm and igg in patients with covid-19 (99) . however, bastos and colleagues also demonstrated that due to excessive discrepancies in test rests, none of the three techniques tested were reliable enough to be recommended for large-scale diagnostic purposes. after the 2002-2003 sars outbreak, chandwani and shuter conducted an in vitro study using an engineered prototype of sars-cov to test lopinavir/ritonavir, protease inhibitors indicated for dual-therapy prophylaxis and treatment of hiv-1 (100). lopinavir has higher potency but is less bioavailable, thus co-administration with ritonavir, which additionally inhibits cytochrome p450 34a, leads to prolongation of its action (101) . they showed that this dual therapy inhibited viral cytopathic activity. furthermore, chu et al. conducted a non-randomized trial in 2004, placing two groups of patients on different regimens: the first group received ribavirin, a nucleoside inhibitor, in dual therapy with a reducing dose of corticosteroids; and the second group received lopinavir/ritonavir/ribavirin. it was shown that treatment group two had a decrease in viral load, fewer nosocomial infections and an increase in circulating lymphocytes, indicating a favorable outcome (102) . after the emergence of sars-cov-2, a randomized trial was conducted involving 199 severe cases. ninety nine of these patients received lopinavir/ritonavir while 100 received standard supportive care. detectable viral load in both groups was the same, and time to improvement after lopinavir/ritonavir was only decreased by 1 day, as compared to the group receiving standard care. the authors concluded that the treatment benefits of lopinavir/ritonavir were not established for severe illness (103) . similarly, an open-label randomized control trial by cao et al. (chictr2000029308), involving severe sars-cov-2 cases, compared lopinavir/ritonavir treatment with standard care alone, and they showed that the antivirals yielded no clinical benefits. further trials are thus recommended to establish any possible benefits for patients suffering from less severe illness (104) . favipiravir, another rdrp inhibitor, is a broad antiviral with a mechanism of action that is hypothesized to either incorporate within viral rna leading to chain termination, or bind to a conserved region of the rdrp and prevent nucleotide addition (105) . interferon-alpha (ifn-α) is an antiviral that binds to interferon receptors and activates signal modulators (jak1/2). the phosphorylated interferon receptor binds to the signal modulators, resulting in immune modulation and antiviral protein transcription. in an open-label control study conducted by cai et al., the antiviral activity of favipiravir + ifn-α was compared to that of lopinavir/ritonavir + ifn-α in patients with confirmed sars-cov-2 infection. they demonstrated that patients receiving favipiravir + ifn-α exhibited faster viral clearance and radiological improvement, compared to the other study group. although yielding positive results, this was not a double-blind placebo-controlled randomized study, and more trials must be implemented before definitive conclusions can be drawn (106) . umifenovir is a broad spectrum antiviral possessing dual properties: direct antiviral and virucidal action, as well as demonstrating virustatic effect through impedance of various stages of the viral life cycle (107) clinical trials-mechanism of action unknown. uprifosbuvir, setrobuvir, balaprevir, 2'-c-methylcytidine, valopectibine bms-986094, mk0608, r7128, r1479, idx-184, yak, psi-6130 and psi-6206 apparent in the umifenovir group, and no side effects were observed in either group (111) . an open-label randomized control trial (chictr2000030254) was conducted by chen et al. in which adult sars-cov-2 patients were administered either favipiravir or umifenovir. they showed that favipiravir significantly improved symptoms associated with cough and pyrexia. however, no clinical benefit could be observed when comparing viral clearance between the two therapies (112) . after isolating the genomic sequence of sars-cov-2, in particular that pertaining to rna dependent rna-polymerase (rdrp), elfiky showed that the rdrp of sars-cov-2 exhibits 90.18% similarity with that of sars-cov. an rdrp model was engineered from the ncbi nucleotide protein database using the sars-cov rdrp genome as a template to test several antiviral drugs. to ensure high reliability of the model, a nucleotide comparison between the rdrp model and the sars-cov-2 rdrp was established, and was shown to yield 97.08% homology. the study sought to establish which of 24 compounds (table 5) could bind to rdrp active sites and elicit inhibitory activity. drug docking site analyses were interpreted using protein-ligand-interaction-profiler (plip). five fda-approved drugs showed very high affinity for rdrp, and could prove to be beneficial against sars-cov-2. additionally, three drugs from the clinical trial by elficky showed high affinity for rdrp, and these include idx-184, setrobuvir and yak. drug side effects and toxicity are yet to be disclosed (117) . additionally, one antiviral drug that sparked interest was remdesivir. in a small cohort study, a 10 day course of remdesivir was administered to 53 patients with severe infection, and clinical improvement was observed in 68%. fewer clinical benefits were observed in patients who received invasive ventilator support, and 13% of the patients, especially those who received invasive ventilation, died after the treatment course. multiple factors impede the accurate measurement of remdesivir efficacy and these include preexisting conditions and duration of intubation. as a result, any clinical benefits need to be further investigated in future placebo-controlled trials (118) . in a double blind placebo-controlled randomized trial (ntc04280705) biegel et al. administered remdesivir to hospitalized sars-cov-2 patients presenting with lower respiratory tract involvement to assess its efficacy and safety. remdesivir was shown to be superior to placebo in decreasing recovery time (119) . however, a multicenter, randomized, placebo-controlled trial conducted by wang et al. to assess the efficacy of remdesivir in patients with severe sars-cov-2 (ntc04257656) showed that remdesivir was of no clinical benefit compared to placebo (120) . lastly, a multicenter analysis involving a double-blind placebo-controlled trial (ntc04280705) was implemented to assess the efficacy and safety of remdesivir in the treatment of hospitalized sars-cov-2 patients. preliminary results from this trial showed that patients receiving remdesivir had a faster time to recovery and a lower mortality rate when compared to the placebo group, and it is on the basis of this that the fda issued authorization of remdesivir for emergency use on may 1, 2020. interferons (ifns) are important cytokines with critical antiviral activity. infected innate immune cells produce ifn which enable the jak-stat pathway, leading to recruitment of more nk cells and macrophages. as with sars-cov and mers-cov, nsp 1 of sars-cov-2 exhibits anti-ifn activity by targeting proteins of the jak-stat signaling pathway (121) . ifn inhibition has been correlated to disease severity (122) . furthermore, ifn treatment has already proved effective against ss-rna viruses, and is widely used in the treatment of hcv and hbv. zhang et al. combined ifn-α and ifn-γ in a study conducted in vivo and in vitro. this combination demonstrated synergy, and smaller dosages were required than in ifn monotherapy. this suggests that reduction in unwanted side effects may be possible with the use of dual therapy (123) . it was shown that sars-cov-2 evades detection from cytosolic rig-i and mda5, preventing the activation of ifn-i and the subsequent stimulation of innate cells. it is at this point that the importance of toll-like receptors (tlrs) should be emphasized. negishi et al. demonstrated that viruses that evade cytosolic safeguards can be inhibited by tlr-3 action. tlr-3 functions to activate ifn-ii, which in turn elicits an antiviral response (124) . the use of tlr-3 agonists in mice by shahabi nezhad et al. showed promising results; they were able to increase levels of ifn-α/β/γ, which compensates for the inhibitory actions of sars-cov on signaling pathways (105) but further research is needed to determine if this may result in toxic overstimulation of the immune system (125) . in an open-label randomized phase ii trial by hung et al., a triple combination of ifn-β-1b, lopinavir/ritonavir and ribavirin was administered to covid-19 patients with mild to moderate disease. this combination proved to be safe and superior to lopinavir/ritonavir + ribavirin, with patients showing alleviation of symptoms, shortened duration of viral shedding, and shortened hospital stay. these promising results mean that future trials utilizing ifn-β-1b are warranted (126) . anti-c5a-antibody, eculizumab, and bevacizumab the discovery of the sars-cov-2 specificity to masp-2 of the mbl pathway opened the potential for prophylactic treatment against cytokine-mediated lung damage. it was previously shown in the literature that acute lung injury due to viral infection could be prevented by the use of anti-c5a-antibody treatment (127) , and on the basis of this, gao et al. used a recombinant c5a-antibody in an open-label trial involving severely ill patients the outcome of the first two recipients of the monoclonal antibody was described. both patients showed improved oxygen saturation, increased lymphocyte count, decrease in inflammatory proteins, improvement in liver function, and alleviation of pneumonia. although the use of anti-c5a antibody shows great promise, the trial is still ongoing and the final efficacy is yet to be disclosed (85) . another trial (ntc0428713) is currently assessing the potential of eculizumab to reduce mortality in 19 patients. in a similar mechanism of anti-inflammatory action, eculizumab inhibits c5 cleavage thus preventing the release of c5a. for the treatment of ards, a clinical trial (nct04275414) is comparing the therapeutic potential and side effects of the monoclonal antibody bevacizumab for critical covid-19 patients. by targeting vascular endothelial growth factor (vegf), an angiogenic factor, bevacizumab may prevent the disruption of the vascular barrier that causes edema and lung injury (128) . a preliminary study conducted by wang et al. (129) identified potential antibodies with the capacity to neutralize sars-cov-2. the s protein ectodomains of both sars-cov and sars-cov-2 were expressed in hek-293t cells using plasmid transduction. similarly, hek-293t cells were transfused with plasmids containing sars-cov1/2 in s protein subdomains tagged with either the mice or human fc portion of igg. h2l2 mice antibodies were produced through gradual immunization with the sars-cov s protein ectodomain. the spleen and lymph nodes were then harvested to produce hybridomas. of the 51 samples, only one of the hybridomas (47d11) exhibited cross-reactivity sars-cov and sars-cov-2 s proteins. the chimeric antibody was reformatted and expressed as a fully human igg. it was shown that this novel igg tightly binds the conserved rbd of the sars-cov-2 s protein in infected cells and neutralized the virus in veroe6 cells. this represents the very first human monoclonal antibody that is able to fully neutralize sars-cov-2 (129) . this cross-neutralizing antibody, due to a conserved epitope region on the spike protein, could be the key to preventing future betacoronavirus outbreaks. 47d11 has recently completed phase i clinical trials (nct04411628) to establish dosing in hospitalized patients with sars-cov-2 under longterm follow up. now undergoing phase ii trials (nct04427501), the monoclonal antibody will be tested on ambulatory patients, with results estimated to be available in september. as stated earlier, the ace-2 receptor is crucial for viral attachment and entry. an in vitro experiment conducted by monteil et al. exposed veroe6 cells to varying concentrations of plaque forming units (pfu) from sars-cov-2 in the presence and absence of human recombinant soluble ace-2 (hrsace-2). the infection was inhibited 15 h after introduction of the virus. the experiment was repeated using human capillary organoids and human kidney organoids, and the same inhibitory actions of hrsace-2 were observed. it is important to note the dosedependent nature of this inhibitory action. hrs-ace-2 has already undergone phase i and ii clinical trials for the treatment of ards (130) , and monteil et al.' findings suggest that hrs-ace-2 could be a potential therapeutic agent against sars-cov-2 and phase ii trials (nct04335136) are currently underway in various european countries (131) . in an open label trial by the recovery collaborative group, 2,014 hospitalized patients received either low dose dexamethasone or standard care alone by random assignment. it was observed that the group of patients on mechanical ventilation who received dexamethasone exhibited lower mortality rates compared to those receiving standard care alone (132) . while other studies further support the role of glucocorticoids in the reduction of mortality (133) some reported conflicting results and showed no clinical benefit or even harm to the patient (134) . a systematic review of 15 studies concluded that while critically ill patients are more likely to benefit from glucocorticoid therapy, their use was associated with increased mortality as it resulted in longer hospital stays and increased tendency toward serious nosocomial infections (135) . clinical trials are currently ongoing to assess the risk vs. benefit for the use of glucocorticoids in the treatment of covid-19. cytokine storm remains the main cause of acute lung injury and organ damage in the course of sars-cov-2 infection. it is chiefly caused by gm-csf and il-6. il-6 receptors exist in two forms: the soluble form (sil-6) and the membrane bound (mil-6). to initiate pro-inflammatory action, il-6 binds to sil-6 and the complex binds to gp130 to complete signal transduction. due to this, the therapeutic use of the il-6 antagonist tocilizumab, which binds to both sil-6 and mil-6, was suggested. xu et al. qualified 21 critical patients in a trial with tocilizumab. clinical symptoms, radiological findings and laboratory values all improved after treatment, and 19 patients were successfully discharged (136) . in an open label study by morena et al., however, 51 critically ill sars-cov-2 patients were treated with tocilizumab. all patients presented with decreased oxygen saturation and an increase in plasma il-6. while positive results were observed with tocilizumab rapidly decreasing inflammatory markers, no clinical benefit was reported as patients quickly developed life-threatening bacterial and fungal infections (137) . jordan et al. conducted another study administering 27 critical patients with a single dose of tocilizumab. this resulted in significant decrease in inflammatory proteins and reduction in fever. twenty two patients receiving mechanical ventilation were able to be extubated and vasopressors discontinued. two patients died, and the authors report that these patients were already in severe septic shock due to sars-cov-2-related pneumonia, and were unresponsive to vasopressors. four patients did not respond to the medication and had poorer outcomes. while the results are promising and in line with the findings by xu et al., limitations of this work include the lower-thanrecommended dose of tocilizumab, chosen by the authors due to drug shortage, and the absence of a control group (138) . a placebo-controlled randomized clinical trial is still necessary before recommendations can be made. historically, il-1 has been shown to act as a pro-inflammatory cytokine with actions on several immune cells (139, 140 (144) . convalescent plasma has been employed and has shown promise in the treatment of sars, mers and influenza (145) (146) (147) . in a study by duan et al., 10 severely ill patients were transfused with 200 ml of convalescent plasma harvested from donors who have recovered from sars-cov-2 and had antibody titers above 1:640. within 1 to 3 days, symptoms had disappeared in all 10 patients, and radiological investigation showed improvement after seven days. viral load was undetectable by rt-pcr in 7 patients and no adverse side effects were detected (148) . shen et al. treated five covid-19 patients with convalescent plasma. all five patients were critically ill and intubated, presenting with ards and pneumonia and experiencing high viral load despite treatment with antivirals. the outcome was largely positive: ards was resolved in four patients within 12 days, and three patients were able to be weaned off mechanical ventilation within 2 weeks. three patients were discharged while two remained stable in recovery (149) . joyner et al. recently made an assessment of the safety of convalescent plasma in 20,000 covid-19 patients. the incidence of serious adverse events including transfusion reactions, cardiac events and thrombotic events was low. mortality rates were shown to be higher in critical patients receiving mechanical ventilation or those in septic shock. the conclusion drawn from the study provided evidence that the administration of convalescent plasma in a hospital setting was safe and that early administration is more likely to reduce fatality rates (150) . it has been previously shown that sars-cov induces viroporin production in the host cell membrane to facilitate virion release. viroporin 3a has been associated with nlrp3 (nod-like r family, pyrin domain 3) inflammasome activation (151) , which induces the production of pro-inflammatory cytokines such as il-1-β and il-18 via the gasdermin d (gsmd) pathway (151, 152) . like sars-cov, sars-cov-2 induces the production of large amounts of il-1-β (33) . on the basis of this, a possible treatment could be il-38, a member of the il-1 family. when placed with activated peripheral mononuclear cells, il-38 demonstrates suppressive and anti-inflammatory effects through the inhibition of il-1, il-6, and tnf production (153) . another member of the il-1 family is il-37. both in vitro and in vivo studies showed that il-37 acts as a negative regulator of inflammation, aiding in the protective actions exhibited by tgfβ on dendritic cells and thus attenuating the t cell response (154) . both il-38 and il-37 could potentially be valuable in the treatment of covid-19 (155) . baricitinib is a janus kinase (jak) 1/2 inhibitor used to treat rheumatoid arthritis. jak 1/2 inhibition prevents activation of pro-inflammatory cytokines such as gm-csf, il-2, il-6, il-12, and il-23. adaptor-related protein complex 2 (ap2)-associated protein kinase i (aak1) induces receptor-mediated endocytosis. baricitinib has been shown to have very high affinity for aak1, thus could feasibly inhibit both cytokine storm and viral entry to the cell (156) . in a small cohort study, titanji et al. administered baricitinib and hydroxychloroquine to 15 patients with moderate to severe covid-19. twelve of the 15 patients recovered, and vitals and inflammatory markers were seen to improve after baricitinib was initiated. two patients however developed serious bacterial or fungal infections due to prolonged icu stay (157) . a phase iii double blind placebo-controlled randomized trial involving baricitinib (nct04421027) is currently ongoing to assess the efficacy and safety of the drug as a potential immune inhibitor preventing cytokine storm and viral entry. sars-cov-2 is the most structurally and genetically similar to sars-cov, thus findings from monoclonal studies on sars-cov have been utilized to target the shared aspects between the strains. the monoclonal antibodies shown in table 6 are engineered to specifically bind to different domains on s1 or s2. though none have progressed to clinical trials, they mechanism of action 80r binds s1 and prevents interaction with ace-2 (158) synergistic action; bind s1 and prevent interaction with ace-2; prevent immune escape (159, 160) f26g18 f26g19 m396 bind linear epitope of s1; bind conformational epitope of s1; inhibit interaction of s1 with ace-2 (161) binds hdr domain of s2 and prevents interaction of receptor in vitro (162) 201 binds s1 and prevents interaction with ace-2 (163) monoclonal antibodies studied for the treatment of sars-cov-2 function igg that binds to a conserved region in the rbd of the s protein and fully neutralizes sars-cov and sars-cov-2 (129) anakinra il-1-inhibitor used to reduce effects of the cytokine storm in sars-cov-2 (143) show promise in vitro and in vivo against sars-cov and sars-cov-2 (164) . the immunomodulatory potential of mesenchymal stem cells (msc) was first identified by luk thromboembolism as a result of endothelial injury in the course of infection is a serious and fatal complication in critically ill patients (168) (169) (170) . tang et al. enrolled 449 covid-19 patients with severe disease for a study in which 99 patients who received low molecular weight heparin for a week or more. these patients were associated with better prognosis than the patients who were not administered anticoagulant therapy (171) . helms et al. followed 150 icu patients in a multicenter prospective cohort study. thromboembolic events were observed in 16.7% of patients despite prophylactic and therapeutic use of anticoagulants. the authors noted that pulmonary embolism was diagnosed a few days after icu admission and was more common in ards patients. they conclude that although other papers have reported the effectiveness of heparin, a higher anticoagulant target should be implemented with other anticoagulants such as anti-xa (172) . the pathogenesis of thromboembolism in the course of covid-19 is still unclear. one of the proposed pathways implicates ards: the profound hypoxemia and vasoconstriction may lead to vascular occlusion (173) . another proposed mechanism is that unlike in a healthy lung, a diseased lung is unable to maintain the balance between fibrinolysis and coagulation, thus resulting in decreased action of tissue plasminogen activators (tpa) (174) . more randomized clinical trials are currently ongoing to assess the efficacy of various anticoagulants. in a 2014 pilot study by fowler et al., critically ill septic patients were given either a vitamin c infusion or a placebo. a dramatic decline in inflammatory markers was observed in the vitamin c group, and their sequential organ failure assessment (sofa) score was decreased compared to the placebo group. there were also no adverse events observed with the vitamin c group (175) . in 2019, fowler et al. published on the use of vitamin c vs. placebo in septic patients with ards, and they concluded that the vitamin c infusion did not improve either inflammatory markers or organ dysfunction score (176) . several clinical trials are ongoing verifying the benefits of vitamin c in the treatment of covid-19. widely used as an antimalarial drug, hydroxychloroquine has been shown to possess broad antiviral action, including effectiveness against hiv-1 and influenza type a and b. its antiviral activity has been tested on sars-cov-2. in preventing the glycosylation of the ace-2 receptor, hydroxychloroquine effectively prevents viral entry (177) . furthermore, it has been shown to alkalinize the organelle, which serves to prevent the formation of mature endosomes required to shield the virus from immune cells, and for replication (178) . apart from its broad antiviral activity, hydroxychloroquine has proven to be adequately anti-inflammatory, interfering with nlrp3 activation and impairing the production of pro-inflammatory cytokines, specifically il-1-β (179) . in an open-label nonrandomized clinical trial by gautret et al., sars-cov-2-positive patients were divided into three groups: group 1 receiving hydroxychloroquine, group 2 receiving hydroxychloroquine + azithromycin (antibiotics were given at the discretion of the physician to prevent opportunistic infections), and group 3 not receiving hydroxychloroquine. the primary outcome in group 1 and 2 was viral clearance within 3 to 6 days, but greater results were achieved when hydroxychloroquine was combined with azithromycin (180) . azithromycin, a bacteriostatic agent, has shown antiviral activity against zika virus, ebola virus (181) and rsv. its antiviral mechanism of action, with regards to rsv, is hypothesized to be in decreasing the expression of fusion proteins in airway epithelium (182) . in a study conducted by million et al., patients suffering from early sars-cov-2 infection were treated with hydroxychloroquine and azithromycin; this combination proved to efficaciously reduce the viral load and was deemed safe with minimal risk of complications (183) . chen et al. also showed that hydrochloroquine was safe and efficacious in the treatment of mild disease (184) . the efficacy of hydrochloroquine with or without azithromycin has since been disputed. in a recent open-label randomized control trial, the efficacy and safety of hydroxychloroquine + standard care vs. standard care alone was assessed by wei et al. in patients with mild to moderate covid-19. the group receiving hydroxychloroquine was shown to suffer more adverse effects, and there was no observed clinical benefit compared to patients given standard care alone (185) . molina et al. also concluded that while hydroxychloroquine proved to be beneficial in past studies, the combination of hydroxychloroquine and azithromycin for severe sars-cov-2 infections resulted in inadequate viral clearance, and the clinical benefits previously seen in patients with mild to moderate illness were not observed in hospitalized patients with severe disease (186) . this broad spectrum antiparasitic agent has been shown to possess antiviral activity. in vitro studies indicate that ivermectin prevents non-structural proteins of both dengue virus (187) and hiv-1 (188) from interacting with the importin α/β1 on the host cell, which prevents viral integration. it has been shown that the sars-cov nucleocapsid (n) protein integrates with the nucleus and nucleolus, and prevents cytokinesis of the host cell via importin-α/β1. the exact role of the n protein in the cell cycle is not known, but it is postulated that this structural protein enters the nucleolus to promote viral replication and encourage suitable conditions for viral packaging (189) . an in vitro experiment by caly et al. showed that a single dose of ivermectin administered to inoculated vero cells effectively controlled viral replication within 1 to 2 days, which may prove beneficial for newly infected patients. it was hypothesized that like in other viruses, ivermectin's antiviral action against sars-cov-2 is derived from the inhibition of importin-α/β1. (190) . alam et al. treated 100 mild to moderately ill patients with a combination of ivermectin and doxycycline. symptom improvement was observed after 72 h following treatment, and no side effects were noted. this study however makes no conclusion on the efficacy and safety of this therapy as it is not a place-controlled randomized clinical trial (191) . similarly, caly et al.' results, while promising, cannot be applied until safety margins have been established in further clinical trials. this glycopeptide antibiotic used in the treatment of grampositive bacterial infections has been shown to possess antiviral activity against ebola, mers, sars and hiv-1. it is believed that teicoplanin interferes with endosome formation through alkalization. cleavage of the s protein by cathepsin in the late endosome is inhibited, which in turn prevents the release of viral rna (192) . baron et al. showed that the cathepsin l sequence is conserved in sars-cov, suggesting that teicoplanin could be a key treatment in patients who are diagnosed early with sars-cov-2 (193) . there have been tremendous advancements in the field of immunotherapy since the development of chimeric antigenreceptor t cells (car-t). these cells differentiate from our basic t-cells by overcoming t-cell control safeguards and therefore express fewer exhaustion markers pd-1, tim3, and lag3. furthermore, they are capable of differentiating into terminal effector t-cells responsible for pathogen and tumor destruction. car-t cell treatment has already shown great advances in oncology, inducing long-term remission in patients suffering from acute lymphoblastic leukemia (194) . it is currently being investigated for the treatment of viral infections such as hiv-1, hbv, and hcv. development of car-t cells specific to hiv-1 infection has now entered clinical trials (195) . crispr-cas (clustered regularly interspaced short palindromic repeats), a mechanism evolved to protect against bacteriophages has shown great promise as a genetic editing tool. in vitro studies have used lentiviral vectors consisting of cas9 (crispr-associated proteins) and sgrna specific ccr5 (single guided rna responsible for the detection of the genome of interest) against cd4+ t cells susceptible to hiv-1 infection. the viral vector was able to disrupt the ccr5 gene in the cd4+ cells thus inhibiting hiv-1 entry (196) . in a novel study, the use of the crispr-cas system against human lung epithelium infected with sars-cov-2 yielded positive results: crispr-cas was able to be transfected in the lung epithelium to degrade the virus (197) . this method was shown to possess protective actions against the known pathogenic coronaviruses. gene editing and car-t cell therapy open a new frontier in future treatment modalities. the rapid spread of sars-cov-2 poses a threat of global proportions. time is of the essence, and the discovery of accurate diagnostic methods and treatment protocols are imperative in preventing further spread of this pathogen. similarities between sars-cov-2 and its predecessor have formed the framework upon which diagnostic and treatment approaches to the novel virus are based. rt-pcr primers, based on sars-cov and mers-cov, have proven to be highly sensitive and specific, though not without their flaws. time consuming and prone to producing false negative results, this has led to the employment of more efficient testing methods such as serologic tests and enzyme-based assays, capable of quantifying infected patients on a large scale. to date, there are still no therapies specifically targeting sars-cov-2. while many fda-approved antivirals on the market have had success in patients presenting with differing degrees of illness severity, the development of specific antivirals remains an area of active research. on the other hand, immunotherapy has been shown to be effective, particularly with the discovery of hrs-ace-2 and other promising immune modulators, the development of the 47d11 monoclonal antibody capable of neutralizing sars-cov-2, as well as msc therapy. the non-traditional use of anti-malarial agents had previously showed great promise but have now proven to lack adequate antiviral action and have been associated with severe complications. until guidelines are updated following the multitude of ongoing clinical trials, standard care remains the main treatment modality. rigorous research with regards to this pandemic not only adds to the scientific literature, but is critical for public health policy surrounding future outbreaks. only with collaborative research efforts and dissemination of knowledge may we interrupt exponential transmission of disease and maintain human losses at the minimum. kk and jc: conceptualization. kk, np, and dh: investigation and resources. kk, np, jc, and dh: writing-original draft preparation. kk, jc, and mk: writing-review and editing. jc and mk: visualization. mk and am: supervision and project administration. all 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perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (covid-19) inactivated mesenchymal stem cells maintain immunomodulatory capacity membrane particles generated from mesenchymal stromal cells modulate immune responses by selective targeting of pro-inflammatory monocytes transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia incidence of thrombotic complications in critically ill icu patients with covid-19 acute fibrinolysis shutdown occurs early in septic shock and is associated with increased morbidity and mortality: results of an observational pilot study coagulation and sepsis anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study the endothelium in hypoxic pulmonary vasoconstriction participation of 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pilot study of hydroxychloroquine in treatment of patients with moderate covid-19 hydroxychloroquine in patients with mainly mild to moderate coronavirus disease: open label, randomised controlled trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection nuclear localization of dengue virus (denv) 1-4 non-structural protein 5; protection against all 4 denv serotypes by the inhibitor ivermectin an alphascreen r -based assay for high-throughput screening for specific inhibitors of nuclear import localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro a case series of 100 covid-19 positive patients treated with combination of ivermectin and doxycycline glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) teicoplanin: an alternative drug for the treatment of covid-19? t cells expressing cd19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial car t cells beyond cancer: hope for immunomodulatory therapy of infectious diseases ccr5 gene disruption via lentiviral vectors expressing cas9 and single guided rna renders cells resistant to hiv-1 infection development of crispr as a prophylactic strategy to combat novel coronavirus and influenza key: cord-016742-y7jgjera authors: bauer, maria title: cardiovascular anatomy and pharmacology date: 2017-07-03 journal: basic sciences in anesthesia doi: 10.1007/978-3-319-62067-1_11 sha: doc_id: 16742 cord_uid: y7jgjera this chapter reviews the cardiovascular anatomy, the effects of medications on the cardiovascular system, and current guidelines. patients in the perioperative period often receive agents that affect hemodynamic variables such as heart rhythm and rate, blood pressure, or cardiac output. are coronary blood flow and arterial o 2 content. coronary blood flow is determined by the patency of the coronaries, coronary perfusion pressure, and coronary vascular resistance. 2. the major determinants of myocardial o 2 demand are heart rate, inotropic state, and wall tension (which is the function of intracavitary pressure, radius and wall thickness, preload and afterload). 3. critical o 2 delivery is the point at which the extraction ratio is maximized, and any further incongruence between demand and supply will lead to tissue hypoxia. 4. in the perioperative setting, continuation of longstanding beta blocker therapy is recommended. 5. beta blockers should not be started on the day of surgery in beta blocker naïve patients. 6 . patients with coronary artery disease undergoing surgical coronary revascularization should be administered a beta blocker before surgery. 7. although recent perioperative guidelines suggested continuing angiotensin converting enzyme (ace) inhibitors/angiotensin receptor blockers (arbs) before non-cardiac surgery, adverse circulatory effects during anesthesia in patients chronically treated with ace inhibitors/arbs has led to the recommendation that these drugs be discontinued 24 h before surgery. 8. epinephrine inhibits uterine contractions especially during the second stage of labor. the heart is a conical hollow muscular organ located between the lungs in the mid-mediastinal portion of the thorax, suspended in the pericardial sac. as a dual pump, it maintains unidirectional blood flow to the body and the lungs by its rhythmical torsion and untwisting throughout the series of cardiac cycles. its receiving chambers (the left and right atrium, composed of 2 myocardial layers), and its pumping chambers (the left and right ventricle, composed of 3 layers of myocardium building up the 2 separate vortices each ventricle) are structurally separated by their respective interchamber septa corresponding with the long axis of the heart, and are electrically isolated by the left and right fibrous rings comprising the fibrous cardiac skeleton in the short axis of the heart, perpendicular to the long axis. the development of the cardiovascular system begins on day 17 of gestation, when mesoderm-derived blood islands, con-sisting of endothelial cells and hemoblasts, begin to form. these blood islands coalesce to form a pair of endothelial heart tubes, which on day 21 fuse into a single primitive heart tube with a cranial inflow (arterial) and a caudal outflow (venous) end. this primitive heart tube is divided into 5 regions: truncus arteriosus, bulbus cordis, primitive ventricle, primitive atrium, and sinus venosus. initial contractions occur on day 21-22, and unidirectional circulation is established by week 4. from weeks 4-7, folding and septation of the heart and the great vessels takes place, critical to the development of the 4 chambers and the normal embryonic vascular circuit. in the developed heart, the atrial chambers lie cephalad and to the right of their corresponding ventricles, and the right-sided chambers lie anterior to their corresponding left-sided chambers. the atrial and ventricular septation is summarized later. the fibrous skeleton of the heart is a framework of 4 dense collagen rings around the atrioventricular (av) and semilunar valves, as well as the left and right fibrous trigones (also known as the central fibrous body), and the membranous portion of the interatrial and interventricular septum. its main component is the central fibrous body, where the leaflets of all 4 cardiac valves converge. the fibrous skeleton reinforces the ostia of the valves and prevents the annuli from overdistension by resisting forces of pressure developing through the cardiac cycle. it provides attachment for the valvular leaflets and cusps, as well as for some of the musculature of the atrial and ventricular wall. it is electrically impermeable, only allowing electrical propagation from the atrioventricular node across the right fibrous trigone to the bundle of his. chronic degenerative changes and calcium deposition into the collagen skeleton results in delayed conduction and depolarization, arrhythmias, rigidity of the valvular ostia, restricted leaflet opening and/or leaflet malcoaptation. the interatrial septum is a blade-shaped wall between the left and right atrium. its development begins during the fifth gestational week with the septum primum growing from the posterior wall of the primary atrium toward the endocardial cushion, formed within the atrioventricular canal. with the incomplete fusion of the septum primum and the endocardial cushion, a progressively diminishing space above the endocardial cushion, the ostium (also known as foramen) primum remains. during this process, small coalescing perforations in the upper portion of the septum primum create the ostium (or foramen) secundum. concurrently, a second septum, the septum secundum begins to form to the right of the septum primum, growing from the anterior wall, partially covering the foramen primum. its growth stops at the seventh week of gestation, leaving a gap known as the foramen ovale. this is essential in reducing pulmonary blood flow through the inactive fetal lungs. the foramen ovale is continuous with the ostium secundum, and is covered by the septum secundum on its left side. as a result of normal adaptive changes in the neonatal circulation, the decreasing pulmonary vascular resistance (pvr) and the resultant reversal of interatrial pressure gradient results in the permanent functional closure of the foramen ovale. in onethird of the population incomplete fusion between the septum primum and the septum secundum results in a residual flap valve and a probe patent foramen ovale (pfo). excessive apoptosis within the septum primum, or incomplete development of the septum secundum results in an atrial septal defect (asd), the most common congenital heart defect to manifest in adulthood: 5 there are 4 types of asd, the most common being the secundum type asd, accounting for 80-90% of all asds. the secundum type asd is located centrally, it is larger than a pfo, and it is commonly associated with mitral valve prolapse. 5 the primum type asd is less common, it represents 2-3% of asds. it is located in the lower portion of the interatrial septum, and it is associated with endocardial cushion defects, av-valve abnormalities, or ventricular septal defects such as in down syndrome. 5 the sinus venosus asd represents 2-10% of asds. it is associated with abnormal pulmonary venous return. 5 the fourth type, the coronary sinus asd or unroofed coronary sinus is the least common. it results from incomplete septation between the inferior portion of the left atrium and the coronary sinus, and is commonly associated with a persistent left superior vena cava. asds often remain clinically silent with preserved normal left atrial size, however, longstanding left-to-right shunting and resultant dilatation of the right-sided chambers along with persistent pulmonary hypertension may reverse the direction of the shunt, resulting in hypoxemia and eisenmenger physiology. the interventricular septum (ivs) separates the left and right ventricles from one another. under physiological pressure and filling conditions its convexity is bowing into the right ventricle. its margins are marked externally by the anterior and posterior interventricular grooves. the upper, posterior part of the interventricular septum is thin, and constitutes the membranous interventricular septum. the greater, anterior portion is the muscular interventricular septum. during cardiogenesis, along with the atrial septation a concurrent ventricular septation is also taking place. with the development of the endocardial cushions and the atrioventricular separation, there is tissue growth between the left and the right sides of the developing atrioventricular canal. this is the muscular portion of the interventricular septum, growing from the inferior portion of the ventricle toward the endocardial cushion. as the muscular ivs reaches the endocardial cushion, a small interventricular foramen remains. this is closed by tissue growth from the endocardial cushion, connective tissue from the muscular ivs, as well as tissue from the septation of the truncus pulmonalis and the conus arteriosus. inadequate contribution from either component results in different types of ventricular septal defects. the right atrium is the cardiac chamber receiving the systemic and cardiac venous return via the superior and inferior venae cavae (svc and ivc) and the coronary sinus. it is divided into 3 components. the venous component receives deoxygenated blood from the svc and the ivc. at the inferior cavoatrial junction lies the eustachian valve, important before birth in directing blood from the ivc to the left atrium across the foramen ovale. a network of fine filamentous strands, the chiari network, may be seen in this region. the variably sized eustachian valve and chiari network are normal variants within the right atrium. the second component is the vestibule, which converges into the leaflets of the tricuspid valve, and the third is the appendage (also known as auricle). the right atrial appendage has a wide junction with both the vestibule and the venous component. the vestibuloauricular junction is identified by the terminal groove, the external marking of the prominent terminal crest on the internal surface. a consistent feature of the right atrial anatomy is the extension of the auricular pectinate muscles beyond the appendage to the atrioventricular junction. the sinus node, a cluster of specialized myocardial cells capable of spontaneous electrical impulse generation, lies at the superior cavoatrial junction at the terminal groove. it is supplied by the nodal artery, arising from the right coronary artery in 55% of cases, or the proximal left circumflex artery in the remaining 45%. the atrioventricular node is located in the floor of the right atrium, near the opening of the coronary sinus. preserving blood flow to the sinus node and other conductive tissues is key to avoiding arrhythmias and other conduction abnormalities. the tricuspid valve is located nearly vertically behind the aortic valve. with a valve area of 4-6 cm 2 , it is the largest of the 4 valves in the heart. its annular plane is saddle-shaped and is apically displaced relative to that of the mitral valve. a displacement index greater than 8 mm/m 2 (body surface area) suggests the presence of ebstein's anomaly. its 3 leaflets are the septal, anterior, and posterior tricuspid valve leaflets, attached by their chordae tendineae to the right ventricular papillary muscles (true chords), or, not uncommonly, directly to the septum (false chords). the subvalvular apparatus of the tricuspid valve is variable. most commonly, the chordae tendineae originate from 2 or 3 papillary muscles, the anterior being the most prominent; the posterior, which is usually less prominent and may have several subdivisions; and the septal, which is the least prominent or may be entirely absent. the right ventricle is the most anterior of the 4 chambers of the heart, located immediately behind the sternum. its geometry is complex: the right ventricle is crescent shaped when viewed in the short axis, and triangular when viewed longitudinally. current guidelines identify its walls as anterior, inferior, and lateral free wall. medially, the right ventricle shares the septum with its left-sided counterpart. for purposes of functional quantification, these walls are further divided into basal, mid, and apical segments. anatomically and functionally, the right ventricle is composed of 3 portions: the smooth muscular right ventricular inflow tract, the apical trabecular, and the right ventricular outflow tract. the inlet portion encircles the tricuspid valve. the trabecular portion of the right ventricle extends into the apical region. the wall of the ventricle is thin here, making it vulnerable to perforation by catheters and electrodes. the most prominent of the trabeculae is the septomarginal trabecula. it carries part of the right bundle branch of the conduction system. it was once wrongly thought to prevent the right ventricle from overdistending, hence the name "moderator band. " the septomarginal trabecula originates from the base of the septum, it divides into an anterior limb that supports the pulmonic valve cusps, and a posterior limb, from which the medial papillary muscle arises. its main mass extends from the base of the septum to the apex and divides into smaller muscle ridges. two of these are particularly prominent, one giving rise to the anterior papillary muscle, and the other crossing the right ventricular cavity as the moderator band. the outlet portion consists of the circumferential muscular infundibulum that supports the pulmonic valve, which is the only one of the cardiac valves with no single ringlike annulus. it is supplied by the conus branch of the right coronary artery. the pulmonic valve separates the right ventricular outflow tract from the pulmonary artery. it is located anteriorly and to the left of the aortic valve. it is a semilunar valve formed by 3 cusps, labeled as anterior (nonseptal), left, and right, each with a fibrous nodule at the midpoint of their free edges. the leaflets coapt via their crescent-shaped surfaces. the cusps of the pulmonic valve are formed by endocardial folds that are supported by internal dense collagenous plates as well as elastic connective tissue, continuous with the fibrous skeleton of the heart. they are thinner than the cusps of the aortic valve, and, lacking fibrous continuity with the septal leaflet of the tricuspid valve, are supported only by a prominent ridge of the posterior subpulmonary infundibular musculature. this is the supraventricular crest that separates the pulmonic and tricuspid valves from one another, and supports the semilunar attachments of the pulmonic valve. surgical incisions made through this crest may run toward the interventricular septum, and jeopardize the right coronary artery. oxygen-rich blood returning from the lungs via the 4 pulmonary veins is received first by the left atrium. the left atrium has 3 basic components: first, the largest, smoothwalled pulmonary venous component; second, the vestibule; and third, the left atrial appendage. the wall of the vestibule is continuous with both the venous component and the posterior (mural) leaflet of the mitral valve, and may affect normal valvular function when left atrial dilation is present. pectinate muscles are confined almost exclusively to the appendage in the left atrium. the flap valve of the fossa ovalis is found on the left atrial side of the interatrial septum. recurrent, symptomatic, drug-refractory atrial fibrillation most often originates from the pulmonary veins or their pulmonary venous ostia. circumferential or segmental ablation of potential triggers is considered effective means to control refractory atrial fibrillation, as scar forming is thought to prevent propagation of abnormal signals to the rest of the atrial musculature. the ablation and mapping catheters are inserted via the femoral or jugular veins, and are placed through separate transseptal punctures. most often, radiofrequency energy is used to make ablation lesions around the pulmonary vein ostia. in the presence of a left atrial appendage thrombus, or inability to anticoagulate in the 30-day peri-and post-procedural period, pulmonary vein isolation for atrial fibrillation ablation is contraindicated. a rare, frequently disabling, sometimes fatal complication of the procedure is an atrio-esophageal fistula resulting from thermal damage. other potential complications are cardiac tamponade, arrhythmias or atrioventricular block, embolic events, valvular complications, or peripheral vascular damage. the bileaflet mitral valve is located between the left atrium and left ventricle, embedded into the saddle-shaped mitral valve annulus. it serves as a unidirectional valve directing blood from the atrium toward the ventricle by passively opening during diastole and closing in systole, as determined by the pressure gradient between the chambers. the mitral valve apparatus consists of the left atrial wall, the annulus, the anterior leaflet attached to the aortic root, the posterior leaflet attached to the left atrial myocardium, the chordae tendineae attached to the ventricular side of the mitral valve leaflets, and the anteromedial and posterolateral papillary muscles. the anterior leaflet of the mitral valve is wide. it occupies about one-third of the annular circumference, and forms the anterior portion of the left ventricular outflow tract. in contrast with the posterior leaflet, its free edge has no indentations. the posterior (mural) leaflet is narrower, it occupies about two-thirds of the annular circumference, and it is further divided into 3 scallops by 2 clefts. because the posterior aspect of the mitral valve annulus contains little fibrous tissue, the posterior leaflet, especially the middle scallop, is more prone to prolapse than the anterior. the circumflex artery courses near the left half of the posterior leaflet, whereas the right half is in close proximity to the coronary sinus and the arterial branch supplying the av node. the mitral valve leaflets are supported by a dense collagen annulus. the most vulnerable portion of the mitral valve annulus is its aspect continuous with the right fibrous trigone, due to the proximity of the atrioventricular node and the penetrating bundle of his. the muscular components of the mitral valve apparatus are the anteromedial and posterolateral papillary muscles. the anteromedial papillary muscle is more prominent and is supplied by the left coronary system. the posteromedial papillary muscle is smaller, and is supplied by the right coronary artery. the mitral valve closes with the contraction of the papillary muscles, and open when they relax. papillary rupture, as a result of ischemia, will result in acute mitral regurgitation. because the anterolateral papillary muscle is supplied by both the left anterior descending (lad) artery and the left circumflex artery, its ischemic dysfunction is relatively uncommon. in about 60-65% of individuals, the posteromedial papillary muscle was found to be perfused by a single vessel, most commonly by the right coronary artery. the left ventricle (lv) is the largest and thickest chamber of the heart. it receives oxygenated blood from the left atrium during diastole, to transfer it to the body during across the aortic valve during systole. relative to the right ventricle, it is located laterally and posteriorly. its superior, widest aspect is termed the base, leaning upward and toward the right shoulder, opposing its apex, pointing down and to the left. by consensus, its 4 walls are termed septal, and the free anterior, inferior, and lateral walls. for purposes of functional quantification, these are further divided into basal, mid, and apical segments. morphologically, the left ventricle also has an inlet, a fine trabecular and an outlet component. the inlet portion extends from the mitral valve annulus to the origins of the papillary muscles, and contains the mitral valve apparatus. in contrast to the coarsely trabeculated right ventricle, the left ventricular free wall is finely trabeculated. the trabeculation extends to the apex, where, due to the progressively decreasing number of myocardial fibers, the wall thickness is thin. its outflow tract differs from that of the right ventricle in that it is anatomically and functionally contiguous with the inlet via the aortic-mitral continuity. the left ventricular outflow tract supports the aortic valve, and its septal portion, in contrast with the primarily muscular right ventricular infundibulum, includes the membranous interventricular septum. the left bundle branch of the conduction system courses posteriorly to the membranous septum between the right and noncoronary cusps of the aortic valve. the interventricular septum is formed by subendocardial myocardial fibers of both the left and the right ventricle, intermingled with circumferentially arranged fibers derived from the left ventricle. for this reason, in a normally func-tioning heart, the septum thickens toward the left ventricle during systole. during contraction, the high left ventricular pressure along with the septum provides a "splint" against which the right ventricle is able to shorten, hereby contributing to the emptying of the right ventricle (systolic ventricular interdependence). under pathologic conditions, for example, during acute right ventricular volume or pressure overload, or due to conduction abnormalities or pacemaker activation, the septum may appear flat, or move paradoxically toward the right ventricle during systole. blood flow to the anterior 2/3 of the septum is supplied by the septal perforators of the left anterior descending artery. the posterior 1/3 is supplied by the distal branches of the right coronary artery. the 2 papillary muscles of the left ventricle arise from the anterolateral and posteromedial portions of the left ventricular free wall. their arrangement ensures parallel alignment of the chordae tendineae with the lv axis during systole, allowing for complete leaflet coaptation and closure of the mitral valve in the normally functioning heart. all 3 coronary branches contribute to the blood supply of the left ventricle, acute ischemic events therefore predictably manifest as regional wall motion abnormalities based on the coronary distribution. the left anterior descending artery and its branches, the septal perforators and the diagonals perfuse the anterior wall, the anterior two-thirds of the septum, and the apex. the obtuse marginal branches of the left circumflex artery supply the lateral wall. the right coronary artery perfuses the medial portions of the inferior wall, as well as the posterior one-third of the interventricular septum. anastomotic connections between the left and the right coronary system allows for preserved myocardial perfusion distal to a significant obstruction or total coronary occlusion. the aortic valve is part of the aortic root consisting of the annulus, the aortic valve cusps, the sinuses of valsalva, the junction between the sinuses and the proximal ascending aorta (the sinotubular junction), and the proximal ascending aorta. it is located at the left ventricular outlet, separating the aorta and the ventricle. owing to its central location, it is the only valve in the heart that is related to all 4 chambers and each of the valves. as a trileaflet semilunar valve, it consists of 3 cusps: the left and right coronary cusps giving rise to their respective coronary arteries, as well as the non-coronary cusp, which faces the interatrial septum. the right coronary cusp is the most anterior and is adjacent to the right ventricular outflow tract. the left coronary cusp is located posteriorly relative to the other 2. histologically, its cusps are composed of dense collagenous plates, which are in continuity with the fibrous skeleton of the heart, covered by endocardium. each cusp has its attachment to the aorta and the left ventricle. immediately behind the cusps, the wall of the proximal ascending aorta bulges outward to form the aortic sinuses of valsalva. the function of these sinuses is to prevent coronary ostial occlusion by creating eddy currents of blood flow during rapid ejection. as with the pulmonic valve, each cusp has a fibrous nodule at the midpoint of their free edges, forming the nodule of arantius. the leaflets coapt via their crescentshaped surfaces. incomplete coaptation of the valve leaflets during diastole results in aortic regurgitation, whereas limited systolic leaflet excursion due to excessive leaflet thickening resulting from degenerative or rheumatic disease process suggests the presence of aortic stenosis. the severity of aortic stenosis can be assessed by various methods. a peak transaortic velocity of greater than 4 m/second, a peak transaortic gradient of at least 70 mm hg and a mean gradient of at least 40-50 mm hg, as well as an aortic valve area of less than 1 cm 2 is consistent with severe aortic stenosis. the first pair of arteries branching off the proximal ascending aorta are the left and right coronaries, originating from their respective sinus of valsalva in the aortic root. while there is some variability in their point of origin and their distribution, the right coronary artery (rca) almost always supplies the right ventricle (rv), whereas the left coronary artery (lca) supplies the anterior and lateral wall of the left ventricle (lv), as well as the anterior portion of the interventricular septum. blood supply to the remainder of the left ventricle is determined by the coronary dominance. this refers to the artery that supplies the posterior descending artery (pda) and the posterior lateral branch (plb). right dominance (when the right coronary artery gives off the pda and the plb) occurs in 80-90% of normal individuals. left dominance (the pda and the plb originating from the left circumflex artery) is slightly more common in men than in women. if the pda is supplied by the rca and the plb branches off the left circumflex artery, the system is codominant. the left coronary artery arises from the left coronary sinus as the left main stem, which then bifurcates into 2 segments: the left anterior descending (lad) and the circumflex (lcx) artery. the relatively short left main stem courses between the left atrial appendage and the pulmonary trunk before giving off the lad and the lcx, and, in 15-30% of cases the variant coronary ramus intermedius, the branch supplying the anterior aspect of the heart. the left anterior descending artery traverses the anterior interventricular groove and perfuses the anterior wall via the diagonal branches, as well as the anterior two-third of the interventricular septum via its septal perforators coursing along the anterolateral wall, and a small portion of the anterior wall of the right ventricle, adjacent to the interventricular groove, via the right ventricular branches. the circumflex artery courses in the left atrioventricular groove. its primary branches are the obtuse marginals, supplying the lateral aspect of the left ventricle, including the posteromedial papillary muscle. in 10% to 15% of individuals, it gives off the posterior descending artery (left dominance). further branches of the left circumflex artery supply the left atrium, and, in 45% of the cases, the sinus node. a left dominant coronary system also supplies the atrioventricular node. the right coronary artery emerges from the right sinus of valsalva and is carried by the right atrioventricular groove. in about half of the cases it gives off the conus branch that supplies the right ventricular outflow tract. the rca perfuses the anterior free wall of the right ventricle via its acute marginal branches, the sinus node via the nodal artery in 55% of individuals, and the atrioventricular node in patients with right-dominant coronary circulation. it bifurcates into the posterior descending artery and the posterolateral branch at the posterior interventricular groove. the posterior descending artery gives off the posterior septal perforators that supply the posterior one-third of the interventricular septum. the posterolateral branch supplies the inferoposterior wall of the left ventricle along with the acute marginal branches of the rca and/or the circumflex artery. the kugel's artery is an anastomotic branch between the rca and the lcx that may give off a branch that supplies collateral circulation to the atrioventricular node. of the cardiac veins, the great cardiac vein courses in the anterior interventricular groove upward from the apex, alongside the lad. it drains into the coronary sinus. the middle cardiac vein courses in the inferior interventricular groove, upward from the apex, accompanying the pda. the coronary sinus courses in the posterior atrioventricular groove, alongside the left circumflex artery. it receives the great cardiac vein proximally and the middle cardiac vein distally, and drains into the right atrium. its orifice is guarded by the thebesian valve. the anterior right ventricular veins course on the anterior surface of the right ventricle, where they may drain directly into the right atrium, or coalesce to form the small cardiac vein. the small coronary vein runs posteriorly through the right av groove. the thebesian veins are small veins draining directly into the cardiac chambers, primarily into the right atrium and the right ventricle. patients in the perioperative period often receive agents that affect hemodynamic variables such as heart rhythm and rate, blood pressure, or cardiac output. the effect of these agents is governed predominantly by transmembrane ion fluxes. drugs used for rhythm and rate control act by modulating na + , k + , and ca 2+ currents. intracellular calcium is a key mediator in coupling electrical excitation to mechanical contraction, and is an important determinant of the contractile state of the myocardium. the vascular tone of resistance vessels is regulated by ion fluxes via different types of k + , ca 2+ and cl − channels. much of human physiology (for example, responses to stimuli by hormones, neurotransmitters, ions, or photons) is regulated by gtp binding (g) protein-linked signal transduction. g-proteins serve as points of communication between the extracellular and intracellular environment. adrenergic receptors are membrane-spanning molecules coupled to adenylate cyclase via a g-protein located on the inner membrane of the cell. their activation ultimately leads to the modulation of downstream effectors. g-proteins consist of 3 (α, β and γ) subunits. the binding of an agonist to the adrenergic receptor replaces guanosine diphosphate (gdp) by guanosine triphosphate (gtp), and causes the α-subunit of the g-protein to break free from the β-γ complex, and act as a primary messenger: in beta receptors, it stimulates adenylate cyclase and triggers cyclic adenosine monophosphate (camp) production, which, as a second messenger in the process of signal transduction, activates its target kinases that phosphorylate regulator proteins and ultimately increases intracellular calcium levels. pure alpha-adrenergic agonists also increase intracellular calcium levels by stimulating phospholipase c, an enzyme that catalyzes hydrolysis of phosphatidyl inositol to diacyl glycerol and inositol triphosphate. inositol triphosphate stimulates the release of calcium from the sarcoplasmic reticulum, and both molecules act as myofilamental calcium sensitizers. under physiologic conditions, electrical impulse generation, conduction, and propagation occurs in specialized excitatory and conductive tissues within the myocardium, and is driven by a sequence of ion fluxes through sarcolemmal ion channels. the pacemaker of the normal cardiac muscle is the sinoatrial (sa) node, located in the right atrium, below and lateral to the ostium of superior vena cava, supplied by the nodal branch of the right coronary artery (rca). its intrinsic rate (60-100 beats per minute) is determined by the resting transmembrane potential, the threshold potential, and the rate of phase 4 spontaneous diastolic depolarization. the sa node has the steepest slope of phase 4 depolarization. for this reason, its intrinsic rate is the highest, therefore this is the dominant, primary pacemaker of the heart. rhythmical, automatic impulses generated by the sinus node spread directly to the right atrium via its own noncontractile sinus fibers that fuse with excitable and contractile atrial fibers, as well as anteriorly to the left atrium via the bachmann's bundle. electrical impulse from the right atrium propagates via 3 other internodal tracts, including the posterior (thorel's) and median (wenkebach's) pathways to the atrioventricular (av) node. the av node, supplied by the av nodal branch of the distal rca and the septal perforators of the left anterior descending (lad) coronary artery, and located in the right atrial side of the interatrial septum behind the tricuspid valve and superior to the coronary sinus, delays impulses from the atria by approximately 70-100 ms before allowing them to pass to the bundle of his. these slow conduction velocities allow the atria to empty before ventricular contraction begins. the av node is the primary regulator of ventricular rate in atrial fibrillation and flutter. its intrinsic rate is 40-60 beats per minute, and it is considered a latent intrinsic pacemaker. the bundle of his is located just above the interventricular septum (ivs) and is supplied by the septal perforators of the lad, as well as the posterior descending artery. as a latent pacemaker, its intrinsic rate is 20-40 beats per minute. from the bundle of his, impulses rapidly travel down the bundle branches on either side of the membranous portion of the interventricular septum, to depolarize the left (left bundle branch, lbb, which also depolarizes the ivs) and the right (right bundle branch, rbb) ventricles. the left bundle branch further divides into the anterior and posterior fascicles. both bundles terminate in the purkinje fibers that penetrate the ventricular myocardium, initiating its contraction from the endocardium toward the epicardium. an organized sequence of impulse generation and conduction is required for the cardiomyocytes to synchronously contract, and, by coordinated chamber contraction, to maintain cardiac output. understanding normal cardiac electrophysiology is the foundation for understanding the basic principles of disease mechanisms and antiarrhythmic pharmacotherapy. the following is a brief review. in the normal resting myocardium, transmembrane potential is determined primarily by potassium conductance, and is maintained by the na + /k + atpase. the resting transmembrane potential is stable around −90 mv (the intracellular compartment being more negative relative to the outside of the cell), approaching the potassium equilibrium potential. an action potential is triggered by either the cardiac pacemaker cells, or by the surrounding cardiomyocytes. this results in a transient increase in na + conductance, which in turn initiates an increase in na + -influx through fast sodium channels. when the threshold potential is reached at around −70 mv, a large enough number of sodium channels have been opened to generate a self-sustaining sodium influx. above −40 mv, long-opening (l-type) ca 2+ -channels open with resultant ca 2+ -influx down its concentration gradient. at around 0 mv, fast na + -channels start to close. this is phase 0 (early rapid depolarization), peaking at +20 mv (na + -equilibrium potential). the increase in na + -conductance is inactivated. this, with the outward movement of k + via transient k + -channels results in a brief period of initial repolarization. the transmembrane potential returns from slightly positive to approximately 0 mv. this is phase 1. following the initial repolarization, a constant ca 2+influx via slow (l-type, "long-opening") ca 2+ -channels, and an increase in k + conductance via delayed rectifiers main-tains the membrane potential just below 0 mv. the electrical countercurrents are balanced. this is phase 2, the plateau. the phase between the initiation of the upstroke and the end of the plateau is the absolute refractory period, during which the cell is unable to be depolarized regardless of the strength of the stimulus, and corresponds with phase between the beginning of the qrs complex and the beginning of the t wave on the electrocardiogram (ecg). the next phase of the cardiac action potential is the rapid repolarization. ca 2+ -conductance via the l-channels is inactivated, but the k + -channels remain open. potassium is rapidly shifted out of the cell, and the transmembrane potential rapidly approaches the k + -equilibrium. this is phase 3. the phase between the initiation of the upstroke and the transition between phase 2 and 3 is the relative refractory period, during which the cell may be able to be depolarized to allow for a non-propagated stimulus. this corresponds with the early upstroke of the t wave. during phase 3, the cell is able to be depolarized by supranormal stimuli (relative refractory period), resulting in an action potential. this corresponds with the dome of the t wave on the ecg. membrane hyperpolarization at the end of this phase results in a hyperexcitable period, during which even weak stimuli can trigger an action potential. this brief phase corresponds with the downward slope of the t wave. during phase 4, na + and ca 2+ channels are closed. there is a constant outflow of potassium ions through inward rectifier channels. this is the resting phase in the contractile cells, or spontaneous diastolic depolarization in the cardiac pacemaker cells. pacemaker cells are found primarily in the dominant and subsidiary rhythm generators (sa node, av node, his-purkinje system) of the heart. however, impulses may originate from other sites; for example, cells at the coronary sinus, atrioventricular valves, or cells at the crista terminalis. abnormally, impulses may originate from around the pulmonary veins. these cells are different from contractile cardiomyocytes in that they share the characteristic to generate spontaneous cardiac action potentials. they exhibit automaticity, have an unstable membrane potential, and have no rapid depolarization phase. automaticity means that pacemaker cells are capable of initiating their own action potential without external stimulus. these cells undergo a spontaneous diastolic depolarization and action potential is triggered when threshold potential is reached. pacemaker cells undergo spontaneous diastolic depolarization during phase 4 (as opposed to the resting phase during phase 4 in contractile cells), and have no rapid depolarization phase. these cells have fewer inward k rectifier channels than contractile myocytes do, and their transmembrane potential is never lower than −60 mv. therefore, the fast na + -channels that need −90 mv to get activated are permanently inactivated in these cells. multiple ion currents are responsible for the generation of spontaneous action potentials. the synergy of 3 different currents-(1) the decay of the k + -efflux; (2) an inward depolarizing "funny" mixed na + -k + inward current, activated by membrane hyperpolarization, and playing a major role in the spontaneous depolarization of the sinoatrial node; and (3) an inward ca 2+ -current in the late phase of the spontaneous diastolic depolarization-determines the rate and shape of action potentials in cardiac pacemaker cells. physiologically, the dominant pacemaker is the sinoatrial node. when its rate drops below the intrinsic rate of a latent, non-dominant pacemaker-for example, as a result of parasympathetic stimulation or sa nodal disease-the removal of the sinus overdrive leads to "escape-activation" of these non-dominant centers. junctional rhythm may also occur when the av junctional pacemaker rate exceeds and suppresses the sinoatrial rate. cardiac arrhythmias are commonly defined as abnormalities of the normal sequence of impulse generation and propagation within the myocardium. while benign arrhythmiasfor example, premature atrial contractions, isolated premature ventricular contractions, or atrial fibrillation in the absence of structural heart disease-are very common, malignant ventricular arrhythmias (monomorphic or polymorphic ventricular tachycardia [vt], ventricular fibrillation) accounted for an estimated 180,000-250,000 sudden cardiac deaths in the united states. underlying provoking factors are, for example, arterial hypoxemia, acidosis or alkalosis, electrolyte abnormalities, ischemia, increased sympathetic activity, atrial or ventricular dilation, or proarrhythmogenic drugs. in some cases, correction of these factors is sufficient to suppress cardiac ectopies. however, when this alone does not control cardiac arrhythmias, and/or hemodynamic compromise ensues, or the presence of a disturbance increases the risk of a life-threatening arrhythmia, administration of antiarrhythmic agents may be warranted. the main mechanisms of cardiac dysrhythmias have been identified as: focal activity due to abnormal impulse generation this results from: 5 enhancement or reduction of normal automaticity (such as in sinus tachycardia and sinus bradycardia, accelerated junctional rhythm). 5 enhanced abnormal automaticity of the purkinje-fibers, atrial or ventricular myocytes; ie, cells that do not normally display automaticity (for example, atrial tachycardia or accelerated idioventricular rhythm. purkinje fibers are catecholamine-sensitive, and their activation can be augmented by increased sympathetic tone, for example during myocardial ischemia). 5 triggered activity. this occurs when early afterdepolarizations and delayed afterdepolarizations initiate spontaneous multiple depolarizations, such as in torsades de pointes, or ventricular arrhythmias in the setting of digitalis toxicity. abnormal conduction delayed conduction (such as in atrioventricular blocks), or re-entrant mechanisms (for example, sa nodal re-entry, atrial flutter, av nodal re-entry tachycardia, or ventricular tachycardia). re-entrant tachycardias can be generated by 3 different mechanisms: reflection, circus movement re-entry, and phase 2 re-entry. circus movement re-entry due to an anatomical or functional block is a major mechanism and the most commonly described. for a re-entrant circuit to occur, 2 roughly parallel conductive pathways must be connected with conductive tissue, capable of forming an electrical circuit. one of these pathways must have a refractory period substantially longer than that of the other pathway (pathway a). finally, the pathway with the shorter refractory period (alternate pathway, pathway b) must conduct impulses more slowly than pathway a. once an extra impulse (for example, premature contraction) encounters these 2 separate pathways of conduction when pathway a is unable to be depolarized due to being in the refractory period but pathway b is capable of depolarization, the impulse will be conducted via pathway b. the signal then travels to the distal end of pathway a to reconnect with it if it is no longer refractory, then it is conducted retrograde to the site where it reconnects with pathway b. pathway b has a shorter refractory period and therefore it recovers faster: the impulse will travel into pathway b where it will reenter that portion of the circuit, completing the loop. micro-re-entry occurs with ventricular tachycardia from conduction around scar tissue such as in myocardial infarction (mi). macro-reentry occurs via conduction through concealed accessory pathways, such as in wolff-parkinson-white syndrome. pharmacological management of arrhythmias is warranted when treatment of the underlying causes does not break the arrhythmia, the arrhythmia results in hemodynamic compromise, or it increases the risk of development of a lifethreatening arrhythmia. the most widely used classification system of antiarrhythmic drugs was proposed by vaughan williams. this system classifies the antiarrhythmic agents based on their ability of abolishing an arrhythmia by blocking specific ion currents during the action potential. ion-specific channels exist in 3 different stages. during the upstroke phase (phase 0) of the action potential the channels are in the activated state. during the plateau phase of repolarization (phase 2), the inactivated state occurs: during the effective refractory period channels are unresponsive to new or continued stimulus. ion channels are closed during the resting phase (phase 4). the effects on the action potential and the effective refractory period of the cardiac action potential determine the clinical effect of antiarrhythmic drugs. drugs blocking inward sodium ion flow will slow conduction and result in suppression of the maximum upstroke velocity of the cardiac action potential. potassium channel blockers prolong repolarization by prolonging the duration qtc prolongation. l and t type calcium channels are present in the myocardium, and are targets of calcium channel blockers. class i class i antiarrhythmic drugs are fast na-channel inhibitors. fast sodium channels are blocked during phase 0 (upstroke) and phase 4 depolarization of the action potential with resultant decreases in the amplitude and rate of the action potential and conduction velocity. class ia for example, quinidine, procainamide, disopyramide. these drugs lengthen both the action potential and the effective refractory period reflecting sodium channel inhibition and lengthening of repolarization reflecting potassium channel blockade. these drugs are considered membrane stabilizers in the treatment of atrial, av nodal, and ventricular arrhythmias. they may prolong the qrs and qt intervals. 5 quinidine -quinidine has mild parasympatholytic and alpha-blocking effects and decreases systemic vascular resistance (svr). 5 procainamide -procainamide is used to suppress premature atrial and ventricular contractions and to prevent precipitation of atrial fibrillation or flutter. it increases refractoriness and can prevent accessory pathway re-entry tachycardias. high serum levels may cause direct myocardial depression and bradycardia requiring temporary pacing or administration of beta-agonists. procainamide and its active metabolite n-acetylprocainamide may induce qt-prolongation and torsades de pointes. long-term therapy may cause lupus-like symptoms. 5 disopyramide -disopyramide has a vagolytic effect that is dose-dependent and reversible by pyridostigmine. it depresses myocardial contractility and increases svr, and may thus precipitate or exacerbate congestive heart failure. class ib for example, lidocaine, tocainide, mexiletine. these are less powerful na-channel blockers. they shorten action potential duration and refractory period in normal ventricular myocytes. class ii drugs have minimal effect on inotropy or svr. in the ischemic tissue, lidocaine may block adenosine triphosphate-dependent channels, preventing ischemicmediated shortening of ventricular depolarization. lidocaine depresses the slope of phase 4 depolarization in purkinje fibers and increases the fibrillation threshold in ventricular myocytes. signs of toxicity are frequent with concentrations above 9 mcg/ml. class ic for example, flecainide, propafenone. class ic drugs are potent sodium channel blockers, indicated for the treatment of ventricular arrhythmias. they markedly decrease the rate of phase 0 depolarization and speed of conduction. they have little effect on the duration of the action potential and the effective refractory period in ventricular muscular cells, but do shorten the duration of the action potentials in the purkinje fibers. this inhomogeneity on the rate of cardiac repolarization plus the slowing of cardiac conduction may contribute to the prodysrhythmic effects of these drugs particularly in patients with history of myocardial infarction, left ventricular dysfunction, or previous sustained ventricular tachycardia. class ic drugs, especially flecainide, significantly depress inotropy and prolong the pr and qt intervals. class ii drugs are beta-adrenergic receptor blockers. betaadrenergic receptor antagonists decrease the rate of spontaneous phase 4 depolarization, important in suppression of ventricular arrhythmias during ischemia and reperfusion. they are effective in the treatment of adrenergically mediated disease states, in which increased phase 4 depolarization, enhanced conduction velocity, and a shorter refractory period all contribute to increased automaticity. beta-blockers decrease the rate of v max of the action potential, prolongs its duration as well as the effective refractory period. druginduced slowing of the heart rate with resulting decreases in myocardial oxygen requirements is desirable in patients with coronary artery disease. beta blockers slow the sinus rate, prolong av-nodal conduction and enhance refractoriness. they prolong the pr interval on the ecg. esmolol is used to convert atrial fibrillation with rapid ventricular response to normal sinus rhythm, or to maintain a slow ventricular rate. it also predictably reverses the fibrillation threshold lowering effects of catecholamines. class iii drugs are potassium channel blockers; for example, amiodarone, bretylium, sotalol. these drugs prolong cardiac repolarization, action potential duration, and the effective refractory period possibly by interference with sodium and calcium exchange. these effects are beneficial in preventing cardiac dysrhythmias by decreasing the proportion of the cardiac cycle during which myocardial cells are excitable and susceptible to a triggering event. reentrant tachycardias may be suppressed if the action potential duration becomes longer than the cycle length of the tachycardia circuit. 5 amiodarone -in addition to class iii effects, amiodarone exhibits class i na + -channel blocking, class ii beta blocking, and class iv ca 2+ -channel blocking properties. it prolongs repolarization and cardiac action potential, it produces negative chronotropy in nodal tissues and as a ca 2+ and k + -channel blocker, it slows sa-nodal conduction speed and prolongs refractory period. it is an αand β-receptor antagonist with potent vasodilating and myocardial depressant potential. it may produce sinus bradycardia or heart block, necessitating administration of positive chronotropes or initiation of temporary pacing. long-term side effects include pulmonary fibrosis, corneal microdeposits, liver cirrhosis, hyperthyroidism, or hypothyroidism. 5 sotalol -sotalol is a mixture of isomers that possess similar class iii effects, used for the treatment of atrial fibrillation or atrial flutter, as well as to treat ventricular arrhythmias. the l isomer of sotalol acts as a beta antagonist, whereas the d isomer may increase mortality in patients with ventricular dysfunction and recent myocardial infarction. sotalol lacks intrinsic sympathomimetic activity or membrane-stabilizing properties. the lower incidence of dysrhythmia effects seen with amiodarone and racemic sotalol may be related to the beneficial class ii effects. 5 bretylium is no longer available in the united states for clinical use. calcium channel blockers: these agents act by inhibiting inward slow inward calcium currents that may contribute to the development of ventricular arrhythmias. these drugs are useful in the treatment of idiopathic ventricular tachycardias. calcium channel blockers prolong neuromuscular blockade. prodysrhythmia effects of antiarrhythmic agents describe bradydysrhythmias or tachydysrhythmias that represent new dysrhythmias associated with chronic antidysrhythmic drug treatment. types of dysrhythmias include torsades de pointes, incessant vt, and wide complex ventricular rhythm. torsades de pointes (polymorphic vt) is the most common dysrhythmia and is triggered by early after-depolarizations in a setting of a delayed depolarization and increased duration of refractoriness manifesting as prolonged qt c interval on the ecg. class ia (especially quinidine) and class iii drugs prolong qt c by potassium channel blockade and provide the setting for torsades de pointes. drug-induced torsades is often associated with bradycardia, because the qt c interval is longer at slower heart rates. exacerbating factors such as hypokalemia, hypomagnesemia, poor lv function, and concomitant administration of other qt-prolonging drugs are important predisposing factors in the development of torsades de pointes. incessant ventricular tachycardia may be precipitated by cardiac antidysrhythmic drugs that slow conduction of cardiac impulses (class ia and class ic) sufficiently to create a continuous ventricular reentry circuit. incessant ventricular tachycardia is more likely to occur with high doses of class ic drugs and in patients with prior history of sustained ventricular tachycardia and poor lv function. ventricular tachycardia due to this mechanism is generally slower because of the drug effect, but may be resistant to drugs or electrical therapy. wide complex ventricular rhythm is usually associated with class ic drugs in the setting of structural heart disease with excessive plasma concentrations or abrupt changes in the dose. wide complex rhythm is thought to reflect a reentrant tachycardia and easily degenerates to ventricular fibrillation. under normal physiological conditions oxygen delivery (do 2 , the amount of oxygen delivered to the cells) is adequate to meet cellular oxygen demand (vo 2 , the amount of oxygen extracted from arterial blood) and maintain aerobic metabolism. the ratio of oxygen consumption to the oxygen available to tissues is the oxygen extraction ratio, which varies for different organs. during periods of increased workload and increased cellular demand, and/or decreased supply, aerobic metabolism can still be maintained independently of blood flow by increasing o 2 extraction. critical o 2 delivery is the point at which the extraction ratio is maximized, and any further incongruence between demand and supply will lead to tissue hypoxia and subsequent activation of anaerobic metabolic pathways. when myocardial oxygen consumption exceeds the reserve in coronary blood supply to meet a given o 2 demand, ischemia is precipitated. the heart's high, 60-70% o 2 extraction ratio (compared to 25-30% for the rest of the body) can make it susceptible to even short periods of ischemia. the major determinants of myocardial o 2 supply are coronary blood flow and arterial o 2 content. coronary blood flow is further determined by the patency of the coronaries, coronary perfusion pressure, and coronary vascular resistance. the 3 major determinants of myocardial o 2 demand are heart rate, inotropic state, and wall tension (which is the function of intracavitary pressure, radius, and wall thickness, as well as preload and afterload). antianginal pharmacotherapy is indicated when the supply/demand imbalance results in ischemia. in accordance with the 2014 american college of cardiology/american heart association (acc/aha) guideline for the management of patients with non-st-elevation acute coronary syndromes, standard medical therapy includes the use of beta blockers, nitroglycerin, calcium channel blockers, analgesics, and cholesterol management. this section will focus on role of nitroglycerin, beta-blockers and calcium channel blockers in the management of angina pectoris. nitroglycerin nitroglycerin (ntg) is an endothelium-independent smooth muscle relaxant that acts predominantly on venous capacitance vessels and large epicardial coronary arteries, maximizing blood flow to the subendocardial areas. peripheral venodilatory effects are prominent even at low doses and are not dose-dependent, whereas dilation of peripheral conductance and resistance vessels occur at higher doses, and further increases in dose result in more pronounced vasodilation. nitroglycerin reduces myocardial o 2 demand and increases oxygen supply by reducing preload and decreasing ventricular dimension and wall tension, dilating normal and atherosclerotic coronary arteries, and enhancing collateral circulation. the degree to which coronaries are able to dilate is dependent on their baseline vascular tone. nitroglycerin produces a dose-dependent systemic and pulmonary arterial vasodilatory effect, and predictably decreases cardiac filling pressures. its use is indicated for initial treatment of nearly all types of myocardial ischemia, as well as for managing hypertension and heart failure. elimination half-life of nitroglycerin is approximately 1.5 min. ntg enters the smooth muscle cells and generates nitric oxide through a glutathione-dependent pathway, which stimulates cyclic gmp production and causes peripheral vasodilation via a sequence of protein phosphorylation and dephosphorylation within the smooth muscle. nitric oxide production via guanylate cyclase stimulation and cgmp production is a sulfhydryl (sh)-group dependent process. depletion of sh-groups by prolonged exposure leads to doseand duration-dependent tolerance, which may manifest within the first 24 hours of treatment. restoring sh-supplies with reducing agents (for example, n-acetylcysteine) does not reliably reverse nitrate tolerance, therefore a drug-free interval of 12-24 hours is recommended to maintain drug efficacy. rebound myocardial ischemia may occur during drug-free intervals. nitroglycerin is available in intravenous (iv), sublingual, or topical formulations. a typical iv dose to treat acute ischemia is 50-10 mcg. for a continuous infusion, the dose range is 0.1-7 mcg/kg/min. nitroglycerin at low doses causes predominantly splanchnic venodilation that results in venous pooling, decreased cardiac filling, and decreased biventricular end-diastolic volume and pressure. excessive decreases in diastolic blood pressure may decrease coronary blood flow and trigger reflex tachycardia and increased contractility mediated by the baroreceptor reflex. the combination of decreased coronary blood flow and increased myocardial o 2 demand may provoke angina pectoris. with administration of intravenous phenylephrine, adequate coronary perfusion pressure can be maintained. in the lungs, nitroglycerin inhibits hypoxic pulmonary vasoconstriction and worsens hypoxia and gas exchange. it has been shown to have platelet-inhibitory effects, the clinical significance of which has not been elucidated. although an uncommon complication of nitroglycerin therapy, the nitrite metabolite of ntg is capable of oxidizing the ferrous (fe 2+ ) ion in the hemoglobin into the ferric (fe 3+ ) state, producing methemoglobin. ferric (oxidated) iron is unable to carry o 2 effectively, and in sufficiently high concentrations (> 40%) it can impair tissue oxygenation. treatment of methemoglobinemia is methylene blue, to facilitate the conversion of methemoglobin to hemoglobin. high doses of ntg is more likely to produce methemoglobinemia in patients with hepatic dysfunction. the 2014 acc/aha guidelines recommend that nitrates should not be administered to patients with non-st-elevationacute coronary syndrome (acs) who recently received a phosphodiesterase inhibitor (class iii, level of evidence: b). nitrates should not be administered to patients with signs of hypovolemia or hypotension, and it should be used with caution in patients with right ventricular infarction. as discussed in the previous section, beta-blockers are class ii antiarrhythmics with a multitude of favorable properties utilized in the treatment of cardiac ischemia. their negative inotropic, negative chronotropic, and antihypertensive properties allow for reduction in o 2 consumption and an increase in blood supply during diastole. beta blockers slow spontaneous diastolic depolarization and shorten the duration of cardiac action potentials. ventricular fibrillation threshold is increased. beta blockers reduce myocardial infarct size. in the absence of contraindications (cardiogenic shock, decompensated heart failure, severe sinus bradycardia, second-or thirddegree atrioventricular block, or active bronchospasm), beta blockers should be administered early in the treatment of myocardial ischemia. while early administration has not been shown to improve short-term survival, beta blockers decrease reinfarction and the frequency of ventricular dysrhythmias, and their use has been associated with mortality benefit after myocardial infarction. perioperative beta blockers should be continued in patients already receiving beta blockers, according to class 1 indications for perioperative beta blockade from the 2014 acc/aha recommendations; patients at high perioperative risk for adverse cardiac events should be started on beta blockers preoperatively, and continued up to 30 days postoperatively. in these patients, it is reasonable to titrate beta blockers to heart rate and blood pressure. beta blockers are competitive, reversible inhibitors of beta adrenergic receptors. beta adrenergic receptors are g-protein coupled receptors. stimulation by their agonists activates adenylate cyclase to produce cyclic amp. cyclic amp in turn activates protein kinases pathways with subsequent phosphorylation of l-type calcium channels and troponin c, the net effect being enhanced inotropy, positive chronotropy and dromotropy. these responses are all blunted by receptor occupancy by beta antagonists. 5 propranolol -propranolol is a non-selective β(beta)-1 and β(beta)-2 receptor blocker with no alpha-receptor activity. as the most soluble beta blocker, its use is associated with the highest frequency of central nervous system side effects. it is well absorbed when taken by mouth. for a comparable effect, higher oral than intravenous doses are required due to its very high (90%) hepatic first-pass metabolism. its active metabolite does not add to the primary clinical effect due to its short half-life. propranolol decreases cardiac output by decreasing the heart rate and reducing myocardial contractility, effects that are especially prominent in sympathetically driven disease states. owing to its β(beta)-2 receptor antagonism, propranolol may increase systemic vascular and coronary vascular resistance. increased airway resistance may be evoked by propranolol in asthmatic patients. renal and hepatic blood flow is reduced with the administration of propranolol. 5 metoprolol -metoprolol was the first β(beta)-1 selective receptor antagonist used in clinical practice to prevent increased chronotropy and inotropy in response to sympathetic stimulation. its receptor selectivity is dose related. metoprolol is lipid-soluble, it diffuses more readily into ischemic regions than hydrophilic drugs. fifty percent of the drug administered is metabolized during first-pass hepatic metabolism. at the intravenous dose of 0.2 mg/kg, maximum beta receptor blockade is achieved. 5 esmolol -esmolol is a short-acting cardioselective agent metabolized rapidly by red blood cell esterases. it is primarily a β-1 blocking agent, producing significant decreases in heart rate and contractility. it lacks the ability to block peripheral vascular β-2 receptors, therefore decreases in blood pressure and cardiac index is more pronounced due to unopposed peripheral vasodilation. esmolol has been safely used in patients with reactive airway disease. 5 sotalol -sotalol is a class iii antiarrhythmic agent with both β-receptor and k + -channel antagonistic effects. a relatively recent cochrane database review found significant reductions in incidence of postoperative atrial fibrillation in the cardiac surgical population. despite the conclusions of this review, the use of sotalol in the post-cardiac surgical population remains limited, owing to sotalol's undesired side effects, such as hypotension, bradycardia, qt-prolongation, and inducing torsadetype ventricular arrhythmias. calcium channel blockers (ccbs) comprise a diverse group of agents that selectively inhibit calcium influx into myocardial and vascular smooth muscle cells. dihydropyridines (such as nifedipine, nicardipine, nimodipine, amlodipine, felodipine, isradipine) exert their effect on the peripheral arteriolar beds (nimodipine favors cerebral vessels), and produce marked peripheral vasodilation with little direct effect on heart rate, av conduction, and inotropy. they may elicit reflex sympathetic activation via the baroreceptor reflex. non-dihydropyridines (phenylalkylamines, for example, verapamil; and benzothiazepines, for example, diltiazem), on the other hand, block av nodal calcium channels, and have significant negative inotropic, chronotropic, and dromotropic effects. their main anti-ischemic effects are due to their ability to reduce myocardial o 2 consumption by depressing contractility, decreasing heart rate and systemic afterload, and increasing o 2 supply by coronary and collateral vasodilation. calcium channel blockers are preferred in vasospastic (variant) angina, as beta-blockers may provoke or aggravate ischemia in some patients. all calcium channel blockers are effective in the treatment and prevention of coronary vasospasm. they may reduce reinfarctions and long-term events in hypertensive patients with acute myocardial infarction. calcium channels display selectivity to calcium ions, and are present among others in myocardial, smooth muscle, skeletal muscle, and neural tissues, as well as in membranes of subcellular organelles, such as the mitochondria or the sarcoplasmic reticulum. calcium channel blockers bind to voltage-gated transient (t), long (l) type channels in the myocardium and smooth muscle, or to neural (n) type channels, rendering them inactive. t-type calcium channels are activated at low voltages, play a major role in the phase 0 depolarization, and are not affected by calcium channel blockers. l-type channels are activated at higher voltages, playing a role in the phase 2 plateau of the cardiac action potential. these are the ca 2+ -channels that are able to be blocked by calcium antagonists. dihydropyridines prevent calcium entry into the vascular smooth muscle cell by extracellular modulation of the l-type channels. the primary target of dihydropyridines is the peripheral arteriolar bed except for nimodipine, which favors cerebral vessels. reflex tachycardia may be elicited with their use. examples include nifedipine, nicardipine, nimodipine, amlodipine, and felodipine. the predominant action of these agents is decreasing systemic, coronary, and cerebrovascular vasomotor tone. dihydropyridine calcium channel blockers, with the exception of the primarily antianginal nifedipine, will be discussed later. 5 nifedipine -nifedipine was the first dihydropyridine calcium channel blocker in clinical use for its coronary and peripheral vasodilator properties. it has greater coronary and peripheral vasodilatory properties than verapamil with negligible effects on venous capacitance vessels. it has little or no effect on cardiac impulse generation and on sa and av nodal conduction. peripheral vasodilation and the resultant decrease of systemic blood pressure activate baroreceptors, leading to reflex sympathetic nervous system activity manifesting as tachycardia. in the absence of concomitant beta-receptor blockade, it may increase the risk of myocardial infarct or recurrent angina. excessive peripheral vasodilation can be antagonized with phenylephrine. it may be combined with beta blockers without increasing the risk of av-block. nifedipine is used in angina pectoris due to coronary artery vasospasm. no iv preparation is available due to its extreme instability when exposed to light. its abrupt discontinuation has been associated with coronary artery vasospasm. phenylalkilamines bind to the intracellular portion of the l-type calcium channel when it is in the open state, and occlude the channel. 5 verapamil -verapamil is a synthetic derivative of papaverine. it is supplied as a racemic mixture, in which the d-isomer lacks ca 2+ -channel blocking properties and acts as a fast na + -channel blocker, accounting for local anesthetic effects, and the l-isomer is specific for slow ca 2+ -channels. the predominance of this action accounts for the classification of this drug as a calcium channel blocker. verapamil decreases the heart rate by depressing sinoatrial and av-nodal activity (hence its utility in the treatment of supraventricular arrhythmias), lowers systemic blood pressure due to myocardial depression and peripheral vasodilation, and produces moderate coronary artery dilation (preferred in essential hypertension and vasospastic angina). its negative inotropy is more pronounced in patients who already have a depressed left ventricular function, therefore verapamil should be avoided in symptomatic heart failure, severe bradycardia, sinus node dysfunction, and av nodal block. these effects of verapamil may be enhanced with concomitant β(beta)-blockade. in the presence of drug-induced heart block, isoproterenol may be useful to increase heart rate. verapamil may also precipitate dysrhythmias in patients with wolff-parkinson-white (wpw) syndrome, and has proven effective in the treatment of hypertrophic cardiomyopathy with or without left ventricular outflow tract (lvot) obstruction. verapamil may be useful in the treatment of premature labor, as well as fetal and maternal tachydysrhythmias. it may decrease uterine blood flow, and should be administered with caution to parturients with impaired uteroplacental perfusion. benzothiazepines block l-type channel via a mechanism that is not well understood. diltiazem may act on the na + /k + pump, decreasing the amount of intracellular na + available for exchange with extracellular calcium, and it may inhibit the calcium-calmodulin binding. 5 diltiazem -diltiazem, like verapamil, blocks the calcium channels at the av node. it is considered first-line treatment for supraventricular tachydysrhythmias. it may also be used for the control of chronic essential hypertension. diltiazem has minimal cardiodepressant effects and is unlikely to interact with β-blockers to decrease contractility. alpha and beta adrenergic receptors are g-protein-coupled receptors present on various types of cells: pre-and postsynaptic sympathetic nerve terminals; as well as cardiac, skeletal, or smooth muscle cells; hepatocytes; pancreatic islets (beta cells); adipose tissue; platelets; and the renal juxtaglomerular apparatus. they bind endogenous and synthetic catecholamines. there are 2 discrete types of adrenergic receptors, each with several subtypes: alpha and beta. alpha receptors are predominant on the peripheral and pulmonary vasculature. they have 2 subtypes, α-1 and α-2. the α-1 subtype is g q protein-coupled, expressed by smooth muscle, adipose tissue, the liver, sweat glands, and the kidneys. g q protein-coupled receptors activate the phospholipase c (plc) pathway: plc cleaves phosphatidylinositol 4,5-bisphosphate (pip 2 ) into diacyl-glycerol (dag) and inositol triphosphate (ip 3 ). ip 3 in turn interacts with intracellular calcium stores and increases the intracellular calcium content. dag activates protein kinase c, which further modulates ion channels. stimulation of α-1 receptor results primarily in vascular smooth muscle contraction. other effects include contraction of the pregnant uterus, contraction of urogenital smooth muscle (bladder neck, prostate), bronchoconstriction, mydriasis, glycogenolysis and gluconeogenesis, secretion from sweat glands, and renal sodium reabsorption. alpha-2 receptors are g i -coupled receptors expressed by central presynaptic nerve terminals. g i -proteins inhibit the production of camp from atp, reduce ca 2+ permeability, and increase k + permeability. presynaptic neuronal α 2 agonists cause inhibition of acetylcholine as well as norepinephrine release by negative feedback from norepinephrine present in the synaptic cleft. stimulation of central presynaptic α 2 receptors inhibits sympathetic nervous system output and causes sedation. peripheral α 2 receptor stimulation causes decreased insulin secretion from pancreatic beta cells, inhibition of lipolysis, platelet aggregation, and vascular smooth muscle contraction. beta-1 adrenergic receptors are found on the sinus node, av-node, and the myocardium. these are g s -protein coupled receptors and are expressed predominantly in the cardiac pacemaker cells, myocardium, salivary glands, as well as eccrine and apocrine sweat glands. their stimulation activates adenylate cyclase, increasing intracellular campsynthesis. camp as a second messenger activates intracellular signaling pathways that result in increased ca 2+ levels during systole. calcium binds to troponin c, facilitates actin-myosin cross-bridge formation, and increases sarcomere contraction. this translates into faster heart rate, increased myocardial excitability, increased conductivity, and more forceful contractions. beta-2 receptors are located on the smooth muscle of the bronchial tree, some coronary vessels, skeletal muscle arteries, gastrointestinal tract, and the urinary bladder. stimulation of a β 2 receptor may activate 2 g-proteins that regulate adenylate cyclase differently: the excitatory g s and the inhibitory g i proteins. stimulation of the gi protein results in decreased smooth muscle tone. epinephrine is an endogenous catecholamine produced by the adrenal medulla. it is a potent direct α 1 -, α 2 -, β 1 -, and β 2adrenergic agonist. in all dose ranges it has strong positive inotropic effects. peripheral vascular resistance varies according to the dominant effect of the receptors activated: at low doses (β > α) svr is usually decreased. at moderate doses (β comparable to α), svr may remain unchanged. at high doses (α > β), svr is increased. it increases myocardial oxygen consumption. 1. hemodynamic effects 1. preload: increased due to α 1 -effect on capacitance vessels 2. contractility: augmented due to β 1 -effect on the myocardium; its lusitropic effects account for an enhanced rate of relaxation. 3. lusitropy: as a β 1 -agonist, epinephrine enhances early diastolic filling by accelerating relaxation, augmenting filling, and reducing end-systolic ventricular size. 4. afterload: dose-dependent effect. pulmonary hypertension may occur. 5. heart rate: tachycardia due to β 1 -effect, increased excitability and conduction, increased risk of arrhythmias. 6. cardiac output: augmented. 2. dopamine is an endogenous catecholamine, a precursor to epinephrine and norepinephrine, found in the adrenal medulla and peripheral nerve terminals. it has effects on alpha, beta, and dopaminergic (da) receptors. at the lowest doses (1-3 mcg/kg/min) its dopaminergic effects are dominant, resulting in the dilation of the cerebrovascular, renal, and mesenteric vascular beds. in low to moderate dose ranges its β 1 -adrenergic effects are predominant, resulting in increased heart rate, increased contractility, and increased cardiac output. at high doses (greater than 10-20 mcg/kg/ min) its α 1 -agonist effects predominate: high-dose dopamine increases svr, pvr, and decreases peripheral perfusion. it is an effective mixed inotrope and vasoconstrictor; however, it is not recommended as first-line therapy in the treatment of septic shock due to the side effects associated with its use. dopamine has positive chronotropic effects in all dose ranges, and in higher dose ranges it may induce arrhythmias due to increased excitability and conduction. dobutamine is a synthetic derivative of dopamine that does not affect endogenous norepinephrine release. it is a racemic mixture of enantiomers of (−) dobutamine with vasoconstrictor (α 1 -agonist), and (+) dopamine with vasodilator (α 1antagonist) properties. it is a myocardial β 1 -agonist with a predominant positive inotropic effect. its β 2 and α 1 effects are limited, and it completely lacks α 2 effects. in the heart, it augments myocardial contractility and o 2 consumption, and increases cardiac output by increasing heart rate and stroke volume. increased myocardial work is compensated by increased coronary blood flow. on all systemic vascular beds and pulmonary blood vessels it acts as a vasodilator and may cause hypotension. this is primarily mediated by its mild β 2 effects, which are partially unopposed by dobutamine's α 1 effects. dopexamine is a synthetic dobutamine-analogue with positive inotropy, positive chronotropy and peripheral vasodilator action, as well as minimal alpha-receptor activity. it is a potent agonist of da1-receptors (and thus decreases renovascular resistance). it is indicated for treatment of low cardiac output states. it is widely used in europe, but it is not approved by the food and drug administration (fda) in the united states. ephedrine is an alkaloid-derivative with sympathomimetic effects. it has mild direct agonist on α 1 -, β 1 -, and β 2 -receptors, and leads to indirect norepinephrine-release from the neurons. it is an easily titratable pressor and inotrope with a short duration of action that does not reduce placental blood flow, and is therefore safe to administer in pregnancy. phenylephrine is a synthetic noncathecolamine with nearly selective postsynaptic α 1 and minimal β effects. due to its vasoconstrictor effects on resistance vessels, it is widely used for the treatment of intraoperative drug-induced hypotension. its vasoconstrictor potency is less than that of epinephrine and norepinephrine. it is a direct α-agonist with short duration of action. it raises both systemic and pulmonary vascular resistance. phenylephrine constricts coronary blood vessels and augments coronary blood flow. it constricts cerebral and renal vessels, but does not compromise blood flow to these organs. phenylephrine may reduce renal, skeletal muscle, mesenteric, and skin blood supply. myocardial o 2 requirement is usually unchanged unless hypertension is present. vagally mediated reflex bradycardia commonly occurs as a response to increased vascular resistance and usually responds well to atropine; this response can be blocked by atropine. its effects on cardiac output appears to be determined by preload-dependency. 1. isoproterenol is a pure direct β 1 -and β 2 -receptor agonist. it increases cardiac output by increasing heart rate, augmenting myocardial contractility as well as by afterload reduc vasopressin is a naturally occurring antidiuretic nonapeptide synthetized as a pro-hormone primarily in the supraoptic, and secondarily in the paraventricular nuclei of the posterior hypothalamus. after binding to carrier protein neurophysin, it is transported via the supraoptic hypophyseal tract to the posterior hypophysis, where it is stored and released into the circulation when plasma osmolality is higher than physiologic, or when profound hypovolemia is present. normal plasma concentrations are less than 4 pg/ml. the half-life of endogenous vasopressin is 10-35 min, and it is metabolized via renal and hepatic pathways. due to trypsin activity in the gastrointestinal tract it must be administered parenterally or intranasally. in concentrations higher than required for its antidiuretic effect, it acts as a non-adrenergic vasoconstrictor on peripheral vascular beds. it completely lacks beta-adrenergic effects; therefore, it produces less tachycardia compared to adrenergic agonists. in severe shock states, or in conditions where refractory vasoplegia is present, a deficiency in vasopressin levels may necessitate restoration of its physiological concentration by administration of low-dose exogenous vasopressin. the effects of vasopressin are mediated by a family of g-protein coupled vasopressin receptors. v 1a and v 1b are g q protein-linked, whereas v 2 is g s protein-linked receptors. v 1a is located primarily on the vascular smooth muscle of the systemic, coronary, splanchnic, and renal vessel bed, mediating pressor response by increasing peripheral vascular resistance. intra-arterial infusion of vasopressin constricts all major celiac artery branches except the hepatic artery. a less well appreciated aspect is vasopressin's pulmonary vasodilatory effect by inducing constitutive endothelial nitric oxide synthase and increasing nitric oxide production. vasopressin's vasodilatory effects are mediated by endothelial oxytocin receptors. v 1a is also expressed in diverse tissues such as hepatic, central neuronal, and myometrial tissues, as well as platelets. v 1a receptors are g q -protein linked, activate phospholipase c, and ultimately increase intracellular calcium, leading to vasoconstriction. v 1b receptors activate adrenocorticotropic hormone (acth) release from the anterior pituitary gland. v 2 receptors are located primarily in the distal tubules and collecting ducts of the kidneys, and play a role in the homeostatic regulation of plasma volume and preservation of serum osmolality. these receptors are gs-protein-linked, and activate adenylate cyclase to ultimately increase intracellular camp levels. this in turn leads to mobilization of aquaporin 2 channels and their insertion into the luminal surface of the collection tubules, and the subsequent increase of water reabsorption. v 2 receptors are also expressed in endothelial cells, where they are involved in the release of von willebrand factor (vwf) and factor viii (f viii). vwf protects f viii from breakdown, and plays an important role in binding platelets to the site of bleeding. 1. hemodynamic effects: adrenergic-independent direct peripheral vasoconstriction via v 1a receptors without affecting chronotropy or inotropy. redistributes blood to the coronaries and the cerebral vasculature without β 1 -induced increase in myocardial o 2 consumption. milrinone is a noncatecholamine and nonglycoside inodilator with potent positive inotropic and vasodilator properties. it acts as a competitive inhibitor of phosphodiesterase-3 (pde-3) in the cardiac and vascular smooth muscle cells, independently of β-adrenergic mechanisms. pde-3 is an enzyme that hydrolyses intracellular camp into its inactive metabolite. inhibition of the camp-breakdown allows for the camp levels to remain elevated. this in turn increases the ca 2+ influx into the myocardium, and increases ca 2+ efflux from the vascular smooth muscle, an effect translating into increased inotropy in the heart, and vasodilation of the peripheral arteries and veins. in the myocardium, milrinone augments contractility, facilitates diastolic relaxation, enhances automaticity, and shortens av-nodal conduction time. afterload reduction in the peripheral vessels usually has little effect on chronotropy. overall, milrinone improves myocardial contractility, cardiac output, and ejection fraction. myocardial o 2 consumption is unchanged or slightly increased. 1. levosimendan is a pde-3 inhibitor that acts as an intracellular calcium-sensitizer to troponin c. it improves contractility in a calcium-dependent manner by binding to cardiac troponin c and stabilizing its calcium-induced conforma-tional changes, and has vasodilatory effects by opening atpsensitive potassium (k atp ) channels of the vascular smooth muscle. k atp channels close as intracellular atp concentration increases, their selective blockade results in arteriolar vasoconstriction. activation of k atp channels may also account for its anti-stunning effects without increasing intracellular calcium concentrations or energy consumption in the myocardium. the effects of levosimendan are greatest during systole when intracellular calcium levels are the highest, and diminish along with the decreasing intracellular calcium availability during diastole. levosimendan acts independently of camp and has been used in beta-blocked patients. it does not increase myocardial oxygen consumption. it does not impair the baseline diastolic function and is tolerated without arrhythmogenicity. it is a systemic and coronary vasodilator used for treatment of acute decompensated congestive heart failure. the levosimendan infusion versus dobutamine (lido) study demonstrated a significant survival benefit of levosimendan over dobutamine, and smaller studies described its benefits for pulmonary hypertension complicated by right ventricular failure. it is not fda-approved for clinical use in the united states. papaverine is a nonspecific phosphodiesterase inhibitor, derived from opium; however, as a benzyl-isoquinolon, it is structurally and functionally unrelated to opioid alkaloids. it is used by surgeons to treat and prevent spasm of the internal mammary artery during cardiac surgery. hypertension is a major risk factor for cardiovascular morbidity and mortality; essential hypertension accounting for the vast majority of adult cases. based on recommendations of the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jnc-7), the classification of blood pressure (bp) for adults has been as follows: diuretics, beta-blockers, angiotensin converting enzyme (ace) inhibitors/angiotensin receptor blockers (arbs), calcium channel blockers (ccbs), and aldosterone antagonists are mainstays of pharmacologic management of essential hypertension. specific antihypertensive classes target specific end-organ damage, hence the difference in drug combinations when coexisting risk factors are taken into consideration: 5 heart failure: diuretics, beta-blockers, ace inhibitors/ arbs, aldosterone antagonists 5 post-myocardial infarction: beta-blockers, ace inhibitors, aldosterone antagonists 5 high coronary disease risk: diuretics, beta-blockers, ace inhibitors, ccbs 5 diabetes: diuretics, beta-blockers, ace inhibitors/ arbs, ccbs 5 chronic kidney disease: ace inhibitors/arbs 5 recurrent stroke prevention: diuretics, ace inhibitors this section will focus on drugs used in the management of systemic hypertension, pulmonary hypertension, and heart failure. the primary determinants of blood pressure are cardiac output and peripheral vascular resistance. cardiac output is maintained by heart rate and stroke volume. stroke volume is determined by preload, contractility, and afterload, as well as the left ventricular and vascular compartmental size. modulation of these factors by controlling input from vasoactive hormones, neurotransmitters, and local endotheliumderived factors will bring about a change in blood pressure: peripheral resistance is decreased by peripheral α-1 blockers, central α-2 agonists, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, diuretics, or direct vasodilators. heart rate is modulated by beta-blockers, calcium channel blockers, or the novel agent ivabradine. stroke volume is reduced by preload-reducer diuretics and venodilators, negative inotropy of calcium channel blockers, or afterload-reducing agents. 5 labetalol -labetalol is a nonselective beta-blocker with selective α 1 receptor blocking properties. the potency of β-blockade is approximately 7-fold greater than α 1 blockade. its partial β 2 agonism promotes peripheral vasodilation. labetalol can be considered a peripheral vasodilator not triggering reflex tachycardia. in a dose-related fashion it decreases heart rate and peripheral vascular resistance, leaving the stroke volume and cardiac output unchanged. after oral administration labetalol is completely absorbed. its significant first-pass metabolism produces inactive metabolites. 5 phentolamine -phentolamine is a reversible nonselective competitive antagonist at the adrenergic α 1 and α 2 receptors, as well as at serotonin receptors. its main action is arterial vasodilation with minimal venodilation. svr and pvr is therefore decreased. heart rate and contractility drastically increases due to both the baroreceptor reflex and phentolamine's direct effect on central presynaptic α 2 receptors, allowing for increased norepinephrine release and decreased reuptake. this results in unopposed β effects that may provoke arrhythmias and myocardial ischemia, and respond well to β-blockers. in patients pre-treated with phentolamine, administration of epinephrine may result in hypotension due to unopposed β 2 effects. phentolamine is a gastrointestinal stimulant, may lower blood glucose, and causes histamine release. it is used for the diagnosis of pheochromocytoma (a drop in sbp >35 mm hg and dbp >25 mm hg is diagnostic for pheochromocytoma), management of hypertensive episodes associated with the resection of pheochromocytoma, as well as local treatment of skin necrosis associated with norepinephrine extravasation. 5 phenoxybenzamine -phenoxybenzamine is a nonselective, noncompetitive adrenergic α 1 -and α 2antagonist at postganglionic synapses in smooth muscle and exocrine glands. its main action is arterial vasodilation with minimal venodilation. it decreases svr and pvr. heart rate and contractility are increased due to both the baroreceptor reflex and phentolamine's direct effect on central presynaptic α 2 receptors, allowing for increased norepinephrine release and decreased reuptake. this results in unopposed β effects that may provoke arrhythmias and myocardial ischemia, and respond well to β-blockers. phenoxybenzamine is available in po form only. it is used for treatment of pheochromocytoma; it is started 10-14 days preoperatively to allow for expansion of blood volume. to avoid unopposed alpha-stimulation and life-threatening hypertensive episodes, β-blockers are not introduced until after adequate alpha-blockade. due to its side effects-for example, orthostatic hypotension and tachycardia-especially after administering the initial dose, its dose should be increased slowly. alpha-and beta-blockers should be continued until the morning of surgery. 5 prazosin, doxazosin, terazosin, tamsulosin -prazosin, doxazosin, terazosin and tamsulosin are selective α 1 -antagonists. their main cardiovascular action is decreasing svr and pvr with only minimal increases in heart rate. prazosin acts on arteries and veins. prazosin and doxazosin are indicated for treatment of chronic hypertension. terazosin blocks vascular α 1b , as well as α 1a receptors in the prostate and bladder neck. it is used for treatment of benign prostate hypertrophy (bph) and hypertension. tamsulosin is a selective α 1a receptor blocker used for symptomatic bph and to help with passage of kidney stones. orthostatic hypotension with syncope may occur with the use of peripheral α-blockers, especially after the first dose. 5 tolazoline -tolazoline is a nonselective competitive α-adrenoceptor antagonist, structurally similar to phentolamine. it acts as a sympathomimetic and also stimulates muscarinergic ach-receptors and causes histamine release. it decreases systemic and pulmonary vascular resistance. it markedly increases heart rate and may provoke arrhythmias due to its sympathomimetic effects and the reflex sympathetic activation associated with its use. although it is used to treat persistent pulmonary hypertension in neonates, it is not a selective pulmonary vasodilator, and may worsen the svr/pvr ratio when fixed pulmonary hypertension is present. central α 2 receptor agonists reduce sympathetic outflow by stimulating bulbar α 2 activity and reducing peripheral norepinephrine release without eliciting reflex tachycardia and increased contractility. these agents potentiate the effects of anesthetics and may substantially reduce anesthetic and narcotic requirements. central α 2 receptor agonists may exacerbate depression and are contraindicated for management of hypertension in patients treated with monoamine oxidase inhibitors. 5 clonidine -clonidine is a centrally acting α-agonist with near selectivity on α 2 -receptors (220:1 α 2 to α 1 effect). as a partial agonist-antagonist, it elicits submaximal response on central pre-and post-junctional α 2 -receptors while blocking the effects of other agonists. it is a coronary vasodilator and acts as a sympatholytic-a property utilized to manage withdrawal symptoms in opioid and alcohol addicts. clonidine effectively reduces svr, pvr, and renal vascular resistance. its half-life is 12 hours. 5 clonidine blocks pain signal transmission in the brain. it may potentiate opioid effects on the central nervous system and has local anesthetic properties. when administered epidurally, it produces dose-dependent analgesia not reversed by opioid antagonists-an effect utilized in the treatment of cancer pain. it doubles the duration of intermediate-duration local anesthetics when used as adjunct to peripheral nerve blocks. due to risk of hypotension and bradycardia, epidural clonidine is not recommended in the perioperative, obstetric, and postpartum setting. it causes sedation in a dosedependent manner. due to lack of adequate data, it should not be administered above the c4 dermatome. 5 clonidine should not be discontinued abruptly, as rebound hypertension is frequently triggered. its immediate-release oral formulations should be discontinued within 4 hours before surgery and resumed as soon as possible in the early postoperative period. 5 methyldopa -methyldopa is a centrally acting antihypertensive. its mechanism of action is not fully elucidated. its antihypertensive effects are most likely due to its metabolism to α-methyl-norepinephrine, which stimulates central inhibitory α-receptors and lowers blood pressure by false neurotransmission and possibly by reducing plasma renin activity. it has no direct effect on cardiac and renal function. reversible thrombocytopenia and leukopenia, hemolytic anemia, a positive coombs test, and liver dysfunction including fatal liver necrosis has been reported with the use of methyldopa. despite these adverse effect, methyldopa is still commonly used for the treatment of pregnancy-induced hypertension due to the lack of adverse effects of long-term treatment on the fetus. 5 guanabenz -guanabenz is an oral antihypertensive active on central α 2 -receptors. its action is mediated by bulbar α 2 activation, leading to attenuated sympathetic outflow at the level of the brain stem. it effectively controls blood pressure without significant effects on renal function. it frequently causes sedation; therefore, additive sedative effects should be considered when used with other centrally acting depressants. its abrupt discontinuation may rarely result in an increased production of endogenous catecholamines and subsequent rebound hypertension. in patients with coexisting liver and kidney disease, careful monitoring of blood pressure is recommended. 5 guanfacine -guanfacine is a selective oral central postsynaptic α 2a -receptor agonist with a duration of action longer than that of clonidine. it reduces sympathetic outflow and with resultant decrease in heart rate and vasomotor tone. guanfacine preferentially binds postsynaptic α 2a -receptors in the prefrontal cortex, and modulates behavioral responses. its immediate release form is used for management of hypertension, whereas its extended release form is used for treatment of attention deficit/hyperactivity disorder as monotherapy or as adjunct to central nervous system stimulants. its dose should be reduced with concomitant use with cyp3a4 inhibitors, and slowly increased with the use of cyp3a4 inducers. 5 dexmedetomidine -dexmedetomidine is a selective central α 2a and peripheral α 2b -agonist with sedative and anesthetic properties. centrally, it reduces sympathetic outflow and inhibits norepinephrine release at regular doses. at high doses, it activates peripheral α 2b receptors with resultant vasoconstriction, increased svr, pvr, pulmonary artery occlusion pressure, and central venous pressure. its use is indicated for procedural sedation for awake fiberoptic intubation, as well as sedation of intubated and mechanically ventilated patients in the intensive care unit. its initial loading dose is 1 mcg/kg over 10 min, followed by a maintenance rate of 0.2-0.7 mcg/kg/hour, administered via a controlled infusion device, and titrated to effect. no dose adjustments are required in patients with severe renal impairment. in patients with severe hepatic impairment, dose reduction is recommended. it may cause episodes of hypotension, bradycardia, or sinus arrest. these adverse effects are enhanced in the presence of beta-blockers and antihypertensive agents, as well as in patients with advanced age, diabetes mellitus, pre-existing heart block, bradycardia, hypovolemia, or depressed ventricular function. limited information is available regarding its use in pregnancy. dexmedetomidine is expected to cross the placenta. perioperative continuation of α 2 agonists are not recommended for prevention of major adverse cardiac events. when the circulating blood volume is low and renal perfusion is reduced (the afferent glomerular arteriolar pressure is decreased), the renal juxtaglomerular apparatus converts proenzyme prorenin into renin, which is then released into the circulation. upon entering the circulation, renin cleaves a decapeptide from plasma protein angiotensinogen to produce angiotensin i, precursor to the potent vasoconstrictor angiotensin ii. angiotensin ii (at-ii) is the primary vasoactive hormone of the renin-angiotensin system. it is generated from angiotensin i by the proteolytic action of angiotensin converting enzyme (ace). it also stimulates aldosterone secretion by the adrenal cortex. at-ii is therefore a potent vasoconstrictor of arteries and veins. it is responsible for increased aldosterone secretion and sympathetic nervous system stimulation. angiotensin ii normally binds to the at1 receptor that ultimately leads to the increased release of calcium from sarcoplasmic reticulum to produce vasoconstriction. besides its effects on vasomotor tone, at ii also mediates alveolar permeability and lung injury: excessive ace inhibition is related to worse lung injury, an important consideration in the management of critically ill patients. decreased generation of angiotensin ii (for example, by inhibition of ace) results in decreased vasoconstrictive effects, usually without eliciting reflex tachycardia or an increase in cardiac output. in addition, decreased concentrations of plasma aldosterone results in decreased sodium and water retention. ace inhibitors block the at-i to at-ii conversion, as well as the breakdown of bradykinin, an endogenous vasodilator substance, which contributes to the antihypertensive effects of these drugs. ace inhibitors reduce activation of low density lipoprotein (ldl) receptors and decrease the concentrations of ldl cholesterol. if the concentration of ldl is already sufficiently low, ace inhibitors may no longer be effective in reducing the rate of cardiovascular events. ace inhhibitors can be classified according to the structural element that interacts with the zinc ion of the enzyme, as well as the form in which the drug is administered (prodrug or active). administration of ace inhibitors (for example, benazepril, fosinopril, ramipril, or quinapril) as prodrugs increases the bioavailability prior to their hepatic metabolism to the active drug. enalapril is the prodrug of the active ace inhibitor enalaprilat, and its conversion may be altered in patients with hepatic dysfunction. captopril, enalaprilat, and lisinopril are not prodrugs. the major difference among the clinically used ace inhibitors is in duration of action. in the absence of contraindications, ace inhibitors and angiotensin receptor blockers are recommended in patients with systolic heart failure to reduce morbidity and mortality. they may be preferred in hypertensive patients with chronic kidney disease and/or diabetes mellitus. unless contraindicated, ace inhibitors are prescribed together with a beta blocker. contraindications to the use of ace inhibitors are prior life-threatening reactions (angioedema) and known or planned pregnancy. caution should be used in the presence of low baseline blood pressure, elevated levels of serum potassium, increased serum creatinine, or bilateral renal artery stenosis. common side effects are cough, upper respiratory congestion, rhinorrhea, and allergic-like symptoms. it is speculated that these airway responses reflect potentiation of the effects of kinins due to drug-induced inhibition of peptidyldipeptidase activity and subsequent breakdown of bradykinin. if respiratory depression develops, prompt injection of epinephrine is advised. angioedema is a potentially lifethreatening complication of treatment with ace inhibitors. decreases in glomerular filtration rate may occur. for this reason, ace inhibitors are used with caution in patients with preexisting renal dysfunction and are not recommended for patients with renal artery stenosis. hyperkalemia is possible due to decreased production of aldosterone. the risk of hyperkalemia is greatest in patients with recognized risk factors (congestive heart failure with renal insufficiency). measurement of plasma concentrations of potassium may be indicated in treated patients. ace inhibitors are to be avoided in pregnancy due to their potential to induce oligohydramnios, other fetal developmental abnormalities, or fetal demise. although recent perioperative guidelines have suggested continuing ace inhibitors/arbs before non-cardiac surgery, adverse circulatory effects during anesthesia have been recognized in patients chronically treated with ace inhibitors/arbs, leading to the recommendation that these drugs be discontinued 24 hours before anesthesia and major, low-risk, urgent, or emergent surgery. prolonged hypotension has been observed in patients undergoing general anesthesia for minor surgery in whom ace inhibitor therapy was maintained until the morning of surgery. surgical procedures with major fluid shifts have also been associated with hypotension in patients treated with ace inhibitors. treatment with ace inhibitors does not increase the incidence of hypotension after the induction of anesthesia in patients with infarction-induced myocardial dysfunction. exaggerated hypotension attributed to continued ace inhibitor therapy has been responsive to crystalloid fluid infusion and/or administration of sympathomimetics such as ephedrine or phenylephrine. if hypotension is refractory to treatment, treatment with vasopressin is usually effective. ace inhibitors may increase insulin sensitivity and hypoglycemia, which is a concern when these drugs are administered to patients with diabetes mellitus. 5 captopril -captopril is an oral vasodilator that inhibits the formation of angiotensin ii in the lung. the subsequent decrease in plasma at-ii levels leads to decreased arterial and venous vasomotor tone, generally without eliciting tachyphylaxis or reflex hemodynamic changes. in common with all ace inhibitors, captopril reduces preload and afterload on the heart. it promotes renal excretion of sodium and water, reducing the circulating blood volume. treatment with captopril is associated with survival benefit after myocardial infarction, congestive heart failure, or hypertension. captopril may delay the development of renal disease in patients with diabetes mellitus, and counteracts ventricular remodeling after myocardial infarction. 5 also in common with all ace inhibitors, captopril may reversibly reduce kidney perfusion and decrease kidney function. patients with bilateral renal artery stenosis are at risk for kidney failure. due to suppressed aldosterone activity, hyperkalemia may occur. a common side effect is chronic nonproductive cough, secondary to the inhibition of the breakdown of bradykinin. angioedema, a life-threatening condition potentially causing airway obstruction, is a rare adverse effect. 5 enalapril and enalaprilat -enalapril is an oral ace inhibitor used for the treatment of hypertension and congestive heart failure. the therapeutic and side effects of enalapril are very similar to those of captopril. as an inactive prodrug, it first must undergo hepatic metabolism into its active form, enalaprilat. 5 enalaprilat is an intravenous ace inhibitor, used primarily to treat severe hypertension. its therapeutic and side effects are similar to those of captopril. it has a duration of action longer than nitrates, eliminating the need for continuous infusion. enalaprilat decreases peripheral vascular resistance without eliciting reflex tachycardia, an increase in cardiac output and myocardial oxygen consumption. 5 lisinopril and ramipril -lisinopril and ramipril are oral ace inhibitors-prodrugs used for the treatment of hypertension. they must first undergo hepatic metabolism into their active form. their therapeutic and side effect profile is very similar to that of captopril. in renal insufficiency, dose adjustment is required. 5 fosinopril -fosinopril is an oral ace inhibitor used for the treatment of hypertension. it must be converted into an active metabolite in the liver and the gastrointestinal tract. it is eliminated by biliary excretion, therefore, unlike other ace inhibitors, it does not need dose adjustment in patients with kidney insufficiency. 5 perindopril, trandolapril, moexipril -perindopril, trandolapril, and moexipril are oral ace inhibitors used for management of left ventricular dysfunction after myocardial infarct, as well as treatment of hypertension. these prodrugs must be hydrolyzed in the liver into their active metabolites. their action and side effect profile is similar to that of captopril. angiotensin ii levels may increase and maintain elevated blood pressure despite adequate treatment with ace inhibitors, due to at-ii production via non-ace-dependent pathways. angiotensin ii receptor blockers produce antihypertensive effects by blocking the vasoconstrictive actions of at ii without affecting ace activity. because these drugs do not inhibit ace, they do not cause an increase in bradykinin, which contributes to some of the side effects, for example, dry cough and angioedema, of ace inhibitors. their effects and indications (hypertension, post-myocardial infarction and heart failure, prevention of renal insufficiency in diabetics) and are similar to angiotensin converting enzyme inhibitors. arbs are preload-and afterload-reducers, they downregulate sympathetic activity by blocking at-ii effect on peripheral norepinephrine release and reuptake, they act as diuretics and natriuretics by blocking aldosterone secretion, and counteract cardiac remodeling associated with hypertension, heart failure and myocardial infarction. arbs may be preferred to ace inhibitors in hypertensive patients with heart failure, ischemic heart disease, and/or post-myocardial infarction. angiotensin receptor blockers are generally well tolerated with a low incidence of side effects and drug interactions. in common with ace inhibitors, arbs are contraindicated in pregnancy. in the presence of bilateral renal artery stenosis, at-ii constricts the efferent glomerular arteriole more than the afferent arteriole, allowing for adequate glomerular capillary pressure and filtration. ace inhibitors and arbs eliminate this effect of at-ii. in the presence of at least 1 unaffected kidney, sufficient filtration can still be maintained even after at-1 receptors are blocked; however, when a solitary kidney or bilateral renal artery stenosis is present, kidney function may worsen. 5 losartan -losartan is an oral arb indicated for the treatment of hypertension and lowering fatal and nonfatal cardiac and cerebrovascular events, especially when left ventricular hypertrophy is present. both losartan and its first metabolite are active and effectively antagonize at-ii action on the at-1 receptor. volume and salt deficit should be corrected before treatment with losartan. in the presence of liver and kidney disease, or in patients whose kidney function depends on the activity of the reninangiotensin-aldosterone system, dosage adjustment, withholding, or discontinuation may be required, and periodic monitoring of kidney function and potassium is necessary. it is contraindicated in pregnancy. 5 valsartan -valsartan is an arb used as monotherapy or in combination with amlodipine and/or hydrochlorothiazide for the initial management of hypertension and heart failure with reduced ejection fraction. it conveys morbidity and mortality benefit in clinically stable patients with symptomatic heart failure or left ventricular dysfunction after mi. valsartan may be given as part of the standard post-mi regimen, along with aspirin, beta-blockers, statins, and thrombolytics. it is contraindicated in pregnancy. 5 irbesartan -irbesartan is an oral arb. its indications, therapeutic and side effect profile is similar to that of losartan and valsartan. 5 olmesartan, candesartan, telmisartan, eprosartan -olmesartan and candesartan are taken as prodrugs, and are metabolized into their active form during their absorption from the gastrointestinal tract. candesartan is the only arb depending on its metabolism for clinical effect. dose adjustment is required in the presence of kidney disease. 5 olmesartan, telmisartan, and eprosartan do not require dose adjustment in the presence of mild to moderate kidney disease. beta blockers have been shown to reduce mortality after myocardial infarction, congestive heart failure, hypertension, and chronic angina. the properties of these antiarrhythmic agents have been discussed previously. this section will briefly review the role of beta blockers in the treatment of hypertension and congestive heart failure. beta adrenoceptor antagonists blunt the effect of sympathetic stimulation on the cardiovascular system. the magnitude of this effect depends on the density of receptors at the effector sites, and the availability and relative concentration of both the agonist catecholamines, and the antagonist receptor blockers. beta blockers inhibit myocardial and peripheral vascular β 1 -receptors to reduce heart rate, contractility, and myocardial o 2 consumption. by decreasing heart rate, beta blockers increase diastolic filling time, and improve o 2 and substrate delivery to the left ventricle. by decreasing contractility, beta blockers reduce left ventricular ejection velocity and decrease shear forces on the aorta in the presence of dissection, and reduce dynamic ventricular outflow tract obstruction; for example, in hypertrophic obstructive cardiomyopathy or the tetralogy of fallot. svr may increase due to inhibition of β 2 vasodilation; beta blockers should therefore be used with caution in patients with peripheral vascular disease. beta blockers are generally not preferred as a first-line agent for initial management of hypertension, but may be considered as add-on therapy in patients who do not respond adequately to treatment with other drug classes. they can precipitate congestive heart failure, especially when used with other myocardial depressants; for example, calcium channel blockers. abrupt perioperative discontinuation of beta blocker therapy may produce rebound tachycardia and hypertension. traditionally, beta-receptor antagonists are classified on the basis of their relative selectivity for β 1 -and β 2 -receptors, the presence or absence of intrinsic agonistic (sympathomimetic) activity and their pharmacokinetic features. the first generation of beta blockers (propranolol) nonselectively blocked both β 1 -and β 2 -receptors. second-generation cardioselective beta blockers have greater affinity for β 1 -than for β 2 -adrenoceptors, and are less likely to produce undesired side effects (bronchoconstriction, increased svr). β 1selectivity is dose-dependent; therefore, caution should be exercised when administering a beta-blocker to a patient with reactive airway disease. examples of cardioselective beta blockers: metoprolol, atenolol, bisoprolol, esmolol, betaxolol, and acebutolol. examples of nonselective beta blockers: labetalol, carvedilol, nadolol, timolol, sotalol, and propranolol. timolol eye drops can produce systemic beta blockade. 5 labetalol -labetalol is a long-acting nonselective beta-receptor blocker with selective alpha 1 -receptor blocking properties. its alpha 1 to beta receptor blocking ratio is 1:3 when administered by mouth, and 1:7 when administered intravenously. it produces dose-related vasodilation without eliciting reflex tachycardia. labetalol decreases blood pressure and systemic vascular resistance. heart rate may slightly increase; stroke volume and cardiac output remain unchanged. due to its long duration of action, it is usually not preferred for intraoperative minute-to-minute control of hemodynamic variables. 5 carvedilol -carvedilol is a nonselective beta-adrenergic blocking agent with selective activity on peripheral alpha 1 receptors. it is used for management of hypertension, either as monotherapy or in combination with other agents. carvedilol has demonstrated survival benefit and is now also part of the standard treatment regimen for clinically stable patients who have survived the acute phase of mi and have a left ventricular ejection fraction of less than 40%. in hypertensive patients with left ventricular dysfunction (those who depend on beta-adrenergic stimulation in order to maintain cardiovascular compensation), the usual (lower) heart failure dosage applies (instead of that for hypertension). contraindications to the use of carvedilol include high-degree atrioventricular block, bronchial asthma or other reactive airway disease, cardiogenic shock, decompensated congestive heart failure (chf) requiring inotropes, severe liver dysfunction, and history of severe hypersensitivity reactions. abrupt discontinuation of carvedilol may precipitate cardiac ischemia or malignant ventricular arrhythmias, as well as thyroid storm in patients with thyrotoxicosis. certain beta blockers may act as a competitive partial agonist-antagonist on peripheral β-receptors, and elicit a submaximal response at maximal occupancy. this phenomenon is referred to as intrinsic sympathomimetic activity, or isa. these agents will prevent a beta-agonist from binding to its receptor, and will decrease blood pressure and systemic vascular resistance while resulting in less resting bradycardia and maintaining resting cardiac output than is observed with beta-blockers without isa. long-term treatment with agents with isa result in a decrease of blood pressure due to decreased vascular resistance, rather than decreased cardiac output. these agents may be useful in patients who are unable to tolerate excessive bradycardia resulting from treatment with beta blockers. agents with isa have not been shown to be beneficial after myocardial infarction and are not included in standard post-mi treatment regimens. they are less effective than other beta blockers in the treatment of angina and tachycardia. examples of beta blockers with isa: pindolol and acebutolol. perioperative cardiac complications are not uncommon, with 2% of patients suffering major cardiac complications, and 8% showing evidence of significant myocardial injury. according to the 2014 acc/aha perioperative clinical practice guideline, continuation of long-standing beta blocker therapy is recommended. beta blockers should not be started on the day of surgery in beta-blocker-naïve patients. in patients with intermediate or high perioperative risk, or in patients with at least 3 revised cardiac risk index (rcri) risk factors, it may be reasonable to initiate beta blocker therapy at least 24 h prior to surgery. it may also be reasonable to initiate perioperative beta blocker therapy long enough in advance to assess safety and tolerability; however, it is uncertain whether starting beta blockers benefits those with long-term indications, but no other rcri risk factors. in the perioperative setting, beta blockers have been confirmed to reduce the incidence of postoperative atrial fibrillation when started before or immediately after surgery. initiation of beta blocker therapy prior to surgery is currently reserved for patients with significant coronary artery disease undergoing surgical coronary revascularization. calcium channel blockers have a multifaceted profile of therapeutic effects. calcium channel blockers are used primarily as anti-ischemic agents for treatment and prevention of stable angina pectoris. common to all calcium channel blockers, but to a different extent in each class, they act as peripheral vasodilators without eliciting reflex tachycardia, they induce coronary vasodilation, they are negative inotropes, and have electrophysiologic depressant properties. the non-dihydropyridine diltiazem and verapamil are used for rate control in acute cardiac ischemia, when beta blockers are contraindicated. most importantly, calcium channel blockers are first-line agents as potent coronary vasodilators in the treatment of prinzmetal (variant, vasospastic) angina. as discussed previously, dihydropyridines act on the peripheral arteriolar beds, and produce marked peripheral vasodilation with direct and indirect effect on heart rate, av conduction, and inotropy. examples are the rapid-acting antianginal nifedipine, the long-acting vasodilator nicardipine, the highly lipid-soluble nimodipine (favoring cerebral vessels), amlodipine, felodipine, or isradipine. 5 nifedipine -nifedipine is primarily indicated in the management of prinzmetal angina. only its extendedrelease formulations are recommended for the treatment of hypertension, as well as the management of raynaud's disease. conventional (immediate-release) formulations are contraindicated in the management of acute myocardial infarction due to its negative inotropy and reflex sympathetic activation. 5 nicardipine -nicardipine is a highly potent peripheral vasodilator that inhibits calcium influx into the myocardium and vascular smooth muscle. nicardipine has no effects on the sa node and av node. it produces clinically insignificant negative inotropy, and may be combined with a beta-blocker for the treatment of angina. this drug has the greatest vasodilating effects of all the ccbs, with vasodilation being particularly prominent in the coronary arteries. because of all the antianginal drugs dihydropyridines produce the greatest peripheral vasodilation, either nifedipine or nicardipine may be useful in patients who have residual hypertension despite adequate beta-adrenergic blockade. nicardipine produces dose-related decrease in both systolic and diastolic blood pressure. nicardipine is frequently used as a tocolytic. when administered, it binds to the inside of the myometrial l-channels causing them to remain closed, and inhibiting uterine contractions. pulmonary edema has been reported when nicardipine was used as tocolytic. its use is contraindicated in patients with severe aortic stenosis: a decrease in diastolic pressure may worsen myocardial oxygen balance. 5 nimodipine -nimodipine is a highly lipid-soluble analogue of nifedipine. this high degree of lipid solubility facilitates its penetration into the central nervous system, where it blocks the influx of extracellular calcium necessary for contraction of large cerebral arteries. this is especially valuable during the treatment and prevention of cerebral vasospasm after subarachnoid hemorrhage. nimodipine has minimal negative inotropic effect on the myocardium. 5 amlodipine -amlodipine is a dihydropyridine calcium antagonist only available in oral form, with minimal cardiodepressant effects. its anti-ischemic effects are comparable to beta-blockers in patients with acute coronary syndrome. the combination of amlodipine and a beta blocker is more effective in the treatment of myocardial ischemia than either drug alone. its actions are resembling those of nifedipine. it is used primarily for oral treatment of hypertension. 5 felodipine -felodipine is primarily a peripheral vasodilator with no clinically significant negative inotropy. its actions are resembling those of nifedipine. it is used for oral treatment of hypertension. 5 isradipine -isradipine is a peripheral vasodilator with no clinically significant negative inotropy. its actions are resembling those of nifedipine. it is used for oral treatment of hypertension. direct vasodilators: hydralazine, nitroglycerine, nitroprusside 5 hydralazine -hydralazine is a direct systemic arteriolar vasodilator, with minimal venodilator (preload and postural) effects. its mechanism of action is not fully understood. it appears to interfere with calcium movements within the cell that initiate and maintain the contractile state. hydralazine decreases systemic blood pressure (systolic less than diastolic). hydralazine may produce reflex sympathetic nervous system stimulation: it increases renin and at-ii activity, which leads to aldosterone stimulation and sodium reabsorption; tachycardia and increased myocardial contractility results in an increase in cardiac output, and may provoke angina. patients with coronary artery disease should be monitored for myocardial ischemia. the use of hydralazine to treat pulmonary hypertension is not recommended since the associated systemic vasodilation may result in systemic hypotension. hydralazine has a relatively slow onset of action with peak effect occurring by 20 min. with chronic po use, a lupus-like reaction may occur. 5 nitroglycerine -nitroglycerine is a direct coronary vasodilator with greater effects on the venous than on the arterial system. its mechanism of action and effects are described in a previous section. 5 nitroprusside -sodium nitroprusside (snp) is a direct-acting, nonselective vasodilator. it is indicated for rapid correction of hypertensive emergencies. it produces vasodilation by directly increasing intracellular nitric oxide levels; its effects on arteries and veins are balanced. nitroprusside lacks effect on nonvascular smooth muscle and the myocardium; however, reflex tachycardia and increased inotropy may occur, making it an undesirable drug of choice for the treatment of aortic dissection, where shear forces should be minimized. its immediate onset of action and short duration allows for iv infusion and precise titration of dosage. as nitroprusside interacts with oxyhemoglobin (hbfe 2+ ), it dissociates immediately and forms methemoglobin (hbfe 3+ ) while releasing nitric oxide (no) and the highly toxic free cyanide ions. in contrast to the organic nitrates (for example, nitroglycerin), nitroprusside does not require the presence of sulfhydryl compounds to generate no, instead it spontaneously produces them and therefore acts as a prodrug. nitric oxide is the active mediator in the vasodilating effects of snp: it activates the guanylate cyclase present in vascular smooth muscle and increases cgmp, which in turn decreases ca 2+ entry into the cell and intracellular ca 2+ concentration. nitroprusside's degradation products are rapidly cleared via non-enzymatic pathways. each snp molecule releases 5 cyanide ions. cyanide ions undergo sulfurilation by the hepatic and kidney enzyme rhodanase (also known as thiosulfate sulfurtransferase, indicating that thiosulfate must be available for the trans-sulfuration to take place) to form thiocyanate, and are excreted in the urine. availability of thiosulfate is the rate-limiting step in cyanide detoxification. excess cyanide ions immediately react with methemoglobin to form cyanmethemoglobin. when sulfur donors and methemoglobin are exhausted, unscavenged free cyanide radicals may accumulate and bind to tissue cytochrome oxidase, to inhibit mitochondrial oxidative phosphorylation. this leads to tissue hypoxia despite adequate levels of available oxygen. the most common adverse effect of snp is hypotension and dysrhythmias. tachyphylaxis may occur, requiring adjustments in dosage for the necessary effect. thiocyanate toxicity is infrequent, and presents with nausea, abdominal pain, hyperreflexia, tinnitus, seizures, and changes in mental status. thiocyanate clearance can be facilitated by dialysis. rarely, cyanide toxicity ensues. signs and symptoms of cyanide toxicity are hypertension (secondary to tachyphylaxis), arrhythmias, st segment changes, altered mental status, seizures, coma, elevated mixed venous po2 (due to inhibition of cellular o 2 utilization), increasing base deficit, and metabolic acidosis. no cyanosis is seen; spo 2 remains high. treatment includes immediate discontinuation of nitroprusside, mechanical ventilation with 100% oxygen, and correction of acidosis with bicarbonate. mild toxicity (stable hemodynamics with discontinuation of snp, base deficit less than 10) can be treated with thiosulfate (150 mg/kg iv bolus over 15 min). severe toxicity (worsening hemodynamics even after discontinuation of snp, base deficit greater than 10) is treated with 3% sodium nitrite (4-6 mg/kg over 5 min). sodium nitrite converts oxyhemoglobin to methemoglobin, which competes with cytochrome oxidase for the cyanide ions. hydroxocobalamin is an alternative to thiosulfate or sodium nitrite. it binds cyanide to form cyanocobalamin, which acts as a nontoxic reservoir and is excreted by the kidneys. trimethaphan dilates peripheral arteries by blocking cholinergic transmission on nicotinergic autonomic ganglia by binding to the postsynaptic ach-receptor. it is a short-acting competitive acetylcholine-antagonist with both sympatholytic and parasympatholytic effects. it causes vasodilation and tachycardia. common side effects are mydriasis, urinary retention, and ileus. its use is very limited; it has been used for inducing controlled hypotension during surgery, reduction of blood pressure during hypertensive emergencies (for example, in patients with a dissecting aortic aneurysm), or for the emergency treatment of pulmonary edema. pulmonary hypertension-sustained elevated pressure within the pulmonary artery-is a heterogenous and frequently progressive disorder of the pulmonary vasculature that ultimately leads to increased pulmonary vascular resistance, right heart failure, and death, due to constrained pulmonary blood flow and vascular remodeling of the resistance arteries. endothelin-1 (et-1) is a proinflammatory mediator-a direct vasoconstrictor that stimulates pulmonary vascular smooth muscle cell proliferation and induces fibrosis. its effects are mediated through the et a receptors that mediate vasoconstriction and smooth muscle proliferation, and the et b receptors that induce production of nitric oxide and prostacyclin, and mediate et-1 clearance. clearance of et-1 is diminished in pulmonary hypertension. 5 bosentan -bosentan is an orally active dual et a and et b antagonist. it is approved for treatment of who group 1 pulmonary hypertension to slow clinical deterioration and enhance functional capacity in patients who are not candidates for treatment with calcium channel blockers. 5 the use of bosentan increases the risk of severe hepatic injury, liver cirrhosis, and liver failure. liver function should be monitored monthly while taking bosentan. bosentan may cause serious birth defects. pregnancy should be ruled out prior to initiation of treatment and monthly thereafter. it should be avoided in patients with elevated transaminases and is contraindicated in pregnancy. angioedema, fluid retention, and possible dose-related decreases in hemoglobin and hematocrit has been reported. prostacyclin (prostaglandin i 2 , pgi 2 , pgx) and thromboxane a 2 are the main metabolites of arachinoid acid with opposing effects on the vascular smooth muscle. thromboxane a 2 induces vasoconstriction and promotes platelet activation, whereas pgi 2 is a vasodilator with platelet inhibitor effects. in pulmonary arterial hypertension, their physiologic balance is shifted toward thromboxane a 2 . this promotes smooth muscle cell proliferation, vasoconstriction, and thrombogenesis. the activity of prostacyclin synthase is decreased in pulmonary hypertension, leading to low levels of the vasodilator and antiproliferative pgi 2 . restoring the balance between vasodilating and vasoconstricting factors, including the administration of prostacyclin analogues, is a mainstay of the medical management of pulmonary hypertension. 5 epoprostenol -pgi 2 -analogue epoprostenol is used for the long-term treatment of idiopathic pulmonary hypertension, and pulmonary hypertension associated with scleroderma spectrum diseases. it improves functional class, exercise tolerance, and hemodynamics. its 2 main pharmacological actions are: (1) direct pulmonary and systemic vasodilation, and (2) platelet inhibition. it is an afterload reducer for both the left and the right ventricle. it produces dose-related decreases in pulmonary vascular resistance; it increases stroke volume and cardiac output. epoprostenol is usually administered as a continuous infusion into a central vein. dosing must be individualized; however, optimal dose range for long-term therapy is believed to be 25-40 ng/kg/minute, when used as monotherapy. alternatively, it may be delivered as an inhaled aerosol, for example for the treatment of acute right ventricular failure in the intraoperative or early postoperative setting. common side effects are headache, flushing, nausea, diarrhea, and musculoskeletal pain. contraindications to its chronic use include congestive heart failure due to severe left ventricular systolic dysfunction, pulmonary edema during initial treatment, or known hypersensitivity to epoprostenol or structurally related agents. abrupt withdrawal or sudden dose adjustments should be avoided. its use should be limited to centers experienced with its administration and with systematic follow-up with patients. 5 iloprost -iloprost is a stable prostacyclin-analogue delivered as an inhaled aerosol to patients with idiopathic pulmonary hypertension, pulmonary hypertension associated with scleroderma spectrum diseases or appetite suppressants, or pulmonary hypertension related to chronic thromboembolic disease. in the acute setting, iloprost decreases pulmonary vascular resistance; with long-term use, in addition to its pulmonary vasodilator effects it suppresses pulmonary vascular smooth vessel proliferation. iloprost is delivered as an inhaled aerosol. it is approved for functional class iii and iv to relieve symptoms, enhance exercise tolerance, and diminish disease progression. its adverse effects are similar to those of epoprostenol. its use should be avoided in pre-existing hypotension. it may induce bronchospasm in patients with reactive airway disease. caution should be used with its administration in the presence of increased risk of bleeding, anticoagulation, or other bleeding disorders. abrupt withdrawal or sudden dose adjustments should be avoided. the use of iloprost should be limited to centers experienced with its administration and with systematic follow-up with patients. 5 treprostinil -treprostinil is a stable prostacyclinanalogue that can be taken by mouth, administered into a central vein, or subcutaneously (preferred). its main actions are pulmonary arterial vasodilation and inhibition of platelet aggregation. it is approved to decrease exercise-related symptoms and diminish clinical deterioration. treprostinil may induce symptomatic hypotension. it may increase risk of bleeding. there appears to be an increased risk of gram-negative bloodstream infections, especially in patients receiving intravenous treatment via a chronic indwelling catheter. its side effects are similar to those of epoprostenol. abrupt withdrawal or sudden dose adjustments should be avoided. its use should be limited to centers experienced with its administration and with systematic follow-up with patients. phosphodiesterase-5 (pde-5) is the intracellular enzyme responsible for degrading cyclic nucleotide monophosphates. it limits signal transduction by camp and cgmp-second messengers implicated in pathogenetic pathways leading to pulmonary hypertension. cgmp is involved in mechanisms of pulmonary vasodilation; its rapid hydrolysis by pde-5 significantly limits its effect on vasomotor tone. 5 sildenafil -sildenafil is a pde-5 inhibitor initially indicated for treatment of erectile dysfunction. it enhances the effect of nitric oxide in the corpus cavernosum and the pulmonary arterial smooth muscle. it is approved for treatment of who group 1 pulmonary hypertension to improve functional capacity and delay disease progression; its unlabeled indication is treatment of pulmonary hypertension after recent left ventricular assist device placement. common side effects associated with the use of sildenafil are headache, flushing, dyspepsia, epistaxis, and disturbances of color discrimination. it may potentiate the effects of antihypertensive agents. the use of sildenafil for pulmonary hypertension should be avoided with strong cyp3a4 inhibitors. the use of sildenafil is contraindicated with concurrent use of organic nitrates and guanylate cyclase stimulant riociguat. 5 tadalafil -tadalafil is a longer-acting pde-5 inhibitor approved for treatment of erectile dysfunction, benign prostate hypertrophy, and who group 1 pulmonary hypertension in patients who are not candidates for treatment with calcium channel blockers. its mechanism of action is similar to that of sildenafil. it is taken by mouth as a once-daily dose. it may be considered for combination therapy with a prostacyclin analogue or et-1 receptor antagonist. its side effects are similar to those of sildenafil. it may potentiate the effects of antihypertensive agents. its use is not recommended in patients with recent myocardial infarction or stroke within 6 months; uncontrolled arrhythmias, hypotension, uncontrolled hypertension, heart failure, or unstable angina. it should be avoided when severe aortic stenosis or dynamic left ventricular outflow tract obstruction is present. concomitant use of organic nitrates is contraindicated. nitric oxide (no) is a potent, endothelium-derived, cgmpdependent direct vasodilator generated from the terminal guanidine nitrogen of l-arginine. it is produced by 3 isoforms of nitric oxide synthase (nos) in response to hypoxia, pulsatile flow, and flow-induced sheer stress on the arterial wall. it exists in the gaseous form and is administered as an inhalational agent. once inhaled, it diffuses to target cells and activates guanylate cyclase to increases cgmp production: an increased cgmp concentration subsequently leads to vasorelaxation. no diffuses across the pulmonary capillary endothelium into the circulation. once in the circulation, it avidly binds to the iron of heme-based proteins; it is rapidly inactivated by hemoglobin. in pulmonary arterial hypertension decreased nos3 activity is present, resulting in decreased no-induced pulmonary vasodilation. the use of nitric oxide is favored in the treatment of pulmonary hypertension for its selective pulmonary vasodilator and antiproliferative effects. it vasodilates the well-ventilated areas, and improves v/q matching. it inhibits platelet activation, aggregation, and adhesion. it is synergistic with pgi2, allowing the endothelium to maintain its antithrombotic properties. nitric oxide is used to treat persistent pulmonary hypertension of the newborn. as with inhaled prostaglandin analogues, it does not appear to improve clinical outcomes in acute respiratory distress syndrome (ards). heart failure is a constellation of clinical symptoms (primarily fatigue and dyspnea) secondary to impaired left ventricular systolic (reduced ejection fraction) or diastolic (preserved ejection fraction) function, and/or elevated intracardiac pressures. the 2013 aha/acc guidelines classify the syndrome by its evolution from asymptomatic preclinical stages to progression to advanced structural heart disease and symptomatology at rest, refractory to maximal medical management. these guidelines also complement the widely accepted functional classification of symptomatic heart failure by the new york heart association (nyha i-iv). chronic heart failure is a condition characterized by vasoconstriction, volume overload, and neurohormonal activation. the goal of its pharmacological management is to reduce vascular tone, reduce sympathetic activation, and improve cardiac function. conventional treatment options include angiotensin-converting enzyme inhibitors, angiotensin ii receptor blockers, aldosterone antagonists, beta blockers, and the combination of isosorbide dinitrate and hydralazine and diuretics. novel approaches are now the use of ivabradine and angiotensin receptor-neprilysin inhibitor/ arb combinations. nesiritide is a recombinant human b-type natriuretic peptide with vasodilatory properties. it binds to vascular endothelial and smooth muscle a-and b-type natriuretic peptide receptors. it increases cgmp levels. cgmp serves as a second messenger to produce arterial and venous dilation. much like endogenous natriuretic peptides, it suppresses the sympathetic nervous system, the renin-angiotensin-aldosterone system, and endothelin. based on the results of initial trials and the observed reduction of pulmonary capillary wedge pressure and symptomatic relief, it was first approved for treatment of patients with acute decompensated heart failure; however, the acute study of clinical effectiveness of nesiritide in decompensated heart failure (ascend-hf) demonstrated that nesiritide did not convey advantage over standard treatment of acute decompensated heart failure. ivabradine is a novel sinoatrial modulator used for the treatment of chronic stable angina pectoris and heart failure with an ejection fraction lower than 35% inadequately controlled by beta blockers, in patients with native sinus rhythm. it reduces the heart rate by inhibiting cardiac pacemaker inward "funny" current in the sinoatrial node, which, unlike beta-blockers or calcium channel blockers, produces selective heart rate control without affecting ventricular repolarization or myocardial contractility. it may prove beneficial for the treatment of heart failure with reduced ejection fraction. it is contraindicated in sick sinus syndrome, as well as concomitant use of cyp3a4 inhibitors; for example, azole antifungals, macrolides, and protease inhibitor antiretrovirals. it is contraindicated with the concomitant use of verapamil or diltiazem. arni combinations have been shown to convey mortality benefit in patients with chronic heart failure, a condition in which neurohormonal activation, volume overload, and vasoconstriction play important roles. natriuretic peptides are potent vasodilators with natriuretic properties. they reduce sympathetic tone and inhibit the renin-angiotensinaldosterone (raas) axis. neprilysin is an endopeptidase that degrades vasoactive and natriuretic peptides. sacubitril is a neprilysin inhibitor, therefore it increases the concentration of natriuretic peptides. its combination with a raasinhibitor angiotensin receptor blocker conveys cardiovascular and renal protection. the paradigm-hf (prospective comparison of arni with acei to determine impact on global mortality and morbidity in heart failure) trial demonstrated significant reduction in cardiovascular and allcause mortality, as well as heart failure-related hospital readmissions in patients with class ii-iv heart failure and reduced ejection fraction when treated with sacubitril/valsartan compared to enalapril alone. it should be avoided in pregnancy and it should not be administered concomitantly with ace inhibitors. the updated 2016 acc/aha/heart failure society of america (hfsa) guidelines for management of heart failure include the addition of ivabradine and sacubitril/valsartan to the list of treatment options for patients with heart failure and reduced ejection fraction. digitalis is a cardiac glycoside that selectively and reversibly inhibits the integral membrane protein na + /k + atpase ion transport system: the na + /k + atpase maintains gradients for na + and k + that determine myocardial excitability and action potential. digoxin is the only commercially available digitalis preparation available in the united states. it binds on the external alpha subunit, increases intracellular na + concentration, decreases ca 2+ efflux, and augments inotropy by releasing calcium from the sarcoplasmic reticulum into the cytoplasm, making more calcium available to generate contraction. digoxin makes resting potential less negative with a resultant increase in myocardial excitability. m vo2 is decreased due to its effects on wall tension, preload, and afterload. common clinical uses treatment of supraventricular tachyarrhythmias, ventricular rate control for atrial fibrillation with rapid ventricular response. it has been used for treatment of paroxysmal supraventricular tachycardia due to av-nodal reentry or av-reentry. historically, it is used for the treatment of symptomatic heart failure associated with left ventricular dysfunction. treatment may be initiated in patients who have not yet responded to the conventional treatment regimen with an ace inhibitor or a beta-blocker. digoxin does not provide overall mortality benefit, but alleviates symptoms and decreases heart failure related hospital admissions. digoxin does not appear to have significant effect on disease progression in asymptomatic individuals. administration po or iv. assuming normal renal function, the loading dose for an adult is typically 0.25-0.5 mg increments iv or im for a total of 1-1.25 mg. maintenance dose is 0.125-0.250 mg/day, based on serum levels and clinical effect. adverse effects dizziness, mental disturbances, diarrhea, nausea, vomiting, cardiac dysrhythmias, heart block, and visual disturbances. warning/contraindication administration of digoxin is not recommended for asymptomatic (nyha class i) patients. digoxin is contraindicated when dynamic lvot obstruction is present and in pre-excitation arrhythmias. caution should be used with concomitant use of beta-blockers, calcium channel blockers, or calcium. digoxin has a very low therapeutic index with a nonlinear dose response. dose should be reduced in hypokalemia, hypothyroidism, extensive myocardial or renal damage, and in geriatric patients. due to its very narrow therapeutic range, increased latency of onset and long duration of action, careful dosage determination is essential to avoid digitalis toxicity. in addition to drug pharmacokinetics and pharmacodynamics, patient characteristics-for example, age, cardiac and renal functional status, other medical comorbidities and their pharmacotherapyshould be considered. doses should be individualized. digoxin toxicity has a relatively high mortality rate. therapeutic digoxin levels fall between 0.5-2.5 ng/ml. levels of lower than 0.5 ng/ml are non-toxic. levels higher than 3 ng/ml are definitely toxic. infants and children appear to be more tolerant of higher digoxin levels without manifesting signs and symptoms of toxicity. signs of toxicity include extracardiac (primarily central nervous system and gastrointestinal) and cardiac effects. extracardiac manifestations are similar in both acute and chronic intoxication. in pediatric patients, drowsiness, nausea, and vomiting are common signs of early toxicity, and may present before or after the manifestation of cardiotoxicity. in neonates and infants, sinus bradycardia is a premonitory sign of digitalis cardiotoxicity. in adults, early gastrointestinal effects include nausea, vomiting, and anorexia. these may present before or after the manifestation of cardiotoxicity. central nervous system effects include headache, drowsiness, generalized weakness, visual disturbances (color vision is commonly affected), disorientation, confusion, delusions, hallucinations, delirium, or amnesia. cardiovascular toxicity may develop in the absence or presence of other signs and symptoms of toxicity, and includes new arrhythmias, especially those that exhibit features of increased automaticity and av-block, premature atrial and ventricular beats, and malignant ventricular arrhythmias. acute toxicity is usually associated with hyperkalemia, whereas chronic toxicity is associated with hypokalemia or normokalemia. factors that predispose to toxicity are electrolyte derangements such as hypokalemia, hypomagnesemia, or hypercalcemia, diuretic use, alkalosis or acidosis, impaired kidney function, hyperventilation, hypocapnia, arterial hypoxemia, treatment with quinidine, and decreased muscle mass. hypokalemia increases myocardial binding of glycosides; binding of cardiac glycosides to the na + /k + atpase enzyme complex is inhibited by elevated potassium levels. treatment of digitalis toxicity immediate discontinuation of digoxin, correction of predisposing factors, treatment of cardiac dysrhythmias (phenytoin, amiodarone, beta-blockers), and temporary pacing. supplemental k decreases the binding of digitalis to myocardial tissue. digoxin fab is a special digoxin-binding antidote that prevents and reverses toxic effects and enhances elimination; as there is no comparable alternative treatment, it should be promptly administered to patients with digitalis toxicity. b. decrease afterload to promote forward flow. c. allow the heart rate to increase above 90 beats per minute to raise aortic diastolic pressure. d. minimize iv hydration to avoid volume overload. 8. the preferred agent to treat drug-induced hypotension in the patient from question 7 is a. ephedrine b. phenylephrine c. epinephrine d. norepinephrine 9. in patients with an intact circulation, rapid infusion of intravenous amiodarone leads to a. rapid restoration of native sinus rhythm and hemodynamic stability b. shortened refractory period c. hypotension and bradycardia d. increased speed of conduction in the sinoatrial node 10. which of the following statements about the cardiovascular effects of norepinephrine is correct? a. the primary effect of norepinephrine is direct β(beta) 2 -agonism. b. norepinephrine increases cardiac output to a greater extent than phenylephrine. c. norepinephrine augments renal and mesenteric blood flow. d. the arrhythmogenicity of norepinephrine is significantly greater than that of epinephrine, dobutamine, dopamine or isoproterenol. flattened t-wave, or the osborne-waves, typically seen in hypothermia. treatment is hydration with normal saline to decrease plasma calcium levels by dilution, administration of non-thiazide diuretics, and avoidance of thiazides. non-depolarizer muscle relaxant doses should be decreased in patients with hypercalcemia and muscle weakness. the cardiac effects of hyperkalemia are peaked t-waves at mildly elevated potassium levels (6-7 meq/l), and wide or flat p wave, prolonged pr interval, wide qrs, prolonged qt, ventricular fibrillation, and asystole at higher serum potassium levels (10-12 meq/l). treatment is: (1) temporary membrane stabilization with iv calcium (commonly administered dose: 500 mg cacl 2 or 1 g ca-gluconate), (2) inducing intracellular k + −shift by administering intravenous insulin and glucose (commonly administered dose: 25 to 50 g intravenous dextrose along with 5 to 10 units of insulin), and (3) promoting excretion (diuretics, hemodialysis, resins). slightly prolonged pr interval, shallow or biphasic t-waves, prominent u-waves, and st-segment depression reflects hypokalemia; whereas prolonged qt c , third degree av-block, torsades de pointes or ventricular tachycardia may reflect hypocalcemia. 3. b. according to the most recent acc/aha guidelines, there is class i recommendation for continuing beta blocker therapy in patients using beta blockers chronically. it may be reasonable to start beta blockers in patients with 3 or more rcri risk factors; however, beta blockers should not be started on the day of surgery. alpha-2 agonists are not recommended for risk reduction of major adverse cardiac events. it is a class i recommendation to wait at least 14 days after percutaneous myocardial revascularization with balloon coronary angioplasty, 30 days after bare metal stent placement, and 365 days after drug eluting stent placement. 4. a. propofol is a gabaergic agent. it causes central nervous system inhibition by decreasing the rate of dissociation of gaba from its receptor, and prolonging the gaba-mediated chloride influx into the cell, causing membrane hyperpolarization. its induction dose for a normovolemic adult patient with normal cardiac function is 2-2.5 mg/kg. propofol decreases mean arterial pressure by producing direct myocardial depression and decreasing svr. it alters the baroreceptor reflex and causes a disproportionally small compensatory increase in heart rate relative to the decrease in blood pressure. it decreases cerebral metabolic rate of oxygen consumption, cerebral blood flow, and icp; however, at high doses, it may significantly decrease cerebral perfusion pressure. propofol may trigger histamine release, and allergic reactions are therefore possible. it is not associated with increased incidence of postoperative nausea and vomiting. a. ketamine is a phencyclidine derivative interacting with numerous receptors: primarily, it acts as a glutamate-antagonist at both nmda and non-nmda receptors; additionally, it is an opioid receptor agonist (opioid-sparing effects) and an m2 muscarinergic acetylcholine receptor antagonist (bronchodilation). it interacts with nicotinergic and monoaminergic receptors, as well as with voltage-dependent na +and l-type ca 2+ channels. its actions on the nmdareceptors account for its psychomimetic, analgesic, and amnestic effects. the incidence of emergence delirium may be reduced by concomitant administration of a benzodiazepine and by minimizing external stimulation during emergence. ketamine produces centrally mediated sympathetic activation: it increases plasma epinephrine levels, heart rate, and mean arterial pressure. it is the only anesthetic that increases peripheral arteriolar tone. in catecholamine-depleted patients, or in those with limited inotropic reserve, its intrinsic cardiodepressant properties predominate, and may cause hypotension. absolute contraindications to the use of ketamine are hypersensitivity to ketamine or chemically related agents, significantly elevated blood pressure, stroke, intracranial hemorrhage, active delirium or psychosis, porphyria, pregnancy, and preeclampsia. 6. c. etomidate is a lipid-soluble nonbarbiturate hypnotic and anesthetic without analgesic effects. it is a carboxylated imidazole, structurally unrelated to other intravenous anesthetics. it is frequently used for rapid intravenous induction of anesthesia at standard doses of 0.2-0.3 mg/kg. its short duration of action is subsequent to its rapid redistribution to peripheral compartments. etomidate causes minimal cardiorespiratory depression even in the presence of cardiac and pulmonary disease. it may increase heart rate and cardiac output by 3% to 4%, and may produce a 10-15% decrease in systemic vascular resistance and mean arterial pressure. it decreases cerebral blood flow, cmro 2 , and icp. even a single induction dose of etomidate suppresses cortisol production by inhibiting the activity of 11-β(beta)-hydroxylase. etomidate causes pain on injection and it is associated with increased incidence of postoperative nausea and vomiting. there are no absolute contraindications to the use of etomidate as an induction agent. 7. a. aortic stenosis, congenital or acquired, is the most common valvular disease in the united states. most common etiologies are senile degeneration and congenital bicuspid aortic valve. asymptomatic patients even with severe aortic stenosis carry a small risk of sudden cardiac death, whereas the occurrence of symptoms are ominous signs of poor outcome. the classic triad of symptomatic aortic stenosis are angina pectoris (life expectancy is about 5 years, unless the aortic valve is replaced), syncope (average life expectancy: 3-4 years), and congestive heart failure (average life expectancy: 1-2 years). treatment is surgical. as the severity of the stenosis progresses, a pressure gradient develops between the left ventricle and the aorta, causing the left ventricular wall tension to increase (fixed increase in lv afterload). this leads to a compensatory increase in wall thickness. according to the law of laplace, the wall tension within a sphere filled to any given pressure depends on the thickness of the sphere: pressure = (2 * wall thickness * wall tension)/radius. consequently, at a constant pressure, wall tension can be decreased by increasing the thickness of the sphere's wall: left ventricular wall tension = (lv pressure * radius)/2* lv wall thickness. wall stress is a major determinant of myocardial o 2 demand. concentric left ventricular hypertrophy with an increasing wall thickness but unchanged chamber size therefore reduces wall stress and o 2 demand. chamber size, contractility, and stroke volume are usually preserved until late in the disease process. the increasing lv wall thickness will eventually lead to impaired left ventricular relaxation and decreased compliance, and eventually a fixed stroke volume. to maintain an adequate stroke volume, preload augmentation is necessary. a thick, noncompliant heart has an increased basal myocardial o2 consumption. in the hypertrophied left ventricle, capillary density does not adapt to the increased wall thickness, and any decrease in coronary perfusion pressure will compromise myocardial o2 supply. maintenance of adequate afterload to ensure adequate systemic diastolic and coronary perfusion pressure is essential in preventing hypotension and the resultant risk of subendocardial ischemia. extremes of heart rate are poorly tolerated in patients with hemodynamically significant aortic stenosis. maintenance of normal rate and sinus rhythm is essential, as a noncompliant left ventricle is unable to augment stroke volume to maintain cardiac output during bradycardic episodes to restore cardiac output, and excessive tachycardia will reduce coronary perfusion during diastole. atrial contraction contributes up to 30% to 40% of the left ventricular filling, maintenance of sinus rhythm is essential, and any supraventricular arrhythmias should be aggressively treated. 8. b. patients with concentric lvh and a hemodynamically significant aortic stenosis are unable to augment cardiac output in response to any sudden decrease in systemic vascular resistance. administration of an α 1 -adrenergic agent-for example, phenylephrine-improves coronary perfusion pressure without adding to the already existing fixed afterload, and increasing myocardial work. concomitant venoconstriction increases venous return and augments preload. reflex bradycardia may improve myocardial o 2 consumption. it is essential not to delay treatment of hypotension, as the thick myocardium is at risk for ischemia, further worsening cardiac output and coronary perfusion, leading to sudden death. agents with the potential to increase heart rate or myocardial o2 consumption are not first choices for treatment of hypotension in this patient with severe aortic stenosis. 9. c. amiodarone is a class iii antiarrhythmic agent used for the treatment of atrial and ventricular arrhythmias. it is used during cardiac arrest to aid defibrillation of recurrent or refractory ventricular fibrillation. amiodarone has na + , k + , ca 2+ − channel, as well as alpha-and beta-adrenergic receptor blocker properties. its rate-dependent (rapid) administration may cause profound hypotension and bradycardia. treatment is volume expansion, administration of vasoconstrictors and positive chronotropes, or temporary pacing. 10. b. norepinephrine is an endogenous catecholamine with peripheral post-synaptic α 1 -and β effects. its peripheral α effects predominate over its myocardial β 1 effects. the effects of norepinephrine mediated by β 2 -receptors are not clinically significant. norepinephrine is a potent vasoconstrictor (map and svr are increased) with mild positive inotropy. it does increase myocardial o2 requirements. its β-induced positive chronotropy is counteracted by the reflex bradycardia resulting from the α-mediated increase in afterload. norepinephrine does not significantly increase cardiac output; an effect different from that of phenylephrine (phenylephrine may decrease cardiac output). norepinephrine is less likely to induce tachyarrhythmias than epinephrine, dobutamine, dopamine, or isoproterenol. inhibits na efflux during phase 0 shortens duration of phase 2. this results in decreased duration of the action potential decreases the slope of phase 3 depolarization. this results in digoxin's characteristic effect on the stsegment of the ekg increases the slope of phase 4 repolarization. this leads to increased automaticity and ectopic beats shortens av conduction time. shortens the refractory period in the atria and the ventricles increases the pr interval due to delayed av-conduction characteristic scaphoid st segment depression due to decreased slope of phase 3 depolarization flat or inverse t-waves. the diminished amplitude or negative deflection does not correlate with serum levels this leads to a more rapid ventricular repolarization. this is independent of parasympathetic activity afterload: decreased 5. heart rate: digoxin reduces ventricular response rate in atrial fibrillation or flutter. it sensitizes arterial baroreceptors in the carotid sinus, increases parasympathetic activity by activating the vagal nuclei and the ganglion nodosum with resultant decreases in sa node activity, prolonged effective refractory period and av conduction time, resulting in slowed heart rate cardiac output: increased chapter 2. surgical anatomy of the heart acc/aha guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery. a report of the american college of cardiology/ american heart association task force on practice guidelines esc guidelines for the diagnosis and treatment of acute and chronic heart failure philadelphia: lippincott williams &wilkins perioperative beta blockade in noncardiac surgery: a systematic review for the 2014 acc/aha guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: a report of the american college of cardiology/american heart association task force on practice guidelines accf/aha 2009 expert consensus document on pulmonary hypertension a report of the american college of cardiology foundation task force on expert consensus documents and the american heart association and the pulmonary hypertension association withholding versus continuing angiotensinconverting enzyme inhibitors or angiotensin ii receptor blockers before noncardiac surgery: an analysis of the vascular events in noncardiac surgery patients cohort evaluation prospective cohort clinical anesthesia key: cord-269776-hj1s3ipp authors: agostoni, angelo; aygören-pürsün, emel; binkley, karen e.; blanch, alvaro; bork, konrad; bouillet, laurence; bucher, christoph; castaldo, anthony j; cicardi, marco; davis, alvin e; de carolis, caterina; drouet, christian; duponchel, christiane; farkas, henriette; fáy, kálmán; fekete, béla; fischer, bettina; fontana, luigi; füst, george; giacomelli, roberto; gröner, albrecht; erik hack, c.; harmat, george; jakenfelds, john; juers, mathias; kalmár, lajos; kaposi, pál n.; karádi, istván; kitzinger, arianna; kollár, tímea; kreuz, wolfhart; lakatos, peter; longhurst, hilary j.; lopez-trascasa, margarita; martinez-saguer, inmaculada; monnier, nicole; nagy, istván; németh, éva; nielsen, erik waage; nuijens, jan h.; o'grady, caroline; pappalardo, emanuela; penna, vincenzo; perricone, carlo; perricone, roberto; rauch, ursula; roche, olga; rusicke, eva; späth, peter j; szendei, george; takács, edit; tordai, attila; truedsson, lennart; varga, lilian; visy, beáta; williams, kayla; zanichelli, andrea; zingale, lorenza title: hereditary and acquired angioedema: problems and progress: proceedings of the third c1 esterase inhibitor deficiency workshop and beyond date: 2004-09-11 journal: j allergy clin immunol doi: 10.1016/j.jaci.2004.06.047 sha: doc_id: 269776 cord_uid: hj1s3ipp hereditary angioedema (hae), a rare but life-threatening condition, manifests as acute attacks of facial, laryngeal, genital, or peripheral swelling or abdominal pain secondary to intra-abdominal edema. resulting from mutations affecting c1 esterase inhibitor (c1-inh), inhibitor of the first complement system component, attacks are not histamine-mediated and do not respond to antihistamines or corticosteroids. low awareness and resemblance to other disorders often delay diagnosis; despite availability of c1-inh replacement in some countries, no approved, safe acute attack therapy exists in the united states. the biennial c1 esterase inhibitor deficiency workshops resulted from a european initiative for better knowledge and treatment of hae and related diseases. this supplement contains work presented at the third workshop and expanded content toward a definitive picture of angioedema in the absence of allergy. most notably, it includes cumulative genetic investigations; multinational laboratory diagnosis recommendations; current pathogenesis hypotheses; suggested prophylaxis and acute attack treatment, including home treatment; future treatment options; and analysis of patient subpopulations, including pediatric patients and patients whose angioedema worsened during pregnancy or hormone administration. causes and management of acquired angioedema and a new type of angioedema with normal c1-inh are also discussed. collaborative patient and physician efforts, crucial in rare diseases, are emphasized. this supplement seeks to raise awareness and aid diagnosis of hae, optimize treatment for all patients, and provide a platform for further research in this rare, partially understood disorder. this supplement, like the 2003 c1 esterase inhibitor deficiency workshop and the many patient and physician remain little known in clinical practice and thus frequently misdiagnosed and inappropriately treated, often resulting in unnecessary suffering. similarities to allergic conditions and inappropriate framing as part of the urticariaangioedema syndrome frequently lead patients with hae to be considered allergic and treated with antihistamines and corticosteroids, ineffective in this disorder. abdominal edema may so closely resemble an acute abdomen that some patients with hae have undergone unnecessary surgical explorations, often more than once. because untreated edema of the larynx may be fatal, inappropriate management may result in death. for many, hae and aae present an ongoing clinical challenge. despite the recurrent nature of angioedema attacks, their acute treatment is often suboptimal, sometimes delayed, and often requires lengthy hospital stays. in some countries, including the united states, no safe and effective acute attack therapy is available. even the prophylactic management of these disorders is inconsistent across centers and nations, and, because of the side effects of antifibrinolytics and steroids currently in use, requires a lifelong, individualized calculation of benefits and risks. these drawbacks are well known to the small community of physicians who deal frequently with these diseases and are a feature of life for those patients who suffer frequent or severe attacks. nonallergic angioedema as a model for the treatment of rare diseases in recognition of these challenges, several national and international physician and patient initiatives have begun in the past 2 decades. in many ways, the field of nonallergic angioedema, and especially hae, is becoming an exemplar for the understanding and management of rare diseases. the estimated frequency of hae is 1:50,000. 7 as in many uncommon conditions, hae's infrequent incidence fosters collaboration, forcing clinicians and researchers to pool their anecdotal experiences and data to attain statistical significance. nonetheless, hae is an attractive field because it offers doctors a chance to improve the lives of their patients dramatically through study but also via educated case management. as such, it has brought together a group of motivated and compassionate physicians. the pharmaceutical industry has also been welcomed to the c1 esterase inhibitor deficiency workshop and other hae initiatives, fostering free exchanges between academia, industry, and patients. indeed, perhaps the most distinctive feature of hae physician initiatives is their inclusion of patients with hae, not only in a traditional capacity of raising awareness and research funding but also as ethical advisors and welcomed guests for the presentation of scientific abstracts and talks. the first c1 esterase inhibitor deficiency workshop, held in hungary in 1999, was the earliest meeting to follow this model. since then, the 2 subsequent workshops and other patient-association gatherings in the united states and canada have followed its inclusive precedent. such a high level of patient involvement reflects not only the close relationship between knowledgeable physicians and their patients but also regional shortcomings in diagnosis and treatment. because of the incapacitating and life-threatening aspects of the disease, patients and their families from areas where hae is largely unknown have been forced to become educated enough to explain the disorder to strangers and, often, emergency department personnel to obtain the proper treatment. even patients whose cases are managed by a competent local practitioner may have attacks while traveling or when their doctor is unavailable and thus may need to articulate their condition to someone entirely unfamiliar with the disease. by incontrovertible necessity, patients with hae are one of the best-educated patient populations, and this is especially true in areas where satisfactory therapy for acute attacks is unavailable. for patients and physicians alike, the internet facilitates increasingly more communication, both personal and scientific. for patients with hae, it can help to reduce the isolation of having a rare disease. many patients first contact their national patient association online and use email to stay in touch with fellow patients. the internet is also being used by physicians and scientists to support a private patient registry and a public, constantly updated human c1-inh gene (c1nh) mutation database. through this online contact and regular meetings open to all, information about nonallergic angioedema is shared rapidly among a small, concerned group. nonetheless, the need to educate more physicians and the general public remains. the rarity of nonallergic angioedema increases the likelihood that clinicians, especially general practitioners or emergency department personnel, may never have seen a case. patient organizations and other groups have thus worked to create emergency passports for patients with known hae to carry and educational materials to distribute to emergency departments. scientific opportunities and current areas of controversy (kayla williams, bs, ma, mfa, cambridge, mass) nonallergic angioedema is a puzzle with relatively welldefined borders: many specific c1nh mutations resulting in hae have been identified, and the symptomatic results are known. however, several central pieces are missing. despite recognition of functional c1-inh deficiency as the cause of most forms of nonallergic angioedema, the specific mechanism of attack generation has not been definitively described. likewise, symptoms similar to those of nonallergic angioedema have now been reported in patients with normal amounts of functional c1-inh. 8 multiple pathways have been proposed for the chemical cause of angioedema attacks. the murine hae model developed by han et al 9 shares similarities with the human form of the disease but diverges from typical hae in the triggering of angioedema. despite homozygous c1-inh deficiency, the mice, with few exceptions, have not been observed to have typical angioedema attacks. attacks, manifesting solely as local increases in vascular permeability, could be provoked by the application of mustard oil. rather than representing a shortcoming of the mouse model, such a high threshold for attacks might parallel the course of those human heterozygotes, identified via a family member with active hae, who nonetheless never have an attack (for documentation of such patients, see agostoni and cicardi 7 ). the absence of spontaneous attacks despite profound c1-inh deficiency suggests that multiple biological events must transpire for angioedema to manifest. equally fascinating is the range of human disorders associated with functional c1-inh deficiency. on the mild end of the spectrum, the american physicians luong and nguyen 10 have reported a group of apparently unrelated vietnamese women presenting to their california clinic with lower extremities discomfort of unknown etiology. all of these women were found to have reduced amounts of serum c1-inh, and danazol treatment resolved both the c1-inh deficiency and the discomfort. at the opposite end of the c1-inh deficiency spectrum, some patients with hae have periods of weekly or nearcontinuous angioedema attacks. in the most severe cases, laryngeal attacks may extend far enough into the thorax that even tracheostomy cannot maintain airway patency. it is unclear whether discerning the mechanism of some forms of hae, aae, and c1-inh deficiency-associated disorders may elucidate others, but the attraction of a unified theory is obvious. however, among other factors, the inhibitory promiscuity of the c1-inh molecule and its predisposition to mutation may not lend themselves to a simple answer. nonetheless, given the many proposed pathways for attack generation, information gained toward a full understanding of nonallergic angioedema attacks may lead to a greater knowledge of 1 or more chemical cascades, including the classical complement pathway, kinin generation, and the intrinsic coagulation pathway. the areas of greatest controversy include which vasoactive peptide is ultimately responsible for the increased vascular permeability that results in angioedema. bradykinin and second component of the complement cascade (c2)-kinin have been proposed, 11, 12 with recent research contributing evidence to the importance of bradykinin. [13] [14] [15] [16] nonetheless, within the current understanding of coagulation, kinin, and complement pathways, neither peptide seems to perfectly explain all of the symptoms of angioedema. although bradykinin is the only candidate mediator for which there is direct clinical evidence, it is possible that yet another system, intermediary, or molecule may be involved in edema-generating vascular leakage. specific triggers for vasoactive peptide release are also unknown. it is proposed that the activation of factor xii is crucial to attack generation, 17 and that factor xii activation may be a result of phospholipids released from damaged or apoptotic cells. recently, endothelial cells have been implicated in the generation, via kallikrein, of bradykinin, both in the presence 18 and absence 19, 20 of factor xii. these hypotheses explain how illness or localized tissue damage may precipitate attacks but do not account for other triggers, which are themselves not well defined. in large part, triggers seem to vary from patient to patient and, in several attacks, may not be apparent. of these, the most scientifically documented and explored are hormonal triggers, made all the more interesting by relatively recent reports of patients with normal c1-inh concentrations and hae-like symptoms provoked or exacerbated by increased levels of estrogen. the importance of hormones in the regulation of nonallergic angioedema has long been acknowledged via its prophylaxis with androgens. increasingly, the effects of estrogen, progesterone, and other sex hormones are being explored. in some women, estrogen results in an increased frequency of angioedema attacks, 21, 22 but others appear unaffected. depending on the patient and trimester, pregnancy may reduce or increase the number and severity of attacks. 23, 24 in this supplement, the role of progesterone is debated, with visy et al finding a positive correlation between serum progesterone values and attack frequency, whereas bork et al note no increase in attack frequency among patients whose oral contraceptive (oc) contained progesterone and estrogen compared with those receiving estrogen alone. indeed, bork et al refer to several published works in which progestins were used, with varying success, to ameliorate hae symptoms. 22a-24a in contrast, danazol, a common prophylaxis, alters multiple biological mechanisms but is known to block progesterone receptors and increase progesterone's metabolic clearance. 25 given these conflicting findings, the influence of progesterone seems a likely area for further study. karen binkley graciously shared her work on very short notice and dr. alvin davis iii provided a valuable review; dr. shih-wen huang's contribution to the us hae association newsletter informed me of the full range of c1-inh deficiencies, dr. alvin schmaier explained the mystery of angiotensin ii receptor blocker-associated angioedema, and dr. erik nielsen, both thorough his online hereditary angioedema thesis and quick correspondence, provided information and inspiration. many thanks go to dr. ineke bos, whose model of the c1 esterase inhibitor molecule graces our cover; chrystal mcdonald who worked tirelessly to secure reprint permissions; dr. brunello wüthrich who provided images of hae attacks; and drs. werner müller and georg dewald for their additions to the text. i would also like to recognize mr. anthony castaldo of the us hae association for his review of text pertaining to the patient experience and his indomitable, sustaining sense of humor. lastly, i would like to dedicate this supplement to its many contributing authors and all the hae, aae, and non-allergic angioedema patients they strive to help. in the first part of this section, cicardi and zingale describe the varied ways in which hae can manifest and discuss other diseases that published case reports and their clinical case series have associated with hae. the symptoms of hae are caused by the extravasation of plasma into the deeper cutaneous or mucosal layers as a result of 1 or more locally released vasoactive peptides. the edema in hae is nonwhealing, nonpruritic, and generally unrelieved by antihistamines, suggesting that histamine is not involved in its induction. 26 the biological characteristics of the vasoactive peptides released in c1-inh-deficient sera indicate that the peptides belong to the kinin family. however, the discussion is not entirely closed on whether bradykinin, released because of contact system activation, or a peptide originated from c2 on classical complement pathway activation and the generation of plasmin, is the main mediator of symptoms in patients with hae. 11, 12 nonetheless, recent lines of evidence coming from c1-inh knockout mice, studies in patients' plasma, and analysis of c1-inh mutants from patients with hae support the bradykinin hypothesis. 9, 27, 28 kinin peptides participate in inflammatory processes and increase vascular permeability, activating intracellular pathways that lead to the release of nitric oxide. 29, 30 vascular leakage can occur without anatomical damage and rapidly revert when the release of mediator molecules ceases. hence, edema usually resolves within 72 hours. in some cases, it may resolve within 12 hours, but in others, it may persist as long as 5 days. urticaria, a condition analogous to angioedema but with plasma leakage into the upper cutaneous layers, is typically absent or minimal and short-lasting in patients with hae. typical symptoms. the recurrence of cutaneous angioedema, abdominal pain, and asphyxia caused by laryngeal edema is the full clinical pattern of hae, present in about 50% of adult patients. 7 attacks usually evolve within a single site, but it is not uncommon for some patients to have simultaneous or closely spaced cutaneous and abdominal involvement. most patients recognize several hours in advance that an attack is coming. they may have sudden mood changes, anxiety, or complete exhaustion. cutaneous symptoms. skin edema is nonpitting and nonerythematous, with ill-defined margins. it typically affects the face (fig 1) , extremities, and genitals (fig 2) . it usually spreads to disfigure the affected site, temporarily depriving it of function. most often, a single site is affected by an extended edema that grows and then regresses within 2 to 5 days. alternatively, edema may persist, although reduced in size, and migrate to different cutaneous locations. in contrast with edema of other etiologies, edema associated with hae does not principally manifest in the perioral region. edema can localize subcutaneously in any body part, including the trunk. abdominal symptoms. recurrent abdominal pain, a consequence of gastrointestinal wall edema, is reported by 70% to 80% of patients with hae. 7, 31, 32 this is a distinguishing feature of c1-inh deficiency because abdominal involvement is rarely seen in angioedema of other origins. it presents with symptoms that may vary from mild discomfort to severe, intractable pain accompanied by vomiting and/or diarrhea. 33 in this setting, hypovolemia can result from a combination of fluid loss, plasma extravasation, and vasodilation and can progress to hypovolemic shock. 34, 35 ascites resulting from extravasation into the peritoneal cavity, edema of the bowel wall, or changes in splenoportal axis caliber have been described during abdominal attacks as detected by ultrasounds or computed tomography. [36] [37] [38] [39] [40] [41] gastrointestinal endoscopy performed during an abdominal attack revealed gastric involvement. interestingly, during the healing process after a prominent gastric edema, several small nodules and raised erosions developed over the entire gastric mucosal surface. within 55 days, the gastric mucosa had returned to normal. 42 the similarity between bowel angioedema and surgical emergencies is confirmed by the fact that approximately e of patients with undiagnosed hae undergo unnecessary surgery during abdominal attacks. 7 however, even after a diagnosis of hae has been established, differentiating angioedema of the bowel from a surgical emergency remains a critical task for the physician. 31 the physical examination can show the presence of an abdominal defense reaction. moderate or sometimes even marked leukocytosis can be part of an angioedema attack. 43 abdominal ultrasounds and computer-assisted tomogra-phy scans demonstrate the presence of free peritoneal fluid and edematous intestinal mucosa. 36, 39, 41 however, all of these signs are clearly not specific to angioedema. the authors note that this symptomatic generality should be borne in mind to avoid the situation that occurred with a patient in their case series. surgery was inappropriately delayed when acute appendicitis was mistaken for intestinal angioedema. the efficacy of c1-inh plasma concentrate in resolving symptoms may help to distinguish angioedema from a true surgical emergency. laryngeal symptoms. laryngeal edema is the most dramatic clinical event for patients with hae. half of them have it at least once in their lives, but a history of recurrent episodes of suffocation caused by laryngeal edema is not uncommon, and deaths still occur as a result. 44 in the past, 25% to 30% of patients with hae died from laryngeal edema. this percentage has dramatically dropped for patients who are appropriately diagnosed because of the availability of effective treatments in several countries. 45 nevertheless, because of previous life-threatening experiences, some patients with hae still carry permanent tracheal cannulae, allowing them to breathe by bypassing the larynx when edema occurs. as mentioned, angioedema without urticaria is the hallmark of c1-inh deficiency. however, a discrete number of patients, 26% in a survey by frank et al, 31 have erythematous mottling, erythema multiforme, or erythema marginatum, always mild and transient, that inconstantly heralds or attends their angioedema. 46 some patients recognize this symptom as announcing an attack, and when on prophylactic treatment, can still have a rash not followed by swelling. fig 3 depicts several erythematous rashes experienced by patients before or during attacks of angioedema. unusual symptoms. reports in the literature suggest that edema caused by c1-inh deficiency could occur in locations other than the characteristic sites of manifestation. [47] [48] [49] [50] frank et al 31 reported transient pleuritic symptoms with pleural effusion in 2 patients. local cerebral edema has been considered responsible for transient seizures and hemiparesis seldom described in patients with hae. 31, 51 this assumption, despite its attraction and its occurrence in other forms of angioedema, 49 has not been confirmed so far. neurologic disorders and the potential manifestation of cerebral edema remain a rarity in patients with hae. although atypical, urinary symptoms mimicking an infection have been described, and in 1 patient, the presence of bladder edema was documented by endoscopy and biopsy. 24, 48 pulmonary edema as a consequence of c1-inh deficiency has occasionally been suggested but never clearly demonstrated. 47 in the authors' experience, such an event was never observed to accompany an angioedema attack. they suggest that the high efficiency of the pulmonary vascular tree in the inactivation of bradykinin accounts for the lungs' protection from its effects. 52 age of onset and frequency of symptoms. c1-inh deficiency is present at birth, and a minority can have perinatal angioedema symptoms. most commonly, symptoms begin at school age. half of patients with hae had symptoms within the first decade of life, and another third had symptoms by the second decade. asymptomatic adults carrying a c1nh mutation, detected because of the presence of offspring with clinically overt disease, have been described and are estimated to account for 5% of all patients with hae. 7 the frequency at which bouts of angioedema recur is extremely variable among subjects and may vary in the same individual during different stages of life. a survey of the italian case list showed that slightly less than e of untreated patients with hae have more than 1 angioedema attack per month, 40% have 6 to 11 swellings per year, and the remaining 30% are infrequently symptomatic or completely symptom-free. this range of phenotypic expression has no significant correlation with plasma concentrations of c1-inh and is usually inconsistent among family groups. it should therefore be concluded that factors other than c1-inh deficiency intervene to determine a subject's tendency to develop angioedema. these factors might be genetic or environmental. the hypothesis that symptom frequency correlates with specific functional polymorphisms of some of the proteins involved in pathogenesis is attractive but thus far unproven. an initial report suggesting that a polymorphism within the bradykinin receptor could distinguish oligosymptomatic from polysymptomatic patients has not been confirmed. 53 farkas et al 54 found that patients with hae infected with helicobacter pylori are more susceptible to symptoms than uninfected patients, and that eradication of the infection reduces the frequency and severity of swellings, particularly angioedema of the bowel. if confirmed in a larger group of patients, these findings could support those of several groups suggesting that infections increase susceptibility to angioedema in the general population as well as in patients with hae. 7, 31, [55] [56] [57] clinical and laboratory criteria for diagnosis are provided in table i ; a severity scale for the evaluation of nonallergic angioedema is provided in table ii . these tools are based on contributions elaborated from experts from 10 european countries who received a grant from the european commission for a project called novel methods for predicting, preventing, and treating attacks in patients with hereditary angioedema (prehaeat), consisting of a concerted action in the framework of the specific research and a technologic development program, quality of life and management of living resources, designed to improve the lives of patients with hae. diseases associated with c1-inh deficiency. most often, patients with hae are substantially healthy apart from problems associated with swelling. however, there are several reports of autoimmune diseases in patients with hae, [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] and systemic lupus, in particular, has been described rather often. 62 in a systematic study, 19 of 157 patients with hae had some kind of autoimmune disorder. 60 moreover, patients with hae, because of defective control of the classical pathway of complement activation, have a deficiency of the fourth component of the complement cascade (c4) and c2, a condition that increases the risk of autoimmune diseases. 69 a large epidemiologic study in 1997 based on 24 major autoimmune diseases estimated the prevalence of autoimmune diseases in americans to be 1 in 31 (3.2%). 70 given that all autoimmune diseases were not evaluated in this general population study, one cannot definitively conclude that patients with hae have a higher risk of autoimmune disease, but it appears likely. the association of hae with other inherited and noninherited conditions has occasionally been reported, but these observations remain isolated. [71] [72] [73] [74] last, patients with hae can be exposed to risk through needed treatments. several cases of hepatitis c virus (hcv) in the italian case series were a result of receiving plasma-derived products. these cases occurred before the introduction of viral inactivating procedures for plasma products. 75 no cases of hiv were reported, but because of hcv, approximately 5% of their patients now have liverrelated problems. in this section, harmat et al describe the results of a study of 70 hungarian patients with hae in whom ultrasonography was used to evaluate acute abdominal attacks of hae. background and rationale. ascites can result from diverse causes. the most common etiology, found in approximately 80% of cases, is the decompensated liver (cirrhosis). the remaining 20% result from other pathologies, such as malignancy in the abdomen (10%); various inflammatory diseases and other disorders, such as nephrotic syndrome, exudative enteropathy, chylous ascites, and mesenteric thrombosis; and others. however, hae is very seldom mentioned as a cause of ascites. this is a real problem, because ascites are a significant diagnostic sign of this uncommon but serious disease. the most common symptoms of hae appear in the form of ascites that cause acute abdominal attacks. for diagnosing this state, ultrasonography is the most potent tool. 39, 76, 77 methods. ultrasonographic assessment is especially well suited to investigating the cause of abdominal symptoms. this study was performed to evaluate the usefulness of ultrasonographic diagnosis and included 70 patients (26 pediatric) from the hungarian hae center database. of these, 60 had hae type i and 10 had hae type ii. the male to female ratio was 32:38, and patient age ranged from 2.5 to 66 years. patient follow-up continued for a decade. in addition to biochemical studies, ultrasound investigations were performed at 6-month intervals. patients with typical symptoms of hae were hospitalized if the presence of other pathologies could be ruled out and if the manifestation was associated with hypovolemia and included recurrent paroxysms of acute colicky pain, nausea and vomiting, or profuse diarrhea, not responding to symptomatic therapy. all hospitalized patients underwent ultrasonography. during each abdominal attack, ultrasound examinations were performed before treatment and repeated at 24 and 48 hours post-treatment. 78, 79 ultrasonographic investigations were performed by using a hitachi 451, a hitachi eub 40 (hitachi medical systems, zug, switzerland), or an aloka ssd-1700 diagnostic system (aloka co, ltd, tokyo, japan), with a 3.5-mhz or -mhz convex transducer or a linear 7.5-mhz transducer. subdiaphragmatic and pelvic regions were scanned with the patient in a supine position. kidneys were explored and the presence of free peritoneal or retroperitoneal fluid was ascertained with the patient in the supine and lateral positions or, when necessary, standing. free fluid, when detected, was classified into 1 of 3 categories, as follows: (1) small-volume free peritoneal fluid was visible only in the subhepatic or subsplenic space, and in every case, in the douglas cul-de-sac. clinical criteria major (1) self-limiting, noninflammatory subcutaneous angioedema without major urticarial rash, often recurrent and often lasting more than 12 hours (2) self-remitting abdominal pain without clear organic etiology, often recurrent and often lasting more than 6 hours (3) recurrent laryngeal edema minor (4) family history of recurrent angioedema and/or abdominal pain and/or laryngeal edema laboratory criteria (1) c1 inhibitor antigenic levels <50% of normal at 2 separate determinations with patient in basal condition and after the first year of age (2) c1 inhibitor functional levels <50% of normal at 2 separate determinations with patient in basal condition and after the first year of age (3) mutation in c1 inhibitor gene altering protein synthesis and/or function diagnosis can be established in presence of 1 major (1-3) clinical criterion and 1 laboratory criterion (2) moderate-volume ascites, in addition to ascites found in these regions, included those identified in the sublienal space and among the intestinal loops. the intestinal walls were also swollen (thickness in excess of 5 mm 80 ). results. an ultrasound image taken during an acute abdominal attack (fig 4) clearly illustrates the abdominal manifestations of hae. in this medial sagittal section of the pelvic area, a large amount of free peritoneal fluid can be observed in the douglas cul-de-sac, distal to and well separated from the urinary bladder. a floating intestinal loop can be seen. during the attack, an edematous thickening of the intestinal wall and a thin, echo-free fluid layer around the bowels also could be observed, as illustrated in edema of the portal veins, biliary ducts, and cholecyst wall, causing gross structural changes in the liver, was also observed. the liver parenchyma generally appeared less echogenic, whereas the walls of the portal vein radicles displayed increased echogenicity, resulting in a so-called starry sky texture that could be observed during the acute phase. because of local edema, the pancreatic region also displayed an increased echogenicity (fig 8) . in addition to the hepatic portal vein, the wall of the cholecyst was also echogenic (fig 9) . after treatment with c1-inh concentrate, the former brightness disappeared, and the echo pattern of the liver returned to normal (sonogram not shown). discussion. early recognition of acute abdominal attacks is of utmost importance because incorrect or delayed diagnosis often leads to unnecessary surgical intervention. in undiagnosed patients, ultrasound examination can be a differential diagnostic means for recognizing hae in the abdominal organs because of its ability to detect nonspecific but sensitive clues such as thickening of the intestinal wall, free peritoneal fluid, intestinal hypermotility or hypomotility, and echo pattern fig 7 . sagittal and transverse sonograms during an hae attack before and after treatment. sagittal sections are shown above, transverse below. a, a large amount of free peritoneal fluid has accumulated in the pouch of douglas and, in the sagittal section, a floating intestinal loop is visible. the urinary bladder appears below. b, soon after treatment with c1-inh concentrate, the amount of peritoneal fluid is somewhat decreased. c, only a minimal amount of fluid is present in the pouch of douglas 24 hours after c1-inh treatment. several sonograms have previously been published in slightly different format. 78, 81 transverse panels b and c reprinted with permission from acta paediatrica. 81 sagittal panels a-c reprinted with permission from the european journal of gastroenterology and hepatology. 78 fig 8. transverse sonogram during an abdominal hae attack: liver and pancreas. increased hepatic reflection (starry sky liver) and thickened, echogenic portal veins (arrow); the pancreatic region is also hyperechoic (double arrows). reprinted with permission from acta paediatrica. 81 changes of the liver and pancreas. ultrasound examination has therefore proven very useful as a complementary, quick, and painless tool for recognizing the early phase symptoms of hae. patients presenting with skin symptoms (erythema marginatum) or acute pains, nausea, vomiting, or profuse diarrhea of unknown origins should be immediately hospitalized and investigated with ultrasound. ultrasound follow-up in known cases of hae is also capable of proving the efficacy and expeditiousness of acute attack treatment. in rare cases in which patients with known hae present with abdominal symptoms unresponsive to c1-inh concentrate, ultrasonography may help distinguish between a refractory hae attack and an unrelated surgical emergency. abdominal and pelvic ultrasound examination is a highly reproducible and informative diagnostic tool and thus is indicated during acute abdominal attacks of hae unresponsive to c1-inh concentrate. conversely, a search for hae is warranted when the typical sonographic features are ascertained in a patient with abdominal symptoms. angioedema may be caused by reasons as various as allergies, inherited or acquired deficiencies of c1-inh, or drug reactions. 82, 83 for the life of a patient presenting with unexplained airway swelling, the most important etiologic distinction is that between angioedema of allergic, histaminergic origins and the far rarer c1-inh-associated or nonallergic angioedema. when allergic angioedema has been ruled out, nonallergic angioedema is next determined to be hereditary or acquired, and subclassification is pursued. allergic angioedema, with histamine as its major mediator, may best be defined by its clinical response to antiallergic drugs such as antihistamines and cortico-steroids. in this type of angioedema, reaction of specific ige antibodies with an allergen induces the release of histamine and other mediators from mast cells. it is often associated with urticaria. in contrast, angioedema caused by c1-inh deficiency is not known to be triggered by an allergic reaction, is not usually associated with hives, and likely has bradykinin as its principal mediator. current systems for classifying hae and aae describe the disorders in terms of c1-inh deficiency type. although observed convention supports the classification of major types, some further classifications, such as aae types, are more fluid. in the case of the more recently described estrogen-sensitive angioedema, a new formal description is suggested here. in the interest of both definition and the elucidation of mechanism that these differences imply, the divisions of hae and aae type are presented. for an example of prevalence, table iii presents agostoni's 573-patient angioedema case series by type. hereditary angioedema related to c1 inhibitor deficiency is a well-defined autosomal dominant trait. its variants include types i (hae-i) and ii (hae-ii), associated with mutations of the c1 inhibitor gene (c1nh or serping1), 84,85 and a newly described type not associated with c1-inh deficiency 8,86,87 further defined and discussed in another section. the disease results from a large variety of mutations of the c1nh gene, located in the q12-q13.1 subregion of chromosome 11. according to the relative concentrations of antigenic and functional c1-inh, 2 types of hae have traditionally been described. 88 the defective gene produces either no c1-inh (hae-i) or a dysfunctional c1-inh (hae-ii). 6, 84, 85, 88 in either case, it is associated with low functional activity of c1-inh, low levels of c4, and normal levels of the third component of the complement in hae-i (;85% of patients with c1-inh-associated hae), defective expression of 1 allele results in low antigenic and functional concentrations of c1-inh. in hae-ii (;15% of patients with c1-inh-associated hae) , concentrations of functional c1-inh are low, but c1-inh antigenic levels are normal or increased, with the presence of a dysfunctional mutant protein. 88 for both, c1-inh function is usually 5% to 30% of normal, instead of the 50% expected if the single normal allele were fully expressed. this difference is ascribed to permanent c1 and contact phase activation, with subsequent c1-inh consumption in the periphery. 89, 90 interestingly, the description of low levels of nonfunctional c1-inh mutants in patients with hae-i has demonstrated that the distinction between hae-i and hae-ii is not absolute. 91 this finding occurred in patients with mutations to exon 8 at the carboxy terminus of the c1nh gene, thought to be responsible for the proper folding necessary for transport outside of the cell and exposure of the reactive site loop. thus, although these patients with low antigenic concentrations of c1-inh appear to have hae-i, they are in fact expressing nonfunctional c1-inh that cannot efficiently exit the cell. estrogen-dependent and estrogen-associated inherited angioedema (previously hae type iii) (karen binkley, md, frcpc, and alvin e. davis iii, md, toronto, canada, and boston, mass) a type of angioedema, to date manifest only in women, has recently been described. 8, 86, 87 its symptoms closely resemble those associated with functional c1-inh deficiency but occur in the presence of normal c1-inh concentrations; the genetic defect responsible is currently unknown. although this type of angioedema has been referred to as hae type iii (online mendelian inheritance in man [omim] 300268), others have argued that this designation is both redundant and misleading. the following piece by binkley and davis explores their work in a kindred with estrogen-dependent inherited angioedema, more fully describes estrogen-sensitive forms of inherited angioedema, and proposes a rational system of nomenclature. overview. the authors investigated a family with symptoms of angioedema restricted to conditions of high estrogen levels. although this investigation was undertaken to provide better care for the affected family members, it also presented a unique opportunity to better understand the effects of estrogen and androgens on c1-inh. 8 however, instead of altered hormonal regulation of c1-inh, this family seemed to possess a completely novel abnormality, as suggested by the absence of identifiable mutations in either the coding or the 5# regulatory regions of the c1nh gene 8 and normal c1-inh function and activity in a pregnant, symptomatic family member. 92 the exact mechanisms responsible for angioedema in these patients have yet to be identified. the importance of kinin degradation pathways and aminopeptidase p (app) in the control of angioedema generation has been independently recognized in studies of angiotensin-converting enzyme (ace) inhibitor-related angioedema. bradykinin and its active metabolite, des-arg-bradykinin, are metabolized largely by 2 enzymes, ace and app. [93] [94] [95] with ace inhibitor administration, app becomes the primary enzyme responsible for inactivating bradykinin and des-arg-bradykinin. in fact, individuals with low plasma concentrations of app appear to be predisposed to developing angioedema during ace inhibitor treatment, when neither ace nor app is available to inactivate these kinins. 96 kinin inactivation pathways might also modulate clinical symptoms in classic hae. for example, decreasing kinin inactivation in patients with hae with the use of ace inhibitors can result in exacerbation of angioedema. [97] [98] [99] [100] given the important contribution of kinin inactivation pathways to the control of angioedema, this may be an avenue for further investigation. case histories and investigation of the index family. the index family presented with histories of episodic, hae-like angioedema. 8 these episodes occurred only during pregnancy, oc use, or estrogen replacement therapy. symptoms began 14 to 21 days after conception, or within 7 to 14 days of starting endogenous hormones. no episodes occurred in the postpartum period. one patient's description was particularly compelling: ''my period was just a day or two late, but when one side of my face swelled up, i knew i must be pregnant, because this is just like what happened to my mother and sisters every time they were pregnant.'' in affected individuals, symptoms occurred in all pregnancies and with each course of estrogen therapy. unaffected individuals had no symptoms at any time. there were 8 affected women in 3 generations and 1 obligate male carrier. transmission was consistent with an autosomal dominant inheritance. complement values, c1-inh, c1-inh function, prekallikrein, factor xii, and high molecular weight kininogen were normal in 3 patients during asymptomatic periods. genetic investigations were undertaken for the following reasons: (1) the patients were asymptomatic at the time of presentation, (2) baseline biochemical investigations were unremarkable, and (3) exposing patients to estrogens for the purpose of detecting resultant biochemical abnormalities was unethical in light of the risk of laryngeal edema. the striking clinical similarity to classic hae focused initial investigations on the c1nh gene. however, no abnormalities in the coding sequences of the c1nh gene or in the 5# regulatory region were detected. 8 when patient iii-24 became pregnant and developed recurrent angioedema, biochemical investigations were undertaken. 92 c1-inh antigen and function were both normal. the mechanism by which increased estrogens precipitate symptoms thus remains under investigation. related phenotypes: hae with normal c1 inhibitor activity in women. most of the 36 women with angioedema in 10 families reported by bork et al 86 appeared to have a phenotype different than that of estrogen-dependent angioedema, because only 1 of 36 patients had attacks exclusively during pregnancy. in 10 of 36 patients, attacks occurred more frequently during oc use but were not limited to these periods. by extrapolation, 15 of these 36 patients had angioedema apparently unrelated to use of ocs or pregnancy. age of onset of symptoms in the patients of bork et al 86 was variable and was not reported as directly correlating with onset of exogenous estrogen use or pregnancy. symptoms in at least 1 patient started as early as 1 year of age, before significant hormonal effects were likely as the authors note. these features are in sharp contrast with those of patients with estrogen-dependent inherited angioedema, in whom episodes of angioedema occurred exclusively during pregnancy or exogenous estrogen therapy, and suggest that a different underlying defect might be responsible for the different phenotypes. in the women described by bork et al, 86 c1-inh and c4 levels were normal in the affected individuals without symptoms. normal measurements of c4 and c1-inh during symptomatic periods were also obtained in some individuals. 86 other pedigrees have also been reported. 87 nomenclature. until further biochemical and molecular genetic studies elucidate the underlying defects in these pedigrees, it remains unclear whether the different pedigrees represent subtle abnormalities in the same underlying pathway or distinct biochemical and clinical entities. therefore, affected patients can currently be classified only on the basis of phenotype, without reference to the underlying defect. this has implications for the nomenclature applied to these conditions. the term hae type iii may be misleading because it implies that these patients have a defect similar to hae-i (inadequate c1-inh concentration) and hae-ii (inadequate c1-inh function). this is clearly not the case, because c1-inh concentration and function are normal in several pedigrees. 92, 101 further confusion arises because the term hae type iii had been previously suggested to apply to a form of angioedema resulting from inadequate c1-inh function caused by a mutation resulting in inappropriate binding to albumin. 102, 103 although hae type iv was suggested for the patients of bork et al 86 to address this latter concern, 103 the term still erroneously implies a defect in c1-inh function. the authors thus suggest that patients should be categorized on the basis of their phenotype and recommend the terms estrogen-dependent inherited angioedema and estrogenassociated inherited angioedema 92,101 until molecular studies suggest an alternate, rational nomenclature. clinical implications. further studies are required to identify the factors that contribute to angioedema in patients with estrogen-dependent angioedema. unaffected family members might then be identified through biochemical or genetic assays so that they might use ocs or plan pregnancies freely. identification of affected family members would allow these individuals to avoid ocs, bypassing a trial of therapy and the attendant risk of laryngeal edema. should effective treatment became available, affected individuals wishing to use ocs or become pregnant could begin treatment prophylactically or, at least, ensure its availability beforehand. if a particular factor is conclusively shown to be reduced in these patients, symptomatic individuals might be treated by replacing the missing factor. other possible treatments include novel strategies to reduce kinin formation or enhance kinin inactivation. prenatal diagnosis of fetal status (affected or unaffected) might also be relevant to the management of pregnancy in these individuals. the reported kindred showed significant variation in symptom severity during pregnancy, with some individuals experiencing relatively mild symptoms. in at least 1 affected individual, it is likely that symptoms during a pregnancy with an affected fetus (identified as such only later in life) were accurately recalled as being particularly severe (binkley, unpublished data, march 2000) . it is interesting to speculate that an affected fetus would not provide the missing factor to the affected pregnant mother, and this might explain the severity of the symptoms. conversely, an unaffected fetus might act as a source of the otherwise missing factor during pregnancy and might mitigate symptoms in an affected pregnant mother. if pregnancies could be identified early as being at high or low risk for severe angioedema on the basis of fetal status, follow-up and management could be guided accordingly. at least 1 direction for further study of the mechanisms responsible for symptoms in patients with estrogenassociated angioedema is suggested by the reduced kinin inactivation in ace inhibitor-associated forms of angioedema. elucidation of the defect responsible for this phenotype would allow better diagnosis and possibly specific treatment. general strategies to reduce kinin formation and/or enhance inactivation might also be helpful for the amelioration of symptoms. concerning hae-i and hae-ii, just as variations in serum concentrations of app appear to determine which individuals in a normal population develop angioedema with a second perturbation of kinin metabolism, such as the use of ace inhibitors, 96 it could be speculated that variations in either kinin activation or inactivation pathways might contribute to the differences in severity of angioedema in individuals with a pre-existing perturbation in kinin metabolism, such as a mutation in c1-inh (as occurs in hae). thus, it is possible that some of the variation in symptom severity seen between different members of the same family, carrying the same c1-inh mutation, comes from variation in other kinin pathways. identification of the defects in estrogen-dependent and estrogen-associated angioedema might illuminate potential candidate factors. knowledge of kinin production and inactivation pathways and how they are influenced by sex hormones may also offer insight into some perplexing issues regarding the effects of sex hormones on c1-inh values and angioedema symptoms in hae. androgens are effective in reducing episodes of angioedema and are used clinically for this purpose in hae. 104, 105 although androgens increase plasma concentrations of c1-inh, 105 the amount of c1-inh increase does not correlate well with symptom diminution. 106 it is tempting to speculate that androgens may also increase kinin inactivation pathways, and this, perhaps in combination with slightly higher amounts of c1-inh, contributes to the observed reduction in angioedema with androgen therapy. further studies will be necessary to explore this possibility as well. use of estrogen therapies typically results in some lowering of plasma c1-inh concentration in normal individuals, 107 and use of estrogen therapy tends to exacerbate angioedema in patients with hae. 31 however, during pregnancy, estrogen concentrations are high, c1-inh concentrations decrease, [108] [109] [110] and paradoxically, episodes of angioedema may decrease, especially in late pregnancy. 31 these puzzling observations have long suggested that a second mechanism is important in controlling angioedema. kinin inactivation pathways may be one such mechanism. speculation about possible mechanisms of symptom reduction in pregnancy suggests potential fruitful areas for further study. for example, are there hormonal factors in pregnancy, not operative during estrogen therapy, that increase kinin inactivation or other factors and reduce angioedema, despite an estrogeninduced lowering of c1-inh? is the fetus or placenta a source of kinin inactivation factors or other factors that mitigate the effects of estrogen-induced lowering of c1-inh? does variation in fetal production of kinin-inactivators or other factors underlie any variation in angioedema symptoms between pregnancies in the same individual, or between individuals in the same families, all with the same c1-inh mutation? acquired angioedema is typically caused by ace inhibitor treatment, and less commonly is caused by autoantibodies directed at c1-inh. general strategies to reduce kinin formation or/and increase kinin inactivation, identified through characterization of the elements of these pathways as well as their regulation, may be applicable to these patients as well. moving ahead. the discoveries of estrogen-dependent and estrogen-associated inherited angioedema are likely to focus attention on mechanisms other than c1-inh that control angioedema. pathways involving kinin production and inactivation may be fruitful areas of further study in these conditions, a better understanding of which might provide new therapeutic opportunities potentially relevant to all types of angioedema. angioedema may be acquired, mainly in association with lymphoproliferative disorders or occasionally with autoimmune, neoplastic, or infectious diseases. 7 aae also includes various other types of secondary c1-inh deficiency, angioedema caused by certain antihypertensive medications, urticaria-associated angioedema, and idiopathic angioedema. 82, 83 in the laboratory, aae is characterized by low functional c1-inh, low amounts of c4, and normal amounts of c3. concentrations of c1q are often very low. angioedema caused by acquired c1-inh deficiency: type i and type ii distinguished. (marco cicardi, md*, andrea zanichelli, laurence bouillet, md, cca,* and emel aygören-pürsün, md, milan, italy, grenoble, france, and frankfurt, germany) in this section, cicardi et al review the current classifications of aae and discuss the possible pathogenic mechanisms on which these distinctions are ostensibly based. angioedema caused by acquired deficiency of the inhibitor of the first component of human complement (c1-inh), usually referred to as acquired angioedema, is a rare, life-threatening disease first described by caldwell et al. 111 characteristic of acquired c1-inh deficiency are the increased consumption of c1-inh and the hyperactivation of the classical pathway of human complement. 112 as a consequence, these patients have almost undetectable serum levels and/or activity of c1-inh, c4, c2, and c1q, r, and s. usually, these abnormalities are constantly present, but temporary normalization of 1 or more of these parameters has been reported. 113 the clinical manifestations of the disease mimic those of the inherited defect of c1-inh and include subcutaneous, nonpruritic swelling without accompanying urticaria; involvement of the upper respiratory tract manifested as dysphagia, voice change, or respiratory stridor; and partial obstruction of the gastrointestinal tract presenting as colicky abdominal pain. 7 angioedema caused by acquired c1-inh deficiency differs from hae by the absence of a family history of angioedema and a late onset of symptoms (in the fourth decade of life or later). response to treatment varies compared with hae caused by the c1-inh hypercatabolism characteristic of acquired c1-inh deficiency. 114 acquired c1-inh deficiency is frequently reported in association with b lymphoproliferative diseases. different forms of b lymphoproliferation can occur, ranging from benign monoclonal gammopathies of undetermined significance (mgus) to true malignancies. 115 in 1986, autoantibodies inactivating c1-inh were first detected in patients with acquired c1-inh deficiency. 127 initially, autoantibodies inactivating c1-inh were identified in otherwise healthy patients. on the basis of this observation, it was proposed that 2 separate forms of acquired c1-inh deficiency existed: type i, paraneo-plastic, mainly associated with lymphatic malignancies; and type ii, autoimmune, caused by autoantibodies to c1-inh. the latter form appeared to be characterized further by elevated serum levels of cleaved c1-inh. 128, 129 because cleaved c1-inh was not invariably found to be present in the serum of patients with so-called autoimmune acquired c1-inh deficiency, 115 this division has been questioned. 130, 131 furthermore, autoantibodies to c1-inh were later described in patients with associated diseases. these autoantibodies were found to be common in patients with mgus and frequently exhibit the same isotype of the m component. 115, 132, 133 autoantibodies to c1-inh impair c1-inh function. although the exact mechanism for such impairment remains controversial, 7,122 the majority of these autoantibodies appear to enhance c1-inh cleavage by target proteases, preventing their inactivation. a recent article on 23 patients with acquired c1-inh deficiency followed for as long as 24 years (median, 8 years) demonstrated that half of the patients with malignancies also had autoantibodies to c1-inh, either at the time of onset of angioedema or later in the course of disease, indicating that autoimmune acquired c1-inh deficiency is not distinct from the acquired c1-inh deficiency that occurs in the setting of malignancies or other diseases. detection of autoantibodies to c1-inh in a patient with acquired c1-inh deficiency should not decrease the importance of considering the possibility of an associated pathologic condition. compared with the general population, patients with acquired c1-inh deficiency presented higher risk for b-cell malignancies. in patients with acquired c1-inh deficiency, the risk for progression of mgus to malignancy was not higher than in other patients with mgus. 134 (angelo agostoni, md, milan, italy) new causes of aae, especially drug-related aae, are still being discovered, posing the question whether all types of aae share a common biomechanism if not a common etiology. in the descriptive sections that follow, agostoni surveys several classes of aae by cause. idiopathic nonhistaminergic angioedema. cicardi et al 135 describe a subset of angioedema patients having normal complement values, no history of provoking drug treatment, and who are unresponsive to antihistamines. this condition, with a clinical presentation similar to that of c1-inh deficiency, is deemed idiopathic nonhistaminergic angioedema. it is possible that this classification might overlap, at least in part, with that of estrogensensitive angioedema. ace inhibitor-related angioedema. angioedema may be a consequence of an adverse drug reaction not induced by an allergic or parallergic mechanism. 136 ace inhibitor-related angioedema occurs in 0.1% to 0.5% of patients taking the drug. decreased bradykinin degradation is implicated because ace, also known as kinase ii, activates both angiotensin i and bradykinin. ace inhibitor-related angioedema may be an underestimated side effect because it can appear after years of ace inhibitor use, thus obscuring its relationship with the drug. unlike patients with c1-inh deficiency, patients who develop ace inhibitor-related angioedema show no evidence of the cleavage products of high molecular weight kininogen (hk) in their plasma, despite high plasma concentrations of bradykinin. because the cleavage of hk generates bradykinin, the pathogenic mechanism of ace inhibitor-related angioedema probably resides in the catabolic side of bradykinin metabolism instead. 16 when ace is inhibited, app plays a major role in plasma bradykinin catabolism. to identify patients at risk of developing angioedema during ace inhibitor treatment, adam et al 96 evaluated blood concentrations of app. their results indicated lower plasma concentrations of app in patients who had previously had ace inhibitorassociated angioedema, suggesting an inverse relationship between app concentration and the tendency to develop angioedema. it is evident that ace inhibitor use should be avoided in patients with hereditary or acquired c1-inh deficiency. angioedema related to other drugs. rare instances of angioedema have been reported with angiotensin ii (at2) receptor antagonsists, 137 although this adverse effect seems to occur less frequently with at2 receptor antagonists than with ace inhibitors. 136 it is unknown whether the 2 adverse drug reactions share the same mechanism. scattered reports have suggested the possibility of angioedema associated with the use of estrogens, fibrinolytic agents, psychotropic agents, and antihypertensives other than ace inhibitors. the molecular diagnosis of angioedema is primarily based on evidence of the decrease or lack of c1-inh function, which is routinely stated by its control capacity toward the target protease c1s in spectrophotometric assays. 138 molecular sizing of circulating c1-inh can subsequently be assessed by sds-page and immunoblot. as discussed in greater detail in the pathogenesis and pathobiology of hae and aae section, c1-inh controls several proteases, including c1r and c1s, the mannosebinding protein associated serine protease (masp) system, kallikrein, coagulation factors xiia and xia, plasmin, and tissue plasminogen activator. [139] [140] [141] [142] [143] [144] therefore, c1-inh plays a key role in regulating the early steps of complement and the contact system of kinin formation. 89 this broad inhibitory ability ensues from a property unique to the serpin class: highly efficient complex formation with target proteases. 145 thus, mutations of the c1nh gene typically affect many pathways. to add further complexity, many different c1nh mutations resulting in hae have been discovered. through the study of these mutations, it is hoped that more complete knowledge of the many functions of c1-inh can be gained, ultimately contributing to a better biochemical knowledge of hae. mutation analysis of the c1nh gene in this section, drouet et al review the methods currently available for detecting c1nh mutations and describe the powerful online mutation database that has grown out of such efforts. c1nh gene. the c1nh gene maps to chromosome 11. theriault et al, 146 by using in situ hybridization in 1990, localized it to 11q11-q13.1; a year later, janson et al 147 mapped it to 11q12-q13.1. it consists of 8 exons distributed over a dna stretch of 17 kb, with introns containing 17 repetitive alu sequences 148 (fig 10) . the structural abnormalities in the c1nh gene in patients with hae have been found to be very heterogeneous. illustrative examples explain the generation of c1nh gene defects: large deletions or, less frequently, partial duplications involving alu repeats distributed along the c1nh gene 149, 150 ; deletions resulting from a peculiar consensus sequence or an alternative secondary structure 151 ; and mutations based on cytosine-phosphateguanine (cpg) methylation and subsequent cytosine deamination to thymine. 91, 152 as shown in table iv , more than 150 mutations have been reported in unrelated patients, with pathogenic amino acid substitutions distributed over the entire length of the coding sequence. 85, 153 in addition, the frequent de novo mutations in the c1nh gene underline the presence of multiple hot spots, including those containing a cpg dinucleotide. 154 all of these lead to an apparent failure to synthesize or secrete functional c1-inh protein. in the context of a normal steady-state c1-inh mrna content in almost all cases of hae-i and hae-ii, defective expression has been related to impaired protein secretion, 91,155 transinhibition of c1-inh translation, 156 or extensive consumption. 157 the consequences of several missense mutations have been determined at the functional and intracellular processing level by transfection of an in vitro mutagenized construct into cos cells. point mutations help map amino acid residues critical for proper molecular folding and processing, with subsequent conversion of the serpin as a substrate, 158 determination of target protease specificity, 28, 159, 160 or spontaneous multimerization. 89, 155, 161 hence, many different mutations can lead to dysfunctional c1-inh, as recently reviewed. 121, 162 strategies for mutation analyses of the c1nh gene. molecular genetic analysis of c1nh gene anomalies in patients serves as a supplementary diagnostic tool for accurate diagnosis at the molecular level. it contributes to the understanding of dna mutagenesis processes, protein folding and processing, and the structure-function relationships of the c1-inh serpin. 85, 153, 154, 163 such studies may also help the collection of population distribution data, potentially deepening understanding of the relationship between de novo mutation formation and distinct, independent founder effects in different geographical locations. c1-inh deficiency is heterogeneous at the gene level and is caused by subtle changes affecting 1 or several nucleotides, large deletions, or duplications. 84, 153, 164 this heterogeneity prompted the authors to develop suitable methods for the detection not only of mutations previously identified in probands but also of as yet unknown mutations. de novo c1nh mutations, including exon deletions, account for nearly 25% of cases of angioedema. 154 this finding has implications relevant to the genetic epidemiology of and genetic counseling for this disease. the following sections describe the principles, advantages, critical parameters, and drawbacks of some technical strategies, including successful developments in the detection of c1nh gene anomalies. scanning methods for point mutations or small deletions or insertions. methods based on heteroduplex dna and single-strand conformation analyses are of proven efficiency in the screening of large segments of genomic or complementary dna. 165, 166 specific pcr amplification is first performed on exons and flanking intronic sequences or cdna. any variation detected is then sequenced to characterize the molecular change. in the following text, whenever possible, methods are accompanied by citations for reviews describing their advantages and limits. chemical cleavage of mismatches (ccm) technology is based on selective reactions of mismatched thymine and cytosine with oso 4 and hydroxylamine, respectively. the modified mismatched bases are subjected to piperidine cleavage reactions, and the resulting fragments are separated and identified by gel electrophoresis. 167 this method is, in principle, well suited to detect mutations independent of the length and sequence composition of the examined region and capable of detecting nearly all single-base mismatches. it has been successfully developed in a large number of studies (reviewed in ellis et al 168 ) , including those of the c1nh gene to detect pathogenic mutations and analyze its polymorphism. 169 even if all mutations could potentially be detected, the efficiency of chemical modification and cleavage of the ccm depends on type of the mispairing and the stability of the adjacent sequences. in consequence, some mismatches are poorly cleaved, with their mutations subsequently undetectable via ccm. 170 the need for a method suited to identifying most anomalies in the c1nh gene, including point mutations and short and medium-sized deletions or insertions, prompted some groups to take advantage of ccm and fluorescent probes with the development of fluorescenceassisted mismatch analysis. 154, 171, 172 denaturing hplc (dhplc) uses an alkylated nonporous poly(styrene-divinylbenzene) matrix and an amphiphilic ion gradient enabling separation of homoduplexes and heteroduplexes by means of ion-pair reversephase liquid chromatography (reviewed in xiao and oefner 173 ) . illustrative examples of its application in c1nh gene studies are given in fig 11. the most important advantage of the dhplc method is its easy automation by using a mechanical sampler. denaturing gradient gel electrophoresis is based on the migration of double-stranded dna molecules through polyacrylamide gels containing linearly increasing concentrations of a denaturing agent (reviewed in fodde and losekoot 174 ). c1nh-specific primers have been designed to amplify genomic dna segments ranging from 171 to 408 bp. 175 the amplified fragments identified as forming heteroduplexes were directly sequenced. single-stranded conformational analysis (ssca) is an electrophoretic method using a nondenaturing gel, in which the mobility of heat-denatured single strands is dependent on their folding according to individual secondary structure formation. since its introduction in 1989, it has gained popularity for its technical simplicity, low cost, and high sensitivity sufficient to detect most mutations (reviewed in nataraj et al 176 ) . the method has been used in c1nh gene studies 84, 177, 178 (fig 12) . analysis of dna rearrangements. because large genomic rearrangements account for nearly 20% of the total spectrum of the observed changes (reviewed in tosi 153 ), methods adapted to analyzing dna rearrangements are required every time the aforementioned strategies are unsuccessful. southern blot analysis. southern blot analysis was first used for c1nh gene analysis by 2 groups who compared dna from multiple members of families with hae against dna from unrelated patients, yielding the first indication that a defective structural gene was responsible for the disease. 179, 180 digestion of genomic dna with bcli can detect most deletion/insertion boundaries, because the 17-kb-long c1nh gene lies within a 20-kb bcli fragment (fig 13) . to localize gene alterations more precisely, other enzymes have been used in single or multiple digestions: bamhi, bglii, ecori, hindiii, saci, sali, pvui, and psti. 150, 181 quantitative exon multiplex pcr. southern analyses are difficult to set up and time consuming. to ensure complete molecular characterization of c1-inh deficien-cies, a fluorescent multiplex assay has been constructed to amplify 5 exons of c1nh and 1 exon of the brca1 gene simultaneously. 182 in brief, after c1nh exon amplification, the fluorescence intensities of c1nh exons are compared quantitatively with those of a control exon (eg, brca1 exon 5) under conditions in which template concentration is rate limiting. the method has been further validated in exploring large deletions and insertions. 183 reliable estimates of relative gene dosage can be obtained by comparing peak levels in the test dna with those of appropriate controls in deletion/insertion situations 182 (fig 14) . c1nh promoter sequence analysis. the promoter of the c1nh gene is unusual because it contains no tata sequence, but instead contains a tdt-like initiator element at nucleotides 23 to 15 and a polypurine-polypyrimidine tract between nucleotides 217 and 245. 184 only a few mutations have been reported to affect promoter sequence transcriptional activity, and among them, an interesting promoter variant (a c-to-t transition at position 2103) was found in an exceptional family with recessive transmission of the disease. 172 however, subsequent transcriptional alteration remains to be demonstrated. these anomalies have been successfully detected via mismatch scanning (fluorescence-assisted mismatch analysis) in reactions with external and internal primers specific for the promoter sequence. sequencing and expression of sequence data. the most widespread, gold standard technique for mutation detection is direct sequencing. this technique is now commonly available with a high degree of automation and a parallel decrease in labor and cost. the principle is based on dideoxy chain termination reactions using fluorescent dideoxy nucleotides followed by automated gel or capillary electrophoresis. direct sequencing identifies the complete nucleotide sequence of a selected and pcr-amplified dna fragment and detects heterozygosity. the resulting data must be carefully interpreted. any newly discovered missense mutation has to meet the following criteria: (1) the complete coding sequence and splice sites of the c1nh gene were screened/sequenced; (2) the new mutation is not present in 100 general population chromosomes; and (3) the new mutation segregates with the disease, ie, affected members carry the mutations whereas healthy relatives do not. novel interactive, locus-specific mutation database of the c1nh gene. published c1nh gene mutations were periodically summarized in reviews, 85, 153 but such collections became quickly outdated when several new studies described a multitude of novel mutations. 154, 175, 178, 185, 186 c1nh gene mutations are also represented in large universal databases (omim 606860; human gene mutation database 119041), but these databases update their contents infrequently, with poor interactions and heavy requests. one of the goals of the european concerted action (prehaeat) is to perform systematic analyses of c1nh gene mutations in several laboratories for structure-function relationships and consequence on disease expression. to achieve this goal, a mutation database (http://hae.biomembrane.hu) was created with the following purposes: (1) to help the comprehensive collection of information on genetic alterations of the c1nh gene, (2) to create a database in which data can be searched and compared, and (3) to provide additional help in deciding whether a new mutation segregates with the disease. c1-inh serpin function defect: contributions from understanding c1nh mutations. the need to ascertain the consequence of c1nh mutation on c1-inh expression and function prompted investigators to develop experimental systems adapted to both transcription and protein expression studies. patient peripheral blood monocytes and fibroblasts were successfully developed to study c1-inh synthesis in pathological conditions. 156, [187] [188] [189] it then became interesting to correlate c1nh gene anomalies with transcription and translation defects, specifically with reference to serpin function, without the need for patient cells. pathological mutations can now be introduced in c1-inh cdna expression systems and transfected into cell lines suitable for biosynthesis experiments. 91, 155, [190] [191] [192] [193] serpin function is routinely assessed in plasma samples (see varga et al in the following laboratory diagnosis section). some type ii mutant c1-inh proteins have been described with respect to their atypical interactions with the target protease. 161 a more complete model of nonfunctional c1-inh mutants can yield insights into the c1-inh inhibitory mechanism and aid in the development of a relevant dynamic 3-dimensional model of the c1-inh molecule. 162 knowledge of these type ii mutant c1-inh proteins allows every mutation to be associated with its corresponding serpin control failure, as proposed in a 3category classification scheme 162 : class i includes mutations that lead to altered exposure of the active site, with consequences on protease specificity (denoted § in table iv) ; class ii mutations convert c1-inh protein into a substrate, with subsequent inefficient protease trapping (denoted à and k in table iv) ; and class iii encompasses mutations with a spontaneous insertion of the reactive loop, either into the same molecule or another molecule entirely (denoted k and { in table iv) . finally, as posited by cumming et al, 183 it is important to correlate mutations with disease expression. however, on the basis of most clinical data, disease expression cannot be attributed to specific mutant proteins. variable clinical presentation is thought to result from genetic or nongenetic elements distinct from the c1nh gene. in laboratory practice. every index case characterized by impaired c1-inh serpin function and ruled not to be of an acquired origin can be submitted to a genetic analysis. to ensure an economical and reliable strategy, an algorithm is proposed in fig 15. currently, to be exhaustive, the conjunction of scanning methods and southern hybridization or exon multiplex technology is required to detect most mispaired or unpaired sequences and rearrangements, respectively. every nucleotide change detected from the index case has to be analyzed in the family members, keeping in mind that de novo mutations are not uncommon. as stated, and in particular when detected in introns, every new mutation without a known significance has to be distinguished from a polymorphism in the c1nh gene after establishing its absence in the relevant healthy population (from at least 100 chromosomes) and its segregation with the disease. the interpretation of biochemical data on c1-inh serpin function and of c1nh gene anomalies is of great importance to establish a better knowledge of the pathogenic effects of c1nh gene mutations. moreover, the possibility of c1-inh polymorphisms affecting disease expression should be considered. 183 generating a c1-inh molecular model should be of great help in the understanding and treatment of hae type ii and discerning which c1-inh residues are essential for efficient control of all its target proteases. 162 the uniform resource locators for data related to this section are as follows: from a pathogenetic perspective, the form of angioedema most extensively studied is that resulting from c1-inh deficiency. nonetheless, the pathogenesis of this clinical condition remains incompletely understood. in the following section, hack briefly reviews the biochemistry and biology of c1-inh as well as the biochemical changes that occur in persons with c1-inh deficiency during both attacks and symptom-free periods. on the basis of these reviewed data, hack then discusses the potential pathogenesis of c1-inh deficiency angioedema. often, hae results from a deficiency of c1-inh. an interesting feature, revisited in the previous clinical manifestation and diagnosis section, is the great variance of the disease's clinical course. some patients are virtually free of attacks even in the absence of treatment, whereas others, despite therapy, have attacks nearly every week. angioedema attacks result from the extravasation of fluid caused by increased vasopermeability. unlike angioedema of allergic origins, angioedema caused by hae typically does not itch. pain is often not a primary feature of such angioedema, although it may manifest secondary to the localization of the angioedema, eg, severe pain caused by obstruction of the bowel lumen. in spite of decades of research, the pathogenesis of hae attacks is still unclear. c1-inh biochemistry and biology. c1-inh belongs to the family of serine protease inhibitors, or serpins, which also includes proteins such as a1-antitrypsin and antithrombin iii [194] [195] [196] [197] (fig 16) . most serpins have only 1 or a few target proteases, but c1-inh is exceptional in this respect. it is the major inhibitor of several proteases, including (1) c1s and c1r-two serine proteases that, together with c1q, constitute the c1 complex of the classical pathway of complement, (2) the mannan-binding lectin (mbl)-associated serine proteases or masps, and (3) the contact system proteases factor xia, factor xiia, and kallikrein. 198 in addition, c1-inh may interact with several proteases such as thrombin, 199 plasmin, 200 and tissue-type plasminogen activator (tpa). 201 thus, c1-inh regulates the activity of several inflammatory, clotting, and fibrinolytic proteases and is therefore an inhibitor of several pathways of inflammation. 198 in comparison with other serpins, c1-inh is a relatively weak inhibitor, approximately 100 times less effective than other serpins. however, the inhibitory activity of c1-inh toward c1r, c1s, and factor xia can be remarkably potentiated by heparin and other glycosaminoglycans. 202, 203 the inhibitory activity of c1-inh to factor xiia and kallikrein cannot be potentiated. dextran sulfate is the most effective glycosaminoglycan, enhancing c1-inh function 130-fold in vitro and 60-fold in plasma. however, the in vivo effect of dextran sulfate is transient, probably because of dissociation of the dextran sulfate-c1-inh complex and the subsequent rapid clearance of dextran sulfate. 204 mature c1-inh consists of 478 amino acids and is heavily glycosylated (approximately 30% by weight). although on sds-page gels it migrates with an apparent molecular weight of 104 kd, its calculated molecular weight is 76 kd. the c1-inh molecule has at least 13 glycosylation sites 205 and possibly 20 glycosylation sites, part of them linked to threonine residues. most carbohydrate groups are located at the n-terminal region; their function is largely unknown. the c1-inh molecule is composed of an n-terminal domain of 113 amino acids and a serpin domain of 365 amino acids. although the function of the n-terminal domain is unknown, recent evidence suggests that it helps to stabilize the central bsheet by forming 2 disulfide bridges linking cysteine 101 to cysteine 406, and cysteine 108 to cysteine 183. 206 the structure of the serpin domain is homologous to that of other serpins and is essential for the inhibitory capacity of the molecule. recently, a novel 3-dimensional model of c1-inh was proposed on the basis of the crystal structure of 4 other inhibitory serpins. 162 similar to other serpins, c1-inh inhibits proteases by binding to their active site via its reactive center. this reaction follows the equation in the first step, a reversible complex is formed between the target protease and c1-inh; c1-inh then undergoes conformational changes caused by insertion of part of its reactive center loop into a 5-stranded b-sheet. this results in the formation of modified c1-inh (c1-inh* in the equation) that is tightly bound to the target protease (step 2). mutations of the residues at the p14, p12, and p10 positions in the reactive center interfere with efficient loop insertion and cause the reaction to proceed to step 3. in that case, c1-inh has become a substrate for its target proteases. such mutations have been found in some patients with hae-ii (characterized by the presence of dysfunctional c1-inh protein). complexes between modified c1-inh* and proteases are very stable and only very slowly dissociate into an inactive, modified c1-inh (with changed conformation caused by a completed insertion of the cleaved active site loop into the 5-stranded b-sheet) and an active target protease (step 3 in the equation). most of the stable complexes will be removed from the circulation before dissociation via receptors specific for complexed serpins. this mechanism ensures efficient removal of biologically active proteases. c1-inh is an acute phase protein, of which plasma concentrations may increase as much as 2-fold during uncomplicated infections. woo et al 207 reported that the synthetic rate of c1-inh may increase as much as 2.5 times the normal rate in patients with rheumatoid arthritis. cytokines such as interferon-g stimulate the synthesis of c1-inh. 208 in normal volunteers, the fractional catabolic rate of c1-inh is 2.5% of the plasma pool per hour, which results in an apparent plasma half-life time of clearance of about 28 hours. 90 the half-life time of clearance of human c1-inh in rabbits is comparable, at 26 hours, 209 whereas that in rats is considerably shorter, at about 4.5 hours. 210 the apparent half-life time of clearance is markedly longer in patients with hae, in whom it may exceed 48 hours, as determined by assessing the course of plasma concentrations after the intravenous administration of exogenous c1-inh. 211 however, at low plasma concentrations of c1-inh, such as occur in untreated patients with hae, c1 is autoactivated 212 and consumes functional c1-inh. at higher c1-inh concentrations such as occur after the administration of exogenous c1-inh, this autoactivation is inhibited, leading to a decreased consumption of c1-inh. hence, monitoring of plasma concentrations after a therapeutic dose of c1-inh may lead to an overestimation of the half-life caused by inhibition of consumption of endogenous c1-inh. removal of sialic acids greatly enhances the clearance of c1-inh from the circulation, yielding an apparent half-life of 3 to 5 minutes, 209 presumably by binding to asialoglycoprotein receptors in the liver. subsequent removal of penultimate galactosyl residues largely restores the clearance rate to a value similar to that of normal c1-inh. 209 activation of cascade systems in hae. as mentioned, c1-inh regulates the activation of at least 3 different so-called plasma cascade systems, ie, the classical and mbl pathways of complement via its effect on c1r, c1s, and the masps; the contact system via its effects on kallikrein and activated factor xii (factor xiia); and the intrinsic pathway of coagulation via its effect on activated factor xi (factor xia). 213 in addition, it may regulate coagulation to some extent via its effects on thrombin, and it may regulate fibrinolysis through its effect on tpa and plasmin. except for the mbl pathway, there is evidence for activation of all of these systems in patients with hae. the classical pathway of complement consists of c1q, c1r, and c1s, together forming the c1 complex; c4 and c2; as well as the inhibitors c4 binding protein and factor i. typically, patients with hae have low c4 and c2 but normal c3 values even during attack-free periods. low c4 and c2 result from uncontrolled activation of activated c1 because the concentration of activation products of c4, c4b/c, or c4d is elevated. 213, 214 activation of c3 is typically low or absent, probably because of efficient control of activation at the level of c4 by c4 binding protein and factor i. this continuous activation of the classical pathway is attributed to enhanced autoactivation of c1, because this autoactivation process is dependent on functional c1-inh values. 212 however, it is now clear that the classic pathway of complement may be activated by apoptotic cells through various mechanisms. [215] [216] [217] hence, one could postulate that the excessive activation of the classic pathway in untreated patients with hae results from activation of this pathway by cell debris. indeed, familian and hack have observed increased amounts of complement activation products in patients with hae (unpublished data, november 2002), reflecting activation via the penetraxin serum amyloid p component, 1 of the proteins that can bind to apoptotic cells. 218 ongoing activation of the classical pathway in untreated patients continuously produces activated c2. the contact system of coagulation consists of the proteins factor xii (also known as hageman factor), prekallikrein, and high molecular weight kininogen. 219 the system is activated in vitro on contact with negatively charged surfaces, but the nature of potential activators in vivo is currently unknown. activation of the system yields factor xiia and kallikrein. c1-inh inhibits both proteases. in addition, kallikrein can also be inhibited to some extent by a2-macroglobulin. kallikrein can cleave hk, yielding cleaved hk and bradykinin. the latter nonapeptide is a potent inducer of vasodilation and increased vasopermeability. activation of this system is often assessed by measuring factor xiia-c1-inh and kallikrein-c1-inh complexes, 220 although these parameters are unsuitable to assess activation in hae. alternatively, activation can be measured by detecting increased amounts of cleaved hk or decreased antigenic quantities of the contact system proteins. activation of the contact system in hae typically occurs during attacks and is hardly detectable during attack-free periods. 221 in particular, increased amounts of cleaved hk are found in most, if not all, patients with attacks. 17, 222, 223 remarkably, nearly all hk is often cleaved during attacks, such that native hk is hardly detectable. it is questionable whether all hk cleaved during an attack gives rise to bradykinin, given its extremely potent activity as a vasodilator. bradykinin is known to cause a significant drop in blood pressure even at low concentrations, yet hypotension is not characteristic for hae. however, there are no data supporting the cleavage of hk via other degradation pathways during hae attacks such as would generate minimal or no bradykinin. it should be noted that the measurement of bradykinin in plasma or other body fluids is hampered by its rapid degradation by carboxypeptidases and is only possible using samples collected in special inhibitor cocktails to prevent enzymatic degradation. few studies have attempted to measure bradykinin according to such protocols in hae, but these studies have shown increased bradykinin generation during attacks and not during symptom-free periods, particularly when samples are drawn from the site of the edematous swelling. 13, 14, 27 c1-inh is a major inhibitor of factor xia of the intrinsic pathway of the intrinsic pathway of coagulation 143, 224 and also inhibits thrombin, 199 although inhibition of this latter enzyme is likely physiologically inconsequential. the role of factor xi in coagulation is not yet resolved. originally, coagulation was considered to proceed via at least 2 different pathways, the intrinsic and the extrinsic, merging at the level of factor x into a common pathway of thrombin generation. the extrinsic pathway is initiated by exposure of factor vii to tissue factor, whereas the intrinsic pathway is triggered by factor xii activation, eg, upon contact of blood with negatively charged surfaces such as glass. in this view, activation of factor xii led to thrombin generation via activation of factor xi, which then activated factor ix, which together with factor viiia activated factor x, which then together with factor va activated prothrombin. this traditional distinction between intrinsic and extrinsic pathway has been left behind, and the intrinsic pathway is now considered to amplify extrinsic pathway activation in that factor ix amplifies factor viia-induced activation of factor x, whereas factor xi, activated by thrombin, activates additional factor ix. according to this revised scheme of coagulation, the activation of factor xi is no longer mediated solely by factor xiia but also may occur with significant thrombin generation. in the traditional scheme of coagulation, c1-inh is expected to have a major effect on intrinsic pathway activation because it blocks the activity of factor xiia, kallikrein, and factor xia, whereas according to the current understanding, c1-inh has only a limited effect on coagulation, with its main effect the inhibition of factor xia-mediated amplification of factor ix activation. clinical observations in hae do not support that c1-inh deficiency is a major risk for thromboembolic disease, although some generation of thrombin may occur during attacks. 222 this mild effect of c1-inh is easily understood through the revised scheme of coagulation. although c1-inh inhibits plasmin and tpa in vitro, and to some extent in vivo, 201 this inhibition is weak. nonetheless, significant plasmin formation, as measured by the formation of plasmin-a2-antiplasmin complexes, occurs during attacks in patients with hae but hardly at all during symptom-free periods. at present there is no evidence for tpa or urokinase-type plasminogen activator involvement in the formation of plasmin during hae attacks. as such, this generation most likely results from factor xii activation. although debated in the literature, convincing in vivo evidence exists to support the activation of plasminogen in human beings by factor xiia. 225, 226 angioedema attack pathogenesis: mediators of angioedema. angioedema is a frequently experienced side effect for patients receiving ace inhibitors. this angioedema does not itch, is not associated with urticarial lesions, and does not respond at all to corticosteroids or antihistamines, features also typical of angioedema associated with c1-inh deficiency. ace inhibitors slow down the processing of c-terminal arginine residues of various vasoactive peptides such as angiotensin and bradykinin, thus prolonging the biological activity of these compounds. the association between ace inhibitors and angioedema is a strong argument that this condition may result from exposure of the endothelium to increased levels of vasoactive peptides. considering the similarities between ace inhibitor-related angioedema and that caused by c1-inh deficiency, the latter form also likely results from enhanced exposure of the endothelium to vasoactive peptides. studies in a few patients indicate that bradykinin may be the vasoactive peptide involved. 13, 14, 27 considering that contact activation in hae mainly, if not exclusively, occurs during attacks, and that activation of this system results in the liberation of bradykinin from hk, most investigators currently believe that c1-inh deficiency angioedema results from the local generation of bradykinin caused by uncontrolled proteolytic activity of factor xiia and kallikrein (fig 17, bottom) . 15 kallikrein has 2 important effects in contact activation: it (1) amplifies factor xii activation via so-called reciprocal activation, and (2) releases bradykinin from hk. this mechanism of contact activation and bradykinin generation as the basis for angioedema is supported by observations in the murine model of c1-inh deficiency, because the enhanced vasopermeability in this model can be blocked with bradykinin receptor antagonists. 9 according to this mechanism, inhibiting kallikrein or blockading bradykinin receptors (bkr-2) would be therapeutic options; as discussed in the treatment section of this supplement, both are currently being investigated in clinical trials. activation of the contact system holds several attractions as an explanation for angioedema attacks. first, fig 17 . different pathways of kinin generation in c1-inhibitor deficiency state. low c1-inhibitor leads to uncontrolled activation of factor xii, which generates kallikrein and plasmin. kallikrein liberates bradykinin from hk, whereas plasmin cleaves off c2-kinin from activated c2. activated c2 is continuously produced during baseline complement activation, which is increased as a result of insufficient control of autoactivation of c1 caused by c1-inh deficiency. factor xii activation can be triggered by chemical activators and may well occur in bouts. second, bradykinin is a potent vasoactive peptide that can enhance vasopermeability. third, ace inhibitors have been shown to result in the delayed breakdown of bradykinin, and part of their hypotensive effects are actually blocked by bradykinin receptor antagonists. 227 however, some strong arguments exist against this mechanism as the sole explanation for angioedema. first, bradykinin is a strong vasodilating agent and among the most hypotensive agents known, whereas hae attacks are not known to be associated with clear hypotensive reactions. nonetheless, one could argue that most bradykinin is generated locally, at the site of edema formation, rather than in the circulation. second, the local injection of bradykinin results in a strong pain sensation, 228 whereas primary, localized pain is not typical for hae attacks. third, contact activation cannot explain the efficacy of antifibrinolytic agents such as tranexamic acid. it has been suggested that these agents are efficacious by reducing plasmin formation during attacks, which would lead to the reduced consumption of functional c1-inh by plasmin. in spite of this, it is difficult to understand how active plasmin would be capable of mediating c1-inh consumption, because it is rapidly inhibited by a potent plasmin inhibitor, ie, a2-antiplasmin. moreover, in most patients with hae, rather than being elevated, the concentration of cleaved c1-inh (free c1-inh* in the equation) is fairly low. finally, although vasopermeability seems to be enhanced and dependent on bradykinin formation in c1-inh-deficient mice, these permeability changes do not appear to occur in bouts and are otherwise atypical for angioedema. for all of these reasons, other mechanisms may be involved in hae attacks. some decades ago, another vasoactive peptide was thought to be involved in angioedema formation. this peptide can be generated by cleavage of the c2b fragment, but not from native c2, by plasmin, and is therefore called c2-kinin. 229 this c-terminal fragment of c2b can be generated by c1s or plasmin. mediation of attacks by this fragment requires activation of multiple plasma cascade systems: (1) complement, to generate activated c2; (2) the contact system, to generate factor xiia; and (3) the fibrinolytic system, to generate the c2-kinin generating enzyme, plasmin. according to this mechanism, attacks are triggered by factor xii activation that then yields plasmin, which in turn cleaves the c2-kinin sequence from c2b (fig 17, top) . the attractive aspects of this controversial mechanism include its explanation of the efficacy of antifibrinolytic agents. furthermore, one may postulate that c2-kinin does not induce vasodilation, explaining why hae attacks are not associated with hypotension and may not induce sensations of pain, and why angioedema in general does not hurt. nevertheless, it should be noted that the biologic properties of c2-kinin are not well established, and thus, it is not truly known whether this kinin's properties are consistent with the description. moreover, receptors for this kinin have not been identified, and it is therefore unknown whether their distribution correlates positively with the localization of attacks. in addition, there are no data regarding concentrations of c2-kinin in patients with hae. finally, if correct, the c2-kinin mechanism provides no explanation for the profound cleavage of hk and the apparent lack of biological activity of bradykinin. these mechanisms are not necessarily exclusive and may be simultaneously involved in attacks. furthermore, the involvement of other, as yet unknown vasoactive peptides in the pathogenesis of attacks cannot be excluded. triggers of angioedema attacks. clinically, hae attacks are often triggered by trauma or emotional stress. attacks of hae are specifically associated with activation of the fibrinolytic and contact systems, whereas complement activation occurs continuously, independent of clinical symptoms. activation of the fibrinolytic system can occur via 3 plasminogen activators: tpa, urokinasetype plasminogen activator, and factor xii. there is no evidence that the former 2 are involved in plasmin formation during attacks; hence, it likely results from factor xii activation. thus, attacks are triggered by activation of factor xii. though factor xii (hageman factor) was discovered in the 1950s, physiological activators of this contact system protein have not yet been identified. in vitro, factor xii is easily activated by glass and other negatively charged compounds such as dextran sulfate. obviously, these compounds are not relevant as activators of the contact system in vivo. the author writes that recently, his group has studied the generation of thrombin by phospholipid microparticles released in vivo from activated platelets and damaged cells. 230 by using various specific mabs against tissue factor, factor vii, factor xi, and factor xii, they noticed that a small but significant part of thrombin generation by microparticles purified from human plasma was dependent on factor xii. indeed, earlier studies have clearly indicated that factor xii can be activated by phospholipids in vitro. 231, 232 apparently, factor xii can be activated by phospholipid microparticles in vivo. damaged or apoptotic cells generate such particles, and one may therefore postulate that these particles constitute triggers for contact activation. under normal conditions, ie, at normal concentrations of c1-inh, this weak activation will never yield substantial contact activation. however, in the absence of sufficient amounts of this inhibitor, the system may become profoundly activated by even small amounts of microparticles via the principle of reciprocal activation (factor xii activates prekallikrein into kallikrein, which in turn activates additional factor xii, and so on). microparticles and damaged cells are likely generated during trauma, which may explain why this is a trigger for angioedema attacks. emotional stress as a trigger for attacks is more difficult to understand, although one may speculate that it predisposes patients to microtrauma. support for this hypothesis is that occasional patients have been described who, in spite of acquired c1-inh deficiency, are free of symptoms. two such patients had detectable antibodies against phospholipids. 125 these antibodies might interfere with the binding of factor xii to phospholipid microparticles or damaged cells. further studies are needed to provide evidence toward this hypothesis of hae attack generation. attack frequency. the angioedema attack rate in persons with c1-inh deficiency may vary widely, ranging from no attacks to an attack every week. the molecular mechanism for the variation in attack rate is unknown. in the previous paragraph, 2 mechanisms to explain the trigger for attacks are discussed. both mechanisms claim that attacks are initiated by activation of factor xii. therefore, one may speculate that the frequency of attacks is strongly linked to an individual's tendency toward factor xii activation. if true, attack rates might be influenced by the concentration of factor xii as well as by the amount of factors that compete with factor xii for binding to its activator molecules. as discussed, in the case of phospholipid microparticles, antiphospholipid bodies may prevent factor xii from binding to the particles, conferring protection against the activation of factor xii. older literature has revealed several proteins capable of competition with factor xii for binding to artificial activators. 233, 234 the concentrations of these competing proteins might well determine whether factor xii may become activated on exposure to an activator and therefore influence hae attack frequency. future research will test this hypothesis. in summary. angioedema attacks caused by c1-inh deficiency are likely mediated by the excessive release of vasoactive peptides such as bradykinin and c2-kinin. this release is initiated by the uncontrolled activation of factor xii, leading to kallikrein and plasmin formation. the trigger for attacks may be phospholipids, released from damaged cells, that activate factor xii. factor xii's ease of activation may be determined by its concentration as well as the concentration of proteins that compete with it for binding to activators, and may explain the variable frequency of attacks in patients. others, assuming bradykinin to be the principal mediator of nonallergic angioedema attacks, have begun work toward bridging the gap between c1-inh deficiency and local angioedema generation. bolstered by the increasing evidence that ace inhibitors [235] [236] [237] can cause aae in a small percentage of individuals, investigators have sought to determine whether-independent of c1nh gene sequence-an increased liberation of bradykinin, a decrease in its catabolism, or a combination of the 2 may contribute to the angioedema experienced in hae, aae, and related disorders. phenotypic characteristics. mating of heterozygousdeficient mice produced the expected 1:2:1 mendelian ratio of c1-inh 1/1 :c1-inh 1/2 :c1-inh 2/2 mice. as observed in human heterozygotes, c1-inh expression was less than 50% of normal in heterozygous mice; homozygous c1-inh knockouts did not produce detectable plasma concentrations of c1-inh. in male but not in female mice, carrying the defective c1-inh gene correlated with a slight but statistically significant decrease in weight. the growth of both heterozygous-deficient and homozygous-deficient mice appeared normal, with no more newborn deaths observed than in wild-type littermates. limited analysis of serum c4 concentration in heterozygous-deficient and homozygous-deficient mice revealed that homozygotes had 38% to 59% as much c4 as their wild-type littermates, constituting a significant mean difference from wild-type; c4 in heterozygotes was more variable, ranging from 49% to 120% of wild-type. the group also reported that total hemolytic complement activity in the sera of null homozygotes was reduced compared with wild-type. although the c1-inh 1/2 and c1-inh 2/2 mice were not observed to have spontaneous attacks of skin edema, 2 heterozygotes and 3 homozygous knockouts developed spontaneous abdominal distension secondary to intestinal wall edema. because these mice were euthanized, it is unclear whether these episodes would have spontaneously resolved. no such episodes of abdominal edema were observed in wild-type littermates. measuring increased vascular permeability. evans blue dye was used to visualize differences in vascular permeability between the 3 types of mice. within 15 to 30 minutes of injection into the tail vein of wild-type mice, the dye caused a faint blue coloration, particularly visible around the nose and eyes and on the feet. after similar injections, c1-inh 1/2 and c1-inh 2/2 mice showed a much more saturated blue color in these areas, with homozygous null mice producing the most saturated blue. these differences could be quantitated spectrophotometrically, as shown in fig 18. the mice were euthanized and the dye extracted from the area in question by using formamide, then read at an absorbance of 600 nm. han et al 9 noted that unlike in the feet and face, there were no apparent vascular permeability differences in the ears of mice of all 3 genotypes after evans blue injection. however, when mustard oil, a local irritant, was applied to the ears of injected mice, a pronounced permeability increase correlating with c1nh mutation could be spectrophotometrically demonstrated. this increase could be reversed if the mice were given human c1-inh concentrate. intestinal vascular permeability was also investigated in a few mice; although of borderline statistical significance because of the sample size, intestinal permeability was also increased in c1-inh 2/2 versus wild-type mice (fig 19) . bradykinin type 2 receptor assays. groups of heterozygous and homozygous c1nh-deficient mice were treated with the plasma kallikrein inhibitor dx-88, the bradykinin receptor 2 antagonist hoe140 (icatibant; jerini, berlin, germany), or the ace inhibitor captopril (figs 19 and 20 ). both interference with plasma kallikrein (and thus contact system-mediated bradykinin generation) and disruption of the bradykinin receptor 2 interaction with its ligand decreased vascular permeability. treatment with the bradykinin receptor 1 antagonist des-arg 9 ,[leu 8 ]-bradykinin had no effect. treatment with captopril dramatically increased vascular permeability. when c1nh knockout mice were crossed with bradykinin receptor 2 knockout mice, the resulting offspring were overtly phenotypically normal. as shown in fig 20, absence of the bradykinin receptor 2 was able to reverse the vascular permeability observed in both c1-inh 1/2 and c1-inh 2/2 mice. in conclusion, although this mouse model does not exclude the possibility of other contributing mechanisms, it strongly supports the hypothesis that bradykinin mediates angioedema. bradykinin-mediated angioedema? (kayla williams, bs, ma, mfa, cambridge, mass) in this section, williams reviews recent information on the role of bradykinin in both drug-related angioedema and hae and explores the contributing factors that may influence the generation of bradykinin-mediated angioedema. drug-related angioedema: a growing problem. as the use of ace inhibitors, at2 receptor antagonists (at2 blockers), and related, next-generation drugs increases, it is projected that the incidence of drug-related angioedema will also increase. 235, 239 although angioedema is a relatively rare adverse effect of such medications, the medications are commonly and increasingly prescribed. the incidence of angioedema during ace inhibitor treatment has been estimated at between 1 and 7 per 1000 patients. 240 these cases of drug-induced angioedema are interesting because, unlike other forms of nonallergic angioedema, the role of bradykinin in ace inhibitor-related angioedema is fairly well established. also of note and concern are reported racial differences in the response to this vasoactive peptide. bradykinin, race, and ace. in 1996, gainer et al published the results of a clinical trial evaluating wheal response to bradykinin among african americans and white americans. 241 in this randomized, double-blind study, increased bradykinin dose, hypertension, and african american race each correlated with an increased wheal response to bradykinin. further investigations by the same group compared the effects of short-term ace inhibition in normotensive and hypertensive individuals of both races. in this study, the hypotensive effects of the ace inhibitor captopril were shown to be greatly reduced in all subjects by concomitant administration of the bradykinin receptor 2 antagonist icatibant. 227 icatibant was also shown to significantly alter the change in plasma renin activity in response to ace inhibition. these results not only confirmed the importance of bradykinin in the short-term effects of ace inhibitors but also suggested that bradykinin contributes to the effects of ace inhibition on the renin-angiotensin system. in addition, when considered together, these results suggest that clinicians prescribing ace inhibitors for patients of afro-caribbean descent should be especially aware of the possibility for drug-related angioedema, a warning beginning to be supported by case analysis. 242, 243 further antihypertensive-related angioedema. initially, at2 blockers were thought to be a safer alternative for patients who had angioedema associated with ace inhibitors. unlike ace inhibitors, at2 blockers have not been shown to increase bradykinin concentrations. nonetheless, further case reports documented angioedema after therapy with at2 blockers. 137, 244 in a survey of the literature, 32% of these patients also had similar episodes in conjunction with ace inhibitor treatment. 245 additional documented cases of at2 blocker-associated angioedema threw bradykinin's role in the pathogenesis of such episodes into doubt 246 because increased concentrations of bradykinin are not associated with at2 blocker use. 245 however, very recent work has shown that increased concentrations of at2, such as would occur during a blockade of at2 receptors, are associated with an increase in the expression of bradykinin receptors that may be almost 3-fold. 247 thus, increased opportunities for bradykinin binding may explain angioedema in the absence of extra bradykinin. the mediator for this receptor crosstalk effect has been shown to be the at2 receptor 1. 247 (presumably, this phenomenon could occur in the presence of at2 receptor 1 blockers only in the absence of a total blockade.) meanwhile, the new drug application for the vasopeptidase inhibitor omapatrilat, a dual inhibitor of both ace and neutral endopeptidase (nep), was voluntarily withdrawn when the food and drug administration raised questions regarding the comparative incidence of angioedema reported within the new drug application database. in response to these questions, a large (25,000-patient) multinational trial was initiated to compare the efficacy and safety of omapatrilat versus the ace inhibitor enalapril in patients with heart failure. 248 the results of this study have recently been reported. 249 angioedema occurred in 2.17% of subjects receiving omapatrilat compared with 0.68% of subjects receiving an ace inhibitor alone. two of the dual inhibitor-treated subjects had angioedema with airway compromise. an editorial 250 published after the trial results were released but before their publication noted that the rates of angioedema were much higher in african americans receiving the dual inhibitor (5.54%) than in those receiving the ace inhibitor alone (1.62%). of smokers receiving the dual inhibitor, 3.93% had angioedema, versus 0.81% of smokers receiving the ace inhibitor alone. the biologic activity of nep must account for this several-fold increase in angioedema. indeed, among its many possible functions, nep has been shown to metabolize bradykinin to an inactive form. 237 a combination of ace and nep inhibition could thus be expected to prevent efficiently the degradation of bradykinin to inactive metabolites, thereby increasing the risk of angioedema. 251 in certain subgroups with altered responses to bradykinin or altered pathways for its generation or degradation, these effects could be especially pronounced, and such predisposing factors may also be involved in the generation of c1-inh deficiency-related angioedema. implications for c1-inh deficiency angioedema. early investigations have confirmed that bradykinin may be at work in c1-inh deficiency angioedema. the transience of bradykinin has complicated measurements of this nonapeptide; however, several small studies of plasma bradykinin concentrations in patients with angioedema have revealed that high concentrations of this vasoactive peptide occur during edematous attacks. 13, 14, 27 this increase in plasma bradykinin was demonstrated both for patients with hae with c1-inh deficiency and for patients with drug-associated angioedema who had an angioedematous attack in conjunction with ace inhibitor treatment. patients receiving ace inhibitors showed elevated plasma bradykinin concentrations throughout ace inhibitor therapy, both during attacks and during periods of remission. 13 as documented in 1 patient, this increase reverted to normal on discontinuation of ace inhibitor therapy. 14 in contrast, patients with c1-inh deficiency were shown to have high concentrations of bradykinin during their attacks 13 but have been documented to have normal or marginally increased amounts of bradykinin during periods of remission. 14 at least in c1-inh deficiency, bradykinin increases may be highly localized; in 2 previously studied c1-inh-deficient patients, bradykinin concentrations in blood draining from an angioedematous site were 3 and 8 times as high as those measured in systemic circulation. thus, bradykinin appears to be important in various currently identified forms of nonallergic angioedema. following the example put forth in the large-scale trial of antihypertensive agents that showed (1) african descent and, to a lesser extent, (2) smoking to correlate with increased angioedema risk, 250 clinicians and researchers may wish to investigate the interaction of these variables with c1-inh deficiency. bradykinin magnified. even if bradykinin is the major mediator of nonallergic angioedema, many questions regarding the triggering and localization of angioedema attacks remain. why should patients with ace inhibitorinduced angioedema have this angioedema in bouts if bradykinin is constantly elevated? what is different about the small percentage of patients taking ace inhibitors who have angioedema? what regulatory pathways are involved? how, chemically, does one proceed from insufficient functional c1-inh to localized, full-blown angioedema? the past decade of research indicates that the answers may reside in a surprisingly multifactorial system. bradykinin-mediated effects can result from increased amounts of bradykinin, the reduced breakdown of bradykinin to inactive metabolites, the increased conversion of bradykinin to active metabolites, and/or the increased uptake of these active metabolites. each of these mechanisms may be involved in at least 1 form of nonallergic angioedema. pathways for the breakdown of bradykinin are depicted in fig 21. decreased or alternative catabolism. molinaro et al 252 have demonstrated that in human plasma, bradykinin is metabolized mainly by 3 metallopeptidases: ace, app, and kininase i (carboxypeptidase n). of these, ace and app, respectively, are the first and second major pathways of inactivation. kininase i represents a minor pathway unless ace is inhibited, whereupon kininase i transforms bradykinin into its active metabolite des-arg9-bradykinin, which, in turn, is inactivated by app and ace, with app the major inactivating metallopeptidase. 253 nep, as discussed in the context of vasopeptidase inhibition, likely represents another minor pathway. once formed, bradykinin and des-arg 9 -bradykinin are short-lived and thus exert their effects locally at the site of their formation, stimulating b2 and b1 receptors, respectively. 254, 255 during ace or vasopeptidase inhibition, patients with low app activity may be subject to angioedema. in fact, adam et al 96 have found a correlation between low app activity with a history of angioedema among patients receiving ace inhibitors. likewise, in their estrogen-dependent and estrogen-associated inherited angioedema piece earlier in this supplement, binkley and davis propose reduced app activity as a contributing element in estrogen-sensitive inherited angioedema. in addition, they note that estrogen may decrease gene expression of ace. 256, 257 to demonstrate further the delicate balance of enzymes and receptors, it should be noted that mathews et al 258 observed angioedema in a subject with familial serum carboxypeptidase n deficiency. because the subject's sister had an equally depressed serum carboxypeptidase value but no reported episodes of angioedema, 259 it is possible that another defective control mechanism, possibly app, contributed to the angioedema. pathways for bradykinin generation and metabolism. on the endothelium, bradykinin generation likely initiates independent of factor xii; however, once kallikrein is generated, it soon catalyzes factor xii activation, thus amplifying the response. increased anabolism. as hack discussed in the pathogenisis and pathobiology of hae and aae section, c1-inh is known to inhibit not just c1 esterase but also other serine proteases, including factor xii (hageman factor). in addition, c1-inh inhibits the serine protease kallikrein. without the controlling effects of c1-inh, it is possible that both of these serine proteases may contribute to an increased generation of bradykinin. this reaction is thought to occur at the endothelial cell surface. the way in which this unchecked bradykinin generation starts has been debated, with recent evidence supporting the secondary role of factor xii, 260, 261 and placing the conversion of prekallikrein to kallikrein, likely by an enzyme such as prolylcarboxypeptidase, 262, 263 foremost, at least under resting physiologic conditions. it is thought that once kallikrein is present, it catalyzes the activation of factor xii. only in the presence of activated platelets, neutrophils, or lymphocytes can the high local concentration of free zinc ions necessary for factor xii to bind to the endothelial cell surface-and thus initiate bradykinin release-occur. 263 this provides yet another example of how infection or tissue damage might aggravate an existing tendency to angioedema. in vitro, kallikrein and plasmin together have been shown to exert a synergistic effect on the release of kinins from hk. of interest, plasmin's digestion of hk in this model could be inhibited by e-aminocaproic acid. 264 because c1-inh is also known to inhibit plasmin, and antifibrinolytics show at least partial efficacy in the treatment of hae, this enhancing mechanism might well be operative in c1-inh deficiency angioedema. in addition, because angioedema has been reported in patients treated for acute ischemic stroke who received recombinant tissue-type plasminogen activator (rtpa; often concomitantly with an ace inhibitor), molinaro et al 252 undertook an in vitro comparison of human plasma incubated with and without rtpa in the presence of an ace inhibitor. their results showed that rtpa generates bradykinin that is further metabolized to des-arg 9 -bradykinin; this effect could be recreated to a similar extent in the absence of rtpa by the addition of negatively charged glass beads, thus mimicking contact activation. joseph et al 19, 20 have identified yet another possible mechanism active at the endothelial cell level, showing that the cytosol of endothelial cells contains a factor capable of converting prekallikrein to kallikrein in the absence of factor xii. they identified this factor as heat shock protein 90. perhaps this mechanism might account for angioedema attacks that occur in the wake of trauma or infection when ruptured cells spill their contents into local circulation. indeed, increased kallikrein may be a feature of mechanical trauma: an experiment conducted more than 20 years ago demonstrated that mechanically generated blisters in 5 of 5 patients with hae contained active kallikrein that could be inactivated by c1-inh concentrate or antibodies to plasma kallikrein. in contrast, blister fluids from 6 of 8 healthy subjects did not contain detectable amounts of plasma kallikrein. 265 last, these anabolic effects might be created or magnified by hormonal interactions. as noted by binkley and davis, 92 estrogens may increase the expression of factor xii 107,266-268 and prekallikrein. 269 increased uptake. various factors may increase gene expression of the b1 and b2 receptors. not normally expressed, the b1 receptor has a high affinity for kinin metabolites. 270 expression of b1 receptors may be induced during inflammation and tissue damage. 271, 272 although b2 receptors are constitutively expressed, 273 their expression may be increased by estrogens 274 and at2 receptor antagonists. 247 in summary. despite increasing evidence that bradykinin is indeed the major mediator of nonallergic angioedema, this knowledge does not represent a simple answer given the many variables governing bradykinin and its metabolites. however, an understanding of this web of chemical interactions may yield more successful management algorithms for the various types of nonallergic angioedema. this recent knowledge also suggests a place for bradykinin receptor antagonists and kallikrein inhibitors for the control of nonallergic angioedema, especially when it occurs in the presence of functional c1-inh. the current scientific understanding of the triggering of angioedema attacks ranges from the anecdotal to that supported by relatively large and rigorous studies. patients with hae, aae, or related forms of angioedema, especially patients who have angioedema attacks in the absence of a formal diagnosis, pay particular attention to the events that precede their angioedema attacks; however, even in the same patient, these events may vary among attacks. various patients have implicated exposure to cold; mechanical trauma sustained during routine activities such as gardening; prolonged sitting or standing; eating certain foods, such as eggs; exposure to pesticides or chemicals in new fabrics and clothing; infection or illness; or excitement or stress in triggering their attacks (k. williams, unpublished data, august 2003). however, as with other, better studied triggering events, these conditions or activities did not always predictably provoke attacks. ethical concerns and the small patient populations available to would-be investigators prevent the systematic investigation of most patient-reported angioedema attack triggers. those triggers more readily quantifiable via laboratory assays without necessitating patients' increased risk of developing attacks have received the most scientific attention, largely restricting the study of attack triggers to the possible role of hormones. (laurence bouillet, md, cca,* grenoble, france) in the first part of this section, bouillet reviews his group's clinical experience and other reports linking sex hormones and hae. on the basis of the literature, he proposes possible pathogenic mechanisms by which these influences might manifest. these relationships are discussed with a final view toward patient care. current literature. it is currently known that hae is influenced by the fluctuation of female hormones, but the effects sometimes appear to vary greatly among women. some patients seem to be estrogen-dependent: puberty, pregnancies, or estroprogestative contraception can precipitate attacks, whereas menopause impairs the disease. however, the course of some women's disease seems to be unaffected by estrogen. reports have highlighted the close relationship between female hormones and angioedema. yip and cunliffe 22 described cases of hae occurring in a mother and her daughter whose symptoms appeared to be estrogen-influenced. their first attack happened around puberty; angioedema worsened premenstrually and when they took ocs. likewise, a woman with hae-ii associated with turner syndrome was reported as experiencing an increase in both the severity and the frequency of her angioedema attacks on beginning estrogen physiological replacement at age 34 years. 275 mcglinchey and mccluskey 276 also described hae manifesting after the initiation of estrogen replacement therapy. bork et al, 277 as discussed later in this supplement, reported their evaluation of the effect of oc use or hormone replacement therapy on the frequency and the severity of hae attacks, finding that 63% of women with hae taking these drugs presented with new or worsened angioedema. some kinin-mediated angioedema appears to be related to estrogen therapy. the first cases of angioedema during oral contraception were described in a 1986 report by warin et al 277a in which 2 sisters presented with hae only after taking oral contraception and during pregnancy: c1-inh concentration and complement components were normal. bouillet's group has also reported 5 women 278 whose angioedema attacks manifested after starting oral contraception. a study of c1-inh biological function revealed a lowered c1-inh activity with marked protein cleavage on the immunoblot. clinical and biological anomalies ceased after oral contraception was discontinued. hypoandrogenism has been associated with insufficient production of c1-inh in women taking cyproterone acetate as well in men with hypogonadism. in 1989, pichler et al 279 reported the cases of 4 women taking the contraceptive drug cyproterone acetate who developed clinical hae reversed by discontinuation of this contraceptive; also described were the cases of 2 men with hypogonadism who had recurrent angioedema successfully cured by androgen treatment. as discussed in previous sections, 3 reports described a new estrogen-influenced hae. 8, 86, 87 only women had angioedema, and their attacks were precipitated when estrogen concentrations were increasing because of pregnancies, oral contraception, or hormone replacement therapy. no c1-inh abnormalities have been reported in these cases. hormone-related pathogenesis. for these clinical events to be understood, they must be placed in a biochemical context. as discussed, the complement, contact, and fibrinolytic systems are involved in the genesis of angioedema attacks, 17, 222, 223 and all are regulated, at least to some extent, by c1-inh. bradykinin is thought by many to be the principal candidate mediator of angioedema genesis, and a mouse model demonstrated that angioedema is mediated by bradykinin via b2 receptor. 9 in human beings, studies have shown local elevations in bradykinin concentration during hae attacks. 13, 14 it is speculated that factor xiia, kallikrein, and thrombin may be increased as well. an elevation of plasmin-a2antiplasmin complexes is also noted. in ovariectomized rats, studies showed that 17b-estradiol favored the increase of factor xii by stimulating its gene transcription. 268, [280] [281] [282] this hormone also increased kininogen and kallikrein concentrations. 283 in addition, estrogens regulate b2 receptor gene expression and function: when reduced in ovariectomized rats, the vasodepressor response to bradykinin and b2 receptor mrna levels could be restored by estrogen substitution. 274 progesterone did not modify factor xii concentration but seemed to increase the amount of kallikrein cdna. 283 in healthy women taking oral contraception, there is an increase of the fibrinolytic proteins: plasmin and factors vii, x, and ix are increased, whereas plasminogen activator inhibitor is decreased. 266, 284, 285 these effects appear to be estrogen dose-dependent. 285 plasma samples from these women showed enhanced in vitro fibrinolysis. 107 the contact system is also altered: factor xii, prekallikrein, kallikrein, and hk increase. [286] [287] [288] last, c1-inh levels are decreased, correlating with the increase in factor xii. 107, 286, 287 hormone replacement therapy (hrt), although using a weaker estrogen dose than oral contraception, appears to have the same effects. fibrinolytic proteins are also increased (plasminogen and tpa), whereas plasminogen activator inhibitor decreases. [289] [290] [291] hrt increases factor xii, prekallikrein, c3, and c4 concentrations. 266, 290, 291 moreover, some studies have shown hrt to influence the bradykinin system: ace activity decreases, whereas bradykinin concentration increases. 256, 292, 293 toward treatment. this imbalance in these 3 systems appears to trigger angioedema attacks in some estrogensensitive women. each physician should identify these estrogen-sensitive patients to manage specifically their contraception needs, pregnancies, and choices regarding hrt. further studies to determine predictive biological, genetic, and clinical parameters would be of use. role of sex hormones in hae, part ii most reported studies have investigated the influence of exogenous sex hormones on the frequency and severity of hae attacks. here, visy et al very briefly summarize a study they undertook measuring serum concentrations of endogenous sex hormones in women with hae and correlating these results with the occurrence of hae attacks. this work has since been published in clinical endocrinology. 294 objective. the fluctuations of sex hormone concentrations at the beginning of adolescence, in the perimenopausal period, and during pregnancy or oc use can precipitate hae attacks. edematous attacks usually disappear after the onset of menopause. the authors undertook their study to establish any relationship between serum concentrations of sex hormones and the incidence of hae attacks. patients and measurements. serum concentrations of luteinizing hormone (lh), follicle-stimulating hormone, progesterone, estradiol, testosterone, prolactin, and sex hormone binding globulin (shbg) were measured in 78 patients (mean age, 30.3 years; range, 4-70 years) with hae. a questionnaire was used to explore the medical history of adult patients to characterize the evolution and properties of attacks. results. twenty-one patients had been symptom-free before menarche. symptoms in 13 of 21 patients first occurred during adolescence, whereas the remaining 8 patients had no attacks in the pubertal period. edema during the perimenstrual period was reported by 42.4%. pregnancy was associated with a higher incidence of attacks in 36%; edema formation was less common in 56%, and 8% had no change in the frequency of symptoms despite pregnancy. of the 11 patients using ocs, 7 reported an increase in the frequency and severity of edematous symptoms compared with the period before hormonal contraception had been initiated. serum concentrations of sex hormones were normal in the majority of patients; however, more than half of the subjects had progesterone values above the upper limit of the normal range. during the 1-year follow-up, the attack rate was 5 times higher in female patients with high progesterone concentrations (above the menopausal threshold of 4 nmol/l) than in women with normal or low serum concentrations. the eminent role of this hormone is also confirmed by the observation that only progesterone was higher in more than 50% of subjects than the serum level considered normal for sex, age, and stage of menstrual cycle. therefore, serum progesterone concentrations above the postmenopausal threshold are predictive of a higher incidence of edematous attacks in female patients with hae. in addition, multiple logistic regression analysis demonstrated a significantly lower attack frequency during 1-year follow-up in patients with higher (40 nmol/l) shbg level (odds ratio, 0.25 confidence interval; 0.07-0.90; p = .034). this difference existed independently of age and danazol dose (fig 22) . looking ahead. in view of these results, the monitoring of progesterone and shbg concentrations can prove useful in the prediction of hae attacks. further inves-tigations might address whether this increased progesterone is a cause or a result of frequent attacks, or whether progesterone itself exerts any direct effects on vascular permeability. last, progesterone's effects on relevant gene expression might represent another valuable avenue of exploration. influence of ocs or hrt on hereditary forms of recurrent angioedema (konrad bork, md,* and bettina fischer, md, mainz, germany) in this section, bork et al write of a systemic study undertaken after they noticed women in their clinic with new or worsened angioedema in response to oral contraception or hrt. their study crosses the barriers of c1-inh deficiency and hae type classifications to investigate the effects of exogenous hormones on hae. the new type of inherited angioedema recently described by their group has clinical features highly similar to classic hae-i and hae-ii; however, it is not associated with a c1-inh deficiency. currently, the genetic defect is unknown. until now, the disease has been observed exclusively in women 8, 86, 87 ; however, in 2 families the existence of clinically unaffected male carriers has been deduced. 8, 86 in the following report, the authors show that estrogens play a similar role in hae-i and this new type of inherited angioedema, herein described as hae with normal c1 inhibitor (hae type iii) in accordance with the original report of this condition by bork et al. 86 among their case series, the authors observed female patients with hae who had newly developed recurrent angioedema after receiving ocs or postmenopausal hrt. in addition, other women had a worsening of their preexisting angioedema after initiating ocs or hrt, suggested that these treatments might trigger hae. however, this possible relationship has not formally been investigated, nor is angioedema listed as an adverse effect of ocs or hrt. [295] [296] [297] information regarding the relationship between hormone administration and the first occurrence or worsening of hae symptoms is limited to anecdotal reports. 8,22-24,31,32,86,87,107,275,276,277a,299 in this study, a systematic approach was used to obtain information about the relationship between the use of oc or hrt and the clinical manifestation of hae-i and hae with normal c1-inh (hae type iii). methods. the case series by bork et al 277 included 228 women with recurrent angioedema who had taken oc, hrt, or both. 277 of these, this study investigated 32 women with hae-i and 39 women with hae with normal c1-inh (hae type iii). the ocs used by all patients were combinations of estrogen (ethinyl estradiol) and progestin. medications used for hrt contained only estrogens, as was standard medical practice in germany until recently. all patients underwent a thorough clinical examination, and a standardized medical and family history was taken. in particular, patients were questioned about the frequency, intensity, and organ involvement of their angioedema since the very first manifestation of their disease. the start date and duration of administration of sex hormones, with special emphasis on oc and hrt, were recorded. patients were asked whether their angioedema occurred shortly after beginning this hormonal medication. if yes, the time between starting oc or hrt and the onset of angioedema was recorded. patients who had recurrent angioedema before the intake of oc or hrt were asked whether their angioedema episodes were more frequent or severe after beginning treatment. concentrations of c1-inh, c4, and c1q were assayed by radial immunodiffusion, and c1-inh functional activity was determined by using the chromogenic substrate c 2 h 5 co-lys(e-cbo)-gly-arg-pna (immunochrom c1-inh; immuno diagnostics, vienna, austria). 300, 301 results. as shown in table v , oc, hrt, or both treatments were associated with angioedema attacks in 63% of women with hae-i and 62% of women with hae type iii. among the group of patients whose angioedema attacks were newly induced by estrogen-containing medications, there was a high preponderance of hae type iii, whereas in the patient group with an exacerbation of symptoms, there were more patients with hae-i (fig 23) . in 23 women-6 with hae-i and 17 with hae type iii-occurrence of skin or gastrointestinal angioedema manifested for the first time after the administration of oc. because of the assumed intolerance, several women tried different oc formulations, and all 33 trials were followed by recurrences of angioedema episodes so severe that all oc had to be discontinued. the interval between starting oc and episode occurrence ranged from 2 to 90 days (mean, 18 days). in 9 of the 23 women, symptoms were limited to the period of oc administration and ended after discontinuation. all 9 had hae type iii. in the other 14 patients, angioedema attacks recurred despite oc discontinuation. episodes occurred less frequently and less severely in 12, whereas symptoms were unchanged in 4 women. there was 1 occurrence of angioedema for the first time after starting postmenopausal hrt. one woman initially developed recurrent angioedema after starting hrt with estradiol (2 mg/d). the patient continued to take oral estradiol over a period of 4 years. after a consultation in the outpatient clinic, hrt was discontinued. symptoms persisted but occurred less frequently and were milder. the patient's daughter had recurrent skin angioedema and gastrointestinal pain attacks since age 12 years; therefore, with a normal c1-inh concentration and activity in both mother and daughter, a diagnosis of hae type iii was assumed. in 18 women, the administration of oc induced a severe exacerbation of their pre-existing angioedematous disease. because of this exacerbation, 4 women switched to another oc, some trying many different preparations. all of these oc doses were followed by recurrences of severe angioedema such that oc had to be discontinued. five women with previous recurrent angioedema of the skin and the gastrointestinal tract developed an exacerbation of their disease after starting hrt. of these, only 2 were eventually able to tolerate such therapy. in 3 of the 5, their angioedema also had exacerbated when on oc several decades before. discussion. the results indicate that in 63% of women with hae-i and in 62% of women with hae type iii, the use of oc or hrt was associated with either the initial appearance of angioedema attacks or with a severe exacerbation of previously existing angioedema. correspondingly, although also taking oc or receiving hrt, 37% of women with hae-i and 38% of women with type iii tolerated these medications without any negative influence on their disease; their disease was neither estrogen-dependent nor estrogen-sensitive. the results demonstrate that estrogen sensitivity is not a feature specific to hae type iii because it occurs in a similar percentage of women with hae-i. results from several case reports have suggested that administration of estrogens may exacerbate clinical manifestations of hae-i. frank et al 31 mentioned 2 young women with an exacerbation of hae associated with the use of oc. similar effects of the administration of oc or hrt on the clinical condition of patients with hae-i have been documented by others. 22-24,32,107,276,277a,299 as in the current study, initiation of hormonal therapy was associated with the onset of clinical disease or a marked increase in the frequency and severity of angioedema attacks. with respect to hae-ii, fletcher and weetman 275 described a patient with hae-ii and coexisting turner syndrome who had a severe exacerbation of angioedema attacks after initiation of a hrt with a combination of conjugated estrogen and progesterone. another type of hae, not associated with a c1-inh deficiency, has been recently described. 8, 86, 87 this disease has been reported exclusively in women and was termed hae type iii by bork et al 86 and estrogen-dependent inherited angioedema or estrogen-associated inherited angioedema by others. 8, 87 ten of the 36 women described as having hae type iii in a previous study by bork et al 86 took estrogen-containing oc and reported either the first appearance of clinical symptoms or a severe exacerbation of the disease in association with this treatment. in 2 additional families with this new type of hae, 8,87 of 11 affected women, 4 developed angioedema after starting oc, as did 3 after initiating hrt. warin et al 277a described 2 sisters with estrogen-associated urticaria/angioedema. with the exception of urticaria, the symptoms in these women closely resemble those of patients bork et al 86 have described as having hae type iii. it is highly probable that the agent responsible for worsening of pre-existing angioedema or provocation of initial symptoms in the women studied was estrogen rather than progestin. six of the women evaluated in their study had new angioedema attacks or exacerbation of preexisting disease after receiving estrogens alone, and their symptoms did not differ from those of women who received estrogens plus progestins. moreover, progestins have even been used for treatment of hae caused by c1-inh deficiency, and improvement of disease has been observed in at least some of the patients treated. [302] [303] [304] most important in the current context, progestin treatment has not been associated with worsening of angioedema. the causative role of estrogens in disease exacerbation suspected in this and other reports is also supported by the fact that attenuated androgens are effective in preventing edema attacks in hae caused by c1-inh deficiency. all of these observations are consistent with the view that estrogens and not progestins are responsible for provocation of angioedema attacks or worsening disease in women with inherited angioedema who take oc or receive hrt. the pathophysiological mechanisms underlying the occurrence of angioedema in association with estrogencontaining medications is not known. as stated, available evidence suggests that bradykinin is involved in the development of symptoms in patients with hae caused by c1-inh deficiency. 9, 14, 30 in addition to its inhibitory effects on c1s and c1r, c1-inh blocks the activity of other plasma proteases including kallikrein and factor xiia/xiif, enzymes that participate in bradykinin formation. bradykinin is generated from hk by kallikrein; the activation of prekallikrein to kallikrein is mediated by activated factor xii. the degradation of bradykinin to des-arg bradykinin and finally vasoinactive peptides is mainly mediated by carboxypeptidase n (kininase i) and angiotensin i converting enzyme (kininase ii). 30 estrogencontaining medications might favor edema formation by influencing both kinin generation and degradation. gordon et al 107 showed that c1-inh levels in the plasma of women taking oc are lowered. administration of estrogens also leads to increased factor xii levels, possibly via a functional estrogen response element in the promoter of the gene encoding factor xii. 30, 107, 280 plasma concentrations of prekallikrein have also been shown to be increased in women using estrogen-containing oc. 269 estrogens might also influence the degradation of bradykinin via their influence on ace. it has been demonstrated that hrt in postmenopausal women leads to a decrease in ace activity. 257, 292, 305 nogawa et al 292 also observed an increase in plasma bradykinin levels in such women, a finding consistent with the action of ace in the breakdown of bradykinin. the mediator responsible for edema formation in hae type iii is not known. however, many clinical similarities exist between hae-i and type iii, and the percentages of women whose disease is negatively affected by estrogencontaining medications is similar in the 2 conditions. these facts could permit speculation that estrogen-associated effects in women with hae type iii may also be related to the kinin pathway. however, whether the influence of estrogen-containing oc or hrt on the kinin pathway actually plays a role in favoring angioedema formation in patients with inherited angioedema is far from being clear today. the current results suggest that among patients with underlying hae, administration of estrogen does not always result in the appearance or worsening of symptoms. for example, other factors that might predispose women with hae to new or exacerbated symptoms when treated with estrogens could include a functionally or quantitatively relevant genetic polymorphism in the kinin system. information about such polymorphisms might be important in differentiating women whose angioedema would be aggravated by administration of estrogen-containing medications. recent guidelines for urticaria and angioedema released by the british association of dermatologists 306 define hae as angioedema without urticaria and highlight the need for a specific treatment for this condition. toward this end, the newly created european prehaeat network proposes a list of diagnostic criteria for angioedema caused by functional c1-inh deficiency ( table i) . because of the tendency of nonallergic angioedema to mimic other disorders, these criteria rely on both clinical and laboratory assessments. in addition, in the case of asymptomatic or newborn family members of relatives with known hae, laboratory measures, and genetic analyses represent the sole means of learning whether they, too, have the disease and might have future attacks. these points underscore the importance of laboratory measures in the definitive diagnosis of hae and related disorders. laboratory findings not only permit more complete diagnosis and classification of angioedema caused by inadequate c1-inh function but also form a key part of the evolving understanding of the genetic and biochemical aspects of these diseases. however, currently, no valuable laboratory criteria are available for the recently detected estrogen-dependent inherited angioedema. 8, 86, 87 in the report that follows, varga et al, in an international collaboration arising out of the third c1 esterase inhibitor deficiency workshop, set forth guidelines for the proper laboratory investigation of suspected hae, aae, and related disorders. for diagnostic purposes, the c1-inh protein phenotypes of the various types of angioedema without an allergic or pseudoallergic background are important (table vi) . mutations in the coding region of the c1-inh gene leading to hae are all heterozygous conditions. (there is 1 known exception, an italian family with a homozygous defect in the gene control region that might have evolved because of multiple consanguinity. however, even as a homozygous mutation, this defect did not lead to a complete deficiency of c1-inh. 171, 238 ) hae-i is defined as the presence of 1 mutant c1-inh gene whose gene product is undetectable in the circulation by routine methods. however, ultrasensitive methods might detect abnormal protein in some patients diagnosed with hae-i by routine analysis. in these patients, a reduced amount of normal c1-inh is found (table vi) . hae-i may result from the production of a missing mutant protein, eg, from a mutation resulting in a stop codon, production of a mutant protein that is subsequently degraded intracellularly, production of a mutant protein that is not secreted and that might accumulate intracellularly, or production of a mutant protein that is released but rapidly catabolized or degraded in the circulation. hereditary angioedema type ii is defined as 1 c1-inh gene producing a dysfunctional protein that is released and coexists with the normal protein in patients' circulation. the current routine analytic tools for detecting c1-inh antigen are unable to distinguish between normal and abnormal forms of circulating c1-inh. as a consequence, normal or elevated concentrations of the protein are found (table vi) . notably, the distinction between hae-i and 332 a transient rise in bradykinin can be measured during attacks. 14 àat least 1 patient with normal c1q level was reported. 114 §female patients and 1 male patient with recurrent edema caused by androgen deficit. 279 hae-ii is not absolute because some mutant protein can be detected in selected patients diagnosed with hae-i by using the most sensitive detection systems rather than routine methods. thus, the classification or diagnosis of hae-i or hae-ii might depend on the sensitivity of the analytic system used. independent of the type of hae, the affected heterozygous individuals show concentrations of native, functional c1-inh far below 50% of normal. estrogen-dependent inherited angioedema patients (formerly hae type iii) show no abnormalities in c1-inh concentration or function (table vi) . tissue swelling in the presence of normal c1-inh concentration and function in this form of inherited angioedema might be understood through the assumption that attacks are related to transient increases in bradykinin concentration. for the classification of the acquired forms of angioedema, the presence of a pathogenic autoantibody to c1-inh is relevant: type i shows no such pathogenic autoantibody, whereas in patients with type ii, such autoantibodies are detectable. however, the distinction between type i and type ii aae may become obsolete over a disease course, because patients might present with symptoms in the early stage of aae without detectable autoantibodies, whereas at later stages, autoantibodies can be detected. 131 regarding the distinction between the 2 types of aae, the diagnostic tools used and their sensitivities or accuracy of assessment might be crucial for classification. 132 the presence of anti-c1-inh autoantibodies causes hypercatabolism of c1-inh; increased amounts of a cleaved c1-inh with a reduced molecular weight are found in the circulation in patients with aae. finally, a few patients with recurrent, nonallergic angioedema in association with androgen deficit have been reported. 279 the report mentions an affected male patient in addition to affected female patients; disease manifestation was reversible by appropriate correction of androgen levels. inheritance was not reported. it remains open whether this form fits best to aae. according to comprehensive family studies and case reports, the onset of hae-i can manifest during an affected child's first years of life. [307] [308] [309] as early as 1966, a high number of colicky babies were reported in families with hae. 310 several authors have observed the onset of the disease beginning in children 1.5 years of age. 311, 312 farkas et al 79 found clinical manifestations of hae in children from 2.5 years. in a large hae-affected family in northern norway, children with the disease had their first typical symptoms at 3 to 4 years of age. however, in 1 case, a mother noticed different phonation, edema of the mouth and tongue, and enlargement of the uvula in her 6-month-old child, arising during a period of primary teeth eruption. 24 even if hae often makes it debut later, the statement in a review on the management of hae in pregnancy in the journal anesthesia that ''hae is always asymptomatic in infancy'' 313 is misleading. early infancy is a likely age for the first manifestations, and physicians dealing with newborns with hae-affected parents should suspect an underlying c1-inh deficiency as a potential cause of symptoms such as unspecific recurrent abdominal pain, diarrhea, upper respiratory disease, or skin rashes. therefore, a reliable diagnosis of children born to hae-affected parents is required as soon as possible. a c1-inh concentration or functional activity <50 % of normal, in conjunction with low c4 concentration, supports a diagnosis of hae. 314 tools for laboratory diagnosis. life-threatening laryngeal edema in association with inadequate c1-inh function in untreated or poorly treated patients has been reported to have a lethality of 14% to 30%. 7,31,315 because c1-inh functional deficit might remain clinically silent for decades, the high lethality because of acute airway obstruction underscores the importance and significance of awareness of this syndrome and the availability of an expert laboratory to assess c1-inh functional deficiency. laboratory analyses should be performed under at least minimal quality assurance conditions. test systems using calibration curves with only 3 points must be considered as not meeting the standard; through 3 points, every curve fitting is possible. the test system must also include 2 quality control specimens: 1 normal to high and 1 low. a test is valid only if the quality control measurements are within a range of predefined values. it is advisable to assess the laboratory's own normal range. the number of normal sera/plasma used (in bern) for such a purpose is >50. a standard specimen is prepared by pooling equal aliquots of each serum/plasma; next, testing individual plasma samples against the pool provides the normal range. material for analysis. for complement protein concentration measurements, edta-treated plasma is best suited. in the event that citrated plasma is used, normal ranges assessed with citrated plasma must be applied to correct for dilution by the citrate. for assessment of c1-inh function, serum or citrated plasma is convenient; heparinized plasma should not be used. 301 a diagnosis of any type of c1-inh deficiency or dysfunction should be confirmed with at least 2 assessments by using samples drawn 1 to 3 months apart with the patient in basal condition. for the early diagnosis of newborns born to parents with hae-i, sufficient blood can easily and painlessly be obtained from the placenta or the umbilical cord at birth. because newborns have a very high hematocrit of 0.6 and a consequently low plasma portion, the amount of protein will be diluted when using citrate as anticoagulant. 314 this should be kept in mind when comparing reference values from newborns; citrate dilution of samples must also be considered with samples from adult patients. c1-inh concentration. the concentration of c1-inh protein can be assessed by several routine methods such as radial immunodiffusion, nephelometry, or elisa. the mean serum concentration in normal individuals is around 0.2 g/l. in the sera of newly diagnosed patients with hae-i or patients with hae-i patients who do not need medication because of mild symptoms, the mean concentration of c1-inh protein is considerably reduced (table vii) . in patients with hae-ii, the serum c1-inh concentration is approximately 100% or may be elevated to 250% to 350%. the latter excess is a consequence of an arg444cys mutation of the reactive site, resulting in a free sulfhydryl group, enabling the mutated c1-inh protein to bind to other serum proteins (table vii) . in the sera of patients with aae, the concentration of c1-inh can be low or normal. normal antigenic amounts of c1-inh can be detected if interaction with autoantibodies against the 105-kd c1-inh protein results in a cleaved, nonfunctional 96-kd protein. 128 c1-inh function. c1-inh function can be assessed by several methods. 316 when analyzing samples from a patient with hae-i before and 30 minutes after c1-inh concentrate infusion, considerable differences emerge in rise of apparent c1-inh function, depending on which method is applied (table viii) . c1-inh function, when assessed by enzyme-based chromogenic assays, results in a linear relationship with the antigenic concentration of the c1-inh protein. the chromogenic substrates used are etco-lys (e-cbo)-gly-arg-pna (formerly from immuno, vienna) or meco-lys (e-cbo)-gly-arg-pna (aventis behring, marburg, germany). some reports favor the assessment of c1-inh function by loss of immunologically reactive c1r. the method uses the natural substrate c1r of c1-inh as the indicator and works exclusively with endogenous complement. by using this inexpensive type of assay system, one can observe a functional threshold value for c1-inh concentration and function. 138 formation of c1-inh-c1r or c1-inh-c1s complexes can be assessed by an elisa test system that also uses the natural but exogenous substrate of c1-inh as an indicator. by commercial elisa tests based on the detection of formed c1-inh-c1s complexes, the median functional concentration is less than 50% of normal at the time of diagnosis (table ix, (table ix) . this observation is somewhat discordant with numerous observations made in bern, reported in table viii . analysis of other complement components. to exclude or to confirm an acquired form of c1-inh dethe concentration of c4 may be assessed by nephelometry or radial immunodiffusion by using polyclonal antibodies. a substantial part of the already diminished c4 actually consists of c4 split products, 317 as can be measured with a monoclonal antibody specific for a neoepitope on c4 split-products. 318, 319 the ratio of c4 splitproducts over c4 was 0.39 for a normal subject and 4.34 for the subject's cousin with hae. 320 functionally active c4 can be determined by hemolytic assay. in aae with diminished c1, the replacement of c1 must be considered, because the low c1 value may hamper c4 assessment. the antigenic or functional concentration of c4 is very low in almost all newly diagnosed or untreated cases of hae (table x) and aae. in hungary, a borderline c4 concentration was found in only 1 of 25 newly diagnosed patients with hae. thus, for an inexpensive follow-up, assessment of c4 concentration might suffice. indeed, increased c1-inh concentration and function because of therapy that resulted in a reduction in edematous attacks was associated with a measurable increase of c4 concentration to nearly normal when using polyclonal antibodies to assess c4 (fig 24) . it should be noted that such a test system is unable to distinguish between natural and cleaved c4. a diminished antigenic level of c3 is extremely rare in hae, but in 10% to 20% of cases, even in 5 of 25 in newly diagnosed patients with hae, a slightly depressed concentration of c3 was measured in hungary. there is no correlation between the concentration of c1q and c3. detection of pathogenic anti-c1-inh autoantibodies. the majority of patients with aae have autoantibodies to c1-inh in their serum (aae type ii). these antibodies bind epitopes within or close to the reactive site of c1-inh. anti-c1-inh autoantibodies that bind to c-inh with different and generally low affinities can belong to each class (igg, iga, or igm). anti-c1-inh autoantibodies are detected by elisa. diagnosis of c1-inh deficiency in newborns at risk. in adults with angioedema caused by c1-inh deficiency, determination of c4 and c1-inh antigenic and functional concentrations nearly always allows a correct diagnosis. however, these complement components show considerable age-specific variations in children. the concentration of c1-inh antigen measured in umbilical cord blood, as a rule, is approximately f the concentration found in adults. [321] [322] [323] [324] [325] a reference range for the common functional c1-inh test in newborn cord blood was published in 1994. 320 the percentiles are presented in table xi . functional assays even allow the prompt detection of the rare form of hae, ie, hae-ii, in which the antigenic concentration of c1-inh is normal or even higher than normal, but mostly consists of a dysfunctional protein. whereas in the 1994 study the mean c1-inh functional activity in healthy newborns was approximately 70% of the mean adult value, sonntag et al 318 found a somewhat higher c1-inh function (;90%) in their study in citrated cord blood. during the child's first days, c1-inh and c4 values rapidly rise and may reach 1.5 times values found in adults. 326 andrew et al 327 found that near-adult values are achieved for c1-inh by 3 months of life. in healthy premature infants, c1-inh concentrations reached the normal adult range by 1 week of age. 328 norman et al 329 found large variation in c4, which in their opinion limits the diagnostic usefulness of quantitative c4 concentrations in children. diagnostic problems and strategy. several consequences result from the aforementioned experiences. to confirm or disprove a suspicion of clinical hae-i, measuring the concentration of c1-inh protein is satisfactory; however, this is not adequate to detect hae-ii or aae type ii. assessment of c1-inh function is therefore required. normal functional c1-inh concentrations can be observed in patients with hae under therapy when measured by assays based on the formation of c1-inh-c1r/c1s complexes. if the analyzing laboratory is unaware of this fact and receives a sample from a patient not known in the laboratory, a diagnosis of hae might be missed. in contrast, c1-inh measurements using commercial enzyme-based chromogenic assays yielded a false diagnosis of hae in 26% of samples. 330 the positive predictive value for a diagnosis of c1-inh deficiency was only 36% with the chromogenic assay, whereas it was 92% with the c1-inh-c1s elisa test kit. the negative measurement of c4 concentration can be used as a screening marker when hae is suspected. however, low c4 is not sufficient for diagnosis, because this is seen in many conditions with classic pathway activation. if c4 and the antigenic concentration of c1-inh are both normal, c1-inh functional deficiency can be excluded. low c4 concentration is also a characteristic feature of aae. diminished amounts of c1 or its subcomponents, preferably c1q, are indicative of an acquired c1-inh deficiency. however, low c1 (q, r, s) together with low c4 is not a proof for aae (table vi) . a normal complement profile (normal antigenic c1-inh, c1q, and c4) was reported in 2 patients with aae. 113, 331 in the latter case, a transient improvement in c1-inh function was associated with a transient loss of anti-c1-inh autoantibodies. on the basis of the listed diagnostic possibilities, the authors suggest an algorithm for the diagnosis of any type of suspected c1-inh deficiency, as shown in fig 25. the use of such an algorithm is strongly advised because, on follow-up of patients. according to the literature, c1-inh concentrations in hae-i patients greater than 35% to 40% of normal are sufficient to provide inhibition of classical pathway activation, a finding that is confirmed by clinical observations. thus, in such patients, the dose of attenuated androgens should not be increased with the aim to reach antigenic c1-inh levels approximately 50% of normal. ignorance of this fact might provoke higher and higher doses of, eg, attenuated androgens that bring no added benefit for c1-inh function and might harm the patient's liver. enzyme-based chromogenic substrate assays are excellent for initial diagnosis of deficient c1-inh function, but seem less well suited for monitoring during therapy (table viii) . measurement of c4 concentration is an inexpensive and generally useful parameter, even to follow up therapy, and especially in aae. concentrations of c4 close to the lower limit of normal, ie, at the lower 95% limit of confidence, have been found to indicate sufficient function of c1-inh to allow patients to be attack-free (fig 24) . in summation. for definitive diagnosis of angioedema associated with c1-inh functional deficiency (hae-i, hae-ii, and aae), the assessment of c1-inh concentration and/or function and, in some cases, the concentration of at least 1 of the c1 subcomponents are the routine diagnostic tools. for laboratory diagnosis, measurements should be performed twice, at an interval of at least 1 month, showing c1-inh concentration and/or function <50% of normal. laboratory diagnosis in newborns is difficult and needs special consideration. assessments of other complement parameters, such as c4 concentration, are to be considered surrogate markers, although excellent ones. no laboratory analyses to reveal estrogen-dependent inherited angioedema are thus far available. because purified c1-inh concentrate is derived from human plasma, virus safety is critical. as mentioned earlier, in 1983 to 1984, 13 patients developed a non-a, non-b hepatitis related to the administration of c1-inh concentrate, probably because of a contaminated batch. 7 since the introduction of viral inactivation steps, transmissions of infectious agents have not been reported. 75 side effects of c1-inh concentrate are rare and include fever and headache. to date, the formation of autoantibodies to c1-inh as a result of c1-inh concentrate administration has not been observed in any patient with hae. c1-inh concentrate is expensive because of high production costs, the low number of patients with hae, and the relatively low dose per patient compared with other blood products, such as those used in hemophilia. c1-inh concentrate is now available in most countries, at least on a named-patient basis, but is not available in the united states. fresh frozen plasma. fresh frozen plasma (ffp) is also effective 338 and can be used if c1-inh concentrate cannot be obtained. because ffp is not virally inactivated, the risk of transmitting infectious agents is relatively high. the administered volume is larger and requires a longer infusion time, which might be critical in emergency situations. the use of ffp is associated with several adverse effects, including urticaria, anaphylactic shock, and hemolysis. other. corticosteroids and antihistamines are usually ineffective. attenuated androgens require at least a few days to become effective and therefore are usually not administered in acute attacks of hae. the same is typically true for antifibrinolytics; however, some patients with mild attacks respond to 1g tranexamic acid by mouth every 3 to 4 hours for 12 to 18 hours. 339 for abdominal pain during attacks, spasmolytics such as butylscopolamine may be used symptomatically and are of benefit in milder attacks. future treatment options for acute edema episodes. kallikrein inhibitors. the candidate mediator of angioedema caused by c1-inh deficiency is bradykinin, released upon kallikrein activation. more than 25 years ago, aprotinin, a kallikrein inhibitor extracted from bovine lungs, was shown to be effective in treating hae attacks. 340 because of the risk of fatal anaphylactic reactions, 341 its use was discontinued after substitutive therapy with c1-inh concentrate became available. 7 a novel kallikrein inhibitor has recently been developed. 342 it is a synthetic kunitz domain protein designed by phage display technology to bind to human kallikrein. it has a high affinity and is highly selective for kallikrein. it is effective in abating acute attacks of hae but is not yet commercially available. 343 bradykinin antagonists. because bradykinin is assumed to be the key mediator of hae, blocking its b2 receptors might be therapeutically useful. icatibant is a synthetic peptide with a structure similar to bradykinin. however, it contains nonproteinogenic amino acids and is not degraded by the 2 main bradykinin cleaving enzymes, carboxypeptidase and ace. a potent antagonist, icatibant has the same affinity for b2 receptors as bradykinin across several tested species, including human beings. it is specific for b2 bradykinin receptors and does not interact with receptors of other peptides. preliminary studies were performed in human beings with asthma and seasonal allergic rhinitis, 344, 345 and icatibant was also shown effective in reversing increased vascular permeability in the murine hae model. 9 recently, the results of a phase ii single-dose trial for the treatment of acute hae attacks in 8 patients were reported. in this european trial conducted by bork et al. 345a icatibant appeared effective and well tolerated. recombinant c1-inh. during recent years, a recombinant c1-inh produced in the milk of transgenic rabbits has been developed. however, because of glycosylation differences, it has a shorter half-life than c1-inh derived from human plasma. initial results in patients with hae concerning tolerability and safety are encouraging. 346 the interim results of a phase ii trial conducted by levi et al 346a in acute attacks of hae appear positive. long-term prophylaxis. long-term prophylactic treatment is indicated for patients with many harmful and disturbing edema episodes, usually patients with more than 1 attack per month. long-term prophylaxis may be performed with attenuated androgens, antifibrinolytic agents, and c1-inh concentrate. all of these medications are associated with potential adverse effects, limiting their use as standard, life-long prophylactic treatment. attenuated androgens. in 1960, spaulding 104 reported the striking efficacy of methyltestosterone in hae, describing both a decrease in the severity and number of attacks. later, further androgens, fluoxymestrone in men and oxymetholone in women (with fewer virilizing effects than methyltestosterone), were tested. in 1959, stanozolol was synthesized, having 1 of the largest anabolic/androgenic ratios. in 1963, danazol, a synthetic analogue of ethinyltestosterone and less virilizing than the early synthetic androgens, was developed. in 1976, the attenuated androgen danazol was shown to considerably reduce the number of hae attacks. 347 since then, attenuated androgens (17-a alkylated androgens), mostly danazol or stanozolol, have been widely used for long-term prophylaxis in hae. 348, 349 although 17-a alkylated androgens are more efficient than other androgens, they are metabolized by the liver. thus, hepatotoxicity and the induction of liver cell adenoma or carcinoma are major concerns. androgens' mechanisms for exerting beneficial effects in patients with hae are unknown. major contraindications for treatment with androgens are pregnancy, lactation, childhood, and prostate cancer. however, as discussed later, there are a few reports on the successful use of androgen treatment in children. danazol. a widely used regimen is to start with an induction dose of 400 to 600 mg daily for 4 weeks and then taper down to the lowest maintenance dose that provides symptomatic relief. most patients' hae is controlled with 50 to 200 mg daily or every other day. long-term administration may be associated with several adverse effects, including weight gain, amenorrhea, decreased libido, menstrual irregularities, virilization in women (hirsutism, deepening of the voice, and decreased breast size), acne, muscle cramps, myalgia, fatigue, headaches, hemorrhagic cystitis, arterial hypertension, and hepatic necrosis or cholestasis. [348] [349] [350] [351] recently, hepatocellular adenoma and liver cell carcinoma have been reported in patients with hae taking danazol for many years. [352] [353] [354] laboratory changes may include decreased sex-hormone binding protein and thyroxine-binding protein, increased aminotransferases and cholesterol, and polyglobulia. stanozolol. stanozolol is used in initial doses of 4 to 6 mg per day for 1 month, then tapered to the minimal effective dose (0.5-2 mg daily). potential adverse effects may be similar to those of danazol but seem to occur less frequently. however, this needs further confirmation, because fewer patients treated with stanozolol have been reported in the literature compared with patients receiving danazol. other androgens. several patients have been treated with an oxandrolone. a small number of patients do not respond to androgens. antifibrinolytic agents. antifibrinolytic agents may also be used for the continuous prophylactic treatment of hae. 355 however, their efficacy is lower than that of androgens. in about 70% of patients, attacks occur less frequently and are usually milder. a considerable reduction of the number and severity of attacks is reached in only approximately 30% of patients. if long-term prophylaxis is necessary in children, tranexamic acid may be preferable to androgen treatment because androgen treatment should be avoided in childhood. presumably, antifibrinolytic agents act through the inhibition of plasmin. all antifibrinolytics bear the risk of thromboembolic events. e-aminocaproic acid is effective and reduces the frequency of attacks. the daily dose is usually 8 to 12 g in 4 equally divided doses. it is associated with various side effects such as thrombosis, postural hypotension, muscular pain and weakness associated with an increase in creatine kinase and aldolase, anal pruritus, and myositis. because of the many adverse effects associated with e-aminocaproic acid, tranexamic acid is the antifibrinolytic of choice. the daily tranexamic acid dose is usually 1 to 3 g per day. side effects are uncommon but include abdominal discomfort, mild transient diarrhea, nausea, headache, and anal pruritus. c1-inh concentrate. not only an acute attack therapy, c1-inh concentrate has been found effective in the longterm prophylaxis of hae. in 1980, the first patients were treated with 500 u c1-inh concentrate 1 or 2 times weekly for 1 year or more. 356 a report of 20 patients who received c1-inh concentrate twice weekly was published in 2002. 357 short-term prophylaxis before surgery. because of the risk of developing angioedema and possibly experiencing life-threatening laryngeal edema after dental surgery, endoscopy, endotracheal intubation, and other surgical procedures, preoperative treatment has been attempted. to date, available information is limited to case reports or small patient series. furthermore, even in the absence of preoperative prophylaxis, not all patients with hae develop edema attacks after surgery. patients who undergo multiple dental surgeries may develop edema episodes after some tooth extractions but not others. it is therefore difficult to assess the efficacy of a recommended preoperative prophylaxis until studies with larger numbers of patients are available. such prophylaxis has been performed with ffp, antifibrinolytics, attenuated androgens, and c1-inh concentrate. in several patients, ffp has been used prophylactically before dental surgery; in some patients, mild attacks occurred despite pretreatment with ffp. [358] [359] [360] antifibrinolytics such as e-aminocaproic acid 361 and tranexamic acid 362, 363 have also been used for preoperative prophylaxis in some patients. because antifibrinolytics are less effective in long-term hae prophylaxis, preoperative treatment focuses on attenuated androgens and c1-inh concentrate. in patients with known hae caused by c1-inh deficiency, preoperative prophylaxis with attenuated androgens, either danazol or stanozolol, has been shown to be effective in many patients, 359, [364] [365] [366] although some patients nonetheless developed swelling. 358, 367 for short-term prophylaxis, attenuated androgens may be administered from 5 days before to 3 days after the event, danazol at a dose of 10 mg/kg/d, maximum 600 mg per day, or stanozolol at a dose of 6 mg per day. currently, c1-inh concentrate has been used as preoperative prophylaxis in only a few patients; for this purpose, the dose of c1-inh concentrate was 500 or 1000 u. 368, 369 the risk of developing angioedema attacks secondary to dental surgery or other oral manipulations cannot be completely avoided by preoperative prophylaxis. as such, it is important to inform the patient that an angioedema attack might occur, to describe the clinical symptoms of laryngeal edema, and to educate the patient about what to do in case a laryngeal edema occurs. in this section, cicardi et al describe the typical treatment of aae and discuss the special considerations unique to this disorder. from pathogenesis to treatment. like other acquired forms of protein deficiencies, the course of acquired c1-inh deficiency can be related to the course of the underlying disease. the possibility of reversing the biochemical and/or clinical abnormalities of acquired c1-inh deficiency by curing the associated disease was reported by cohen et al 105 and subsequently confirmed. 122, 132, [370] [371] [372] however, the response can be temporary, even without evidence of relapse of the associated disease. 373 analogous to hae, patients with acquired c1-inh deficiency have received attenuated androgens for prophylaxis and c1-inh concentrate to treat acute attacks. 334, 347 nevertheless, these patients are frequently resistant to attenuated androgens, 114, 371 whereas they tend to benefit from antifibrinolytic agents. 114, 133, 374 antifibrinolytics are thus more effective for long-term prophylaxis in this population and represent the first choice for patients with acquired c1-inh deficiency. 134 replacement therapy with c1-inh plasma concentrate is the treatment of choice for life-threatening laryngeal attacks. however, patients with aae partially resistant to this treatment have been reported. 114, 129, 356, 375 the response to treatment differs from hae because of the rapid catabolism of c1-inh that characterizes aae. higher doses of c1-inh plasma concentrate were required in patients with a slow response to treatment. 112 slow responsiveness is a high-risk condition because it seems to increase with subsequent treatment. a new peptide, dx-88, a synthetic kallikrein inhibitor based on the same functional domain (kunitz domain) of aprotinin, is under study for treating angioedema in patients with hae. 342, 343 by inhibiting kallikrein activation, this peptide can stop generation of the vasoactive peptide bradykinin from hk, 18 for hae and related conditions, plasma-derived c1-inh concentrate remains the acute attack therapy of choice. in this section, juers and gröner detail the steps that are taken to ensure the high margin of virus safety for berinert p, produced at aventis behring gmbh, a zlb behring company. before the advent of specific therapy, the hae mortality rate caused by laryngeal edema was as high as 30%; even today, rates as high as 40% mortality associated with hae have been published. 31, 44 the introduction of c1-inh concentrate (berinert in 1979 in germany) was followed by the german registration of a pasteurized c1-inh concentrate (berinert p) in 1985. these drugs significantly changed the treatment procedures for acute attacks in patients with hae, becoming the treatment standard in the countries where berinert p is available. in countries where berinert p or another c1-inh concentrate was not available (for example, the united states), treatment of acute attacks was and sometimes still is usually performed with ffp. however, during the preparation of ffp, no reduction of potentially present viruses is achieved, in contrast with the plasma-derived c1-inh concentrate berinert p. similarly, although solvent-detergent treated ffp might seem an alternative, this treatment does not inactivate nonenveloped viruses. the following sections describe the current status of virus safety of berinert p. methods. berinert p is produced from pooled human plasma, primarily from source plasma, ie, plasma collected by plasmapheresis. in general, plasma-derived products may potentially transmit infectious and/or pathogenic viruses. this risk is minimized by careful selection of donor centers with regard to virus marker rates (epidemiology) and careful selection of donors; each donor, including qualified donors (ie, repeat donors), is evaluated before each donation for vital signs including temperature and blood pressure and subjected to an intensive questionnaire. details have been described recently. 376 furthermore, every donation is tested with serologic methods for hepatitis b surface antigen (hbsag) and antibodies against hcv, hiv-1, and hiv-2 as well as elevated alanine aminotransferase levels. in addition, sample pools of donations are tested by using nucleic acid amplification technique (nat)/pcr for genomic material of hepatitis a virus (hav), hepatitis b virus (hbv), hcv, hiv-1, and high titers of parvovirus b19 (b19v). reactive donations are discarded. furthermore, the plasma pool for fractionation is tested and released for further processing only if the pool is nonreactive (negative) for hbsag and nonreactive for antibodies against hcv, and hiv-1, and hiv-2 as well as nonreactive for hav rna, hbv dna, hcv rna, and hiv-1 rna, and for b19v dna not exceeding 10 5 iu/ml. the third main step ensuring virus safety is the elimination and inactivation of possible viral contaminants by the manufacturing procedure, whose capacity for virus reduction is tested in virus validation studies. the virus inactivation/removal steps used in the manufacture of berinert p use treatment in aqueous stabilized solution at 60°c for 10 hours (pasteurization) and specific chromatography. results. in virus validation studies designed to assess the capacity of the manufacturing process to inactivate and/or eliminate intentionally added virus, high overall virus reduction factors were demonstrated (table xii) . these virus validation studies were performed in accordance with the applicable committee for proprietary medicinal products (cpmp) notes for guidance. viruses covering a wide range of physicochemical properties were used in these studies (hiv-1 and hav are relevant viruses, bovine viral diarrhea virus [bvdv] and canine parvovirus [cpv] are specific model viruses for hcv and b19v, respectively, and pseudorabies virus (prv) is a nonspecific model virus for large enveloped dna viruses). the results demonstrate that the pasteurization and the manufacturing process of berinert p effectively inactivate and/or eliminate relevant human pathogenic viruses. to demonstrate that the manufacturing steps explored in the virus validation studies were very robust and that variations in the manufacturing procedure had no effect on the virus reduction capacity, specific studies covering parameters beyond production specification were performed. these studies showed that even under these conditions, a very effective virus inactivation occurred. regarding analysis of postmarketing surveillance for berinert p from 1985 until now, no cases of proven virus transmission, according to the assessment of the global pharmacovigilance department at aventis behring, were reported, although a total of almost 100 million units of berinert p were administered. discussion. the outbreak of aids in the 1980s and the resulting infections caused by blood transfusion and treatment with certain plasma derivatives, mainly factor viii concentrates, showed that virus safety in plasma proteins is a field of permanent attention, screening, and continuous research. since the former behringwerke ag introduced pasteurization in the 1980s (developed by heimburger primarily for hemophilia products), no hiv transmission via the company's pasteurized products has occurred. [377] [378] [379] this pasteurization procedure has been adapted to aventis behring's plasma products, including c1-inh concentrate. the pasteurization procedure has been shown to have an excellent track record of virus safety, and the validity of the pasteurization for virus inactivation was confirmed in scientific articles. [380] [381] [382] pasteurization is a very effective and robust virus inactivation method, but there are also limits. animal parvovirus, for example, is inactivated by pasteurization only to a certain degree. however, because of recent findings, it should be kept in mind that the human b19v is heat-sensitive and can be effectively inactivated by pasteurization; preliminary data from the group's own experiments (data not shown) also confirm this observation for stabilized aqueous solutions. 383 in addition, antibodies against b19v in the plasma pool for fractionation and in intermediates derived from that pool will neutralize b19v to a high degree. furthermore, regarding cpv, the specific chromatographic step was validated for its capacity to remove cpv, and a very effective and robust removal could be demonstrated. in conclusion, on the basis of the data from virus validation studies, 2 independent effective virus reduction steps in the production procedure for berinert p, effective for a wide range of enveloped and nonenveloped viruses, could be demonstrated. as outlined in the cpmp guideline (cpmp/bwp/268/ 95), the aim of virus validation studies is ''(i) to provide evidence that the production process will effectively inactivate/remove viruses which are either known to contaminate the starting materials, or which could conceivably do so, and (ii) provide indirect evidence that the production process might inactivate/remove novel or unpredictable virus contamination.'' so far, all new emerging viruses or members of the same virus family, which may be of severe concern in a blood transfusion setting, were shown to be reduced through manufacturing procedures. this was especially demonstrated for west patients had medical, psychologic, and economic difficulties attributable to hae or aae. among patients with a home supply of c1-inh concentrate, 3 patients had 4 avoidable adverse effects. one developed severe anxiety and depression requiring outpatient psychiatric care on being diagnosed with aae. another was unable to work as a baker on several occasions because of severe hand edema. the third had frequent angioedema and longstanding severe temperomandibular pain, which resolved when she was started on regular c1-inh concentrate. this patient required frequent absences from work. of patients who did not have access to c1-inh concentrate at home, 7 patients had 16 avoidable adverse effects. as fig 26 illustrates, 2 patients had 6 avoidable admissions to hospital, attributable to c1-inh concentrate treatment not being given or being delayed, resulting in suboptimal response to therapy. one man required intubation and a 5-day stay in the intensive care unit when no c1-inh concentrate could be found in the department. the same man was listed for surgery (tracheotomy for laryngeal edema and laparotomy for acute abdominal pain and guarding) on 2 occasions despite carrying a letter giving advice concerning the emergency management of his aae. on both occasions, after calls to his regular physician, he responded to treatment with c1-inh concentrate, and surgery was canceled. in addition, 3 patients were treated with ffp, a product that is likely to be less safe and possibly less efficacious than c1-inh concentrate. 75, 382 one woman received an average of 2 units of ffp each month at her local emergency department, where personnel were unwilling to prescribe c1-inh concentrate. one patient had severe anxiety and depression and was unable to work. she improved when started on the home therapy program. however, she had a severe exacerbation of her psychiatric condition when funding difficulties put the future of her home therapy in doubt. four additional patients had 1 day or more per month of absence from work or school because of symptoms, and 1 patient requested a private prescription of c1-inh concentrate to enable him to travel abroad safely, despite being entitled to c1-inh concentrate funded by the uk national health service. discussion. many uk patients have difficulty in accessing appropriate emergency treatment for hae or aae, with avoidable medical, psychiatric, and economic consequences. the events recorded here are the most serious. many patients have ongoing anxiety concerning access to treatment. some are reluctant to travel; many accept frequent painful attacks and disruption of education or work without complaint (price; uk hae network; personal communication, june 2004). this audit suggests that patients who carried their own supplies of c1-inh concentrate were less likely to have difficulties in accessing appropriate care and were less likely to have ill effects. reasons for pcts' reluctance to fund home c1-inh concentrate included lack of familiarity with the use of c1-inh, reluctance to use a product not licensed in the uk, lack of national management guidelines for hae and aae, lack of agreement among uk specialists of the necessity for a home c1-inh supply, expense of the product, and the mistaken belief that the hospital had funds to prescribe c1-inh concentrate. on the basis of their experience, the author's group recommends that all patients with hae or aae should carry their own supply of c1-inh concentrate and that their local hospital should stock additional supplies so that this can be replaced immediately after use. to improve access in the uk, the author recommends negotiation with uk primary care trusts to fund costs of hae or aae treatment for all patients, development of national and international guidelines concerning hae management and supply of c1-inh concentrate, and improved education for medical staff. in this companion piece, longhurst and o'grady describe a program allowing patients with hae to selfinfuse c1-inh concentrate and detail its outcome. the benefit and safety of home therapy is well established for many patient groups, including those requiring blood products such as igs for antibody deficiency and factor viii for hemophilia a. 391, 392 in the uk, patients with hae are advised to attend their local emergency department for treatment of severe or potentially life-threatening angioedema attacks. patients with frequent attacks are also offered prophylaxis with attenuated androgens, such as danazol, or tranexamic acid. 83, 339 despite prophylaxis, a minority of patients with c1-inh deficiency require repeated emergency hospital visits for administration of c1-inh concentrate to treat life-threatening laryngeal swelling and severe abdominal pain. waiting times in uk emergency departments can be several hours, and personnel may be unfamiliar with managing hae. as a result, hospital attendance is disruptive to the patient, who may be tempted to wait until the attack is severe, with potentially serious consequences. methods. the authors have introduced a home therapy program to improve access to treatment, thereby improving quality of life for people with severe hae and reducing the use of emergency services. patients entering the home therapy program must fulfill the following criteria: (1) proven c1-inh deficiency (2) prophylactic therapy optimal (3) required infusions of c1-inh concentrate at least every 3 months (4) infusion partner available (5) commitment to program of 34 patients considered for the program, 4 patients from the same family were already self-administering (under the care of their general practitioner) and received a refresher course. six further patients' symptoms were not controlled after optimizing prophylactic therapy. of these, 4 patients met the criteria for inclusion, 1 patient was refused funding, and 1 patient did not wish to have home therapy. all 4 patients were trained and successfully use c1-inh home therapy. a minimum of 6 hours training over a period of 3 visits was followed by a formal assessment of patient and infusion partner. patients kept a record sheet of all attacks and contacted the specialist nurse if they infused. records were reviewed and infusion technique (using sterile saline) and knowledge were assessed at 3-month intervals. case reports. patient a, a 30-year-old female patient, was initially treated with danazol. this was stopped because of persistently abnormal liver function. tranexamic acid was only partially effective. she had attacks of severe angioedema affecting the skin, abdomen, and occasionally the larynx, requiring treatment with 1500 iu of c1-inh concentrate at least once a week. intractable temperomandibular pain developed. she was started on c1-inh concentrate (berinert p) 1000 iu twice weekly. her temperomandibular pain improved, and she had fewer angioedema attacks. patient b, a 51-year-old female patient, had discontinued danazol because of excessive weight gain and voice abnormalities. she had subsequently received depot methyltestosterone 100 mg subcutaneously every 3 months without side effects for many years, in combination with tranexamic acid 1g twice daily. however, she had cutaneous and severe abdominal angioedema every 3 to 4 days. her mother had died of hae despite knowledge of the diagnosis, and the patient developed severe anxiety, necessitating specialist psychiatric treatment. after inclusion on the home therapy program, the patient infused 1000 to 1500 iu c1-inh concentrate approximately once a month, as required for severe symptoms, and reported an improvement in her quality of life. however, the threat of suspension of her home therapy because of funding problems led to an increase in her anxiety and the number of angioedema attacks, a well-recognized consequence of psychologic distress, 24 such that 1000 iu of c1-inh concentrate was required once or twice per week. resolution of the funding problems led to an immediate restoration of well-being and reduction in use of medical services, but she continued to require 4 to 5 infusions of 1000 iu c1-inh concentrate per month. patient c, a 33-year-old female patient, had frequent abdominal hae attacks, requiring an average of 2 days off work per month. she attended her local hospital emergency service approximately once per month and on each occasion received 2 units of ffp. after inclusion in the home therapy program, she reported a greatly improved quality of life, no time off work, and no further exposure to ffp. she infused 500 to 1000 iu of c1-inh concentrate approximately once per month. patient d, a 29-year-old female patient who had infrequent attacks without prophylaxis, developed severe abdominal attacks, occurring once or twice a week from the 13th week of her first pregnancy. she was treated with 1000 iu of c1-inh concentrate twice weekly from week 16 until 5 weeks after delivery of a healthy boy. she had only 1 mild abdominal attack during her pregnancy and continued full time work until the 36th week. after delivery, her attacks were infrequent, despite discontinuation of c1-inh concentrate. she chose to withdraw from the home therapy program but plans to retrain if necessary during any future pregnancies. family e, a 59-year-old mother, her 2 sons (30 and 20 years), and her daughter (31 years), had been trained in the use of c1-inh concentrate by their family doctor, and had been using this for treatment of acute attacks for 20 years without significant side effects. this family was the inspiration for the establishment of home therapy service. all 4 members of this family have been able to complete tertiary education, pursue demanding professional careers, and travel extensively, including to regions where medical care is not readily available, despite, in 1 case, severe hae. results. in all cases, home therapy resulted in improved quality of life as a result of improved access to treatment and reduced use of emergency services. no patient used c1-inh concentrate inappropriately. no adverse incidents occurred. discussion. despite the existence of successful home therapy programs for ig and factor viii, 391 patients also retain the option of hospital treatment if conditions for home therapy are not optimal. these results show that use of c1-inh concentrate is appropriate and that, in general, c1-inh concentrate usage is not increased by home therapy. use of emergency and routine medical services and time off work is reduced, and all patients report great improvements in their quality of life. (jan h. nuijens, md, phd, and c. erik hack, md, phd,* leiden and amsterdam, the netherlands) as discussed, recombinant c1-inh is among the therapies currently being investigated for the future treatment of hae. in this section, nuijens and hack describe the potential advantages of such a therapy and the steps being taken toward realizing this means of treatment. functional deficiency of the plasma protein c1-inh in patients with hae and aae accounts for the recurrent attacks of localized submucosal and/or subcutaneous swelling that can cause serious discomfort and are sometimes life-threatening. 7 intravenous supplementation with human plasma-derived c1-inh is the preferred treatment of severe attacks of angioedema, and this treatment appears very effective and well tolerated. 335, [393] [394] [395] however, plasma-derived products are of limited availability for reasons related to their human blood origin, supply, safety concerns, and/or development costs. three different plasma-derived c1-inh preparations are licensed by regulatory authorities in 1 to 3 european countries each. even where licensed and/or available, treatment with plasma-derived c1-inh often is restricted to laryngeal attacks with risk for suffocation, severe abdominal attacks, and preoperative prophylactic use. these restrictions have grown out of viral safety concerns, although the risks of transmission of human pathogenic viruses may nowadays be considered minimal. 75 long-term prophylactic treatment with plasmaderived c1-inh, although apparently safe and efficacious, occurs only on a very small scale. 356 plasma-derived c1-inh has also been tried in several serious pathological conditions such as sepsis, vascular leakage syndromes, and acute myocardial infarction, ie, in conditions associated with excessive activation of the complement and contact systems and/or absolute or relative deficiency of functional c1-inh. 198, 396 although its application in these conditions appeared safe, its efficacy was not yet demonstrated unequivocally. rationale for production of recombinant human c1-inh. until very recently, all animal and clinical studies with c1-inh used human blood plasma-derived c1-inh. although the advantage of this c1-inh preparation is that it has normal clearance kinetics, normal specificity, and no antigenicity, the apparent disadvantages are that the source material is limited and potentially contaminated with blood-borne pathogens. in particular, the limited source is an obstacle to develop c1-inh for indications other than hae because these indications require more c1-inh than for the treatment of hae (eg, approximately 15-25 u/kg body weight for an hae attack vs 200 u/kg for the treatment of myocardial infarction). hence, availability of recombinant c1-inh is warranted. major advantages of production of human c1-inh via recombinant dna technology are that the recombinant material is not derived from a human source, that the production can more easily be controlled, and that the production can be scaled up to meet the requirements for the investigational treatment of hae as well as other potential indications. 198 on cloning of c1-inh by bock et al, 205 recombinant functional c1-inh was expressed in cos-1 cells by eldering et al 190 in the late 1980s. the c1-inh expressed in this way was fully active, but expression levels were low (approximately 1 mg/l), thus allowing only structurefunction relationship studies. 190 expression in escherichia coli was also attempted. though much larger quantities of protein were expressed, only a fraction (a few percent) was active, whereas the majority was denatured (hack et al, unpublished data, january 1990). similar observations have been made by lamark et al, 397 although these investigators could obtain more functional c1-inh preparations by using different vectors and different strains of e coli. hence, other expression systems are needed to produce recombinant c1-inh for clinical applications. two such systems, expression in transgenic animals and a yeast system, are discussed here. generation of recombinant human c1-inh transgenic rabbits and pharmaceutical development of transgenic recombinant human c1-inh. expression of c1inh via the mammary gland of transgenic animals was first attempted in rabbits, among others, because of rabbits' short generation time. in addition, a lactating rabbit may produce 10 l milk per year; because of this, transgenic rabbits may yield sufficient quantities of recombinant human c1-inh (rhc1-inh) for the hae indication. a mammary gland-specific expression vector containing a bovine milk-specific promoter sequence (as1 casein) functionally linked to the gene encoding human c1-inh was introduced into the rabbit germ line by microinjection of fertilized oocytes isolated from superovulated female donor animals. a production line was selected with a relatively low transgene copy number and that expressed rhc1-inh in milk at levels exceeding 10 g/l. to ensure continuous availability of a herd of transgenic animals from the selected founder line and to restrict potential rearrangements of the transgene through subsequent generations, a master transgenic bank and a manufacturing working transgenic bank have been established. production herds are housed in specially designed and validated facilities subject to stringent environmental monitoring. animal health is continuously monitored, and the rabbits are regularly tested for the presence of a wide range of rabbit and human pathogens. rabbits showing any abnormality are excluded from being milked. this health monitoring system ensures that the production herd is a specified pathogen-free rabbit population. milk is collected by using a specially designed rabbit-milking device. the raw milk is analyzed for compliance with internal criteria before release for downstream processing. the purification of rhc1-inh from raw milk is composed of skimming the milk, cationexchange chromatography, viral inactivation by solventdetergent incubation, anion-exchange chromatography, zinc-chelating chromatography, virus removal by nanofiltration, ultrafiltration, final filtration, filling, and lyophilization. stability studies of vialed rhc1-inh have indicated a shelf life of at least 2 years. the downstream process of rhc1-inh has been the subject of a virus removal or inactivation validation study using a panel of viruses representing different viral classes and sizes (enveloped and nonenveloped, single-stranded and double-stranded rna and dna viruses). characterization of transgenic rhc1-inh protein. nonreduced and reduced sds-page of rhc1-inh showed rather broad (single-chain) protein bands with an estimated relative molecular mass (mr) of approximately 90,000. the observed heterogeneity in mr on sds-page of rhc1-inh as well as the difference in mr (approximately 15,000 and 10,000 on nonreduced and reduced sds-page) of rhc1-inh and plasma-derived c1-inh most likely results from differential glycosylation. this follows from the observation that both the n-terminus and c-terminus of the protein are intact as determined by n-terminal and c-terminal amino acid sequence analyses. moreover, liquid chromatographymass spectrometry peptide mapping data revealed approximately 70% recovery of the amino acid sequence of rhc1-inh and conformity with the expected amino acid sequence. the theoretical molecular mass of the polypeptide backbone of c1-inh is approximately 53 kd. the use of matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed a molecular mass of approximately 67 kd and approximately 73 kd for rhc1-inh and plasma-derived c1-inh, respectively, and suggests that approximately 21% and approximately 28% of the molecular mass of rhc1-inh and plasmaderived c1-inh can be ascribed to carbohydrate structures. monosaccharide analysis of the major monosaccharides in glycoproteins (fuc, galnac, glcnac, gal, man, and neu5ac) confirmed that rhc1-inh consists of approximately 21% carbohydrates. n-glycolyl neuraminic acid, a sialic acid that is not found in normal human tissue, as well as a-galactosyl were not detected on monosaccharide analysis of rhc1-inh. mass spectrometric and nuclear magnetic resonance analyses of the glycans released from rhc1-inh indeed showed the presence of at least 4 different o-linked and at least 7 different n-linked carbohydrate structures, including oligomannose and hybrid and complex-type carbohydrates. 398 n-linked glycan profiling indicated that most of the n-linked complex structures of rhc1-inh appeared to be monosialylated. the inhibitory potency of rhc1-inh toward the target proteases c1s, kallikrein, factor xia, and factor xiia was found to be comparable with that of plasma-derived c1-inh. in addition, no difference in the inhibition of plasmin and thrombin was observed. physicochemical characterization to demonstrate identity, purity, potency, and consistency between different batches of rhc1-inh revealed that it was consistently being produced as an active, intact molecule, with purity exceeding 99.98 %, containing no detectable amounts of cleaved rhc1-inh, endogenous rabbit c1-inh (less than 1 ppm), and rabbit milk proteins (less than 20 ppm). summary of experiments in animals with transgenic rhc1-inh. the purpose of the preclinical safety program was to assess the initial safe dose in human beings and to identify safety parameters for clinical monitoring. the package contained safety pharmacology in dogs (625 u/ kg), acute single-dose toxicity in rats and dogs (25-1250 u/kg), 2-week subchronic toxicity in rats (25-625 u/kg), repeated dose toxicity in dogs (625 u/kg/day for 5 days), and local tolerance in rabbits (intravenous, perivenous, or intra-arterial microinfusions). overall, no adverse clinical signs were observed and no or negligible effects were reported on clinical laboratory parameters. on the macroscopic level, some minor findings were reported, but histopathological analyses revealed no treatment-related findings. rhc1-inh was cleared more rapidly from the circulation of the rat and the dog in comparison with plasma-derived c1-inh. in rat serum samples from animals in the highest dosage group (single-dose, 1250 u/kg; subchronic, 625 u/kg), a mild specific immune response was noted. there was no evidence for the generation of neutralizing antibodies. on the basis of these studies, it was concluded that rhc1-inh may be considered safe. phase i study of rhc1-inh in asymptomatic subjects with hae. an open-label phase i study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending intravenous doses of rhc1-inh (6.25-100 u/kg) in 12 asymptomatic subjects with hae was conducted at the centre for human drug research (leiden, the netherlands; cohen et al). the study subjects were invited to participate by levi (department of internal medicine, academic medical centre, amsterdam, the netherlands) in close communication with the dutch association of patients with hae and quincke's edema. all subjects were without angioedema symptoms at the time of the trial and received rhc1-inh on 2 occasions with an interval of at least 5 weeks (table xiii) . the subjects were assessed clinically, and blood samples were taken to evaluate laboratory safety parameters and immunogenicity as well as the clearance and biological effects of rhc1-inh. assays of pharmacokinetics and pharmacodynamics parameters were performed at sanquin research (hack, amsterdam, the netherlands). safety results were as follows. rhc1-inh appeared safe and well tolerated: no probable product-related adverse events (eg, allergic reactions) or changes in electrocardiogram, vital signs, or routine laboratory parameters were observed. neither clinically significant increases in anti-c1-inh or antirabbit milk protein nor evidence of c1-inh neutralizing antibodies was noted. pharmacokinetics results were as follows. infusion of rhc1-inh resulted in dose-dependent increases of functional c1-inh response parameters maximum concentration (above baseline), area under effect curve above baseline, dose-normalized area under effect curve, and of time above 0.4 u/ml, whereas dose-normalized maximum concentration appeared constant (approximately 0.02 u/ml/u/kg). the profiles of functional c1-inh showed a full initial recovery and a dose-dependent clearance of rhc1-inh that indicated a saturable mechanism of elimination. after the infusion of rhc1-inh at 100 u/kg, a clearance of approximately 13 ml/min, a half-life of about 3 hours, a volume of distribution of about 3 l, and an endogenous infusion rate of about 2 u/min were observed by using a standard 1-compartment model of analysis. application of a 1-compartment model with michaelis-menten elimination provided dose-independent estimates of v max , k m , volume of distribution, and endogenous infusion rate of approximately 45 u/min, approximately 0.6 u/ml, approximately 3 l, and approximately 12 u/min, respectively. thus, the half-life of rhc1-inh in the subjects with hae appeared reduced compared with historical data of plasma-derived c1-inh. 211, 334, 335 this likely relates to the differential glycosylation and lower degree of sialylation in rhc1-inh. the increased clearance of rhc1-inh from the bloodstream likely occurs mainly through endocytic asialoglycoprotein and mannose receptors on hepatocytes. 209 after dosing with rhc1-inh at 100 u/kg, functional c1-inh was at least 2-fold the normal level for about 2 hours and remained above 0.4 u/ml for about 9 hours. activation of the cascade systems releasing inflammatory mediators responsible for the generation of edema is thought to be sufficiently controlled with functional c1-inh levels exceeding 0.4 u/ml. 138 pharmacodynamics results were as follows. rhc1-inh displayed biological activity in the subjects as evidenced by plasma c4 increases and inhibition of c4 cleavage. the dose-dependent increase in mean normalized c4 was highly variable within dosage groups and was approximately 2-fold increased after the infusion of rhc1-inh at 100 u/kg. c4 peak levels occurred at approximately 12 hours postinfusion, similar to c4 responses after plasmaderived c1-inh, and thereafter gradually declined to baseline. 211, 335 an immediate dose-dependent effect of rhc1-inh on plasma c4b/c was observed. the magnitude and the duration of the decrease in c4b/c appeared dependent on the dose of rhc1-inh. cleavage of c4 appeared to occur once functional c1-inh dropped below a level of approximately 70% of normal. taken together, the phase i results suggest that an adequate dosage of rhc1-inh will be able to correct c1-inh activity in blood for a sufficiently long period to halt the progression of a swelling episode and to allow the resolution of edema. that is, the results warrant clinical studies of the safety and efficacy of rhc1-inh in symptomatic patients with hae. phase ii and phase ii/iii studies of transgenic rhc1-inh for the treatment of acute attacks in patients with hae. a phase ii exploratory open-label study of the safety, tolerability, and efficacy as well as pharmacokinetics and pharmacodynamics of rhc1-inh at 100 u/kg in symptomatic patients with hae with severe attacks of angioedema in the abdomen, the facial-oro-pharyngeal region, and/or the genito-urinary region is currently being performed in the netherlands (levi) . on the basis of the initial encouraging results of the phase ii study, a multicenter phase ii/iii study of rhc1-inh at sites in several european countries is being initiated. expression of wild-type c1-inh in the yeast pichia pastoris. the yeast pichia pastoris is increasingly used as an expression system to produce recombinant proteins on a large scale. 399 hence, recombinant c1-inh has been expressed in this system as well. however, development of yeast-expressed rhc1-inh for clinical use is still at an early stage. initial expression of c1-inh was achieved by using the vectors ppiczaa and ppic9. 400 without optimization, expression of as much as 2 g of recombinant protein per liter was observed. though some of the c1-inh preparations produced in p pastoris were fully active, compared with plasma c1-inh, other preparations were inactive because of proteolytic inactivation of the recombinant molecule by contaminating proteases. inactivation in particular was observed when the yeast was cultured for more than 3 days. the recombinant protein was secreted in the culture supernatant and constituted at least 20%, in most cases >50%, of the total amount of protein, which may make this system attractive for commercial production applications. recombinant c1-inh was purified to >95% homogeneity on sds-page on laboratory scale by using hplc cationexchange chromatography. plasma c1-inh is a heavily glycosylated molecule, owing approximately 28% of its molecular mass to carbohydrate groups. in the original publication by bock et al 205 describing the nucleotide sequence of cdna coding for human c1-inh, 13 glycosylation sites were identified. however, the number of potential glycosylation sites (n-linked and o-linked) may be as high as 28. the function of these carbohydrate groups is not completely clear, although there is conclusive evidence that incomplete glycosylation and/or undersialylation of the carbohydrate groups is involved in the clearance of the molecule in vivo. 209 on the basis of migration patterns of yeast-expressed c1-inh on sds-page as well as current knowledge of glycosylation by yeast (mainly mannose), it is expected that carbohydrate groups on yeast-expressed c1-inh will need to be modified to achieve normal clearance in vivo. indeed, in a rat model, accelerated clearance of pichia-expressed c1-inh was observed (half-life, 20 minutes for pichia-c1-inh vs 3.5 hours for plasma derived c1-inh; bos et al). 400, 401 expression of c1-inh mutants. c1-inh mutants have been made for different purposes, ie, to study the structure and function, to study glycosylation, and to modify function toward applicability in inflammatory conditions. regarding application in inflammatory conditions, c1-inh, like many other serpins, can be catalytically inactivated by neutrophil elastase, which may limit its use in inflammatory diseases. to improve the potential performance of c1-inh at sites of inflammation, it may be beneficial to enhance the resistance of the molecule to catalytic inactivation by elastase, because this would reduce the amount of inhibitor needed. c1-inh mutants with amino acid replacements at the so-called p3 and p5 positions of the reactive center (this is the domain interacting with target and nontarget proteases) have been developed. 402 these mutants have a nearly normal function but are much more resistant to inactivation by elastase than wild-type c1-inh. until now, these mutants have never been tested in animal models to prove the principle that they indeed perform better in inflammatory conditions. as indicated, pichia-expressed c1-inh is glycosylated differently (mainly mannose) than plasma-derived c1-inh (mainly complex-type carbohydrate groups). most glycosylation sites are located in the n-terminal part of the molecule. the function of this part is currently unknown, although according to the latest data, the most c-terminal amino acids of this n-terminal part serve to stabilize the so-called central b-sheet of c1-inh, which is essential for serpin function. 206 to evaluate the role of the n-terminal portion in the functional activity of c1-inh, deletion mutants lacking 76 or 98 amino acids of the n-terminus have been expressed in pichia. the functional activity of these mutants has yet to be studied in detail but at first glance looks normal. in contrast, a mutant lacking the complete n-terminal part was inactive. the mutant lacking 98 amino acids is interesting in that it seems to have normal functional activity despite having only 3 carbohydrate groups left. experiments with endoglycosidase h and concanavalin a reveal that these groups are n-linked (asn 216; asn 231 and asn 330). for the future. recombinant protein technology not only offers the possibility of producing large amounts of protein at acceptable costs but also allows for mutation of the protein to adapt its function. expression of wildtype human c1-inh has been successfully achieved in transgenic rabbits, and this recombinant inhibitor is now in phase ii clinical development for the treatment of acute attacks in patients with hae. pichia-expressed c1-inh may constitute another alternative to plasmaderived c1-inh but is still at a very early stage of development. kallikrein inhibition and nonallergic angioedema. the kallikrein inhibitor dx-88 was generated via phage display, a technique for rapidly identifying target-specific protein binders. the phage display process involves generating many possible binders specific for each desired target, in this case human plasma kallikrein, and then selecting the binder with the highest affinity. as such, this process resulted in a molecule with an extremely high (44pmol/l) binding affinity for human plasma kallikrein. kallikrein is known to catalyze the conversion of kininogen to bradykinin. 254 assuming bradykinin is the major mediator of nonallergic angioedema, 28 plasma kallikrein inhibition should be beneficial during angioedema attacks. kallikrein has also been shown to influence the renin-angiotensin system by promoting the conversion of prorenin to renin 262 ; however, as discussed in the pathogenesis section of this supplement, the renin-angiotensin system likely plays a much lesser role in angioedema generation. phase i normal volunteer study. in a study conducted in scotland, dx-88 was administered to 12 healthy male or female volunteers; pharmacokinetic and safety parameters were assessed. two patients each received 10 or 20 mg dx-88, and 4 patients each received 40 or 80 mg dx-88. half-life measurements ranged from 0.55 to 1.71 hours. phase ii european study. an open-label study of dx-88 for the treatment of nonallergic angioedema attacks was conducted in 4 centers in europe. of the 9 treated patients, 7 patients had hae, and 2 had aae. patients presented with facial/lip, hand, genital, or abdominal attacks; 1 patient was treated for simultaneous hand and abdominal edema. in this dose-ranging study, 3 patients each received 10 mg, 40 mg, or 80 mg intravenous dx-88. one patient (40 mg dx-88; genital edema) had a drugrelated anaphylactoid reaction. patient-reported times to the start of attack resolution ranged from 25 to 240 minutes (mean, 92 minutes); patient-reported times to complete resolution ranged from 2 to 72 hours (mean, 38.6 hours). the authors note that dx-88 could potentially advance the treatment of hae by reducing patients' reliance on anabolic steroids or plasma-derived products. attenuated anabolic steroids are the most common prophylaxis for hae and associated disorders. although these agents are effective in many patients, they carry a wide range of side effects, many of which are undesirable. their mechanism of action in these diseases, and indeed the full extent of their systemic effects, is not entirely known. as illustrated by the following investigations, attenuated androgens such as danazol may have other, at least transiently positive effects in addition to their better-documented risks. attenuated androgens effectively prevent swelling attacks in hae. 351 the attenuated anabolic steroid danazol is mainly used in the therapy of many estrogendependent diseases such as endometriosis, cystic fibrotic mastopathy, primary menorrhagia, primary pubertas praecox, and gynecomastia. 403, 404 however, its other, less known clinical indication is the prophylaxis of hae. 351 the high androgen and low estrogen levels caused by this drug 25, 405 are responsible for some side effects. among these, seborrhea, acne, hirsutism, weight gain, hair loss, voice changes, clitoral hypertrophy, vaginitis, irregular menstruation or amenorrhea, and reduced breast mass are the most common. 349 methods. this study investigated the effects of danazol on bone metabolism. low estrogen concentrations have previously been shown to play a role in the development and progression of osteoporosis. the authors' current investigation sought to discover how reduced estrogen production caused by danazol therapy influences bone turnover. according to available data, danazol's effects on bone metabolism have been investigated only in patients with endometriosis. [406] [407] [408] [409] these investigations gave contradictory results. in addition, the authors note that the danazol doses given to treat endometriosis in these studies far exceed the doses administered in hae. rock et al 410 have reported a temporary increase in bone mineral density (bmd) lasting only for the 24 weeks of treatment, which then decreased such that bmd values measured at the 72nd week were lower than the initial values. according to dodin et al, 411 the lumbar bone mass slightly increased by the end of the third and sixth months. 366, dmowski et al 412 found no significant change in bmd during the danazol treatment. the current investigation measured the degree to which the minimum effective dose of danazol for hae control influenced bone turnover. all patients cared for at the hungarian hae center were involved in this study. the control group contained 50 patients not treated with danazol. concentrations of alkaline phosphatase, calcium, phosphate, parathyroid hormone, dehydroepiandrosterone sulfate, b-cross laps, and osteocalcin were measured in blood serum. dual-energy x-ray absorptiometry was also performed in patients older than 20 years. results. significant correlations have been found by using multiple logistic regression analysis between the variable of b-cross laps, osteocalcin values, z score values detected by the femoral neck, and danazol treatment. b-cross laps and osteocalcin concentration (markers of bone resorption) have shown a negative correlation, whereas z score values (markers of bone formation) have shown a positive correlation with danazol treatment. in the authors' study, it was evident that b-cross lap concentration was lower in the danazol-treated group than in the control group. changes in serum osteocalcin concentrations, like those in b-cross lap values, showed a negative correlation. osteocalcin concentrations in patients treated with danazol were considerably decreased. however, the authors note that both osteocalcin and b-cross lap concentrations oscillated in the normal range. z scores measured by the femoral neck showed that positive results were much more frequently detected in the danazoltreated group, whereas values in the untreated control group were always between 0 and 20.5. thus, bone mineral density was significantly higher in the danazoltreated group. the authors propose that the increased androgen level may have compensated for the deleterious effect of decreased estrogens, citing that the anabolic effect of androgens on bone metabolism have been well documented in the literature. discussion. comparing the results of this study with those reported in the literature on the effects of high-dose danazol treatment in endometriosis, the authors suggest that the minimum effective dose treatment in hae does not cause osteoporosis and, indeed, may protect the bone from mineral loss. ovulation is the result of complex, interrelated mechanisms initiated by the surge of lh and characterized by resumption of meiosis and germinal vesicle breakdown, initiation of luteinization of the granulosa cells, rupture of the follicle wall, and release of a fertilizable ovum. 413 hormonal control of ovulation and biochemical and morphologic changes of the preovulatory follicle, including collagenolysis and vascular changes, have been extensively studied. ovarian follicular fluid (ff) is an enzyme reservoir controlling the permeability of the capillaries of the antrum and proteolytically degrading collagen, thus leading to the rupture of the follicle wall. the plasminogen-activator-plasminogen hypothesis is the most likely explanation of the mechanism initiating the cascade that leads to follicular rupture. the preovulatory follicle, stimulated by gonadotropins, secretes urokinasetype plasminogen activator, which can convert plasminogen to plasmin. a latent collagenase activated by plasmin attacks collagen, and the resulting telopeptidefree collagen can be attacked and degraded by nonspecific proteases. the decrease of the tensile strength of the follicle wall then allows the rupture of the follicle by the action of the existing intrafollicular pressure. 413 in addition to several multifactorial systems (eg, kinins and fibrinolysis) that have been demonstrated and characterized in human ff, the presence of active complement has also been demonstrated in amounts similar to those present in normal human serum. 414 plasmin's activation of complement in ff is very important for the function of the enzymatic multifactorial mechanism of ovulation. after complement activation in ff, biologically active peptides such as c3a, c4a, and c5a are released via the complement cascade. complement activation is also important for the assembly of the membrane attack complex on the follicle wall. 414 complement activation and ovulation: consequences in hae. it has been clearly demonstrated that women of reproductive age with hae who were not under danazol treatment and off any therapy, including ocs, frequently had cystic ovaries with an ultrasound pattern of polycystic ovaries (pcos; approximately 35%) or of multifollicular ovaries (mfos; approximately 55%). 415 although pcos in women not affected with hae are associated with increased lh and testosterone concentrations, and mfos with almost normal values of these hormones, in patients with hae, normal concentrations of lh and testosterone (and of prolactin, cortisol, and acth) are almost always demonstrable independently from the ultrasound pattern (pco, mfo, normal) but in the presence of significantly reduced follicle stimulating hormone and slightly increased lh:follicle stimulating hormone ratio. menstrual irregularities and oligomenorrhea are frequent features of the patients studied, and hirsutism is sometimes present in patients with hae both with pco and mfo. neuroendocrine connection? in hae, the role of c1-inh deficiency in the development of clinical symptoms and in the management of the disease has been explored. 31 nevertheless, some interesting questions remain. the proposed relationship between physical and/or psychologic stress and the onset of acute episodes has yet to be proven fully. likewise, the effects of repeated stressful situations, such as the attacks themselves, on patients' neuroendocrine systems have yet to be completely explained. however, some possible links between the neuroendocrine system and hae have been studied and reported. perricone et al 416 have previously found high plasma bendorphin concentrations without simultaneous elevation of acth during hae attacks as well as during symptomfree periods. this provides evidence of a possible massive release of b-endorphin from the readily disposable pool present in the pituitary and/or for an increase in the turnover of the peptide as evaluated by b-endorphin: b-lipotropin ratios and, in turn, of a pituitary abnormality in the patients. also in the group of patients with hae affected with cystic ovaries (studied off therapy and in symptom-free periods), significantly high plasma b-endorphin concentrations and normal acth have been detected. 415 the very high prevalence of pco and mfo in hae again emphasizes the possible presence of hypothalamic-pituitary abnormality in these patients. in fact, both pco and mfo are linked to hypothalamic-pituitary dysfunction, 417 and in hae, 1 of the features of such a dysfunction, ie, high plasma b-endorphin concentrations and normal acth, is present in many patients, including men, postmenopausal women, and girls who are not of reproductive age. 416 influences on ovulation. the presence of both complement and ovarian abnormalities in patients with hae and the role of ff complement in ovulation induced the authors to study complement function in the ff of women of reproductive age affected with hae. they found that impaired complement function is present in ff obtained from female patients of reproductive age affected with hae. in fact, contrary to what had been observed both in previously studied women not affected with hae 415 and in controls, hae patients' ff showed a dramatically decreased classic pathway activity as well as undetectable functional and antigenic c1-inh, and very low c4. in addition, the authors observed a slightly reduced or within normal serum range values for alternative complement pathway activity, including c3 and factor b. this complement profile was also detected 415 in the patients' sera. 418 it is well known that the multifactorial mechanism of ovulation involves 2 relevant steps modulated by c1-inh: (1) the formation of plasmin, and (2) the plasmin-dependent complement classical pathway activation. 414 activation of ff complement is important for the correct continuation of the ovulatory process, and the lack of c1-inh, which facilitates the formation of plasmin and plasmin-dependent complement activation, can, in theory, be beneficial. on the contrary, when the activation of complement is massive, as occurs during hae attacks, a significant release of biologically active complement products can induce consistent inflammation and, in turn, pain. abdominal pain is frequently encountered in female patients with hae during attacks and during ovulation as well. however, because complement is chronically consumed during remissions and dramatically consumed during hae attacks, the potential of complement activation in hae ff is indeed the result of complex interrelated phenomena. if significant consumption of complement components occurs as a result of hae attacks shortly before the periovulatory period, ff complement will represent a defective reservoir of components unable to support further activation during ovulation. such a situation occurred in 1 patient: a few days before ovulation, an attack characterized by edema of the extremities and abdominal pain occurred; thereafter, the patient manifested neither abdominal pain nor detectable ff complement activities and c3 cleavage fragments during the periovulatory period. in another patient, the periovulatory period (as well as the time of the study) was far away from attacks (30 days); at this time, abdominal pain (without other symptoms of the disease) in the presence of reduced but detectable ff complement activities and of c3 cleavage fragments occurred. notwithstanding the presence of some important mechanisms of proven efficacy that can compensate for the complement deficiency (eg, the activation of a latent collagenase and the action of lysosomal enzymes), 413 it is possible that, in the presence of impaired complement function, the rupture of the follicle wall is more difficult. however, once significant time has elapsed since the last hae attack, complement function can ameliorate; if ovulation coincides with such a condition, it will meet an only mildly reduced complement that will be capable of participating in the rupture of the follicle wall. these considerations can explain the apparently undiminished reproductive capacity of female patients with hae. on the other hand, hae patients' ff complement supports a certain degree of activation by seminal plasma, 419 possibly caused by the relative integrity of the alternative pathway supporting its participation in the phenomenon. it is nonetheless possible that the more difficult rupture of some follicles in hae and the consequent altered intraovaric regulation by atresic follicles can result in the pco and mfo frequently observed in the disease. 415 it is known that in cystic ovaries, the rupture of the cysts is very painful; thus, the authors cannot exclude that in patients with hae, the rupture of ovarian cysts might contribute to the genesis of the dramatic abdominal pain during attacks. danazol and ovulation: conclusions. one of the mainstays in the management of hae is long-term therapy with danazol, a mild androgen capable of correcting low c1-inh concentrations and of greatly reducing the typical symptoms of the disease. 350 the authors therefore studied the effects of danazol on ovarian ff c1-inh and on ovarian ultrasound pattern. six patients with hae affected with cystic ovaries (3 with pco and 3 with mfo) who had been off any therapy for at least 1 year were treated with danazol (200 mg daily). as a result of danazol treatment, hae clinical symptoms consistently improved, and c1-inh serum concentrations increased. after 6 months of therapy, these patients were monitored by pelvic ultrasound scanning, and their ovaries were found normal. serum c1-inh and ff c1-inh also increased. furthermore, when the ultrasound pattern of the ovaries was studied in another group of patients, women with hae of reproductive age who were usually under danazol treatment, cystic ovaries were found in only 1 of 9 patients (11%). taken together, these data suggest a further link between the neuroendocrine and immune systems, describing a pathology involving both hypothalamic-pituitary dysregulation and an immunologic disorder. recent research has contributed to a less anecdotal and more systematic appreciation of the differences between adult and pediatric hae and the special age-related concerns that attend hae from the first months of life through puberty. here, 2 prospective follow-ups of pediatric case series are reported. most patients have the first manifestation of hae during childhood. unfortunately, treatment of acute attacks that manifest with circumscribed swelling of the skin and the subcutaneous tissues can be performed successfully only by administering c1-inh concentrate. 420 other medications that are usually helpful in edema of other origins (eg, antihistamines or corticoids) are not effective here. 421 the authors report that f of their patients, when anamnestically well evaluated, had edema of the mucous membranes of the respiratory tract at least once; remarkably high lethality rates (as high as 30%) have been associated with this disease in the past. 31, 44, 421 even recently, a mortality rate as high as 12.5% was published in an article summarizing experiences with austrian, swiss, and german patients with hae. 422 as previously discussed, bradykinin seems to be the major mediator of edema, but discussions have not yet been finalized. 9 because this disease still bears unknown details thus far identified, the authors chose to examine the baseline parameters of patients with hae to broaden the general database available and to enhance the understanding of the medical and scientific community. methods. data from 245 patients with hae have been collected in the database at the department for pediatric hematology, oncology and hemostaseology of the johann wolfgang goethe university, frankfurt/main, germany. as of this march 2003 analysis, this database contained a total of 56 pediatric patients (age <18 years) with diagnosed hae. these patients had been followed up for a period of as long as 12.9 years. the baseline characteristics of a total of 45 patients from 35 different families with hae were analyzed. the authors further monitored c1-inh plasma activity and c1-inh plasma antigen and supplemented these values with analyses of c3 and c4 and measurement of the total hemolytic activity (ch 50 ) in the patients' plasma. finally, the frequency and localization of attacks, the history of each patient, and the efficacy and safety of replacement therapy with c1-inh concentrate (berinert p) were investigated. antigenic c1-inh levels were determined by radial immunodiffusion with nor-partigen plates (dade behring, deerfield, ill). functional c1-inh concentration was determined by chromogenic assay with berichrom c1-inhibitor (dade behring). results and discussion. all patients showed low c1-inh activity, with a median activity of 22% of normal (normal value, 64% to 146%; range, 6.4% to 47%). all acute hae attacks were successfully treated with 10 to 30 units c1-inh concentrate per kilogram body weight. in these pediatric hae patients, c1-inh concentrate was effective and well tolerated. no side effects or seroconversions for hav, hbv, hcv, hepatitis g virus, hiv-1, or hiv-2 were observed. the gold standard of treatment for acute attacks in pediatric patients with hae is the administration of a c1-inh concentrate, preferably 1 that is specifically virus-inactivated. the authors always used a pasteurized c1-inh concentrate (berinert p) and did not observe any problems regarding safety or efficacy. in 1998, the transmission of hepatitis g virus in patients with angioedema treated with a steam-heated concentrate of c1-inh was reported. 423 the authors have not observed a seroconversion with regard to hepatitis g virus in their patients treated with pasteurized c1-inh concentrate. 386, 423 as treatment of pediatric patients with hae with c1-inh concentrate is not registered all over the world, some pediatric patients have to be treated with danazol therapy. 312 introduced in 1976 by gelfand et al, 347 this treatment seems to have a rationale in long-term prophylaxis of patients with hae. 347 however, because this treatment sometimes loses efficacy over time despite increased doses of danazol 424 and is often associated with severe side effects, 352 especially in children, the authors recommend the treatment of pediatric hae exclusively with c1-inh concentrate if possible on a named-patient basis through personal importation programs. in their experience, this approach should also be feasible for women of childbearing age and all other patients with hae who cannot tolerate the side effects associated with danazol treatment. methods. between 1987 and 2000, ninety patients were registered in the hungarian hae center database. what follows is a review of the clinical data of 26 pediatric patients from 19 families. the male to female ratio was 11:15, and the age at time of diagnosis ranged from 2.5 to 15 years (mean, 7.3 years). ch 50 total complement levels were measured by the standard hemolytic titration method, using sensitized sheep erythrocytes. 425 the c4 complement fraction was determined by single radial immunodiffusion using anti-c4 antibodies (atab, stillwater, minn), and results were expressed as the percentage of standard, normal serum values. 426 the c1-inh concentration was also measured by radial immunodiffusion; c1-inh activity was measured by kinetic assay using a commercially available diagnostic kit (behringwerke ag, marburg, germany). the activity of the c1-inh in the serum was determined by photometry and expressed as the percentage of standard, normal values. in the follow-up protocol, the first occurrence, frequency, and localization of clinical manifestations, duration of the disease, as well as potential precipitating factors of edematous attacks were ascertained. d a pedigree analysis was performed in all cases. d long-term prophylaxis was elected if edematous attacks recurred frequently (!1 attack per month) or a life-threatening episode could be identified in the history. d the following tests were performed in patients receiving long-term danazol prophylaxis: -anthropometric assessment of growth (comparison of body height and weight development to agespecific normal values). -bone age determination (hand x-rays). -development of secondary sexual characteristics and time of puberty compared with control population. -mental development (performance at school). d short-term prophylaxis was administered before surgical or diagnostic procedures performed on the head and neck. d changes in serum complement fractions and clinical manifestations were monitored during therapy. potentially drug-related adverse effects of treatment were also recorded. d in patients undergoing long-term prophylaxis, laboratory tests (complete blood count, liver function tests, creatine kinase activity, and urinalysis) were repeated at 3-month to 4-month intervals, and abdominal ultrasound was performed semiannually during the first 2 years of treatment. subsequently, asymptomatic individuals or patients with mild symptoms were checked every 6 months to detect potential liver damage. d patients with an acute attack of submucosal (laryngeal or severe abdominal) edema were always hospitalized and abdominal ultrasound performed if the attack involved the gastrointestinal tract. hospitalization for an acute abdominal attack was considered justified when the presence of other acute abdominal pathologies could not be ruled out with certainty and/or manifestations were severe-that is, the attack was associated with obvious signs of hypovolemia (paleness of skin, prostration, dehydration, tachyarrhythmia) and included recurrent paroxysms of acute colicky pain unresponsive to symptomatic therapy, nausea and vomiting, or profuse diarrhea. d the c1-inh concentrate used in this study was screened for hepatitis b and c by the manufacturer. nevertheless, in observance of the safety precautions pertinent to blood products, hbsag and hcv specific antibody were tested by using commercially available immunoassays in all patients who had received c1-inh concentrate. acute edematous attacks were treated by administering c1-inh concentrate (berinert p injection; centeon, vienna, austria), and e-aminocaproic acid (acepramin; pannon pharma, pecsvarad, hungary), tranexamic acid (exacyl; sanofi synthe labo-chinoin, budapest, hungary), and danazol (danoval; krka, novo mesto, slovenia) were used for prophylaxis. results. the initial manifestations of hae were observed within an age range of 2.5 to 12 years (median, 5.9 years). six children were asymptomatic at the time of diagnosis, which was established only after hae was ascertained in their symptomatic parents. pedigree anal-ysis revealed familial occurrence of the disease in 24 cases (from 19 families) but failed to identify hae in firstdegree relatives of 2 children; therefore, in these patients, c1-inh deficiency must have resulted from a new c1nh mutation (table xiv) . the localization of clinical signs was variable. in the majority of patients (20 children; 76%), edema formation involved subcutaneous tissues, whereas gastrointestinal manifestations occurred in 14 patients (53%) and laryngeal edema developed in 7 patients (26%). subcutaneous edema of the extremities or genitals usually persisted for 2 to 3 days, then resolved spontaneously. edematous swelling of the skin was not accompanied by itching but caused an unpleasant sensation of distension within the involved region. in 8 children, generalized, nonpruritic skin rash (erythema marginatum) was observed before and during edematous attacks of hae. the exploration of potential precipitating causes invariably identified mechanical trauma; however, upper airway infection was another important factor (table xv) . according to the results of serum complement studies, 21 children had hae type i, whereas 5 had hae type ii (table xvi) . all children with an acute edematous attack manifesting as laryngeal edema or severe acute abdominal complaints were hospitalized. emergency treatment was implemented by the parenteral administration of 500 u c1-inh concentrate exclusively (table xvii) . the injection accomplished substantial symptomatic relief within 30 minutes to 1 hour. this was accompanied by the regression of edematous swelling; however, complete resolution of clinical signs and symptoms took 24 to 48 hours. administration of an additional 500 u dose (ie, a 1000 u cumulative dose) was necessary in 2 patients (1 with laryngeal and diffuse facial edema, 1 with acute abdominal attack) to achieve a satisfactory rate of improvement. all patients hospitalized with an acute abdominal attack underwent abdominal ultrasound examination. ascites was invariably ascertained in all cases, whereas edema of the intestinal wall was detected in 80% of patients (fig 7, a) . as evidenced by follow-up ultrasound, appropriate treatment (c1-inh concentrate) achieved complete disappearance of free peritoneal fluid and edematous swelling of the intestinal wall within 48 hours. long-term drug prophylaxis with antifibrinolytic agents (tranexamic acid or e-aminocaproic acid) was initiated in 11 cases because of frequent (weekly or monthly) recurrence of edematous attacks or the presence of life-threatening attacks in the history. complete remission was achieved in 3 girls maintained on 1 to 2 g/d tranexamic acid; however, antifibrinolytic agents were ineffective in 8 cases. transferring these patients to treatment with 100 to 200 mg danazol/d completely eliminated serious edematous attacks (table xvii) . to taper the cumulative dose, the dosage interval of danazol (100 mg) was increased to 2 to 3 days after 6 months of clinical remission. switching to this intermittent regimen (ie, doses repeated every other day or at 3-day intervals) from continuous dosage was successful in 4 cases. in patients maintained on long-term prophylaxis, doubling the daily dose for several days aborted prodromal symptoms or prevented the progression of mild clinical manifestations to full-blown attacks. a similar protective effect was observed in cases exposed to a precipitating factor (eg, upper airway infection or mechanical trauma). no adverse effects potentially related to long-term prophylaxis necessitating the withdrawal of treatment were observed. the bone age, skeletal growth, and weight development were not substantially different from the agespecific averages of the hungarian pediatric population. the age at appearance of secondary sexual characteristics and the onset of puberty were also similar to the national average in 7 children, but in 1 girl, menarche was delayed (to the age of 19 years), and her menstruation was irregular. hirsutism was not observed in patients receiving long-term danazol prophylaxis. monitoring of laboratory parameters revealed normal values in all 11 children tested, and abdominal ultrasound showed no abnormalities. the efficacy of danazol treatment was demonstrated by the significant increases of c4 and c1-inh concentrations in the serum (fig 27) . nine children had only mild, subcutaneous symptoms, and even these resolved spontaneously within 2 to 3 days. accordingly, long-term prophylaxis was withheld, and treatment was administered only when prodromal symptoms and signs occurred. in 4 patients, tranexamic acid (2 g/d) given for 2 to 3 days reduced the severity and duration of subcutaneous manifestations. follow-up visits were scheduled at 6-month intervals for these children. short-term prophylaxis was necessary in 2 boys (age 11 and 14 years) undergoing dental extraction, because both had facial and laryngeal edema precipitated by similar procedures in their history. danazol (300 mg/d) initiated 4 days before and continued 4 days after dental treatment successfully prevented edema formation. the elimination of known precipitating factors also substantially reduced the incidence of edematous attacks. discussion. in 80% of hae cases, a known family trait of the disease facilitates diagnosis. in the remaining 20%, in which no afflicted relatives can be identified, hae results from a new gene mutation. 393 in the hungarian pediatric population analyzed by the authors, the proportion of such cases was 93% and 7%, respectively. according to the literature, 85% of patients with hae have type i and 15% have type ii disease. 31 the percentage distribution of type i and type ii cases was 81% and 19% in the hungarian series. in general, the clinical manifestations of hae first develop before 6 years of age 7 ; the median age of the hungarian study population was 5.9 years. however, no occurrences have been observed in newborns, and only 1 infant (19-month-old) case has been reported in the literature. 320 according to the experience of the authors, adolescence is often associated with substantial changes in the activity of the disease, particularly in girls, warranting closer follow-up during this period. in particular, clustering of edematous attacks during the menstrual period was observed in 4 girls. nowadays, contraception is initiated in early adolescence. however, ocs can precipitate acute attacks, and therefore, these agents are not recommended for girls with hae. 7 the incidence and severity of characteristic manifestations show substantial interindividual variation. less than 5% of patients with hae are asymptomatic, and 25% had symptoms only sporadically. manifestations of the disease are common in 70% of patients and severe in 30% of this subpopulation. 7 eleven of the 26 children followed up in this study had frequent and severe attacks. usually, mechanical trauma is reported as the most common precipitating factor. through their experience, the authors confirm this and identify upper airway infection as another potential triggering factor. considering the importance of physical activity and sports on growth and development, restrictions should be determined carefully and individually. in children, as in adults, edema of the extremities is the dominant manifestation; furthermore, abdominal attacks are more common than laryngeal edema. 7,31 nevertheless, even rare occurrences of laryngeal edema should be considered real emergencies. because of the small diameter of upper airways in children, relatively mild swelling of the mucosal lining causes substantial obstruction, and suffocation can therefore rapidly ensue. in view of these risks, referral to intensive care is justified to monitor the clinical course and ascertain the efficacy of treatment. in some patients, repeated administration of c1-inh concentrate may be necessary to achieve satisfactory control of edematous manifestations. this fact and the ineffectiveness of conventional agents (eg, antihistamines, corticosteroids, and epinephrine) account for the invariably high mortality of the disease and emphasize the importance of accurate diagnosis. the striking similarity between the clinical manifestations of abdominal hae attacks and surgical emergencies is an apparent diagnostic pitfall. failure to evade this trap often leads to unnecessary interventions on patients already prostrated by the consequences of the acute edematous attack. 36, 427 the authors reviewed the medical history of their patients before diagnosis, revealing that 2 children had undergone exploration or laparoscopy during an acute abdominal attack, 1 of them twice. these interventions were clearly unwarranted, because no abnormality other than the edematous swelling of intestinal wall was ascertained. to avoid this diagnostic pitfall, the authors recommend close monitoring of the patient, volume replacement, parenteral medication, and assessment of therapeutic efficacy, all achievable by observing the patient for 24 to 48 hours. abdominal ultrasound is a noninvasive and readily accessible diagnostic test that yields reproducible results, features particularly advantageous in pediatric practice. 39 the authors identify free peritoneal fluid and edema of the intestinal wall as invaluable sonographic clues for distinguishing abdominal hae attacks from other pathologies commonly associated with ascites formation (such as intra-abdominal inflammation, autoimmune disease, tumors, and so forth). they recommend that, when all other acute abdominal disorders had been ruled out, c1-inh concentrate should be administered to afebrile patients without laboratory abnormalities, particularly to those with known hae. prompt symptomatic relief (or lack thereof) after c1-inh concentrate administration can then confirm or refute the tentative diagnosis. during life-threatening hae attacks, appropriate measures can include the administration of ffp. this preparation contains c1-inh; however, its use may be associated with alloimmunization or the transmission of infections. 7 the most appropriate therapy is the intravenous administration of c1-inh concentrate. its dosage is the same for children and adults (ie, 500-1000 u). 320 in this study, acute hae attacks were treated with c1-inh concentrate exclusively. this approach was highly effective, resolving clinical symptoms within an hour, and was not associated with adverse effects. c1-inh concentrate has been suggested as an ideal agent for permanent substitution, particularly for pediatric patients. unfortunately, the short (72-hour) half-life and high cost ($310 per vial) of the concentrate preclude its use for continuous administration. 428 furthermore, c1-inh concentrate is a blood product, and the theoretical risk of blood-borne infections cannot be completely excluded. nevertheless, viral (hiv, hbv, hcv) safety of the concentrate is promoted by heat treatment as well as the establishment and continuous validation of safe plasma pools. 75, 334 in the hungarian in pediatric patients, antifibrinolytic agents are the first choices for long-term prophylaxis because their safety profile is more favorable than that of attenuated androgens. 7, 31, 36, 429 nevertheless, antifibrinolytic agents can cause adverse effects such as muscle weakness, myalgia, elevation of creatine kinase activity, vascular thrombosis, postural hypotension, and myonecrosis. 31 the experience of the authors showed tranexamic acid to be better tolerated than e-aminocaproic acid, which often caused gastrointestinal discomfort. however, when these agents fail to achieve satisfactory improvement, treatment with attenuated androgens (eg, danazol, stanozolol) is necessary. 312 numerous studies performed on adult patients have demonstrated the efficacy of 17a-ethyltestosterone (danazol) manifested by the substantial relief of symptoms as well as the elevation of serum c4 and c1-inh concentrations. 347 their mechanism of action has not yet been fully elucidated, and limited experience with their pediatric use has been obtained only in patients treated for idiopathic thrombocytopenic purpura. 430 potential adverse effects of danazol include weight gain, myalgia, headache, tremor, libido changes, elevation of serum transaminase levels, microhematuria, menstruation irregularities, and hirsutism. 349 in addition, 2 reports have been published on hepatocellular adenoma formation observed in adults undergoing long-term, high-dose danazol therapy. 353 nevertheless, using the lowest effective maintenance dose and intermittent dosage regimens can prevent adverse effects that would require the withdrawal of treatment. in the study population, the longest duration of danazol prophylaxis was 11 years. no drug-related adverse reactions or impairment of growth was recorded in these pediatric patients, with the exception of 1 case of delayed menarche with subsequent irregular menstruation probably caused by long-term treatment with 200 mg/ d danazol. compared with age-specific percentiles of the hungarian population, somatomental development was normal in all cases. treatment prevented edematous attacks and elevated c4 and c1-inh concentrations significantly. effective therapy eliminated the need for frequent hospitalizations for acute symptoms-along with their unfavorable psychologic sequelae-and thereby improved the patients' quality of life. the authors emphasize the essential importance of close follow-up, with appropriate tests performed at regular intervals. the diagnosis of hae can be made as early as at birth by assessing c1-inh antigenicity and function. 320 prenatal screening is not yet feasible. importantly, baseline complement concentrations are of no use for predicting the severity and frequency of future attacks; for example, some patients are asymptomatic during a patient's first 2 years of long-term prophylaxis, laboratory tests should be performed every 3 to 4 months and abdominal ultrasounds scheduled at 6-month intervals. the clinical course of hae and reactions to treatment should be monitored to recognize potential adverse effects and ascertain the rate of somatomental development as well as the need for therapy modification. in view of their favorable experience, the authors suggest that danazol administered in the lowest effective dose seems to be well tolerated by pediatric patients and is probably appropriate for long-term therapy. nevertheless, adverse reactions can occur sporadically. in particular, 1 case of delayed menarche and irregular menstruation was observed in this series; however, the abnormality was readily reversible on discontinuing danazol treatment. short-term prophylaxis is indicated for patients undergoing surgical or diagnostic interventions of the head and neck region. such procedures most commonly include dental procedures and tonsillectomy, but the potential consequences of endotracheal intubation for general anesthesia must also be emphasized. 365, 431 although short-term prophylaxis is required less frequently for children than for adults, antifibrinolytic agents or attenuated androgens provide adequate protection if administered in higher than usual doses. nevertheless, prophylaxis with c1-inh concentrate is the method of choice in patients with a history of severe attacks precipitated by similar procedures. 432 in addition, follow-up should also include the education of patients and their parents, as well as advice on the most suitable means of lifestyle modification. the activity of patients' self-help groups can lend vigor to such efforts. early health education and meticulous follow-up initiated in childhood can successfully prevent social stigmatization and guarantee an improved the quality of life for adulthood. it is prudent to provide patients with hae with a medical information card summarizing essential knowledge and methods of emergency help in several languages. when justified by their history, patients should be supplied with c1-inh concentrate to be kept at hand, ie, in the refrigerator at home, for emergencies. the data contained on the medical information card can prove an extremely useful aid for medical professionals relatively unfamiliar with hae and help to ensure patients' safety. this article first appeared in a slightly different form as although all patient and physician groups share the common goals of increased knowledge of hae and related disorders and optimized therapy, patient and physician experiences differ among countries. the following accounts demonstrate the range of perspectives, treatment options, and concerns found in europe and the united states, concluding with a short history of past c1-inh deficiency workshops in which representatives from these and other countries have met to further their common aims. an international approach to patient registries (marco cicardi, md,* and john jakenfelds, md, milan, italy, and chalfont st. giles, uk) the concept of a european patient registry grew out of the first c1-inh deficiency workshop. in this section, cicardi and jakenfelds describe the process of creating the european hae register and how the register will work. in 1999, the european group of patients, clinicians, and scientists interested in hae met for the first c1-inh workshop in visegrád, a hungarian town near budapest. workshop members identified the creation of a register containing the clinical and laboratory data of patients with hae as an obvious first step. there is a real need for a better insight into the full presentation of hae, the different ways it is managed, and the various outcomes (including clinical, functional, and economical variables) associated with different disease management strategies. currently, there are significant differences in the way patients with hae are treated in various hae expert centers. there is no consensus on the state-of-the-art treatment of hae, and there is a lack of data to develop uniform, evidence-based treatment guidelines. as a result, an enormous gap exists between resources already or potentially available and their exploitation. furthermore, the low awareness of this disease causes only a minority of patients to reach correct diagnosis and treatment. small and dispersed case lists and the lack of epidemiologic data lessen the value of clinical studies and discourage public and private investment in research aimed at developing new therapeutic strategies or tailoring existing ones to hae. given this situation, an international patient register could play a significant role in the development of a better understanding of hae and its management. first steps. two pharmaceutical companies, pharming and baxter, encouraged the establishment of an hae register in 2001. baxter already had a product for the treatment of the disease (plasma-derived c1-inh) in some european countries, and pharming was developing a recombinant version (rhc1-inh). pharming became the major contributor until 2002, when a group of hae experts from 10 different european countries received a grant from the european commission for a project called prehaeat, consisting of a concerted action in the framework of the specific research and technologic development program, quality of life and management of living resources. establishing a european register for hae was the first work package of the project and was started accordingly by using the pharming-funded register as a starting point for further refinement. in this new register, physicians will enter completely anonymized patient data into a web-based data capture tool. princeton healthcare will be responsible for the central server and software, accessible by all sites and individuals wishing to enter data (fig 28) . princeton is registered under the uk data protection act to store personal information as part of its ongoing work with clinical systems, which have been in use for several years. the register will work by using the internet. by connecting to the web and entering the site's name, the password-protected entry screen will be viewable (fig 29) . on filling out details, the user will be taken to a menu of options, including the entry of new data or the viewing of reports covering anonymized patient records (either individual or for the group under treatment at the clinic). only the treating physician, by knowing a code, will be able to link an individual report to an individual patient. access initially will be restricted to the physicians participating in the concerted action and eventually will be open to any hae-treating physician on request. efforts will be taken to publicize maximally the existence of the register. access to data: ownership and management. each patient owns his or her own data. in order for the data to be entered into the register, patients must give consent to share their anonymized data. all clinicians contributing to the register will have access to their own data as individual or group reports. the board, consisting of the participants to the concerted action and of 2 patient association representatives, will manage the anonymized database and may access aggregated reports but not individual data sets. no single member of the board can use or publish the data without the consent of the board and the contributors as a whole. the goal of the register is to develop a better understanding of the current benchmarks of care for hae and the outcomes that result to improve the ways in which patients are treated. for each patient, there will be an initial entry form and 1 or more follow-up visit forms (fig 30) . the initial entry form is structured to capture the natural course of hae in a patient. it will contain the past history before diagnosis of hae was recognized and/or specific treatment established. it will contain anagraphic data, information on characteristics and severity of symptoms, presence of associated diseases, and laboratory data at the time of diagnosis. the follow-up visit forms can be replicated indefinitely. they will collect the same data, updated according to the changes occurring in the course of the disease, and detailed data on the different therapies. specific care has been taken to record effectiveness and side effects of hae treatments. expected achievements and perspectives. this register is expected to become a reference database in hae. efforts have already begun to promote the harmonization of similar data collection systems on this topic around the world such that compatible formats will allow pooled data analysis. information describing the natural history of hae and the effect of treatment should be obtained. this database should also be used to provide policy makers and the pharmaceutical industry with data showing exactly how the disease affects the lives of patients who have it. the absence of an approved hae acute attack therapy in the united states of america represents a catastrophic unmet medical need. patients are suffering needlessly and in some cases are dying. indeed, the united states hae association (haea) is aware of 4 hae-related deaths over the period of the past 40 months. the most pressing near-term goal of the haea is to work with industry, the research community, and regulators to encourage usbased clinical trials that will result in licensure of an acute attack therapy. clearly, the lack of a therapy to treat an hae attack once it has begun poses a tragic and unnecessary risk to every us hae patient's life. fortunately, a phase ii clinical trial for an acute hae attack therapy sponsored by dyax corp is now up and running in the united states. the patient community is excited about the prospects for dyax's therapy on the basis of some promising safety and efficacy data developed during phase i and ii trials conducted in europe. the haea is working closely with dyax to recruit patients and complete this vital clinical trial. if the us phase ii results are as promising as results seen in phase i/ii european trials, the researchers hope that dyax will request an expedited licensing review by the food and drug administration. the haea is committed to ensuring that patients, industry, and the food and drug administration work together and strike an intelligent balance that permits an expeditious path to meeting a catastrophic unmet medical need while simultaneously protecting the public health and safety. meetings such as the one in budapest have provided the opportunity for patients with hae to interact and share information. this networking has spawned a movement that should soon result in the creation of a legally recognized international hae patient organization. clearly, an international hae organization can provide the world's hae groups with information, analysis, and guidance on key issues regarding hae management, diagnosis, and the direction of future research. indeed, researchers active in establishing an international hae group have already begun the process of examining the state-of-the-art for hae treatment in different parts of the world. this process has uncovered some strongly held views among the world's patients who are not satisfied with the current treatment options and standards. this drumbeat of concern is prompting international leaders to re-examine the hae treatment paradigm that almost always features androgens for prophylaxis, with acute attack therapy (c1-inh concentrate) administered only for gastrointestinal and/or life-threatening attacks. there is some very exciting and innovative work going on in germany that quantifies patient dissatisfaction with the current treatment norm. specifically, the german data show that when given an option, many patients discontinue androgens and opt for on-demand (usually homebased) treatment with an acute attack therapy that they infuse during the attack prodrome. in sum, the treatment of hae in the united states is best described as medieval. without access to an acute attack therapy, patients must rely exclusively on prophylaxis with androgens. these drugs are highly toxic and have harsh side effects, particularly in women. the haea is working hard to assist companies interested in licensing an acute attack therapy in the united states. there is much hope that once formally established, an international hae organization will help to create and rationalize a partnership between the world's hae patients, researchers, regulators, and industry to ensure new therapies can be expeditiously tested, approved, and marketed. the hungarian experience: assisting the few to live a better life (istván nagy, arianna kitzinger, and henriette farkas, md, phd,* székesfehérvár, sopron, and budapest, hungary) in this section, hungarian physician farkas and patientresearchers nagy and kitzinger describe the importance of the hungarian hae center and its infrastructure, as well as their case series and approach to hae. creating an infrastructure. the hungarian complement laboratory in budapest was founded by prof. george füst in 1970. the first article detailing 3 hae patients' cases was published in 1972. 433 these achievements were followed by several milestones leading to the establishment of the hungarian hae center: d accumulating relevant knowledge from the international literature, adapting foreign experiences to domestic conditions, and developing hungarian protocols for clinical work-up and treatment d undertaking retrospective screening for patients by using archived laboratory test results (complement values) d communicating pertinent knowledge to medical professionals, graduate and postgraduate education programs, and others via publications d establishing a results-based referral system: if laboratory complement measurements are suggestive of hae, accessibility information for the hungarian hae center is printed on the laboratory test slip the development of research ran in parallel with the increasing number of patients diagnosed with hae. fig 31 illustrates the rate of case accrual. currently, there are 96 patients in hungary (total population: 10 million people), of whom 28 live in budapest and its suburbs. hungarian hae center. the headquarters of the hungarian hae center are located on the premises of semmelweis university in budapest. each facility has its assignments, as shown in fig 32. assignments of the allergy and angioedema outpatients' clinic at semmelweis university include the following: the complement laboratory functions as a unit of the hae center and is properly equipped to perform total complement profiling (ch 50 , c4, c1q, c1-inh concentration and functional activity, anti-c1-inh antibodies, anti-c1q antibodies). the molecular biology laboratory and the research laboratory of the third department of internal medicine conduct genetic testing and mutation analysis on all patients with hae. furthermore, the center participates in undergraduate and postgraduate education and also pursues scientific activities. case management standards. the hungarian algorithm for hae treatment, in the light of its symptoms, therapy, and management is as follows: d mild or infrequent subcutaneous edema does not require special treatment. self-management of symptoms will suffice, and medical monitoring can consist of yearly complement measurements. d severe or frequent subcutaneous and submucosal edematous symptoms are treated by either long-term prophylaxis (danazol, e-aminocaproic-acid, tranexamicacid) or short-term prophylaxis (danazol, e-aminocaproic-acid, tranexamic-acid, c1-inh concentrate). such patients require regular medical and self-control. medical control involves liver function monitoring and blood cell and complement measurements twice yearly. d in case of acute abdominal or laryngeal edematous attacks, inpatient therapy with c1-inh concentrate is needed. patients experiencing these symptoms also require regular medical and self-control. postattack follow-up is necessary. hungarian hae patients' association. established in 1996, the hungarian hae patients' association organizes annual meetings at which patients can meet doctors and exchange views with each other. a foundation has been established to support patients with hae, and a hungarian hae web site is now being created. the hungarian hae patients' association recently collected 26 patient diaries, drawing attention to topics important among the patient population. the association is now beginning to make a systematic survey of social background and its effect on the disease, which ideally will lead to a deeper understanding of hae and an improvement in patients' quality of life. the patients' association maintains fruitful relationships with foreign patients' self-help groups. as with all uncommon diseases, the elucidation of its epidemiologic features and pathomechanism, the establishment of current diagnostic algorithms, and the development of effective and safe treatment protocols all require international effort. this recognition led to the establishment of the european c1-inh deficiency working group in 1998. during its first conference held in rhodes, greece, this professional organization identified accomplishing these goals as its ultimate objective. since that time, the hungarian hae work group has been the main organizer of the european c1-inh deficiency workshops, the basis for this supplement. much progress has been made since the first steps taken in the 1970s. in addition to the results described, the association is glad to welcome several humanitarian changes. patients with the diagnosis of hae are now under regular medical control, in contrast with perpetual uncertainty of the past. as a result of patient education and counseling (ie, sufficient familiarity with the disease), patients are understood and helped more effectively by those in their environment. instead of isolation, patients feel integration, trust, and confidence: a major change and an impressive outcome in the case of this life-threatening disease. as shown in table xviii , all of this progress has helped patients to find an appropriate lifestyle. however, without doctors' professional knowledge and its perpetual updating, these developments in hungarian hae research could not have been achieved. many doctors regard their job as their hobby and also feel a commitment to help at both the domestic and international levels. their compassionate care for patients is both noticed and appreciated by those in need. portions of this article appeared in farkas h, varga l. the hungarian hae experience. transfus apheresis sci 2003;29:229-33. they are reprinted with permission. the german experience: hae and selfmedication with berinert p (ursula rauch, aldenhoven/siersdorf, germany) in this section, rauch, a member of the german hae patient association, hae vereinigung e. v., writes of her experiences as she presented them at the 2003 c1 esterase inhibitor deficiency workshop in budapest. the german hae patient association's motto is nicht mehr allein, ''no longer alone.'' to introduce myself, i would like to tell you a little bit about my career as an hae patient. i think all the hae-affected here have had the same or similar experiences. so, here is the short version of my story. i am 44 years old and have hae-i. i had my first attack at the age of 1½, diagnosed as allergy; it was followed by tests and experiments with antiallergic remedies and corticoids, which were, of course, useless. there was an increase in my attacks during puberty, and i received the correct diagnosis at age 16. there was no remedy at that time, but it was a relief to know it was hae and to stop the antiallergy experiments. sometimes i went for 2 months without an attack; sometimes i would spend 2 weeks with a permanent attack; but to a certain degree i managed to arrange my life around my disease. but after my second pregnancy, the attacks greatly increased. i often needed help to care for my children. i became depressed and could not hold down a job because i was ill too often. not 1 week went by without an attack, and i often had nonstop attacks that lasted 8 to 10 days. in 2000, i finally found our support group, got a lot of information, and was told about the hae center in frankfurt. i made an appointment at the university hospital in frankfurt, where i received very good counseling, and 4 weeks later i went to my first hae group meeting. with the help of the physicians of the university hospital in frankfurt and my practitioner, i learned to infuse myself in an emergency. so i was much more independent and felt much safer than before. after training with normal saline, when i tried my first injection, i went to my practitioner in order to have a supervisor-and it did work! and today? no endless pain, vomiting, and breakdowns of my circulation. no waiting for emergency medical personnel, especially on saturday or sunday, or at night. no more useless claims that corticoids always help, even though my emergency passport says differently. no hurrying to a hospital, breathless and in panic, because of a laryngeal edema. no waiting there for a tired physician who already has 30 hours of work behind him. no enduring questions about whether my husband could have beaten me because my face looks just like a boxer's. i was able to participate in further professional education. i look after my children myself. i can again go on vacations, which always seemed too dangerous before. and i am active in the patient group as much as i can be. i learned how helpful and important it is to work together and fight for a better and more effective therapy. to me, it is absolutely incredible that in most countries, families do not have even 1 package of c1 inhibitor at home for emergency cases, and that there are doctors who give androgens to children, even though they are still growing and the doctors should know how dangerous these androgens can be. and it is horrible that a lot of affected people are still misdiagnosed and therefore constantly living in danger. we must create the opportunity for every hae-affected person to get the right diagnosis and the same help, so that children can grow up as normally as possible, without missing school very often, and so that we all can go to work and enjoy life-just like the nonaffected, without horrible pains and horrible fears about the risk of suffocating to death or dying of pain. we are a minority-in every country-but together we are strong enough not to be stopped. in recent years, patient associations have had a fundamental role in the fight against rare diseases. many organizations now exist in various parts of the world, even for hae, to perform the important task of sharing information and maintaining a connection between patients and doctors. such associations perform indispensable functions, useful to both doctors and researchers. aims. aaee was founded as a nonprofit voluntary association in milan on march 29, 1980 , and has the following aims: d to spread knowledge of the disease and allow correct diagnosis d to make high-quality medical care and adequate treatment available to all patients d to issue an officially recognized health identification document describing the disease and its emergency therapy and containing the contact information of doctors who can be called for further information d to promote social and scientific meetings and congresses at a national and international level d to encourage meetings and exchanges among patients, between patients and doctors, and among doctors services. aaee represents approximately 400 members and, in more than 20 years of activity, has created a series of services to benefit patients: d in collaboration with the doctors of the diagnosis and cure center in milan, a 24-hour emergency telephone number was activated. d all patients have an emergency identification document indicating their diagnosis and its suggested treatment in more than 1 language. d a direct link has been established with the pharmaceutical company that produces c1-inh concentrate to locate this life-saving medicine at any time. d the minister of health and welfare has added hae to the list of rare diseases for which patients are exempted from paying for medicine. d a multilingual internet site (http://www.angioedemaereditario.org) offers medical and scientific information, useful addresses, and contact information for all hae associations worldwide. d regional representatives permit a more direct connection between patients and the association. d gynecologic and dental consultations are available. d to spread medical and scientific information as well as the association's activities, radio and television broadcasts have been organized. d every year, a national meeting is held in addition to regional meetings to exchange information between doctors and patients and between patients and the association. d some patients who have social roles in the community offer their experience and assistance to the association to deal correctly with various bureaucratic problems. d there are now 3 other centers in addition to the 1 in milan available to patients who need treatment, in rome, palermo, and civitanova marche. recent initiatives. recently, the association prepared a very simple form to collect data on the frequency of hae attacks in italian patients. information will be collected over the period of the next 6 months as a brief trial. this will help aaee evaluate replies received from patients to make all necessary modifications and adapt the survey as much as possible to each involved country's needs and requirements. data will be collected in a proposed specific database. to make better use of the information, the aaee intends to put their internet site at everyone's disposal. in the last few weeks, the international pages have been modified, transforming them into a very simple but functional place where hae information and links throughout the world can easily be found. with the representatives of other associations, the members of aaee are working to create an international umbrella organization. after last year's short meeting on hae in palermo, where the first ideas were elaborated and after the budapest workshop, an international statute proposal now exists. during the last meeting, which took place in milan on october 4 and 5, 2003, an international committee and a medical panel of doctors and researchers were formed. hereditary angioedema and related disorders are characterized by acute episodes of unexplained swelling that are not responsive to antihistamines or corticosteroids. attacks may manifest in 1 or more locations, most frequently the upper airways, face, hands, feet, genitalia, or intra-abdominal contents. untreated laryngeal swelling can result in death; angioedema of the small intestine can result in obstruction and mimic an acute abdomen. ultrasound may be useful to distinguish between abdominal pain attributable to hae and a true surgical emergency. occasionally, erythema marginatum or a similar, nonwhealing, nonurticarial rash may precede angioedema attacks; at other times, there may be no warning. episodes of hae may present as early as late infancy and may vary, even within the same individual, in frequency, severity, and location. attacks typically resolve within 72 hours but may persist as long as 5 days. aae, although much rarer than hae, often follows lymphoproliferative disorders or occasionally autoimmune, neoplastic, or infectious diseases. drug-induced angioedema may closely follow initiation of a drug regimen (often an antihypertensive agent) or appear many months later, whereas estrogendependent angioedema seems to follow initiation of pregnancy, ocs, or hrt more immediately. hereditary angioedema types i and ii are associated with various mutations to the c1nh gene, located on chromosome 11. a frequently updated online database of these mutations is available to the public at http:// hae.biomembrane.hu; at the time of writing, 150 different c1nh mutations were known. the progression from low functional c1-inh concentrations to attack genesis is still under debate; however, bradykinin has been identified as a mediator of angioedema attacks, and, unlike other proposed mediators, has been found to correlate with attack localization in early studies. nonetheless, this nonapeptide is unstable and cannot be measured via routine laboratory analyses. it is hypothesized that the local generation of bradykinin results in regional increases in vascular permeability. the presence of other mechanisms, intermediaries, and moderating factors has not been discounted. case reports have shown that in some women, the occurrence or frequency of hae attacks appears to be influenced by estrogen. in these patients, puberty, pregnancy, or taking estrogen-containing ocs or hrt may initiate attacks. patients have also been described with normal concentrations of functional c1-inh who nonetheless have angioedema attacks in response to increases in estrogen. bork et al found that approximately 62% of women with hae-i or hae type iii have a worsening of their attacks because of pregnancy or exogenous estrogen. laboratory diagnosis c1-inh antigenic and functional assays are sufficient to diagnose hae-i, hae-ii, and aae. low c4 concentration is often a reliable confirmation but is not exclusive to c1-inh deficiencies. currently, no routine laboratory methods can detect estrogen-dependent angioedema (formerly hae type iii). ideal prophylactic regimens are highly individualized and should consider the severity and frequency of the patient's attacks. 17-a alkylated androgens, antifibrinolytics, or c1-inh concentrate have been used successfully for hae maintenance prophylaxis and/or short-term prophylaxis surrounding surgical or dental procedures. bork et al describe c1-inh as an effective, if less frequently used, prophylactic agent. in some patients, antifibrinolytics may be sufficient to prevent attacks; however, attenuated androgens more often reduce the frequency and severity of attacks. patients receiving longterm androgens, especially pediatric patients, should be monitored for undesirable hepatic effects and other consequences such as virilization or delayed menarche; farkas et al suggest laboratory work-ups at intervals of 3 to 4 months. androgen overdosing is to be avoided; varga and other members of the workshop laboratory diagnosis panel note that, in hae-i, antigenic c1-inh concentrations approaching 35% to 40% of normal are often sufficient to prevent attacks, and therefore, aiming steroid prophylaxis to achieve a 50% antigenic concentration subjects patients to an unnecessary risk. for the prophylaxis of aae, antifibrinolytic agents are recommended because patients with this condition are frequently resistant to attenuated androgens. danazol is a common prophylaxis for patients with hae who have frequent, severe angioedema attacks. despite known associated adverse effects such as virilization, weight gain, and decreases in hepatic function, the administration of danazol may result in an improvement of some conditions. contrary to findings in patients receiving high-dose danazol treatment for endometriosis, kollár et al reported that patients with hae receiving the minimum effective dose of danazol necessary to control their angioedema attacks did not have osteoporosis. indeed, their data suggest that such a danazol regimen may have a protective effect against bone loss. perricone et al noted a higher prevalence of polycystic or multifollicular ovaries in women with hae but found that danazol administered in hae-management doses, at least in a small sample of patients, improved ovarian condition. treatment varies depending on the location and severity of angioedema. peripheral edema often requires no treatment. for attacks involving the airways or severe abdominal manifestations, c1-inh concentrate is the current acute attack therapy of choice for patients with hae. in countries where this is not available, personal importation of c1-inh concentrate may be an option; alternatively, the administration of ffp or an antifibrinolytic, although less effective, may be of some benefit in emergencies. supportive measures such as rehydration, antiemetics, and pain control may be necessary depending on the site and severity of the attack. new uses for existing therapy, such as prophylactic or home administration of c1-inh concentrate, have been studied on a small scale and proven successful. new therapies, including 2 versions of recombinant c1-inh, a kallikrein inhibitor, and a bradykinin receptor-2 antagonist, are in development. (marco cicardi, md,* milan, italy) since the first comprehensive clinical description of hae in 1888, knowledge of the disease has been expanding. as have many other genetic defects, hae has benefited in the last 20 years from the spread of molecular biology, which brought a tremendous advance in the understanding of the molecular basis of the disease. however, what appears remarkably unique to hae is that within the last 4 years, this bulk of information has been directly transferred into therapeutic approaches. the dream of research from the bench to the bedside is becoming real in hae, and the rule that there is no interest in developing drugs for treating a small number of patients seems to have vanished. revolutionary new techniques for identifying and producing drugs, such as peptide selection from phage display libraries and transgenic animals as source of recombinant proteins, have been implemented to obtain agents active in hae. currently, 3 completely new compounds, a kallikrein inhibitor, a bradykinin receptor antagonist, and a recombinant c1-inh, are under clinical evaluation for the treatment of hae. this disease is facing the new pharmacology. chemicals antagonizing biologic systems are being substituted with disease-tailored agents, and in hae, this does not necessarily mean gene therapy. this close relationship between a pharmacologic agent and a disease results in the need for a close relationship between patients and industry. in 1999, the first c1 esterase inhibitor deficiency workshop was held in hungary with the idea of having patients, scientists, physicians, and industries contributing with equal authority. since that time, a real hae community has been created, and patient support groups are actively involved in designing and carrying clinical studies and are supporting initiatives, such as the hae register, aimed to improve the understanding of the disease and its treatment. aside from the new scientific knowledge or the new therapeutic compounds, recent years' frank and open discussion between different parties is a major achievement for the advancement of the hae cause. many sections of this supplement have repeatedly highlighted how disabling and risky it has been for most patients living with hae, but patients can now have great hopes for the future. the major concern remains the difficulty that many patients still have in reaching correct diagnosis and treatment. deaths caused by hae and similar angioedematous disorders still occur in developed countries because the majority of physicians, ignoring the existence of hae, still label these patients as allergic. we hope that this supplement will help bring more hae, aae, and nonallergic edema patients to the correct diagnosis. institute in the netherlands, and has consulting agreements with genmab and lev pharmaceuticals. m. juers is an employee of aventis behring gmbh, a zlb behring company, and has aventis stock options. h. longhurst has received support/funding from dyax corp., pharming, aventis behring, and jerini. j. nuijens is employed by pharming group nv, a biotechnology company developing recombinant c1nh. c. o'grady has received support/funding from dyax corp., pharming, aventis behring, jerini, and baxter. p. späth is a collaborator of zlb behring. k. williams is an independent writing contractor paid for this and other projects by dyax corp.; her husband is a senior-level employee of dyax corp. and owns shares of dyax corp. stock. all other authors-none. hereditary angioneurotic edema: report of a large kindred with defect in c'1 esterase inhibitor and review of the literature the house of the seven gables on giant urticaria ü ber akutes umschriebenes hautö dem hereditary angio-neurotic oedema a biochemical abnormality in hereditary angioneurotic edema: absence of serum inhibitor of c' 1-esterase hereditary and acquired c1-inhibitor deficiency: biological and clinical characteristics in 235 patients clinical, 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of c1 inhibitor deficiency with cellular abnormality acquired c1 inhibitor deficiency in a case of lymphosarcoma of the spleen: reversal of complement abnormalities after splenectomy acquired c1 inhibitor deficiency with angioedema symptoms in a patient infected with echinococcus granulosus acquired angioedema associated with chronic hepatitis c angioedema due to acquired c1-esterase inhibitor deficiency in a patient with helicobacter pylori infection angiooedema due to acquired deficiency of c1-esterase inhibitor associated with leucocytoclastic vasculitis acquired deficiency of the inhibitor of the first component of complement: report of five additional cases with commentary on the syndrome recurrent febrile panniculitis and hepatitis in two patients with acquired complement deficiency and paraproteinemia acquired c1 inhibitor deficiency associated with systemic lupus erythematosus affecting the central nervous system acquired c1 esterase inhibitor deficiency in two patients presenting with a lupus-like syndrome and anticardiolipin antibodies angioedema with acquired deficiency of the c1 inhibitor: a constellation of syndromes an igg autoantibody which inactivates c1-inhibitor autoantibody facilitated cleavage of c1-inhibitor in autoimmune angioedema acquired c1 inhibitor (c1-inh) deficiency type ii: replacement therapy with c1-inh and analysis of patients' c1-inh and anti-c1-inh autoantibodies autoimmune c1-inhibitor deficiency acquired angioedema with c1 inhibitor deficiency: is the distinction between type i and type ii still relevant? prevalence of monoclonal gammopathy in patients presenting with acquired angioedema type 2 clinical and biological distinctions between type i and type ii acquired angioedema autoantibodies and lymphoproliferative diseases in acquired c1-inhibitor deficiencies idiopathic nonhistaminergic angioedema drug-induced angioedema without urticaria valsartan-induced angioedema quantification of c1-inhibitor functional activities by immunodiffusion assay in plasma of patients with hereditary angioedema-evidence of a functionally critical level of c1-inhibitor concentration interaction of 125i-labelled complement subcomponents c-1r and c-1s with protease inhibitors in plasma inactivation of factor xii active fragment in normal plasma: predominant role of c-1-inhibitor distribution of plasma kallikrein between c-1 inactivator and alpha 2-macroglobulin in plasma utilizing a new assay for alpha 2-macroglobulin-kallikrein complexes on the role of c1-inhibitor as inhibitor of tissue-type plasminogen activator in human plasma inactivation of factor xia in human plasma assessed by measuring factor xia-protease inhibitor complexes: major role for c1-inhibitor proteolytic activities of two types of mannose-binding lectin-associated serine protease the serpin superfamily of proteinase inhibitors: structure, function, and regulation regional assignment of the human c1-inhibitor gene to 11q11-q13 detailed physical map of human chromosomal region 11q12-13 shows high meiotic recombination rate around the men1 locus complete nucleotide sequence of the gene for human c1 inhibitor with an unusually high density of alu elements recombinations between alu repeat sequences that result in partial deletions within the c1 inhibitor gene recombinational biases in the rearranged c1-inhibitor genes of hereditary angioedema patients contiguous deletion and duplication mutations resulting in type 1 hereditary angioneurotic edema cpg mutations in the reactive site of human c1 inhibitor molecular genetics of c1 inhibitor frequent de novo mutations and exon deletions in the c1inhibitor gene of patients with angioedema cooh-terminal substitutions in the serpin c1 inhibitor that cause loop overinsertion and subsequent multimerization transinhibition of c1 inhibitor synthesis in type i hereditary angioneurotic edema the catabolism of c1(-)-inhibitor and the pathogenesis of hereditary angio-edema c1 inhibitor. functional analysis of naturally-occurring mutant proteins c1 inhibitor hinge region mutations produce dysfunction by different mechanisms unique c1 inhibitor dysfunction in a kindred without angioedema, ii: identification of an ala443-val substitution and functional analysis of the recombinant mutant protein a hinge region mutation in c1-inhibitor (ala436-thr) results in nonsubstrate-like behavior and in polymerization of the molecule structural and functional aspects of c1-inhibitor c1 inhibitor gene sequence facilitates frameshift mutations c1 inhibitor gene and hereditary angioedema the rapid detection of unknown mutations in nucleic acids enzymatic methods for mutation scanning reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations chemical cleavage of mismatch: a new look at an established method c1 inhibitor mutations which affect intracellular transport and secretion in type i hereditary angioedema current methods of mutation detection efficient detection of point mutations on color-coded strands of target dna exhaustive mutation scanning by fluorescence-assisted mismatch analysis discloses new genotype-phenotype correlations in angiodema denaturing high-performance liquid chromatography: a review mutation detection by denaturing gradient gel electrophoresis (dgge) five novel mutations in the c1 inhibitor gene (c1nh) leading to a premature stop codon in patients with type i hereditary angioedema single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection molecular defects in hereditary angioneurotic edema detection of c1 inhibitor (serping1/c1nh) mutations in exon 8 in patients with hereditary angioedema: evidence for 10 novel mutations molecular basis for the deficiency of complement 1 inhibitor in type i hereditary angioneurotic edema altered c1 inhibitor genes in type i hereditary angioedema restriction fragment length polymorphism analysis of the c1-inhibitor gene in hereditary c1-inhibitor deficiency rapid detection by fluorescent multiplex pcr of exon deletions and duplications in the c1 inhibitor gene of hereditary angioedema patients the effect of sequence variations within the coding region of the c1 inhibitor gene on disease expression and protein function in families with hereditary angio-oedema the promoter of the c1 inhibitor gene contains a polypurine.polypyrimidine segment that enhances transcriptional activity mutation screening of the c1 inhibitor gene among hungarian patients with hereditary angioedema hereditary angioedema with a de novo mutation of exon 8 in the c1 inhibitor gene showing recurrent edema of the hands around the peripheral joints: importance for the differential diagnosis of joint swelling type i c1 inhibitor deficiency with a small messenger rna resulting from deletion of one exon synthesis of c1 inhibitor in fibroblasts from patients with type i and type ii hereditary angioneurotic edema impaired production of both normal and mutant c1 inhibitor proteins in type i hereditary angioedema with a duplication in exon 8 expression of functional human c1 inhibitor in cos cells characterization of recombinant c1 inhibitor p1 variants characterization of c1 inhibitor-ta: a dysfunctional c1inh with deletion of lysine 251 c1 inhibitor: analysis of the role of amino acid residues within the reactive center loop in target protease recognition a surprising new protein superfamily containing ovalbumin, antithrombin-iii, and alpha 1-proteinase inhibitor primary structure of the reactive site of human c1-inhibitor molecular cloning of human c1 inhibitor: sequence homologies with alpha 1-antitrypsin and other members of the serpins superfamily the serpins are an expanding superfamily of structurally similar but functionally diverse proteins: evolution, mechanism of inhibition, novel functions, and a revised nomenclature c1-esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema in vitro interaction of c1-inhibitor with thrombin c1 inactivator inhibition by plasmin plasminogen activator in normal subjects after exercise and venous occlusion: t-pa circulates as complexes with c1-inhibitor and pai-1 potentiation of c1 inhibitor by glycosaminoglycans: dextran sulfate species are effective inhibitors of in vitro complement activation in plasma modulation of contact system proteases by glycosaminoglycans: selective enhancement of the inhibition of factor xia the potentiation of human c1-inhibitor by dextran sulphate is transient in vivo: studies in a rat model human c1 inhibitor: primary structure, cdna cloning, and chromosomal localization the functional integrity of the serpin domain of c1-inhibitor depends on the unique n-terminal domain, as revealed by a pathological mutant simultaneous turnover of normal and dysfunctional c1 inhibitor as a probe of in vivo activation of c1 and contact activatable proteases regulation of c1 inhibitor synthesis the role of sialic acid in the functional activity and the hepatic clearance of c1-inh clearance of human native, proteinase-complexed, and proteolytically inactivated c1-inhibitor in rats a randomized, controlled trial to study the efficacy and safety of c1 inhibitor concentrate in treating hereditary angioedema a new role for c-1-inhibitor in homeostasis: control of activation of the first component of human complement activation of the complement, coagulation, fibrinolytic and kallikrein-kinin systems during attacks of hereditary angioedema activation of the fourth component of complement (c4): assessment by rocket immunoelectrophoresis and correlation with the metabolism of c4 i-pla(2) activation during apoptosis promotes the exposure of membrane lysophosphatidylcholine leading to binding by natural immunoglobulin m antibodies and complement activation c-reactive protein binds to apoptotic cells, protects the cells from assembly of the terminal complement components, and sustains an antiinflammatory innate immune response: implications for systemic autoimmunity c1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited chromatin-independent binding of serum amyloid p component to apoptotic cells surface-mediated defense reactions: the plasma contact activation system quantification of plasma factor xiia-c1-inhibitor and kallikrein-c1-inhibitor complexes in sepsis prekallikrein activation and high-molecular-weight kininogen consumption in hereditary angioedema activation of the coagulation cascade in c1-inhibitor deficiencies generation of plasmin during acute attacks of hereditary angioedema inactivation of factor xia in vivo: studies in chimpanzees and in humans reduction of contact activation related fibrinolytic activity in factor xii deficient patients: further evidence for the role of the contact system in fibrinolysis in vivo the role of factor xii in contact system activation effect of bradykinin-receptor blockade on the response to angiotensin-converting-enzyme inhibitor in normotensive and hypertensive subjects roles for the kallikrein-kinin system in inflammatory exudation and pain: lessons from studies on kininogen-deficient rats angioedema induced by a peptide derived from complement component c2 microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms binding and activation properties of human factor xii, prekallikrein, and derived peptides with acidic lipid vesicles dextran sulphate inhibits phospholipid and sulphatide mediated autoactivation of factor xii inhibition of the activation of hageman factor (factor xii) by platelet factor 4 inhibition of the activation of hageman factor (factor xii) by beta 2-glycoprotein i angioneurotic edema of the upper airways and antihypertensive therapy vasopeptidase inhibition: a double-edged sword? hereditary angioedema: genealogic studies and clinical considerations on 2 genetic forms in a case study of 10 patients late angioedema caused by ace inhibitors underestimated angioedema due to ace inhibitors: common and inadequately diagnosed increased sensitivity to bradykinin among african americans angioedema and cough in nigerian patients receiving ace inhibitors angioedema due to ace inhibitors: increased risk in patients of african origin angiotensin ii receptor blocker-associated angioedema: on the heels of ace inhibitor angioedema angiotensin ii receptor blockers in patients with ace inhibitor-induced angioedema angioedema associated with angiotensin ii receptor antagonists: challenging our knowledge of angioedema and its etiology mechanisms of angiotensin ii-induced expression of b2 kinin receptors omapatrilat: bristol-myers squibb omapatrilat and enalapril in patients with hypertension: the omapatrilat cardiovascular treatment vs. enalapril (octave) trial omapatrilat-the story of overture and octave vasopeptidase inhibitors-concepts and evidence biochemical basis of angioedema associated with recombinant tissue plasminogen activator treatment: an in vitro experimental approach bradykinin and des-arg(9)-bradykinin metabolic pathways and kinetics of activation of human plasma bioregulation of kinins: kallikreins, kininogens, and kininases bradykinin receptors effects of hormone replacement therapy on serum angiotensin-converting enzyme activity and plasma bradykinin in postmenopausal women according to angiotensin-converting enzyme-genotype hormone replacement therapy and serum angiotensin-convertingenzyme activity in postmenopausal women decreased synthesis of serum carboxypeptidase n (scpn) in familial scpn deficiency familial carboxypeptidase n deficiency factor xii does not initiate prekallikrein activation on endothelial cells activation of the plasma kallikrein/kinin system on endothelial cell membranes the plasma kallikrein-kinin system counterbalances the renin-angiotensin system the physiologic basis of assembly and activation of the plasma kallikrein/kinin system mechanism of enhanced kinin release from high molecular weight kininogen by plasma kallikrein after its exposure to plasmin detection of active kallikrein in induced blister fluids of hereditary angioedema patients dose-dependent effects of postmenopausal estrogen and progestin on antithrombin iii and factor xii increased euglobulin fibrinolytic potential in women on oral contraceptives low in oestrogen-levels of extrinsic and intrinsic plasminogen activators, prekallikrein, factor xii, and c1-inactivator molecular basis of estrogen regulation of hageman factor xii gene expression contact factors in plasma from women on oral contraception-significance of factor xi for the measured activity of factor xii biochemical and molecular pharmacology of kinin receptors transcription factor nuclear factor kappab regulates the inducible expression of the human b1 receptor gene in inflammation receptors for kinins: from classical pharmacology to molecular biology mechanisms regulating the expression, self-maintenance, and signaling-function of the bradykinin b2 and b1 receptors regulation of bradykinin b2-receptor expression by oestrogen coexistence of hereditary angioedema and turner's syndrome hereditary angioedema precipitated by estrogen replacement therapy in a menopausal woman recurrent episodes of skin angioedema and severe attacks of abdominal pain induced by oral contraceptives or hormone replacement therapy recurrent angioedema: familial and oestrogen-induced angioedema and oral contraception recurrent angioedema associated with hypogonadism or anti-androgen therapy estrogen induction and contact phase activation of human factor xii enhanced expression of factor xii (hageman factor) in isolated livers of estrogenand prolactin-treated rats the influence of estrogen and prolactin on hageman factor (factor xii) titer in ovariectomized and hypophysectomized rats differential regulation of kininogen gene expression by estrogen and progesterone in vivo the effect of oestrogen dose and progestogen type on haemostatic changes in women taking low dose oral contraceptives effects of a very low-estrogen oral contraceptive on clotting factors, carbohydrate metabolism and plasma lipids and lipoproteins contact activation factors in plasma from women using oral contraceptives-increased levels of factor xii, kinin-free high molecular weight kininogen and acetone-activated kallikrein estrogen-associated thromboembolism contact factor mediated fibrinolysis is increased by the combined oral contraceptive pill prospective randomized study of effects of unopposed estrogen replacement therapy on markers of coagulation and inflammation in postmenopausal women positive impact of hormone replacement therapy on the fibrinolytic system: a long-term randomized controlled study in healthy postmenopausal women postmenopausal hormone replacement therapy increases coagulation activity and fibrinolysis effect of long-term hormone replacement therapy on angiotensin-converting enzyme activity and bradykinin in postmenopausal women with essential hypertension and normotensive postmenopausal women estrogen regulation of angiotensin-converting enzyme mrna sex hormones in hereditary angioneurotic oedema the complete drug reference bethesda: american society of health-system pharmacists drugdexò system hereditary angioedema and oral contraception effects of methylamine and heparin on a rapid chromogenic assay of c1-esterase inhibitor in plasma effect of time, temperature and additives on a functional assay of c1 inhibitor effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women management and diagnostic guidelines for urticaria and angio-oedema hereditary angioneurotic oedema in three families: symptomatic heterogeneity, complement analysis and therapeutic trials hereditary angioneurotic oedema in finland: clinical, immunological and genealogical studies hereditary angioedema due to deficit of c1 esterase inhibitor hereditary angioneurotic edema: a clinical survey c1-inh defect as an example of deficiency disease hereditary angioedema: danazol therapy in a 5-year-old child hereditary angioneurotic oedema: current management in pregnancy a multicentre evaluation of the diagnostic efficiency of serological investigations for c1 inhibitor deficiency inherited and acquired deficiencies of c1 esterase inhibitor in humans c3 is activated in hereditary angioedema, and c1/c1-inhibitor complexes rise during physical stress in untreated patients contact system in healthy term newborns: reference values in cord blood application of a monoclonal antibody against a neoepitope on activated c4 in an elisa for the quantification of complement activation via the classical pathway c1 inhibitor and diagnosis of hereditary angioedema in newborns complement components in 100 newborns and their mothers determined by electroimmunoassay angioedema: a review on the acquired, allergic or non-allergic, and the hereditary forms serum complement levels in infancy: age related changes influence of age and sex on serum complement components in children development of the human coagulation system in the full-term infant plasma protease inhibitors in premature infants: influence of gestational age, postnatal age, and health status serum complement profiles in infants and children misdiagnosis of hereditary angio-oedema type 1 and type 2 acute consumption of c1 inhibitor in a patient with acquired c1-inhibitor deficiency syndrome normal complement c4 values do not exclude hereditary angioedema hereditary angioneurotic oedema treatment of hereditary angioedema with a vapor-heated c1 inhibitor concentrate replacement therapy in hereditary angioedema: successful treatment of acute episodes of angioedema with partly purified c1 inhibitor c1 inh concentrate in the therapy of hereditary angioedema c1-esterase inhibitor transfusions in patients with hereditary angioedema replacement therapy in hereditary angioedema: successful treatment of two patients with fresh frozen plasma c1-inhibitor deficiency and angioedema treatment of hereditary angioedema letter: anaphylactic reaction to aprotinin dx-88 and hae: a developmental perspective the synthetic kunitz domain protein dx-88 to treat angioedema in patients with hereditary angioedema role of kinins in seasonal allergic rhinitis: icatibant, a bradykinin b2 receptor antagonist, abolishes the hyperresponsiveness and nasal eosinophilia induced by antigen efficacy and tolerability of icatibant (hoe 140) in patients with moderately severe chronic bronchial asthma jerini announces positive phase ii study results with icatibant for the treatment of hereditary angioedema a phase i study of recombinant human c1-inh in asymptomatic patients with hereditary angioedema-hae pharming features clinical results of c1 inhibitor at investigator meeting [press release treatment of hereditary angioedema with danazol: reversal of clinical and biochemical abnormalities morphologic evaluation of the liver in hereditary angioedema patients on long-term treatment with androgen derivatives side effects of long-term prophylaxis with attenuated androgens in hereditary angioedema: comparison of treated and untreated patients the long-term safety of danazol in women with hereditary angioedema long-term treatment of hereditary angioedema with attenuated androgens: a survey of a 13-year experience danazol therapy: an unusual aetiology of hepatocellular carcinoma hepatocellular adenomas in patients taking danazol for hereditary angio-oedema danazol-induced hepatocellular adenoma in patients with hereditary angio-oedema a case of hereditary angioneurotic oedema, successfully treated with epsilonaminocaproic acid: studies on c'1 esterase inhibitor, c'1 activation, plasminogen level and histamine metabolism long-term prophylaxis with c1-inhibitor (c1 inh) concentrate in patients with recurrent angioedema caused by hereditary and acquired c1-inhibitor deficiency pharmacokinetic parameters of c1-inhibitor concentrate in 40 patients with hereditary angio-oedema (hae)-a prospective study acute airway obstruction following tooth extraction in hereditary angioedema oral manifestations and dental management of patients with hereditary angioedema hereditary angioedema: report of case prophylactic use of epsilon aminocaproic acid for oral surgery in a patient with hereditary angioneurotic edema tranexamic acid: preoperative prophylactic therapy for patients with hereditary angioneurotic edema intravenous tranexamic acid in the management of hereditary angio-oedema potentially fatal hereditary angioedema: a review and case report the efficacy of short-term danazol prophylaxis in hereditary angioedema patients undergoing maxillofacial and dental procedures successful off-pump coronary artery bypass graft surgery in a patient with hereditary angioedema preoperative prophylaxis for c1 esterase-inhibitor deficiency in patients undergoing oral surgery: a report of three cases hereditary angioedema: uncomplicated maxillofacial surgery using short-term c1 inhibitor replacement therapy c1-esterase inhibitor concentrate prevents upper airway obstruction in hereditary angio-oedema acquired angioedema as the presenting feature of lymphoproliferative disorders of mature b-lymphocytes spontaneous regression of acquired c1 esterase inhibitor deficiency associated with splenic marginal zone lymphoma presenting with recurrent angio-oedema acquired angioedema associated with rectal carcinoma and its response to danazol therapy: acquired angioedema treated with danazol activation of the contact system and fibrinolysis in autoimmune acquired angioedema: a rationale for prophylactic use of tranexamic acid angioneurotic edema with acquired c1-inhibitor deficiency and autoantibody to c1-inhibitor: response to plasmapheresis and cytotoxic therapy purity, activity, and virus safety of a pasteurized antithrombin concentrate a factor viii concentrate, highly purified and heated in solution virus-safe plasma proteins: elimination of viruses of risk by the manufacturing procedure inactivation of hepatitis a virus by pasteurization and elimination of picornaviruses during manufacture of factor viii concentrate absence of anti-human immunodeficiency virus types 1 and 2 seroconversion after the treatment of hemophilia a or von willebrand's disease with pasteurized factor viii concentrate absence of hepatitis after treatment with a pasteurized factor viii concentrate in patients with hemophilia and no previous transfusions effectiveness of alternative treatments for reducing potential viral contaminants from plasma-derived products inactivation of parvovirus b19 during pasteurization of human serum albumin a novel coronavirus associated with severe acute respiratory syndrome virus safety of pasteurized clotting factor concentrates: an eleven year follow up hepatitis c and pasteurized c1-inhibitor concentrate prospective virus safety follow-up after therapy with a pasteurized c1-inhibitor concentrate prophylactic treatment with pasteurised c1 inhibitor in herditary angioedema (hae)-a prospective 32 months follow up prospective follow up of clinical manifestation and therapy in children with hereditary angioedema (hae) high-titer screening pcr: a successful strategy for reducing the parvovirus b19 load in plasma pools for fractionation prospective audit of adverse reactions occurring in 459 primary antibody-deficient patients receiving intravenous immunoglobulin state of care for hemophilia in pediatric patients hereditary angioedema treatment of acute attacks of hereditary angioedema with c1-inhibitor concentrate clinical studies of sudden upper airway obstruction in patients with hereditary angioedema due to c1 esterase inhibitor deficiency proteolytic inactivation of plasma c1-inhibitor in sepsis expression of active human c1 inhibitor serpin domain in escherichia coli n-and o-glycans of recombinant human c1 inhibitor expressed in the milk of transgenic rabbits production of complex human glycoproteins in yeast recombinant human c1-inhibitor produced in pichia pastoris has the same inhibitory capacity as plasma c1-inhibitor recombinant human c1-inhibitor produced in pichia pastoris has the same inhibitory capacity as plasma c1-inhibitor recombinant c1 inhibitor p5/p3 variants display resistance to catalytic inactivation by stimulated neutrophils the effect of danazol in the treatment of chronic cystic mastitis danazol-induced pseudomenopause in the management of endometriosis effects of danazol on pulsatile gonadotropin patterns and on serum estradiol levels in normally cycling women cortical and trabecular bone mineral content in women with endometriosis: effect of gonadotropin-releasing hormone agonist and danazol a comparison of the skeletal effects of goserelin and danazol in premenopausal women with endometriosis evidence of similar increases in bone turnover during nafarelin and danazol use in women with endometriosis the effects of nafarelin and danazol on vertebral trabecular bone mass in patients with endometriosis zoladex (goserelin acetate implant) in the treatment of endometriosis: a randomized comparison with danazol. the zoladex endometriosis study group bone mass in endometriosis patients treated with gnrh agonist implant or danazol excretion of urinary n-telopeptides reflects changes in bone turnover during ovarian suppression and indicates individually variable estradiol threshold for bone loss mechanism of mammalian ovulation complement, complement activation and anaphylatoxins in human ovarian follicular fluid cystic ovaries in women affected with hereditary angioedema increased plasma beta-endorphin levels in hereditary angioedema elevated levels of plasma beta-endorphin and gamma 3-melanocyte stimulating hormone in the polycystic ovary syndrome impaired human ovarian follicular fluid complement function in hereditary angioedema functionally active complement is present in human ovarian follicular fluid and can be activated by seminal plasma inherited and acquired deficiency of c1 esterase inhibitor in humans transmission of hepatitis g virus in patients with angioedema treated with steam-heated plasma concentrates of c1 inhibitor loss of danazol responsiveness in angioedema with c1 inhibitor deficiency complement and complement fixation immunochemical quantitation of the third, fourth and fifth components of human complement: concentrations in the serum of healthy adults recurrent abdominal pain as the sole manifestation of hereditary angioedema in multiple family members danazol therapy for hereditary angio-oedema in children epsilon aminocaproic acid therapy of hereditary angioneurotic edema: a double-blind study danazol for children with immune thrombocytopenic purpura hereditary angio-oedema in children tonsillectomy in a patient with hereditary angioedema after prophylaxis with c1 inhibitor concentrate a cluster of mutations within a short triplet repeat in the c1 inhibitor gene dysfunctional c1 inhibitor ta: deletion of lys-251 results in acquisition of an n-glycosylation site characterisation of nucleotide sequence variants and disease-specific mutations involving the 3# end of the c1-inhibitor gene in hereditary angio-oedema an rna splice site mutation in the c1-inhibitor gene causes type i hereditary angio-oedema a point mutation in exon 7 of the c1-inhibitor gene causing type i hereditary angioedema a novel donor splice site mutation in the c1 inhibitor gene of a patient with type i hereditary angioneurotic edema nonsense mutations affect c1 inhibitor messenger rna levels in patients with type i hereditary angioneurotic edema identification of a novel mutation of c1 inhibitor gene in a chinese family with hereditary angioedema a single base deletion from the c1-inhibitor gene causes type i hereditary angio-oedema c1-inhibitor gene nucleotide insertion causes type ii hereditary angio-oedema substrate properties of c1 inhibitor ma (alanine 434-glutamic acid): genetic and structural evidence suggesting that the p12-region contains critical determinants of serine protease inhibitor/substrate status identification of type i hereditary angio-oedema (hae) mutations type ii hereditary angioneurotic edema that may result from a single nucleotide change in the codon for alanine-436 in the c1 inhibitor gene a dysfunctional c1 inhibitor protein with a new reactive center mutation type ii hereditary angio-oedema associated with two mutations in one allele of the c1-inhibitor gene around the reactive-site coding region c1-inhibitors and their genes: an update identification of a c/t mutation in the reactive-site coding region of the c1-inhibitor gene and its detection by an improved mutation-specific polymerase chain reaction method identification of a new p1 residue mutation (444arg-ser) in a dysfunctional c1 inhibitor protein contained in a type ii hereditary angioedema plasma a mutation unique in serine protease inhibitors (serpins) identified in a family with type ii hereditary angioneurotic edema a de novo deletion in the c1 inhibitor gene in a case of sporadic hereditary angioneurotic edema a point mutation in the c1-inhibitor gene causes type i hereditary angiooedema clusters of intragenic alu repeats predispose the human c1 inhibitor locus to deleterious rearrangements structural and functional aspects of c1-inhibitor n, none; r, rare (<1 attack/mo); f, frequent (>1 attack/mo). *e-aminocaproic acid is not shown because it had no effect or caused intolerance, and therefore, it was replaced by tranexamic acid.cover. carbon backbone of a 3-dimensional model of c1-inhibitor, with amino acids involved in hae. this model is made by homology modeling and is based on the crystal structure of 4 other serpins. the a-helices are depicted in red, the b-strands in light blue. amino acids mutated in hae patients are depicted in yellow ball-and-stick. the reactive site, p1-arg, is depicted in magenta ball-and-stick. ineke bos. adapted with permission from bos et al. 456 