








key: cord-297178-moxhk2e0
authors: Novaes Rocha, Vinicius
title: Viral replication of SARS-CoV-2 could be self-limitative - the role of the renin-angiotensin system on COVID-19 pathophysiology
date: 2020-10-01
journal: Med Hypotheses
DOI: 10.1016/j.mehy.2020.110330
sha: 
doc_id: 297178
cord_uid: moxhk2e0

Currently, the world is suffering with one of the biggest pandemics of recent history. Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus disease 2019 (COVID-19) is provoking devastating consequences on economic and social fields throughout all continents. Therefore, pathophysiological knowledge about COVID-19 is imperative for better planning of preventive measures, diagnosis, and therapeutics of the disease. Based on previous studies, this work proposes new hypothesis related to the role of the renin-angiotensin system on the pathophysiology of COVID-19, and its purpose is to enrich the discussion and to offer alternative ways for experimental and clinical studies aiming at the formulation of new diagnosis and / or treatment methods.

The renin-angiotensin system (RAS) is currently one of the focuses of worldwide research due to coronavirus 2019 (COVID-19), a pandemic that has destabilized the world and created devastating consequences for economic and social areas [1] [2] [3] . Amongst the components of rennin-angiotensin system (RAS), the angiotensin-converting enzyme 2 (ACE2) has gained great prominence for being directly associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the coronavirus related to COVID-19 [4, 5] . Thus, the protagonism of ACE2 is being debated amongst researchers with the aim of establishing a role of RAS on the pathophysiologic context of COVID-19 [6] [7] [8] .

RAS was initially described as a regulating component of blood pressure and hydric balance, being called "classic pathway". Subsequently, with the discovery of new components and finding of a "local tissue RAS", the existence of a more complex system was observed with endocrine, paracrine and intracrine characteristics [9] [10] [11] . RAS elucidation continues to expand, new components are being discovered and also new ways of interaction between the local and systemic pathways, as well as interactions with the external environment [12] .

Nowadays, RAS is known as a complex system, with systemic and local (tissue) activities which communicate between themselves and involve different sign pathways, leading to different results depending on the chosen pathway [9, 12] .

This system's balance is imperative for homeostasis, in which the angiotensinconverting enzyme (ACE) and ACE2 are the main weights in this scale ( Figure   1 ). When the scale plate tends to the ACE side, the production of angiotensin II (Ang II) and the activation of its respective receptors (AT1) increases. The 3 activation of the detrimental pathway (ACE -Ang II -AT1R) stimulates actions such as cell proliferation, inflammation, fibrosis, and thrombosis. On the other hand, when the scale plate tends to the ACE2 side, the production of angiotensin 1-7 (Ang 1-7) increases, as well as the activation of its respective receptors (Mas) [13] . The activation of the protective pathway (ACE2 -Ang 1-7 -Mas) neutralizes the detrimental pathway, stimulating actions such as antiinflammatory, anti-fibrotic, and anti-thrombotic effects [9, 14] .

Coronaviruses are classified into four different genus, in which 3 species (α HCoV-NL63, SARS-CoV, and SARS-CoV-2) have ACE2 as a receptor [15] . The process of cell invasion occurs similarly amongst the different species. Briefly, the virus-cell interaction occurs through the ACE2 receptor coupling, and the internalization of the virus then occurs through fusion or endocytosis, with the participation of other components (clathrin, TMPRSS2, and others) [16] [17] [18] .

Although they belong to the same subfamily Coronavirinae and share the same receptor, these viruses have significant differences, as presented in Table 1 .

Amongst these differences, it is important to highlight in SARS-CoV-2 (the virus related to COVID-19) the longer incubation period and the faster viral peak, observed a few days after the beginning of symptoms.

Such characteristics might be related to the fact that SARS-CoV-2 presents the most extreme deficiency of CpG amongst the known betacoronavirus genomes.

The reduction of the CpG index may have allowed the virus to escape from immune response mediated by human ZAP (zinc finger antiviral protein), thus 4 becoming a severe human pathogen [24] . Furthermore, SARS-CoV-2 has a much higher infection and replication capacity than SARS-CoV, however without inducing in a significant way the increase in interferons type I, II or III, and inflammatory mediators (pro-inflammatory cytokines / chemokines) [25] .

Such characteristics of SARS-CoV-2 are imperative for the understanding of COVID-19 pathophysiology. The high replication rate associated with low inflammation leads to the viral peak even at the onset of symptoms, which makes early disease identification difficult, and as a consequence, makes treatment with antivirals ineffective because the effectiveness of their action occurs during the rise of the replication curve, the most recommended phase for such medicine [25] . Although SARS-CoV and SARS-CoV-2 use ACE2 as cell coupling receptor, the pathogenic characteristics of SARS-CoV-2 provide a great difference on disease progression, as shown in Figure 2 .

ACE2 is a fundamental piece in the pathophysiology of COVID-19, since the high replication capacity of SAR-CoV-2 is directly related to the coupling to ACE2 and cell infection. Studies show that the virus causes a reduction of ACE2 in the infected organs [26] . Therefore, the higher the amount of virus, the higher will be the use of ACE2 by it, creating an inverse correlation between the amount of SARS-CoV-2 and ACE2. At a certain time, the constant and gradual reduction of ACE2 will cause a RAS imbalance, increasing the action of ACE in detriment of the decrease in the action of ACE2, thus prevailing the harmful factors ( Figure 3 ).

The ACE2 level reduction caused by SARS-CoV-2 infection may be directly related to the pathogenesis of COVID-19 [26] . The Ang 1-7 decrease and the lower activation of Mas receptors and / or the Ang II production increase, with 5 subsequent activation of AT1 receptor, is widely known for the triggering of inflammation and fibrosis [27] . For example, a study with mice has shown that acute lung lesion results in a marked decrease of ACE2, and the use of recombinant ACE2 has a protective effect against the lung lesion. The same study noted that ACE2, Ang II, and the Ang II receptor 1 (AT1) provoke the disease pathogenesis. Thus, the important role of RAS on acute lung lesion pathogenesis is demonstrated [28] . Histologically, patients with COVID-19 present a pattern of diffuse alveolar damage and perivascular lymphocyte infiltration, similar to what is observed in influenza cases. A surprising histopathological finding on COVID-19 was the lung angiogenesis, which is 2.7 times greater than on the lungs of patients with influenza [29] . Angiogenesis, a complex process by which new blood vessels are formed from existing, is induced by hypoxia. Hypoxia is a condition in which tissues are not properly oxygenated, resulting in considerable cell stress and adaptive responses. The transcriptional responses to hypoxia are mostly mediated by the hypoxiainducible factor (HIF), a transcription factor that acts as oxygen sensors and are related to the activation of VEGF-A expression [30, 31] . Under hypoxia, HIF influences ACE and ACE2 in different ways, positively regulating the expression of ACE and negatively of ACE2 [32, 33] . In COVID-19, such circumstances may worsen the clinical condition, for stimulating even more the inflammatory, fibrotic, and thrombogenic pathways of RAS. Although COVID-19 is clinically classified as an acute respiratory syndrome, the increased angiogenesis suggests a chronic cell adaptation towards progressive hypoxia. The chronic adaptation to hypoxia could explain the higher resistance of some patients towards low O2 saturation, in which such patients do not present an involuntary increase of ventilation, even when great lung damage is present ("ground-glass opacity").

The replicative cycle of SARS-CoV-2 causes a local immune response as a result of cell death and tissue damage. In some cases, the immune response occurs in an unregulated manner, triggering a cytokine storm, and consequently, generalized pulmonary inflammation [34] . As with COVID-19, other acute respiratory diseases (SARS, MERS, Influenza) can also present the clinical picture of cytokine storm [35] . High virus titers and dysregulation of the cytokine / chemokine response cause an inflammatory cytokine storm, mainly related to the influx of inflammatory mononuclear macrophages. The activation of these macrophages by interferon-α / β produces more chemotactic factors for monocytes (CCL2, CCL7 and CCL12), resulting in the additional accumulation of mononuclear macrophages, and subsequently, an increase in the levels of pro-inflammatory cytokines (TNF, IL-6, IL1-β and inducible nitric oxide synthase), thus increasing the severity of the disease [35] .

In severe cases of COVID-19, patients demonstrate elevated plasma levels of IL-2, IL-7, IL-10, granulocyte colony stimulating factor (G-CSF), IP-10, MCP1, macrophage inflammatory protein 1α (MIP1α) and tumor necrosis factor (TNF) [34] . Viral infection and replication in airway epithelial cells can cause high levels of pyroptosis. Pyroptosis is a highly inflammatory form of programmed cell death seen in cytopathic viruses such as SARS-CoV-2. IL-1β, an important cytokine released during pyroptosis, is elevated in infection by SARS-CoV-2, thus being a possible trigger for the cytokine storm [34] .

The excessive or uncontrolled release of pro-inflammatory cytokines, characteristic of the cytokine storm, is also associated with non-infectious 7 diseases, such as autoimmune diseases [36] . Yiguo Qiu et al. analyzed SARS in mice with autoimmune uveitis. The study demonstrated that the administration of Ang 1-7 antagonist reversed the protective effects of ACE2 on inflammatory signals and on the production of inflammatory cytokines, as well as on the regulation of local immune responses. The inhibition of Ang 1-7 increased the production of the pro-inflammatory cytokines IL-6, IL-1β, TNF-α, and MCP-1 [37] . This study demonstrates the importance of Ang 1-7 in mediating the inflammatory process, as its inhibition can cause an increase in the production of pro-inflammatory cytokines, similar to that observed in the cytokine storm in COVID-19. ACE2 plays a crucial role in RAS because it neutralizes ACE activity by degrading Ang I in Ang (1-9), as well as hydrolyzing Ang II, producing Ang (1-7) [38] . Thus, ACE2 is essential to stimulate the beneficial effects of the protective axis of RAS, ACE2 / Ang (1-7) / Mas, and to mitigate the deleterious effects of the harmful axis of RAS, ACE / Ang II / AT1.

Maintaining normal levels of ACE2 in the lung is beneficial for combating inflammatory lung disease. The reduction in ACE2 expression may be related to pulmonary inflammation and subsequent cytokine storm seen in patients with severe COVID-19. ACE2 maintains the proper function of the heart and kidneys, and the negative regulation of ACE2 by SARS-CoV-2 can compromise this protective characteristic and contribute to the damage caused by the infection of these organs [38] . Renin-angiotensin system and the self-limitative replication of SARS-CoV-2 RAS is classified as systemic (classic) and local, and the interaction between them is fundamental to homeostasis [9,10,39]. This interaction between local 8 and systemic RAS probably occurs through the circulation of soluble ACE and ACE2. Although they are transmembrane proteins, ACE and ACE2 may undergo a cleavage process by ACE secretase and ADAM17, respectively, releasing an active extracellular domain. This soluble component is capable of converting angiotensin I into Ang II (ACE) and Ang II into Ang 1-7 (ACE2) [39, 40] . The balance between the amount of transmembrane ACE and soluble ACE is imperative, because the excessive release of the soluble form to the extracellular environment could lead to local tissue injury, since it will cause misbalance on the production of local Ang II and Ang 1-7. The local RAS misbalance makes the harmful pathway effects prevail (ACE -Ang II -AT1R) on the referred organ or tissue [41] . In an ACE2 receptor-dependent viral infection (Coronavirus NL63), the reduction of ACE2 cellular level occurs without changes in ACE levels [42] . This finding, plus the fact that SARS-CoV-2 elicits a low immune and inflammatory response, even when compared to SARS-CoV [25] , is more evidence that COVID-19 pathogenesis is more related to RAS misbalance than to the viral action itself. Mossel Although the protease activity of ADAM17 over ACE2 is observed, the same is 11 not observed in ACE [55] [56] [57] . Interestingly, ACE can regulate the activity of ADAM17, since it can be triggered from the activation of the AT1 receptor by

Ang II [58] . Thus, ACE directly regulates the formation of Ang II from Ang I, and

interferes via the action of ADAM17 in ACE2, in the tissue formation of Ang 1-7 from Ang II. Therefore individuals with greater expression of tissue ACE, and consequently, greater production of Ang II, will present a decrease in tissue ACE2 and an increase in soluble ACE2 (systemic), due to greater activation of ADAM17 and ACE2 cleavage. This interaction between ACE, ADAM17 and ACE2 can explain the difference in COVID-19 involvement between men and women. Androgens increase renin levels and ACE activity, while estrogen decreases renin levels, ACE activity, and AT1 receptor density, and is also related to increased levels of Ang 1-7 [59] , an important actor in the protective axis of RAS. This explains the fact that men have higher circulating (soluble)

ACE2 levels than women [60], due to the greater action of ADAM17 via androgens / ECA / Ang II / AT1. Thus, due to higher levels of circulating ACE2, men may present a lower level of tissue ACE2, becoming more vulnerable to local SARS imbalance after SARS-Cov-2 infection. This argument gains more strength when we observe that children [61] and the elderly [62] do not show a difference in the involvement of COVID-19 between genders, probably due to the lack of hormonal interference before puberty and also due to the loss of protection from estrogen in women after menopause. show similar responses to the disease, presenting a great increase in the 13 production of Ang 1-7 when compared to the control group [67, 71] . In sick adults, the Ang 1-7 level is also high when compared to healthy subjects [68, 69] , but the response intensity is much lower when compared to dogs and 

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The author has no conflict of interest or commercial sponsor for this work

Thanks to Isabela Tanuri Bessa for her helpful comments. Thanks also to the researchers from different knowledge areas for all their work undertaken as the basis for the construction of this article.

The author has no conflict of interest or commercial sponsor for this work. 16 

