J Arthropod-Borne Dis, March 2017, 11(1): 19–26 M Sefikogullari et al.: Increased Levels of … 19 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Increased Levels of VEGF-A and HIF-1α in Turkish Children with Crimean- Congo Hemorrhagic Fever Murat Sefikogullari 1, Ali Kaya 2, Huseyin Aydin 3, Enver Sancakdar 3, Veysel Kenan Celik 3, *Gokhan Bagci 3 1Pediatric Clinic, Private Samandağ Medical Center, Hatay, Turkey 2Department of Pediatrics, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey 3Department of Biochemistry, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey (Received 22 Feb 2015; accepted 23 Sep 2015) Abstract Background: Crimean-Congo Hemorrhagic Fever (CCHF) is a disease characterized by serious course, including acute viral fever, ecchymosis, thrombocytopenia, liver dysfunction and high rate of mortality. Hypoxia Inducible Factor-1α (HIF-1α) and Vascular Endothelial Growth Factor-A (VEGF-A) play an important role both in the in- flammatory process and plasma leakage. The aim of this study was to define HIF-1α and VEGF-A serum levels obtained from CCHF patients and control group and to investigate whether these factors were correlated with the pathogenesis of this disease. Methods: Thirty cases younger than 17yr confirmed by RT-PCR and/or ELISA for CCHF were included in this study. Thirty age and sex matched healthy peoples were enrolled as controls. Blood samples collected from the pa- tient and control groups. Serum levels of HIF-1α and VEGF-A were measured with ELISA. Results: Levels of HIF-1α and VEGF-A were statistically significantly increased in CCHF patients compared to the control group (P< 0.05). A significant positive correlation was found between the levels of HIF-1α and VEGF-A in the patient group (P< 0.01). The levels of ALT, AST, CK, aPTT, WBC and Thrombocyte count were significantly higher in the patients than in the control group (P< 0.001). A positive correlation was found among the levels of AST and CK from biochemical parameters and VEGF and HIF-1α in the patient group (P< 0.05) Conclusion: HIF-1α and VEGF-A might play an important role in CCHF pathogenesis. Keywords: Crimean congo hemorrhagic fever, VEGF, HIF-1α, Sepsis, Children Introduction Crimean-Congo Hemorrhagic Fever (CCHF) is a serious disease occurring in case of in- fection by tick-borne virus (Nairovirus) genus from Bunyaviridae family (Ergonul 2008). It begins with fever, severe headache, nausea, weakness, vomiting and progresses with bleed- ing in different parts of the body. “The bleed- ing findings emerge as subcutaneous bleed- ing, nasal bleeding, gingival bleeding and visceral bleeding” (Bakir et al. 2005). Mon- onuclear phagocytic cells, hepatocytes and endothelial cells are known as main target of CCHF virus (Whitehouse 2004, Ergonul et al. 2004). CCHF cases have been reported from more than 30 countries of Asia, South-Eastern Eu- rope and Africa until now (Hubalek and Rudolf 2012). In Turkey, annually more than 1,000 human CCHF cases are reported (Maltezou et al. 2010). Like sepsis, endothelial damage plays an important role in the pathogenesis of CCHF. Endothelium may be indirectly targeted by virus mediated host derived soluble factors or directly by viral factors (Schnittler and Feldmann 2003). Hypoxia inducible factor (HIF-1α) is an oxygen sensitive transcription factor facilitating oxygen distribution and cel- lular adaptation to oxygen deprivation. HIF- 1α activates transcription of genes, which en- *Corresponding authors: Dr Gokhan Bagci, E- mail: gokhanbagci@hotmail.com.tr J Arthropod-Borne Dis, March 2017, 11(1): 19–26 M Sefikogullari et al.: Increased Levels of … 20 http://jad.tums.ac.ir Published Online: March 14, 2017 code angiogenic growth factors, including vas- cular endothelial growth factor (VEGF), an- giopoietin 1 (ANGPT1), (ANGPT2), placen- tal growth factor (PGF) and platelet-derived growth factor B (PDGFB), playing a critical role in angiogenesis. HIF-1α regulates expres- sion of certain genes related with erythropoi- esis, energy metabolism, angiogenesis, cell pro- liferation and apoptosis (Ferrara et al. 2003, Podar and Anderson 2005, Manalo et al. 2005). Vascular Endothelial Growth Factor (VEGF- A), a member of platelet-derived growth fac- tors super family is specific for the endo- thelial cells and has important effects. Blood vessel formation is regulated mainly by vas- cular endothelial growth factor (VEGF-A) in health and disease. Human VEGF gene fam- ily consists of five members of VEGF A-E, among which VEGF-A is also commonly referred to as VEGF (Ferrara 2003). VEGF-A was also known as vascular per- meability factor (VPF) due to its substantial effect on increasing of vascular permeability, resulting in a leakage out of blood vessels. One of the main aberrations in pathogenesis of CCHF is vascular dysfunction resulting in leak- age driven hemorrhagic manifestations (Dvorak et al. 1995, Hoeben et al. 2004, Ergonul 2006). In this study, we aimed to demonstrate whether VEGF-A and HIF-1α was correlated with pathogenesis of sepsis in serum samples obtained from CCHF patients and control group. Materials and Methods Study design Thirty patients diagnosed with CCHF in Department of Pediatrics, Faculty of Medi- cine, Cumhuriyet University, from 2010 to 2011 and 30 healthy controls were enrolled in the study. All of the patients with CCHF used to live in the same endemic region (Si- vas and neighbor’s cities) which showed high prevalence in CCHF. Blood samples col- lected from the hospitalized patients with pre- sumed diagnosis of CCHF were sent to Refik Saydam Hifzisihha Center, Virology Labor- atory. Thirty patients having positive CCHFV RNA with Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and/ or positive CCHFV-specific IgM with ELISA were in- cluded in the study. Thirty age and sex matched healthy individuals who had not any infectious or metabolic diseases were enrolled as the controls. This study was approved by the Local Ethics Committee of Cumhuriyet University, Faculty of Medicine. Measurement of HIF-1α and VEGF-A levels Blood samples of 5 ml were collected from patient and control groups. The sam- ples were centrifuged at 3000 rpm for 5 min and obtained serums were portioned and kept at -80 °C until the analysis time. Then these samples were cooled to the room tem- perature at the same time and HIF-1α and VEGF-A levels were measured through ELI- SA method. Human/Mouse Total HIF-1 al- pha Cell-Based ELISA (R and D Systems, Inc.) and VEGF-A Human (BioVendor) kits were used to measure the levels of HIF-1α and VEGF-A. MicroELISA (sandwich) meth- od was performed using full automatic TRIT- URUS device. Statistical analysis All statistical analyses were conducted with IBM SPSS Statistics for Windows, Ver- sion 20.0 (Chicago, IL, USA) computer pro- gram. Descriptive statistics were expressed as arithmetic mean (min-max). Mann–Whitney U test was performed to determine the sig- nificance of independent continuous varia- bles. For bivariate correlation analysis, Pear- son’s or Spearman’s test was used. In evalu- ation of the categorical data, Chi-square test was used in the statistical analyses. P< 0.05 values were considered as statistically signif- icant for all the tests. J Arthropod-Borne Dis, March 2017, 11(1): 19–26 M Sefikogullari et al.: Increased Levels of … 21 http://jad.tums.ac.ir Published Online: March 14, 2017 Results Demographic features and laboratory find- ings of the controls and patient groups are given in Table 1. Mean age was found as 11.30±4.41 in the patients and 10.40±4.49 in the controls. Nineteen of the patients were male (63.3%) and 11 were female (36.7%), whereas 20 (66.7%) of the controls were male and 10 (33.3%) were female. The differences between the controls and patient groups in terms of age and gender were not significant. A statistically significant increase was ob- served in the levels of HIF-1α and VEGF in the patients compared to control groups (P< 0.05, Table 2). A significant positive correla- tion was found between the levels of HIF-1α and VEGF-A in patient group (P< 0.01, Table 2). A positive correlation was found among the levels of AST and CK from biochemical parameters and VEGF and HIF-1α (P< 0.05), while this was not significant in ALT (P= 0.086, Table 3). The levels of ALT, AST and CK were statistically significantly higher in CCHF patients than in the control group (P< 0.001). There was a significant increase in the levels of aPTT in coagulation tests compared to controls (P=0.022), increase in the prothrom- bin time and INR was not statistically sig- nificant (P> 0.05 Table 1). WBC (P< 0.05) and thrombocyte counts (P< 0.001) were sta- tistically lower in the patients than in the con- trol group, while no significant difference was observed in the amount of hemoglobin. Table 1. Comparison of demographic and laboratory findings of the patients and controls Variable Control (n=30) CCHF (n=30) P Age (mean±SD) 10.40±4.49 11.30±4.41 0.05 Age range 2-17 2-17 0.05 Male (%) 20 (66.7%) 19 (63.3%) 0.05 Female (%) 20 (66.7%) 19 (63.3%) 0.05 ALT (IU/L) 35 (12-48) 83 (28-335) <0.001 AST (IU/L) 38 (11-54) 171 (26-357) <0.001 CK (IU/L) 107 (68-172) 899 (140-1235) <0.001 Prothrombin time (s) 14.4 (9.6-14.0) 15.9 (9.6-20.1) >0.05 aPTT (s) 34.7 (13.5-45.7 42.4 (23.8-55.1) 0.022 INR 1.2 (0.8–2.2) 1.3 (0.9–2.4) >0.05 WBC (x109 cells/L) 5.7 (3.5–7.0) 3.17 (1.2-9.0) <0.05 Hemoglobin (g/dL) 14.2 (8.3-15.8) 13.6 (11.8-15.8) >0.05 Thrombocyte count (x103 cells/L) 176 (69-214) 110 (38-152) <0.001 ALT: Alanine aminotransferase AST: Aspartate aminotransferase CK: Creatine kinase aPTT: activated partial thromboplastin time INR: International normalized ratio WBC: White blood cell Table 2. Serum levels of VEGF-A and HIF-1α in the patient and control groups Patients Control P VEGF-A (pg/ml) 312.66±87.31 150.70±78.48 0.001 HIF-1α (pg/ml) 195.93±63.49 68.20±36.11 0.001 J Arthropod-Borne Dis, March 2017, 11(1): 19–26 M Sefikogullari et al.: Increased Levels of … 22 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 3. HIF-1α and VEGF-A correlation between biochemical parameters in patients with CCHF HIF-1α VEGF-A Variable Correlation Coeffi- cient P Correlation Coefficient P ALT 0.181 0.086 0.162 0.103 AST 0.256 0.020 0.266 0.022 CK 0.214 0.012 0.292 0.048 VEGF-A 0.969 <0.01 - - ALT: Alanine aminotransferase AST: Aspartate aminotransferase CK: Creatine kinase Discussion We aimed to demonstrate whether HIF-1α and VEGF, related to sepsis and viral infec- tion, were correlated with pathogenesis of CCHF disease. Despite numerous studies conducted about CCHF, pathogenesis of the disease is not ful- ly understood. There was a relationship be- tween sepsis and viral infection, and HIF-1α and VEGF (Irwin et al. 2009). In infected humans by the CCHFV, damaging of en- dothelial cells and vascular leakage may be a direct result of an immune mediated indirect effect (Schnittler and Feldmann 2003). A common characteristic of viral hemor- rhagic fevers is viruses to enter macrophages and dendritic cells, making a cytopathic ef- fect. VEGF produced by monocytes and en- dothelial cells is known to play an important role in breakdown of coagulation, leukocyte adhesion, angiogenesis and increased vascu- lar permeability (Burt et al. 1997, Geisbert and Jahrling 2004). It is known as a deterio- rated coagulation, leukocyte adhesion, angi- ogenesis and increased vascular permeability in CCHF patients (Bodur et al. 2010). Clinical symptoms of CCHF are similar to sepsis and endothelial structure of vessels is retrograded (Elson et al. 2001). The cor- relation of sepsis and viral infection with HIF-1α and VEGF-A was shown (Kilani et al. 2004, Zinkernagel et al. 2007). At hypoxic conditions, hypoxia-inducible factor (HIF) regulates expression of certain genes. In in vivo and in vitro studies, oxygen tension affects virus production either up regulating or down regulating viral replica- tion depending on virus type and method used for to study viral replication. Viral rep- lication is modulated mainly by HIF-1α in normal and hypoxic conditions (Morinet et al. 2013). HIF-1α is known to activate transcription of the genes including VEGF and other an- giogenic growth factors and plays a critical role in the development of angiogenesis in hypoxic conditions (Manalo et al. 2005). HIF- 1α mRNA expression was suppressed and con- versely associated with disease severity in sepsis (Schaefer et al. 2013). HIF-1α expres- sion was statistically significantly higher in the shock patients compared to the controls and the reasons of shock were sepsis (78%), hemorrhage (18%), and cardiac dysfunction (4%) (Textoris et al. 2012). In sepsis, microvascular permeability close- ly associates with the increased plasma VEGF concentration (Pickkers et al. 2005). ROS– HIF-1α–VEGF pathway is partly responsible for hypoxia-induced permeability (Irwin et al. 2009). The levels of VEGF increased in sepsis, and this increase correlated with se- verity of the disease and mortality (van der J Arthropod-Borne Dis, March 2017, 11(1): 19–26 M Sefikogullari et al.: Increased Levels of … 23 http://jad.tums.ac.ir Published Online: March 14, 2017 Flier et al. 2005, Yano et al. 2006, Karlsson et al. 2008). Inhibition of VEGF signaling contributes the development of sepsis-induced organ dys- function via blocking endothelial survival and increasing apoptosis. Furthermore, in patho- logical situations like sepsis and cancer, VEGF participates to mobilizing of endo- thelial progenitor cells (Jesmin et al. 2012). The main target of sepsis-induced events is generally endothelium, so ability to ame- liorate damaged endothelium is one of the main determiners of clinical outcome in septic patients. The angiogenic factors and their soluble receptors have a dual role and seem to play both beneficial and harmful effects during sepsis development and therapy. While normal levels of VEGF is required for protection of endothelial function, both ex- tremely high or low levels of VEGF have disruptive effect on endothelial barrier (Zhang et al. 2013). In our study, a strong correlation was seen between the levels of HIF-1α and VEGF-A in CCHF patients (Table 3). Besides, a pos- itive correlation was found between HIF-1α and VEGF-A, suggesting that angiogenesis could be triggered and, pathogenesis of the disease could be influenced. Accordingly, we recommend that comprehensive studies related to angiogenesis should be conducted in CCHF patients. Despite the absence of studies that in- vestigated HIF-1α levels in patients with CCHF in the literature, the studies that eval- uating VEGF levels have increased in recent years. All of the studies that investigate se- rum levels of VEGF have been conducted with Turkish CCHF patients. Serum levels of VEGF was statistically higher in the adult patients having CCHF compared to the con- trols (Bakir et al. 2013), and in another study, levels of VEGF was found statistically high- er in fatal CCHF patients compared to the non-fatal patients (Ozturk et al. 2010). In contrast to these findings, level of VEGF was significantly decreased in CCHF pa- tients (Bodur et al. 2010). Our study was not consistent with the study by Bodur et al. while it was consistent with the other two studies. In studies conducted with other hemorrhag- ic fevers such as dengue hemorrhagic fever (DHF) (Tseng et al. 2000, Srikiatkhachorn et al. 2007, Furuta et al. 2012, del Moral-Her- nández et al. 2014) and hemorrhagic fever with renal syndrome (HFRS) (Ma et al. 2012), also VEGF level was statistically significant- ly higher in patient groups. Consequently, all of these studies indicate that VEGF regulates vascular permeability and VEGF is a good marker of the severity of the infection by viral diseases such as CCHF and DHF. Our study has some limitations. First, we could not linked the levels of HIF-1α and VEGF-A together with disease severity. Se- cond, HIF-1α and VEGF-A serum levels were not compared between patients with survival and non-survival. Lastly, we did not evaluate the levels of VEGF-A and HIF-1α in another viral disease -especially the other hemorrhagic fevers- or patients with sepsis. Increased levels of the HIF-1α which is the main transcriptional factor of the regula- tion of oxygen homeostasis and VEGF-A which has a crucial role in the regulation of angiogenesis and vascular permeability sug- gested that these parameters might contrib- ute to the development of vascular endothe- lial damage. Nevertheless, because of the in- sufficient information about molecular mech- anism on pathogenesis of CCHF, our results indicate that VEGF and HIF-1α should be one of the markers need to be focused on the elucidation on the pathogenesis of CCHF disease. 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