Pathogenesis and Host Response
Important of Angiopoietic System in Evaluation of Endothelial Damage in Children with Crimean–Congo Hemorrhagic Fever Enver Sancakdar, MD,* Ahmet S. Guven, MD,† Elif B. Uysal, MD,‡ Köksal Deveci, MD,* and Esra Gültürk, PhD§ Background: Crimean–Congo hemorrhagic fever (CCHF) causes endothelial activation and dysfunction by affecting the endothelium directly or indirectly. In maintaining the vascular integrity, vascular endothelial growth factor (VEGF-A) and its receptor (VEGFR1) and angiopoietin-2 (Ang-2) and its receptor (Tie-2) are very important mediators. For this reason, we aimed at studying the association of Ang-2 and VEGF and their receptors Tie-2 and VEGFR1 with CCHF infection. Methods: Thirty one CCHF patients and 31 healthy controls (HC) were included in the study. CCHF patients were classified into 2 groups in terms of disease severity (severe and nonsevere). VEGF-A, VEGFR1, Ang-2 and Tie-2 levels were measured in all groups. Result: Serum levels of Tie-2, Ang-2, VEGF-A and VEGFR1 were significantly increased in CCHF patients compared with the HC. Furthermore, serum Tie-2, Ang-2, VEGF and VEGFR1 levels were found to be significantly higher in the severe group than in the nonsevere and HC groups (P < 0.05 and P < 0.001, respectively). Also, Tie-2, Ang-2, VEGF-A and VEGFR1 levels were significantly higher in the nonsevere group than in the HC group (P < 0.05). Conclusion: Having statistically significant higher Ang-2, Tie-2, VEGFA and VEGFR1 levels in the severe group when compared with the other groups suggested that VEGF-related Ang-2/Tie-2 system played a critical role in the pathogenesis of the disease, and these markers could be used as the severity criteria. Key Words: Crimean–Congo hemorrhagic fever, angiogenesis, angiopoietin-2, Tie-2, VEGF (Pediatr Infect Dis J 2015;34:e200–e205)
C
rimean–Congo hemorrhagic fever (CCHF), which is a fatal viral hemorrhagic disease, is caused by a tick-borne virus [CCHF virus (CCHFV)] belonging to the genus Nairovirus in the Bunyaviridae family.1 Despite increasing knowledge about hemorrhagic fever viruses, the specific mechanisms underlying the pathogenesis of the CCHF infection have not been clearly explained.1,2 Major targets of the CCHFV during the course of the infection are mononuclear phagocytes, hepatocytes and endothelial cells.1,3 Endothelial damage is the most accepted view explaining the pathogenesis of CCHF.1,3,4 Inflammatory cytokines and angiogenic proteins are important mediators of vascular integrity.3,5 Vascular endothelial growth factor (VEGF) and angiopoietins (Ang) are 2 of the most important regulators for neovascularization.6 VEGF, the most potent angiogenic factor, promotes endothelial proliferation and increases
Accepted for publication February 20, 2015. From the *Department of Biochemistry, †Department of Pediatrics, ‡Department of Microbiology, and §Department of Biostatistics, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey. The authors have no conflicts of interest or funding to disclose. Address for correspondence: Enver Sancakdar, MD, Department of Medical Biochemistry, Faculty of Medical, Cumhuriyet University, 58140 Sivas, Turkey. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0891-3668/15/3408-e200 DOI: 10.1097/INF.0000000000000706
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vascular permeability by binding to its specific receptors in endothelial cells such as Flt-1 (VEGFR1).6 Ang are cytokines that have a major role in regulating both the quiescent and the angiogenic microvasculature. The Ang–Tie system consists of 2 cellsurface tyrosine kinase receptors (Tie-1 and Tie-2) and 4 ligands (Ang-1 to Ang-4). Tie-2 is primarily expressed on endothelial cells and binds all the known Ang, whereas Tie-1 is an orphan receptor that can regulate Tie-2 activity via heterodimerization with it.7 Ang2, which is produced in endothelial cells and stored in Weibel–Palade bodies,8 acts as a modulator of the inflammatory response by promoting vascular leakage.9 Interaction of VEGF with Ang/Tie-2 system has an important role in the development of new vessel and arrangement of the endothelial structure.10 However, the role of Ang and its receptor, Tie-2, which are another class of angiogenic proteins, is unknown in CCHF. Therefore, this study was conducted to investigate Ang-2 and VEGF-A and their receptors Tie-2 and soluble VEGFR1 levels in blood as potential biomarkers and the role of endothelial dysfunction/damage in children with CCHF.
MATERIALS AND METHODS The consecutive patients under 18 years of age and having a laboratory confirmed diagnosis of CCHF were (n = 31; mean age 12.5 ± 2.7 years, 21 male) enrolled to the study between 2013 and 2014 at Department of Pediatrics, Cumhuriyet University Medicine School. As a healthy control (HC) group, age and gender matched individuals (n = 31; mean age 11.3 ± 3.2 years, 14 male) with no history of health problems are involved in the study. The first 7 days after the onset of disease were considered as the acute phase. The cases that were referred to our hospital in a very late stage of the disease course were excluded. The HC group consisted of age and gender matched healthy children free of any inflammatory disease (such as infectious diseases and connective tissue diseases) in same period of time. The diagnosis of CCHF infection was based upon typical clinical and epidemiological findings and confirmed serological tests with enzyme-linked immunosorbent assay (ELISA; anti-CCHF IgM and IgG antibodies) and/or of genomic segments of the CCHFV by reverse transcription-polymerase chain reaction (RT-PCR) either in the acute or convalescent phase of the disease. Serum samples of the patients were sent to the Virology Laboratory of Refik Saydam Hygiene Central Institute, Ankara, Turkey, for microbiological testing for CCHFV infection. Subjects were excluded when the CCHF diagnosis could not be confirmed by ELISA and/or RT-PCR. The patients had positive serological tests with ELISA (anti-CCHF IgM antibodies), and/or CCHFV genomic segments by real-time RT-PCR were recruited for the study. Serum samples of the patients were sent to the Refik Saydam National Public Health Agency (RSNPHA), Virology Reference and Research Laboratory of the Ministry of Health, Ankara, Turkey. TaqManbased one-step real-time RT-PCR was used to reveal and quantify CCHF viral RNA at RSNPHA. CCHF IgM in-house ELISA testing was performed at the RSNPHA, Virology Research and Reference Laboratory, following the recommendations of the Centers for Disease Control and Prevention (Atlanta, GA). CCHF IgM was identified on IgM antibody capture-ELISA prepared with inactivated native CCHF viral antigens (Strain IbAr 10200) grown in Vero E6
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cells on serum samples.11,12 Our study protocol was approved by the local Human Ethics Committee (Decision Number: 2014-02/17), and informed consent was obtained from the parents of children. The serum samples of the patients were obtained during the acute phase and/or convalescent phase of the disease, and HCs’ serum samples were stored at −80°C. Patient’s baseline characteristics were recorded, and baseline complete blood count, biochemical parameters and homeostasis parameters were determined at the Clinical Laboratory of Cumhuriyet University Medical Faculty Hospital. Previous CCHF studies were conducted mostly on adult patients13–16 using fatality rate as a marker for disease severity14–17; hence, such marker could not be used for disease severity among children in all time. The classification of disease severity in our study was based on the study of Deveci et al18 because of the fact that there was no fatality in our study, and furthermore, the other studies suffered much less fatality in children19,20 than in adults with CCHF.13–17 Deveci et al18 had based their classification of disease severity criteria on several laboratory and clinical findings. In our study, planned in the light of the aforementioned cited studies, the CCHF patients were classified into 2 groups based on disease severity (severe and nonsevere groups). Patients with at least one of the following were considered severe cases: somnolence, melena, activated partial thromboplastin time (aPTT) ≥60 seconds and thrombocyte count ≤20 × 109/L during their hospital stay.13,15,18 VEGF, VEGFR1 and Tie-2 were measured in duplicate with quantitative ELISA technique (Boster Biological Technology Co., Ltd., Wuhan, China), and Ang-2 was measured with ELISA technique (USCN life Science Inc.,Wuhan, China) according to the manufacturer’s guidelines. Descriptive statistical analysis was performed for all the studied variables. The mean ± SD was used for the homogeneous parameters, whereas the arithmetic median (min. − max.) was used for the nonhomogeneous parameters. In intergroup comparisons, the Student’s t-test, which is a parametric test, and the Mann–Whitney U test and Kruskal–Wallis test, which is a nonparametric test, were used. The difference between the categorical variables was assessed using the χ2 test. Categorical data were expressed as numbers (%). P < 0.05 was regarded as statistically significant. Statistical analyses were performed using SPSS 11.0 statistical package (SPSS Inc., Chicago, IL).
RESULTS Thirty one [mean age 12.5 ± 2.7 years, 21 (67.7%) male] patients and 31 [mean age 11.3 ± 3.2 years, 14 (45.2%) male] healthy children were enrolled in the study. Groups were similar for age and gender (P = 0.129 and P = 0.073, respectively). When the patients were grouped as severe and nonsevere, the 3 groups (severe, nonsevere and HC groups) were similar for age and gender (mean age ± SD, 11.8 ± 3.4 vs. 12.8 ± 2.4 vs. 11.3 ± 3.2, P = 0.217; males/ females, 6/5 vs. 15/5 vs. 14/17, P = 0.110, respectively). None of the patients died during the study. On admission, clinical findings, such as nausea or vomiting and facial-conjunctival hyperemia, were statistically significant in the severe group when compared with the nonsevere group (P < 0.05). Diarrhea and headache were more frequent in the severe patient group but the difference was not statistically significant. In terms of other clinical findings, such as myalgia, fever, tonsillopharyngitis, abdominal pain, bleeding, petechiae, purpura or ecchymosis, there was no difference between the severe and nonsevere groups. In comparison with the HC group, laboratory tests revealed that the patients in the severe group and nonsevere group demonstrated significantly lower median platelet count (PLT), white blood cell (WBC) and higher median values for aspartate aminotransferase (AST), alanine aminotransferase © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Angiopoietic System
(ALT) and lactate dehydrogenase (LDH; P < 0.001) on admission. Also, in comparison with the nonsevere group, laboratory tests revealed that the patients in the severe group demonstrated significantly lower median PLT and fibrinogen, longer mean aPTT(s) (P < 0.001), prothrombin time(s), international normalized ratio (P < 0.05) and higher median values for AST, ALT, LDH, creatine phosphokinase (P < 0.001), C-reactive protein and D-dimer (P < 0.05) on admission. However, when compared with the severe group, median WBC and hemoglobin values were higher in the nonsevere group, but the difference between the groups was not significant (P > 0.05). The demographic and clinical characteristics of the severe and nonsevere CCHF groups are shown in Table 1. Serum levels of Tie-2, Ang-2, VEGF-A and VEGFR1 were significantly increased in CCHF patients group, severe and nonsevere group when compared with the HC group. Furthermore, serum Tie-2, Ang-2, VEGF and VEGFR1 levels were found to be significantly higher in the severe group than in the nonsevere group (P < 0.05; Table 2). Cut-off points of Ang-2, Tie-2, VEGF and VEGFR1 levels in CCHF patients, which may be predictive of death and severity, were evaluated. In addition, area under the receiver operating characteristic (ROC) curve (AUROC), sensitivity and specificity of those cut-off points were evaluated. Following evaluation of the Ang-2, Tie-2, VEGF and VEGFR1 levels using the ROC curve method that displayed a statistically significant difference, the cutoff points were determined for these parameters in patients of the severe disease group: Tie-2: 4.6 ng/mL (95% confidence interval: 0.547–0.953), Ang-2: 1.4 ng/mL (95% confidence interval: 0.588– 0.944), VEGF: 0.6 ng/mL (95% confidence interval: 0.656–0.989) and VEGFR1: 0.6 ng/mL (95% confidence interval: 0.574–0.922). The specified cut-off points and the sensitivity, specificity and AUROC of those cut-off points are shown in Table 3. In addition, Figures 1 and 2 show the ROC curves of the levels of Ang-2, Tie-2, VEGF and VEGFR1 in severe patients group.
DISCUSSION In this study, we found that blood levels of Ang-2, Tie-2, VEGF and VEGFR1 were significantly increased in children with CCHF patients. The said increase was more prominent in children with severe CCHF. Endothelial cells are one of the main targets of CCHFVs. Infection of the endothelium plays an important role in CCHF pathogenesis.1,3,21 The involvement of endothelium in CCHF and other viral hemorrhagic fever diseases has been explained by the hypothesis that virus infection activates endothelial cells directly or indirectly via infected leukocytes and releases soluble mediators with concomitant activation of the endothelium.3,22 Endothelial damage stimulates platelet aggregation and degranulation and thus contributes to hemostatic failure and activates the intrinsic coagulation cascade.1 There is no published data on the role of the Ang-2/Tie-2 system in CCHF. For this reason, our aim was to investigate the role of angiogenic factors in the development of endothelial damage. Angiogenesis is regulated by several peptides and nonpeptide molecules. VEGF and its receptor VEGFR1 and the Ang family of molecules, Ang-1 and Ang-2 and their receptor Tie-2, are among the most widely studied molecules.23–25 Ang-1 and Ang-2 and their endothelial tyrosine kinase receptor Tie-2 form a central signaling system in endothelial permeability.24,25 VEGF and its 2 receptors, VEGFR1 and VEGFR2, are expressed in the endothelium of blood vessels and contribute to vascular development and regulation of vascular permeability.25,26 The angiogenic protein VEGF is a strong inducer of vascular permeability, and several studies have reported circulating www.pidj.com | e201
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TABLE 1. Demographic, Clinical and Laboratory Characteristics of Patients with CCHF Severe Group (n = 11)
Characteristics Age (years) Gender (male/female) Days from symptoms to admission Hospitalization days Symptoms (n, %) and clinical findings (n, %) Myalgia Headache Nausea or vomiting Fever (>38°C) Tonsillopharyngitis Fatigue/weakness Diarrhea Abdominal pain Bleeding (epistaxis or hemoptysis) Petechiae, purpura or ecchymosis Facial-conjunctival hyperemia Selected laboratory findings PLT (×109/L) WBC (×109/L) AST (IU/L) ALT (IU/L) LDH (IU/L) Hb (g/dL) PT (seconds) aPTT (seconds) INR CPK (IU/L) CRP (mg/dL) D-dimer (×103) Fibrinogen
Nonsevere Group (n = 20)
11.8 ± 3.4 6/5 5.3 (2.0–10.0) 11.8 ± 3.8
12.8 ± 2.4 15/5 6.9 (0.0–15.0) 7.7 ± 2.2
1 (9.1%) 4 (36.4%) 9 (80.8%) 10 (90.9%) 5 (45.5%) 4 (36.4%) 3 (30.0%) 3 (27.3%) 1 (9.1%) 1 (9.1%) 5 (45.5%)
1 (5.3%) 3 (15.8%) 3 (15.0%) 17 (85.0%) 10 (50.0%) 5 (26.3%) 1 (5.0%) 3 (15.0%) 0 (0%) 1 (5.0%) 1 (5.0%)
79.0 (8.0–137.0)*† 2.5 (1.2–5.8)† 191.0 (47.0–557.0)*† 50.0 (21.0–329.0)*† 706.0 (306.0–1435.0)*† 12.4 (11.8–15.0) 18.1 (9.7–26.1) 49.0 (34.2–65.1) 1.7 (0.9–2.3) 310.0 (89.0–18,765.0) 25.0 (2.5–86.0) 6.6 (0.4–39.2) 218.0 (96.0–254.0)
135.0 (50.0–446.0)‡ 3.7 (1.7–12.2)‡ 40.0 (20.0–129.0)‡ 21.5 (9.0–96.0) 271.5 (137.0–454.0)‡ 13.6 (8.3–15.8) 12.6 (9.6–17.0) 31.1 (16.0–47.3) 1.1 (0.9–1.5) 118.0 (26.0–1420.0) 4.3 (0.5–81.3) 0.6 (0.2–16.0) 254.0 (168.0–412.0)
HC (n = 31)
P Value
11.3 ± 3.2 14/17 — —
0.217 0.110 0.286 0.002
— — — — — — — — — — —
0.685 0.199 0.001 0.639 0.809 0.563 0.058 0.408 0.170 0.657 0.006
298.0 (189.0–503.0) 6.5 (3.4–12.4) 18.0 (6.0–49.0) 19.0 (6.0–49.0) 205.0 (119.0–283.0) 13.4 (9.2–14.8) — — — — — — —
0.001 0.001 0.001 0.004 0.001 0.159 0.023 0.001 0.023 0.009 0.032 0.036 0.006
*P < 0.001 vs. nonsevere group. †P < 0.001 vs. HC group. ‡P < 0.01 vs. HC group. Hb indicates hemoglobin; PT, prothrombin time; INR, international normalized ratio; CPK, creatine phosphokinase; CRP, C-reactive protein.
TABLE 2. Comparison Study Test Levels Between in Study Groups
Tie-2 (ng/mL) Ang-2 (ng/mL) VEGF-A (ng/mL) VEGFR1 (ng/mL)
Severe Group (n = 11)
Nonsevere Group (n = 20)
CCHF Patients (n = 31)
HC (n = 31)
4.9 (1.8–9.0)*† 2.0 (0.8–5.7)*† 1.2 (0.3–2.4)*† 2.0 (0.5–4.6)*†
2.4 (1.1–4.6)‡ 0.9 (0.1–1.4)‡ 0.5 (0.1–1.9)‡ 1.1 (0.2–3.3)‡
3.3 (1.1–9.0)† 1.2 (0.1–5.7)§ 0.8 (0.1–2.4)† 1.4 (0.2–4.6)§
1.5 (0.2–3.2) 0.6 (0.1–1.1) 0.3 (0.1–1.1) 0.7 (0.1–3.4)
*P< 0.05 vs. nonsevere group. †P < 0.001 vs. HC group. ‡ P < 0.05 vs. HC. §P < 0.01 vs. HC group.
TABLE 3. Cut-off Points for Tie-2, Ang-2, VEGF-A and VEGFR1 Levels in CCHF Patients, Which May be Predictive of Severity, and AUROC, Sensitivity and Specificity for the Cut-off Points 95% Confidence Interval
Tie-2 Ang-2 VEGF-A VEGFR1
Cut-off Point
Sensitivity (%)
Specificity (%)
AUROC
Lower Bound
Upper Bound
PPV (%)
NPV (%)
P Value
4.6 1.4 0.6 0.6
54.5 45.5 81.8 81.8
100 100 85 60
0.750 0.766 0.823 0.748
0.547 0.588 0.656 0.574
0.953 0.944 0.989 0.922
100 100 75 52.9
80 80 89.5 85.7
0.0158 0.0034 0.0001 0.0053
Bold values indicate P < 0.05. PPV indicates positive predictive values; NPV, negative predictive values.
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Angiopoietic System
FIGURE 1. ROC curves of the levels of Tie-2 and Ang-2 in patients in the severe group.
FIGURE 2. ROC curves of the levels of VEGF-A and VEGFR1 in patients in the severe group. VEGF levels in viral hemorrhagic fever’s patients with contradicting results.4,27–30 In one study, plasma VEGF levels in patients with dengue hemorrhagic fever (DHF) were significantly higher than the ones obtained in dengue fever (DF) patients, and it was suggested that VEGF could be contributing to the pathogenesis of DHF as a factor increasing vascular permeability.29 In contrast, Sathupan et al30 demonstrated that serum and plasma levels of VEGF were significantly lower in patients with DHF than DF at the toxic stage. Similar to the studies on dengue hemorrhagic virus, VEGF levels have been reported to differ in mild and fatal CCHF cases.4,27,28 Shapiro et al31 demonstrated that the circulatory VEGFR1 level was increased in septic patients, and this increase was correlated with disease severity. Furuta et al32 demonstrated that VEGF and VEGFR1 levels were significantly increased in patients with DHF and dengue shock syndrome compared with the levels in those © 2015 Wolters Kluwer Health, Inc. All rights reserved.
with DF and in control subjects. Bakir et al28 and Ozturk et al27 reported that VEGF levels were significantly higher in fatal cases when compared with the nonfatal cases. On the other hand, Bodur et al4 reported that VEGF levels were significantly reduced in fatal cases and severe group. Our results support the results obtained by Bakir et al28 and Ozturk et al.27 High levels of VEGF and VEGFR1 determined especially in severe patients support the plasma leakage and can be used as a prognostic factor for children with CCHF. VEGF and Ang-2 may mediate endothelial proliferation, not just in angiogenesis but also in endothelial repair.33 Ang-2 provokes inflammation and vascular hyperpermeability, whereas Ang-1 has a protective effect.7,24 Indeed, VEGF has already been shown to stimulate endothelial proliferation and accelerate reendothelialization after angioplasty-induced endothelial denudation in rabbit carotid arteries.33 Ang-2 antagonizes the effects of Ang-1; it destabilizes www.pidj.com | e203
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Sancakdar et al
the endothelium by disrupting cell–cell adhesion and primes the endothelial cells to the effects of proinflammatory cytokines and VEGF. Ang-2 is almost exclusively produced in endothelial cells and stored in Weibel–Palade bodies, from which it can be rapidly released upon activation of the endothelium.8 Ang-1 is not only produced in pericytes and smooth muscle cells, but platelets also contain high quantities of Ang-1.25 Thus, the number and activation status of circulating platelets may influence plasma Ang-1 levels. Evidence is increasing that platelets are important cells for maintaining vascular stability and platelet-derived Ang-1 may be one of the factors involved.34 The Ang/Tie-2 system controls the responsiveness of the endothelium to inflammatory, hyperpermeability, apoptosis and vasoreactive stimuli.25 In humans with severe sepsis, Parikh et al35 reported that circulating Ang-2 was elevated, and the endothelial barrier function was weakened by the acute disruption of Tie-2. In another study, Michels et al36 reported that Ang-1 levels decreased, and Ang-2 levels were increased in severe group than nonsevere group in DF. Ang-1-mediated Tie-2 activation maintains the quiescent state of the endothelium by stabilizing endothelial cell–cell junctions and by countering the permeabilizing effects of VEGF.7 They commented this finding as an Ang/Tie imbalance and inducing the plasma leakage.36 Our results supports this statement. Similar to the Bakir et al,28 this study supported the present study demonstrated that both VEGF and sVEGFR1 biomarkers are elevated in patients with CCHF, suggesting the involvement of the VEGF-A axis in the control of microvascular permeability and in the pathogenesis associated with endothelial barrier disruption in CCHF. Impaired Ang-1/Ang-2 balance because of endothelial activation1,4 and decrease in PLT1,4 in CCHF causes impairment of endothelial barrier function and vascular leakage in CCHF patients. Elevated Ang-2 and Tie-2 levels causing impairment of the vessel’s endothelial functions may be evidencing the clinical hemorrhage in CCHF patients and may have an important role in the pathogenesis of the disease. Further studies are needed for confirmation. Elevated Ang-2, Tie-2, VEGF and VEGFR1 levels correspond to increased sensitivity, specificity and AUROC (Table 3, Figs. 1 and 2). Therefore, it is suggested that Ang-2, Tie-2, VEGF and VEGFR1 are important biomarkers in determining the risk of developing severe disease.
CONCLUSION Although possible pathways and interrelations among angiogenesis mediators remain obscure, our data analysis shows that VEGF, VEGFR1, Tie-2 and Ang-2 are upregulated, reflecting a destabilization of blood vessels. Furthermore, very high sensitivity, specificity and AUROC values were noted in Ang-2, Tie-2, VEGF and VEGFR1 levels, especially in the severe group. Related markers being elevated in severe CCHF patients suggests that VEGF-A, VEGFR1, Tie-2 and Ang-2 are effective in the pathogenesis of the disease and could be used as useful markers in determining the severity.
ACKNOWLEDGMENTS Authors are grateful to Asst. Prof. Ziynet Çinar for the contributions as a statistician in evolutions of the results. REFERENCES 1. Ergönül O. Crimean–Congo haemorrhagic fever. Lancet Infect Dis. 2006;6:203–214. 2. Whitehouse CA. Crimean–Congo hemorrhagic fever. Antiviral Res. 2004;64:145–160. 3. Akıncı E, Bodur H, Leblebicioglu H. Pathogenesis of Crimean–Congo hemorrhagic fever. Vector Borne Zoonotic Dis. 2013;13:429–437.
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4. Bodur H, Akinci E, Ongürü P, et al. Evidence of vascular endothelial damage in Crimean–Congo hemorrhagic fever. Int J Infect Dis. 2010;14:e704–e707. 5. Chaturvedi UC, Agarwal R, Elbishbishi EA, et al. Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis. FEMS Immunol Med Microbiol. 2000;28:183–188. 6. Yamaguchi R, Yano H, Nakashima Y, et al. Expression and localization of vascular endothelial growth factor receptors in human hepatocellular carcinoma and non-HCC tissues. Oncol Rep. 2000;7:725–729. 7. Augustin HG, Koh GY, Thurston G, et al. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol. 2009;10:165–177. 8. Fiedler U, Scharpfenecker M, Koidl S, et al. The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel–Palade bodies. Blood. 2004;103:4150–4156. 9. Roviezzo F, Tsigkos S, Kotanidou A, et al. Angiopoietin-2 causes inflammation in vivo by promoting vascular leakage. J Pharmacol Exp Ther. 2005;314:738–744. 10. Zhang ZL, Liu ZS, Sun Q. Expression of angiopoietins, Tie2 and vascular endothelial growth factor in angiogenesis and progression of hepatocellular carcinoma. World J Gastroenterol. 2006;12:4241–4245. 11. Yapar M, Aydogan H, Pahsa A, et al. Rapid and quantitative detection of Crimean–Congo hemorrhagic fever virus by one-step real-time reverse transcriptase-PCR. Jpn J Infect Dis. 2005;58:358–362. 12. Tuygun N, Tanir G, Caglayik DY, et al. Pediatric cases of Crimean–Congo hemorrhagic fever in Turkey. Pediatr Int. 2012;54:402–406. 13. Yilmaz G, Koksal I, Topbas M, et al. The effectiveness of routine laboratory findings in determining disease severity in patients with Crimean–Congo hemorrhagic fever: severity prediction criteria. J Clin Virol. 2010;47: 361–365. 14. Swanepoel R, Gill DE, Shepherd AJ, et al. The clinical pathology of Crimean–Congo hemorrhagic fever. Rev Infect Dis. 1989;11 (Suppl 4):S794–S800. 15. Cevik MA, Erbay A, Bodur H, et al. Clinical and laboratory features of Crimean–Congo hemorrhagic fever: predictors of fatality. Int J Infect Dis. 2008;12:374–379. 16. Bakir M, Engin A, Gozel MG, et al. A new perspective to determine the severity of cases with Crimean–Congo hemorrhagic fever. J Vector Borne Dis. 2012;49:105–110. 17. Barut S, Dincer F, Sahin I, et al. Increased serum ferritin levels in patients with Crimean–Congo hemorrhagic fever: can it be a new severity criterion? Int J Infect Dis. 2010;14:e50–e54. 18. Deveci K, Oflaz MB, Sancakdar E, et al. Evaluation of the serum levels of soluble IL-2 receptor and endothelin-1 in children with Crimean–Congo hemorrhagic fever. APMIS 2013;10:12209 19. Tezer H, Sucakli IA, Sayli TR, et al. Crimean–Congo hemorrhagic fever in children. J Clin Virol. 2010;48:184–186. 20. Dilber E, Cakir M, Acar EA, et al. Crimean–Congo haemorrhagic fever among children in north-eastern Turkey. Ann Trop Paediatr. 2009;29:23–28. 21. Fisman DN. Hemophagocytic syndromes and infection. Emerg Infect Dis. 2000;6:601–608. 22. Connolly-Andersen AM, Moll G, Andersson C, et al. Crimean–Congo hemorrhagic fever virus activates endothelial cells. J Virol. 2011;85:7766–7774. 23. Engin H, Üstündağ Y, Tekin İÖ, et al. Plasma concentrations of angiopoietin-1, angiopoietin-2 and Tie-2 in colon cancer. Eur Cytokine Netw. 2012;23:68–71. 24. van Meurs M, Kümpers P, Ligtenberg JJ, et al. Bench-to-bedside review: angiopoietin signalling in critical illness - a future target? Crit Care. 2009;13:207. 25. Brouwers J, Noviyanti R, Fijnheer R, et al. Platelet activation determines angiopoietin-1 and VEGF levels in malaria: implications for their use as biomarkers. PLoS One. 2014;8:e64850. 26. Demir R, Seval Y, Huppertz B. Vasculogenesis and angiogenesis in the early human placenta. Acta Histochem. 2007;109:257–265. 27. Ozturk B, Kuscu F, Tutuncu E, et al. Evaluation of the association of serum levels of hyaluronic acid, sICAM-1, sVCAM-1, and VEGF-A with mortality and prognosis in patients with Crimean–Congo hemorrhagic fever. J Clin Virol. 2010;47:115–119. 28. Bakir M, Bakir S, Sari I, et al. Evaluation of the relationship between serum levels of VEGF and sVEGFR1 with mortality and prognosis in patients with Crimean–Congo hemorrhagic fever. J Med Virol. 2013;85:1794–1801.
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29. Tseng CS, Lo HW, Teng HC, et al. Elevated levels of plasma VEGF in patients with dengue hemorrhagic fever. FEMS Immunol Med Microbiol. 2005;43:99–102. 30. Sathupan P, Khongphattanayothin A, Srisai J, et al. The role of vascular endothelial growth factor leading to vascular leakage in children with dengue virus infection. Ann Trop Paediatr. 2007;27:179–184. 31. Shapiro NI, Yano K, Okada H, et al. A prospective, observational study of soluble FLT-1 and vascular endothelial growth factor in sepsis. Shock. 2008;29:452–457. 32. Furuta T, Murao LA, Lan NT, et al. Association of mast cell-derived VEGF and proteases in dengue shock syndrome. PLoS Negl Trop Dis. 2012;6:e1505.
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33. Callow AD, Choi ET, Trachtenberg JD, et al. Vascular permeability factor accelerates endothelial regrowth following balloon angioplasty. Growth Factors. 1994;10:223–228. 34. Nachman RL, Rafii S. Platelets, petechiae, and preservation of the vascular wall. N Engl J Med. 2008;359:1261–1270. 35. Parikh SM, Mammoto T, Schultz A, et al. Excess circulating angiopoietin-2 may contribute to pulmonary vascular leak in sepsis in humans. PLoS Med. 2006;3:e46. 36. Michels M, van der Ven AJ, Djamiatun K, et al. Imbalance of angiopoietin-1 and angiopoietin-2 in severe dengue and relationship with thrombocytopenia, endothelial activation, and vascular stability. Am J Trop Med Hyg. 2012;87:943–946.
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Important of Angiopoietic System in Evaluation of Endothelial Damage in Children with Crimean–Congo Hemorrhagic Fever Enver Sancakdar, MD,* Ahmet S. Guven, MD,† Elif B. Uysal, MD,‡ Köksal Deveci, MD,* and Esra Gültürk, PhD§ Background: Crimean–Congo hemorrhagic fever (CCHF) causes endothelial activation and dysfunction by affecting the endothelium directly or indirectly. In maintaining the vascular integrity, vascular endothelial growth factor (VEGF-A) and its receptor (VEGFR1) and angiopoietin-2 (Ang-2) and its receptor (Tie-2) are very important mediators. For this reason, we aimed at studying the association of Ang-2 and VEGF and their receptors Tie-2 and VEGFR1 with CCHF infection. Methods: Thirty one CCHF patients and 31 healthy controls (HC) were included in the study. CCHF patients were classified into 2 groups in terms of disease severity (severe and nonsevere). VEGF-A, VEGFR1, Ang-2 and Tie-2 levels were measured in all groups. Result: Serum levels of Tie-2, Ang-2, VEGF-A and VEGFR1 were significantly increased in CCHF patients compared with the HC. Furthermore, serum Tie-2, Ang-2, VEGF and VEGFR1 levels were found to be significantly higher in the severe group than in the nonsevere and HC groups (P < 0.05 and P < 0.001, respectively). Also, Tie-2, Ang-2, VEGF-A and VEGFR1 levels were significantly higher in the nonsevere group than in the HC group (P < 0.05). Conclusion: Having statistically significant higher Ang-2, Tie-2, VEGFA and VEGFR1 levels in the severe group when compared with the other groups suggested that VEGF-related Ang-2/Tie-2 system played a critical role in the pathogenesis of the disease, and these markers could be used as the severity criteria. Key Words: Crimean–Congo hemorrhagic fever, angiogenesis, angiopoietin-2, Tie-2, VEGF (Pediatr Infect Dis J 2015;34:e200–e205)
C
rimean–Congo hemorrhagic fever (CCHF), which is a fatal viral hemorrhagic disease, is caused by a tick-borne virus [CCHF virus (CCHFV)] belonging to the genus Nairovirus in the Bunyaviridae family.1 Despite increasing knowledge about hemorrhagic fever viruses, the specific mechanisms underlying the pathogenesis of the CCHF infection have not been clearly explained.1,2 Major targets of the CCHFV during the course of the infection are mononuclear phagocytes, hepatocytes and endothelial cells.1,3 Endothelial damage is the most accepted view explaining the pathogenesis of CCHF.1,3,4 Inflammatory cytokines and angiogenic proteins are important mediators of vascular integrity.3,5 Vascular endothelial growth factor (VEGF) and angiopoietins (Ang) are 2 of the most important regulators for neovascularization.6 VEGF, the most potent angiogenic factor, promotes endothelial proliferation and increases
Accepted for publication February 20, 2015. From the *Department of Biochemistry, †Department of Pediatrics, ‡Department of Microbiology, and §Department of Biostatistics, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey. The authors have no conflicts of interest or funding to disclose. Address for correspondence: Enver Sancakdar, MD, Department of Medical Biochemistry, Faculty of Medical, Cumhuriyet University, 58140 Sivas, Turkey. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0891-3668/15/3408-e200 DOI: 10.1097/INF.0000000000000706
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vascular permeability by binding to its specific receptors in endothelial cells such as Flt-1 (VEGFR1).6 Ang are cytokines that have a major role in regulating both the quiescent and the angiogenic microvasculature. The Ang–Tie system consists of 2 cellsurface tyrosine kinase receptors (Tie-1 and Tie-2) and 4 ligands (Ang-1 to Ang-4). Tie-2 is primarily expressed on endothelial cells and binds all the known Ang, whereas Tie-1 is an orphan receptor that can regulate Tie-2 activity via heterodimerization with it.7 Ang2, which is produced in endothelial cells and stored in Weibel–Palade bodies,8 acts as a modulator of the inflammatory response by promoting vascular leakage.9 Interaction of VEGF with Ang/Tie-2 system has an important role in the development of new vessel and arrangement of the endothelial structure.10 However, the role of Ang and its receptor, Tie-2, which are another class of angiogenic proteins, is unknown in CCHF. Therefore, this study was conducted to investigate Ang-2 and VEGF-A and their receptors Tie-2 and soluble VEGFR1 levels in blood as potential biomarkers and the role of endothelial dysfunction/damage in children with CCHF.
MATERIALS AND METHODS The consecutive patients under 18 years of age and having a laboratory confirmed diagnosis of CCHF were (n = 31; mean age 12.5 ± 2.7 years, 21 male) enrolled to the study between 2013 and 2014 at Department of Pediatrics, Cumhuriyet University Medicine School. As a healthy control (HC) group, age and gender matched individuals (n = 31; mean age 11.3 ± 3.2 years, 14 male) with no history of health problems are involved in the study. The first 7 days after the onset of disease were considered as the acute phase. The cases that were referred to our hospital in a very late stage of the disease course were excluded. The HC group consisted of age and gender matched healthy children free of any inflammatory disease (such as infectious diseases and connective tissue diseases) in same period of time. The diagnosis of CCHF infection was based upon typical clinical and epidemiological findings and confirmed serological tests with enzyme-linked immunosorbent assay (ELISA; anti-CCHF IgM and IgG antibodies) and/or of genomic segments of the CCHFV by reverse transcription-polymerase chain reaction (RT-PCR) either in the acute or convalescent phase of the disease. Serum samples of the patients were sent to the Virology Laboratory of Refik Saydam Hygiene Central Institute, Ankara, Turkey, for microbiological testing for CCHFV infection. Subjects were excluded when the CCHF diagnosis could not be confirmed by ELISA and/or RT-PCR. The patients had positive serological tests with ELISA (anti-CCHF IgM antibodies), and/or CCHFV genomic segments by real-time RT-PCR were recruited for the study. Serum samples of the patients were sent to the Refik Saydam National Public Health Agency (RSNPHA), Virology Reference and Research Laboratory of the Ministry of Health, Ankara, Turkey. TaqManbased one-step real-time RT-PCR was used to reveal and quantify CCHF viral RNA at RSNPHA. CCHF IgM in-house ELISA testing was performed at the RSNPHA, Virology Research and Reference Laboratory, following the recommendations of the Centers for Disease Control and Prevention (Atlanta, GA). CCHF IgM was identified on IgM antibody capture-ELISA prepared with inactivated native CCHF viral antigens (Strain IbAr 10200) grown in Vero E6
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cells on serum samples.11,12 Our study protocol was approved by the local Human Ethics Committee (Decision Number: 2014-02/17), and informed consent was obtained from the parents of children. The serum samples of the patients were obtained during the acute phase and/or convalescent phase of the disease, and HCs’ serum samples were stored at −80°C. Patient’s baseline characteristics were recorded, and baseline complete blood count, biochemical parameters and homeostasis parameters were determined at the Clinical Laboratory of Cumhuriyet University Medical Faculty Hospital. Previous CCHF studies were conducted mostly on adult patients13–16 using fatality rate as a marker for disease severity14–17; hence, such marker could not be used for disease severity among children in all time. The classification of disease severity in our study was based on the study of Deveci et al18 because of the fact that there was no fatality in our study, and furthermore, the other studies suffered much less fatality in children19,20 than in adults with CCHF.13–17 Deveci et al18 had based their classification of disease severity criteria on several laboratory and clinical findings. In our study, planned in the light of the aforementioned cited studies, the CCHF patients were classified into 2 groups based on disease severity (severe and nonsevere groups). Patients with at least one of the following were considered severe cases: somnolence, melena, activated partial thromboplastin time (aPTT) ≥60 seconds and thrombocyte count ≤20 × 109/L during their hospital stay.13,15,18 VEGF, VEGFR1 and Tie-2 were measured in duplicate with quantitative ELISA technique (Boster Biological Technology Co., Ltd., Wuhan, China), and Ang-2 was measured with ELISA technique (USCN life Science Inc.,Wuhan, China) according to the manufacturer’s guidelines. Descriptive statistical analysis was performed for all the studied variables. The mean ± SD was used for the homogeneous parameters, whereas the arithmetic median (min. − max.) was used for the nonhomogeneous parameters. In intergroup comparisons, the Student’s t-test, which is a parametric test, and the Mann–Whitney U test and Kruskal–Wallis test, which is a nonparametric test, were used. The difference between the categorical variables was assessed using the χ2 test. Categorical data were expressed as numbers (%). P < 0.05 was regarded as statistically significant. Statistical analyses were performed using SPSS 11.0 statistical package (SPSS Inc., Chicago, IL).
RESULTS Thirty one [mean age 12.5 ± 2.7 years, 21 (67.7%) male] patients and 31 [mean age 11.3 ± 3.2 years, 14 (45.2%) male] healthy children were enrolled in the study. Groups were similar for age and gender (P = 0.129 and P = 0.073, respectively). When the patients were grouped as severe and nonsevere, the 3 groups (severe, nonsevere and HC groups) were similar for age and gender (mean age ± SD, 11.8 ± 3.4 vs. 12.8 ± 2.4 vs. 11.3 ± 3.2, P = 0.217; males/ females, 6/5 vs. 15/5 vs. 14/17, P = 0.110, respectively). None of the patients died during the study. On admission, clinical findings, such as nausea or vomiting and facial-conjunctival hyperemia, were statistically significant in the severe group when compared with the nonsevere group (P < 0.05). Diarrhea and headache were more frequent in the severe patient group but the difference was not statistically significant. In terms of other clinical findings, such as myalgia, fever, tonsillopharyngitis, abdominal pain, bleeding, petechiae, purpura or ecchymosis, there was no difference between the severe and nonsevere groups. In comparison with the HC group, laboratory tests revealed that the patients in the severe group and nonsevere group demonstrated significantly lower median platelet count (PLT), white blood cell (WBC) and higher median values for aspartate aminotransferase (AST), alanine aminotransferase © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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(ALT) and lactate dehydrogenase (LDH; P < 0.001) on admission. Also, in comparison with the nonsevere group, laboratory tests revealed that the patients in the severe group demonstrated significantly lower median PLT and fibrinogen, longer mean aPTT(s) (P < 0.001), prothrombin time(s), international normalized ratio (P < 0.05) and higher median values for AST, ALT, LDH, creatine phosphokinase (P < 0.001), C-reactive protein and D-dimer (P < 0.05) on admission. However, when compared with the severe group, median WBC and hemoglobin values were higher in the nonsevere group, but the difference between the groups was not significant (P > 0.05). The demographic and clinical characteristics of the severe and nonsevere CCHF groups are shown in Table 1. Serum levels of Tie-2, Ang-2, VEGF-A and VEGFR1 were significantly increased in CCHF patients group, severe and nonsevere group when compared with the HC group. Furthermore, serum Tie-2, Ang-2, VEGF and VEGFR1 levels were found to be significantly higher in the severe group than in the nonsevere group (P < 0.05; Table 2). Cut-off points of Ang-2, Tie-2, VEGF and VEGFR1 levels in CCHF patients, which may be predictive of death and severity, were evaluated. In addition, area under the receiver operating characteristic (ROC) curve (AUROC), sensitivity and specificity of those cut-off points were evaluated. Following evaluation of the Ang-2, Tie-2, VEGF and VEGFR1 levels using the ROC curve method that displayed a statistically significant difference, the cutoff points were determined for these parameters in patients of the severe disease group: Tie-2: 4.6 ng/mL (95% confidence interval: 0.547–0.953), Ang-2: 1.4 ng/mL (95% confidence interval: 0.588– 0.944), VEGF: 0.6 ng/mL (95% confidence interval: 0.656–0.989) and VEGFR1: 0.6 ng/mL (95% confidence interval: 0.574–0.922). The specified cut-off points and the sensitivity, specificity and AUROC of those cut-off points are shown in Table 3. In addition, Figures 1 and 2 show the ROC curves of the levels of Ang-2, Tie-2, VEGF and VEGFR1 in severe patients group.
DISCUSSION In this study, we found that blood levels of Ang-2, Tie-2, VEGF and VEGFR1 were significantly increased in children with CCHF patients. The said increase was more prominent in children with severe CCHF. Endothelial cells are one of the main targets of CCHFVs. Infection of the endothelium plays an important role in CCHF pathogenesis.1,3,21 The involvement of endothelium in CCHF and other viral hemorrhagic fever diseases has been explained by the hypothesis that virus infection activates endothelial cells directly or indirectly via infected leukocytes and releases soluble mediators with concomitant activation of the endothelium.3,22 Endothelial damage stimulates platelet aggregation and degranulation and thus contributes to hemostatic failure and activates the intrinsic coagulation cascade.1 There is no published data on the role of the Ang-2/Tie-2 system in CCHF. For this reason, our aim was to investigate the role of angiogenic factors in the development of endothelial damage. Angiogenesis is regulated by several peptides and nonpeptide molecules. VEGF and its receptor VEGFR1 and the Ang family of molecules, Ang-1 and Ang-2 and their receptor Tie-2, are among the most widely studied molecules.23–25 Ang-1 and Ang-2 and their endothelial tyrosine kinase receptor Tie-2 form a central signaling system in endothelial permeability.24,25 VEGF and its 2 receptors, VEGFR1 and VEGFR2, are expressed in the endothelium of blood vessels and contribute to vascular development and regulation of vascular permeability.25,26 The angiogenic protein VEGF is a strong inducer of vascular permeability, and several studies have reported circulating www.pidj.com | e201
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TABLE 1. Demographic, Clinical and Laboratory Characteristics of Patients with CCHF Severe Group (n = 11)
Characteristics Age (years) Gender (male/female) Days from symptoms to admission Hospitalization days Symptoms (n, %) and clinical findings (n, %) Myalgia Headache Nausea or vomiting Fever (>38°C) Tonsillopharyngitis Fatigue/weakness Diarrhea Abdominal pain Bleeding (epistaxis or hemoptysis) Petechiae, purpura or ecchymosis Facial-conjunctival hyperemia Selected laboratory findings PLT (×109/L) WBC (×109/L) AST (IU/L) ALT (IU/L) LDH (IU/L) Hb (g/dL) PT (seconds) aPTT (seconds) INR CPK (IU/L) CRP (mg/dL) D-dimer (×103) Fibrinogen
Nonsevere Group (n = 20)
11.8 ± 3.4 6/5 5.3 (2.0–10.0) 11.8 ± 3.8
12.8 ± 2.4 15/5 6.9 (0.0–15.0) 7.7 ± 2.2
1 (9.1%) 4 (36.4%) 9 (80.8%) 10 (90.9%) 5 (45.5%) 4 (36.4%) 3 (30.0%) 3 (27.3%) 1 (9.1%) 1 (9.1%) 5 (45.5%)
1 (5.3%) 3 (15.8%) 3 (15.0%) 17 (85.0%) 10 (50.0%) 5 (26.3%) 1 (5.0%) 3 (15.0%) 0 (0%) 1 (5.0%) 1 (5.0%)
79.0 (8.0–137.0)*† 2.5 (1.2–5.8)† 191.0 (47.0–557.0)*† 50.0 (21.0–329.0)*† 706.0 (306.0–1435.0)*† 12.4 (11.8–15.0) 18.1 (9.7–26.1) 49.0 (34.2–65.1) 1.7 (0.9–2.3) 310.0 (89.0–18,765.0) 25.0 (2.5–86.0) 6.6 (0.4–39.2) 218.0 (96.0–254.0)
135.0 (50.0–446.0)‡ 3.7 (1.7–12.2)‡ 40.0 (20.0–129.0)‡ 21.5 (9.0–96.0) 271.5 (137.0–454.0)‡ 13.6 (8.3–15.8) 12.6 (9.6–17.0) 31.1 (16.0–47.3) 1.1 (0.9–1.5) 118.0 (26.0–1420.0) 4.3 (0.5–81.3) 0.6 (0.2–16.0) 254.0 (168.0–412.0)
HC (n = 31)
P Value
11.3 ± 3.2 14/17 — —
0.217 0.110 0.286 0.002
— — — — — — — — — — —
0.685 0.199 0.001 0.639 0.809 0.563 0.058 0.408 0.170 0.657 0.006
298.0 (189.0–503.0) 6.5 (3.4–12.4) 18.0 (6.0–49.0) 19.0 (6.0–49.0) 205.0 (119.0–283.0) 13.4 (9.2–14.8) — — — — — — —
0.001 0.001 0.001 0.004 0.001 0.159 0.023 0.001 0.023 0.009 0.032 0.036 0.006
*P < 0.001 vs. nonsevere group. †P < 0.001 vs. HC group. ‡P < 0.01 vs. HC group. Hb indicates hemoglobin; PT, prothrombin time; INR, international normalized ratio; CPK, creatine phosphokinase; CRP, C-reactive protein.
TABLE 2. Comparison Study Test Levels Between in Study Groups
Tie-2 (ng/mL) Ang-2 (ng/mL) VEGF-A (ng/mL) VEGFR1 (ng/mL)
Severe Group (n = 11)
Nonsevere Group (n = 20)
CCHF Patients (n = 31)
HC (n = 31)
4.9 (1.8–9.0)*† 2.0 (0.8–5.7)*† 1.2 (0.3–2.4)*† 2.0 (0.5–4.6)*†
2.4 (1.1–4.6)‡ 0.9 (0.1–1.4)‡ 0.5 (0.1–1.9)‡ 1.1 (0.2–3.3)‡
3.3 (1.1–9.0)† 1.2 (0.1–5.7)§ 0.8 (0.1–2.4)† 1.4 (0.2–4.6)§
1.5 (0.2–3.2) 0.6 (0.1–1.1) 0.3 (0.1–1.1) 0.7 (0.1–3.4)
*P< 0.05 vs. nonsevere group. †P < 0.001 vs. HC group. ‡ P < 0.05 vs. HC. §P < 0.01 vs. HC group.
TABLE 3. Cut-off Points for Tie-2, Ang-2, VEGF-A and VEGFR1 Levels in CCHF Patients, Which May be Predictive of Severity, and AUROC, Sensitivity and Specificity for the Cut-off Points 95% Confidence Interval
Tie-2 Ang-2 VEGF-A VEGFR1
Cut-off Point
Sensitivity (%)
Specificity (%)
AUROC
Lower Bound
Upper Bound
PPV (%)
NPV (%)
P Value
4.6 1.4 0.6 0.6
54.5 45.5 81.8 81.8
100 100 85 60
0.750 0.766 0.823 0.748
0.547 0.588 0.656 0.574
0.953 0.944 0.989 0.922
100 100 75 52.9
80 80 89.5 85.7
0.0158 0.0034 0.0001 0.0053
Bold values indicate P < 0.05. PPV indicates positive predictive values; NPV, negative predictive values.
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FIGURE 1. ROC curves of the levels of Tie-2 and Ang-2 in patients in the severe group.
FIGURE 2. ROC curves of the levels of VEGF-A and VEGFR1 in patients in the severe group. VEGF levels in viral hemorrhagic fever’s patients with contradicting results.4,27–30 In one study, plasma VEGF levels in patients with dengue hemorrhagic fever (DHF) were significantly higher than the ones obtained in dengue fever (DF) patients, and it was suggested that VEGF could be contributing to the pathogenesis of DHF as a factor increasing vascular permeability.29 In contrast, Sathupan et al30 demonstrated that serum and plasma levels of VEGF were significantly lower in patients with DHF than DF at the toxic stage. Similar to the studies on dengue hemorrhagic virus, VEGF levels have been reported to differ in mild and fatal CCHF cases.4,27,28 Shapiro et al31 demonstrated that the circulatory VEGFR1 level was increased in septic patients, and this increase was correlated with disease severity. Furuta et al32 demonstrated that VEGF and VEGFR1 levels were significantly increased in patients with DHF and dengue shock syndrome compared with the levels in those © 2015 Wolters Kluwer Health, Inc. All rights reserved.
with DF and in control subjects. Bakir et al28 and Ozturk et al27 reported that VEGF levels were significantly higher in fatal cases when compared with the nonfatal cases. On the other hand, Bodur et al4 reported that VEGF levels were significantly reduced in fatal cases and severe group. Our results support the results obtained by Bakir et al28 and Ozturk et al.27 High levels of VEGF and VEGFR1 determined especially in severe patients support the plasma leakage and can be used as a prognostic factor for children with CCHF. VEGF and Ang-2 may mediate endothelial proliferation, not just in angiogenesis but also in endothelial repair.33 Ang-2 provokes inflammation and vascular hyperpermeability, whereas Ang-1 has a protective effect.7,24 Indeed, VEGF has already been shown to stimulate endothelial proliferation and accelerate reendothelialization after angioplasty-induced endothelial denudation in rabbit carotid arteries.33 Ang-2 antagonizes the effects of Ang-1; it destabilizes www.pidj.com | e203
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the endothelium by disrupting cell–cell adhesion and primes the endothelial cells to the effects of proinflammatory cytokines and VEGF. Ang-2 is almost exclusively produced in endothelial cells and stored in Weibel–Palade bodies, from which it can be rapidly released upon activation of the endothelium.8 Ang-1 is not only produced in pericytes and smooth muscle cells, but platelets also contain high quantities of Ang-1.25 Thus, the number and activation status of circulating platelets may influence plasma Ang-1 levels. Evidence is increasing that platelets are important cells for maintaining vascular stability and platelet-derived Ang-1 may be one of the factors involved.34 The Ang/Tie-2 system controls the responsiveness of the endothelium to inflammatory, hyperpermeability, apoptosis and vasoreactive stimuli.25 In humans with severe sepsis, Parikh et al35 reported that circulating Ang-2 was elevated, and the endothelial barrier function was weakened by the acute disruption of Tie-2. In another study, Michels et al36 reported that Ang-1 levels decreased, and Ang-2 levels were increased in severe group than nonsevere group in DF. Ang-1-mediated Tie-2 activation maintains the quiescent state of the endothelium by stabilizing endothelial cell–cell junctions and by countering the permeabilizing effects of VEGF.7 They commented this finding as an Ang/Tie imbalance and inducing the plasma leakage.36 Our results supports this statement. Similar to the Bakir et al,28 this study supported the present study demonstrated that both VEGF and sVEGFR1 biomarkers are elevated in patients with CCHF, suggesting the involvement of the VEGF-A axis in the control of microvascular permeability and in the pathogenesis associated with endothelial barrier disruption in CCHF. Impaired Ang-1/Ang-2 balance because of endothelial activation1,4 and decrease in PLT1,4 in CCHF causes impairment of endothelial barrier function and vascular leakage in CCHF patients. Elevated Ang-2 and Tie-2 levels causing impairment of the vessel’s endothelial functions may be evidencing the clinical hemorrhage in CCHF patients and may have an important role in the pathogenesis of the disease. Further studies are needed for confirmation. Elevated Ang-2, Tie-2, VEGF and VEGFR1 levels correspond to increased sensitivity, specificity and AUROC (Table 3, Figs. 1 and 2). Therefore, it is suggested that Ang-2, Tie-2, VEGF and VEGFR1 are important biomarkers in determining the risk of developing severe disease.
CONCLUSION Although possible pathways and interrelations among angiogenesis mediators remain obscure, our data analysis shows that VEGF, VEGFR1, Tie-2 and Ang-2 are upregulated, reflecting a destabilization of blood vessels. Furthermore, very high sensitivity, specificity and AUROC values were noted in Ang-2, Tie-2, VEGF and VEGFR1 levels, especially in the severe group. Related markers being elevated in severe CCHF patients suggests that VEGF-A, VEGFR1, Tie-2 and Ang-2 are effective in the pathogenesis of the disease and could be used as useful markers in determining the severity.
ACKNOWLEDGMENTS Authors are grateful to Asst. Prof. Ziynet Çinar for the contributions as a statistician in evolutions of the results. REFERENCES 1. Ergönül O. Crimean–Congo haemorrhagic fever. Lancet Infect Dis. 2006;6:203–214. 2. Whitehouse CA. Crimean–Congo hemorrhagic fever. Antiviral Res. 2004;64:145–160. 3. Akıncı E, Bodur H, Leblebicioglu H. Pathogenesis of Crimean–Congo hemorrhagic fever. Vector Borne Zoonotic Dis. 2013;13:429–437.
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Angiopoietic System
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