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Role of functional polymorphisms of the IL-10 gene promoter in the risk of ischemic stroke in Chinese Uyghur population: One case–control study
International Journal of Cardiology 211 (2016) 58–60
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Correspondence
Role of functional polymorphisms of the IL-10 gene promoter in the risk of ischemic stroke in Chinese Uyghur population: One case–control study Yeqing Tong a,b,1, Li Cai c,b,1, Yan-wei Zhang d,1, Liangqiang Lin d, Shaochu Liu d, Shuangyi Hou a, Yang Wu a, Shenhong Han a, Qing Lu b,⁎, Jiafa Liu a,b,⁎⁎ a
Center for Disease Control and Prevention, Hubei 430079, China School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Wuhan Center for Disease Control and Prevention, Wuhan 430015, PR China d Key Laboratory of Molecular Biology of Guangdong Province, Center for Disease Control and Prevention, Shenzhen 518055, China b c
a r t i c l e
i n f o
Article history: Received 29 January 2016 Accepted 28 February 2016 Available online 2 March 2016
Keywords: Ischemic stroke Functional variants IL-10 Uyghur population Case–control
Ischemic stroke (IS), a common multifactor neurological disease with a variety of etiologies affected by a number of genetic mutations and environmental factors, is manifested by atherosclerosis or gradual cholesterol deposition. Chronic low-grade inflammation and activation of the innate immune system are closely involved in the pathogenesis of IS [1–2]. Cross-sectional studies have provided support for the hypothesis that chronic subclinical inflammation may be associated with atherosclerosis and precede the progression of clinically overt IS [3–4]. Interleukin-10 (IL-10), a cytokine with both pro-inflammatory and anti-inflammatory functions, is thought to be involved in the downregulation of cell-mediated and cytotoxic inflammatory responses by downregulating the synthesis of proinflammatory cytokines. IL-10 also downregulates the human leukocyte antigen II expression in antigen-presenting cells, and inhibits the proliferation and cytokine production of T cells responding to an antigen [5]. Abbreviations: IL-10, interleukin-10; IS, ischemic stroke; BMI, Body Mass Index; WHR, Waist Hip Ratio; FBG, fasting blood glucose; TC, total cholesterol; TG, triglyceride; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author. ⁎⁎ Corresponding author at: Center for Disease Control and Prevention, Hubei 430079, China. E-mail addresses: [email protected] (Q. Lu), [email protected] (J. Liu). 1 These authors have equally contributed to this work.
http://dx.doi.org/10.1016/j.ijcard.2016.02.151 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Twin and family studies have suggested that about 75% of the level of IL-10 production is genetically determined. IL-10 gene expression is largely depended on three SNPs located in the promoter −592, −819 and − 1082 and the haplotypes composed of them. These SNPs form haplotypes that have been associated with high or low level production of IL-10. Several studies done on IL-10 promoter polymorphisms suggest that IL-10 may be associated with the risk of atherosclerosis, and other inflammatory diseases [6–7]. However, the association of genetic polymorphisms of IL10 with IS remains poorly understood. Based on these, our aim for the study was to determine whether the promoter region polymorphism of IL10-1082 G N A, − 819 C N T and − 592 C N A are associated with IS in the Chinese Uyghur population and to procure wider genetic inferences of locus effects across Caucasians and black persons with different geographic origins and lifestyles. In the present study, the study protocols were carried out with prior approval from the local Ethics Committee. We enrolled inpatients attending the Stroke Unit of two large general hospitals in Xinjiang which lasted for four years. Due to religious belief and the harsh medical environment as well as the living environment in which this ethnic group is clustered, the collection of blood sample was extremely difficult. All subjects provided written informed consent. A total of 100 typical Uyghur subjects presenting with IS were enrolled into this case–control study. The diagnosis of IS was established according to the World Health Organization, and TOAST classification. All stroke cases was first-ever IS determined by physical examination and head imaging (CT/MRI). Concurrently, a total of 100 age and gender and ethnically-matched normal healthy controls with established medical checkup reports were randomly selected consecutively from two local community-based populations. Subjects with a history of stroke, Alzheimer's disease, brain aneurysm, dementia, dystonia or Parkinson's disease were excluded from the control group. A structured questionnaire was applied to investigate associated clinical parameters and epidemiological features. Details regarding genomic DNA isolation and genotyping procedures as well as quality control programs are available in the previous studies [8]. FBG, TC, HDL, LDL and TG were detected according to the standard laboratory procedures in the clinical laboratory. Comparisons between groups were made with the χ2 test (nominal data) or Student's t-test (interval data) with SPSS software package
Correspondence
SPSS12.0 (SPSS Inc., Chicago, Illinois, USA). Forest plots was carried out using the Stata software 10.0 (STATA Corp, College Station, Texas). Pairwise linkage disequilibrium (LD) between SNPs were evaluated as D′ and r2 using Haploview version 3.0 (http://www.broad.mit.edu/ mpg/haploview/). All genotype data for each sample were then taken to infer the haplotypes by using the PHASE 2.0 program (http://www. stat.washington.edu/stephens/software.html), haplotype data were then given to a permutation analysis (with 100,000 Monte Carlo simulations) using Haploview software. A total of 100 IS patients (mean age, 64.12 ± 9.36 years) and 100 age-, sex- and ethnicity-matched healthy controls (mean age, 63.18 ± 10.19 years) were evaluated for the IL-10 gene promoter polymorphisms study. The results suggest that WHR, hypertension, and history of heart diseases may increase the risk of IS. However, there was a trend for HDL to be a protective factor for IS. The triglyceride, cholesterol, LDL, and other parameters are summarized in Supplementary Table 1. We analyzed the linkage disequilibrium (LD) patterns among IL101082 G N A, −819 C N T and −592 C N A. The data were derived from the genotyping data of the Uyghur IS (Fig. 1a, c) and control (Fig. 1b, d) population, respectively. The pairwise correlations among SNPs were measured as D′ (Fig. 1a, b) or r2 (Fig. 1c, d) and are shown in each diamond of Fig. 1. All pairwise LD measures D′ for these three investigated SNPs ranged from 0.41 to 1, whereas r2 ranged from 0.17 to 0.55. All the data show that IL-10-819 C N T(rs1800871) and IL-10-592 C N A(rs1800872) are in middle linkage disequilibrium (0.52 ≤ r2 ≤ 0.55, 0.96 ≤ D′ ≤ 1) in the Uyghur patients and controls. However, IL-10-1082 G N A (rs1800896) and IL-10-819
59
C N T(rs1800871) are suggested to be in extremely low linkage disequilibrium (0.52 ≤ r2 ≤ 0.5, 0.96 ≤ D′ ≤ 1). Table 1 shows the distribution of genotypes and allelic frequencies for IL-10 − 1082 G N A, − 819 C N T and − 592 C N A polymorphisms. For each genotype frequency of the three SNPs, there was no significant difference in genotype distribution between either IS or controls in the Uyghur ethnic group, which were different from the results either observed in the Asian population or American(Caucasian)/European populations. The comparative distribution of genotype in patients versus controls showed that IL-10 gene promoter polymorphism maybe not a risk factor for the risk of IS in a any genetic model (all P N 0.05). In regard to allele frequency and carriage rate, there was not a significant association among patients and controls in three SNPs (all P N 0.05) [Table 1]. Moreover, it is worth noting that the CC genotype in −592 C N A and AA genotype in −1082 G N A are all extremely low in cases and controls which are different from observed previously in American/European populations [9,10]. After adjustment for confounders, there was still not a more significant difference in carriage rate of these SNPs among patients and controls using a multivariate logistic regression model (all P N 0.05) [Table 1]. An extended SNP haplotype structure analysis was conducted, which may provide some insights into the relationship between SNP patterns and IS that is beyond what single point SNP analysis can reveal. A total of four high-frequency haplotypes were estimated in the IS and control groups using Haploview software to reconstruct haplotypes based on the observed genotypes. No risk haplotype was found in the cases and controls [Supplementary Table 2].
Table 1 Association between SNPs and risk of IS between cases and controls for Chinese Uyghur population. Uyghur
Cases Controls P (n = 100) (n = 100) value rs1800871 CC 26 CT 56 TT 18 CT + 74
Adjusta
Crude OR(95%CI)
P value
OR(95%CI)
30 48 22 70
1.00 0.371 1.35(0.70–2.58) 0.367 1.37(0.73–2.60) 0.890 0.94(0.42–2.13) 0.887 0.95(0.43–2.15) 0.529 1.22(0.66–2.26) 0.525 1.23(0.69–2.28)
108 92 36.00%
−
24 76 0 76
1.00 0.766 1.11(0.57–2.14) 0.761 1.12(0.58–2.15) 0.489 − − − 0.737 1.12(0.58–2.16) 0.731 1.13(0.59–2.18)
124 76 38.00%
0.758 1.07(0.71–1.59) −
TT Alleles C T MAF
108 92 46.00%
rs1800872 AA 22 AC 77 CC 1 AC + 78
−
−
−
CC Alleles A C MAF
121 79 39.50%
rs1800896 GG 6 GA 94 AA 0 GA + 94
8 92 0 92
−
0.579 1.36(0.46–4.08) 0.577 1.37(0.47–4.10) − − − − 0.579 1.36(0.46–4.08) 0.577 1.37(0.47–4.10)
AA
Fig. 1. Analysis of linkage disequilibrium (LD) patterns among rs1800872, rs1800871, and rs1800896 in the Uyghur IS (a, c) and control (b, d) populations, respectively. The pairwise correlations between SNPs were measured as D′ (a, b) or r2 (c, d) and are shown (×100) in each diamond.
Alleles G A MAF
106 94 47.00%
108 92 46.00%
0.841 1.04(0.70–1.54) −
−
MAF: Minor Allele Frequency. a Multivariate logistic regression analysis after adjustment for hypertension, diabetes, smoking, alcohol drinking, tea drinking, BMI, WHR.
60
Correspondence
In general, we found that the CC genotype in − 592 C N A and AA genotype in − 1082 G N A are all extremely low in Chinese Uyghur cases and controls which are significantly different from American and European populations. In addition, our results suggest that IL10 promoter −1082 G N A, −819 C N T and −592 C N A SNPs on chromosome 1q31–32 may not be associated with IS in the Chinese Uyghur population. Further explorations with larger and more ethnically diverse population are required to better shed light on the association of IL-10 polymorphisms with IS, as well as the effects of IL-10 in IS. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments This study was supported by funds (81302497 & 81573237) from National Natural Science Foundation of China and Hubei Natural Science Foundation (No. 2013CFB056) as well as Hubei Province's Outstanding Medical Academic Leader Program as well as China Postdoctoral Scientific Foundation (nos. 2014M550394 & 2015T80807). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2016.02.151.
References [1] T. Xu, K. Ke, C-reactive protein and ischemic stroke risk in general population: a dose–response meta-analysis of prospective studies, Int. J. Cardiol. 190 (2015) 264–267. [2] X. Li, X.W. Cheng, L. Hu, H. Wu, P. Guo, C.N. Hao, et al., Cathepsin S activity controls ischemia-induced neovascularization in mice, Int. J. Cardiol. 183 (2015) 198–208. [3] Z. Fan, J. Yang, C. Yang, X. Guo, IL-17: a promising therapeutic target for atherosclerosis, Int. J. Cardiol. 202 (2016) 930–931. [4] S.A. Anderson, J.A. Barry, P.J. Hardiman, Risk of coronary heart disease and risk of stroke in women with polycystic ovary syndrome: a systematic review and meta-analysis, Int. J. Cardiol. 176 (2014) 486–487. [5] G. Karabela, G. Karavolias, A. Chaidaroglou, C. Theleritis, D. Degiannis, D. Kremastinos, et al., Is IL-10 a predictor of in-stent restenosis in stable and unstable angina patients undergoing coronary interventions? Int. J. Cardiol. 176 (2014) 1156–1157. [6] R.G. Westendorp, J.A. Langermans, T.W. Huizinga, A.H. Elouali, C.L. Verweij, D.I. Boomsma, et al., Genetic influence on cytokine production and fatal meningococcal disease, Lancet 349 (1997) 170–173. [7] D.M. Turner, D.M. Williams, D. Sankaran, M. Lazarus, P.J. Sinnott, I.V. Hutchinson, An investigation of polymorphism in the interleukin-10 gene promoter, Eur. J. Immunogenet. 24 (1997) 1–8. [8] Y. Tong, Y. Zhang, F. Zhan, Z. Wang, X.H. Guan, J. Liu, et al., Association of endothelial nitric oxide synthase gene mini-satellite marker VNTR polymorphism with ischemic stroke in the Chinese Uyghur population, Int. J. Cardiol. 174 (2014) 812–814. [9] C. Heeschen, S. Dimmeler, C.W. Hamm, S. Fichtlscherer, E. Boersma, M.L. Simoons, et al., Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes, Circulation 107 (2003) 2109–2114. [10] W. Ambrosius, R. Kazmierski, S. Michalak, W. Kozubski, Anti-inflammatory cytokines in subclinical carotid atherosclerosis, Neurology 66 (2006) 1946–1948.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Correspondence
Role of functional polymorphisms of the IL-10 gene promoter in the risk of ischemic stroke in Chinese Uyghur population: One case–control study Yeqing Tong a,b,1, Li Cai c,b,1, Yan-wei Zhang d,1, Liangqiang Lin d, Shaochu Liu d, Shuangyi Hou a, Yang Wu a, Shenhong Han a, Qing Lu b,⁎, Jiafa Liu a,b,⁎⁎ a
Center for Disease Control and Prevention, Hubei 430079, China School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Wuhan Center for Disease Control and Prevention, Wuhan 430015, PR China d Key Laboratory of Molecular Biology of Guangdong Province, Center for Disease Control and Prevention, Shenzhen 518055, China b c
a r t i c l e
i n f o
Article history: Received 29 January 2016 Accepted 28 February 2016 Available online 2 March 2016
Keywords: Ischemic stroke Functional variants IL-10 Uyghur population Case–control
Ischemic stroke (IS), a common multifactor neurological disease with a variety of etiologies affected by a number of genetic mutations and environmental factors, is manifested by atherosclerosis or gradual cholesterol deposition. Chronic low-grade inflammation and activation of the innate immune system are closely involved in the pathogenesis of IS [1–2]. Cross-sectional studies have provided support for the hypothesis that chronic subclinical inflammation may be associated with atherosclerosis and precede the progression of clinically overt IS [3–4]. Interleukin-10 (IL-10), a cytokine with both pro-inflammatory and anti-inflammatory functions, is thought to be involved in the downregulation of cell-mediated and cytotoxic inflammatory responses by downregulating the synthesis of proinflammatory cytokines. IL-10 also downregulates the human leukocyte antigen II expression in antigen-presenting cells, and inhibits the proliferation and cytokine production of T cells responding to an antigen [5]. Abbreviations: IL-10, interleukin-10; IS, ischemic stroke; BMI, Body Mass Index; WHR, Waist Hip Ratio; FBG, fasting blood glucose; TC, total cholesterol; TG, triglyceride; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author. ⁎⁎ Corresponding author at: Center for Disease Control and Prevention, Hubei 430079, China. E-mail addresses: [email protected] (Q. Lu), [email protected] (J. Liu). 1 These authors have equally contributed to this work.
http://dx.doi.org/10.1016/j.ijcard.2016.02.151 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Twin and family studies have suggested that about 75% of the level of IL-10 production is genetically determined. IL-10 gene expression is largely depended on three SNPs located in the promoter −592, −819 and − 1082 and the haplotypes composed of them. These SNPs form haplotypes that have been associated with high or low level production of IL-10. Several studies done on IL-10 promoter polymorphisms suggest that IL-10 may be associated with the risk of atherosclerosis, and other inflammatory diseases [6–7]. However, the association of genetic polymorphisms of IL10 with IS remains poorly understood. Based on these, our aim for the study was to determine whether the promoter region polymorphism of IL10-1082 G N A, − 819 C N T and − 592 C N A are associated with IS in the Chinese Uyghur population and to procure wider genetic inferences of locus effects across Caucasians and black persons with different geographic origins and lifestyles. In the present study, the study protocols were carried out with prior approval from the local Ethics Committee. We enrolled inpatients attending the Stroke Unit of two large general hospitals in Xinjiang which lasted for four years. Due to religious belief and the harsh medical environment as well as the living environment in which this ethnic group is clustered, the collection of blood sample was extremely difficult. All subjects provided written informed consent. A total of 100 typical Uyghur subjects presenting with IS were enrolled into this case–control study. The diagnosis of IS was established according to the World Health Organization, and TOAST classification. All stroke cases was first-ever IS determined by physical examination and head imaging (CT/MRI). Concurrently, a total of 100 age and gender and ethnically-matched normal healthy controls with established medical checkup reports were randomly selected consecutively from two local community-based populations. Subjects with a history of stroke, Alzheimer's disease, brain aneurysm, dementia, dystonia or Parkinson's disease were excluded from the control group. A structured questionnaire was applied to investigate associated clinical parameters and epidemiological features. Details regarding genomic DNA isolation and genotyping procedures as well as quality control programs are available in the previous studies [8]. FBG, TC, HDL, LDL and TG were detected according to the standard laboratory procedures in the clinical laboratory. Comparisons between groups were made with the χ2 test (nominal data) or Student's t-test (interval data) with SPSS software package
Correspondence
SPSS12.0 (SPSS Inc., Chicago, Illinois, USA). Forest plots was carried out using the Stata software 10.0 (STATA Corp, College Station, Texas). Pairwise linkage disequilibrium (LD) between SNPs were evaluated as D′ and r2 using Haploview version 3.0 (http://www.broad.mit.edu/ mpg/haploview/). All genotype data for each sample were then taken to infer the haplotypes by using the PHASE 2.0 program (http://www. stat.washington.edu/stephens/software.html), haplotype data were then given to a permutation analysis (with 100,000 Monte Carlo simulations) using Haploview software. A total of 100 IS patients (mean age, 64.12 ± 9.36 years) and 100 age-, sex- and ethnicity-matched healthy controls (mean age, 63.18 ± 10.19 years) were evaluated for the IL-10 gene promoter polymorphisms study. The results suggest that WHR, hypertension, and history of heart diseases may increase the risk of IS. However, there was a trend for HDL to be a protective factor for IS. The triglyceride, cholesterol, LDL, and other parameters are summarized in Supplementary Table 1. We analyzed the linkage disequilibrium (LD) patterns among IL101082 G N A, −819 C N T and −592 C N A. The data were derived from the genotyping data of the Uyghur IS (Fig. 1a, c) and control (Fig. 1b, d) population, respectively. The pairwise correlations among SNPs were measured as D′ (Fig. 1a, b) or r2 (Fig. 1c, d) and are shown in each diamond of Fig. 1. All pairwise LD measures D′ for these three investigated SNPs ranged from 0.41 to 1, whereas r2 ranged from 0.17 to 0.55. All the data show that IL-10-819 C N T(rs1800871) and IL-10-592 C N A(rs1800872) are in middle linkage disequilibrium (0.52 ≤ r2 ≤ 0.55, 0.96 ≤ D′ ≤ 1) in the Uyghur patients and controls. However, IL-10-1082 G N A (rs1800896) and IL-10-819
59
C N T(rs1800871) are suggested to be in extremely low linkage disequilibrium (0.52 ≤ r2 ≤ 0.5, 0.96 ≤ D′ ≤ 1). Table 1 shows the distribution of genotypes and allelic frequencies for IL-10 − 1082 G N A, − 819 C N T and − 592 C N A polymorphisms. For each genotype frequency of the three SNPs, there was no significant difference in genotype distribution between either IS or controls in the Uyghur ethnic group, which were different from the results either observed in the Asian population or American(Caucasian)/European populations. The comparative distribution of genotype in patients versus controls showed that IL-10 gene promoter polymorphism maybe not a risk factor for the risk of IS in a any genetic model (all P N 0.05). In regard to allele frequency and carriage rate, there was not a significant association among patients and controls in three SNPs (all P N 0.05) [Table 1]. Moreover, it is worth noting that the CC genotype in −592 C N A and AA genotype in −1082 G N A are all extremely low in cases and controls which are different from observed previously in American/European populations [9,10]. After adjustment for confounders, there was still not a more significant difference in carriage rate of these SNPs among patients and controls using a multivariate logistic regression model (all P N 0.05) [Table 1]. An extended SNP haplotype structure analysis was conducted, which may provide some insights into the relationship between SNP patterns and IS that is beyond what single point SNP analysis can reveal. A total of four high-frequency haplotypes were estimated in the IS and control groups using Haploview software to reconstruct haplotypes based on the observed genotypes. No risk haplotype was found in the cases and controls [Supplementary Table 2].
Table 1 Association between SNPs and risk of IS between cases and controls for Chinese Uyghur population. Uyghur
Cases Controls P (n = 100) (n = 100) value rs1800871 CC 26 CT 56 TT 18 CT + 74
Adjusta
Crude OR(95%CI)
P value
OR(95%CI)
30 48 22 70
1.00 0.371 1.35(0.70–2.58) 0.367 1.37(0.73–2.60) 0.890 0.94(0.42–2.13) 0.887 0.95(0.43–2.15) 0.529 1.22(0.66–2.26) 0.525 1.23(0.69–2.28)
108 92 36.00%
−
24 76 0 76
1.00 0.766 1.11(0.57–2.14) 0.761 1.12(0.58–2.15) 0.489 − − − 0.737 1.12(0.58–2.16) 0.731 1.13(0.59–2.18)
124 76 38.00%
0.758 1.07(0.71–1.59) −
TT Alleles C T MAF
108 92 46.00%
rs1800872 AA 22 AC 77 CC 1 AC + 78
−
−
−
CC Alleles A C MAF
121 79 39.50%
rs1800896 GG 6 GA 94 AA 0 GA + 94
8 92 0 92
−
0.579 1.36(0.46–4.08) 0.577 1.37(0.47–4.10) − − − − 0.579 1.36(0.46–4.08) 0.577 1.37(0.47–4.10)
AA
Fig. 1. Analysis of linkage disequilibrium (LD) patterns among rs1800872, rs1800871, and rs1800896 in the Uyghur IS (a, c) and control (b, d) populations, respectively. The pairwise correlations between SNPs were measured as D′ (a, b) or r2 (c, d) and are shown (×100) in each diamond.
Alleles G A MAF
106 94 47.00%
108 92 46.00%
0.841 1.04(0.70–1.54) −
−
MAF: Minor Allele Frequency. a Multivariate logistic regression analysis after adjustment for hypertension, diabetes, smoking, alcohol drinking, tea drinking, BMI, WHR.
60
Correspondence
In general, we found that the CC genotype in − 592 C N A and AA genotype in − 1082 G N A are all extremely low in Chinese Uyghur cases and controls which are significantly different from American and European populations. In addition, our results suggest that IL10 promoter −1082 G N A, −819 C N T and −592 C N A SNPs on chromosome 1q31–32 may not be associated with IS in the Chinese Uyghur population. Further explorations with larger and more ethnically diverse population are required to better shed light on the association of IL-10 polymorphisms with IS, as well as the effects of IL-10 in IS. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments This study was supported by funds (81302497 & 81573237) from National Natural Science Foundation of China and Hubei Natural Science Foundation (No. 2013CFB056) as well as Hubei Province's Outstanding Medical Academic Leader Program as well as China Postdoctoral Scientific Foundation (nos. 2014M550394 & 2015T80807). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2016.02.151.
References [1] T. Xu, K. Ke, C-reactive protein and ischemic stroke risk in general population: a dose–response meta-analysis of prospective studies, Int. J. Cardiol. 190 (2015) 264–267. [2] X. Li, X.W. Cheng, L. Hu, H. Wu, P. Guo, C.N. Hao, et al., Cathepsin S activity controls ischemia-induced neovascularization in mice, Int. J. Cardiol. 183 (2015) 198–208. [3] Z. Fan, J. Yang, C. Yang, X. Guo, IL-17: a promising therapeutic target for atherosclerosis, Int. J. Cardiol. 202 (2016) 930–931. [4] S.A. Anderson, J.A. Barry, P.J. Hardiman, Risk of coronary heart disease and risk of stroke in women with polycystic ovary syndrome: a systematic review and meta-analysis, Int. J. Cardiol. 176 (2014) 486–487. [5] G. Karabela, G. Karavolias, A. Chaidaroglou, C. Theleritis, D. Degiannis, D. Kremastinos, et al., Is IL-10 a predictor of in-stent restenosis in stable and unstable angina patients undergoing coronary interventions? Int. J. Cardiol. 176 (2014) 1156–1157. [6] R.G. Westendorp, J.A. Langermans, T.W. Huizinga, A.H. Elouali, C.L. Verweij, D.I. Boomsma, et al., Genetic influence on cytokine production and fatal meningococcal disease, Lancet 349 (1997) 170–173. [7] D.M. Turner, D.M. Williams, D. Sankaran, M. Lazarus, P.J. Sinnott, I.V. Hutchinson, An investigation of polymorphism in the interleukin-10 gene promoter, Eur. J. Immunogenet. 24 (1997) 1–8. [8] Y. Tong, Y. Zhang, F. Zhan, Z. Wang, X.H. Guan, J. Liu, et al., Association of endothelial nitric oxide synthase gene mini-satellite marker VNTR polymorphism with ischemic stroke in the Chinese Uyghur population, Int. J. Cardiol. 174 (2014) 812–814. [9] C. Heeschen, S. Dimmeler, C.W. Hamm, S. Fichtlscherer, E. Boersma, M.L. Simoons, et al., Serum level of the antiinflammatory cytokine interleukin-10 is an important prognostic determinant in patients with acute coronary syndromes, Circulation 107 (2003) 2109–2114. [10] W. Ambrosius, R. Kazmierski, S. Michalak, W. Kozubski, Anti-inflammatory cytokines in subclinical carotid atherosclerosis, Neurology 66 (2006) 1946–1948.