Abstract
Background
The association between genetic variations of vascular endothelial growth factor (VEGF) gene and the risk for atherosclerosis has been hypothesized. We aimed to assess the relationship between rs2010963 (+405 C/G) polymorphism and presence, severity, and outcome of coronary artery disease (CAD) in an Iranian cohort.
Methods
Genotyping of VEGF rs2010963 polymorphism was performed on 520 individuals, comprising 347 patients with documented coronary artery disease based on angiography report and 173 individuals with normal coronary arteries, using the TaqMan real‐time PCR method. In final, 484 subjects were followed up over a 5‐year period for cardiovascular‐related outcomes.
Results
C allele of VEGF rs2010963 polymorphism was related to increase risk for CAD and also slightly to 5‐year cardiovascular mortality. The 5‐year survival in C and G allele subgroups were 92.3% and 94.3% in CAD group and 95.7% and 98.0% in non‐CAD group, respectively.
Conclusions
Vascular endothelial growth factor rs2010963 polymorphism may be associated with the presence of CAD and its long‐term survival, but not with its severity. To the best of our knowledge, this is the first report of genetic association between rs2010963 SNP and CAD‐related death. It can be thus suggested that rs2010963 VEGF gene can be considered as a genetic risk predictor for CAD and its outcomes.
Keywords: coronary artery disease, gene, Gensini score, mortality, vascular endothelial growth factor
1. INTRODUCTION
Coronary artery disease (CAD) is a common form of atherosclerotic vascular disease which can be considered as the leading cause of approximately one‐third of deaths worldwide.1 CAD is a complex disease involving several risk factors such as genetic variants, advanced age, male gender, smoking, hyperlipidemias, hypertension, obesity, and diabetes mellitus. Previous studies on the interaction between these factors have revealed that some genetic variations of the genes with angiogenesis function play an important role in the development of atherosclerosis.2, 3
Vascular endothelial growth factor (VEGF), a glycoprotein in the endothelial signaling pathway with multiple functions including promotion of angiogenesis and formation of blood vessels,4, 5 is produced and secreted from endothelial cells and also from smooth muscle cells of arteries walls.6 In addition, VEGF influences the activation and migration of monocytes and vascular smooth muscle cells, which can accelerate atherosclerotic plaque formation.7, 8, 9 VEGF‐A belonged to VEGF family has been more studied in CAD patients than other members such as VEGF‐B, VEGF‐C, VEGF‐D, and placental growth factor (PLGF).10
Recent studies have shown conflicting results regarding the role of VEGF‐A in atherosclerosis. Several studies have suggested that VEGF‐A is implicated in both the proliferation of vascular endothelial cells and provide protection against atherosclerotic plaque formation.11, 12, 13, 14, 15, 16 On the other hand, there is some evidence indicating that VEGF induces the neovascularization of the atherosclerotic plaque and extension of the aortic calcified lesion. These reports have suggested that VEGF protein may play important modulating roles in endothelial dysfunction and atherosclerotic pathogenesis via mediating the intimal hyperplasia, which improves atherosclerotic plaque progression in coronary arteries in both human and animal models.17, 18, 19, 20
The VEGF gene is located on chromosome 6P12 and spans the 4 kb coding region with eight exons and seven introns.10 The VEGF gene encodes a 45 kDa protein with 165 amino acids and N‐terminal signal peptide. Several gene polymorphisms of VEGF‐A have been found and investigated so far. Previous studies have described that −2578 C/A and +405 C/G polymorphisms of VEGF located in the promoter and 5' untranslated region of the gene could be more effective on susceptibility to atherosclerosis, CAD, and myocardial infarction.21, 22, 23, 24
There are, however, a few reports on the association between VEGF polymorphisms and CAD risk and its clinical outcomes. In this scenario, this study focused on a single nucleotide polymorphism (SNP) of the VEGF‐A gene (NG_008732.1), namely +405 C>G (rs2010963: p.ser338phe) (www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=2010963), in order to investigate whether rs2010963 polymorphism in VEGF‐A could predispose CAD and its related mortality Moreover, Major Adverse of Cardiovascular Events (MACE) were assessed during a 5‐year follow‐up among an Iranian population.
2. MATERIALS AND METHODS
2.1. Study population
Five hundred and twenty subjects, who were randomly selected from the persons undergoing coronary angiography at Tehran Heart Center (a hospital affiliated with Tehran University of Medical Sciences), were recruited in the present cohort study. On the basis of the angiography results, the participants were divided into two groups: 347 CAD‐positive patients and 173 individuals without angiographic signs of the disease. A 7‐year follow‐up, via either clinical visits or telephone calls at least once a year, was commenced in October 2009. In this study, the first data gathering among a cohort of 484 participants (93% of study subjects) was performed successfully by the physicians during the 5‐year follow‐up period (i.e., from the dates of coronary angiography until October 31, 2014) and events including cardiovascular mortality, myocardial infarction, and restenosis were recorded.
To precisely evaluate the severity of CAD in the subjects, both Gensini score (GS) was calculated by cardiologists grading narrowing of the epicardial coronary artery and reduction in the lumen diameter.25 The number of involved coronary vessels was also determined. Coronary artery stenosis with more than 50% luminal narrowing was regarded as CAD‐positive condition.26 Baseline information, including gender, age, as well as history of diabetes, hypertension, and cigarette smoking, was collected by interviewing the participants or reviewing their historical medical records. Individuals with blood pressure ≥140/90 mm Hg and/or history of antihypertensive drug consumption were considered hypertensives.27 According to the American Diabetes Association (ADA) definition, diabetes mellitus was defined as the use of insulin or a hypoglycemic agent, a fasting plasma glucose level of 126 mg/dL or more, or a 2‐hour post‐load plasma glucose level of 200 mg/dL or more.26 Patients with cardiovascular diseases (except for CAD), renal insufficiency, arthritis, gastrointestinal disorders, and malignancies were excluded from this study. The research protocol was approved by the institutional ethics committee at the Tehran Heart Center, and the participants signed informed consents.
2.2. Laboratory measurements, extraction of genomic DNA, and single nucleotide polymorphism genotyping
The concentrations of serum glucose, cholesterol, triglyceride, and high‐density lipoprotein cholesterol of all the blood samples collected from the subjects following a 12‐ to 14‐hour fast, were measured. The Friedewald equation was used to calculate the concentration of low‐density lipoprotein cholesterol.28 DNA samples were obtained from 10 mL of blood via a standard salting‐out method.29 The quality of the extracted DNA was evaluated via the spectrophotometric method and calculation of the optical density ratio (A260/A280).30 Genotypes were determined using the TaqMan real‐time PCR technique on Rotor‐Gene 6000 (Corbett Life Science Inc., Sydney, Australia) and an allelic discrimination assay kit (ABI; Applied Biosystems Inc., California, USA).
2.3. Statistical analysis
SPSS software version 18 (SPSS Inc., Chicago, USA) was employed as a statistical package in the analysis and comparison of the variables between the two groups. The continuous variables were analyzed using Student's t test or non‐parametric Mann‐Whitney U test. The categorical variables were analyzed using the chi‐squared test or Fisher's exact test if required. The genotype association between the two different groups was determined via the logistic regression analysis and odds ratio (OR) and 95% confidence interval (CI) for OR were also expressed. A P value <.05 was considered significant. The relative hazard ratio (HR) and 95%CI were determined for cardiovascular events using the COX proportional hazard analysis. According to the Hardy‐Weinberg equilibrium law, the balance of the allele frequencies of the polymorphism was investigated. G power 3.1 software (Heinrich‐Heine‐Universität, Düsseldorf, Germany) was used to calculate the power of this study.
3. RESULTS
3.1. Laboratory findings and clinical characteristics
In this cohort study, a total of 520 subjects (353 males, 167 females; mean age=57.8±10.7 years for the males and 59.8±9.0 years for the females) were divided into two groups according to their angiography results: 347 patients with positive CAD and 173 with angiographically normal coronary arteries. In total, 484 of 520 individuals were followed up to assess Major Adverse Cardiovascular Events (MACE).
Table 1 compares the demographic data and laboratory findings between the two groups with and without CAD. The majority of cardiovascular risk factors were more frequent in CAD group than those with normal coronary artery. In this regard, the frequencies of male gender, as well as the prevalence of smoking, hyperlipidemia, and diabetes mellitus were higher in the CAD group compared to non‐CAD group. The mean age in CAD patients was also higher when compared to the normal controls (P=.001). However, the two groups were similar in family history of CAD, hypertension, and body mass index (BMI).
Table 1.
Comparing baseline characteristics between CAD and non‐CAD groups
| Non‐CAD group (n=173) | CAD group (n=347) | P value | |
|---|---|---|---|
| Age (y) | 56.3±10.1 | 59.6±10.2 | .001 |
| Sex (Male/female) | 91/82 | 262/85 | <.0001 |
| Body mass index (kg/m2) | 28.3±4.8 | 29.7±5.2 | .077 |
| Hypertension | 68 (39.0) | 138 (39.9) | .466 |
| History of hypertriglyceridemia | 65 (38.0) | 176 (50.7) | .004 |
| History of hypercholesterolemia | 49 (28.7) | 132 (38.0) | .022 |
| Triglyceride level (mg/dL) | 178.1±90.1 | 178.2±99.8 | .984 |
| Cholesterol level (mg/dL) | 184.1±40.7 | 189.0±46.5 | .105 |
| HDL‐cholesterol level (mg/dL) | 46.4±10.7 | 41.9±10.3 | .001 |
| LDL‐cholesterol level (mg/dL) | 103.8±38.3 | 112.0±39.9 | .003 |
| Diabetes mellitus | 29 (16.9) | 96 (27.7) | .004 |
| Current smoking | 59 (35.5) | 166 (47.8) | .006 |
| Family history of CAD | 51 (30.1) | 115 (33.5) | .199 |
| Gensini score | 1.8±3.2 | 63.1±43.8 | <.0001 |
CAD, coronary artery disease; HDL‐C, high‐density lipoprotein cholesterol; LDL‐C, low‐density lipoprotein cholesterol. Data are presented as mean±SD or n (n %).
3.2. Prevalence of +405 G/C gene polymorphism
The genotypic distribution in our study population agreed with the Hardy‐Weinberg equilibrium (chi‐squared=3.30). Among the 520 study subjects, 53 (10.2%) had VEGF +405 CC, 198 (38.1%) had GC, and 269 (51.7%) had GG genotype. Univariate analysis showed an association between mutated variants of VEGF +405 C/G polymorphism (CC) and presence of CAD (OR=3.43, 95%CI=1.55–7.58, P=.002; Table 2). The association analysis was then adjusted for hypercholesterolemia, diabetes mellitus, and hypertension in a multivariable logistic regression model. The results after adjusting persisted with a strong association between the CC genotype of VEGF +405 C/G polymorphism and CAD (OR=3.65, 95%CI=1.53–8.72; P=.003). No significant association was found between the heterozygote model (GC) of VEGF +405 C/G polymorphism and CAD risk (P=.175). In addition, CC/CG genotype in C allele carriers greatly increased the susceptibility of CAD (P=.027).
Table 2.
Adjusted and unadjusted difference in genotype variants between CAD and non‐CAD groups
| rs2010963 (+405 C/G) | Non‐CAD group (n=173) | CAD group (n=347) | OR (95%CI) | P value |
|---|---|---|---|---|
| Genotypes | ||||
| CC | 8 (4.6) | 45 (13.0) | 3.43 (1.55–7.58) | .002 |
| Adj: 3.65 (1.53–8.72) | .003 | |||
| GC | 63 (36.4) | 135 (38.9) | 1.30 (0.88–1.92) | .173 |
| Adj: 1.34 (0.87–2.05) | .175 | |||
| CC+GC | 71 (41.0) | 180 (51.9) | 1.54 (1.07–2.23) | .020 |
| Adj: 1.56 (1.03–2.31) | .027 | |||
| GG | 102 (59.0) | 167 (48.1) | Ref. | |
| Alleles | ||||
| C allele (Minor allele) | 79 (22.8) | 225 (32.4) | 1.62 (1.20–2.18) | .001 |
| Adj: 1.66 (1.20–2.30) | .002 | |||
| G allele | 267 (77.2) | 469 (67.6) | Ref. | |
Ref., reference; GG, homozygous carriers of G allele; GC, heterozygous carriers of G and C allele; CC, homozygous carriers of C allele; CC+GC, C allele carriers; OR, odds ratio; CI, 95% confidence interval; P, P value; Adj, after adjustment; P values were calculated by logistic regression before and after adjustment for diabetes mellitus, hyperlipidemias, age, and gender.
Allelic analysis of the SNP after adjustment for covariates showed that the presence of C allele as the minor allele tended the subjects to have higher susceptibility to CAD in comparison with the G allele (OR=1.66, 95%CI=1.20–2.30; P=.002; Table 2).
In this study, the association between +405 C/G genotype distributions and CAD severity was evaluated by two scoring tools: the number of involved coronary vessels and Gensini score. The frequency of +405 C/G VEGF polymorphism in the patients with one, two, and three affected vessels and the mean of the GSs are presented in Table 3.
Table 3.
The association between gene variants and CAD severity
| rs2010963 (+405 C/G) | Vessel score (n (%)) | P | Gensini score | P | ||
|---|---|---|---|---|---|---|
| SVD (n=93) | 2VD (n=105) | 3VD (n=149) | ||||
| Genotypes | ||||||
| CC | 14 (15.1) | 14 (13.3) | 17 (11.4) | 58.7±50.8 | ||
| GC | 33 (35.5) | 39 (37.1) | 63 (42.3) | 50.6±40.2 | ||
| GG | 46 (49.5) | 52 (49.5) | 69 (46.3) | 52.3±44.2 | ||
| χ2 test | .812 | .657 | ||||
| Alleles | ||||||
| C allele (Minor allele) | 61 (32.8) | 67 (31.9) | 97 (32.6) | 53.8±44.7 | ||
| G allele | 125 (67.2) | 143 (68.1) | 201 (67.4) | 51.8±43.0 | ||
| χ2 test | .980 | .418 | ||||
P, P value (P significant <.05); P values were calculated by chi‐squared test. SVD, single‐vessel coronary artery disease, 2VD, two‐vessel coronary artery disease; 3VD, three‐vessel coronary artery disease. Grades of CAD severity were based on vessel score and Gensini score.
The analysis of +405 C/G genotypes in these subgroups of CAD revealed that the distribution of the CC and GC genotypes and also C allele did not have a relationship with the rise in the number of the involved vessels and also with the increase in Gensini score.
In this study, the association between VEGF +405 C/G polymorphism and mortality due to CAD was investigated. Overall, 484 individuals from 520 study subjects were followed up for a median follow‐up time of 64 months. Table 4 lists the frequency of the genotypes and CAD‐related outcomes. At the end of the follow‐up period, 30 patients died due to CAD. The 5‐year follow‐up results suggested that VEGF rs2010963 (+405C/G) polymorphism could be associated with the cardiovascular mortality. After adjusting age, BMI, the associations of GC genotype (P=.082) and CC/GC genotype (P=.004) were revealed with increased risk of CAD‐related death (Table 4).
Table 4.
The association between gene variants and CAD outcome
| rs2010963 (+405 C/G) | Cardiovascular events | Hazard ratios of CV death | ||||
|---|---|---|---|---|---|---|
| No events (n=308) | Restenosis (n=138) | AMI (n=8) | CV death (n=30) | HR (95%CI) | P | |
| Genotypes | ||||||
| CC | 27 (8.8) | 8 (5.8) | 1 (12.5) | 3 (10.0) | 2.29 (0.89–5.82) | .082 |
| GC | 107 (34.7) | 61 (44.2) | 3 (37.5) | 17 (56.7) | 2.35 (1.31–4.22) | .004 |
| GG | 174 (56.5) | 69 (50.0) | 4 (50.0) | 10 (33.3) | Ref. | |
| Alleles | ||||||
| C allele (Minor allele) | 161 (26.1) | 77 (27.9) | 5 (31.3) | 23 (38.3) | 1.72 (0.99–2.98) | .053 |
| G allele | 455 (73.9) | 199 (72.1) | 11 (68.7) | 37 (61.7) | Ref. | |
Ref., reference; CI, 95% confidence interval; P, P value (P significant <.05); AMI, acute myocardial infarction; CV death, cardiovascular death. Hazard ratios were calculated by Cox regression after adjustment for age, body mass index, and presence of CAD.
Analysis of allele frequencies data revealed that minor allele (C allele) of the polymorphism could be as a slight high‐risk allele for cardiac death with hazard ratio 1.720 (P=.056). Regarding association between patients' survival and patterns of genotypes, the 5‐year survival in C and G alleles subgroups were 92.3% and 94.3% in CAD group and 95.7% and 98.0% in non‐CAD group, respectively (Fig. 1 and Fig. 2).
Figure 1.
The survival curve in non‐coronary artery disease patients according to vascular endothelial growth factor genotyping
Figure 2.
The survival curve in coronary artery disease patients according to vascular endothelial growth factor genotyping
Minor allele frequency in this study was calculated to be 0.23 for the normal group and 0.32 for the CAD group. The sample size of 520 individuals with a 2:1 case: control ratio made the statistical power of this study more than 90%. The overall genotyping error rate between the replicates was 1.7%.
4. DISCUSSION
This study investigated the correlation between VEGF rs2010963 (+405 C/G) polymorphism and the presence and severity of CAD and searched the potential interactions between this gene variation and risk of cardiovascular death. Our main findings showed that +405 C/G polymorphism is associated with the increased risk for CAD but not with CAD severity. To the best of our knowledge, this is the first study suggested that VEGF rs2010963 polymorphism could be related to worse survival among patients with CAD. According to our results on association between lower long‐term survival and presence of C allele in both CAD and non‐CAD groups, the role of this allele for predicting long‐term lower survival in both groups is obvious.
Vascular endothelial growth factor has been known as a multifunctional glycoprotein in the endothelial signaling pathway which is involved in angiogenesis, monocyte activation and migration, as well as vascular smooth muscle cell migration.6, 7 Although the role of VEGF in atherosclerotic changes remains ambiguous in the literature, most previous studies have reported that the role of VEGF in the progression of CAD could be link to angiogenesis and vasoactivation activity, proliferation of smooth muscles cells, pathways of coagulation, fibrinolysis and renin–angiotensin–aldosterone and inflammation factors.21, 22, 23, 24 Some studies have also observed that VEGF acts as an endogenous mediator in the endothelial signaling pathway and a regulator of endothelial integrity in the arterial wall. According to their reports, VEGF acts as an anti‐atherosclerotic factor and VEGF administration could extend the re‐endothelialization after arterial damage and also contribute to the reduction of intimal thickening and/or mural thrombus formation.11, 12, 13, 14, 15, 16
A number of studies have recently demonstrated the effect of VEGF on the occurrence of coronary artery atherosclerosis. Two polymorphisms of VEGF at positions +405 (−634) and −2578 have been the principal focus of the investigations into cardiovascular diseases, and the findings have shown that of these two polymorphisms, VEGF +405 C/G could be considered a more potential genetic variant for atherosclerotic CAD in different racial populations and that −2578 C/A could be more allied to myocardial infarction.20, 21, 22, 23, 24, 31
Lin et al.1 in a study on +405 C/G polymorphism in the VEGF gene analyzed the genotypes of 398 Chinese patients with advanced CAD. The authors utilized two methods to score the severity of CAD: the clinical vessel score and the diffuse score (relating to the number of the affected vessels and the circulation rate in the coronary artery). Their study results showed a significant association between +405 C/G SNP and the two severity score models. In addition, they found a synergistic effect of this polymorphism on diabetes mellitus among patients with multi‐vessel coronary disease. Recently, Moradzadegan et al.32 presented fresh evidence of the efficacy of this polymorphism on the risk of CAD among Iranian population. They have studied the association between angiotensin‐converting enzyme (ACE) insertion/deletion (I/D) (rs4646994), and VEGF polymorphism (+405 G/C) in Iranian patients with type II diabetes and showed synergistic interaction between VEGF and ACE variants indicated to pathophysiological significance of VEGF in progression of atherosclerosis by influence on the vascular wall growth processes.
In this study, the results for VEGF +405 C/G genotyping and allele frequencies were analyzed both before and after adjusting for hyperlipidemia, diabetes mellitus, age, and gender. Our data showed that the subjects carrying CC genotype and also those carrying C allele had a higher susceptibility to CAD, which pointed toward the potential role of VEGF in CAD development and clinical complications of atherosclerosis. In spite of this strong association, further analysis of the data in two CAD severity models showed no dose‐dependent effect of the polymorphism on the CAD severity (assessed by the Gensini score) and the degree of arterial obstruction (assessed by the number of involved coronary vessels) in our study population.
We drew on the Gensini score as a precise method for the assessment of CAD severity according to angiographic findings. The Gensini score calculation is based on the evaluation of the number of epicardial coronary artery segments with stenosis, reduction in the lumen diameter, and localization of stenotic changes.25 In addition to the Gensini scoring system, we used vessel score model (based on the number of diseased coronaries) as a confirmatory analysis mode.
Although this study did not examine the possible association between the genotypes and expression of VEGF, there are some evidence implying that VEGF genotypes at sites of +405, −2578, and +936 may be related to the amounts of VEGF protein production. Previous studies have suggested that the presence of +405 C allele and −2578 A allele in the VEGF gene is associated with elevated VEGF levels and development of CAD, whereas a correlation has been observed between +936 T allele VEGF polymorphism and lower VEGF serum levels but not with the severity of CAD.33, 34, 35, 36 The association between VEGF expression and susceptibility to atherosclerosis may imply that VEGF protein plays an important role in modulating atherosclerosis changes.
In this study, the results of a 5‐year follow‐up period demonstrated that VEGF and its genetic variations of rs2010963 could predict the mortality and survival of CAD patients. The high hazard ratios (more than 2 for genotypes and 1.73 for C allele) of death due to atherosclerotic changes among our SNP carriers indicated that VEGF +405 C/G polymorphism could be deemed a mortality risk factor in this atherosclerosis‐related disease.
There are some limitations in our cohort study. Although our sample size provide power more than 90%, we only analyzed the +405 C/G polymorphism regardless of other polymorphisms related to susceptibility of CAD and its clinical outcomes. Moreover, the complete elimination of interaction between the use of drugs and genetic markers was impossible.
Finally, results from these studies are highlighted this possibility that +405 C/G VEGF genetic variations could act as a pro‐atherosclerotic factor in CAD probably through the changes it makes in the metabolism of lipids and the endothelial integrity in the wall of arterial vessels. In addition, VEGF +405 C/G polymorphism might be regarded as genetic factor for atherosclerotic CAD and risk of sudden cardiovascular death. Further studies should be conducted to identify VEGF molecular basis and pathogenesis of the atherosclerotic changes leading to future cardiovascular events.
Regarding the risky or bystander status of the pointed polymorphism, we first examined the association between SNP and CAD in univariate analysis indicating a significant difference. Then, by adjusting baseline variables in multivariable regression modeling, the pointed relationship remained significant showing the risk role of the SNP for triggering CAD.
Reviewing the literature shows that the presence of both polymorphisms is significantly associated with both CAD and myocardial ischemic events, however, the triggering role of +405 (G/C) SNP as a risk factor was shown to be higher than −2578 (C/A) to predict CAD. As revealed by Lin et al. (2010), the odds ratio for predicting significant CAD was higher than for +405 (G/C) SNP when compared to −2578 (C/A). In another survey by Lin et al. (2010), there was a significant independent association of +405 C/G genotype with CAD and its diffuse score in the uni‐ and multivariate regression analysis, but no association was found between VEGF‐2578C/A polymorphism and both atherosclerotic parameters. Because of stronger evidence on stimulatory role of +405 (G/C) SNP in CAD, we preferred to assess this polymorphism among our population.
5. CONCLUSIONS
In conclusion, our cohort study suggested that VEGF +405C/G polymorphism could be considered as a genetic marker for predicting CAD in Iranian population without a causality effect on CAD severity. In addition, our study hinted this hypothesis that the risk of mortality due to CAD can be predicted by confirming +405 C/G VEGF genetic variations.
ACKNOWLEDGMENTS
We gratefully acknowledge all the patients, cardiologists, and laboratory staff of Tehran Heart Center, who cooperated in this study. This study was supported by Tehran Heart Center, Affiliated to Tehran University of Medical Sciences.
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