Angiotensin converting enzyme insertion/deletion (I/D) (rs4646994) and Vegf polymorphism (+405G/C;rs2010963) in type II diabetic patients: Association with the risk of coronary artery disease

Abstract

Hypothesis:

Little is known about the concomitant presence of the angiotensin-converting enzyme (ACE) (rs4646994) D allele and vascular endothelial growth factor(VEGF) (+405G/C; rs2010963) G allele on the susceptibility of coronary artery disease (CAD). Here we examined the hypothesis that ACE-D and VEGF-G alleles act synergistically to increase the severity of CAD in patients with type II diabetes mellitus (T2DM).

Materials and methods:

The VEGF (rs2010963) and ACE (rs4646994) genotypes were detected by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and PCR, respectively in 510 T2DM patients undergoing their first coronary angiography. Diabetic patients were classified as T2DM patients with and without CAD (control).

Results:

The crude odds ratio (OR) for the presence of CAD in ID+DD and D allele carriers were 1.98 (p=0.01) and 1.55 (p=0.001), respectively. Also, adjusted ORs in the presence of normolipidemia and the absence of history of hypertension for the risk of CAD in the either ACE(rs4646994) D allele or VGEF(rs2010963)-G alleles were 2.08 (p=0.004) and 1.75 (p=0.024), respectively. In addition, the concomitant presence of the ACE-D and VEGF-G alleles increased the risk of CAD 2.25-fold (p=0.043).

Conclusion:

Our results indicated that ACE(rs4646994)-D allele alone and in the presence of VEGF(rs2010963)-G allele can be an important independent risk factor for susceptibility of CAD in T2DM patients even after correcting for conventional risk factors in a population of Iran.

Keywords

Angiotensin converting enzyme (rs4646994)vascular endothelial growth factor (rs2010963)coronary artery disease diabetes mellitus genetic polymorphism

Introduction

Coronary artery disease (CAD) is the major complication of type II diabetes mellitus (T2DM),^1,2 that accounts for nearly 50% of all deaths per year in the Iranian population.³ Coronary artery disease (CAD) is a complex disorder resulting from the interaction between genetic and environmental factors.^4,5 Several genes have been investigated for heir involvement in the development of CAD.

Studies indicate that the presence of angiotensin-converting enzyme (ACE) gene polymorphism resulting in increased ACE activity might be associated with the atherosclerotic process and consequently cardiovascular diseases and mortality.⁶ Renin-angiotensin system blockade has been considered a cornerstone of strategies to reduce cardiovascular risk, through antihypertensive, anti-inflammatory, anti-proliferative and oxidative stress lowering actions.⁶ The ACE gene is located on chromosome 17q23 and consists of 26 exons and 25 introns. ACE polymorphism (rs4646994) is due to the presence of the insertion (I) allele or absence (deletion (D) allele of a 287 bp Alu repeat sequence within intron 16 resulting in three genotypes DD, II and ID.⁷

Many studies, including a meta-analysis, have reported an association between the DD genotype and increased risk of developing coronary artery disease.^8–10 However, some studies failed to confirm these findings.^11–13

On the other hand, the gene encoding vascular endothelial growth factor (VEGF) has also been investigated in coronary disease studies.^14–16 VEGF, a mitogen that promotes vascular endothelial cell proliferation and angiogenesis, is a 45 kDa glycoprotein secreted by endothelial cells and smooth muscle cells.^17,18 The VEGF gene is located on chromosome 6p21.3 and consists of eight exons exhibiting alternate splicing to form a family of proteins.¹⁹

Some of SNPs in the VEGF gene such as, the +405G/C; rs2010963 polymorphism in the 5 /–untranslated region has a significant effect because the GG genotype is correlated with higher VEGF protein production in healthy individuals.^{20, 21} Some studies point to a protector effect of VEGF on atherosclerotic plaque development, acting as regulator of endothelial integrity of the coronary artery wall. It was observed that recombinant VEGF administration for artery disease leads to a reduction of intimal thickening and/or thrombus formation.^22,23 There are studies showing that neovascularization, mediated by VEGF, influences the pathogenesis of arterial diseases.²⁴ The understanding of the role of VEGF in atherosclerosis, as well as the factors that can quantitatively and qualitatively modify the production of this protein becomes, then, of great importance in the clarification of the mechanisms that lead to the development of CAD.

The aim of the present study was to investigate the association between ACE (rs4646994) I/D and VEGF (rs2010963) polymorphisms and their possible synergistic effects on the risk and severity of CAD in T2DM patients from Tehran, Iran.

Materials and methods

Patients

In a case–control study we investigated VEGF (rs2010963) and ACE (rs4646994) gene polymorphisms in 510 T2DM patients (141 with CAD and 369 without CAD). T2DM patients were recruited from Imam Hospital of Tehran University of Medical Sciences. All studied patients were from Tehran, the capital and a cosmopolitan city. A detailed medical history of each patient was obtained. The following variables were determined for each patient: age, sex, smoking habits, body mass index (BMI), total cholesterol, low-density lipoprotein cholesterol (LDL–C), high-density lipoprotein cholesterol (HDL-C), and triacylglycerol (TG). The diagnosis of diabetes was based on criteria suggested by American Diabetes Association ²⁵. The diagnosis of CAD was confirmed by coronary angiography. After a 12 h overnight fasting, 10 ml of 15% ethylenediaminetetraacetic acid (EDTA) anticoagulated blood was obtained from each patient. Normolipidemic individuals were selected if they had total cholesterol (TC), TG, and LDL-C levels less than 5.18, 2.26 and 3.36 mmol/l, respectively.²⁶ Hypertension was defined as resting systolic blood pressure≥130 mm Hg and diastolic blood pressure≥85 mm Hg or using anti-hypertension drugs.

Chemical analysis

Serum TC, TG, LDL-C and HDL-C levels were measured by standard enzymatic methods. Very-low-density lipoproteins (VLDLs) were calculated by Friedewald’s formula.²⁷

Genomic DNA analysis

Five milliliters of peripheral blood samples was collected in tubes containing EDTA. Genomic DNA was isolated from peripheral blood leukocytes by standard methods and stored at −20ºC.²⁸ Genotyping of the +405G/C; rs2010963 polymorphism in the VEGF gene and I/D (rs4646994) polymorphism in the ACE gene was determined by employing the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and PCR, respectively.^29,30

Statistical analysis

The SPSS statistical software package version 16 was used for the statistical analyses. A p value <0.05 was considered significant. The gene counting method calculated for allelic frequencies. The χ²-test was used to verify the agreement of observed genotype frequencies with those expected according to the Hardy–Weinberg equilibrium. The genotypes and alleles frequencies of VEGF and ACE were compared between T2DM patients with and without CAD using the χ²- test. Odds ratios (ORs) were calculated as estimates of relative risk for disease and 95% confidence intervals (CIs) obtained by SPSS logistic regression. The interaction between the ACE D and VGEF G alleles was determined using a logistic regression model. A two-tailed Student’s t test, analysis of variance (ANOVA) and nonparametric independent sample Mann-Whitney analysis were used to compare quantitative data.

Results

The clinical and laboratory features of the CAD patients and controls (patients without CAD) are reported in Table 1. Among the 510 patients with T2DM, 141 patients had CAD and 369 patients were classified as without CAD. The mean age of CAD patients was slightly higher than that of the control group, although, it did not reach to a statistical significance. In addition, cholesterol, TG, and LDL concentrations were significantly higher, in the T2DM patients with CAD than in patients without CAD. The frequency of genotypes did not deviate from the Hardy-Weinberg equilibrium in total sample and in two studied groups (p>0.05). The prevalence of hypertension was 63.8% in diabetic patients with CAD compared to 37.4% in patients without CAD (p<0.001). Distribution of VEGF (rs2010963) and ACE (rs4646994) genotypes and alleles in T2DM with CAD and without CAD are presented in Table 2. Distribution of ACE (rs4646994) genotypes and alleles, except for I/D genotype, were significantly different in T2DM patients with CAD compared with patients without CAD. There were no statistically significant differences between the VEGF (rs2010963) genotypes and allele frequencies (as calculated using the actual allele number) in the diabetic patients with CAD compared with patients without CAD.

Table 1.

Demographic characteristics and distribution of risk factors in diabetic patients with and without coronary artery disease (CAD). FBS; Fasting blood sugar

Parameter	Diabetic patients with CAD (n=141)	Diabetic patients without CAD (n=369)	p value
Sex, M/F (%)	76/65(53.9/46.1)	170/199(46.1/53.9)	0.113
Age (years)	60.2±8.6^a	58.6±7.5	0.25
Smoking (%)	30 (21.3)	71(19.2)	0.266
Diabetic duration (years)	13.52±6.6	13.94±10.7	0.59
Systolic blood pressure (mm Hg)	144.8±22.1	136.4±20.7	<0.001
Diastolic blood pressure (mm Hg)	89.5±10.6	84.7±10.3	<0.001
FBS (mg/dl)	205.2±66.7	201.3±61.9	0.53
Cholesterol (mg/dl)	245.1±119.7	206.2±60.1	<0.001
Triglyceride (mg/dl)	193±83.5	170.5±67.5	0.004
HDL (mg/dl)	42.5±8.9	44.5±10.6	0.036
LDL (mg/dl)	126±34.1	118.35±10.5	0.015
VLDL (mg/dl)	39.6±12.4	35.59±10.5	0.001
BMI (kg/m²)	27±4.8	26.24±4.0	0.078
History of hypertension	82(58.2%)	158(42.6%)	<0.002

BMI: body mass index; HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very-low-density lipoprotein.

Data are presented as mean±standard deviation (SD) unless otherwise stated. Comparisons were made using student’s t test (for continuous variables). Statistically significant if p<0.05.

Table 2.

The distribution of angiotensin-converting enzyme (ACE) and vascular endothelial growth factor (VEGF) genotypes in diabetic patients with and without coronary artery disease (CAD).

	CAD patients	Patients without CAD
ACE genotypes
I/I	(n=21, 14.9%)	(n=95, 25.7%)
I/D	(n=69, 48.9%)	(n=180, 48.8%)
D/D	(n=51, 36.2%)	(n=94, 25.5%)
D/D	χ²=9.387, df=2, p=0.009	(n=94, 25.5%)
I/D+D/D	(n=120, 85.1%)	(n=174, 74.3%)
I/D+D/D	χ²=6.8, df=1, p=0.009	(n=174, 74.3%)
ACE alleles
I	(n=111, 39.4%)	(n=370, 50.1%)
D	(n=171, 60.6%)	(n=368, 49.9%)
D	χ²=9.5, df=1, p=0.002	(n=368, 49.9%)
VEGF genotypes
C/C	(n=33, 23.4%)	(n=87, 23.6%)
G/C	(n=65, 46.1%)	(n=197, 53.4%)
G/G	(n=43, 30.5%)	(n=85, 23.0%)
G/G	χ²=3.319, df=2, p=0.190	(n=85, 23.0%)
G/C+G/G	(n=108, 76.6%)	(n=182, 74.4%)
G/C+G/G	χ²=0.02, df=1, p=0.96	(n=182, 74.4%)
VEGF alleles
C	(n=131, 46.5%)	(n=371, 50.3)
G	(n=151, 53.5%)	(n=367, 49.7)
G	χ²=1.189, df=1, p=0.275	(n=367, 49.7)

Note: The distribution and comparisons of genotype frequencies were made using χ² analysis.

The OR for the presence of ACE and VEGF genotypes and ACE D and VEGF G alleles are shown in Table 3. The ORs for the presence of ACE (I/D+D/D) genotype and D allele in the CAD patients were found to be 1.98 (95% CI=1.18–3.3, p=0.01) and 1.55 (95% CI=1.2–2.1, p=0.001), respectively. The ORs for the presence VEGF (G/C+G/G) genotype and G allele in the CAD patients were detected to be 1.01 (95% CI=0.64–1.6, p=0.97) and 1.17 (95% CI= 0.89–1.53, p=0.276), respectively.

Table 3.

Odds ratios of angiotensin-converting enzyme (ACE) and vascular endothelial growth factor (VEGF) genotypes and alleles with respect to I/I genotype or I allele and C/C genotype or C allele respectively, in diabetic patients with and without coronary artery disease (CAD).

	CAD patients OR (95% CI, p)	Patients without CAD
ACE genotypes
I/I	Reference group (n=21)	Reference group (n=95)
I/D	1.73 (1.32–2.74, p=0.049, n=69)	n=180
D/D	2.45 (1.37– 4.4, p=0.003, n=51)	n=94
(I/D+D/D)	1.98 (1.18–3.3, p=0.01, n=120)	n=274
ACE alleles
I	Reference group (n=111)	Reference group (n=370)
D	1.55 (1.2–2.1, p=0.001, n=171)	n=368
	CAD patients	Patients without CAD
VEGF genotypes
C/C	Reference group (n=33)	Reference group (n=87)
G/C	0.87 (0.53–1.4, p=0.57, n=65)	n=197
G/G	1.33 (0.87–2.3, p=0.3, n=43)	n=85
G/C+G/G	1.01 (0.64–1.6, p=0.97, n=108)	n=182
VEGF alleles
C	Reference group (n=131)	Reference group (n=371)
G	1.17 (0.89–1.53, p=0.276, n=151)	n=367

CI: confidence interval; OR: odds ratio.

Furthermore, we calculated adjusted OR according to the age, sex, normolipidemia and the absence of history of blood pressure for all the patients. Also, the adjusted OR was calculated for ACE (rs4646994) and VGEF (rs2010963) alleles that demonstrated in Table 4. We found a statistically significant OR of 2.08 (95% CI=1.26–3.4, p=0.004) and OR of 1.75 (95% CI=1.08–2.8, p=0.024), respectively for the presence of ACE D and VGEF alleles in CAD subjects with normolipidemia and without hypertension. We observed that the ACE (rs4646994) D and VGEF (rs2010963) G alleles are risk factors for CAD after correcting for conventional risk factors.

Table 4.

The distribution and odd ratio of angiotensin-converting enzyme (ACE) and vascular endothelial growth factor (VEGF) alleles (allele count) with respect to I and C allele in diabetic patients with and without coronary artery disease (CAD) and after adjustment for sex, age, normolipidemia and absence of history of blood pressure.

	CAD patients	Patients without CAD
ACE alleles
I	Referent group (n= 28, 33.7 %)	Referent group (n=204, 51.4%)
D	(n=55, 66.3%)	(n=193, 48.6 %)
	OR=2.08 (1.26–3.4, p=0.004)
	^aχ²=8.6, df=1, p= 0.003
VEGF alleles
C	Referent group (n=32, 38.6%)	Referent group (n=208, 52.3%)
G	(n=51, 61.4%)	(n=189, 47.7 %)
	OR=1.75 (1.08 −2.8, p=0.024)
	^aχ²=5.2, df=1, p=0.023

OR: odds ratio.

The distribution and comparisons of allele frequencies were made using χ² analysis.

The gene counting method calculated for allelic frequencies.

As shown in Table 5, logistic regression analysis as defined by Vaisi-Raygani et al.,³¹ demonstrated a strong and significant synergism between ACE (rs4646994) D and VGEF (rs2010963) G alleles on the increased risk of CAD in diabetic patients after adjustment for the presence of normolipidemia and absence of history of blood pressure with OR=2.25 (95% CI= 1.03–4.9, p=0.043). The presence of ACE (rs4646994)-D allele increased the risk of CAD 2.08-fold (p=0.004). Also, the presence of VEGF (rs2010963)-G allele increased the risk of CAD 1.75-fold (p=0.024). We observed the more increased risk of CAD by 2.25-fold (p=0.043) in the presence of both alleles of ACE-D and VEGF-G.

Table 5.

Interaction of vascular endothelial growth factor (VEGF) G/C and angiotensin-converting enzyme (ACE) I/D polymorphism on the risk of coronary artery disease (CAD) in diabetic patients after adjustment for sex, age, normolipidemia and absence of history of blood pressure.

VEGF G	ACE D	CAD patients	Patients without CAD
		Reference group	Reference group
_	_	(n=8, 9.6%)	(n=67, 16.9%)
+	_	(n=0)	(n=42, 10.6%)
+	_	^aχ²=4.8, df=1, p=0.028	(n=42, 10.6%)
_	+	(n=8, 9.6%)	(n=38, 9.6%)
		1.76 (0.61– 5, p=0.29)
		^aχ²=1.1, df=1, p=0.29
+	+	(n=67, 80.7%)	(n=250, 63%)
		2.25 (1.03– 4.9, p=0.043)
		^aχ²=4.3, df=1, p=0.038
		^bχ²=14.4, df=3, p=0.002

Distribution of corresponding alleles in control group has been compared with CAD patients.

Distribution of the VEGF G, ACE D (–, –), VEGF G, ACE D (+, –), VEGF G, ACE D (–, +) and VEGF G, ACE D (+,+) has been compared between CAD and non CAD patients.

These data suggest that diabetic patients carrying both ACE (rs4646994)-D and VGEF (rs2010963)-G alleles have a synergistically increased risk of CAD after adjustment for conventional risk factors (the presence of hypertension and dyslipidemia) in Tehran population of Iran.

Table 6 indicates the distribution of ACE (rs4646994) I/D and VEGF (rs2010963)-G/C polymorphisms in males and females separately. Distribution of ACE (rs4646994) I/D variants were not significantly different between males and females patients with and without CAD. No significant difference was observed between males and females patients with and without CAD regarding the frequency of ACE (rs4646994) I/D genotypes. Also, the absence of association between the VEGF (rs2010963) G/C polymorphism and gender was detected among diabetic patients with and without CAD (Table 6).

Table 6.

The comparison of angiotensin-converting enzyme (ACE) and vascular endothelial growth factor (VEGF) genotypes between males and female of diabetic patients with and without coronary artery disease (CAD).

	CAD patients		Patients without CAD
	Male	Female	Male	Female
ACE genotypes
II	13(17.1%)	8(12.3%)	37(21.8%)	58(29.1%)
#χ²=3.7, df=1, p=0.055
ID	40(52.6% )	29(44.6%)	83(48.8%)	97(48.7%)
#χ²=2.8, df=1, p=0.094
DD	23(30.3%)	28(43.1%)	50(29.4%)	44(22.1%)
#χ²=0.9, df=1, p=0.35
*χ²=2.6, df=2, p=0.27			*χ²=3.9, df=2, p=0.15
VGEF genotypes
CC	23(30.3%)	10(15.4%)	44(25.9%)	43(26.1%)
#χ²=3.56, df=1, p=0.06
CG	30(39.5%)	35(53.8%)	80(47.1%)	117(58.8%)
#χ²=0.6, df=1, p=0.43
GG	23(30.3%)	20(30.8%)	46(27.1%)	39(19.6%)
#χ²=0.05, df=1, p=0.94
*χ²=4.9, df=2, p=0.087			*χ²=5.3, df=2, p=0.071

a: defined as distribution and comparisons of II, ID and DD genotypes of ACE and CC, CG and GG genotypes of VGEF between male and female in diabetic patients with CAD; b: defined as overall distribution of ACE and VGEF genotypes between males and females in diabetic patients with CAD and without CAD separately.

Discussion

We observed that the ACE D allele alone and in the presence of the VEGF (rs2010963)-G allele increased the risk of CAD in T2DM patients. Also, we clearly demonstrated that the ACE (rs4646994) D and VEGF (rs2010963) G alleles act in synergy to increase the risk of CAD in patients after adjustment for conventional risk factors including hyperlipidemia and hypertension. Recent studies suggest that the ACE (rs4646994) D and VEGF (rs2010963) G alleles are genetic factors that may increase the risk of developing CAD and T2DM.^32–35 In the present study, we examined the relationship between these two risk factors and CAD, in T2DM patients of Iran. This study indicates that the presence of the ACE (rs4646994) D allele increases the risk of CAD in T2DM patients. However, the allele and genotype frequencies of VEGF (rs2010963) G/C polymorphism were not different between patients with and without CAD. The frequency of the ACE (rs4646994) D allele was found to be significantly higher in CAD patients (60.6%) than in the control group (49.8%). Consistent with data obtained from different studies,^{26, 36–38} we found that the ACE (rs4646994) D allele increases the risk of CAD in T2DM patients. In contrast, several investigators were not able to find an association between the ACE D allele and the risk of CAD in the populations that they had investigated.^39
–41 It is conceivable that a gene-environment interaction may play a significant role in the predisposition to CAD.^38,42

The insertion/deletion (I/D) (rs4646994) polymorphism in intron 16 of the ACE gene is considered an important genetic determinant of CAD and hypertension.^43,44 There is controversy related to the role of the ACE D/I polymorphism in the risk of CAD and myocardial infarction (MI).³²

The mechanism by which the ACE I/D genotypes may predispose an individual to the development of CAD and MI remains unclear. ACE (rs4646994) D allele has been correlated to increase ACE activity that is responsible for the conversion of angiotensin I to the peptide precursor angiotensin II that induces aldosterone production. Elevation of circulating levels of aldosterone influences on arterial hypertension, cardiac fibrosis and, consequently, both diastolic dysfunction and ventricular remodeling, is implicated in the pathogenesis of atherosclerosis.⁴⁵

The role of VEGF in the pathogenesis of atherosclerosis remains a matter of debate in the literature. Some studies observed that VEGF acts as an anti-atherosclerotic factor, leading to accelerated re-endothelialization, which in turn leads to reduction of intimal thickening and thrombus formation.^11,46 Others have reported that administration of recombinant human VEGF enhances atherosclerotic plaque progression^34,47–49 It was observed that atherosclerotic plaque neovascularization, mediated by VEGF, makes available nutrients and components to the plaque, increasing its volume. In artery wall with total occlusion, microvessels called vasa vasorum were related, in anatomy and amount, to degree of plaque intimal layer inflammation.⁵⁰ Moulton et al.⁵¹ observed the atherosclerosis progression reduction secondary to inhibition of the neovascularization is mediated by VEGF. In this regard, more studies are necessary to clarify the real role of the VEGF protein in CAD.

Interestingly, our study demonstrates a significant association between possessions of both ACE D and VEGF G alleles in diabetic patients with CAD after adjustment for the presence of normolipidemia and absence of history of blood pressure that increased the risk of CAD in T2DM 2.25 times. This study indicates that in the presence of either ACE D or VEGF G allele alone the risk of CAD is lower (2.08-fold and 1.75-fold, respectively) compared with concomitant presence of both ACE-D and VEGF-G.

The interaction of ACE with VEGF is of special interest in view of the important role played by the renin-angiotensin system in many cardiovascular disorders in which angiogenesis is induced. This includes myointimal proliferation after vascular injury, atherosclerosis, and diabetic angiopathy. Association of enhanced vascular ACE expression with the development of coronary atherosclerosis in humans has been reported.^52,53 Furthermore, ACE inhibitors have vascular protective effects that may contribute to the prevention of coronary atherosclerosis in humans and animal models.^54,55 On the other hand, induction of VEGF in human atherosclerotic lesions and in animal models of arterial injury has been described.^56
–59

Saijonmaa et al.⁶⁰ observed VEGF increased ACE at mRNA and protein levels in cultured human endothelial cells. Saijonmaa et al.⁶¹ in 2004, observed atorvastatin completely inhibited VEGF-induced ACE upregulation in endothelial cells probably by inhibiting PKC activation. This effect was mediated via the mevalonate pathway, and inhibition of both FPP and GGPP were involved. These findings may be an additional beneficial effect of statins in the prevention and treatment of cardiovascular disease.

Our observations suggested, the interaction of VEGF and ACE may play a role in the pathophysiological processes of the CAD and the synergistic interaction between VEGF and ACE variants may enforce detrimental growth processes in the vascular wall. If true, such a synergy could be a future target of pharmacological intervention and combined treatment with ACE inhibitors and inhibitors of VEGF, if made available, may be of value in the prevention of atherosclerosis, but additional analysis is needed to clarify the real contribution of ACE and VEGF to the development of CAD in different Iranian populations.

Conclusion

We have demonstrated that ACE D allele alone and synergistically with VEGF G allele increases the risk of CAD in T2DM patients even after adjustment for normolipidemia and absence of history of blood pressure. Because of the small sample size in the present study and because Iranians show wide genetic diversities, additional analysis on a larger samples is needed to clarify the contribution of ACE D and VEGF G alleles to the development of CAD in different Iranian populations and various ethnic groups in the world.

Limitations of the study

One of the limitations of this study is the lower number of diabetic patients with CAD available for the analysis of VEGF +405 G/C and ACE I/D genotypes. The influence of some common gene polymorphisms in the promoter region of the VEGF gene on the susceptibility to diabetes, myocardial infarction and impaired prognosis in patients with chronic heart failure has been reviewed by Petrovic et al.⁵⁹ It has been suggested that CC genotype of the VEGF +405 G/C gene promoter is associated with the impaired prognosis in patients with chronic heart failure.⁵⁹ However, the influence of VEGF +405 G/C polymorphism on the gene expression was not examined in our study that may also be considered as another limitation of our study.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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