The Protective Effects of the VEGF −2578C>A and −1154G>A Polymorphisms Against Hypertension Susceptibility

Abstract

Aims: It has been reported that plasma vascular endothelial growth factor (VEGF) levels are elevated in hypertensive patients. The aim of this study was to investigate the association of −2578C>A and −1154G>A polymorphisms in the VEGF gene with susceptibility to hypertension. Methods: A total of 640 subjects (320 hypertensive patients and 320 healthy normotensive subjects) were enrolled in the study. Genotyping of the −2578C>A and −1154G>A polymorphisms was performed by polymerase chain reaction and restriction fragment length polymorphism analysis. Results: The CA and AA genotypes and dominant and recessive models of the −2578C>A polymorphism were protective against MetS susceptibility (p<0.0001, p=0.001, p<0.0001, and p=0.015, respectively). The GA genotype and dominant model (GG vs. GA+AA) of the −1154G >A polymorphism were found less frequently in patients with hypertension compared to the controls (p=0.0001, p<0.0001, respectively). Haplotypes of the −2578C>A and −1154G>A polymorphisms were also different between the patients and controls. Conclusions: The A allele of the −2578C>A polymorphism and haplotypes of the −2578C>A and −1154G>A polymorphisms in the promoter region of the VEGF gene might be protective against the development of hypertension in Koreans. Further studies examining these associations in other populations are warranted.

Introduction

In light of the importance of a potent angiogenic factor, vascular endothelial growth factor (VEGF) has been considered as a candidate contributing to the pathogenesis of vascular diseases. Plasma VEGF levels are quite variable among healthy individuals (Renner and Pilger, 1999). Furthermore, the levels of plasma VEGF are considered to be increased under both physiological and pathological conditions (Gargett and Rogers, 2001). A previous study showed that patients with hypertension have elevated levels of plasma VEGF (Stumpf et al., 2009). Blood pressure and inflammation are also correlated with plasma VEGF levels (Siervo et al., 2010, 2012).

The gene encoding human VEGF has been cloned and assigned to chromosome 6p21.3 (Tischer et al., 1991). A number of polymorphisms have been reported in the promoter at 5′- and 3′-untranslated regions of the VEGF gene. Several polymorphisms of the VEGF gene were associated with plasma VEGF levels. Among them, healthy subjects with the −2578A alleles of the −2578C>A and −1154G>A polymorphisms were associated with lower plasma VEGF levels, compared to the noncarriers (Shahbazi et al., 2002; Steffensen et al., 2010).

Although the VEGF polymorphisms were elucidated to be associated with vascular diseases in many studies, they are neither strong nor consistent in all populations studied. Furthermore, the allelic frequencies of the −2578C>A and −1154G>A polymorphisms have been reported to be different among the populations studied. A number of studies have suggested that hypertension is a multifactorial disease in which the genetic and environmental factors, such as diet and life style, play important roles (Kato, 2002). These factors may differ among racial or ethnic groups. Therefore, we investigated whether the VEGF −2578C>A and −1154G>A polymorphisms are associated with Korean patients with hypertension.

Materials and Methods

Study population

We planned the case-control study on a total of 640 individuals. Three hundred twenty patients who were diagnosed with hypertension were enrolled from the Biobank of Jeju National University Hospital, which is a member of Korea Biobank Network supported by the Ministry of Health and Welfare in South Korea. Blood pressure was measured in the seated position after 10 min of rest. Anthropometric and biochemical levels, including the body-mass index (BMI), triglyceride (TG), waist circumference (WC), fasting blood sugar (FBS), and high-density lipoprotein (HDL)-cholesterol, were also measured. The control subjects consisted of 320 healthy individuals who were randomly selected following a health screening, and the individuals with a history of vascular diseases were excluded from this study. The hypertensive patients had significantly higher values for all the conventional risk factors, including the BMI, TG, systolic blood pressure, diastolic blood pressure, FBS, and WC (p<0.001), but they had lower HDL-cholesterol levels (p<0.001) than the controls (Table 1). Written informed consents were provided from all study participants. The study protocol was approved by the Institutional Review Board of Jeju National University Hospital.

Table 1.

Demographic Characteristics of the Study Subjects

	Control (n=320)	HTN patients (n=320)	p-Value
Age, years	50.94±8.43	52.29±10.46	0.073
BMI, kg/m²	23.87±2.715	26.03±2.962	<0.001
FBS, mg/dL	87.35±10.09	96.99±17.77	<0.001
TG, mg/dL	86.53±39.57	135.66±87.73	<0.001
HDL-C, mg/dL	57.01±13.57	51.38±12.38	<0.001
SBP, mmHg	117.99±12.03	137.09±14.94	<0.001
DBP, mmHg	70.84±8.53	83.84±10.51	<0.001
WC, cm	72.07±9.160	83.86±10.40	<0.001

Values are mean±standard deviation.

BMI, body-mass index; DBP, diastolic blood pressure; FBS, fasting blood sugar; HDL-C, high-density lipoprotein-cholesterol; HTN, hypertension; SBP, systolic blood pressure; TG, triglyceride; WC, waist circumference.

VEGF genotyping

Genomic DNA was extracted from peripheral blood leukocytes using an extraction kit (Intron Biotechnology, Seongnam, South Korea). Genotypes of the VEGF −2578C>A and −1154G>A polymorphisms were determined by the polymerase chain reaction (PCR) and restriction fragment length polymorphism techniques, under the conditions previously described (Park et al., 2012).

Briefly, the VEGF −2578C>A polymorphic site was amplified using a sense primer 5′-GGA TGG GGC TGA CTA GGT AAG-3′ and an antisense primer 5′-AGC CCC CTT TTC CTC CAA C-3′. Then, the PCR-amplified products were digested with AvaII restriction enzyme. Alleles of the −2578C>A polymorphic site were classified depending on the presence (A allele, 308 and 18 bp) or absence (C allele, 326 bp) of a restriction enzyme-cutting site. For the −1154G>A polymorphism, it was amplified with the primers of sense 5′-CGC GTG TCT CTG GAC AGA GTT TCC-3′ and antisense 5′-CGG GGA CAG GCG AGC TTC AG-3′. After the digestion of PCR products with MnlI restriction enzyme, the −1154G allele was divided into two fragments of 141 and 32 bp, whereas the −1154A allele remained uncut (173 bp).

Statistical analyses

Baseline data for the cases and controls were compared using the student's t-test. To compare the genotype and allele frequencies of the VEGF polymorphisms between the controls and cases and to identify deviations from the Hardy-Weinberg equilibrium, the chi-square test was used. Associations between the VEGF polymorphisms and hypertension susceptibility were calculated using adjusted odds ratios (AORs) and 95% confidence intervals (CI) by multivariate logistic regression analysis. Statistical analyses were performed using GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA). Haplotype frequencies for VEGF −2578C>A and −1154G>A loci were estimated using the expectation-maximization algorithm with SNPAlyze (Version 5.1; DYNACOM Co, Ltd., Yokohama, Japan). The p-value <0.05 was considered statistically significant.

Results

The genotype distributions of the VEGF −2578C>A and −1154G>A polymorphisms are summarized in Table 2. The genotype distributions of the VEGF −2578C>A and −1154G>A polymorphic loci met the Hardy-Weinberg equilibrium condition, in both hypertensive patients and normotensive control groups.

Table 2.

VEGF Genotype Distributions in the HTN Patient and Control Groups

Genotype	Control (n=320)	HTN (n=320)	AOR (95% CI)	p-Value
VEGF −2578C>A
CC	164 (51.3)	223 (69.7)	1.000 (reference)
CA	134 (41.9)	90 (28.1)	0.455 (0.320-0.647)	<0.0001
AA	22 (6.9)	7 (2.2)	0.237 (0.095-0.589)	0.002
CC versus CA+AA			0.427 (0.303-0.601)	<0.0001
CC+CA versus AA			0.326 (0.132-0.807)	0.015
VEGF −1154G>A
GG	200 (62.5)	258 (80.6)	1.000 (reference)
GA	104 (32.5)	62 (19.4)	0.473 (0.323-0.693)	0.0001
AA	16 (5.0)	0 (0.0)	N/A	N/A
GG versus GA+AA			0.406 (0.279-0.590)	<0.0001
GG+GA versus AA			N/A	N/A

AOR adjusted odds ratio; CI, confidence interval; N/A, not assessed; VEGF, vascular endothelial growth factor.

The CA and AA genotypes of the −2578C>A polymorphism were significantly associated with a decreased risk of hypertension (AOR 0.455, 95% CI 0.320-0.647, p<0.0001; AOR 0.237, 95% CI 0.095-0.589, p=0.002, respectively). The dominant (CC vs. CA+AA) and recessive (CC+CA vs. AA) models were also protective against hypertension susceptibility (p<0.0001, p=0.015, respectively). There was a significant difference in allele distribution of the VEGF −2578C/A polymorphism between the patient and control groups (A allele, 0.206 and 0.278, respectively, p=0.002). For the −1154G>A polymorphism, the GA genotype and dominant model (GG vs. GA+AA) were significantly lower in patients with hypertension, compared with the control (AOR 0.473, 95% CI 0.323-0.693, p=0.0001; AOR 0.406, 95% CI 0.279-0.590, p<0.0001, respectively). The −1154A allele frequency was 0.223 in controls and 0.213 in patients, which did not show a significant difference between the two groups.

To evaluate for any association between a particular genotype of the VEGF −2578C>A and −1154G>A polymorphisms and gender, we subgrouped the hypertensive and normotensive subjects according to gender (Table 3). The CA and AA genotypes and the dominant model (CC vs. CA+AA) of the −2578C>A polymorphism in the male group (p=0.003, p=0.027, p=0.0001, respectively) and the CA genotype and dominant model (CC vs. CA+AA) of the −1154G>A polymorphism in the female group (p=0.034, p=0.017, respectively) were less frequently found in patients with hypertension, compared with the controls. On the other hand, for the −1154G>A polymorphism, the GA genotype and dominant model (GG vs. GA+AA) were significantly lower in patients with hypertension, compared with the controls in both subgroups.

Table 3.

VEGF Genotype Distributions in the HTN Patient and Control Groups According to Gender

	Male				Female
Genotype	Control (n=152)	HTN (n=240)	OR (95% CI)	p-Value	Control (n=168)	HTN (n=80)	OR (95% CI)	p-Value
VEGF −2578C>A
CC	73 (48.0)	164 (68.3)	1.000 (reference)		91 (54.2)	59 (73.8)	1.000 (reference)
CA	70 (46.1)	70 (29.2)	0.447 (0.290-0.688)	0.0003	64 (38.1)	20 (25.0)	0.509 (0.272-0.952)	0.034
AA	9 (5.9)	6 (2.5)	0.297 (0.101-0.873)	0.027	13 (7.7)	1 (1.3)	0.200 (0.025-1.624)	0.132
CC versus CA+AA			0.430 (0.283-0.653)	0.0001			0.477 (0.260-0.876)	0.017
CC+CA versus AA			0.408 (0.142-1.173)	0.096			0.286 (0.036-2.287)	0.238
VEGF −1154G>A
GG	99 (65.1)	192 (80.0)	1.000 (reference)		101 (60.1)	66 (82.5)	1.000 (reference)
GA	44 (28.9)	48 (20.0)	0.563 (0.350-0.905)	0.018	60 (35.7)	14 (17.5)	0.311 (0.155-0.627)	0.001
AA	9 (5.9)	0 (0.0)	N/A	N/A	7 (4.2)	0 (0.0)	N/A	N/A
GG versus GA+AA			0.468 (0.295-0.741)	0.001			0.290 (0.145-0.580)	0.001
GG+GA versus AA			N/A	N/A			N/A	N/A

The C-G haplotype of the VEGF −2578C>A and −1154G>A polymorphisms was most commonly found in both groups (Table 4). There were significantly low frequencies of C-A, A-G, and A-A haplotypes in the cases, compared with the controls (p=0.002, p=0.027, and p<0.0001, respectively). Also, the frequency of C-G haplotype was significantly higher in the controls, compared with the cases (p=0.006). When the haplotype distributions were stratified by the gender, the haplotypes of the female group showed the same pattern with overall subjects, except for the A-G haplotype. In the male group, however, only the A-A haplotype was associated with the decreased risk of hypertension susceptibility (p<0.0001).

Table 4.

VEGF Haplotype Distributions in the HTN Patient and Control Groups

Haplotype	Control (2n=640)	HTN (2n=640)	OR (95% CI)	p-Value
−2578C>A/−1154G>A
Overall
C-G	388 (60.7)	496 (77.5)	1.278 (1.076-1.519)	0.006
C-A	74 (11.5)	40 (6.3)	0.541 (0.362-0.806)	0.002
A-G	116 (18.1)	82 (12.8)	0.707 (0.522-0.957)	0.027
A-A	62 (9.7)	22 (3.4)	0.355 (0.216-0.584)	<0.0001
Male	(2n=304)	(2n=480)
C-G	192 (63.2)	368 (76.8)	1.214 (0.968-1.522)	0.097
C-A	24 (7.9)	30 (6.2)	0.792 (0.454-1.380)	0.471
A-G	50 (16.4)	64 (13.2)	0.811 (0.545-1.206)	0.307
A-A	38 (12.5)	18 (3.9)	0.300 (0.168-0.535)	<0.0001
Female	(2n=336)	(2n=160)
C-G	197 (58.5)	127 (79.7)	1.354 (1.011-1.813)	0.044
C-A	49 (14.7)	11 (6.6)	0.471 (0.239-0.931)	0.027
A-G	65 (19.5)	19 (11.6)	0.614 (0.356-1.058)	0.096
A-A	25 (7.3)	3 (2.2)	0.252 (0.075-0.847)	0.019

OR, odds ratio.

Discussion

As a potent angiogenic factor, VEGF could be a candidate contributing to the development of diseases such as cancers and metabolic or vascular disorders. Hypertensive patients are associated with increased plasma VEGF levels (Stumpf et al., 2009). However, little is known, to date, about the associations between the VEGF polymorphisms and hypertension. This study aimed to elucidate the associations between the VEGF −2578C>A and −1154G>A polymorphisms and patients with hypertension in the Korean population.

In the present study, the A allele and the CA and AA genotypes of the VEGF −2578C>A polymorphism were significantly associated with a decreased risk for hypertension susceptibility. The nucleotide change of the −2578C>A polymorphism may reduce the risk of hypertension through the regulation of VEGF expression. In fact, the AA genotype of the VEGF −2578C>A polymorphism was associated with a reduced VEGF expression in mononuclear cells from peripheral blood, compared with other allele carriers (Shahbazi et al., 2002). However, the −2578C>A polymorphism was not associated with systemic hypertension or gestational hypertension in the patients of Brazilian population (Sandrim et al., 2009; Lacchini et al., 2014). Yang et al. (2011) have also reported that there was no association between the VEGF −2578C>A polymorphism and hypertensive nephropathy in a Hispanic population, suggesting that there are interethnic differences in the susceptibility of hypertension. On the other hand, Howell et al. (2005) have reported that the AA genotype of the VEGF −2578C>A polymorphism is a risk factor, and the CC genotype is protective against atherosclerosis development in an English population.

So far, the association studies of the −1154G>A polymorphism regarding hypertension have been conducted more than those for the −2578C>A polymorphism. In the present study, the GA genotype and dominant model (GG vs. GA+AA) of the −1154G>A polymorphism were protective against hypertension susceptibility. In the previous study, the VEGF −1154A allele was particularly known to be associated with a decreased VEGF expression, compared with the −1154G allele (Shahbazi et al., 2002). This suggests that the mutant of the −1154G>A polymorphism could influence VEGF levels. With regard to the associations between the −1154G>A polymorphism and hypertension in the populations studied, inconsistent results have existed. The GA+AA genotype for the −1154G>A polymorphism was associated with a reduced ejection fraction in patients with systemic hypertension, in the Brazilian population (Shahbazi et al., 2002). On the other hand, Palmirotta et al. (2010) have reported that the −1154G>A polymorphism was correlated to hypertension; and especially, the −1154A allele was an independent predictor for microvascular complications in patients with essential hypertension, in the Italian population. The AA genotype of the −1154G>A polymorphism had a higher frequency in the hypertensive nephropathy patients than in controls, in a Hispanic population (Yang et al., 2011). However, Sandrim et al. (2009) have reported that the −1154G>A polymorphism was not associated with gestational hypertension in the Brazilian population.

We constructed the haplotypes by combining the −2578C>A and −1154G>A polymorphisms. The C-A, A-G, and A-A haplotypes were significantly lower in the cases than in controls, which suggests the existence of a linkage disequilibrium (LD) between the two polymorphisms. In the previous studies, the A-A-G and A-G-G haplotypes of the VEGF −2578C>A, −1154G>A, and −634G>C polymorphisms were associated with a reduced and increased ejection fraction, respectively, in the patients with systemic hypertension in the Brazilian population (Lacchini et al., 2014). On the other hand, the C-A-G haplotype showed a protective effect against hypertension in the Brazilian population (Sandrim et al., 2013). Yang et al. (2011) have reported that the C-T-G-C and C-A-T-C haplotypes of the VEGF −2578C>A, −1154G>A, −460C>T, and 936C>T polymorphisms were associated with hypertensive nephropathy in a Hispanic population. They found a high degree of LDs among the VEGF −2578C>A, −1154G>A, and −460C>T polymorphisms. However, Sandrim et al. (2009) have reported that the haplotypes of the VEGF −2578C>A and −1154G>A polymorphisms were not associated with gestational hypertension in the Brazilian population. On the other hand, Mohammadi et al. (2009) have reported that the combinations of −2578CC/ −1154GG and −2578CA/ −1154GG genotypes were significantly associated with higher VEGF production, in an Iranian population. Therefore, in the association studies, haplotype analysis can provide much more useful information than the information derived from a single polymorphism analysis (Crawford and Nickerson, 2005; Sandrim et al., 2009).

This inconsistency between the studied populations, regarding the VEGF polymorphisms and risk of hypertension susceptibility, may be due to the differences in genetic background, sample numbers, and diagnostic criteria of hypertension. This demonstrates the importance of using a homogeneous population in the selection of the study samples. Therefore, an exact evaluation on the associations between the VEGF polymorphisms and hypertension is not yet conclusive. Thus, further studies are required to be conducted on the efficiently large and homogeneous ethnic populations, to be able to identify more exactly the distributions of the VEGF genotypes among the populations.

In conclusion, the A allele and haplotypes of the −2578C>A and −1154G>A polymorphisms of the Korean population were associated with a decreased risk in hypertensive patients, compared with the controls. However, the association patterns were different among other ethnic populations. Therefore, to confirm the associations of the VEGF polymorphisms with hypertensive patients, further studies are required in various racial or ethnic groups.

Footnotes

Acknowledgment

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (NRF-2012R1A1A4A01012216).

Author Disclosure Statement

No competing financial interests exist.

References

Crawford

, Nickerson

(2005) Definition and clinical importance of haplotypes. Annu Rev Med, 56:303-320.

Gargett

, Rogers

(2001) Human endometrial angiogenesis. Reproduction, 121:181-186.

Howell

, Ali

, Rose-Zerilli

, Ye

(2005) VEGF polymorphisms and severity of atherosclerosis. J Med Genet, 42:485-490.

Kato

(2002) Genetic analysis in human hypertension. Hypertens Res, 25:319-327.

Lacchini

, Luizon

, Gasparini

, et al. (2014) Effect of genetic polymorphisms of vascular endothelial growth factor on left ventricular hypertrophy in patients with systemic hypertension. Am J Cardiol, 113:491-496.

Mohammadi

, Bazratshani

, Day

, Ollier

(2009) Vascular endothelial growth factor production is regulated by gene polymorphisms. Iran J Immunol, 6:119-129.

Palmirotta

, Ferroni

, Ludovici

, et al. (2010) VEGF-A gene promoter polymorphisms and microvascular complications in patients with essential hypertension. Clin Biochem, 43:1090-1095.

Park

, Jeon

, Kim

, et al. (2012) The Role of VEGF and KDR polymorphisms in moyamoya disease and collateral revascularization. PLoS One, 7:e47158

Renner

, Pilger

(1999) Simultaneous in vitro quantitation factors of vascular endothelial growth factor mRNA splice variants. J Vasc Res, 36:133-138.

10.

Sandrim

, Luizon

, Izidoro-Toledo

, et al. (2013) Functional VEGF haplotypes affect the susceptibility to hypertension. J Hum Hypertens, 27:31-37.

11.

Sandrim

, Palei

, Cavalli

, et al. (2009) Vascular endothelial growth factor genotypes and haplotypes are associated with pre-eclampsia but not with gestational hypertension. Mol Hum Reprod, 15:1151-1120.

12.

Shahbazi

, Fryer

, Pravica

, et al. (2002) Vascular endothelial growth factor gene polymorphisms are associated with acute renal allograft rejection. J Am Soc Nephrol, 13:260-264.

13.

Siervo

, Ruggiero

, Sorice

, et al. (2010) Angiogenesis and biomarkers of cardiovascular risk in adults with metabolic syndrome. J Intern Med, 268:338-347.

14.

Siervo

, Ruggiero

, Sorice

, et al. (2012) Body mass index is directly associated with biomarkers of angiogenesis and inflammation in children and adolescents. Nutrition, 28:262-266.

15.

Steffensen

, Waldstrøm

, Brandslund

, Jakobsen

(2010) The relationship of VEGF polymorphisms with serum VEGF levels and progression-free survival in patients with epithelial ovarian cancer. Gynecol Oncol, 117:109-116.

16.

Stumpf

, Jukic

, Yilmaz

, et al. (2009) Elevated VEGF-plasma levels in young patients with mild essential hypertension. Eur J Clin Invest, 39:31-36.

17.

Tischer

, Mitchell

, Harrman

, et al. (1991) The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem, 266:11947-11954.

18.

Yang

, Hutchinson

, Shah

, et al. (2011) Gene polymorphism of vascular endothelial growth factor −1154G>A is associated with hypertensive nephropathy in a Hispanic population. Mol Biol Rep, 38:2417-2425.