Eur J Clin Pharmacol DOI 10.1007/s00228-015-1872-5

PHARMACOGENETICS

Polymorphisms in VEGFA gene affect the antihypertensive responses to enalapril G. H. Oliveira-Paula 1 & R. Lacchini 2 & V. Fontana 3 & P. S. Silva 4 & C. Biagi 5 & Jose E. Tanus-Santos 1

Received: 2 March 2015 / Accepted: 17 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Purpose Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that affects blood pressure by promoting vasodilation mediated by nitric oxide. Angiotensinconverting enzyme inhibitors (ACEi) up-regulate the VEGF expression; thus, genetic polymorphisms in the VEGFA gene could affect the antihypertensive responses to these drugs. Methods Hypertensive patients (n=102) were prospectively treated only with the ACEi enalapril for 60 days. We compared the effect of VEGFA polymorphisms on changes in blood pressure after enalapril treatment. In addition, multiple linear regression analysis was carried out to assess the effect of covariates on blood pressure. Genotypes for g.-2578C>A (rs699947), g.-1154G>A (rs1570360), and g.-634G>C (rs2010963) VEGFA polymorphisms were determined, and haplotype frequencies were estimated.

Results Individuals carrying the CA and AA genotypes for the g.-2578C>A polymorphism and the AGG haplotype showed more intense decrease in blood pressure in response to enalapril 20 mg/day. A multiple linear regression analysis showed that the AA genotype for the g.-2578C>A polymorphism and the AGG haplotype are associated with more intense decrease in blood pressure in response to enalapril 20 mg/day, while the CC genotype for the g.-2578C>A polymorphism and the CGG haplotype are associated with the opposite effect. Conclusions These findings suggest that polymorphisms in VEGFA gene may affect the antihypertensive responses to enalapril.

Electronic supplementary material The online version of this article (doi:10.1007/s00228-015-1872-5) contains supplementary material, which is available to authorized users.

Introduction

* Jose E. Tanus-Santos [email protected]; [email protected] 1

Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirao Preto, SP, Brazil

2

Department of Psychiatric Nursing and Human Sciences, Ribeirao Preto College of Nursing, University of Sao Paulo, Ribeirao Preto, SP, Brazil

3

Brazilian Biosciences National Laboratory, CNPEM, Campinas, SP, Brazil

4

Department of Pharmacy, Faculty of Pharmacy, Federal University of Juiz de Fora, Governador Valadares, MG, Brazil

5

Santa Casa de Araçatuba, Araçatuba, SP, Brazil

Keywords Enalapril . Haplotypes . Hypertension . Polymorphisms . VEGFA

Hypertension is a multifactorial disease that affects approximately one billion subjects and is a major risk factor associated with cardiovascular events [1]. Angiotensin-converting enzyme inhibitors (ACEi), known to exert many beneficial effects that reduce mortality and morbidity in patients with cardiovascular disorders, are widely used in the therapy of hypertension [2]. The main mechanisms of action for these drugs include reduction of angiotensin II formation and prevention of bradykinin degradation [3]. Moreover, it was shown that ACEi affect other relevant mediators, such as vascular endothelial growth factor (VEGF) [4–9]. VEGF is a potent angiogenic factor expressed mostly by endothelial cells and has many biological roles, including proliferation, migration, and survival of endothelial cells [10]. Importantly, VEGF stimulates endogenous nitric oxide (NO)

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production, resulting in vasodilation and thereby contributing to blood pressure regulation [11–18]. Interestingly, the administration of recombinant VEGF reduced blood pressure in humans and animal models [11, 13, 14, 17] and this effect was blunted by NO synthase inhibitors [12, 14]. In addition, hypertensive patients showed lower VEGF and NO-related marker levels compared with healthy subjects [19]. Therefore, considering the effect of VEGF on blood pressure, it is expected that it may be affected by antihypertensive drugs, including ACEi [5]. Indeed, ACEi treatment increased VEGF expression in several experimental studies [4, 6–9]. ACEi treatment also increases tissue bradykinin levels, stimulating both bradykinin B1 receptor (BR1) and bradykinin B2 receptor (BR2) on endothelial cells [20]. BR1 signaling, in turn, stimulates tissue VEGF expression [6], while BR2 stimulates endothelial NO production [21]. Additionally, Li et al. have suggested that ACEi may increase tissue VEGF levels through a possible interaction of BR2 with the angiotensin II type 2 receptor [6]. Since ACEi up-regulate VEGF expression, which promotes NO formation and vasodilation, it is possible that variations in the VEGFA gene could affect the antihypertensive response to ACEi. Among several polymorphisms in VEGFA gene, the singlenucleotide polymorphisms (SNPs) g.-2578C>A (rs699947), g.-1154G>A (rs1570360), and g.-634G>C (rs2010963) have been widely studied due to their functional implications and clinical relevance [22–30]. In fact, studies demonstrated that these SNPs affect VEGF expression [27, 29]. Moreover, these three SNPs were associated with many pathological conditions, such as diabetic retinopathy [22], left ventricular hypertrophy [26], renal allograft rejection [29], and hypertensive disorders [28, 31, 32]. Taking all these findings into account, we hypothesized that genotypes or haplotypes of VEGFA polymorphisms modulate the antihypertensive response to the ACEi enalapril.

Methods Subjects and study design Approval for use of human subjects in this study was obtained from the Institutional Review Board at the Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil, and each subject provided written informed consent. The study participants included a total of 102 hypertensive patients with systolic blood pressure (SBP) ≥140 and ≤179 mmHg and diastolic blood pressure (DBP) ≥90 and ≤109 mmHg, recruited from Cardiology Division of the Araçatuba Health Center (Araçatuba, SP, Brazil). Office blood pressure measurement was obtained from the average of three blood pressure readings on at least two office visits with the individuals in the

seated position, according to the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure [1]. Office SBP and DBP were recorded with a semiautomatic blood pressure monitor (OMRON® HEM-433 INT, Bannockburn, IL, USA) before and after the use of enalapril. Patients were never treated (n=75) or underwent a washout period for at least 2 weeks (n=27). This washout period has been widely used [33–36] and described to be sufficient to eliminate the effects of previous antihypertensive treatments on physiological parameters [37]. Patients underwent a complete medical history, physical examination, and laboratory analysis to exclude subjects with evidence of severe or secondary hypertension; other concomitant cardiovascular diseases; or respiratory, hepatic, renal, or hematological dysfunction. After the initial evaluation, patients were treated for 60 days with enalapril 10 mg/day (n=48) or 20 mg/day (n=54), according to the judgment of the physician (taking into account the number of risk factors and the previous history of treatment of patients that underwent washout). Mean blood pressure (MBP) was calculated using the formula MBP = (SBP + 2 × DBP) / 3. Values of systolic, diastolic, and MBPs were used to calculate changes in blood pressure related to drug response. Changes in blood pressure were calculated as follows: (BP at baseline−BP after enalapril treatment)×(−1). After blood pressure measurements, venous blood samples were collected and genomic DNA was extracted by salting-out method and stored at −20 °C until analysis.

Genotyping Genotypes for the g.-2578C>A (rs699947), g.-1154G>A (rs1570360), and g.-634G>C (rs2010963) polymorphisms were determined by TaqMan Allele Discrimination assays ( a s s a y I D s : C _ 8 3 116 0 2 - 1 0 , C _ 1 6 4 7 3 7 9 - 1 0 , a n d C_8311614-10, respectively; Applied Biosystems, Foster City, CA, USA). TaqMan polymerase chain reaction (PCR) was performed in a total volume of 10 μl (5 ng of template DNA, 1× TaqMan Genotyping Master Mix and 1× TaqMan Allele Discrimination Assay) placed in 96-well PCR plates. Thermal cycling was performed in standard conditions, and fluorescence was recorded by the StepOne Plus Real-Time PCR equipment (Applied Biosystems, Foster City, CA, USA). Genotyping quality was checked using in each experiment both previously determined positive controls (representative of all three genotypes) and negative controls (samples containing no template). Moreover, we have also repeated 10 % of the whole sample randomly and have obtained 100 % of consistency. Results were analyzed with the manufacturer’s software.

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Haplotype inference The Bayesian statistical-based program PHASE (version 2.1, http://www.stat.washington.edu/stephens/software.html) was used to estimate the haplotype frequencies [38]. The possible haplotypes including the three VEGFA genetic polymorphisms (g.-2578C>A, g.-1154G>A, and g.634G>C) were CGG, CGC, CAG, CAC, AGG, AGC, AAG, and AAC. Only the haplotypes with observed frequencies>1 % were included in subsequent analysis. Statistical analysis The clinical and laboratory characteristics of the studied groups were compared by unpaired t test (parametric data), by Mann-Whitney test (non-parametric data), by chi-square test, or Fisher’s test (categorical variables), when appropriate. Differences in genotype/allele distributions and deviation from the Hardy-Weinberg equilibrium were assessed by chisquare test. The effects of genotypes and haplotypes on changes in SBP, MBP, and DBP were tested by ANOVA. In addition, we carried out a multiple linear regression analyses to account for possible confounding factors that could influence changes in blood pressure. Age, gender, ethnicity, smoking status, VEGFA genotypes, and VEGFA haplotypes were included as independent variables in multiple linear regression models to explain changes in SBP, MBP, and DBP after enalapril treatment using JMP® software (SAS Institute, Cary, NC). A probability value 0.05). Baseline SBP, DBP, and MBP did not differ among the genotypes/alleles

(P>0.05; data not shown). Likewise, there were no differences in baseline SBP, DBP, and MBP stratified by genotypes/alleles between the group of patients treated with enalapril 10 mg/day and the group of patients treated with enalapril 20 mg/day (P>0.05; data not shown). Figure 1a–c shows, respectively, the effects of g-2578C>A, g.-1154G>A, and g.-634G>C genotypes on changes in blood pressure of hypertensive patients after treatment with enalapril 10 mg/day, while Fig. 1d–f shows, respectively, the effects of g-2578C>A, g.-1154G>A, and g.-634G>C genotypes on changes in blood pressure of hypertensive patients in response to enalapril 20 mg/day. Although the g.-2578C>A genotypes have not affected blood pressure response to enalapril 10 mg/day (Fig. 1a; P>0.05), individuals carrying the CA and AA genotypes showed more intense decrease in MBP after treatment with enalapril 20 mg/day compared with individuals carrying the homozygous ancestral genotype CC (Fig. 1d; PA (Fig. 1b, e) and g.-634G>C (Fig. 1c, f) polymorphisms did not significantly affect the

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Fig. 1 Effects of VEGFA polymorphisms on changes in blood pressure of hypertensive patients in response to enalapril treatment. a Effects of CC (n=15), CA (n=28), and AA (n=5) genotypes for the g.-2578C>A polymorphism on changes in systolic, mean, and diastolic blood pressure in response to enalapril 10 mg/day. b Effects of GG (n=30), GA (n=16), and AA (n=2) genotypes for the g.-1154G>A polymorphism on changes in systolic, mean, and diastolic blood pressure in response to enalapril 10 mg/day. c Effects of GG (n=17), GC (n=25), and CC (n=6) genotypes for the g.-634G>C polymorphism on changes in systolic, mean, and diastolic blood pressure in response to enalapril 10 mg/day. d Effects of

CC (n=21), CA (n=25), and AA (n=8) genotypes for the g.-2578C>A polymorphism on changes in systolic, mean, and diastolic blood pressure in response to enalapril 20 mg/day. e Effects of GG (n=33), GA (n=19), and AA (n=2) genotypes for the g.-1154G>A polymorphism on changes in systolic, mean, and diastolic blood pressure in response to enalapril 20 mg/day. f Effects of GG (n=25), GC (n=25), and CC (n=4) genotypes for the g.-634G>C polymorphism on changes in systolic, mean, and diastolic blood pressure in response to enalapril 20 mg/day. Data are shown as means ± SEM. *P < 0.05 versus CC genotype for the g.2578C>A polymorphism

changes in blood pressure of hypertensive patients in response to enalapril treatment (P>0.05). To improve genetic information, we studied VEGFA haplotypes. Figure 2 shows the effects of VEGFA haplotypes on changes in blood pressure of hypertensive patients after enalapril treatment. Interestingly, although haplotypes have not affected the changes in blood pressure after treatment with enalapril 10 mg/day (Fig. 2a; P>0.05), subjects carrying the AGG haplotype showed more intense decrease in SBP, MBP, and DBP after treatment with enalapril 20 mg/day compared with carriers of the reference haplotype CGG (Fig. 2b; P0.05; Table S2), while the CC and AA genotypes for the g.-2578C>A polymorphism were associated, respectively, with lower and higher decrease in MBP in response to enalapril 20 mg/day, after adjusting for selected variables (P=0.002 for CC genotype and P=0.024 for AA genotype; Table 2). Moreover, in accordance with the results of Fig. 2, we observed no

associations between VEGFA haplotypes and changes in blood pressure after treatment with enalapril 10 mg (P>0.05; Table S3). However, consistent with the results of Fig. 2, the AGG haplotype was associated with more intense decrease in SBP, MBP, and DBP in response to enalapril 20 mg/day, after adjusting for selected variables (P = 0.005 for SBP, P = 0.002 for MBP, and P = 0.001 for DBP; Table 3). On the other hand, the CGG haplotype was associated with lower decrease in MBP after treatment with enalapril 20 mg/day (P=0.021; Table 3).

Discussion To our knowledge, this is the first study to evaluate whether functional polymorphisms of the VEGFA gene may affect the antihypertensive response to enalapril in hypertensive patients as a single therapy. The main findings of this study were that (i) the variant genotypes for the g.-2578C>A polymorphism and the AGG haplotype in VEGFA gene were associated with better responses to enalapril 20 mg/day and (ii) the CC genotype for the g.-2578C>A polymorphism and the CGG haplotype were associated with worse responses to enalapril 20 mg/day.

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Fig. 2 Effects of VEGFA haplotypes on changes in blood pressure of hypertensive patients in response to enalapril treatment. a Effect of CGG (n = 21), AGG (n = 19), AAG (n = 19), and CGC (n = 37) haplotypes on systolic, mean, and diastolic blood pressure after

treatment with enalapril 10 mg/day. b Effect of CGG (n=34), AGG (n=18), AAG (n=23), and CGC (n=33) haplotypes on systolic, mean, and diastolic blood pressure after treatment with enalapril 20 mg/day. Data are shown as means±SEM. *PA or g.-1154G>A polymorphisms [29]. Consistently, the g.-1154A allele was associated with reduced VEGFA promoter activity in vitro compared with the g.-1154G allele [27]. On the other hand, the C allele for the g.-634G>C polymorphism was associated with increased expression of a VEGF isoform (the L-VEGF) [27]. Additionally,

Eur J Clin Pharmacol Table 2 Effects of VEGFA genotypes on changes in blood pressure of hypertensive patients in response to enalapril 20 mg/day after adjusting for selected variables Change in SBP (mmHg)

Change in MBP (mmHg)

Change in DBP (mmHg)

R2 =0.24

R2 =0.37

R2 =0.32

RMSE=10.16

RMSE=8.00

RMSE=10.13

Source

B

P

B

P

B

P

Age (years) Gender (male) Ethnicity (non-Whites) Smoking status (current smoker) g.-2578C>A

0.499 0.998 0.344 0.230

P 0.934 0.338

+0.25 +2.03 +1.37 −3.83 P=0.054 B +7.42 −1.15 −6.27 P=0.064 B −8.17 −1.01 +9.19 P=0.701 B −2.18 −1.22

0.067 0.199 0.423 0.116

P 0.277 0.357

+0.15 +2.21 +1.28 −2.52 P=0.009a B +7.62 −1.53 −6.09 P=0.172 B −5.24 −1.73 +6.97 P=0.605 B +0.18 −1.95

0.149 0.080 0.345 0.188

GG GC

−0.09 −0.01 +1.63 −2.91 P=0.102 B +6.48 −0.72 −5.76 P=0.145 B −6.97 −2.12 +9.09 P=0.425 B −3.10 −2.38

CC

+5.48

0.194

+1.77

0.592

+3.40

CC CA AA g.-1154G>A GG GA AA g.-634G>C

P 0.036 0.784 0.089 P 0.051 0.548 0.119

P 0.002a 0.457 0.024a P 0.062 0.534 0.129

P 0.017 0.660 0.064 P 0.023 0.773 0.115 P 0.441 0.633 0.416

SBP systolic blood pressure, MBP mean blood pressure, DBP diastolic blood pressure, R2 portion of variability explained by the model, RMSE root mean square error, B parameter estimate a

Statistically significant

Table 3 Effects of VEGFA haplotypes on changes in blood pressure of hypertensive in response to enalapril 20 mg/day after adjusting for selected variables Change in SBP (mmHg)

Change in MBP (mmHg)

Change in DBP (mmHg)

R2 =0.15

RMSE=9.96

R2 =0.27

RMSE=7.98

R2 =0.25

RMSE=9.87

Source Age (years) Gender (male) Ethnicity (non-Whites)

B −0.05 −0.10 +2.26

P 0.600 0.924 0.040a

B +0.21 +2.16 +2.17

P 0.004a 0.013a 0.015a

B +0.28 +1.89 +2.00

P 0.001a 0.077 0.067

Smoking status (current smoker) Haplotype

−0.76 P=0.036a B +2.88 −5.80 +1.32 +1.60

0.572

−0.45 P=0.016a B +1.55 −5.09 +0.49 +3.04

0.681

−2.26 P=0.013a B +2.52 −6.65 +2.15 +1.98

0.096

CGC AGG AAG CGG

P 0.079 0.005a 0.464 0.326

P 0.236 0.002a 0.733 0.021a

P 0.120 0.001a 0.232 0.221

SBP systolic blood pressure, MBP mean blood pressure, DBP diastolic blood pressure, R2 portion of variability explained by the model, RMSE root mean square error, B parameter estimate a

Statistically significant

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individuals carrying the A or G allele for the g-2578C>A and g.-634G>C polymorphisms, respectively, showed lower VEGF serum levels compared with non-carriers, corroborating the functional implications of these polymorphisms [22, 30]. We found that the g-2578C>A polymorphism affects the antihypertensive response to enalapril. Individuals carrying the CA and AA genotypes for the g.-2578C>A polymorphism showed more intense decrease in MBP after treatment with enalapril 20 mg/day. Likewise, a multiple linear regression showed that AA genotype for this polymorphism is associated with more intense decrease in MBP in response to enalapril 20 mg/day. Conversely, the CC genotype was associated with the opposite effect. Interestingly, although only the MBP has been statistically affected for the g-2578C>A polymorphism, the enhanced antihypertensive response to enalapril 20 mg/ day seems to be dependent on the variant allele for this polymorphism. Our findings are in line with a previous study that found that the g.-2578A allele is associated with earlier onset of hypertension in preeclampsia, an important hypertensive disorder of pregnancy [31]. Indeed, lower VEGF levels were found in hypertensive patients compared with normotensive subjects [19], thus contributing to impaired vasodilation observed in hypertension [16]. Moreover, our results are in line with the functional implication of AA genotype for the g2578C>A polymorphism, which was shown to decrease VEGF expression [29]. Therefore, it seems reasonable to suspect that the g.-2578A allele that apparently predisposes to hypertensive disorders and reduces VEGF expression can also enhance the responses to drugs that increase VEGF expression, such as the ACEi enalapril. However, the molecular mechanisms supporting this suggestion remain to be proved. Although we found an effect of g.-2578C>A on blood pressure after enalapril treatment, it is possible that this polymorphism may interact with the other VEGFA polymorphisms. Therefore, we analyzed VEGFA haplotypes, which may offer improved genetic information than the analysis of genetic markers individually [28]. Indeed, we consistently found that the AGG haplotype affects the antihypertensive responses to enalapril, resulting in more intense decrease in SBP, MBP, and DBP after treatment with enalapril 20 mg/day. In an opposite direction, the CGG haplotype was associated with lower decrease in MBP in response to enalapril 20 mg/ day. In fact, the AGG haplotype was showed to express less VEGF than CGG haplotype [41]. Moreover, the AGG haplotype was associated with stroke [42] and affected the ejection fraction in hypertensive patients with left ventricular hypertrophy [25]. Taking these findings into consideration, we could speculate that a reduced VEGF expression could increase the risk for cardiovascular damages in AGG haplotype carriers through mechanisms suggested by experimental studies [16, 43]. On the other hand, the AGG haplotype carriers could show enhanced response to drugs that can reverse

cardiovascular damages by increasing VEGF expression. Despite these suggestions, the molecular basis for our findings remains to be elucidated. Interestingly, although the antihypertensive effect of enalapril did not differ between the doses used, VEGFA polymorphisms affected blood pressure only in patients taking enalapril 20 mg/day. Indeed, some genetic polymorphisms seem to modify the effects of antihypertensive drugs mainly in higher doses [44]. Therefore, although a previous study has showed that the eNOS and BDKRB2 genotypes affected the enalapril response independently of the doses used [45], our findings indicate that dose approach is important to evidence the effect of VEGFA polymorphisms on the antihypertensive response to enalapril, suggesting a gene-dose relationship. Indeed, antihypertensive treatment with lower doses of enalapril could have no effects on VEGF expression, and therefore, the effects of this drug could not be affected by VEGFA polymorphisms. On the other hand, a higher dose of enalapril could effectively increase VEGF expression and this would be especially important in individuals carrying genetic polymorphisms that lead to lower VEGF expression, resulting in more intense decrease in blood pressure. However, these are speculations requiring confirmatory studies. One important limitation of our study is the small number of subjects included. However, it is important to highlight that we include only hypertensive patients under no current antihypertensive treatment. Nevertheless, further studies in external populations are required to confirm our findings. Another issue that has not been addressed in this study includes several other genes related to VEGF that may contain important polymorphisms that were not determined here, such as VEGFR and others. Therefore, further studies should address this possibility. In conclusion, our findings indicate that VEGFA polymorphisms affect the antihypertensive responses to enalapril. The variant genotypes for the g-2578C>A polymorphism and the AGG haplotype predispose to better antihypertensive responses to enalapril, while the CC genotype for the g2578C>A polymorphism and the CGG haplotype are associated with worse responses to this drug. These results may help to drive the antihypertensive therapy. Compliance with ethical standards

Acknowledgments This study was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Fundaçao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP). Conflict of interest The authors declare that they have no conflict of interest. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the

Eur J Clin Pharmacol institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study.

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