The C Allele of −634G/C Polymorphism in the VEGFA Gene Is Associated with Increased VEGFA Gene Expression in Human Retinal Tissue

Abstract

Purpose.: The purpose of this study was to evaluate the effect of the −634G/C polymorphism on VEGFA gene expression in the human retina.

Methods.: A cross-sectional study was performed to analyze the frequency of the −634G/C polymorphism (rs2010963) in 190 cadaveric cornea donors. Individuals with diabetes mellitus, eye/retinal disease, or both were not included in this study.

Results.: A total of 53 retinal samples were analyzed (18 GG, 17 GC, and 18 CC). VEGFA gene expression was measured by reverse transcription–quantitative polymerase chain reaction. Donor age ranged from 13 to 79 years (mean, 55.8 ± 15.8 years), and 49.1% (n = 26) were male. Subjects carrying the C allele (CC or GC genotypes, 5.15 ± 4.47 arbitrary units [AU] or 3.72 ± 3.25 AU, respectively) presented higher VEGFA gene expression than subjects with the GG genotype (2.62 ± 2.56 AU; P = 0.045).

Conclusions.: This study indicates that the C allele of the −634G/C polymorphism is associated with higher VEGFA gene expression in the human retina.

Introduction

Vasculogenesis, or the spontaneous formation of blood vessels during embryogenesis, as well as angiogenesis, a complex sequence of events leading to the formation of new blood vessels from preexisting vessels, ^1–6 plays a fundamental role in specific physiological processes. However, vascular turnover is extremely low, and angiogenesis rarely occurs in healthy adults. In fact, the etiology and pathogenesis of some diseases are determined by a persistent angiogenic response, as in diabetic retinopathy (DR). ^7,8  

VEGF belongs to a group of dimeric glycoproteins, which includes the placental growth factor (PlGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and VEGF-F. ^6,9 VEGF-A is a potent, multifunctional cytokine that acts on the endothelium. ^5,10  

The gene that encodes VEGFA in humans is organized into eight exons and seven introns located in chromosome 6p21.3. ¹¹ The alternative splicing process results in four main isoforms containing 121, 165, 189, and 206 amino acids, respectively: VEGFA 121, VEGFA 165, VEGFA 189, and VEGFA 206. ^5,12–14 VEGF165, the main VEGFA isoform, ^5,6 is a 45-kDa homodimeric glycoprotein, which acts directly and selectively through receptors VEGFR-1 and VEGFR-2 expressed in the vascular endothelium. ⁵ It causes increased vascular permeability, promotes angiogenesis, and stimulates endothelial cell proliferation and migration in a variety of physiological and pathologic processes. ^5,13  

VEGFA promotes the growth of vascular endothelial cells from arteries, veins, and lymphatic vessels, prevents endothelial apoptosis induced by nutrient deprivation, and is considered an endothelial survival factor. ^10,15 Although it is a major mediator of ischemia-mediated ocular neovascularization, VEGFA is expressed in normal retinas and has an endogenous role in retinal preservation in adults. ^16,17  

In a previous study from our group, the CC genotype of the −634G/C polymorphism (rs2010963) in the VEGFA gene was associated with proliferative DR (PDR) in type-2 diabetic patients. ¹⁸ The C allele was observed more often in patients with DR ^19–22 or with diabetic macular edema (DME) ²⁰ in some studies, but not in others. ^23,24 Furthermore, serum and vitreous levels of VEGFA were elevated in CC genotype carriers, independent of the presence of DR. ²⁵  

We therefore hypothesized that the −634C allele is associated with risk of PDR owing to an increased VEGFA gene expression in C allele carriers. Thus, the purpose of this study was to evaluate the effect of −634G/C polymorphism on VEGFA gene expression in retinas from cadaveric cornea donors. 

Methods

Samples

One hundred and ninety eyes were obtained from cornea donors identified through the Central de Transplantes do Rio Grande do Sul (a Brazilian organization that regulates organ donations in Rio Grande do Sul [RS], Brazil). The collection took place at two hospitals in Porto Alegre (RS), Hospital de Clínicas de Porto Alegre (HCPA), and Hospital Santa Casa de Misericórdia (HSCM), from May 2009 to May 2010. A standard questionnaire was used to collect information from medical records about age, sex, presence of arterial hypertension, and diabetes mellitus (DM), smoking status, occurrence of other diseases, and cause of death. Individuals with DM, ocular/retinal disease, or both, and those without sufficient information were not included in the study. The CC genotype (minor genotype) for the −634G/C polymorphism was present in 18 subjects. Also, the eye tissue of 18 subjects with GG and 17 with GC was included in the VEGFA gene expression study. 

After enucleation and separation of the corneas for donation, retinas were visually separated from the remaining intraocular structures, immediately snap-frozen in liquid nitrogen, and then conserved at −80°C until analyses. The mean duration (±SD) from death of the donor to dissection and conservation of the retinal tissue was 5.54 ± 2.2 hours. A 10-mL sample of peripheral blood was also collected from each donor for DNA extraction and genotyping of the VEGFA −634G/C polymorphism. Following genotyping, a subset of subjects were divided into groups according to the presence of the different genotypes of the analyzed polymorphism (18 CC homozygous, 17 heterozygous, and 18 GG homozygous), and the VEGFA gene expression in retinal tissue from this group was measured as described below. 

The relatives of the donors signed a Letter of Informed Consent authorizing the use of the material that would otherwise have been discarded. The project was approved by the Committee of Ethics in Research at HCPA and HSCM. All subjects were treated in accordance with the Declaration of Helsinki. 

Genotyping of VEGFA −634G/C Polymorphism (rs2010963)

DNA was extracted from peripheral blood leukocytes using a standardized salting-out procedure. Genotyping of the −634G/C polymorphism located in the promoter region of the VEGFA gene was performed using primers and probes contained in the Human Custom TaqMan Genotyping Assay 40× (Life Technologies – Applied Biosystems – ABI, Foster City, CA). The following sequences of primers and probes were used: VEGFA-5′- GAGAGAAGT CGAGGAAGAGAGAGA-3′ (forward primer), VEGFA-5′- CCCAAAAGCAGGTCA CTCACTT-3′ (reverse primer), VEGFA-FAM-5′- CCTGTCCCTTTCGC-3′, and VEGFA-VIC-5′- CCTGTCGCTTTCGC-3′. Real-time PCR (RT-PCR) reactions were performed on 96-well plates, with a total volume of 5 μL containing 2 ng genomic DNA, TaqMan Genotyping Master Mix 1× (ABI), and Custom TaqMan Genotyping Assay 1× (ABI). RT-PCR experiments were performed in a 7500 Fast Real Time PCR System (ABI). The thermocycling condition was as follows: an initial cycle of 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Fluorescence data of each plate were analyzed by the System Sequence Detection Version 2.1 program (ABI). All amplification reactions were performed in duplicate, with an error rate of 0.01% based on the results of the duplicates. 

RNA Isolation

Retinal tissues (250 mg) were homogenized in phenol-guanidine isothiocyanate (Invitrogen Life Technologies, Foster City, CA). RNA was extracted with chloroform and precipitated with isopropanol by centrifugation (12,000g) at 4°C. RNA pellet was washed twice with 75% ethanol and resuspended in 10 to 50 μL diethylpyrocarbonate-treated water. The concentration and quality of total RNA samples were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Scientific Inc., Waltham, MA). Only total RNA samples that achieved adequate purity ratios (A260/A280 = 1.9–2.1) were used for subsequent analyses. ²⁶ In addition, RNA integrity and purity were also checked using agarose gel containing GelRed Nucleic Acid Gel Stain (Biotium, Inc., Hayward, CA). 

Quantification of VEGFA Gene Expression by Quantitative RT-PCR

Real-time reverse transcription-PCR was performed in two separate reactions: first, RNA was reverse transcribed into cDNA, then cDNA was amplified by quantitative real-time PCR (RT-qPCR). Reverse transcription of 1 μg RNA into cDNA was carried out using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies), following the manufacturer's protocol for oligo(dT) method. 

RT-qPCR experiments were performed in a 7500 Fast Real-Time PCR System Thermal Cycler with 7500 Fast System Sequence Detection 1.4 Software (ABI). Experiments were performed by monitoring, in real time, the increase in fluorescence of the SYBR Green dye. ²⁷ PCR reactions were performed using 10 μL of 2× Fast SYBR Green Master Mix (ABI), 1 μL (1 ng/μL) forward and reverse primers for VEGFA or β-actin, and 1 μL cDNA template (0.25 μg/μL), in a total volume of 20 μL. Each sample was assayed in triplicate, and a negative control was included in each experiment. The thermocycling conditions for these genes were as follows: an initial cycle of 95°C for 20 seconds, followed by 45 cycles of 95°C for 3 seconds and 60°C for 30 seconds, each cycle. The specificity of the RT-qPCR was determined using melting-curve analyses, and all primers generated amplicons that produced a single sharp peak during the analyses. 

Quantification of the VEGFA mRNA was performed using the relative standard curve method, ^26,28 and β-actin as the reference gene. Relative standard curves were generated for both target and reference genes by preparing serial dilutions of the same cDNA sample with a known relative quantity. Then, relative amounts of each VEGFA mRNA sample were obtained by normalizing their results using β-actin and were presented as arbitrary units (AU). 

Primers for VEGFA and β-actin genes were designed using published human sequences and the Primer Express 3.0 Software (ABI), and they were projected to target two consecutive exons of a gene in order to prevent the amplification of any contaminated genomic DNA. The following primer sequences were used: VEGFA-5′- GGCGAGGCAGCTTGAGTTAA-3′ (forward primer), VEGFA-5′-CACCGCCTCGGC TTGTC-3′ (reverse primer), β-actin-5′-GCGCGGCTACAGCTTCA-3′ (forward primer), and β-actin-5′-CTTAATGTCACGCACGATTTCC-3′ (reverse primer). 

Statistical Analyses

Data are described as mean ±SD or as percentages. Clinical characteristics and VEGFA mRNA abundance were compared between genotypes and alleles using Student's t-test, one-way ANOVA, or χ² test, as appropriate. Pearson's correlation test was used to assess the correlation between VEGFA gene expression and age. Variables with skewed distribution were logarithmically transformed before analyses. A linear regression was performed to evaluate the independence of the association between −634G/C polymorphism and VEGF expression. The mRNA levels (log transformed) were entered as the dependent variable. Age, smoking status, sex, and hypertension were the independent variables. Statistical analysis was performed using statistical software, SPSS 16.0 (Statistical Package for Social Sciences, Armonk, NY). 

Results

Fifty-three retina tissue samples from cornea donors were included in the gene expression analyses. Donor age ranged from 13 to 79 years (mean, 55.8 ± 15.8 years), and 49.1% (n = 26) were male. Among these subjects, 18 (34%) were CC homozygous, 18 (34%) were GG homozygous, and 17 (32%) were heterozygous. The clinical characteristics of the three genotype groups were similar regarding age, sex, proportion of hypertensive individuals, and smokers (Table 1). 

Table 1. 

 

Clinical Characteristics and VEGFA Gene Quantification in Retina from Cadaveric Cornea Donors According to the VEGFA –634G/C Polymorphism

	–634G/C Genotype			P
	CC (n = 18)	GC (n = 17)	GG (n = 18)
Age, y	60.6 ± 10.42	53.71 ± 16.53	53.44 ± 19.24	0.375
Male sex, n (%)	12 (66.67)	5 (29.41)	9 (50)	0.088
Arterial hypertension, n (%)	2 (11.11)	3 (17.65)	6 (33.33)	0.251
Smoker status, n (%)	7 (38.89)	4 (23.53)	4 (22.22)	0.354
VEGFA (AU)	5.15 ± 4.47*	3.72 ± 3.25*	2.62 ± 2.56	0.045

 Data are expressed in mean ± SD or number of subjects (percentage). P values were computed by ANOVA one-way test or by χ² test.

*   P ≤ 0.05 in relation to the GG genotype group (LSD post hoc test).

When comparing the CC (5.15 ± 4.47 AU) and GC genotypes (3.72 ± 3.25 AU), both had similar gene expression of VEGFA (P = 0.87), while GG (2.62 ± 2.56 AU) had lower expression when compared with CC (P = 0.025) and GC (P = 0.04). 

No significant differences were observed when gene expression was analyzed by sex (men, 3.97 ± 3.69 AU, versus women, 3.7 ± 3.6 AU; P = 0.87), hypertensive status (hypertensives, 3.09 ± 3.28 AU, versus normotensives, 4.9 ± 4.2 AU; P = 0.237), or smoking status (smokers, 3.9 ± 4.1 AU, versus nonsmokers, 5.71 ± 4.08 AU; P = 0.259). VEGFA gene expression did not correlate with age (r = 0.293; P = 0.911). 

VEGFA gene expression in retinal samples analyzed according to the presence of different genotypes of the −634G/C polymorphism is depicted in Table 1 and the Figure. VEGFA gene expression was higher in both CC and GC genotype groups when compared with the GG genotype group (P = 0.045). Assuming a dominant model, the VEGFA mRNA levels were higher in −634C allele carriers (CC and GC) when compared with GG carriers (4.46 ± 3.94 AU versus 2.62 ± 2.56 AU; P = 0.031). 

Figure. 

 

VEGFA gene expression in human retinal samples stratified according to different genotypes of the VEGFA −634G/C polymorphism. P = 0.045 (1-way ANOVA). Data are presented as mean ± 2 SD.

In a multilinear regression model, the presence of the C allele remained associated with higher VEGF gene expression (P = 0.045). The only other factor statistically associated with VEGF mRNA levels was smoker status (P = 0.02) (Table 2). 

Table 2. 

 

Multiple Linear Regression and C Allele −634 VEGF Gene Expression

	Unstandardized Coefficients		P
	BETA	SE
C allele	−0.317	0.155	0.045
Age	0.003	0.004	0.530
Male sex	−0.081	0.151	0.596
Arterial hypertension	0.134	0.128	0.302
Smoker status	−0.267	0.133	0.02
Constant	0.555	0.272	0.049

 VEGF expression (AU) as the dependent variable.

Discussion

In this study, we observed that in nondiabetic individuals without retinal disease, the VEGFA −634C allele is associated with increased retinal expression of the VEGFA gene. This finding was independent of age, sex, smoking status, or hypertension status. 

It is well known that functional polymorphisms can influence gene expression and regulate the final amount of protein in a given disease. The growing interest in researching the VEGFA −634G/C polymorphism is justified by its association with DR. ^18,29 Previous studies suggested that the C allele of the VEGFA −634G/C polymorphism is a risk factor for DR ^18,19,21,22 or DME, independent of the presence of DR, ²⁰ and the presence of the C allele in homozygosity is an independent risk factor for the development of PDR in type 2 DM patients. ¹⁸ In addition, the C allele was also associated with elevated serum and vitreous levels of VEGFA in patients with type-2 DM. ²⁵ These reports are in agreement with the results presented here. 

Although much is known about changes involving VEGFA in eye diseases, and the inhibition of this protein through the therapeutic use of intravitreous anti-VEGFA drugs, little is known about its role in the normal human retina. In experimental studies of rats and monkeys, VEGFA was shown to be constitutively expressed in vascularized tissues of normal eyes. ^16,30 In rats, retinal VEGFA expression is associated with age and is found to be higher in older animals. ³⁰ This suggests greater susceptibility to neovascularization and may be related to the etiology, for instance, of the choroidal neovascular membrane and more severe DR in older patients. However, in the present study, no association was seen between VEGFA gene expression and donor age. 

The increased VEGFA gene expression in the retina of donors carrying CC or GC genotypes is compatible with the role of this protein in DME and DR, because it is an important factor in increased vascular permeability, stimulus of mitosis of endothelial cells and in angiogenesis. ^5,31 Our findings suggest that this polymorphism should be investigated as a risk factor for greater VEGFA production in the retina of diabetic patients and consequent greater predisposition to revascularization and increased vascular permeability from exposure to low oxygen tension and ischemia. ^5,6  

A functional effect of the −634 G/C polymorphism cannot be inferred from this study. It should be noted that we genotyped and analyzed the retina of individuals without DM and/or eye/retinal disease—there was no hypoxia inducing VEGF production. Therefore, a greater effect on VEGF expression might be supposed in subjects exposed to DM or eye/retinal disease. Previous functional data regarding the −634G/C polymorphism indicate that this variant could be directly associated with increased VEGFA gene expression. ^18,19,32 We also cannot rule out the possibility that this polymorphism is not itself responsible for the observed changes in VEGFA gene expression, but rather a surrogate that is in linkage disequilibrium with an unknown causative polymorphism in a distal regulatory site. 

Although our sample may seem small, it is adequate for the purposes of this study. Human retinal tissue is hard to obtain, which complicates gene expression research. We believe that our results provide a new and original insight for the investigation of retinal neovascular diseases, especially DR. The identification of putative functional DNA variants will support the development of new treatment options in the future. 

We must be careful and critical in the interpretation of genetic association data. Therefore, additional genotype studies measuring VEGF levels in retinal disease are required to confirm the present findings. Even though this is a difficult task, owing to the variety of clinical and genetic factors involved in DM complications such as DR, the recognition of genetic predisposing factors to increased VEGF production could help in the identification of susceptibility to ischemic damage. The VEGFA −634 G/C polymorphism might be one such factor. 

In conclusion, the C allele of the VEGFA −634G/C polymorphism is associated with increased VEGFA gene expression in retinas of nondiabetic cornea donors. This provides evidence of increased risk of DR in DM patients carrying this allele. Further studies will be necessary in diabetic patients to elucidate our findings. 

Acknowledgments

The authors thank Rosana R. Nothen and Fernando Pagnussato from Hospital de Clínicas de Porto Alegre and Alexandre S. Marcon from Hospital Santa Casa de Misericórdia de Porto Alegre for their support with sample collection and preparation. Luis Henrique Canani is the guarantor of this work, had full access to all the data, and takes full responsibility for the integrity of data and the accuracy of data analysis. 

Footnotes

 Supported in part by grants from Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundo de Incentivo à Pesquisa e Eventos (FIPE) at Hospital de Clínicas de Porto Alegre. The authors alone are responsible for the content and writing of the article.

Footnotes

 Disclosure: F.B. Vailati, None; D. Crispim, None; D.A. Sortica, None; B.M. Souza, None; L.A. Brondani, None; L.H. Canani, None