|Molecular Vision 2006;
Received 9 May 2005 | Accepted 28 March 2006 | Published 11 April 2006
Association of VEGF and eNOS gene polymorphisms in type 2 diabetic retinopathy
Balasubbu Suganthalakshmi,1 Rajendran Anand,2
Ramasamy Kim,2 Rajendran Mahalakshmi,3 Sundaramoorthi
Karthikprakash,3 Perumalsamy Namperumalsamy,2
1Department of Genetics, Aravind Medical Research foundation, 2Technology Information Forecasting & Assessment Council - Centre of Relevance and Excellence (TIFAC-CORE) in Diabetic Retinopathy, 3Department of Biostatistics, Lions Aravind Institute of Community Ophthalmology (LAICO), Aravind Eye Care System, Madurai, India
Correspondence to: Dr. P. Sundaresan, Department of Genetics, Aravind Medical Research Foundation, Aravind Eye Hospital, #1, Anna Nagar, Madurai-625 020, India; Phone: +91-452-2532653 ext-423; FAX: +91-452-2530984; email: firstname.lastname@example.org
Purpose: Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis, and nitric oxide (NO) is an upstream and downstream regulator of VEGF mediated angiogenesis. VEGF and NO have been suggested to play an important role in the pathogenesis of microvascular complications in diabetic retinopathy (DR). The objective of this study was to examine the genetic variations of the VEGF and eNOS gene and assess their possible relationship to DR in type 2 diabetic patients in the Indian population.
Methods: In this study, 210 unrelated patients were enrolled and categorized into two study groups: a DR group, consisting of patients with proliferative diabetic retinopathy, and a diabetic without retinopathy (DWR) group comprised of patients with type 2 diabetes of more than 15 years duration who showed no signs of DR or had fewer than five dots or blot hemorrhages. Association of the genetic polymorphisms in the promoter and 5' UTR region of VEGF and the intron4 region of eNOS were studied. Total genomic DNA was isolated from peripheral blood leukocytes. PCR-RFLP analysis was performed for all samples to evaluate the genotypes. The distributions of the genotypes were compared using the χ2 test. Haplotype estimation and multiple logistic regression analysis were carried out to analyze the significance of polymorphisms.
Results: We investigated four reported polymorphisms in the VEGF (5' UTR, promoter) and one reported polymorphism (intron 4) in the eNOS gene in Type 2 diabetes patients with (n=120) and without (n=90) retinopathy. The genotype distribution of the C(-7)T, T(-1498)C, and C(-634)G polymorphisms of VEGF differed significantly between patients with DR and DWR (p=0.001, p=0.0001, and p=0.021, respectively). Allele C in the -1498 region (p=0.0001) and T in -7 region (p=0.002) were also found to be significantly increased in patients with retinopathy. Calculated odds ratios (OR) for three heterozygous genotypes of C(-7)T, T(-1498)C, and C(-634)G regions were 4.17 (95% CI: 1.90-9.18, p=0.0001), 4.37 (95% CI: 2.44-7.84, p=0.0001), and 2.33 (95% CI: 1.24-4.36, p=0.008), respectively, and was found to be significantly higher in the DR group when compared with the DWR group. Multiple logistic regression analysis revealed that the nongenetic parameters, age (p=0.024) and duration of diabetes (p=0.009), and the genetic parameters, like VEGF C(-7)T (p=0.002) and T(-1498)C (p=0.001) polymorphisms, were significantly associated with DR. The frequencies of haplotype consisting of the majority of alleles in VEGF were found to be significantly associated with DR. The genotype distribution of eNOS did not differ significantly between the two study groups, and therefore the eNOS intron 4 polymorphism was considered to be less significant.
Conclusions: This is the first study to report VEGF and eNOS gene polymorphisms in patients with DR in the Indian population. The data suggest that the polymorphisms in the 5' UTR and promoter region of VEGF could be regarded as a major genetic risk factor for DR.
Diabetic retinopathy (DR), a devastating microvascular complication in the eye, is one of the leading causes of blindness in the western world but its frequency varies in different ethnic groups. As the prevalence of diabetes is high among asian Indians, studies on diabetic complications are of considerable clinical significance [1,2].
Most diabetic patients, especially those with poor glycemic control, develop DR, which remains the major cause of onset of blindness among diabetic adults . The Wisconsin Epidemiologic Study of Diabetic retinopathy (WESDR) showed that 28.8% of diabetic patients develop retinopathy early, whereas 22.2% with the history of diabetes irrespective of glycemic exposure do not develop retinopathy. This study suggested that genetic factors could promote the onset of retinopathy in diabetic patients . DR is characterized by vascular permeability, increased tissue ischemia, and angiogenesis. VEGF is a 45 KDa homodimeric glycoprotein , which has been shown to be an important mediator of retinal ischemia-associated intraocular neovascularization . Many cell types within the eye produce vascular endothelial growth factor (VEGF). In patients with proliferative diabetic retinopathy (PDR), VEGF levels are markedly elevated in the vitreous and aqueous fluids [7,8]. Retinal VEGF expression is high in DR [9,10] and appears to be an attractive candidate gene for DR. The human VEGF gene is organized into eight exons separated by seven introns and is located on chromosome 6 .
Another possible factor for diabetes is nitric oxide (NO). The gene encoding eNOS (endothelial nitric oxide synthase) is located on chromosome 7q35-36. NO causes increased oxidative stress due to free radicals and could play an important role in the pathogenesis of microvascular complications in humans [12,13]. NO production has been reported to be either increased or decreased in the presence of high glucose concentrations [14,15]. NO is a short-lived, highly reactive intercellular signaling molecule. In addition to its antithrombogenic and antiplatelet regulatory activities, NO plays a major role in the regulation of vascular tone, including retinal circulation and vascular remodeling [16,17]. The angiogenic and inflammatory effects of VEGF can be mediated by NO, which is produced by VEGF-activated eNOS in vascular endothelial cells. This suggests that eNOS is involved in inflammation, ischemic processes, and the pathogenesis of DR [18-20]. Evidence suggests that VEGF and eNOS are associated with DR in different ethnic groups. To our knowledge, no information about this association is available in the Indian population. Hence we investigated the VEGF and eNOS to assess their possible relationships to DR in type 2 diabetic patients in the Indian population.
This study was conducted over 2003-2004 with the approval of our Institutional Review Board and in accordance with the guidelines of the Declaration of Helsinki. Informed consent was given by all patients. All patients had type 2 diabetes, as defined by age of onset being later than 30 years of age, and who controlled their diabetes exclusively with oral hypoglycemic agents. All were subjected to a clinical evaluation which involved fundus examination by a binocular indirect ophthalmoscope and a slit lamp biomicroscopic examination with a 90 D lens in the retina clinic. Patients with either no signs of DR or with fewer than five dots or blot hemorrhages coupled with type 2 diabetes of >15 years duration were assigned to the diabetic without retinopathy (DWR) study group. Patients with PDR and more severe hemorrhages were allocated to the DR study group. Other patient details such as age, sex, body weight, duration of diabetes, age at onset of diabetes, family history of diabetes, other systemic illness, and treatment details were also documented. All the patients in addition underwent 5-field 50° color fundus photography.
Total genomic DNA was extracted from peripheral blood leucocytes by the salt precipitation method  from all participants. DNA was suspended in Tris-EDTA (TE) buffer, pH 8, and stored at -20 °C until further investigation.
Polymerase chain reaction
The promoter, 5' UTR region of VEGF, and the intron4 region of eNOS were PCR amplified and conditions adopted as per previous protocol [22,23]. The eNOS polymorphic genotypes were assessed directly through the Amplification Refraction Mutation System (ARMS) PCR and the products were electrophoresed on 1.5% agarose gel. Two types of PCR products were obtained, a 420 bp band which indicates five repeats of the 27 bp allele (b allele), and a 393 bp band that indicates four repeats of the 27 bp allele (a allele).
Restriction fragment length polymorphism analysis
The PCR products were restriction digested using 5 units of enzymes Fnu4HI for -T(-1498)C, DdeI for -G(-1190)A and C(-7)T, and BsmFI for -C(-634)G (New England Biolabs, Beverly, MA) in a 10 μl setup and visualized on 3% agarose gel electrophoresis to detect the reported polymorphisms in VEGF.
Allele frequencies were calculated from the observed numbers of genotypes. Differences in the allele frequencies between the groups were tested by Pearson's χ2 test and Fisher's exact test. Monte-Carlo methods were used to confirm the estimations of haplotype differences due to concern over sparse contingency tables. Continuous clinical data were compared by unpaired Student's t-test. Multiple logistic regression analysis was performed to assess the role of the VEGF genotype and other variables, including age, sex, and duration of diabetes. Stata 8.1 (Stata Corporation, College station, TX) was used to do the statistical analyses.
We recruited, 120 DR and 90 DWR patients for genetic analysis. Their clinical characteristics are given in Table 1. Restriction-fragment length polymorphism (RFLP) analysis was performed to evaluate the genotypes of polymorphisms C(-7)T and C(-634)G in the 5' UTR and T(-1498)C and G(-1190)A in the promoter region of VEGF. The alleles -634G (Figure 1), -7T (Figure 2), and -1498C (Figure 3) resulted in gains of Fnu4HI, BsmFI, and DdeI restriction sites, respectively. The PCR products and allele variations of VEGF are presented in Table 2. A comparison of genotype and allele frequencies between both the DWR and DR group revealed that genotype distribution of C(-7)T, T(-1498)C, and C(-634)G polymorphisms were statistically significant in the DR group (Table 3). In addition, the allele distribution also revealed that the C allele of the -1498 region (p=0.0001) and T allele of the -7 (p=0.002) region in VEGF were significantly increased in the DR group (Table 4). However, calculated odds ratios estimating the presence of CT genotype in C(-7)T region (OR=4.17, 95% CI: 1.90-9.18, p=0.0001), the TC genotype in T(-1498)C region (OR=4.37, 95% CI: 2.44-7.84, p=0.0001) and the CG genotype in C(-634)G region (OR=2.3, 95% CI: 1.27-4.45, p=0.008) were significantly higher in the DR group compared with the DWR group. The power of the study calculated to detect the odds ratio of 4.37 for TC genotype in VEGF T(-1498)C between DR and DWR is more than 80%.
Multiple logistic regression analysis was performed to analyze the relevance of selected parameters (age, sex, duration of diabetes, and genotypes of VEGF polymorphisms) as given in Table 5. Age, duration of diabetes, and -7CT, -1498TC genotypes proved to be significant predictors of DR (p=0.024, p=0.009, p=0.002, and p=0.001, respectively). Based on this logistic regression analysis, the duration is one of the significant predictors of diabetes for DR. We have divided the duration of diabetes into two groups: lower (less than or equal to 15 years) and higher (>15 years) duration. If the diabetes duration is less than or equal to 15 years, the risk of developing DR is 1.91 times higher when compared with DWR and it was statistically significant (p=0.02).
As shown in Table 6, six major haplotypes were estimated and each estimate was compared between DR and DWR using the Monte Carlo simulation test. We found that the -1498C/-1190A/-634C/-7C (p=0.012) and -1498C/-1190A/-634G/-7C (p=0.000) haplotypes were significantly increased in patients with DR. The significance of the haplotype is due to VEGF T(-1498)C, C(-634)G, and C(-7)T polymorphisms. Taken together, the data suggest that the VEGF polymorphisms are associated with DR.
We have also identified one reported polymorphism (intron 4) in eNOS in patients with DR and DWR, but the eNOS polymorphism did not differ significantly between the two study groups (data not shown). This study shows VEGF polymorphisms have a high significant association with DR in the Indian population.
As VEGF is involved in the process of new blood vessel formation, it seems to be a potential candidate gene for DR. In this study, we have focused on the 5' UTR, promoter region of VEGF for genetic analysis as it has been shown to be highly polymorphic. In addition, most of the hypoxia responsive elements are present in this region and retinal hypoxia has been observed in DR. During hypoxia, hypoxia-inducible factors bind to the hypoxia-response elements and induce the expression of VEGF [24-26], which leads to the stimulation of angiogenesis. The polymorphisms in this gene have been studied in different ethnic groups but not in the Indian population. Hence we chose to examine VEGF for possible association with DR.
In the VEGF promoter and 5' UTR region, three polymorphisms were identified (C(-7)T, T(-1498)C, and C(-634)G). Comparison of the allele frequencies of these VEGF polymorphisms and their genotype combinations, haplotype frequencies between DR and DWR groups revealed significant results. In the Japanese population Takuya Awata et al.  identified C(-7)T, T(-1498)C, G(-1154)A, C(-634)G, and G(-1190)A polymorphisms in VEGF. They identified the CC genotype of the C(-634)G region to be significantly associated with DR (OR=3.20, 95% CI: 1.45-7.05, p=0.0046), whereas the CG genotype of the C(-634)G region was significantly associated with DR (OR=2.33, 95% CI: 1.24-4.36, p=0.008) in the Indian population. In addition to that, other genotypes of CT in the C(-7)T region and TC in the T(-1498)C region of VEGF were also found to be statistically significant in DR, whereas these genotypes were not reported to be significant in the Japanese population. Multiple logistic regression revealed nongenetic parameters such as diabetes mellitus duration and age, and genetic parameters like T(-1498)C and C(-7)T polymorphisms in VEGF as significant predictors of DR.
Comparison of distribution of genotype combinations between DR and DWR groups revealed significant differences. Combinations largely consisting of a majority of alleles in the -7C/T, -1498T/C, and -634C/G positions were found to be significantly associated with DR.
When the results obtained from our study were analyzed, the frequencies of heterozygous genotypes of VEGF C(-7)T, T(-1498)C, and C(-634)G were found to be significantly higher in patients with DR compared with DWR. The other genotypes in VEGF did not differ significantly between the two study groups. We suggest that the three heterozygous genotypes may be the high risk factors for DR. The association of polymorphism related to DR still remains unclear. Therefore further studies are required to conclusively establish the gene link between the genotype and DR.
The eNOS4b/b genotype was significantly higher in severe DR in caucasian populations . In our study the genotypes of eNOS did not differ significantly between the DR and DWR study groups. Our data suggest that the VEGF polymorphisms might be a major risk factor in the development of DR in the Indian population.
This study was supported by a grant from Aravind Medical Research Foundation and was carried out as a part of the activities of TIFAC-CORE in Diabetic Retinopathy, New Delhi, India. We would like to thank Dr. V. R. Muthukkaruppan, Director of Research, Aravind Medical Research Foundation for his guidance and valuable suggestions. We thank Dr. Rohini, G. Neethirajan, J. Kanagavalli, and R. Ramya for critical reading of manuscript, Mr. Jayakrishnan and Mr. Rajkumar for the photographic work, J. Nallathambi, B. Hema, and P. Murugeswari for their encouragement, and V. R. Muthulakshmi and T. P. Vasanthi for technical assistance.
1. Sulochana KN, Ramakrishnan S, Rajesh M, Coral K, Badrinath SS. Diabetic retinopathy: molecular mechanisms, present regime of treatment and future prospectives. Curr Sci 2001; 80:133-42.
2. Rema M, Deepa R, Mohan V. Prevalence of retinopathy at diagnosis among type 2 diabetic patients attending a diabetic centre in South India. Br J Ophthalmol 2000; 84:1058-60.
3. Balasubramanyam M, Rema M, Premanand C. Biochemical and molecular mechanisms of diabetic retinopathy. Curr Sci 2002; 83:1506-14.
4. Klein R, Klein BE, Moss SE, Davis MD, DeMets DL. The Wisconsin epidemiologic study of diabetic retinopathy. III. Prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Arch Ophthalmol 1984; 102:527-32.
5. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997; 18:4-25.
6. Duh E, Aiello LP. Vascular endothelial growth factor and diabetes: the agonist versus antagonist paradox. Diabetes 1999; 48:1899-906.
7. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD, Shah ST, Pasquale LR, Thieme H, Iwamoto MA, Park JE, Nguyen HV, Aiello LM, Ferrara N, King GL. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994; 331:1480-7.
8. Adamis AP, Miller JW, Bernal MT, D'Amico DJ, Folkman J, Yeo TK, Yeo KT. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 1994; 118:445-50.
9. Boulton M, Foreman D, Williams G, McLeod D. VEGF localisation in diabetic retinopathy. Br J Ophthalmol 1998; 82:561-8.
10. Amin RH, Frank RN, Kennedy A, Eliott D, Puklin JE, Abrams GW. Vascular endothelial growth factor is present in glial cells of the retina and optic nerve of human subjects with nonproliferative diabetic retinopathy. Invest Ophthalmol Vis Sci 1997; 38:36-47.
11. Lutty GA, McLeod DS, Merges C, Diggs A, Plouet J. Localization of vascular endothelial growth factor in human retina and choroid. Arch Ophthalmol 1996; 114:971-7.
12. Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, Shi XM, Tsui LC, Schappert KT. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993; 268:17478-88.
13. West IC. Radicals and oxidative stress in diabetes. Diabet Med 2000; 17:171-80.
14. Li H, Forstermann U. Nitric oxide in the pathogenesis of vascular disease. J Pathol 2000; 190:244-54.
15. Colasanti M, Suzuki H. The dual personality of NO. Trends Pharmacol Sci 2000; 21:249-52.
16. Sheng H, Ignarro LJ. Biochemical and immunohistochemical characterization of nitric oxide synthase in the rat retina. Pharmacol Res 1996; 33:29-34.
17. Goldstein IM, Ostwald P, Roth S. Nitric oxide: a review of its role in retinal function and disease. Vision Res 1996; 36:2979-94.
18. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney M, Chen D, Symes JF, Fishman MC, Huang PL, Isner JM. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest 1998; 101:2567-78.
19. Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1997; 100:3131-9.
20. Parenti A, Morbidelli L, Cui XL, Douglas JG, Hood JD, Granger HJ, Ledda F, Ziche M. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium. J Biol Chem 1998; 273:4220-6.
21. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16:1215.
22. Awata T, Inoue K, Kurihara S, Ohkubo T, Watanabe M, Inukai K, Inoue I, Katayama S. A common polymorphism in the 5'-untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes. Diabetes 2002; 51:1635-9.
23. Taverna MJ, Sola A, Guyot-Argenton C, Pacher N, Bruzzo F, Chevalier A, Slama G, Reach G, Selam JL. eNOS4 polymorphism of the endothelial nitric oxide synthase predicts risk for severe diabetic retinopathy. Diabet Med 2002; 19:240-5.
24. Treins C, Giorgetti-Peraldi S, Murdaca J, Van Obberghen E. Regulation of vascular endothelial growth factor expression by advanced glycation end products. J Biol Chem 2001; 276:43836-41.
25. Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D, Brusselmans K, Van Dorpe J, Hellings P, Gorselink M, Heymans S, Theilmeier G, Dewerchin M, Laudenbach V, Vermylen P, Raat H, Acker T, Vleminckx V, Van Den Bosch L, Cashman N, Fujisawa H, Drost MR, Sciot R, Bruyninckx F, Hicklin DJ, Ince C, Gressens P, Lupu F, Plate KH, Robberecht W, Herbert JM, Collen D, Carmeliet P. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 2001; 28:131-8.
26. Stevens A, Soden J, Brenchley PE, Ralph S, Ray DW. Haplotype analysis of the polymorphic human vascular endothelial growth factor gene promoter. Cancer Res 2003; 63:812-6.