Age-related macular degeneration (AMD) is a progressive chronic inflammation that affects the cells of the retinal pigment epithelium (RPE) and promotes the formation and deposition of phospholipid material retinal drusen. This maculopathy progresses to degeneration in two forms: “wet AMD” and “Dry AMD”. Indeed, most visual loss occurs in the late stages of the disease due to one of two processes: choroïdal neovascularization (CNV) and geographic atrophy [1, 2]. Etiological research suggests that AMD is a complex disease, caused by the interactions of several genetic and environmental factors. Different evidences have supported that in exudative AMD form, inflammatory lesions are associated with a high expression and synthesis of growth factors such as vascular endothelial growth factor (VEGF) which is a key regulatory factor in angiogenesis and vascular permeability in both physiological and pathological states [3]. VEGF expression has been shown in experimental choroidal neovascularization and shown to induce CNV growth in animal models [4]. Therefore, it would be involved in the development of CNV in this disease.

More recently, attention has been focused on the function of VEGF in light of its role as a therapeutic target and VEGF-inhibitors have been used in successful therapy of exudative AMD [5, 6]. The most important advance in the treatment of neovascular AMD is the development of anti-vascular endothelial growth factor (anti-VEGF) therapeutic agents that preserve and improve visual acuity by arresting choroidal neovascular growth and reducing vascular permeability. Thus, VEGF is a potential candidate for genetically influencing AMD susceptibility, based on its functional relevance to AMD pathophysiology.

VEGF gene exhibits many single nucleotide polymorphisms (SNPs) that might influence qualitatively and/or quantitatively its expression. Previous studies have shown that the genetic variations in the VEGF gene influence the rate of the VEGF protein synthesis [7–9]. The human VEGF-A gene is located on chromosome 6 (6p21.3) and is organized into eight exons [10]. Several different isoforms of VEGF are generated by alternate splicing of the VEGF-A gene [11]. Of these, the VEGF₁₆₅ isoform (named according to the number of amino acids), is the most abundant and corresponds to a 23 kDa polypeptide, constituting a monomer of homodimeric human VEGF-A the role of vascular endothelial growth factor (VEGF) in inflammatory bowel disease [12].

In this context, three common VEGF SNPs (C-2578A, rs699947), (G + 405C, rs2010963) and (C + 936 T, rs3025039), in the promoter, the 5′Untranslate and the 3′Untranslated regions respectively, were investigated in Tunisian exudative AMD patients in order to determine their association with the disease susceptibility, their influence to the level of production of this glycoprotein and the response to intravitreal bevacizumab therapy.

The current study showed that (+405) G/C and (+936) C/T VEGF polymorphisms were statically associated with exudative AMD in the Tunisian patients corroborating the large study of Haines et al., who attributed to the some SNPs of VEGF, VLDLR and LRP6 genes, a role in the risk of AMD development [15]. These results were in close agreement with those previously published by Janik-Papis et al., in a Polish population [16] and by Lin et al., in Taiwan Chinese population [17]. Inversely, it has been reported in other studies that the positive association between VEGF polymorphisms of the gene and susceptibility to the occurrence of AMD could not be confirmed in the Rotterdam study and in Anglo-Celtic subpopulation [18–20]. These controversial results are due to differences in the size of the study, patients heterogeneity, the choice of analyzed SNPs located in the promoter region and/or in the coding regions and the methods used for their genotyping. It is now well established that genetic susceptibility to complex diseases such as AMD, is rather due to the cumulative effect of several predictive alleles identified in the full haplotype information [17, 18]. Among combined haplotypes observed in this cohort, the risk ACT variant was more frequently found in patients with the active form of the disease (G1patients) compared to those with the scar form (G2 patients), but the difference was not statistically significant. The small numbers of subjects in G2 could induce a loss of statistical power to detect this difference.

The angiogenesis process is highly controlled through the balance of pro- and anti-angiogenic factors. VEGF is the key pro-factor in this process. Specific polymorphisms in the VEGF gene have been associated with a variation of protein levels [7, 21, 22]. The data from this study support this hypothesis by showing that both in controls and G2 patients compared to the G1 subjects, a significant decrease in s-VEGF was observed, confirming once again that VEGF is the major stimulus for the development and growth of choroidal neovascularization in the exudative AMD because wet AMD involves neovascularization, the best approach for the treatment of CNV appears to be the use of anti-VEGF drugs. At present, several molecules have been used in intravitreal therapies approved by Food and Drug Administration (FDA) and the European Medecines Agency (EMEA).

Among these anti-VEGF agents, several studies report that the bevacizumab (Avastin*; Genentech Inc) gives the best results for the treatment of neovascular AMD [23–26].

The effectiveness of such treatments depends on their molecular weight, the binding affinity of the various isoforms of VEGF and their pharmacokinetics lesional prior retinal barrier. In this study, taking into account of the availability of bevacizumab exposure in plasma, this molecule would be more effective in patients with a high concentration of plasmatic VEGF levels compared to those with the rate of protein relatively low. Indeed, a difference in the distribution of plasma levels of VEGF was found among good responders compared with poor responders. These results corroborate those of Carneiro et al., who show that bevacizumab significantly reduced the plasma levels of VEGF after 28 days after intravitreal injections in patients with exudative AMD compared to injections of ranibizumab, with two main potential determinants of their systemic adverse effects including the blood levels of plasmatic VEGF and the degree of systemic anti-VEGF inhibition.

About the relationship between SNPs of the VEGF gene studied and response to intravitreal injection of bevacizumab, this study revealed, when each SNP was analyzed individually, that the patients mutated homozygous AA (−2578) genotype were rather classified as good responders whereas those with risk homozygous genotype TT(+936) belonged to the group of poor responders. Nevertheless, theses SNPs lose their significance in analysis of combined haplotypes. In the literature, unanimity is not yet fully established for the use of these genetic biomarkers to predict the visual evolution in response to anti-VEGF therapy in the exudative AMD. Indeed, if in the promoter region of VEGF gene, the SNP (C-2578A, rs699947) seems to be also, a predictor of success to bevacizumab treatment in Japan population [27], our results, concerning the SNP (C936T, rs3024039) in the 3′Untranslated region, are in discordance with a Qu Y. in Chinese population and Boltz A. studies that provide no evidence for an association between this SNP and the response to bevacizumab treatment [28, 29]. In Korean neovascular AMD cohort, Un Chul Park et al., do not find statistically significant effect of VEGF-SNPs (C936T, C-2578A and C-460 T) on visual outcome after ranibizumab treatment [30]. In 5′Untranslate region, the SNP (G405C, rs2010963), despite the fact that it is associated with susceptibility to AMD, the response to bevacizumab was independent of this polymorphisme. This is in agreement with study of Imai et al. found that VEGF (−2578; rs699947) and PDEF (+rs113628) were associated with vision changes at 1 month and 3 months post-bevacizumab therapy respectively but VEGF (+405; rs2010963) may not be genetic biomarker to estimate visual outcomes in response to intravitreal bevacizumab treatment for neovascular AMD [27].

In view of these results, the question is what about the genetic profile associated with long-term visual outcome? The follow-up of further study with larger sample sizes and standard intervals to record the results of visual acuity should be performed to confirm these results and to answer this question.

In conclusion, this study found that VEGF genetic variants may contribute to the susceptibility to neovascular AMD in Tunisian patients and were also associated with vision changes at 3 months of anti-VEGF therapy. However, our findings need to be replicated in additional studies. Further expression studies are needed to investigate the potential pharmacologic role of these variants in antiangiogenesis AMD therapy at long-term.