Inhibition of experimental choroidal neovascularization in mice by anti-VEGFA/VEGFR2 or non-specific siRNA

Abstract

Choroidal neovascularization (CNV) is one of the severe pathological consequences of the end-stage of age-related macular degeneration (AMD). Several lines of evidence implicate increased levels of vascular endothelial growth factor (VEGF) in the retinas of AMD patients. Current available agents for the inhibition of VEGF protein such as bevacizumab show significant promise for the treatment of exudative AMD. However, this compound still has limited efficacy and requires multiple administrations; thus, it is associated with a variety of ocular complications, including endophthalmitis and retinal detachment. In this study, we used anti-VEGFA/VEGFR2 or non-specific siRNA and evaluated their suppression of laser-induced choroidal neovascularization (CNV) in mice. Male adult C57BL/6J mice were used in the study. The mice were subjected to laser rupture of Bruch’s membrane to induce CNV and then randomized to five groups with six mice per group. The five groups were blank control, vehicle control (5% glucose solution, GS), VEGFA.siRNA, VEGFR2.siRNA, and non-specific siRNA. Two days after laser photocoagulation, each group, with the exception of the blank control group, received 1 μl of the appropriate agent by intravitreal injection to both eyes. Seven days later, after taking fundus photography and fundus fluorescein angiography (FFA), the mice were killed for tissue sampling. Six eyes from three mice in each group were used for choroidal flatmounts to examine CNV. Six eyes from three mice in each group were subjected to RNA extraction for VEGF mRNA quantification by qRT-PCR. Retinal tissue from 2 mice without laser treatment was harvested as the assay reference. The incidence of burns which showed fluorescein leakage was 80.0% in blank control, 75.0% in GS, 55.0% in VEGFA.siRNA, 40.0% in VEGFR2.siRNA and 30.0% in non-specific siRNA group. The flatmounted specimens showed that the retinal pigment epithelium (RPE) was visualized as a uniform hexagonal array in nonlasered areas. On day 7 after laser burn, well-defined isolectin-B4 labeled CNV networks were shown within the burn spots of the 2 control groups. The inhibitory effects of the 3 siRNAs on CNV formation were statistically significant as compared to the 2 control groups. Compared to the control groups, the ocular expression of VEGF mRNA was decreased significantly in the 3 siRNA treated groups. The areas of CNV correlated with the expression of VEGF mRNA. Anti-VEGFA/VEGFR2 or non-specific siRNA can inhibit CNV and attenuate VEGF mRNA expression in a laser-induced mouse model of CNV. We conclude that the siRNAs may be an option for treating patients with exudative AMD, and more studies are needed to test the possible side-effects of the treatment.