Effect of intravitreal injection with small interfering RNA targeting hypoxia inducible factor-1α on vascular endothelial growth factor protein expression in diabetic rats

Abstract

AIM: To evaluate the inhibitory effect of hypoxia inducible factor-1α(HIF-1α)small interfering RNA(siRNA)on the expression of vascular endothelial growth factor(VEGF)protein and explore the feasibility of potential therapeutic approach for diabetic neovascular disease.METHODS: Using pSilencer2.1-U6neo for plasmid vector, HIF-1α siRNA recombinant plasmid was constructed. There was totally 54 healthy Sprague Dawley rats in which 15 rats were chosen as normal group and 39 rats were constructed for diabetic retinopathy model by streptozotocin(STZ)which was divided into three subgroups randomly including control model group(DR group, 15 rats), vector group(12 rats)and gene therapy group(HIF-1α siRNA group, 12 rats). Nothing was transfected into DR group and normal group. The vector plasmid and HIF-1α siRNA were injected into the vitreous in vector group and HiF-1α siRNA group respectively. The retinal morphology was observed by hematoxylin-eosin(HE)staining and the expression of VEGF protein was measured by immunohistochemical staining. The inhibition efficiency of VEGF was calculated at 24, 48, 72h and 1wk after injected. Significant differences between groups were evaluated by one-way analysis of variance, followed by LSD-t analysis. RESULTS: HIF-1α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. HE staining showed that the retinal cells at each layers in normal control group were arranged regularly, and cell's morphology was roughly normal. The retinal cells at each layers arranged in disorder in diabetic rat And the inner limiting membrane was not complete with neovascular buds and neovascularization cluster growing out of the inner limiting membrane vertically. Immunohistochemical staining showed that the positive expression of VEGF was brown yellow granules, which was mainly located in ganglion cell layer. It also revealed the expression of VEGF protein was weakly positive in normal control group, while the DR group and empty vector group were significantly increased. Compared with DR group and the empty vector group, gene therapy group was significantly decreased, the difference was statistically significant(PCONCLUSION: Intravitreal injection with HIF-1α siRNA can efficiently inhibite VEGF protein in retina of diabetic rats, which may be a new method for the treatment of diabetic neovascular disease.

Keywords

• hypoxia inducible factor-1
• vascular endothelial growth factor
• RNA interfering
• retinal neovascularization
• gene therapy