Inhibitory effect of VEGF-targeted RNA interference on retinal neovascularization in mice with oxygen-induced retinopathy

Abstract

Objective To investigate the inhibitory effect of vascular endothelial growth factor (VEGF)-targeted RNA interference on the retinal neovascularization of mice with oxygen-induced retinopathy (OIR).Methods This was an experimental study.OIR mice models were induced by exposing mice to a 75％ concentration of oxygen from postnatal day 7 (P7) to P12 and then returned to room air from P12 to P17.VEGF-siRNA was designed,screened and packaged in 293T cells to produce VEGF-RNAi-lentivirus.Sixty C57BL/6J mice were divided into 4 groups (every group n=15):normal control group,OIR group,OIR+lentivirus (OIR-K-virus) group,and OIR+VEGF-RNAi-lentivirus (OIR-Ri-virus) group.Intravitreal injection of lentivirus was performed at P5 in the OIR-K-virus group and OIR-Ri-virus group.After FITC-Dextran perfusion,a retinal flat-mount was used to observe the area of retinal neovascularization.The distribution of Claudin-5 and Occludin was detected by double immunofluorescence staining of the retinal flat-mount.The expression of VEGF,phosphotylinosital 3 kinase (PI3K),tyrosine kinase SRC,extracellular signal-regulated kinase (ERK) and phosphorylation ERK (p-ERK) proteins was analyzed by Western blot.Data were analyzed with a one-way ANOVA.Results A study of the retinal flat-mounts after FITC-Dextran perfusion showed that the distribution of retinal vessels was uniform and there was no neovascularization in the normal group.The area of retinal neovascularization in the OIR group was 1.212 782 mm2,OIR-K-virus group was 1.152 504 mm2,and OIR-Ri-virus group was 0.271 399 mm2,the difference between groups was significant (F=449.924,P＜0.01).There was no significant difference in the areas of retinal neovascularization between the OIR group and OIR-K-virus group.The areas of retinal neovascularization in the OIR-Ri-virus group were reduced significantly (P＜0.01) compared to the OIR group or the OIR-K-virus group.The distribution of Claudin-5 and Occludin in the retinal vessels of the OIR-Ri-virus group was more uniform and smoother than distribution in the OIR group or OIR-K-virus group.The expression of VEGF,PI3K,tyrosine kinase SRC and p-ERK protein in the OIR-Ri-virus group was significantly decreased compared to expression in the OIR and OIR-K-virus groups and was similar to the expression of the normal group.Conclusion The expression of VEGF and its signal pathway and development of retinal neovascularization in the OIR mice model were significantly inhibited by the intravitreal injection of lentivirus-mediated VEGF RNA interference,which may provide a new approach for the treatment of retinopathy of prematurity (ROP) in the clinic. Key words: Vascular endothelial growth factor A; RNA interference; Retinal neovascularization; Retinopathy of prematurity; Models,animal