Small interference RNA targeting vascular endothelial growth factor gene effectively attenuates retinal neovascularization in mice model.

BACKGROUND: The mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF). METHODS: In vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified. RESULTS: In vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals. CONCLUSIONS: VEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

[Expression of vascular endothelial growth factor and pigment epithelium derived factor in mouse oxygen-induced retinopathy and its significance].

OBJECTIVE: To investigate the expression and significance of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in oxygen-induced mouse retinopathy. METHODS: This experiment was a control experiment study. Newborn C57BL-6 mice were exposed to hyperoxia, and then returned to normoxia to induce retinal neovascularization. Mice were sacrificed at postnatal days 12, 14 and 17 and the retina were processed for RT-PCR, Western Blot and fluorescein angiography. Analysis of variance and Dunnett's t3 was used to compare the mRNA and protein level of VEGF and PEDF between experiment and control groups. Statistical difference was considered significant at a P value less than 0.05. RESULTS: At any of the time points tested, there was significant difference at VEGF protein level (A value) between the experiment (0.47 +/- 0.12, 2.15 +/- 0. 46, 5.49 +/- 0.97) and control (l.81 +/- 0.50, 0.90 +/- 0.05, 0.88 +/- 0.91) groups (P = 0.009, 0.010, 0.000, respectively); the same situation occurred at PEDF protein level (P = 0.002, 0.046, 0.000, respectively). At postnatal day 12, 14 and 17, a significant difference at VEGF protein level was observed among the different experiment groups (P = 0.002, 0.001, 0.000, respectively); the same situation occurred at PEDF protein level (P = 0.009, 0.010, 0.000, respectively). There was significant difference at VEGF mRNA level between the experiment and control groups (P = 0.001, 0.000, 0.001); at postnatal day 12 and 14, the same situation occurred at PEDF protein level (P = 0.001, 0.000, respectively), but there was no significant difference at postnatal day 17 (P = 0.612). At postnatal day 12, 14 and 17, a significant difference at VEGF mRNA level was observed among different experiment groups (P = 0.000, 0.001, 0.000, respectively); the same situation occurred at PEDF mRNA level (P = 0.000, 0.001, 0.000, respectively). The time course of the decrease of PEDF was consistent with the increase of VEGF expression. VEGF/PEDF ratio change was correlated with the development and progression of retinal neovascularization. The time course of PEDF mRNA down-regulation was consistent with the VEGF mRNA up-regulation, similar with the changes of the protein. The change of VEGF and PEDF mRNA was prior to that of the protein. CONCLUSIONS: One of the mechanisms for development of retinal neovascularization is the changes of VEGF\PEDF level in the retina.