Suppression of retinal neovascularization by shRNA targeting HIF-1alpha

PURPOSE: To investigate whether vector-based HIF-1alpha -targeted shRNA expression system (pSUPER(siHIF-1alpha)) can inhibit HIF-1alpha and VEGF expression in vitro and suppress retinal neovascularization in the murine model of oxygen-induced retinopathy.

METHODS: pSUPER(siHIF-1alpha) from which siRNA targeting HIF-1alpha could be generated was constructed and transfected to human umbilical vein endothelial cell lines (HUVECs). Then the expression levels of HIF-1alpha and VEGF in the cultured cells were measured by RT-PCR, immunoblot, and ELISA assays. Subsequently, pSUPER(siHIF-1alpha)was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated by angiography using fluorescein-labeled dextran and quantitated histologically. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of pSUPER(siHIF-1alpha)could affect the expression levels of HIF-1alpha and VEGF in murine retinas.

RESULTS: HIF-1alpha and hypoxia-induced vascular endothelial growth factor (VEGF) increase in cultured cells were greatly abolished by pSUPER(siHIF-1alpha). Meanwhile, retinal neovascularization in the eye with pSUPER(siHIF-1alpha)injection was significantly reduced compared with that of the contralateral control eye. Histological analysis indicates that neovascular nuclei protruding into the vitreous cavity were decreased by nearly 65%. Furthermore, HIF-1alpha and VEGF expression levels were down-regulated in the murine retinas treated with pSUPER(siHIF-1alpha).

CONCLUSIONS: RNAi targeting HIF-1alpha could inhibit the retinal neovascularization by approximately 65% through down-regulating the expression of HIF-1alpha and VEGF in the murine retinas, which may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.