Suppression of Retinal Neovascularization by shRNA Targeting HIF-1α

Abstract

Purpose: To investigate whether vector-based HIF-1α -targeted shRNA expression system (pSUPER^siHIF-1α) can inhibit HIF-1α and VEGF expression in vitro and suppress retinal neovascularization in the murine model of oxygen-induced retinopathy. Methods: pSUPER^{siHIF − 1α} from which siRNA targeting HIF-1α could be generated was constructed and transfected to human umbilical vein endothelial cell lines (HUVECs). Then the expression levels of HIF-1α and VEGF in the cultured cells were measured by RT-PCR, immunoblot, and ELISA assays. Subsequently, pSUPER^{siHIF − 1α}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-1αcould affect the expression levels of HIF-1α and VEGF in murine retinas. Results: HIF-1α and hypoxia-induced vascular endothelial growth factor (VEGF) increase in cultured cells were greatly abolished by pSUPER^siHIF-1α. Meanwhile, retinal neovascularization in the eye with pSUPER^siHIF-1αinjection 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-1α and VEGF expression levels were down-regulated in the murine retinas treated with pSUPER^siHIF-1α. Conclusions: RNAi targeting HIF-1α could inhibit the retinal neovascularization by approximately 65% through down-regulating the expression of HIF-1α and VEGF in the murine retinas, which may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.

KEYWORDS:

• hypoxia inducible factor 1α
• retinal neovascularization
• small hairpin RNA
• vascular endothelial growth factor