Unique Homologous siRNA Blocks Hypoxia-Induced VEGF Upregulation in Human Corneal Cells and Inhibits and Regresses Murine Corneal Neovascularization

Abstract

PURPOSE: To determine whether RNA interference (RNAi) could block hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) in human corneal epithelial cells in vitro and inhibit and regress injury-induced murine corneal neovascularization in vivo. METHODS: siRNA selected on the basis of target sequence homology between mouse and human VEGF was placed into expression cassettes and transfected into human corneal epithelial cells. Hypoxia-induced VEGF synthesis was assayed. Also, the effect of a plasmid capable of directing the expression of an siRNA against VEGF when injected into mouse corneas 8 hours before alkali-mechanical trauma was studied. Leukocyte count, VEGF protein levels, and degree of neovascularization in corneas were compared with that of a control siRNA plasmid. Plasmids were injected 1 week after injury to assess the ability of RNAi to regress corneal neovascularization. RESULTS: Hypoxia-induced VEGF mRNA synthesis and protein secretion by human corneal epithelial cells was efficiently suppressed by an siRNA targeted against a sequence uniquely identical for the mouse and human VEGF genes. Intrastromal delivery of a plasmid expressing this siRNA before murine corneal injury suppressed corneal VEGF by 55.7% versus control (P = 0.014), leukocyte infiltration by 69.5% (P < 0.001), and neovascularization 1 week after injury by 72.3% (P = 0.001). At the regression time point, treated corneas had 72.8% less neovascularization (P < 0.001). CONCLUSIONS: RNAi significantly suppresses expression of VEGF induced by hypoxia in human corneal epithelial cells in vitro. In vivo, intrastromal delivery of a plasmid expressing siRNA against VEGF suppresses injury-induced VEGF expression, leukocyte infiltration, and angiogenesis and was able to regress corneal neovascularization.