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.
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.
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).
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.
From the *Department of Ophthalmology, Medical College of Georgia, Augusta, GA; the †Department of Cell Biology and Anatomy, Medical College of Georgia, Augusta, GA; the ‡Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY; and the §Department of Ophthalmology, Veterans Administration Medical Center, Augusta, GA.
Received for publication March 29, 2005; revision received July 12, 2006; accepted August 22, 2006.
Supported in part by the Veterans Administration CDA-VISN-7 (B.K.A.), Knights-Templar Eye Foundation (B.K.A.), and University of Kentucky Physician-Scientist Award (J.A.).
The materials presented herein are part of a provisional patent application filed with the US Patent Office.
Reprints: Balamurali K. Ambati, 1120 15th St., BA 2723, Augusta, GA 30912 (e-mail: firstname.lastname@example.org).