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

Singh, Nirbhai PhD*; Higgins, Eric MD*; Amin, Shivan BS*; Jani, Pooja BS*; Richter, Elizabeth BS*; Patel, Anup BS*; Kaur, Rajwinder MD*; Wang, Jin BS; Ambati, Jayakrishna MD; Dong, Zheng PhD; Ambati, Balamurali K MD

doi: 10.1097/ICO.0b013e31802b4201
Basic Investigation

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.

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: bambati@mail.mcg.edu).

© 2007 Lippincott Williams & Wilkins, Inc.