Inhibition of Retinal Neovascularization (NV) by AAV-mediated Gene Transfer of Endostatin in a Mouse Model of Retinopathy of Prematurity (ROP)

Abstract

Abstract: : Purpose: Endostatin (-/-) mutant mice show delayed regression of hyaloid vessels after birth and abnormal outgrowth of retinal vessels suggesting that endostatin is important in regulating normal vascular development. It was also demonstrated that expression of endostatin mediated by adenoviral vector inhibited choroidal neovascularization (NV) in mice. The purpose of this experiment is to evaluate whether AAV-mediated gene expression of endostatin can inhibit retinal NV in a mouse model of ROP. Methods: An AAV vector was constructed by standard procedures containing the CBA (chicken ß-actin) promoter and the cDNA for human endostatin. A woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was also included at the 3’ end of endostatin to enhance its expression. Postnatal day 0 (P0) neonatal mice were injected in one eye with the AAV vector expressing endostatin. The mice were placed in a 73% oxygen chamber from P7 to P12, and then returned to room air for another 5 days. At P17, the extent of retinal NV was evaluated by comparing the number of vascular endothelial nuclei immediately above the inner limiting membrane of the retina sections spanning the whole eye in virus injected and contralateral uninjected or PBS injected eyes. Results: The number of vascular endothelial nuclei was reduced 50-90% in rAAV virus injected eye compared to contralateral uninjected or PBS injected eye in this mouse model of ROP. Care was taken to avoid any vascular endothelial cells associated with persistent hyaloid vasculature. By ELISA, ocular endostatin levels, undetectable in control eyes, were elevated to an average of 1.2ng per eye, consistent with endostatin expression accounting for the improvement in retinal NV. Conclusions: These results, in addition to other recent evidence suggesting that endostatin can prevent VEGF-mediated vascular leakage, imply that endostatin may have therapeutic potential for treating retinal NV and macular oedema caused by diabetic retinopathy and other ischemic retinopathies.

Keywords: retinal neovascularization • retinopathy of prematurity • ischemia