The Collagen-Binding Protein Hsp47 as a Therapeutic Target for Inhibition of Retinal Neovascularization

Abstract

Purpose:: Retinal neovascularization and fibrovascular proliferation underlie a number of blinding disorders. Previous studies showed a consistent increase in expression of the collagen-binding protein Hsp47 during maximal retinal neovascularization. In models of non-ocular disease, Hsp47 has been implicated in experimental angiogenesis, and inhibitors of Hsp47 have been shown to reduce the extent of experimental fibrosis. The present work was undertaken to analyze the differential cellular expression of Hsp47 in a rat model of retinal neovascularization and to examine the in vitro and in vivo effects of Hsp47 inhibition.

Methods:: Litters of newborn rats and their nursing mothers were exposed to alternating 24-hour cycles of 50% oxygen and 10% oxygen for a total of 14 days and then removed to room air. Control newborn rats and their nursing mothers were reared in room air. Retinal flat-mounts were prepared from both groups at P15, P17, and P20. Double-labelling immunohistochemistry was performed using a monoclonal anti-Hsp47 antibody in conjunction with antibodies to glial fibrillary acidic protein, CD90, and glutamine synthetase and isolectin. A small interfering RNA (siRNA) against rat hsp47 was synthesized, and a robust reduction of RNA and protein expression in endothelial cells and Muller cells was confirmed. Hsp47 involvement in endothelial cell proliferation and tube formation was assessed in vitro using rat retinal microvascular endothelial cells transfected with hsp47 siRNA. For in vivo studies, intravitreal injection of 1 nM, 10 nM, or 100 nM of siRNA was given to the right eye of experimental rats at P15, while vehicle alone was administered to the left eye. Following sacrifice at P20, retinal flatmounts were stained with ADPase for quantitative evaluation of retinal neovascularization.

Results:: Immunohistochemical studies showed: (1) Hsp47 expression in normal retinal vasculature and in neovascular tufts; (2) Hsp47 expression in Muller cells, and, during retinal neovascularization, more abundant expression at the vitreoretinal interface; (3) no Hsp47 expression in astrocytes. Eyes of oxygen-exposed rats treated with Hsp47 siRNA had 50% fewer clock-hours of retinal neovascularization than vehicle-treated eyes. No gross toxicity to native retinal vasculature was seen.

Conclusions:: These preliminary findings support the hypothesis that Hsp47 is involved in experimental retinal neovascularization and that inhibition of hsp47 may reduce the extent of neovascularization in vivo.

Keywords: retinal neovascularization • neovascularization • retina