Abstract
Abstract: :
Purpose: Retinal neovascularization and fibrovascular proliferation underlie a number of blinding disorders, such as diabetic retinopathy and retinopathy of prematurity. Previous DNA microarray analysis using a murine model of oxygen–induced retinopathy 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 expression of Hsp47 in a rat model of oxygen–induced retinopathy. 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. Total protein was extracted from retinas harvested from both groups immediately upon return to room air and 1, 2, 3, and 6 days later. Western blotting with anti–Hsp47 antibody was performed for all timepoints. Immunohistochemistry with anti–Hsp47 antibody was performed on transverse retinal sections from 1–day, 3–day, and 6–day post–exposure and corresponding controls. Hsp47 protein expression was measured in cultured human retinal microvascular endothelial cells (HRMECs) treated with vascular endothelial growth factor (VEGF), as well as in cultured Muller cells exposed to hypoxia (<2% oxygen) for 24 hours. Results: Retinal expression of Hsp47 protein progressively increased in the six days following oxygen exposure. At one, three, and six days post–exposure, Hsp47 protein levels were significantly higher than in corresponding controls. In control eyes, Hsp47 expression was observed in retinal vascular endothelium and in the inner nuclear layer. In oxygen–exposed eyes, Hsp47 expression was increased at the junction of Muller cells and the internal limiting membrane and was observed in native retinal vascular endothelium and in retinal neovascular tufts. No significant change in Hsp47 expression was observed in cultured HRMECs following stimulation with VEGF or in Muller cells following hypoxia. Conclusions: In the rat model of oxygen–induced retinopathy, Hsp47 expression is increased. The protein appears to accumulate at the vitreoretinal interface and may participate in extraretinal proliferation through its functions as a collagen–binding protein. Hsp47 may constitute a rational target of inhibition to reduce the extent of vitreoretinal proliferation.
Keywords: neovascularization • retinal neovascularization • retinopathy of prematurity