Localization of angiotensin converting enzyme in rabbit cornea and its role in controlling corneal angiogenesis in vivo

PURPOSE: The renin angiotensin system (RAS) has been shown to modulate vascular endothelial growth factor and angiogenesis. In this study we investigated (i) the existence of the RAS components angiotensin converting enzyme (ACE) and angiotensin II receptors (AT(1) and AT(2)) in the rabbit cornea using in vitro and ex vivo models and (ii) the effect of enalapril, an ACE inhibitor, to inhibit angiogenesis in rabbit cornea in vivo. METHODS: New Zealand White rabbits were used. Cultured corneal fibroblasts and corneal epithelial cells were used for RNA isolation and cDNA preparation using standard molecular biology techniques. PCR was performed to detect the presence of ACE, AT(1), and AT(2) gene expression. A corneal micropocket assay to implant a vascular endothelial growth factor (VEGF) pellet in the rabbit cornea was used to induce corneal angiogenesis. Rabbits of the control group received sterile water, and the treated group received 3 mg/kg enalapril intramuscularly once daily for 14 days starting from day 1 of pellet implantation. The clinical eye examination was performed by slit-lamp biomicroscopy. We monitored the level of corneal angiogenesis in live animals by stereomicroscopy at days 4, 9, and 14 after VEGF pellet implantation. Collagen type IV and lectin immunohistochemistry and fluorescent microscopy were used to measure corneal angiogenesis in tissue sections of control and enalapril-treated corneas of the rabbits. Image J software was used to quantify corneal angiogenesis in the rabbit eye in situ. RESULTS: Our data demonstrated the presence of ACE, AT(1), and AT(2) expression in corneal fibroblasts. Cells of corneal epithelium expressed AT(1) and AT(2) but did not show ACE expression. Slit-lamp examination did not show any significant difference between the degree of edema or cellular infiltration between the corneas of control and enalapril-treated rabbits. VEGF pellet implantation caused corneal angiogenesis in the eyes of vehicle-treated control rabbits, and the mean area of corneal neovascularization was 1.8, 2.8, and 3.2 mm(2) on days 4, 9, and 14, respectively. Enalapril treatment caused a notable decrease in corneal neovascularization of 44% (1 mm(2)), 28% (2.1 mm(2)), and 31% (2.2 mm(2)) on the three tested time points, respectively. The immunostaining of corneal tissue sections with collagen type IV and lectin confirmed the presence of blood vessels, with enalapril-treated rabbit corneas showing a lesser degree of blood vessel staining. CONCLUSIONS: Corneal cells show expression of tissue RAS components, such as ACE, AT(1), and AT(2). Treatment with ACE inhibitor enalapril markedly decreased corneal angiogenesis in a rabbit model of VEGF-induced corneal neovascularization, suggesting that ACE inhibitors may represent a novel therapeutic strategy to treat corneal angiogenesis.