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A.V. Mezentsev, F. Seta, M.W. Dunn, M. Laniado–Schwartzman; TRANSFECTION OF CYP4B1 INTO THE CORNEA INDUCES ANGIOGENIC ACTIVITY IN THE LIMBAL VESSELS . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2348.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Injury to the ocular surface provokes an inflammatory response that is mediated, at least in part, by corneal epithelial–derived 12–hydroxyeicosanoids. These eicosanoids, which exhibit potent inflammatory and angiogenic properties, are formed by a cytochrome P450 enzyme, presumably CYP4B1. We have isolated and cloned rabbit corneal CYP4B1 and examined the effect of CYP4B1 overexpression on corneal inflammation and neovascularization in vivo and in vitro. Methods:The expression vectors pIRES2–EGFP or pIRES2–CYP4B1 (2 µg/4 µl) were administered to the left eye using a 30G1/2in. needle dipped in the plasmid solution and inserted repeatedly into the limbus over 360o. Inflammatory response was examined by slit lamp microscopy over a 6–day period. Eyes were excised and corneas processed for GFP detection by fluorescence and immunohistochemistry 3 and 6 days after treatment. Corneal–limbal explants were placed in Matrigel–coated 24–well plates and their angiogenic capacity was determined over time (1–7 days) by measuring the length of the neovascular sprouts; the lectin Ulex europeus was used to identify the sprouts as endothelial cells. The culture medium was collected for determination of 12–hydroxyeicosanoid levels. Results:Plasmid inserted into the limbus effectively transduced GFP expression in the corneal epithelial cells primarily at the limbus and in the corneal endothelial cell layer. Slit lamp microscopy in vivo revealed a significant inflammatory response that included limbal vasodilation and corneal neovascularization in eyes transfected with the pIRES2–CYP4B1 but not with pIRES2–EGFP. Corneal limbal explants from eyes transfected with pIRES2–CYP4B1 exhibited a marked 3–fold increase in neovascular sprouting composed of GFP positive endothelial cells. This angiogenic activity was associated with increased levels of 12–HETrE, the angiogenic eicosanoid formed by CYP4B1. Conclusions: The results further implicate corneal CYP4B1 as a component of the inflammatory cascade initiated by injury to the ocular surface. Neovascularization was associated with increased levels of the angiogenic eicosanoid 12–HETrE. These finding suggest a novel strategy for control of ocular surface inflammation and neovascularization.
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