The Corneal Epithelial CYP4B1 Produces Angiogenic Eicosanoids and Induces Inflammation of the Ocular Surface

Abstract

Abstract: : Purpose: We have previously shown that inflammation and neovascularization in response to injury to the ocular surface is mediated by 12–hydroxyeicosanoids (12–HETE and 12–HETrE) produced in the corneal epithelium by cytochrome P450 monooxygenase. This enzyme has been isolated and identified as the corneal CYP4B1, however its relationship to the production of 12–hydroxyeicosanoids and the inflammatory response has not been demonstrated. We have constructed expression plasmids containing the corneal CYP4B1 cDNA and evaluated its catalytic activity in vitro and in vivo and its role in ocular surface inflammation and neovascularization. Methods: Rabbit corneal epithelial (RCE) cells were transfected with the expression vectors pIRES2–EGFP or pIRES2–EGFP–CYP4B1. Arachidonic acid metabolism by transfected cells was determined using HPLC and GC/MS analyses. Plasmids were also administered in vivo by repeated injection into the limbus of the rabbit eye. Transfection efficiency was monitored by following GFP fluorescence. Inflammatory response was examined by pachymetry and slit lamp microscopy over a 6–day period. Angiogenic response was determined ex vivo by measuring the length of the neovessel sprouts formed by corneal–limbal explants placed in Matrigel–coated culture dishes. The angiogenic response was correlated with the levels of 12–hydroxyeicosanoids. Results: RCE cells transfected with the CYP4B1 cDNA metabolized arachidonic acid to a product that had the HPLC elution profile of authentic 12–HETrE. 12–HETrE constituted the main metabolite in cell transfected with the CYP4B1 and was formed at a rate of 5.33 nmol/10⁷ cells/h as compared to a rate of 1.22 nmol/10⁷ cells/h in cells transfected with the control plasmid, pIERS2–EGFP. Eyes transfected with pIRES2–EGFP–CYP4B1 showed significant inflammation. Corneal–limbal explants from eyes transfected with pIRES2–EGFP–CYP4B1 demonstrated marked angiogenic activity which was correlated with increased levels of 12–HETrE. Conclusions: The results indicate that the corneal CYP4B1 is the enzyme catalyzing the synthesis of the pro–inflammatory and angiogenic eicosanoid, 12–HETrE and further implicate it as a component of the inflammatory cascade initiated by injury of the ocular surface.

Keywords: eicosanoids • inflammation • cornea: epithelium