Transfection of cytochrome P4504B1 into the cornea increases angiogenic activity of the limbal vessels

Injury to the ocular surface induces the production of the corneal epithelial-derived 12-hydroxyeicosatetrienoic acid (12-HETrE), which exhibits stereospecific potent inflammatory and angiogenic properties and is formed by a cytochrome P450 (P450) enzyme, CYP4B1. We have cloned the rabbit corneal CYP4B1 into the expression plasmid pIRES2-enhanced green fluorescent protein (EGFP) and examined the effect of CYP4B1 overexpression on corneal inflammation in vivo and limbal vessel sprouting ex vivo. Cultured rabbit corneal epithelial cells transfected with pIRES2-EGFP-CYP4B1 metabolized arachidonic acid to 12-HETrE at a rate five times higher than that of pIRES2-EGFP-transfected cells (3.53 +/- 0.08 versus 0.62 +/- 0.10 nmol/h/10(6) cells; mean +/- S.E.M., n = 6, p < 0.05), indicating a functional expression of the CYP4B1. Injection of either plasmid into the rabbit cornea resulted in EGFP fluorescence in the corneal epithelium. However, corneal neovascularization, as measured by the length of penetrating blood vessels, was significantly greater in the corneas of eyes transfected with the pIRES2-CYP4B1 compared with pIRES2-EGFP. Corneal-limbal explants from eyes transfected with pIRES2-CYP4B1 showed a marked angiogenic activity (46 +/- 10 versus 12 +/- 3 mm capillary length, n = 6, p < 0.05), which correlated with increased levels of 12-HETrE, the CYP4B1-derived angiogenic 12-hydroxyeicosanoid (0.93 +/- 0.18 versus 0.15 +/- 0.02 pmol/explant, n = 6, p < 0.05), and was inhibited (76 +/- 5%) by the P450 inhibitor 17-octadecynoic acid. The results further implicate the corneal CYP4B1 as a component of the inflammatory and angiogenic cascade initiated by injury to the ocular surface and raise the possibility of a new therapeutic target for preventing corneal neovascularization, namely, the CYP4B1-12-HETrE system.