Hypoxia stimulates the synthesis of cytochrome P450-derived inflammatory eicosanoids in rabbit corneal epithelium

The corneal epithelium metabolizes arachidonic acid by a cytochrome P450-(C YP) mediated pathway to 12(R)hydroxy-5,8,10,14-eicosatrienoic acid [12(R)-H ETE] and 12(R)hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE]. Both metabo lites possess potent inflammatory properties with 12(R)-HETrE being a power ful angiogenic factor and assume the role of inflammatory mediators in hypo xia- and chemical-induced injury in the cornea, in vivo. We developed an in vitro model of corneal organ culture to characterize the biochemical and m olecular events involved in the increased synthesis of these metabolites. T hese cultured corneas exhibit epithelial cytochrome P450 CYP-dependent 12(R )-HETE and 12(R)-HETrE synthesis as indicated by chiral analysis and by the ability of CYP enzyme inhibitors to repress their synthesis. Hypoxia great ly and selectively stimulated the synthesis of 12(R)-HETE (7-fold over cont rol normoxic conditions) and 12(R)-HETrE. The bacterial endotoxin, lipopoly saccharide, also increased the synthesis of these eicosanoids, substantiati ng the notion that this activity may function as an inflammatory pathway. T hese metabolites were detected in the culture medium by gas chromatography/ mass spectroscopy (GC/MS) analysis and their levels significantly increased in hypoxia-treated corneas, further indicating their endogenous formation in response to injury. This in vitro model provides an excellent preparatio n for studying factors regulating the synthesis of these inflammatory eicos anoids and for isolating, identifying and characterizing the CYP protein re sponsible for their synthesis.