This is a proposal to test the hypothesis that injury to the cornea increases the activity of an epithelial cytochrome P450 (CYP) isozyme(s) which metabolizes arachidonic acid (AA) to 12(R)-hydroxy-5,8,10,14- eicosatetraenoic acid (12(R)-HETE) and 12(R)-hydroxy-5,8,14-eicosatrienoic acid (12(R)-HETrE) and that 12(R)-HETrE acts directly on the adjacent limbal vessel endothelial cells to promote neovascularization of the cornea. The following findings form the basis of our hypothesis: 1) Corneal epithelium from several species, including human, possesses a CYP monooxygenase(s) capable of metabolizing AA stereospecifically to 12(R)- HETE and 12(R)-HETrE; 2) injury to the corneal epithelium via closed eye- contact lens wear results in the time-dependent formation of CYP-AA metabolites which correlates strongly with the in situ inflammatory response; 3) inhibition of CYP-AA metabolism in this model dramatically reduces in situ inflammatory response indicating a cause-effect relationship between CYP-AA metabolism and inflammation; 4) 12(R)-HETrE, possesses potent biological activities in vitro and in vivo indicative of a pro-inflammatory factor (e.g., vasodilation, increased capillary permeability, neutrophil chemotaxis and angiogenesis); and 5) the amount of 12(R)HETrE produced by the injured corneal epithelium is sufficient for the expression of its proinflammatory properties implicating it as a major pathophysiological mediator of such responses in the eye.
The specific aims fall into two research areas: (A) The biochemical and molecular identification of the CYP-AA metabolizing enzyme(s) in the corneal epithelium under control and inflamed conditions. In achieving this goal, CYP enzymatic activity and endogenous levels of metabolites will be assessed in normal and injured corneas under CYP-induced/suppressed conditions. This type of characterization will provide the basis for comparison with the next studies in which the protein and mRNA levels of several CYP isoforms will be measured under the same conditions. The results derived from both studies should provide substantial information with regard to the isoform(s) whose expression (protein and mRNA levels) correlates to CYP-AA activity and metabolite levels following injury. We will then proceed with the molecular cloning of this isoform. (B)The elucidation of the cellular and molecular mechanisms underlying the pro- inflammatory properties of 12(R)-HETrE, in particular, its angiogenic activity. This will include characterization of its cellular receptor in limbal endothelial cells and signaling pathways including activation of transcriptional factors, early immediate genes and genes that are crucial for the process of angiogenesis. This will allow us to fully understand the pathophysiologic ramifications of this pathway and its metabolite and may offer insight into the interplay between the corneal epithelium and the surrounding limbal microvasculature following corneal epithelial injury. Understanding the role of this new player in the pathogenesis of corneal inflammatory reaction and neovascularization will permit the development of therapeutics targeted at inhibiting the synthesis of a pro- inflammatory mediator (metabolic inhibitors) as well as preventing its action (receptor/functional antagonists) for the treatment of inflammation associated with corneal injury, infection and surgery.
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