This renewal application proposes to continue to test novel hypotheses about lipid mediators in corneal inflammation and remodeling after injury. It addresses the critical balance between signaling mechanisms that promote cellular damage and repair, which is fundamental for the maintenance of corneal integrity and function. The hypothesis is that while platelet-activating factor (PAF) plays a key role during excessive corneal inflammation (e.g., chemical burns) and contributes to tissue destruction and neovascularization, the12-1ipoxygenase (12-LOX) derivative, 12-(S)-hydroxyeicosatetraenoic acid (12-[S]-HETE), contributes to repair of the cornea. We propose to test four specific hypotheses: that during sustained inflammation, PAF induces impaired wound healing by (a) activation of specific transcription factors in the promoter of MMP-9 and (b) modulating apoptotic signals through the expression or silencing of PAF-R in stromal cells and that PAF increases corneal neovascularization by activating VEGF signals and MMP-2 through induction of the membrane type-1 MMP (MT1-MMP). Also, 12-(S)-HETE acts as a second messenger to promote repair and wound healing through (c) MAPK-mediated pathways and expression of cell-cycle regulatory proteins, and (d) increased induction of its enzyme, 12-LOX, that allows the transcellular synthesis of lipoxins. These compounds are suppressors of inflammation. Selective PAF-R antagonists and 12-LOX inhibitors as well as PAF-R knockout mice will be used to determine the site of action. The debridement of corneal epithelium will be employed as a model of wound healing and the corneal mouse micropocket as an in vivo model of neovascularization. Studies on activation of regions of the MMP-9 promoter will be done in transiently transfected cells. To determine the activation of transcription factors, EMSA and supershift assays will be used. Quantification of specific mRNA will be accomplished by real-time PCR. Identification of pathway components will be done by RP-HPLC, Western blot, ELISA, and immunochemistry. The results obtained will define the involvement of PAF and 12-(S)-HETE in corneal damage and repair and could lead to the development of drugs that target selective steps in the corneal inflammatory response.
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