METALLOPROTEINASE EXPRESSION IN CORNEAL WOUNDS

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Extracellular matrix remodelling, known to be mediated by matrix metalloproteinases (MMPs) in normal and pathological conditions, may determined the visual outcome of patients undergoing excimer keratectomy. The aims of the proposed research are to obtain information about the expression in corneal tissue of MMPs and their inhibitors following excimer wounds, to determined if intrastromal epithelial migration is associated with MMP expression, and to correlate MMP levels with stromal scarring and remodelling. A. MMP expression following excimer corneal wounds: Following excimer keratectomy, histologic examination shows absence of leukocyte infiltration, and relative delay in the reformation of corneal epithelial basement membrane and adhesion structures. MMPs, held responsible for basement membrane and matrix degradation in other systems, are detectable in corneal epithelium and stroma after excimer wounds. Tissue inhibitors of metalloproteinases (TIMPs) regulate MMP activation and may guard against extracellular matrix degradation. In vivo wound-healing experiments will be performed to correlate the expression of MMPs and TIMPs with the time, size, and depth of excimer wounds using zymography and serial immunoblot assays. These data will be compared to those after manual keratectomy wounds. Immunohistochemical techniques will also be used for MMP and TIMP localization. B. MMP Expression in vitro: An organ culture wound-healing system will be used to determined if corneal cells can synthesize MMP de novo in the absence of leukocytes. In addition, effect of TIMPs and synthetic MMP inhibitors on short-term MMP expression will be determined. C. Correlation of Intrastromal Epithelial Migration with MMP Synthesis: MMP-mediated extracellular matrix degradation facilitated cell migration. We have observer, following deep annular excimer keratectomy, migration and proliferation of epithelial cells into the central corneal stroma. This phenomenon appears to be excimer-specific and may be influence by MMPs. Immunolocalization of MMP and TIMP in the cornea will be compared with that in manual annular keratectomy, migration and proliferation of epithelial cells into the central corneal stroma. This phenomenon appears to be excimer-specific and may be influence by MMPs. Immunolocalization of MMP and TIMP in the cornea will be compared with that in manual annular keratectomy wounds. In addition, the effect of topical synthetic MPP inhibitors on intrastromal epithelial migration in vivo will be studied. D. Correlation of MMP and TIMP expression with long-term stromal scarring and clearing following excimer keratectomy: The process of stromal scarring and subsequent clearing, clinically observer 1-6 months post-excimer keratectomy, may be related to MMP-2 and TIMP expression. The degree of scarring may depend on the balance of these and other proteins involved in matrix regulation. Zymographic analysis of stromal MMP will be determined before, during, and after scar formation and clearing. This will be correlated with corneal light scattering and with newly-synthesized collagen. These experiments will increase our understanding of corneal MMP expression and of corneal wound healing.