Corneal Metalloproteinase-Matrix Interactions

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DESCRIPTION (provided by applicant): Loss of corneal clarity is a common endpoint of many blinding conditions. Corneal opacification is a major cause of blindness worldwide. Our laboratory is studying the mechanisms associated with corneal wound repair that regulate coneal clarity. Specifically, we have identified an endogenous matrix metalloproteinase within the cornea which helps prevent corneal opcification after an excimer laser wound. Matrilysin (MMP-7) deficient mice have exuberant scarring following laser injuries which are innocuous to their wild-type counterparts. In this application we will investigate the interaction of MMP-7 with extracelluar matrix (ECM) components localized to native and wounded corneas. Specific Aim A will use a combination of immunohistochemistry, western blotting, zymography, and transgenic animals to localize the sites and quantify the amount of MMP-7 expressed in wounded and unwounded corneas. Specific Aim B extends these studies to determine the matrix substrate specificities of corneal epithelium- and keratocyte-derived MMP-7 in vitro. In this aim we will examine corneal celI-ECM interactions by assaying the migration of epithelium on 2-dimensional matrix gels, and the proliferation of keratocytes in 3-dimensional matrix gels. Specific Aim C will translate the in vitro results to MMP-7 knockout mice. The expression of xtracellular matrix components in wild-type and matrilysin knockout mice after excimer laser wounding will be evaluated by immunohistochemistry, western blotting and real-time PCR. The second portion of Aim C will carefully examine the MMP-7 dependent breakdown products of corneal extracellular matrix in these mice. Lastly, using MMP-7/ECM double knockout mice, the requirement of specific extracellular matrix components for reduced corneal clarity will be investigated. Our studies of protease-matrix interactions in wounded corneas will further our understanding of the mechanisms for maintaining corneal clarity. Results of these studies in the cornea may ultimately be helpful in understanding healing in other organs as well.