There is a need for effective treatments which enhance healing of corneal wounds caused by surgery, trauma or disease. Our long-term goals are to determine the roles peptide growth factors and their receptors play in spontaneous corneal wound healing and develop treatments based on this knowledge to enhance this process. Our hypothesis is that peptide growth factors and their receptors regulate key aspects of corneal wound healing by the autocrine and paracrine mechanisms. We will investigate and further enlarge this concept by focusing on three aspects of corneal wound healing: (1) determine the profiles of growth factors and their receptors during spontaneous healing of corneal wounds; (2) evaluate a new synthetic inhibitor of matrix metalloproteinases (SIMMP) for treatment of alkali and bacterial induced corneal ulcers; (3) synthesize a chimeric fusion protein consisting of EGF and the collagen binding domain of fibronectin, and measure the biochemical properties and effect on corneal wound healing. For the first project, we will focus on the changes which may occur during corneal wound healing in the TGF-alpha, TGF-beta, and FGF systems. We recently reported that corneal epithelial and endothelial cells synthesize TGF-alpha mRNA and that levels of TGF-alpha protein in aqueous fluid of cats increased 12-fold after endothelial injury. We will determine if changes occur in the levels of TGF-alpha mRNA of epithelial and endothelial cells during healing of wounds in organ cultured human and bovine corneas by quantitative in situ hybridization with 32P-labeled TGF-alpha sense and antisense RNA probes. Changes in TGF-alpha protein levels epithelial and endothelial will be assessed by immunohistochemistry of specimens using a specific TGF-alpha antibody we have generated, and changes in TGF-alpha receptor levels will be determined by quantitative autoradiography 125I-TGF-alpha binding to sections of wounded corneas. We will also determine if addition of a TGF-alpha neutralizing antibody will inhibit corneal wound healing in vitro. In the second part of this project, we will extend our recent finding that topical treatment with SIMMP immediately after severe alkali injury completely blocked corneal ulceration. We will determine if treatment with SIMMP can halt ulceration of alkali injured eyes once ulceration has begun, and we will evaluate SIMMP in the treatment of corneal ulcers induced by Pseudomonas or Staphylococcus. We will determine the effect of SIMMP on chemotactic and phagocytic responses of inflammatory cells and measure pharmacokinetic parameters of 14C labeled SIMMP. In the third project, we will address a major limitation of growth factor treatment of cornea wounds: rapid loss of growth factors from a wound site To overcome this problem, we will synthesize a chimeric protein consisting of the human EGF gene coupled with the human collagen binding domain (EGF-FnCol). We will measure biochemical properties of the EGF-FnCol fusion protein including EGF receptor binding, mitogenic activity, chemotactic activity, and reversible binding to collagen. Biological effects of the chimera fusion protein will be evaluated in epithelial and endothelial wound healing models.
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