The long-range goal of this project is to understand the molecular, supramolecular and cellular basis of corneal wound healing following injury and refractive surgery, including photorefractive keratectomy (PRK) and laser assisted in-situ keratomileusis (LASIK). It is well recognized that the major complications of PRK and LASIK are refractive regression, and the development of corneal haze after PRK causing significant loss of visual acuity in tens of thousands of the millions of patients undergoing this surgery annually world wide. Progress studying the cellular mechanisms of corneal wound healing and PRK during the last funding cycle strongly suggest that TGF-beta-1 plays a major role in modulating the keratocyte to myofibroblast phenotype, which ultimately controls corneal haze and refractive regression. In order to test this overall hypothesis we have developed novel and unique experimental techniques to assess the effects of in vivo and in vitro myofibroblast differentiation. We have developed an excimer laser phototherapeutic keratectomy (PTK) model in mice, a serum-free culture system and a human telomerase infected keratocyte cell line, and a biophysical model of matrix organization and wound contraction that allows quantitative measurement of mechanical forces exerted by cells. Using these techniques we propose the following specific experimental aims. (1) Determine the in vivo, ex vivo and in vitro phenotypic characteristics of corneal keratocytes from (i) TGF-beta-1 over-expressing and (ii) TGF-beta-1 knockout transgenic mice before and after excimer laser PTK. (2) Evaluate and identify specific PDGF protein and receptor expression in human and rabbit keratocytes cultured under the following conditions: i) TGF-beta compared to PDGF alone, ii) TGF-beta compared to PDGF in combination with RGD peptides, and iii) TGF compared to PDGF in combination with other inhibitors, e.g. decorin, TGF-beta-3, and FGF2. (3) Determine the mechanical force transduction, actin filament assembly, orientation and matrix organization associated with interconnected human and rabbit corneal keratocytes exposed to: i) serum/serum-free, ii) inhibitors of gap junction communication (e.g. oleamide), iii) stimulation with growth factors (TGF-beta, PDGF, FGF, TGF-beta-3, decorin). And (4) Test the therapeutic effects of specific blockers of TGF-beta-1-mediated keratocyte differentiation on the development of corneal haze following PRK in rabbit eyes using confocal microscopy, including, a) RGD peptides, b) decorin, c) TGF-beta-3 and d) anti-FGF2 blocking antibodies.
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