The long range goal of this research is to elucidate cellular and biochemical bases of corneal transparency based on the hypothesis that stromal proteoglycans play a central role in corneal transparency and its loss during scarring. Confirmation of this hypothesis demands understanding (a) how proteoglycans are changed in scars, (b) the biological stimuli that induce these changes, and (c) functional roles of normal and abnormal proteoglycans in the cornea. The proposed research will address these 3 questions using 4 Specific Aims. (1) Characterize changes in proteoglycan expression and molecular basis for these changes in an in vitro model of stromal wound healing. Recent studies with cultured keratocytes have shown it possible to reproduce in vitro phenotypes of cells in healing wounds.
The first Aim will examine proteoglycan protein and mRNA expression in keratocytes exhibiting differentiated, remodeling, and myofibroblastic phenotypes. Glycosyltransferase and sulfotransferase enzymes will also be examined. (2) Elucidate the signals determining keratocyte phenotype. A model of the wound healing process was formulated predicting roles for cytokines and extracellular matrix in the phenotypic transition of keratocytes during wound healing. This model will be tested using exogenous cytokines, antibodies to cytokines, and transient overexpression of growth factors and intergrins in cultured keratocytes. (3) Define the involvement of specific proteoglycans in the cellular events of wound healing. It is hypothesized that corneal proteoglycans and hyaluronan interact directly with corneal cells, influencing the biology of wound healing. To test this hypothesis, first, the recently discovered receptor for the proteoglycan lumican will be purified and characterized as to its function. Secondly, induction of keratocyte movement by biglycan and hyaluronan (molecules upregulated in corneal wound healing) will be examined. Participation of the CD44 hyaluronan receptor in keratocyte motility will be examined. (4) Characterize the mechanism by which proteoglycans affect matrix self assembly. To achieve this aim native recombinant proteoglycan proteins and recombinant protein fragments will be employed to map sites within the proteoglycan proteins involved in collagen binding and collagen fibrillogenesis. These experiments will increase our knowledge of the molecular basis of corneal transparency, presenting information that may be valuable in formulating avenues of therapeutic intervention in corneal scarring.
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