CHARACTERIZATION OF CORNEAL CELL-SURFACE ANTIGENS

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Injury or disorders of the cornea, including corneal dystrophies, are common causes of impaired vision and eye discomfort. The interactions between the cell surface, cytoskeleton and the extracellular matrix (ECM) are important during wound healing. An insight into the mechanisms of these interactions will lead to devising better treatments for corneal problems. Using hybridoma techniques, three cytoskeletal associated proteins (CAPs) were found to be involved in the activation of quiescent corneal stromal cells from GO to Gl to S phase during wound healing. The proposed study will evaluate the functional of these CAPs (one associated with actin and two with vimentin filaments) in corneal wound healing. The mechanisms of interactions of the CAPs with actin or vimentin will be analyzed by using purified proteins in vitro and by studying the pattern of their distribution in cells using immunoelectron microscopy. Their possible functions during GO to Gl to S transition, will be studied in vitro using a tissue culture model of wound healing. The effects of single cell microinjections of these CAPs and their monoclonal antibodies on the cellular behavior, cytoskeletal organization and cellular functions will be evaluated. Recombinant DNA technology will be used to develop cDNA clones containing sequences complementary to the mRNAs for these CAPs and to develop DNA and RNA probes. These probes will be employed to study the regulation of the gene expression of theses CAPs during would healing, using nucleic acid hybridization techniques. From previous studies, a basement membrane and cell surface associated unique protein in corneal epithelial cells has been identified. This protein is postulated to be involved in cell attachment and migration during wound healing. The mechanisms of its involvement in cell-cell and cell-ECM will be studied by analyzing its interactions with laminin, heparan sulfate proteoglycan, type IV collagen and fibronectin. The regulatory changes in the expression of this protein during epithelial healing will be studied in tissue culture models of wound healing in vitro and in rabbit in vivo. Recombinant DNA techniques will be used for this study as described for CAPs. The third aspect of this study will characterize two corneal epithelial cell surface differentiation antigens which are associated with corneal epithelial healing problems (recurrent corneal epithelial erosion and incomplete transdifferentiation of conjunctivally derived cells to corneal epithelial cells). The effect of growth factors on the expression of these antigens in cultured cells and their biochemical characteristics will be carried out.