Surgical techniques are widely used to correct for myopia, and many of these techniques, such as photorefractive keratectomy and laser in situ keratomileusis, disturb the integrity of the epithelium. Injury to the epithelium results in a subsequent loss of cells from the corneal stroma. Non-surgical conditions characterized by impaired epithelial healing include neurotrophic keratopathy, chemical burn injury, and recurrent corneal erosion syndrome. Persistent epithelial defects result in pain and significant visual impairment and sometimes, complicated by infection, corneal perforation and loss of the eye. The long term objective of the present proposal is to determine the regulation of the corneal epithelial and stromal fibroblast cell proliferation. A mediator of many intracellular signaling pathways, is protein kinase C, of which there are eleven isoforms. The first major objective is to determine the protein kinase C isoforms that are involved in regulating the proliferation of corneal epithelial and stromal fibroblast cells. The second objective is to determine the cell cycle proteins that are regulated by these specific protein kinase C isoforms. To achieve these goals, primary cultures of human corneal epithelial and stromal fibroblast cells will be used. The activity of specific protein kinase C isoforms will be inhibited by transducing the cells with adenoviruses expressing dominant-negative mutants of these isoforms. A second method of inhibiting specific isoforms of protein kinase C will be employed using antisense oligonucleotides. The proliferation, in response to growth factors, of corneal epithelial cells and corneal fibroblasts will be analyzed after the inhibition of specific protein kinase C isoforms. We will then analyze the expression of cell cycle proteins (cyclins) and cell cycle inhibitors using biochemical and molecular biology techniques.
