Proper healing of corneal wounds is vital to maintaining a clear, healthy cornea and preserving vision. Our long-term goal is to understand the mechanisms involved in corneal epithelial wound repair and how the stromal substrate affects these mechanisms. In the last four years, a component of focal contacts; alpha-enolase, a glycolytic enzyme; and epidermal growth factor (EGF) receptor were found in higher concentrations in the epithelium during wound repair. Immunohistochemically, vinculin localized as punctate spots along the cell-substrate border during migration, and alpha-enolase and EGF receptor localized to the corneal epithelial stem cells. These data suggest three mechanisms involved in wound repair: 1) focal-contact formation, 2) glycolysis, and 3) stem-cell mitosis. Synthesis of vinculin, alpha-enolase, and EGF receptor are known to be stimulated by EGF and/or fibroblast growth factor (FGF), suggesting that growth factors may regulate these three mechanisms.
The specific aims of the current application are to determine if members of the EGF or FGF families regulate these three mechanisms; if glycolytic activity is required for DNA synthesis; and if levels of EGF or FGF families are altered in response to wounding. To determine if EGF or FGF regulate focal-contact formation, Western blotting will be used to determine levels of EGF receptor, FGF receptor and vinculin during epithelial migration and if these proteins are phosphorylated. Vinculin mRNA levels will be assayed with Northern blots. To determine if EGF or FGF regulate glycolysis during wound healing, Western and Northern blots will be used to determine if EGF or FGF stimulates changes in alpha- enolase protein and mRNA levels. Spectrophotometric assays will determine if enzymatic activities of alpha-enolase, aldolase, and phosphofructokinase are altered during migration and in response to EGF or FGF. To determine if growth factors stimulate stem-cell mitosis, mitotic rates will be assayed by [3h]-thymidine incorporation, and Western blotting will be used to assay levels of phosphorylation of EGF and FGF receptors, following application of EGF or FGF to healing corneas. To determine if glycolytic activity is required for corneal epithelial DNA synthesis, EGF and FGF will be used to stimulate the entry of epithelial cells in culture into the cell cycle. The following will be assayed: receptor phosphorylation, alpha-enolase protein and mRNA, activity of glycolytic enzymes and DNA synthesis. To determine if levels of EGF or FGF are altered during wound repair, EGF and FGF in epithelium, stroma, tear fluid and plasma will be determined using radioimmunoassays, heparin-affinity chromatography, Western blotting and in situ hybridization. The proposed studies will determine if EGF and FGF are involved in wound repair, and the timing of their involvement. These data may be useful in developing therapeutic uses of growth factors to enhance wound repair.
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