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K. Xu, Y. Ding, F.X. Yu; Requirement of HB-EGF Ectodomain Shedding for Corneal Epithelial Wound Healing . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3814.
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Purpose: Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)-ligand interactions. The study sought to identify endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells. Methods: Using a porcine corneal organ culture model, epithelial debridement wounds of 4 mm in diameter were made and allowed to heal for up to two days ex vivo. In a cell culture model, a wound was created by scraping an epithelial monolayer of SV40 adenovirus transformed human corneal epithelial (THCE) cells and allowed to heal for 24 h. To determine the role of heparin binding-EGF like growth factor (HB-EGF) shedding in epithelial migration, wounded corneas or cell monolayer were allowed to heal in the presence of lysophosphatidic acid (LPA), tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a non-selective matrix metalloproteinase, MMP, inhibitor) or CRM197 (a non-toxic diphtheria toxin mutant) with or without HB-EGF. Activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies and Western blotting with phosphotyrosine antibody. Results: In both organ and cell culture models, wounded epithelial cells migrated as a sheet to cover the wound. Addition of human recombinant HB-EGF accelerated the rate of corneal epithelial wound closure. LPA accelerated wound closure is inhibited by EGFR inhibitor AG1478, HB-EGF inhibitor CRM197 and MMP inhibitor GM6001. LPA prolonged EGFR phosphorylation that induced by wound. Tyrphostin AG1478 inhibited cell migration with or without HB-EGF. GM6001 inhibited epithelial migration and delayed wound closure; its effects diminished in the presence of exogenous HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. Treatment of wounded porcine corneas or THCE cells with CRM197 resulted in a reduced rate of epithelial migration, which can be overcome by the addition of a high concentration of exogenous HB-EGF. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR tyrosine phosphorylation in cultured THCE cells. Conclusions: HB-EGF released upon wounding acts as an auto/paracrine EGFR ligand. LPA induces EGFR transactivation via induction of shedding of HB-EGF. Shedding of HB-EGF represents a critical event during corneal epithelial migration and wound healing, suggesting a possible manipulation of wound healing at early phases.
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