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J. He, H.E. P. Bazan; Requirement of Epidermal Growth Factor (EGF) in Corneal Keratocyte Proliferation and Differentiation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5040.
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During corneal stromal wound healing, the quiescent keratocytes at the wound periphery are metabolically activated and differentiate to fibroblasts and/or myofibroblasts. They produce a fibrotic extracellular matrix (ECM) important to close the wound. Early studies have shown that topical application of EGF significantly increases the tensile strength of stromal wound in vivo, but little is known about the cellular and molecular mechanisms by which EGF exerts this action. Here, we investigated the action of EGF on proliferation, differentiation, and expression of ECM components in corneal keratocytes and the involvement of the signaling pathways activated by the EGF receptor.
Keratocytes isolated from rabbit corneas were cultured in serum–free DMEM/F12 containing antibiotics. Five– to seven–day cultures were treated with EGF, TGF–ß1 or EGF plus TGF–ß1 in the presence or absence of inhibitors of EGF–receptor and of signaling kinases including AG1478, LY294002, PD98059 and SB203580 for 2 days to 1 week. Immunofluorescence was performed by using monoclonal anti–CD34 and α–SMA antibodies to determine the keratocyte differentiation status. Cell proliferation was evaluated with Ki–67 antibody. ECM components including keratan sulfate, chondroitin sulfate and fibronectin were assayed by immunochemistry and Western Blotting.
Addition of EGF (50ng/mL) to keratocytes induced 100% transformation into proto–myofibroblasts, with the loss of dendritic shape and expression of α–SMA, chondroitin sulfate and fibronectin. EGF also induced significant cell proliferation during the first 2 days that decreased by day 7. Treatment with TGF–ß1 (10ng/mL) for 1 week induced the cells to transform into proto–myofibroblasts (88%) and into differentiated–myofibroblasts (12%). However, TGF–ß1 plus EGF induced 90% of the cells to transform into differentiated–myofibroblasts, with significant increase in the expression of chondroitin sulfate and fibronectin. AG1478 not only blocked the effect of EGF on keratocyte proliferation but also prevented the effect of TGF–ß1 on keratocyte differentiation. Similar inhibitory effects were observed in the presence of LY294002, but not in the presence of PD98059 or SB203580.
EGF through activation of its receptor and a PI–3K pathway may have an important role in proliferation, differentiation, and ECM expression of corneal keratocytes. Hence, this process contributes to the acceleration of wound closure and scar tissue formation during stromal wound healing.
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