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H. He, E.M. Espana, T. Kawakita, D. Tseng, C.–Y. Liu, S.C. G. Tseng; Suppression of TGF–ß Signaling and Myofibroblast Differentiation in Human Keratocytes Maintained by Amniotic Membrane Stromal Matrix . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3782.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Recently, we showed that amniotic membrane (AM) stroma can, while plastic cannot, preserve the keratocyte phenotype as defined by keratocan expression in a serum–containing medium (Espana EM. et.al., IOVS, 2003; 44: 5136–5141). We hypothesize that suppression of TGF–ß signaling is involved in maintaining such keratocyte phenotype, and would like to investigate whether myofibroblasts induced by TGF–ß on plastic can be reversed by subculturing them on AM. Methods: Human keratocytes isolated by collagenase were cultured on plastic or AM in DMEM plus 10% FBS as described (Espana EM. et.al., IOVS, 2003; 44: 5136–5141). Keratocytes maintained by AM were subcultured on plastic or AM, and cells containing myofibroblasts on plastic were also subcultured on AM or plastic. Cells cultured on AM or plastic in DMEM with 1% FBS were added with or without 10 ng/ml TGF–ß1. Immunostaining and western blotting were performed with antibodies against α–smooth muscle actin (α–SMA) for myofibroblast differentiation. Total RNAs were extracted for RT–PCR to measure TGF–ß1, 2, 3 and receptor II transcripts. Results: In 10% FBS, immunostaining and western blot consistently showed that cells continuously subcultured on AM up to P3 did not express α–SMA. In contrast, cells subcultured on plastic (P2 or P3) expressed α–SMA. Cells cultured on AM started expressing α–SMA when subcultured on plastic, however, cells expressing α–SMA on plastic at P2 lost the expression of α–SMA when subcultured on AM. Addition of TGF–ß1 markedly upregulated α–SMA expression on plastic, but failed to do so on AM. Compared to the plastic control, AM suppressed the expression of TGF–ß2 (2.4 fold), TGF–ß3 (2 fold) and TGF–ß receptor II (1.6 fold) transcripts, but upregulated that of TGF–ß1 (3.4 fold) transcript. Conclusions: AM suppresses myofibroblast differentiation in human keratocytes even after exposure to serum or exogenous TGF–ß1 and reverses myofibroblast phenotype promoted by plastic cultures in a serum–containing medium. These findings, correlating well with AM’s suppression of TGF–ß signaling, explain why amniotic membrane transplantation exerts an anti–scarring effect in vivo.
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