May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Suppression of TGF–ß Signaling and Myofibroblast Differentiation in Human Keratocytes Maintained by Amniotic Membrane Stromal Matrix
Author Affiliations & Notes
  • H. He
    Ocular Surface Res & Edu Foundat and TissueTech, Inc., Miami, FL
  • E.M. Espana
    Ocular Surface Res & Edu Foundat and TissueTech, Inc., Miami, FL
  • T. Kawakita
    Ocular Surface Res & Edu Foundat and TissueTech, Inc., Miami, FL
  • D. Tseng
    Ransom Everglades School, Coconut Grove, FL
  • C.–Y. Liu
    Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL
  • S.C. G. Tseng
    Ocular Surface Res & Edu Foundat and TissueTech, Inc., Miami, FL
  • Footnotes
    Commercial Relationships  H. He, TissueTech, Inc. E; E.M. Espana, TissueTech, Inc. E, P; T. Kawakita, TissueTech, Inc. E; D. Tseng, None; C. Liu, None; S.C.G. Tseng, TissueTech, Inc. I, C, P.
  • Footnotes
    Support  NIH EY06819 (to S. C. G. Tseng), NIH EY12486 (to C–Y. Liu), a research grant from TissueTech, Inc.,
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3782. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      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.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Abstract: : 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.

Keywords: cornea: stroma and keratocytes • growth factors/growth factor receptors • gene/expression 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×