May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Effects of DNA Methylation Inhibitor on RPE Transdifferentiation Induced by Transforming Growth Factor-β
Author Affiliations & Notes
  • S. He
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California
    Pathology,
  • S. Kase
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California
    Ophthalmology,
  • Z. Wang
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California
    Ophthalmology,
  • J. Xu
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California
    Ophthalmology,
  • S. J. Ryan
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California
    Ophthalmology,
  • D. R. Hinton
    Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California
    Pathology,
  • Footnotes
    Commercial Relationships  S. He, None; S. Kase, None; Z. Wang, None; J. Xu, None; S.J. Ryan, None; D.R. Hinton, None.
  • Footnotes
    Support  Supported by NIH grants EY01545, EY03040 and grants from Research to Prevent Blindness and the Arnold and Mabel Beckman Foundation
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 479. doi:https://doi.org/
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      S. He, S. Kase, Z. Wang, J. Xu, S. J. Ryan, D. R. Hinton; Effects of DNA Methylation Inhibitor on RPE Transdifferentiation Induced by Transforming Growth Factor-β. Invest. Ophthalmol. Vis. Sci. 2008;49(13):479. doi: https://doi.org/.

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

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Abstract

Purpose: : Retinal pigment epithelial (RPE) cell transdifferentiation plays a critical role in the pathogenesis of proliferative vitreoretinopathy (PVR). The mechanisms regulating RPE transdifferentiation are not fully understood. The purpose of this study was to investigate the effects of a DNA methylation inhibitor on RPE cell transdifferentiation induced by TGF-β in vitro.

Methods: : Cultured early passage human fetal RPE cells and surgically excised human PVR membranes (10 cases) were used. The expression of smooth muscle actin (SMA) was evaluated by flow cytometry and Western blot after treatment with TGF-β2 (10 ng) in the presence of DNA methylation inhibitor-5-aza-2’-deoxycytidine (5-AZA, 0.1,1,2,6 uM) for 24 hours before and during treatment with TGF-β.The regulation of SMA and fibronectin by 5-AZA in RPE and the expression of methyl-CpG- binding protein (MeCp) in human PVR membranes were analyzed using immunohistochemistry. The effects of 5-AZA on RPE cell migration was evaluated using a modified Boyden chamber assay. MTT assay was used to measure RPE cell proliferation after 5-AZA exposure.

Results: : Treatment with 5-AZA (1, 2, 6 uM) significantly inhibited expression of SMA and fibronectin induced by TGF-β in RPE. Reduced migration of RPE cells was seen after exposure to 5-AZA (0.1, 1, 2, and 6 uM, P<0.05) for 72 hours. RPE cell proliferation induced by hepatocyte growth factor (20 ng/ml) was significantly inhibited by the treatment with 5-AZA at doses of 1 uM or more (P<0.05). MeCp expression was detected in 7 of 10 human PVR membranes; MeCp was abundantly expressed in 3 of the PVR specimens examined.

Conclusions: : 5-AZA is able to downregulate expression of SMA and fibronectin and inhibits RPE cell migration and proliferation. Genomic methylation status appears to be involved in RPE transdifferentiation.

Keywords: retinal pigment epithelium • gene/expression • growth factors/growth factor receptors 
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