May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
The Effect of Glutathione Depletion in Human RPE on Expression of HGF and VEGF
Author Affiliations & Notes
  • R.E. Burton
    Doheny Eye Institute, Los Angeles, CA, United States
  • R. Kannan
    Doheny Eye Institute, Los Angeles, CA, United States
  • S. He
    Departments of Pathology and Ophthalmology, Keck School of Medicine at the University of Southern California, Los Angeles, CA, United States
  • S.J. Ryan
    Doheny Eye Institute and Department of Ophthalmology, Keck School of Medicine at the University of Southern California, Los Angeles, CA, United States
  • D.R. Hinton
    Doheny Eye Institute and the Departments of Ophthalmology and Pathology, Keck School of Medicine at the University of Southern California, Los Angeles, CA, United States
  • Footnotes
    Commercial Relationships  R.E. Burton, None; R. Kannan, None; S. He, None; S.J. Ryan, None; D.R. Hinton, None.
  • Footnotes
    Support  EY02061, EY03040, RPB, and Arnold & Mabel Beckman Foundation
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1630. doi:
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      R.E. Burton, R. Kannan, S. He, S.J. Ryan, D.R. Hinton; The Effect of Glutathione Depletion in Human RPE on Expression of HGF and VEGF . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1630.

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

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Abstract

Abstract: : Purpose: To study changes in mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor-beta2 (TGF-ß2) in human fetal retinal pigment epithelial cells (RPE) in response to oxidant stress induced by dl-buthionine-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis. Methods: RPE cells cultured in DMEM containing 1% FBS (passage 3) were treated with 500 µM BSO for 0-48h. After total RNA isolation, cDNA was synthesized for real-time polymerase chain reaction (PCR) analysis. Gene specific primers for HGF, VEGF, and TGF-ß2 were used to quantitate and compare mRNA samples from control and BSO- treated RPE. Cell viability of RPE was evaluated by Trypan Blue exclusion staining. The effect of BSO on expression of the above growth factors was determined using immunocytochemical staining. Results: BSO treatment for 6 and 48 hours caused a 2-fold and a 5-fold increase in VEGF mRNA expression from control levels in RPE, respectively. In the initial 6 hours of treatment, HGF mRNA decreased 60% followed by a return to control levels in 24 hours and a 4-fold increase in 48 hours. TGF-ß2 mRNA levels were not significantly changed by BSO treatment. Viability of RPE (93-95%) was little affected with BSO treatment for up to 48 hours. Immunochemical staining generally confirmed the pattern observed in mRNA studies. Conclusions: HGF and VEGF are differentially regulated in oxidant stress induced by GSH depletion in RPE while TGF-ß2 expression is not affected.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • growth factors/growth factor receptors 
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