April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Redox Regulation of Low Molecular Weight PTP in Retinal Endothelial Cells
Author Affiliations & Notes
  • M. A. Abdelsaid
    Clinical and Experimental Therapeutics, University of Georgia, Augusta, Georgia
  • A. B. El-Remessy
    Clinical and Experimental Therapeutics, University of Georgia, Augusta, Georgia
  • Footnotes
    Commercial Relationships  M.A. Abdelsaid, None; A.B. El-Remessy, None.
  • Footnotes
    Support  SDG from AHA and CDA from JDRF
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1435. doi:
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      M. A. Abdelsaid, A. B. El-Remessy; Redox Regulation of Low Molecular Weight PTP in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1435.

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Abstract

Purpose: : Over-expression of the angiogenic vascular endothelial growth factor (VEGF) plays a key role in retinal neovascularization. Our previous studies in retinal endothelial cells have shown that physiological low levels of peroxynitrite (PN) transduces VEGF’s angiogenic signal. Focal adhesion assembly is regulated by the balance between activation of focal adhesion kinase (FAK) and its specific phosphatase, low molecular weight protein tyrosine phosphatase (LMW-PTP). The activity of LMW-PTP is redox regulated via cysteine oxidation of its active site. The purpose of this study is to test the hypothesis that PN regulate VEGF-induced FAK activation via altering cellular redox state of LMW-PTP.

Methods: : Human and bovine retinal endothelial cells were treated with VEGF (20 ng/ml) or PN (1,100 or 500 µM). Cellular redox state was determined by GSH levels using DTNB-assay. Phosphatase activity was measured using fluorometric assay. Western blot is used to determine phosphorylation of FAK and LMW-PTP. Thiol oxidation of LMW-PTP was assessed in 5-IAF labeled cell lysate.

Results: : VEGF causes transient oxidation of GSH levels and FAK activation that was comparable with the low levels of PN (1-100 µM). High levels of PN (500 µM) caused permanent oxidation of GSH levels. PN caused a dose dependant reduction of the LMW-PTP phosphatase activity. Decomposing peroxynitrite with FeTPPS (2.5 µM) prevented LMW-PTP thiol oxidation.

Conclusions: : VEGF-induced PN formation transiently alters the redox state of endothelial cells to inactivate LMW-PTP via thiol oxidation and hence activate FAK. Treatments that target PN formation and thiol oxidation should be considered for anti-angiogenic therapy.

Keywords: retinal neovascularization • vascular endothelial growth factor • oxidation/oxidative or free radical damage 
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