December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Protective Effect of Thionein Peptide in 4-Hydroxynonenal -Induced Apoptosis in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • S Choudhary
    Human Biol Chem & Genetics Univ Texas Med Branch Galveston TX
  • T Xiao
    Human Biol Chem & Genetics Univ Texas Med Branch Galveston TX
  • NH Ansari
    Human Biol Chem & Genetics Univ Texas Med Branch Galveston TX
  • Footnotes
    Commercial Relationships   S. Choudhary, None; T. Xiao, None; N.H. Ansari, None. Grant Identification: NIH Grant EY 13014-02
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 713. doi:
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      S Choudhary, T Xiao, NH Ansari; Protective Effect of Thionein Peptide in 4-Hydroxynonenal -Induced Apoptosis in Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):713.

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

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Abstract

Abstract: : Purpose:Age-related macular degeneration (ARMD), a disease involving progressive degeneration of photoreceptors and their underlying retinal pigment epithelium (RPE) is the leading cause of blindness in the developed world. Although, the clinical and pathological pictures of ARMD are well known, the molecular events initiating the disease remain elusive, and no effective therapy or prevention exists to date. Since retina, which contains high levels of polyunsaturated fatty acids (PUFAs), is vulnerable to undergo oxidative stress and the fact that in ARMD, there is an age related decrease in metallothionein in human macular retinal pigment epithelium, the present study was carried out to investigate the protective effect of thionein peptide in 4-hydroxynonenal (HNE) and H2O2 - induced apoptosis in retinal pigment epithelial (RPE) cells. Methods: Human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H2O2 and HNE. Apoptosis was monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry using JC-1 cationic dye and Hoechst staining. Since proteolysis is a requirement in apoptosis, we investigated the activation of various caspases such as caspase-1 (ICE-1), caspase-2 (Nedd-2), caspase-3 (CPP-32), caspase-8 (Mch-1) and caspase-9 by employing their specific fluorogenic substrates. Cells were pre-incubated with a broad spectrum caspase inhibitor (Z-VAD) or thionein peptide one hour before addition of H2O2 or HNE to study their protective effect on apoptosis in these cells. Results:A concentration- and time-dependent increase in HNE-induced apoptosis was observed with an LD50 of approximately 30 µM. Initiation of caspase activation preceded the DNA fragmentation. Caspase-1 and caspase-9 were not activated. Z-VAD inhibited the activation of caspases as well as apoptosis. Treatment of RPE cells with thionein peptide prior to the exposure to H2O2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis in H2O2 or HNE treated cells indicating its potential efficacy to scavenge the lipid derived aldehydes (LDA) and thereby preventing oxidative stress-induced RPE degeneration. Conclusion:It appears that preventing HNE formation by antioxidants, scavenging HNE by thionein peptide and inhibiting apoptosis by caspase inhibitors, in combination, may be a potential therapeutic strategy to prevent retinal degeneration such as in ARMD.

Keywords: 308 age-related macular degeneration • 567 retinal pigment epithelium • 341 cell death/apoptosis 
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