July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Regulation of oxidative stress in corneal endothelial cells by PRDX-6.
Author Affiliations & Notes
  • Gary S L Peh
    Ocular Tissue Eng & Stem Cell Group, Singapore Eye Research Institute, Singapore, Singapore
    Eye-ACP, Duke-NUS Graduate Medical School, Singapore, Singapore
  • Matt Lovatt
    Ocular Tissue Eng & Stem Cell Group, Singapore Eye Research Institute, Singapore, Singapore
    Eye-ACP, Duke-NUS Graduate Medical School, Singapore, Singapore
  • Khadijah Adnan
    Ocular Tissue Eng & Stem Cell Group, Singapore Eye Research Institute, Singapore, Singapore
  • Jodhbir S Mehta
    Ocular Tissue Eng & Stem Cell Group, Singapore Eye Research Institute, Singapore, Singapore
    Singapore National Eye Center, SINGAPORE, Singapore
  • Footnotes
    Commercial Relationships   Gary Peh, None; Matt Lovatt, None; Khadijah Adnan, None; Jodhbir Mehta, None
  • Footnotes
    Support  National Research Foundation Translational and Clinical Research (TCR) Programme (NMRC/TCR/008-SERI/2013)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5108. doi:https://doi.org/
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    • Get Citation

      Gary S L Peh, Matt Lovatt, Khadijah Adnan, Jodhbir S Mehta; Regulation of oxidative stress in corneal endothelial cells by PRDX-6.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5108. doi: https://doi.org/.

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

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Abstract

Purpose : To evaluate the role and characterize the function of PRDX-6 in human corneal endothelial cells (HCEnCs).

Methods : Primary HCEnCs were established from cadaveric corneal tissue unsuitable for transplantation, procured through Lions Eye Institute for Transplant and Research. Isolated HCEnCs were expanded using a dual media approach until the second or third passage in order to obtain sufficient cell numbers for experiments. Other cell types used in this study includes corneal stromal fibroblasts, human retinal microvascular endothelial cells (RMECs), SV40 transformed human corneal endothelial cell line (B4G12), human lung adenocarcinoma cell line (A549), human embryonic kidney cells (HEK293T), and spontaneously arising retinal pigmented epithelial cell line (ARPE19). To address the role of PRDX-6 in HCEnCs, we first characterized the expression of PRDX-6 on HCEnCs by cell fractionation and western blotting. This was followed by a combination of biochemical studies including RNAi knockdown of PRDX-6, and Click-iT lipid peroxidation assay to evaluate its role in cells. Following treatment, cellular viability were assessed via flow cytometry, xCELLigence, and mitochondrial membrane potential.

Results : Our data suggests that PRDX-6 is expressed at unusually high levels at the plasma membrane of HCEnCs. Western blot analysis of the cell surface fraction form HCEnCs treated with tert-butyl hydroperoxide suggested that PRDX-6 levels were diminished in HCEnCs under oxidative stress. RNAi mediated knockdown of PRDX-6 revealed a role for PRDX-6 in lipid peroxidation. Furthermore, in response to oxidative stress induced by menadione, PRDX-6 deficient cells had defective mitochondrial membrane potential and were more sensitive to cell death.

Conclusions : Our data suggest that in HCEnCs, PRDX-6 is compartmentalized and has multiple functions to preserve cellular integrity.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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