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