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Thore Schmedt, Cecily Hamill, Yuming Chen, Ula Jurkunas; Oxidative Stress Causes Mitochondrial Dysfunction in Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1670.
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Human corneal endothelial cells (HCEnCs) form a single monolayer of hexagonal cells that are non-proliferative in vivo and enter rapid cellular senescence in vitro, rendering them of limited use in the study of endothelial cell biology. Introduction of telomerase (hTERT) has been shown to extend the life span of HCEnCs, but it is unclear whether hTERT expression protects mitochondria against oxidative stress. The purpose of this study was to investigate the effect of hTERT on mitochondrial integrity and apoptosis in response to the pro-oxidant menadione in HCEnCs.
Highly uniform subpopulations of HCEnCs that exhibited increased proliferative capacity were isolated from a 21-year-old donor (HCEnC-21), and hTERT was introduced creating the HCEnC-21T cell line. Cells were grown to confluence and treated with 25 and 100 µM of menadione sodium bisulfite for 1 hour. Non-treated cells served as controls. Cell viability was measured by Trypan blue exclusion using an automatic cell counter. Mitochondrial integrity was determined using the JC-1 dye and red-to-green fluorescence ratios to detect depolarized (damaged) mitochondria.
Mitochondrial polarization of both cell lines decreased with increasing concentrations of menadione. At baseline, the fluorescence ratios of in HCEnC-21T and HCEnC-21 were 2.63±0.41 and 3.01±0.31, respectively. At 25 µM, the ratios dropped to 1.32±0.27 in HCEnC-21T and 1.91±0.04 in HCEnC-21. This is an average decrease of 49.9% (p=0.04) in HCEnC-21T and 36.5% (p=0.014) in HCEnC-21. Cell viability tended to decrease, but was not significantly lower after treatment with 25 µM. At 100 µM, the fluorescence ratios were found to be 0.73±0.07 in HCEnC-21T and 0.76±0.09 in HCEnC-21. This corresponds to an average decrease of 72.3% (p<0.001) in HCEnC-21T and 74.6% (p<0.001) in HCEnC-21. Similarly, cell viability was reduced by 26.2% (p=0.0014) in HCEnC-21T and 14.9% (p=0.01) in HCEnC-21. There were no significant differences between HCEnC-21T and HCEnC-21 at any dosage.
Oxidative stress induced loss of mitochondrial integrity in a dose-dependent manner, which preceded cell death in both HCEnC-21T and HCEnC-21. Telomerase overexpression did not augment resistance to the pro-oxidant in HCEnC-21T as opposed to HCEnC-21. Both cell lines provide suitable models for the study of HCEnC biology and endothelial disease pathogenesis.
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