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Christian F Mueller, Sangita P Patel; Metabolism of primary human corneal endothelial cell (HCEnC) cultures grown at ambient and 2.5% oxygen. Invest. Ophthalmol. Vis. Sci. 2021;62(8):803.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress is central to the pathophysiology of Fuchs endothelial corneal dystrophy. Typically, HCEnC cultures are grown in 5% CO2, room air incubators ([O2]A). However, [O2] in the anterior chamber is approximately 2.5% ([O2]2.5). The purpose of our study was to investigate in vitro metabolism of HCEnC grown at [O2]A and [O2]2.5. We hypothesized that cells grown at [O2]2.5 would have greater glycolytic activity and decreased oxidative respiration compared to cells grown at [O2]A.
Protocols were approved by the Univ at Buffalo and VA WNY IRBs. Donor eyes (n=5 [O2]A and n=6 [O2]2.5, mean age 74) were obtained within 24 hrs of death for initiating HCEnC cultures. [O2]A cells were grown in a humidified, 5% CO2, 37°C incubator. [O2]2.5 was created in a humidified chamber (Billups-Rothenberg, Inc., Del Mar, CA) with 2.5% O2, 5% CO2, balance N2 at 37°C. Mitochondrial respiration (O2 consumption rate [OCR]) and glycolysis (extracellular acidification rate [ECAR]) were measured with the Seahorse XFe24 (Agilent Technologies, Inc., Santa Clara, CA). OCR and ECAR measurements were performed at baseline (B) and following additions of oligomycin (O), FCCP, rotenone/antimycin A (RA) and 2DG. We calculated basal respiration (B-RA for OCR and B-2DG for ECAR), ATP-linked respiration (B-O), proton leak (O-RA), max respiration (FCCP-RA), spare capacity (FCCP-B), non-mitochondrial O2 consumption (RA), and glycolytic reserve capacity (O-B). Mean values were compared between [O2]A and [O2]2.5 using unpaired two-tailed t-tests with significance at p<0.05.
OCR analysis showed significant differences between [O2]A and [O2]2.5 in proton leak, spare capacity, and non-mitochondrial O2 consumption (Table). There were no significant differences between groups for basal respiration, ATP linked respiration, and max respiration. ECAR analysis showed significant differences between [O2]A and [O2]2.5 in basal glycolysis and glycolytic reserve capacity.
Our results demonstrate that in HCEnC, glycolytic activity is greater but ATP-linked respiration the same at physiologic [O2]2.5 compared to [O2]A. Increased proton leak and non-mitochondrial O2 consumption at [O2]2.5 compared [O2]A suggest alternative metabolic pathways are prominent under physiologic conditions. [O2] is an important consideration in HCEnC metabolism and FECD pathophysiology.
This is a 2021 ARVO Annual Meeting abstract.
OCR and ECAR data for [O2]A and [O2]2.5 groups
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