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Kohsaku Numa, Morio Ueno, Munetoyo Toda, Atsushi Mukai, Kazuko Asada, Asako Uehara, Chie Sotozono, Shigeru Kinoshita, Jyunji Hamuro; Mitochondria as platforms for dictating differentiation/maturation of human corneal endothelial cells with distinct energy metabolism and metabolomic signatures. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3450.
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© ARVO (1962-2015); The Authors (2016-present)
Cultured human corneal endothelial cells (cHCECs) are composed of subpopulations (SPs) that are heterogeneous in their differentiation and maturation status. The purpose of this study was to clarify the existence of a distinctive energy metabolism in terms of the dysfunction of cHCECs.
cHCEC cell extracts or corresponding culture supernatants were obtained for metabolome analysis using a capillary electrophoresis (CE)-connected mass-spectrometry (MS) CE-MS/MS system. Mitochondrial respiration was assessed by use of the Seahorse XF Mito Stress Test Kit (Agilent Technologies, Santa Clara, CA) to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) and the metabolic fuel properties; glucose (Glc), glutamine (Gln), fatty acids (FA). The cells' dependency on, and flexibility for, using each of the three fuel sources was assessed by measuring the decrease in fuel oxidation upon the addition of one or more inhibitors.
Hierarchical clustering of metabolomic profiles of the culture media identified clusters of metabolites that correlated with the differentiation/maturation of cHCECs and the presence of cell-state transition (CST). SPs with CST exhibited a disposition toward anabolic glycolysis, instead of mitochondria-dependent oxidative phosphorylation. Basal and maximum respiration was significantly reduced in SPs with CST (baseline OCR; mature SPs 134.2±8.9 / CST-SPs 89.3±3.8, maximum OCR; 371.9±28.3 and 178.7±11.8 pmol/min, respectively) (P<0.01). In mature cHCEC SPs sharing the common phenotype with fresh-tissue HCECs, the mean % fuel dependency and fuel flexibility of Glc, Gln, and FA was 34.1±6.1%, 26.6±8.1%, and 0%, and 36.5±14.6%, 2.2±11.9%, and 82.0±14.2%, respectively. In CST-SPs, the former was 26.6±8.6%, 13.8±7.8%, and 30.1±16.3%, and the latter was 58.29±21.6%, 10.9±10.7%, and 69.0±16.3%, respectively. Mature SPs barely used FA-based respiration, whereas CST-SPs showed FA-use potential (P<0.01), depending on the nutrients in microenvironments.
The metabolic state of cHCECs and nutrients availability possibly dictate HCEC differentiation/maturation, implying the pivotal roles of mitochondrial respiration and energy metabolism in the pathogenesis of functionally impaired HCECs in bullous keratopathy and Fuchs corneal dystrophy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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