MICROVASCULAR ENDOTHELIAL CELL ADAPTATION TO HYPOXIA IS ORGAN-SPECIFIC AND CONDITIONED BY ENVIRONMENTAL OXYGEN

S ummary Microvascular endothelial cells (MVEC) are plastic, versatile and highly responsive cells, with morphological and functional aspects that uniquely match the tissues they supply. The response of these cells to oxygen oscillations is an essential aspect of tissue homeostasis, and is finely tuned to maintain organ function during physiological and metabolic challenges. Primary MVEC from two continuous capillary networks with distinct organ microenvironments, those of the lung and brain, were pre-conditioned at normal atmospheric (∼ 21 %) and physiological (5 and 10 %) O 2 levels, and subsequently used to compare organ-specific MVEC hypoxia response. Brain MVEC preferentially stabilise HIF-2α in response to hypoxia, whereas lung MVEC primarily accumulate HIF-1α; however, this does not result in significant differences at the level of transcriptional activation of hypoxia-induced genes. Glycolytic activity is comparable between brain and lung endothelial cells, and is affected by oxygen pre-conditioning, while glucose uptake is not changed by oxygen pre-conditioning and is observed to be consistently higher in brain MVEC. Conversely, MVEC mitochondrial activity is organ-specific; brain MVEC maintain a higher relative mitochondrial spare capacity at 5% O 2 , but not following hyperoxic priming. If maintained at supra-physiological O 2 levels, both MVEC fail to respond to hypoxia, and have severely compromised and delayed induction of the glycolytic shifts required for survival, an effect which is particularly pronounced in brain MVEC. Oxygen preconditioning also differentially shapes the composition of the mitochondrial electron transport chain (ETC) in the two MVEC populations. Lung MVEC primed at physioxia have lower levels of all ETC complexes compared to hyperoxia, an effect exacerbated by hypoxia. Conversely, brain MVEC expanded in physioxia display increased complex II (SDH) activity, which is further augmented during hypoxia. SDH activity in brain MVEC primed at 21 % O 2 is ablated; upon hypoxia, this results in the accumulation of near-toxic levels of succinate in these cells. Our data suggests that, even though MVEC are primarily glycolytic, mitochondrial integrity in brain MVEC is essential for metabolic responses to hypoxia; these responses are compromised when cells are exposed to supra-physiological levels of oxygen. This work demonstrates that the study of MVEC in normal cell culture environments do not adequately represent physiological parameters found in situ , and show that the unique metabolism and function of organ-specific MVEC can be reprogrammed by external oxygen, significantly affecting the timing and degree of downstream responses. Graphical Abstract In brief Hypoxia sensing by microvascular endothelial cells (MVEC) is organ-specific, and efficacy of response is affected by external oxygen. While glycolytic capacity is mostly maintained in MVEC regardless of organ or origin, mitochondrial function is required for adequate sensing and timely metabolic shift to glycolysis. Hyperoxygenation of MVEC compromises mitochondrial function, glycolytic shift and survival to hypoxia. Highlights Environmental O 2 influences MVEC hypoxia response in an organ-specific fashion Brain MVEC are unable to respond and survive to hypoxia if hyperoxygenated prior to stress MVEC glycolytic capacity is not affected by O 2 , but the increase in glucose uptake and shift to glycolytic metabolism stifled and delayed in hyperoxidized MVEC High O 2 ablates activity of mitochondria complex II in brain MVEC, significantly disturbing succinate levels Disruption of mitochondrial integrity compromises hypoxia sensing irrespective of glycolytic capacity

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