Hypoxia-induced reprogramming of glucose-dependent metabolic pathways maintains the stemness of human bone marrow-derived endothelial progenitor cells

Abstract: Background The benefits of hypoxia for maintaining the stemness of cultured human endothelial progenitor cells (EPCs) have previously been demonstrated but the mechanisms responsible remain unclear. There is growing evidence to suggest a role for cellular metabolism in the regulation of stem cell fate and self-renewal. This study aimed at exploring changes in glucose metabolism and roles in maintaining EPC stemness under hypoxia. Methods Extracellular flux analysis, LC-MS/MS and ¹³C tracing HPLC-QE-MS were used to establish EPC metabolic status. Then inhibitors of glucose metabolism were used to assess the impact of the dependent pathways on cell stemness. The key enzymes of glycolysis, tricarboxylic acid cycle (TCA), pentose phosphate pathway (PPP) and mitochondrial respiration were inhibited, and the cell survival rate, clone formation rate, mRNA expression of stemness markers, Nanog, Oct4, Klf4 and Sox2, and adenosine triphosphate (ATP) level were compared. Results Reprogramming of pathways concerned with glucose metabolism was found under hypoxic conditions, including increased rates of flux through glycolysis and the pentose phosphate pathway (PPP), together with decreased flux through the tricarboxylic acid (TCA) cycle and mitochondrial respiration. Use of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and the mitochondrial electron transport chain (ETC) revealed that inhibition of glycolysis or the PPP impaired cell proliferation under both normoxic and hypoxic conditions. By contrast, inhibition of pyruvate oxidation, via targeting of the TCA cycle or ETC, increased cell stemness under normoxic conditions. Moreover, promotion of pyruvate oxidation reversed the effect of hypoxia in maintaining cell stemness. Although hypoxia decreased mitochondrial ATP level, total cellular ATP level was unchanged, indicating that energy production does not play a major role in hypoxic effects. Conclusion In summary, hypoxia-induced reprogramming of pathways involved in glucose metabolism maintained EPC stemness. Artificial manipulation of cell metabolism may be a way to regulate EPC stemness, thereby facilitating in vitro cell expansion.