Promotion Of NAD~+ Recycling By The Hypoxia-induced Shift In The Lactate Dehydrogenase Isozyme Profile Reduces The Senescence Of Human Bone Marrow-derived Endothelial Progenitor Cells

Background:Human bone marrow-derived endothelial progenitor cells(EPCs)serve to repair blood vessels and show therapeutic potential in various ischemic diseases.Short of resource,EPCs expanded in vitro to gain relevant cell numbers for therapeutic applications has to face with growing senescence under traditional normoxic culture.We found hypoxia was the optimized condition to attenuate cell senescence.In this study,we demonstrated the metabolic differences between normoxic and hypoxic EPCs and further emphasized the role of NAD+recycling in attenuating senescence,providing a basis for future study on the mechanism of hypoxia and optimizing in vitro culture condition.Object:Explore the phenotype and metabolic differences of EPCs under normoxia or hypoxia,interfere the expression and activity of LDHs to modulate NAD+recycling and detect the cell survival and senescent performance.Method:EPCs were isolated from human bone marrow and cultured under normoxia or hypoxia for identification and tube forming analysis.Cells were then used for assessment of proliferative and senescent performance,energy metabolic level,organic acid and NAD+concentration,LDH isozyme profile,LDH activity and mitophagy.LDHA and/or LDHB was knocked down under hypoxia and cultured for further detection of knocking-down effectiveness,LDH isozyme profile alternation,organic acid and NAD+concentration,mitophagy,cell proliferative and senescent performance.Result:Isolated cells presented endothelial cell characters,expressed specific surface markers and performed tube forming capacity.Compared with traditional normoxic culture,EPCs expanded under hypoxia showed favorable proliferative capacity and attenuated senescence,LDH isozyme profiles were shifted under normoxia and hypoxia for hypoxia expressed more LDHA-rich LDHs and normoxic LDHs showed more balanced profile.Besides,hypoxic LDHs exceled in forward and reverse reaction activity and NAD+recycling,including less NADH,more NAD+and total NAD and a higher NAD+/NADH ratio.Hypoxic EPCs presented higher mitophagy as well.Knock-down of LDHA under hypoxia shifted the LDH isozyme profile to normoxic mode and impaired LDH activity in both reactions.Knock-down of LDHB shifted the profile to LDHA-rich LDHs.Single knock-down did no influence on NAD+ recycling.However,knock-down of LDHA and LDHB simultaneously impaired expression of all isozymes and NAD+recycling,and further reduced mitophagy,promoted senescence and impaired tube forming.Conclusion:In general,shift of LDH isozymes under hypoxia promoted NAD+ recycling and cell proliferation and attenuated cell senescence,providing a novel strategy for research on relationship between hypoxia and senescence and optimizing in vitro culture.