Superoxide induced by a high-glucose concentration attenuates production of angiogenic growth factors in hypoxic mouse mesenchymal stem cells

Previous reports have shown that the paracrine system may be an important mediator in bone-marrow-derived mesenchymal stem cell (MSC) therapy for ischemic diseases. Hyperglycemia and hypoxia have been associated with increased levels of reactive oxygen species; oxidative stress may therefore influence the paracrine effects of MSCs under hypoxic conditions in diabetic patients, although the mechanism underlying this effect remains unknown. Hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates the transcription of hypoxia-inducible genes. We determined the effect of high-glucose concentrations on the production of angiogenic growth factors via HIF-1 alpha induction in hypoxic MSCs. MSCs were cultured with different glucose concentration (5.6, 11, 20, or 30 mM) for 24 h. The cells were then incubated in a hypoxic chamber (5% O-2) or under normoxia (21% O-2) for 6 or 24 h. Protein levels of HIF-1 alpha, vascular endothelial growth factor A(165) (VEGF-A(165)), and platelet-derived growth factor B (PDGF-B) were attenuated by glucose in hypoxic MSCs in a dose-dependent manner. Treatment with MG132, a specific inhibitor of proteasome activity, significantly reversed the inhibitory effect of high-glucose concentrations in hypoxic MSCs. 4-Hydroxyl-tetramethylpiperidin-oxyl (a cell-permeable superoxide scavenger) or Apocynin (a NADPH oxidase inhibitor) significantly reversed glucose-induced attenuation of VEGF-A(165), PDGF-B, and HIF-1 alpha protein levels. Stimulation with a high-glucose concentration (30 mM) significantly increased intracellular superoxide levels in hypoxic MSCs. Our results suggest that in hypoxic MSCs the increase in intracellular superoxide levels induced by high-glucose concentrations may attenuate hypoxia-induced HIF-1 alpha expression, which in turn attenuates hypoxia-induced VEGF-A(165) and PDGF-B transcription. Journal of Endocrinology (2011) 208, 147-159