BackgroundThis study tested the hypothesis that type 2 diabetes restricts multipotency of db/db mesenchymal stem cells (MSCs), promotes their terminal differentiation into adipocytes rather than endothelial cells, thereby promotes adipocytic infiltration into ischemic muscles, and reduces their capacity to participate in postischemic neovascularization.Methods and ResultsTo test this hypothesis, we transplanted MSCs from db/db or wild-type (WT) mice into WT recipients after induction of hind limb ischemia. WT recipients of db/db MSCs demonstrated adipocyte infiltration of ischemic muscle and impaired neovascularization; WT recipients of WT MSCs showed no intramuscular adipocyte infiltration and had significantly enhanced neovascularization (P<0.05; n=6). Confocal microscopy showed that the percentage of MSCs that differentiated into an adipocyte phenotype was greater and into an endothelial cell was less in WT recipients transplanted with db/db MSCs than those transplanted with WT MSCs (P<0.05; n=6). In vitro, db/db MSCs exhibited greater oxidant stress, greater adipocyte differentiation, and less endothelial differentiation than WT MSCs, and these differences were reversed by treatment with N-acetylcysteine or Nox4 siRNA (P<0.05; n=6). Insulin increased Nox4 expression, oxidant stress, and adipocyte differentiation in WT MSCs, and these insulin-induced effects were reversed by Nox4 siRNA (P<0.05; n=6). Reversal of db/db MSC oxidant stress by in vivo pretreatment with Nox4 siRNA before transplantation reversed their impaired capacity to augment postischemic neovascularization.ConclusionsType 2 diabetes–induced oxidant stress restricts the multipotency of MSCs and impairs their capacity to increase blood flow recovery after the induction of hind-limb ischemia. Reversal of MSC oxidant stress might permit greater leverage of the therapeutic potential of MSC transplantation in the setting of diabetes.

BackgroundClinical studies suggest that hypercholesterolemia may cause ageing in hematopoietic stem cells (HSCs) because ageing‐associated alterations were found in peripheral blood cells and their bone marrow residing precursors in patients with advanced atherosclerosis. We hypothesized that hypercholesterolemia induces oxidant stress in hematopoietic stems cells that accelerates their ageing.Methods and ResultsHere we show that HSCs from ApoE−/− mice, as well as HSCs from C57Bl/6 mice fed a high cholesterol diet (HCD) accumulated oxLDL and had greater ROS levels. In accordance, the expression pattern of the genes involved in ROS metabolism changed significantly in HSCs from ApoE−/− mice. Hypercholesterolemia caused a significant reduction in phenotypically defined long‐term HSC compartment, telomere length, and repopulation capacity of KTLS cells, indicating accelerated ageing in these cells. Gene array analysis suggested abnormal cell cycle status, and the key cell cycle regulators including p19ARF, p27Kip1 and p21Waf1 were upregulated in KTLS cells from hypercholesterolemic mice. These effects were p38‐dependent and reversed in vivo by treatment of hypercholesterolemic mice with antioxidant N‐acetylcysteine. The oxidant stress also caused aberrant expression of Notch1 that caused loss of quiescence and proliferation leading to the expansion of KTLS compartment in hypercholesterolemic mice.ConclusionTaken together, we provide evidence that hypercholesterolemia can cause oxidant stress that accelerates the ageing and impairs the reconstitution capacity of HSCs.

Rationale: We have recently shown that experimental type 2 diabetes (db/db mouse) restricts mesenchymal stem cell’ (MSCs) differentiation capacity through a hyperinsulinemia-dependent induction of Nox 4 expression that promotes adipocytic and inhibits endothelial cell differentiation both in vitro and in vivo. We also showed that transplantation of type 2 diabetic MSCs into wild type (WT) recipients reduced post-ischemic neovascularization significantly below that of WT recipients receiving WT MSCs. Objective: This study tested the hypothesis that pretreatment of db/db MSCs with siRNA Nox4 prior to their transplantation and infusion 24 hrs after the induction of hindlimb ischemia restores their differentiation capacity in vivo and rescues their impaired capacity to promote neovascularization in wild type mice. Methods and Results: To reverse oxidative stress in db/db MSCs, we knockdown NOX 4 expression by a siRNA under in vitro conditions. In vitro inhibition of Nox 4 expression of db/db MSCs significantly increased their differentiation capacity towards endothelial cells and reduced their propensity to differentiate into adipocytes. Db/db MSCs also showed significantly increased tubular formation ability after Nox 4 knockdown. We transplanted NOX 4 siRNA transduced db/db MSCs into WT recipients 24 hours after induction of hindlimb ischemia. Blood flow recovery was measured at different time points by laser Doppler scanning. Reversal of oxidative stress in db/db MSCs by pretreatment with Nox4 siRNA prior to transplantation reversed their impaired capacity to augment post-ischemic neovascularization as evidenced by blood flow recovery that increased to that of WT mice. In addition, knockdown of NOX 4 expression in db/db MSCs also increased collateral artery diameter and capillary density by 50%. Conclusions: Pretreatment of db/db MSCs with siRNA against Nox 4 significantly increased their differentiation capacity towards endothelial cells rather than adipocytes and also their capacity to augment neovascularization after induction of hindlimb ischemia. Reversal of MSC oxidant stress induced by type 2 diabetes might permit greater leverage of the therapeutic potential of MSC transplantation to treat cardiovascular diseases.