People with type 2 diabetes mellitus (T2DM) have a 25-fold higher risk of limb loss than non-diabetics due in large part to impaired wound healing. Here, we show that the impaired wound healing phenotype found in T2D mice is recapitulated in lethally irradiated wild type recipients, whose hematopoiesis is reconstituted with hematopoietic stem cells (HSCs) from T2D mice, indicating an HSC-autonomous mechanism. This impaired wound healing phenotype of T2D mice is due to a Nox-2-dependent increase in HSC oxidant stress that decreases microRNA let-7d-3p, which, in turn, directly upregulates Dnmt1, leading to the hypermethylation of Notch1, PU.1, and Klf4. This HSC-autonomous mechanism reduces the number of wound macrophages and skews their polarization towards M1 macrophages. These findings reveal a novel inflammatory mechanism by which a metabolic disorder induces an epigenetic mechanism in HSCs, which predetermines the gene expression of terminally differentiated inflammatory cells that controls their number and function.

Objective We sought to directly compare the effects of type 1 and type 2 diabetes on post-ischemic neovascularization and evaluate the mechanisms underlying differences between these groups. We tested the hypothesis that type 2 diabetic mice have a greater reduction in eNOS expression, greater increase in oxidative stress, and reduced arteriogenesis and angiogenesis resulting is less complete blood flow recovery than type 1 diabetic mice after induction of hindlimb ischemia. Methods Hindlimb ischemia was generated by femoral artery excision in streptozotocin-treated mice (model of type 1 diabetes), in db/db mice (model of type 2 diabetes), and in control (C57BL/6) mice. Dependent variables included markers of arteriogenesis and angiogenesis, as well as eNOS and markers of oxidative stress. Results Post-ischemia recovery of hindlimb perfusion was significantly less in type 2 than in type 1 diabetic mice; however, neither diabetic group demonstrated a significant increase in collateral artery diameter or collateral artery angioscore in the ischemic hindlimb. The capillary/myofiber ratio in the gastrocnemius muscle decreased in response to ischemia in control or type 1 diabetic mice, but remained the same in type 2 diabetic mice. Gastrocnemius muscle eNOS expression was lower in type 1 and 2 diabetic mice than in control mice; this expression decreased after induction of ischemia in type 2, but not type 1 diabetic mice. The percentage of endothelial progenitor cells (EPC) in peripheral blood failed to increase in either diabetic group after induction of ischemia, whereas this variable significantly increased in the control group in response to ischemia. EPC eNOS expression decreased after induction of ischemia in type 1, but not type 2 diabetic mice. EPC nitrotyrosine accumulation increased after induction of ischemia in type 2, but not type 1 diabetic mice. EPC migration in response to VEGF was reduced in type 1 and type 2 diabetic mice than in control mice. EPC incorporation into tubular structures was less effective in type 2 diabetic mice. Extensive fatty infiltration was present in ischemic muscle of type 2, but not type 1 diabetic mice. Conclusion We conclude that type 2 diabetic mice displayed a significantly less effective response to hindlimb ischemia than type 1 diabetic mice.

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.

Obesity will soon surpass smoking as the most preventable cause of cancer. Hypercholesterolemia, a common comorbidity of obesity, has been shown to increase cancer risk, especially colorectal cancer. However, the mechanism by which hypercholesterolemia or any metabolic disorder increases cancer risk remains unknown. In this study, we show that hypercholesterolemia increases the incidence and pathologic severity of colorectal neoplasia in two independent mouse models. Hypocholesterolemia induced an oxidant stress-dependent increase in miR101c, which downregulated Tet1 in hematopoietic stem cells (HSC), resulting in reduced expression of genes critical to natural killer T cell (NKT) and γδ T-cell differentiation. These effects reduced the number and function of terminally differentiated NKT and γδ T cells in the thymus, the colon submucosa, and during early tumorigenesis. These results suggest a novel mechanism by which a metabolic disorder induces epigenetic changes to reduce lineage priming of HSC toward immune cells, thereby compromising immunosur-veillance against cancer.

Introduction Most current animal models of hindlimb ischemia use acute arterial occlusion that does not accurately reflect the pathogenesis of gradual arterial occlusion in humans. We therefore developed the first mouse model of gradual arterial occlusion and tested the hypothesis that the mechanisms regulating blood flow recovery are critically dependent on the rate of arterial occlusion. Methods Gradual arterial occlusion was induced by placing ameroid constrictors on the proximal and distal left femoral artery, and ligating the femoral arterial branches (n=36). Acute arterial occlusion was accomplished by excising the left femoral artery (n=36). The blood flow recovery was studied by laser Doppler imaging. Differential gene expression between these two models was assessed by quantitative real time PCR. Inflammatory and progenitor cells recruitment were determined by immunohistochemistry. Results We found that hypoxia-related genes increased significantly in the calf, but not in the thigh, after gradual and acute femoral arterial occlusion (p<0.05). Shear-stress dependent genes and inflammatory genes were upregulated immediately in the thigh only after acute femoral arterial occlusion (p<0.05). These differences in gene expression were consistent with increased SDF-1α expression, recruitment of macrophages and hemangiocytes, and higher blood flow recovery after acute arterial occlusion than after gradual arterial occlusion (p<0.05). Conclusion This is the first study to show the mechanisms that regulate blood flow recovery are critically dependent on the rate of arterial occlusion. This novel model of gradual arterial occlusion may more closely resemble the human diseases, and may provide more accurate mechanistic insights for creating novel molecular therapies.

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.

We tested the hypothesis that oxidized low-density lipoprotein (oxLDL)-induced inactivation of Akt within endothelial progenitor cells (EPCs) is mediated at the level of Phosphoinositide 3-kinase (PI3K), specifically by nitrosylation of the p85 subunit of PI3K, and that this action is critical in provoking oxLDL-induced EPC apoptosis. Hypercholesterolemic ApoE null mice had a significant reduction of the phosphorylated Akt (p-Akt)/Akt ratio in EPCs, as well as a greater percentage of apoptosis in these cells than EPCs isolated from wild-type (WT) C57Bl/6 mice. EPCs were isolated from WT spleen and exposed to oxLDL in vitro. oxLDL increased O₂^– and H₂O₂ in these cells and induced a dose- and time-dependent reduction in the p-Akt/Akt ratio and increase in EPC apoptosis. These effects were significantly reduced by the antioxidants superoxide dismutase, L-NAME, epicatechin and FeTPPs. oxLDL also induced nitrosylation of the p85 subunit of PI3K and subsequent dissociation of the p85 and p110 subunits, an effect significantly reduced by all the antioxidant agents tested. EPC transfection with a constitutively active Akt isoform (Ad-myrAkt) significantly reduced oxLDL-induced apoptosis of WT EPCs. The present findings indicate that oxLDL disrupts the PI3K/Akt signaling pathway at the level of p85 in EPCs. This dysfunction can be reversed by ex vivo antioxidant therapy.

Mesenchymal stem cells (MSCs), due to their paracrine, transdifferentiation, and immunosuppressive effects, hold great promise as a therapy for peripheral arterial disease. Diabetes is an important risk factor for peripheral arterial disease; however, little is known of how type II diabetes affects the therapeutic function of MSCs. This review summarizes the current status of preclinical and clinical studies that have been performed to determine the efficacy of MSCs in the treatment of peripheral arterial disease. We also present findings from our laboratory regarding the impact of type II diabetes on the therapeutic efficacy of MSCs neovascularization after the induction of hindlimb ischemia. In our studies, we documented that experimental type II diabetes in db/db mice impaired MSCs’ therapeutic function by favoring their differentiation towards adipocytes, while limiting their differentiation towards endothelial cells. Moreover, type II diabetes impaired the capacity of MSCs to promote neovascularization in the ischemic hindlimb. We further showed that these impairments of MSC function and multipotency were secondary to hyperinsulinemia-induced, Nox4-dependent oxidant stress in db/db MSCs. Should human MSCs display similar oxidant stress-induced impairment of function, these findings might permit greater leverage of the potential of MSC transplantation, particularly in the setting of diabetes or other cardiovascular risk factors, as well as provide a therapeutic approach by reversing the oxidant stress of MSCs prior to transplantation.