Synergistic Effects Of Transplanted Endothelial Progenitor Cells And P38 MAPK Inhibitor In Diabetic Ischemic Stroke Models

Part IThe effects of p38 MAPK inhibitors on the function of endothelial progenitor cells in high glucoseObjective The aim of this study was to investigate whether p38 MAPK inhibitors could reverse the adverse effects of high glucose on bone marrow-dereived endothelial progenitor cells (EPCs) and to compare the efficacy of two inhibitors, RWJ 67657 and SB 203580, in protecting EPCs.Materials and Methods Mononuclear cells were isolated from bone marrow of C57BL/6J mice using ficoll density gradient centrifugation. EPCs were obtained from adherent cells and characterized by morphology, immunofluorescent staining, flow cytometry analysis, and tube formation assay. EPCs were cultured with different concentration of glucose. RWJ 67657 (1μM) and SB 203580 (1μM) were added to the medium with high glucose. Cell proliferation, senescence, apoptosis, adherence, migration, tube formation, and ELISA assays were performed to observe the function of EPCs.Results The cultivated cells were positive for CD133, CD34, and VEGFR2. Glucose significantly reduced the proliferation of the EPCs in a concentration-dependent manner. Western Blot showed that the glucose concentration dose-dependently increased the phosphorylation of p38 MAPK in the EPCs, which was significantly reduced by both RWJ 67657 and SB 203580. Cell apoptosis and tube formation assays showed that the p38 MAPK inhibitors could significantly improve EPC survival and tube fomation ability, and RWJ 67657 exhibited a high efficacy in protecting EPC than SB 203580. Furthermore, the p38 MAPK inhibitors also significantly reduced the effects of high glucose on EPC senescence, adherence, migration, and VEGF secretion.Conclusion Most of our cultured cells have features of hematopoietic stem cells and endothelial cells, which can differentiate into endothelial cells in vitro. The p38 MAPK inhibitor could reverse the adverse effects of high glucose on EPC number and function. The novel inhibitor RWJ 67657 was more effective than SB 203580 at protecting EPCs from being damage by high glucose levels in vitro.Part IINon-invasive monitoring of transplanted endothelial progenitor cells in diabetic ischemic stroke modelsObjective The objectives of this study were to establish a non-invasive imaging strategy to monitor the homing of transplanted EPCs in diabetic stroke mice and to assess the effect of RWJ 67657, an inhibitor of p38 MAPK, on the homing ability of exogenous EPCs.Materials and Methods Bone marrow-derived EPCs were labeled in vitro with a multi-functional nanoprobe modified with paramagnetic chelators and fluorophores before being infused into wild-type and diabetic stroke mice via ipsilateral internal carotid artery. Magnetic resonance imaging (MR1) and near-infrared fluorescence imaging (NIRFI) were performed at selected time pointsResults The signal of the nanoprobe reached its peak on day 5 in both MRI and NIRFI after EPC transplantation in wild-type stroke models. A few of rodamine positive cells were observed in the peri-infarct areas via immunofluorescence staining, indicating that the transplanted cells were indeed homing to the ischemic brain. The signal enhancement of diabetic stroke models was significantly lower than that of wild-type controls. However, the signal intensity of diabetic stroke models significantly increased after oral administration of RWJ 67657, indicating that more transplanted EPCs migrated to the ischemic brain.Conclusion These results suggest that this dual-modal imaging strategy is feasible for non-invasively monitoring transplanted cells in vivo. Diabetes reduced the number of transplanted EPCs homing to the ischemic area, which was increased by the consecutive intragastric administration of RWJ 67657.Part IIISynergistic Effects of Transplanted Endothelial Progenitor Cells and RWJ 67657 in Diabetic Ischemic Stroke ModelsObjective The aim of this study was to investigate the combined effects of EPC transplantation and p38 MAPK inhibitor administration on diabetic stroke outcomes.Materials and Methods Bone marrow-derived endothelial progenitor cells (EPCs) were injected intra-arterially into db/db mice after ischemic stroke induction. RWJ 67657, a p38 MAPK inhibitor, was administered orally for 7 consecutive days, with the first dose given 30 min before stroke induction. Functional outcome was determined at days 0,1,7,14, and 21. Angiogenesis, neurogenesis, infarct volume, and Western Blot assays were performed on day 7, and white matter remodeling was determined on day 14.Results Neither EPC transplantation nor RWJ 67657 administration alone significantly improved diabetic stroke outcome, although RWJ 67657 displayed a potent anti-inflammatory effect. By both improving the functioning of EPCs and reducing inflammation, EPC transplantation plus RWJ 67657 administration in vivo synergistically promoted angiogenesis and neurogenesis after diabetic stroke. In addition, the white matter remodeling, behavioral scores, and expressions of VEGF and BDNF were significantly increased in diabetic mice treated with both EPCs and RWJ 67657.Conclusion The combination of EPC transplantation and RWJ 67657 administration accelerated recovery from diabetic stroke, which might have been caused by increased levels of pro-angiogenic and neurotrophic factors.