Nicorandil Promotes High-glucose-suppressed Bone Marrow-derived Endothelial Progenitor Cell Functions

Aim:Hyperglycemia aggravates vascular endothelial dysfunction,leading to diabetes mellitus patients being more prone to coronary heart disease(CHD).Endothelial progenitor cells(EPCs)can directly participate in the repair of endothelial injury and improve endothelial function;however,EPCs function is significantly attenuated in a high-glucose environment.Thus,a strategy to enhance the biological functions of EPCs is required.Nicorandil is a hybrid agent with both nitrate-like and ATP-sensitive potassium(Katp)channel-activating properties;it also functions as a cardiovascular protective agent.However,whether nicorandil can improve EPCs function in a high-glucose environment and the possible underlying mechanisms remain to be determined.In this study,we aimed to study the effect of nicorandil on EPCs in a high-glucose environment.Methods:Flirstly,rat bone marrow mononuclear cells were isolated by density gradient centrifugation,and EPCs was purified continuously by combining differential adhesion method and improved colony selection method.EPCs was identified by uptake test,immunofluorescence,flow cytometry,proliferation,viability,migration and in vitro tube formation assay.Secondly,investigate the proliferation,migration and angiogenesis of EPCs under condition of higher glucose in different concentrations in rats,and provide reference for study on pathophysiological mechanism of EPCs in high glucose environment.Thirdly,EPCs were treated with nicorandil in a hyperglycemic environment(25 mM glucose),.A mitochondrial ATP-sensitive potassium channel(mitoKA-Tp)antagonist 5-hydroxydecanoate(5-HD),a C-X-C motif chemokine receptor 4(CXCR4)antagonist(AMD3100),a phosphatidylinositol 3-kinases(PI3K)antagonist(LY294002),and an endothelial nitric oxide synthase(eNOS)antagonist(L-NAME)were used to characterize the underlying mechanisms.CCK-8 and MTT assays were used to investigate the proliferation and viability of EPCs.The concentrations of stromal cell-derived factor 1?(SDF-1?)were examined by an ELISA assay.Cell migration and angiogenesis were analyzed.High-glucose-induced apoptosis was detected by Annexin V-FITC/PI and Western blotting.Protein expression was examined by Western blotting.Results:1.The early EPCs appeared with spindle shape and the late EPCs showed a typical characteristic endothelial 'cobblestone' morphology.95.7±3.8%EPCs were double-positive for the uptake of Dil-acLDL and bound to FITC-UEA-I.2.The purified EPCs immunofluorescence showed that the expression of CD31,VEGFR2,vWF and eNOS,VE-cadherin,indicating that EPCs has the characteristics of endothelial cells.3.The purified EPCs flow results showed that after the first,second and third time purification,which was significantly higher than non-purified cells(P<0.01)which means high purity of EPCs in vitro.4.The 1×105cells/ml had the best proliferation and vitality(P<0.01),which were applied to the subsequent experiments.The cultured cells have the ability of migration and in vitro tube formation.5.In the established high glucose model,the concentration of glucose at 25 mM reached the maximum inhibitory concentration for EPCs function.At this time,compared with the control group,the ability of proliferation(p<0.01),the ability of migration(p<0.01),and the ability of angiogenesis in vitro the lowest.6.Nicorandil significantly increased the viability,proliferation,migration,and angiogenesis of EPCs in a high-glucose environment(p<0.01).The administration of nicorandil increased the concentration of SDF-la(p<0.01),and inhibited high-glucose-induced EPCs apoptosis(p<0.01).7.The protective effects of nicorandil were attenuated by 5-HD(p<0.01).Nicorandil improved the function of EPCs by opening the mitoKA-TP channel.In addition,nicorandil increased the expression of CXCR4(p<0.05),phospho-Akt(p<0.05),phospho-eNOS(p<0.05),Bcl-2(p<0.05),and decreased the expression of cleaved caspase3(p<0.01).8.Inhibition of the signaling pathway with AMD3100,LY294002,and L-NAME subsequently reduced nicorandil-induced SDF-la/CXCR4/PI3K signaling activity(p<0.01).SDF-la/CXCR4/PI3K is the downstxeam signaling pathway of the mitoKATP channel.Conclusions:1.Morphological changes during cell growth were observed by morphology,proliferation and viability assay to observe the growth of cells,identification of uptake test and cell surface markers to make sure that EPCs has the characteristics of endothelial cells.Migration tests determine cell migration ability,In vitro tube formation assay determined that the cells have the ability of angiogenesis.The purity of the purified cells was detected by flow cytometry,and the cultured cells were determined to be EPCs.2.To provide a modified,effective EPCs method for isolation,cultivation,purification and identification of bone marrow-derived endothelial progenitor cells.3.EPCs was inhibited in a concentration dependent manner with the increase of glucose concentration in high glucose environment,and its proliferation,migration and angiogenesis in vitro decreased significantly.4.To establish a high glucose model is suitable for exploring the biological function of EPCs in high glucose environment.5.Nicorandil improved EPCs activity,proliferation,migration and angiogenesis in a concentration dependent manner under high glucose environment,while inhibiting the apoptosis induced by high glucose,and promoted the secretion of SDF-la.6.Nicorandil is through opening nutoKATP channels and activate SDF-la/CXCR4 signaling pathway,further activation of PI3K/Akt/eNOS signaling pathway to improve high glucose inhibit bone marrow-derived EPCs function.