Effects And Mechanisms Of Sitagliptin On Proliferation?Migration Of High-glucose-treated Endothelial Progenitor Cells From Rat Bone Marrow

Backgroud and ObjectiveDiabetes mellitus is associated with an increased risk of atherosclerosis due toits negative impact on the vascular endothelium. The damaged endothelium isrepaired by resident cells also through the contribution of a population of stem cellsderived from bone marrow. These cells?termed endothelial progenitor cells?EPCs?are involved in maintaining endothelial homeostasis and contributes to the formationof new blood vessels with a process called postnatal vasculogenesis. Consistentevidences have shown that impairment and reduction of EPCs are hallmark featuresof type1and type2diabetes. Type2diabetes?especially in the presence ofmacrovascular complications?is associated with reduced circulating EPCs?whichin turn has been linked to incident cardiovascular disease. Therefore?EPC alterationsmight have a pathogenic role in diabetic complications?The study of endothelialprogenitor cells is a hot topic.Glucagon-like peptide-1(GLP-I) is one of the incretin hormones?which has aglucose-dependent release and promotes the secretion function of postprandialinsulin. The serum levels or activities of GLP-1usually dccrcase in diabetic patientsand the endogenous GLP-1could be dissolved rapidly by dipeptidyl peptidase (DPP-IV).Dipeptidyl-peptidase-? ?DPP-?? inhibitor sitagliptin is a kind drugs toregulate blood glucose? mainly by improving the incretin levels in vivo and thefunction of islet beta cells.Stromal cell-derived factor-1(SDF-1) have been shown to possess the potential toincrease the number of circulating EPCs and improve endothelial function. The mice ofcritical limb ischemia after oral sitagliptin?increase angiogenesis. However?whetherDDP-? inhibitors can regulate SDF-1? whether DDP-? inhibitors regulatevasculogenesis as a result of regulation of EPC function is still unclear.To investigate the effects of sitagliptin on the proliferation and migration ability ofhigh-glucose-treated EPCs in vitro?to confirmed that sitagliptin can improve thefunction of EPCs?and explore its possible mechanism.Methods1?The culture and identification of rat bone marrow-derived endothelialprogenitor cells in vitroUsing density gradient centrifugation and differential velocity adherent methodsto culture cells?With the identification of endothelial progenitor cells in vitro usingDil-ac-LDL and FITC-UEA-1double dyeing?using flow cytometry to detect theexpression of cell surface antigen CD34and CD133, using matrigel to detect themicrovascular formation in vitro.2?Effects of high glucose on the proliferation?migration and secretion ofEPCs.EPCs were cultured in vitro after7days?EPCs were cultured48h in normal?different concentrations of glucose(5mmol/L?10mmol/L?20mmol/L?40mmol/L)?the effects of high glucose on the proliferation?migration and secretion capacity ofEPCs were detected by MTT assay?Boyden chamber assay and ELISA.3?Effects of sitagliptin on the proliferation?migration and secretion ofEPCs. EPCs were cultured in40mmol/L high glucose after48h?EPCs were cultured48h in normal?different concentrations of sitagliptin(0.01?mol/L?0.1?mol/L?1?mol/L?10?mol/L?50?mol/L)?the effects of sitagliptin on the proliferation?migration and secretion capacity of EPCs were detected by MTT assay?Boydenchamber assay and ELISA.4?Mechanisms of sitagliptin on proliferation?migration of high-glucose-treated EPCsHigh-glucose-treated EPCs were divided into3groups?respectively added to1?mol/L Sitagliptin?1?mol/L Sitagliptin and5?g/ml SDF-1/CXCR4channelblocker AMD3100?and setting up a blank control group only added mediem. Cellproliferation and migration was determined also by MTT assay and Boyden chamberassay.Results1?Bone marrow mononuclear cells after primary vaccination were in round anduniform in size?four days after plating?adherent cells increased?the cells werespindle-shaped?seven days after plating?the colony-forming unit of center for roundcell?around for spindle cell could be seen?Flow cytometry analysis showed thatEPCs expressed CD34, CD133?and most of the cells were double positive toDiI-ac-LDL and FITC-UEA-I?cells can form microvessel-like structures on thematrigel. The EPCs exhibited cobblestone morphology on about2w.2?Compared with the control group?high glucose dose-dependently degradedthe proliferation?migration of EPCs?and degraded the release of SDF-1by EPCs?and the strongest effect was glucose at40mmol/L.3? Compared with the control group? When sitagliptin at1?mol/Lconcentration?it can improve endothelial progenitor cell propagate?migrate andsecret SDF-1at the greatest degree.4?The effects disappear when there is SDF-1/CXCR4channel blockersAMD3100exists. Conclusions1?High glucose inhibits proliferation and migration of EPCs?and degraded therelease of SDF-1by EPCs.2? DPP-? inhibitor Sitagliptin promotes high-glucose-treated endothelialprogenitor cell to secrete SDF-1?and improves endothelial progenitor cell propagateand migrate in vitro?this effect may be mediated by SDF-1/CXCR4signal pathway.