The Effect Of Adiponectin On EFCs Treated With High Glucose And Its Impact To Oxidative Stress

Objective:Endothelial Progenitor Cells(EPCs) also called into the blood essel cells, not onlyparticipate in the embryonic angiogenesis, but participates in the first angiogenesis andendothelial injury repair. Diabetes combined with cardiovascular complications caused thedecline of EPCs numbers and function, resulting in re-endothelialization injured.Adiponectin is one kind of fat plasma protein, which has the roles of anti-atherosclerosisand protecting the heart and blood vessels; APN could anti-inflammatory and adjust theway such as cell metabolism, regulate the heart ischemia-reperfusion injury, inhibition ofmyocardial hypertrophic reconstruction. Recent study had show that incubation of humanperipheral blood mononuclear cells with adiponectin led to an increase of the number ofEPCs. However, whether APN can improve the function of EPCs in a high glucoseenvironment is not clear.Therefore it is important for us to study the factors that influenceabout APN. In this experiment, we treated the EPCs by glucose with differentconcentration, observed the numbers and function of EPCs. On this basis, given gifferentdestiny APN intervention to observe the numbers and function of EPCs injuried by highglucose. In this way, we can identified the protective effects on EPCs, explored thepossible mechanisms and provided necessary scientific basis for clinical treatment.Methods:1. Isolation, culture and identification of EPCs: Peripheral blood mononuclear cellswere isolated from peripheral blood by Ficoll density gradient centrifugation. After7daysin M199medium containing rh-VEGF, rh-b-FGF and10%Fetal Bovine Serum, Themorphology, flow cytometric measured cell and molecular markers (CD34CD133, andKDR) and confocal laser inverted microscope culture cells absorb ac-LDL and combinedwith UEA-I identified cells for income.2. The effects of glucose on EPCs: The cells were collected, then treated withdifferent concentration glucose(5.5,20,25,30,50mmol/l)for48h. The proliferation wereassayed using MTT, the rate of apoptosis by Flow Cytometry and the detection of reactiveoxygen species(ROS) by Fluorescent probe(DCFH).3. The influences of APN on EPCs injured by high glucose: Collected adherent cellswhich were cultured for4days, then divided into7groups: three of them treated with5.5 mmol/l glucose,30mmol/l glucose, hypertonic control group for96h and the rests treatedwith30mmol/l glucose48h and then with different concentrations adiponectin intervention(1.25,2.5,5,10μg/ml). After2days in vitro culture, the proliferation, migration wereassayed using MTT, Transwell chamber respectively. The rate of apoptosis by FlowCytometry and the detection of reactive oxygen species(ROS) by Fluorescentprobe(DCFH).4. Statistical approach: The data were expressed as mean±standard deviation amonggroups were compared using single factor analysis of variance, homogeneity of variancewith LSD method; heterogeneity of variance using Dunnett’s method. The data wereanalyzed using SPSS19.0statistical software, P<0.05was considered statisticallysignificant.Results:1. Isolation, culture and identification of EPCs: Peripheral blood mononuclear cellswere isolated from peripheral blood by Ficoll density gradient centrifugation round andbright. Under microscope, morphological characteristics of cells: training after4days,some adherented cells appearing short spindle and typical cell group; Training7days, cellgroups increase obviously, spindle cells more growth;The expression of CD34, CD133andKDR was positive, expression rates were as follows:23.3%,10.7%,92.32%. By confocalmicroscopy, cells uptake of DiI-Ac-LDL, can also be combined with FITC-UEA-I. Doublestaining positive rate of cells proves that the cultured cells are EPCs.2. The effects of glucose on EPCs: The A value of MTT assay about control groupwas(0.287±0.031), and the apoptosis rate was(8.495±1.279). The level of reactive oxygenspecies(ROS) by Fluorescent probe(DCFH) was(0.361±0.050). When the glucoseconcentration at20mmol/l,25mmol/l,30mmol/l,50mmol/l respecrivly, the A value ofMTT assay were(0.252±0.034),(0.242±0.019),(0.215±0.024),(0.208±0.017). Theapoptosis rate was(12.942±1.177),(14.770±1.438),(16.393±1.216),(17.502±1.259) andthe level of reactive oxygen species(ROS) by Fluorescent probe(DCFH) was(0.468±0.090),(0.483±0.053),(0.518±0.081),(0.613±0.098). The results indicated that,with the increase of the concentration of the sugar, EPCs proliferation droped, andapoptosis, ROS levels increased (P<0.01). When the glucose concentration at50mmol/l,the effects on EPCs were no statistical difference compared with30mmol/l(P>0.05).3. The influences of APN on EPCs injured by high glucose: Collected adherent cellswhich were cultured for4days, divided into7groups: control group; high glucose; high glucose (30mmol/l) with different concentrations adiponectin intervention (1.25,2.5,5,10μg/ml). A hypertonic control group was set up. The results indicated that, with theincrease of the concentration of the APN, EPCs proliferation increased and apoptosis, ROSlevels droped(P<0.01). As a result, when the APN concentration at10μg/ml, the effects onEPCs were no statistical difference compared with5μg/ml(P>0.05). The osmotic pressuredid not affect the result(P>0.05).Conclusion:1. Compared with the control group, the injured EPCs induced by high glucose had alower proliferation, migration and had a higher apoptosis.2. EPCs incubated with adiponectin showed the capacities higher than that incubatedby high glucose.3. High sugar can cause EPCs ROS levels rised and function damaged, while APNcan decline high sugar induced ROS levels, protecting EPCs function. The osmoticpressure did not affect the result.