Effect of adiponectin on high-glucose-treated endothelial progenitor cells and its possible mechanisms

AIM:To investigate the effect of adiponectin(APN) on hyperglycemia-induced oxidative damage of endothelial progenitor cells(EPCs).METHODS:Blood mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation.After 7 days in M199 medium containing rh-VEGF,rh-b-FGF and 10%fetal bovine serum,the attached cells were identified as EPCs by cell morphology.Cell molecular markers(CD34 CD133,and KDR) were measured by flow cytometry and the capabilities of cultured cells in absorbing ac-LDL and combining with UEA-I were observed by confocal laser-inverted microscope.Cells were synchronized and then divided into seven groups:control group(5.5 mmol/L),high glucose group(30 mmol/L),four adiponectin-treated intervention groups,respectively,with different concentrations of adiponectin(1.25,2.5,5 or 10 μg/ml) and hypertonic control group.After 7 days of culture,adherent cells were collected and incubated with adiponectin for 48 h.Proliferation,migration,apoptosis rate and reactive oxygen species were tested by MTT,transwell chamber,flow cytometry and fluorescent probe(DCFH).RESULTS:In peripheral blood mononuclear cells isolated from peripheral blood by Ficoll density gradient centrifugation following the 7-day treatment,an obvious increase in cell groups and growth of spindle cells were observed.The expressions of CD34,CD133 and KDR were positive and the expression rates were 23.3%,10.7%and 92.32%,respectively.By confocal microscopy,cell uptake of DiI-Ac-LDL was combined with FITC-UEA-I.Double-staining positive rate of cells proved that the cultured cells were EPCs.With the increase of the sugar concentrations,EPC proliferation decreased,whereas apoptosis and ROS levels increased(P 0.01).No statistical difference was observed in the effects on EPCs between glucose concentrations of 50 and 30 mmol/L.With the increase of APN concentration,EPC proliferation increased,whereas apoptosis and ROS levels dropped(P 0.01).No statistical difference was observed in the effects on EPCs between APN concentrations of 10 and 5 μg/ml.The osmotic pressure did not affect the results.CONCLUSION:Compared with the control group,the injured EPCs induced by high glucose showed lower proliferation and migration and a higher rate of apoptosis.EPCs incubated with adiponectin have higher capacities than those incubated by high glucose.High sugar increases ROS levels of EPCs and impairs the function of EPCs,whereas APN lowers high sugar-induced ROS levels and protects EPC function.The osmotic pressure has no effect on EPC function.