The Role Ancd Mechanism Of Oxidative Stress In Endothelial Progenitor Cells Of Patients With Stable Coronary Artery Disease

Coronary artery disease(CAD)in recent years has become one of the major causes of death in China.Recent studies suggest that endothelial dysfunction play a key role in The mechanism of occurrence and development of CAD.Endothelial dysfunction is the essence of dynamic balance between endothelial injury and repair of endothelial injury.EPCs involved in angiogenesis of ischemic tissue and promote repair of damaged vascular endothelium,maintaining intact endothelial function.Recent studies indicate that the oxidative stress plays an important role in the function of EPCs.Oxidative stress is defined as excessive reactive oxygen species(ROS)or metabolic disorders exceeds the endogenous antioxidant defense system to eliminate the reactive oxides,including superoxide anion(O2-),hydrogen peroxide(H2O2)and the hydroxy product(-OH),by interacting with DNA,proteins and lipids,produce excessive DNA damage,protein oxidation and lipid peroxidation and cause lysosomes,mitochondria damage.Studies have shown that oxidative stress reducing mobilization of EPCs,impairing the adhesion,proliferation,migration functions of EPCs and promote EPCs senescence.Studies indicate that in endothelial cells and stem/progenitor cells,ROS mainly derived from NADPH oxidase.NADPH oxidase is a major ROS regulation enzymes that can be used as an indicator of oxidative stress,but the role of oxidative stress in EPCs number and function of stable coronary artery disease is unclear.In addition,NADPH oxidase activation requires the combination of cytosolic subunits and the cell membrane subunits p22phox and gp91phox(Nox2).Cytosolic subunits phosphorylation and subsequent translocation to the plasma membrane is a critical step of NADPH oxidase activation.Many studies have confirmed that PKC play an important role in the process.NADPH oxidase activation can promote P38MAPK and ERK phosphorylation,P38MAPK phosphorylation can result in decreased EPCs numbers and EPCs dysfunction.Activation of ERK1/2 is related to EPCs migration,whereas ERK2 activation is related to EPCs adhesion.NADPH oxidase activation can also promote phosphorylation of Akt and PI3K/Akt/eNOS signal transduction pathway play an important role in the regulation of EPCs functions and senescence.We have confirmed in our previous studies that SDF-1,thymosin β4,homocysteine,nicotine and puerarin were all through PI3K7Akt/eNOS signal transduction pathways to affect EPCs function and senescence.Based on the above theory,we hypothesize:(1)EPCs dysfunction in patients with coronary heart disease is closely related to oxidative stress;(2)PKC plays an important role in NADPH oxidase activation of EPCs in patients with coronary heart disease;(3)NADPH oxidase-mediated oxidative stress may affecting EPCs function and senescence through PI3K/Akt/eNOS,ERK or P38MAPK signaling pathway.Part 1 Oxidative stress levels of EPCs increased and EPCs function impaired in stable coronary artery diseaseObjectiveTo observe the levels of oxidative stress and to evaluate EPCs functions in stable coronary artery disease.MethodsEPCs were isolated from peripheral blood of 40 patients with or without stable coronary artery disease.ROS levels,NADPH oxidase activity,antioxidant enzymes activity,NO bioavailability,mRNA and protein expression of Gpx,Cu/ZnSOD,MnSOD were detected respectively.Paracrine function of VEGF,IL-8,SDF-1,HGF were detected by ELISA.The in vivo angiogenesis capacity was evaluated using immunofluorescence by transplanting EPCs into a rat hindlimb ischemia model.ResultsThe ROS,MDA levels and NADPH oxidase activity were significantly increased in EPCs with CAD,while decreased antioxidant enzyme activity of CAT,SOD were found.Intracellular concentration of NO and paracrine function of EPCs were seriously damaged.The in vivo angiogenesis capacity was impaired in EPCs obtained from CAD subjects.ConclusionOxidative stress levels of EPCs in patients with stable coronary artery disease were elevated.Part 2 Activation mechanism of NADPH oxidase within EPCs in patients with stable coronary artery diseaseObjectiveThe study was designed to investigate the process of NADPH oxidase activation and the underlying mechanisms.Methods EPCs were isolated from the peripheral blood of 40 patients with or without stable CAD.NADPH oxidase activation was detected by measuring the expression of each subunit using western blotting and qPCR analyses and the membrane translocation of p47phox using immunofluorescence.The PKC inhibitor GO-6983 was used to determine the role of PKC in NADPH oxidase activation.ResultsThe expression of NADPH oxidase subunit p47Pphox、p22Pphox、gp91phox and Nox4 weare increased in EPCs with coronary heart disease,p47Pphox membrane translocation was significanly increased in CAD-EPCs.In addition,up-regulation of PKC/β2 was found and PKC inhibition GO6983 can reduce the expression and activity of NADPH oxidase.ConclusionNADPH oxidase activation via p47phox membrane translocation plays a critical role in the initiation and progression of CAD,and the PKCa/β2 signaling pathway may be involved.Part 3 Mechanisms of NADPH oxidase affecting EPCs function in coronary artery diseaseObjectiveThis study is expected to further clarify the mechanism of NADPH oxidase affecting EPCs function in coronary artery disease。MethodsEPCs were isolated from peripheral blood of 40 patients with or without stable coronary artery disease.Cell migration and adhesion were determined using the modified Boyden chamber assay and adhesion assay;Senescence of EPCs were detected by SA-β-galactosidase staining kit.PKC and Akt,eNOS,ERK,p38MAPK phosphorylation levels and corresponding chemical inhibitors were used for pathway validation of activation mechanism and downstream effects of NADPH oxidase.ResultsNADPH oxidase activity,ROS activity and NO concentration of EPCs in patients with coronary artery disease were deceased when NADPH oxidase were inhibited by NAC,apocynin and subunit siRNA.The phosphorylation of Akt,eNOS,ERK and P38MAPK were weakened and migration,adhesion function of EPCs were impaired while cell senescence accelerated in patients with coronary artery disease.NADPH oxidase inhibition can reversed these effects,and signaling pathways AKT/PI3K/eNOS,ERK and P38MAPK may be involved.ConclusionThe impaired function of EPCs in CAD may be achieved through activation of NADPH oxidase and followed by activation of downstream signaling pathways such as AKT/PI3K/eNOS,ERK and P38MAPK.