Effects And Mechanisms Of Resveratrol On Number And Activity Of Endothelial Progenitor Cells From Peripheral Blood

Endothelial progenitor cells (EPCs) are a cell population which could circulate,proliferate,and differentiate into mature endothelial cells,but do not acquire matureendothelial lineage markers or form a lumen yet.EPCs have the capacity to incorporateinto the sites of physiological and pathological neovascularization in vivo.Recently,circulating EPCs have been identified as a marker of endothelial function andcardiovascular risk.Endothelial dysfunction is usually one of the earlier markers ofatherosclerosis,and predisposes to increased vascular tension and thrombosis.EPCsmay play an important role as an endogenous repair mechanism to maintain the integrityof the endothelial monolayer by replacing denuded parts of the vessel.The maintenanceof endothelial monolayer could inhibit thrombotic complication and atheroscleroticlesion development.The beneficial potential of EPCs is attractive for cell therapy whichtargets the endothelial regeneration.However,various risk factors for coronary arterydisease(CAD),such as aging,diabetes,hypertension,hypercholesterolemia,hyperhomocystenemia and smoking,attenuate the number and functional activity of EPCs in healthy individuals and CAD patients.Thus,in most cases,in vitro expansionof EPCs appears to be necessary for the autologous EPCs-based cell therapy.Epidemiological studies have indicated that there is a low prevalence of ischaemicheart disease in the population of southern France,despite a diet rich in saturated fat andcholesterol.This so-called"French paradox"has been attributed to moderateconsumption of red wine.Resveratrol (3,5,4'-trihydroxystilbene),a polyphenolcompound found in significant amounts in grapes and red wine,has been designated theactive agent.However,the mechanisms of action of resveratrol are,as yet,unclear.Anemerging area of investigation has demonstrated critical and multiple actions ofresveratrol on CAD,such as inhibition of the early progression of atherosclerotic lesions,protection of cardiomyocytes against ischaemia-reperfusion injury and reversal ofneointimal formation of injured arteries.Moreover,a number of recent studies hasdemonstrated that resveratrol functioned to inhibit platelet aggregation and/or adhesion,lower oxidative stress in platelets,prevent low-density lipoprotein oxidation,suppressproliferation or hypertrophy of smooth muscle cells and increase high-densitylipoprotein.As far as we know,little study has been performed to investigate the directmodulatory effects of resveratrol on the number and function of EPCs,moreover,norelevant mechanism has been mentioned regarding these effects..On the basis of these considerations,we hypothesized that resveratrol affectedEPCs number and function,thus influenced endothelial repair process and maintainedthe balance between the magnitude of injury and the capacity for repair,whichprevented thrombotic compiication and atherosclerotic lesion development.To test thishypothesis,we measured the number and functional activity of EPCs exposed toresveratrol in vitro at first.Then,we studied the mechanisms by which resveratrol actedon EPCs. The aim of this study is to investigate the effects of resveratrol on number andfunctional activity of EPCs from peripheral blood.Total mononuclear cells (MNCs)were isolated from peripheral blood by Ficoll density gradient centrifugation,and thenthe cells were plated on fibronectin-coated culture dishes.After 4 days cultured,attached cells were stimulated with resveratrol (to make a series of final concentrations:10μmol/L,25μmol/L,50μmol/L,100μmol/L) or vehicle control.EPCs werecharacterized as adherent cells double positive for DiLDL-uptake and lectin binding bydirect fluorescent staining under a laser scanning confocal microscope.EPCs werefurther documented by demonstrating the expression of CD34,CD45,VE-cadherin,VEGFR-2 and AC133 with flow cytometry.EPCs proliferation,migration,in vitrovasculogenesis and secretion were assessed with MTT assay,modified Boyden chamberassay,in vitro tubule forming assay and ELISA assay respectively.EPCs adhesionassay was performed by replating those on fibronectin-coated dishes,and then adherentcells were counted.We showed that incubation of isolated human MNCs withresveratrol dose dependently increased the number of EPCs with the maximum at50μmol/L (to compare with that of control,85.8±26.0 vs 44.8±9.6/hpf,P＜0.01).Resveratrol significantly increased the proliferative,migratory,adhesive andvasculogenic capacities of EPCs,maximum at 50μmol/L (to compare with that ofcontrol,proliferative capacities 0.296±0.028 vs 0.196±0.015;migratory capacities 24.0±5.2 vs 10.7±3.2/hpf;adhesive capacities 49±6.3 vs 30±5.0/hpf;vasculogeniccapacities 49±6.3 vs 26±4.2/hpf,;secretion capacity 377±28.8 vs 282±20.1pg/mL;P＜0.01 for each comparision). Part 2:Resveratrol reduces senescence of endothelial progenitorcells through telomerase activationEmerging evidence has suggested that reduced EPCs number and activity wasassociated with EPC senescence which involved with telomerase activity.Resveratrolhas been shown to augment a variety of cellular functions of EPCs and subsequentlycontribute to ischemic neovascularization.Therefore,we investigated whetherresveratrol might be able to prevent senescence of EPCs through telomerase activation.EPCs were isolated from peripheral blood and characterized.After ex-vivo prolongedcultivation,EPCs became senescence as determined by acidicβ-galactosidase staining.Resveratrol dose-dependently inhibited the onset of EPC senescence in culture,maximum at 50μmol/L.The effect of resveratrol on senescence was significantlyattenuated by the highly selective phosphoinositide 3-kinase (PI3K) inhibitor(LY294002),but not by the eNOS inhibitor N~G -mono-methyl-L-arginine (L-NAME) orthe estrogen receptor (ER) blocker faslodex (ICI182780).Resveratrol increasedtelomerase activity dose-dependently,which was accompanied with up-regulation of thecatalytic subunit,telomerase reverse transcriptase (TERT),and these effects were alsosignificantly attenuated by LY294002.Immunoblotting analysis showed that treatmentof EPCs with resveratrol resulted in time and dose-dependent Akt phosphorylation.Inaddition,resveratrol.increased proliferative as well as migratory activity of EPCs asassessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation assay,colony-forming assayand modified boyden chamber assay.But 50μmol/L resveratrol failed to reduce EPCs apoptosis significantly.In conclusions,resveratrol delays the onset of EPC senescence,which may be related to telomerase activation by the Akt-dependent mechanism...