Effects And Mechanisms Of Stromal Cell-derived Factor-1a On Number And Activity Of Endothelial Progenitor Cells From Peripheral Blood

Endothelial progenitor cells(EPCs)are a cell population that have the capacity to circulate,proliferate,and differentiate into mature endothelial cells,but have neither acquired characteristic mature endothelial markers nor formed a lumen.EPCs have been shown to be incorporated into sites of physiological and pathological neovascularization in vivo.More recently,it has been suggested that circulating EPCs may also be a marker of endothelial function and cardiovascular risk.Endothelial dysfunction is usually one of the earlier markers of atherosclerosis,and predisposes to vasoconstriction and thrombosis.EPCs may exert an important function as an endogenous repair mechanism to maintain the integrity of the endothelial monolayer by replacing denuded parts of the artery.The maintenance of endothelial monolayer may prevent thrombotic complication and atherosclerotic lesion development.These beneficial properties of EPCs are attractive for cell therapy that targets the endothelial regeneration.However,various risk factors for coronary artery disease,such as aging,diabetes,hypercholesterolemia, hypertension and smoking,affect the number and functional activity of EPCs in healthy volunteers and in patients with coronary artery disease.Ex vivo expansion of EPCs appears to be necessary.Stromal cell-derived factor-1a(SDF-1a)is a member of the CXC chemokine family,which was originally isolated from murine bone marrow stromal cells and characterized as a pre-B-cell growth stimulating factor.CXCR4,a 7-transmembranespanning G protein-coupled receptor,is the receptor for SDF-1a and is also a coreceptor for HIV type 1 infection.Recent studies have shown that SDF-1a/CXCR4 interaction also plays an important role in the regulation of a variety of cellular functions such as migration,proliferation,survival and angiogenesis.On the basis of these considerations,we hypothesized that SDF-1a affected EPCs number and functional activity,thus influenced endothelial repair process and maintained the balance between the magnitude of injury and the capacity for repair, which prevented thrombotic complication and atherosclerotic lesion development.To test this hypothesis,we measured the number and functional activity of EPCs exposed to SDF-1a at first.Then,we studied the mechanisms by which SDF-1a affected EPCs number and functional activity. Part 1:Effects of Stromal cell-derived factor-1a on number and activity of endothelial progenitor cells from peripheral bloodThe aim of this study is to investigate the effects of SDF-1a on number and functional activity of EPCs from peripheral blood.Total mononuclear cells(MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.After 7 days cultured,attached cells were stimulated with SDF-1a(to make a series of final concentrations:1ng/mL,10 ng/mL,50 ng/mL,100 ng/mL)or vehicle control.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of KDR,VEGFR-2 and AC133 with flow cytometry. EPCs proliferation,migration were assayed with 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,modified Boyden chamber assay respectively.EPCs adhesion assay was performed by replating those on fibronectincoated dishes,and then adherent cells were counted.We showed that incubation of isolated human MNCs with SDF-1a dose dependently increased the number of EPCs maximum at 100ng/mL,24h(110.8±26.0 vs 61.8±12.6,P＜0.01).SDF-1a significantly increased the proliferative,migratory and adhesive capacities of EPCs, maximum at 100ng/ml(to compare with that of control subjects,proliferative capacities 0.37±0.06 vs 0.23±0.03,migratory capacities 102.0±22.5 vs 21.5±6.2, adhesive capacities 95.7±21.0 vs 32.2±8.3).Part 2:SDF-1a/CXCR4 decreases endothelial progenitor cells apoptosis under serum deprivation by PI3K/Akt/eNOS pathwayRecent studies have demonstrated that SDF-1a/CXCR4 interaction regulates multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation,survival and angiogenesis.In present study,we aimed to determine the effect of SDF-1a on EPC apoptosis induced by serum deprivation and the implication of phosphoinositide 3-kinase(PI3K)/Akt and mitogen-activated protein kinases(MAPKs) signaling in this effect.EPCs were isolated from peripheral blood and characterized.We showed that EPCs expressed CXCR4 by flow cytometric analysis and reverse transcription-PCR.SDF-1a decreased EPC apoptosis induced by serum deprivation in a dose-dependent manner and the inhibitory effect was CXCR4 dependent as confirmed by the total abolishment by AMD3100,a CXCR4-specific peptide antagonist.SDF-1a treatment also significant decreased caspase-3 expression and activity.The inhibitory effect of SDF-1a on EPC apoptosis was nearly completely abolished by PI3K inhibitors (either Wortmannin or LY294002)and partially abolished by NOS inhibitor,N~G-nitroarginine methyl ester,whereas inhibitors of MAPKs had no significant effect on this inhibitory effect.The treatment of EPCs with SDF-1a resulted in time-dependent Akt, eNOS,ERK1/2,p38 MAPK and c-Jun N-terminal kinase(JNK)phosphorylations.In addition,SDF-1a increased proliferative activity of EPCs as assessed by MTT assay. These findings suggested that PI3K/Akt/eNOS activation,but not MAPKs activation,is required for this inhibitory effect of SDF-1a on EPC apoptosis.Part 3:Stromal cell-derived factor 1a reduces senescence of endothelial progenitor cells through telomerase activationEmerging evidence has suggested that reduced EPCs number and activity was associated with EPC senescence which involved with telomerase activity.Stromal cell-derived factor-1a(SDF-1a)has been shown to augment a variety of cellular functions of EPCs and subsequently contribute to ischemic neovascularization.Therefore,we investigated whether SDF-1a might be able to prevent senescence of EPCs through telomerase activation.EPCs were isolated from peripheral blood and characterized. After ex-vivo prolonged cultivation,EPCs became senescence as determined by acidicβ-galactosidase staining.SDF-1a dose-dependently inhibited the onset of EPC senescence in culture.SDF-1a increased telomerase activity dose-dependently,which was accompanied with up-regulation of the catalytic subunit,telomerase reverse transcriptase(TERT).Whereas these effects of SDF-1a on telomerase activity and expression of hTERT mRNA were significantly attenuated by CXCR4-specific peptide antagonist(AMD3100)and phosphoinositide 3-kinase(PI3K)inhibitor(LY294002). Immunoblotting analysis showed that treatment of EPCs with SDF-1a resulted in time and dose-dependent Akt phosphorylation.In addition,SDF-1a increased proliferative activity of EPCs as assessed by MTT assay and colony-forming activity.In conclusions, SDF-1a delays the onset of EPC senescence,which may be related to telomerase activation.