Effects Of Actived TLR4 And ADMA On The Proliferation Of Human Umbilical Cord Blood-Derived Endothelial Progenitor Cells

Chapter 1 The Possible Mechanisms Responsible for the effects of the Activation of Toll-Like Receptor 4 expression on Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsPart 1 Differentiation in vitro of two types of human umbilical cord blood-derived endothelial progenitor cellsObjective:to investigate the method for culturing two types of endothelial progenitor cells from human umbilical cord blood and study their differentiation traits.Methods:Mononuclear cells were isolated from fresh cord blood by 6%HES and density gradient centrifugation,Isolated cells were cultured in medium supplemented with VEGF and bFGF,The growth situation and biological features of the cells were observed at different time point and identified by morphology,immunofluorescence staining,RT-PCR and flow cytometry.Results:Attached cells were divided into two types of enthelial progenitor cells:early EPCs and Late EPCs,early EPCs changed from small sized round cells to spindle shape cells,Late EPCs formed a typical cobblestone-like cells.Fluorescence microscopy show that EPCs are positive for both DiI-acLDL uptake and FITC-ulex-lectin binding.RT-PCR and FACS showed the difference of endothelial cell-specific,gene expression and changed AC133,CD34 and KDRin two different EPCs.MTT assay showed the different proliferation kinetics in two different EPCs. Conclusions:There are two different types of EPCs from a source of human umbilical cord blood.They have different phenotype,gene expression and proliferation kinetics.This provide a basis for further research..Part 2 The Possible Mechanisms of Proliferation in Early Endothelial Progenitor Cells Induced by the activation of Toll-Like Receptor 4 expressionObjective:To investigate whether the Toll-like receptors(TLRs)are expressed on EPCs and the effects of lipopolysaccharide(LPS)on the proliferation EPCs and analysis the possible mechanisms of it.Methods:RT-PCR is selected to detect the gene expression in EPCs.FACS is used to detect the protein expression on the surface of EPCs.By using of accounting of the quantity of EPCs and using PI-Annexin V staining tests the apoptosis level of EPCs induced by LPS.Western blot is used to assay the activation of signal transduction molecules such as P38,ERK and NF-kB in EPCsResults:1,RT-PCR results showed that the TLRs except TLR7 were expressed on the surface of EPCs.Proteins of CD14,TLR2 and TLR4 was expressed on early EPCs by FACS,and the expression of these differnt cytokines(IL—8,IFN-α,IFN-β,TNFα)were up-regulated when the EPCs were stimulated with TLR4 ligand LPS.2,The activation of TLR4 by LPS increased the quantity of EPCs,but not induced apoptosis,and pheotype development of EPCs showed that AC133,CD34 mRNA and protein expression were up-regulated.3,LPS induced the phophorylation of NF-kB and MAPK family protein P38,ERK,which belong to the TLR4 signal transduction pathway.Conclusion:1,We first report that TLRs were expressed on early EPCs and that TLR4 was highly expressed on early EPCs.2,Actived TLR4 could not induce the apoptosis of EPCs but the proliferation of it.3,The possible mechanism of the proliferation of early EPCs have a relationship with TLR4 signal transduction pathway.Chapter 2 The Possible Mechanisms Responsible for the effects of Asymmetric Dimethylarginine on Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsPart 1 The Relationship between Asymmetric Dimethylarginine and Circulating Endothelial Progenitor Cells in Patients with Coronary Artery Disease and Its Clinical SignificanceObjective:To investigate the correlation between Asymmetric Dimethylarginine and circulating endothelial progenitor cells(EPCs)in patients with coronary artery disease(CAD)and its clinical significance. Methods:80 cases were divided into2 groups:CAD group(n=50) and control group(n=30),and CAD patients were divided into 3 groups according to the result of coronary angiography and the CAD Gensini cumulative index.The concentration of serum Asymmetric Dimethylarginine was measured.Total mononuclear cell were isolated from peripheral blood by Ficoll density gradient centrifugation,and were cultured in medium supplemented with 10%fetal bovine serum,VEGF and bFGF.After 7-10 days cultured,the number of colony-forming units(CFU)of EPCs were counted by phase-contrast microscope.Results:1,The concentration of plasma Asymmetric Dimethylarginine level in multitude vessels disease group was significantly higher than those in control group(P＜0.01),and in Gensini scores≥60 group,Asymmetric Dimethylarginine level was significantly higher than those in control group(P＜0.01).2,meanwhile,The number of colony-forming units of circulating EPCs in multitude vessels disease group was significantly lower than those in control group(P＜0.01),and in Gensini scores≥60 group the circulating EPCs level were significantly lower than that of control group (P＜0.01).It was also observed there was a strong negative correlation between the concentration of serum Asymmetric Dimethylarginine and the number of colony-forming units of circulating EPCs (r=-0.416,P＜0.01).Conclusions:1.Correspond with the degree of coronary atherosclerosis made heavyer,The serum Asymmetric Dimethylarginine concentration,in CAD group was higher than that of control group,The levels of circulating EPCs of CAD patients were lower than those of healthy person.Asymmetric Dimethylarginine accumulation may contribute to endothelial progenitor cell depletion,with consequent impairment of endothelial progenitor cell-mediated endothelial repair,which can promote atherogenesis.2.Asymmetric Dimethylarginine was negatively correlated with the number of colony-forming units of circulating EPCs..These data indicate that Asymmetric Dimethylarginine may impair EPCs-mediated reendothelialization through some mechanisms.Part 2 Influence of asymmetric dimethylarginine on the proliferation of endothelial progenitor cells and the antagonism of L-arginineObjective:To investigate the effects of asymmetric dimethylarginine (ADMA)on the proliferation of endothelial progenitor cells(EPCs)of human umbilical cord blood and observe whether L-arg can antagonize the effects of ADMA,Furthermore,this study aims to explore the possible mechanisms responsible for ADMA on EPCs.Methods:Mononuclear cells were isolated from fresh cord blood and cultured for 7days,Attached cells were incubated with different concentration of ADMA(1μmol/l,5μmol/l,10μmol/l)among different times(24,48,72hr).MTT assay was used and colony forming units (CFU)were quantified to evaluate the proliferation of EPCs after treated with ADMA.Meanwhile,MTT assay was used and DiI-acLDL uptake cells under high-power field of fluorescence microscopy were quntified to assay effects of L-arg on ADMA-induced EPCs proliferation.EPCs were randomly assigned to five groups:control,ADMA(10μmol/L),L-arg (1mmol/L)pre-teatment,intracellular antioxidant pyrollidine dithiocarbama(PDTC 50μmol/L)pre-teatment and L-arg(1mmol/L). 2' 7'-dichlorofluorescein(DCF).was used to measured the cellular reactive oxygen species(ROS).RT-PCR was employed to detect mRNA expression of NADPH oxidase subunit p22 phox,gp91phox and the intracellular antioxidative enzymes manganese superoxide dismutase(MnSOD).Nitric oxide(NO)and nitric oxide synthase(NOS)in the supernatant were measured nitrate reductase assay and chemical colorimetry assay.Results:1,Incubation of EPCs with ADMA dose and time-dependently decreased the number and the proliferation of EPCs.2,In addition,the depressant effect of ADMA on EPCs were dismissed after giving the inceasing dosage of L-arg.3,ADMA significantly induced the production of intracellular ROS.L-arg and PDTC inhibited ADMA-induced ROS generation.4,ADMA stimulated NADPH oxidase subunit gp91phox and p22phox mRNA expression.L-arg and PDTC inhibited the up-regulation of gp91phox and p22phox mRNA expression.5,ADMA had no effect on the intracellular antioxidative enzymes MnSOD mRNA expression.Conclusions:1,It is suggested that ADMA can suppress the reendothelialization by means of depressant EPCs proliferation,decreased the concentration of NO,induced the production of intracellular ROS and NADPH oxidase expression.2,Exogeno L-arg maybe antagonize the effects of ADMA on the proliferation and inhibit ADMA-induced oxidative stress in EPCs.L-arg have a protective roles on EPCs.