The Effect Of P38 MAPK On The Endothelial Progenitor Cell Induced By OxLDL

To generate the precursor cell which has the phenotypic characteristics of mature vascular endothelial cell with the endothelial progenitor cell(EPC),which can circulate,proliferate and differentiate into vascular endothelial cell.The previous studies found that EPC is involved not only in the human embryonic angiogenesis but also in the vascular neogenesis after birth and the reparative process after endothelial injuries.The stem cell factor(SCF),vascular endothelial cell growth factor(VEGF),HMG-CoA reductase inhibitor,granulocyte macrophage colony stimulating factor(GM-CSF),etc can mobilize the EPC in marrow and stimulate its abilities of proliferation,differentiation, adhesion and migration.The oxidized low density lipoprotein is an independent risk factor of coronary artery disease and its biological activity is mainly to affect the functions of endothelial cell to further result in artherosclerosis.It has been proved that EPC plays an important role in the maintenance of endothelial structure and the functional integrity and it is also involved in multiple physiological and pathological reconstruction of blood vessel of the organism,meanwhile,the proportion of endothelial cells from EPC is 25%of the total endothelial cells in the new vessel.The function of EPC to stimulate angiogenesis and endothelium regeneration is because that it can be mobilized from the marrow into the peripheral circulation and home to the areas with injury of blood vessel and angiogenesis.The homing of EPC is accomplished through the coordination of multiple steps,which include chemotaxis, adhesion,migration through the endothelim and the final differentiation into endothelial cell.Recently,the clinical studies indicated that the number and the ability of migrated EPC from the patients with coronary disease are significantly decreased.The oxidized low density lipoprotein (oxLDL) is an important and independent risk factor of cardiovascular disease,and the plasma level of oxLDL in the patients with coronary disease and diabetes is significantly increased.The previous studies indicated that oxLDL can induce the endothelial cellular apoptosis, increase the expression of endothelial cell adhesion molecules and inhibit the epithelial cell migration to inhibit the angiogenesis.Thus,we presumed that oxLDL might be the factor which affects the number and function of EPC.The purpose of this study is to observe that whether oxLDL affect the number of EPC in the peripheral blood or change the EPC abilities of proliferation,migration and adhesion,to detect the impact degree of oxLDL to the EPC ability of angiogenesis and to observe that whether oxLDL affect the expression of the P38 signal in EPC to affect the number and function of EPC.This study includes two parts and the main methods and results are as the following:ChapterⅠ.The effects of the oxidized low density lipoprotein to the survival and function of epithelial progenitor cellObject:to investigate the effects of the oxidized low density lipoprotein (oxLDL) to the survival and function of epithelial progenitor cell.Methods:1.The separation and culture of EPC:harvest the peripheral venous blood from the healthy adults at the state of empty stomach, obtain the mononucleated cells through density gradient centrifugation, culture for 7 days and collect the adherent cells.The adherent cells are randomly assigned into 5 groups:1) the control group;2) the groups treated with oxLDL of different concentrations(3 groups):add 25,50, 100 and 200μg/ml oxLDL into the medium,respectively,and culture for 24 h;and 3) LDL group:culture in the conditioned medium containing 100μg/ml LDL for 24 h.2.Staining and identification of the cells:the separated mononucleated cells transformed into spindle-shaped endothelioid cells after 7 d of culture.The cells were identified by the luorescence microscope after the staining by acLDL-Dil and FITC-UEA-I,in which, the positive cells of double staining are proliferating EPCs.Count the cells of each well under the fluorescence microscope(×200).3.Examination of the cellular phenotype:incubate 2×10~5 cells with VEGFR-2,CD31 and monoclonal antibody of CD34 labeled by FITC and KDR labeled by PE for 30 min at 4℃,respectively,and transfer 300μl PBS suspension cells for examination.4.Examination of the adhesion ability of EPC:collect the adherent cells and suspended in 500μl medium for counting,and then inoculate the same amount of cells into the culture plate covered by human fibronectin and count the adherent cells after 30 min of culture.5.Examination of the migration ability of EPC:collect the adherent cells and count.Transferred 25μl medium with VEGF(50 ng/mL) into the lower chamber of the modified Boyden chamber,inject 2×10~4 EPC suspended in 200μl medium into the upper chamber,culture for 24 h,scrape off the cells without migration on the filter membrane and count the cells migrated into the lower part.6.Examination of the proliferation ability of EPC:apply the Non-Radioactive Cell Proliferation Assay(Promega company) kit to detecte the cell proliferation rate by MTS/PMS chromatometry and operate according to the procedures which were simplified as the following:suspended the digested cells of various EPC groups in the EBM-2 containing 0.5%BSA(containing 50 ng/ml VEGF) with the concentration of 1×10~5 cells/ml,respectively,transfer 50μl cell suspension from each group into the 96-well plate in triplication as control,culture for 72 h,add the 15μl prepared staining solution into each well and culture for another 4 h,add the stop solution and incubate overnight.Examine each well by the enzyme-labeled instrument at the wavelength of 570 nm and take the mean value of the triplication as the test value of each group.7.Examination of the in vitro angiogenesis ability:use the in vitro angiogenesis assay kit to detect the angiogenesis ability of EPC. Mix the ECMa t r i x" glum-solution and 10×dilution of ECM after freeze thawing at the proper ratio to form the gum.Digest the adherent cells with 0.250%trypsin to obtain the EPC,re-suspend in the medium and adjust the concentration to 5 x 104cells/ml.Inoculate the EPC on the ECMa t r i x" glum.Culture for 24 h,observe the formation of tubules under the 200×inverted microscope,choose 5 fields(×200) randomly and count the number of tubules.Results:1.Identification of the EPC:the separated mononucleated cells turned to the adherent growth after 3 d of culture and transformed into the spindle-shaped endothelioid cells after 7 d.The cells,which with double staining of UEA-I and DiLDL under the fluorescence microscope after the staining by acLDL-DiI and FITC-UEA-I,were recognized as the proliferating ECP which composed 90%of the adherent cells.The surface markers of the adherent cells were detected by the flow cytometer and the results indicated that the cells expressed KDR(VECFR-2)(68.8±7.5)%, CD34(25.4±9.1%),CD31((77.1±7.2%) and CD144:(73.9±6.3)%, which further confirmed that the cells were EPC.2.The effect of oxLDL to the number of EPC in peripheral blood:after 24 h-exposure of EPC to different concentrations of oxLDL, the results indicated that oxLDL significantly decreased the number of EPC and the number of EPC decreased with the concentration increase of oxLDL.However,the LDL had no effect to the number of EPC.The numbers of EPC in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 46.3±4.85,34.2±3.59 and 22.7±2.38, respectively,which were all significantly lower than the number of EPC in the control group which was 70.7±7.41(P＜0.05).The numbers of EPC in the groups treated by 25μg/ml oxLDL and LDL were 63.8±6.69 and 68.3±7.16,respectively,and there was not significant difference between them(P＞0.05).3.The effect of oxLDL to the adherent ability of the EPC in peripheral blood:oxLDL significantly decreased the number of adherent cells and the number of adherent cells decreased with the concentration increase of oxLDL.However,the LDL had no effect to the adherence. The numbers of adherent EPC in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 21.7±2.28,16.3±1.71 and 10.2±1.07,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 30.2±3.17(P＜0.05).The numbers of adherent EPC in the groups treated by 25μg/ml oxLDL and LDL were 25.8±2.71 and 28.3±2.97,respectively,and there was not significant difference between them(P＞0.05).4.The effect of oxLDL to the migration ability of EPC in peripheral blood:oxLDL of different concentrations all decreased the migration ability of the EPC significantly,but the LDL had no effect to the migration of the EPC.The number of migrated cells in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 16.5±1.73, 10.8±1.13 and 6.2±0.65,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 25.5±2.67(P＜0.05).The numbers of migrated EPC in the groups treated by 25μg/ml oxLDL and LDL were 22.3±2.34 and 23.8±2.50, respectively,and there was not significant difference between them (P＞0.05).5.The effect of oxLDL to the proliferation ability of the EPC in peripheral blood:the proliferation ability of EPC in the groups treated by oxLDL of different concentrations were all inhibited and the inhibitory effect increased with the increase of the concentration.The LDL had no effect to the proliferation of the EPC.The EPC proliferation rates of the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 0.73±0.762,0.45±0.048 and 0.31±0.033,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 1.29±0.135(P＜0.05).The EPC proliferation rates in the groups treated by 25μg/ml oxLDL and LDL were 1.12±0.118 and 1.198±0.126,respectively,and there was not significant difference between them(P＞0.05). 6.The effect of oxLDL to the in vitro angiogenesis ability of EPC in peripheral blood:the in vitro angiogenesis experiment was applied to simulate the in vivo angiogenesis in order to detect the angiogenesis ability of EPC.The results indicated that oxLDL of different concentrations all decreased the angiogenesis ability of the EPC significantly and the inhibitory effect was most significant in the group treated by 200μg/ml oxLDL.However,the LDL had no effect to the angiogenesis ability of the EPC.The numbers of the generated blood vessels in the groups treated by 50μg/ml,100μg/ml and 200μg/ml oxLDL were 19.2±2.01,13.2±1.38 and 9.5±1.00,respectively,which were all significantly lower than the number of adherent EPC in the control group which was 26.2±2.75(P＜0.05).The EPC proliferation rates in the groups treated by 25μg/ml oxLDL and LDL were 23.0±2.41 and 25.8±2.70,respectively,and there was not significant difference between them(P＞0.05).Conclusions:1.OxLDL can decrease the number of EPC in vitro and the effect is concentration dependent;2.OxLDL can impair the EPC abilities of proliferation,migration and adherence and the effect is concentration dependent.3.OxLDL can decrease the in vitro angiogenesis ability of EPC.These results indicated that oxLDL is one of the factors leading to the decreases of EPC amount and the function. ChapterⅡThe effect of p38 MAPK on the endothelial progenitor cell induced by oxLDLObjective:to study that whether oxLDL lead to the abnormal expressions of P38 and P-P38 in EPC and whether the effect of oxLDL to EPC be related to the P38 signal in the cells.Methods:1.The separation,culture and grouping of EPC:see chapterⅠfor the separation and culture of EPC and collect the adherent cells.The adherent cells are randomly assigned into 3 groups:1) the control group; 2) oxLDL group:the medium contains 100μg/ml oxLDL;and 3) SB203580 group:add SB203580 in the medium to 0.5μM at half an hour before adding oxLDL to 100μg/ml.2.The detection of p38 and p-p38 expression by Western blot: detect the p38 and p-p38 expressions in the EPC at 0,5,15,25 and 35 min when the cells were treated by oxLDL and preteated by SB203580, meanwhile,the p38 and p-p38 expressions were also detected in the cells treated by 25,50,100 and 200μg/ml oxLDL and 100μg/ml LDL for 30 min.3.EPC apoptosis analysis:Operate according to the instruction of BD Biosciences company:digest the EPC of each group by 0.1%trypsin after the treatments,wash twice by 4℃PBS and re-suspend in the binding buffer and adjust the concentration to 1×10~6 cells/ml.Transfer 100μl cell suspension into the 5 ml test tube and add 5μl Annexin V-FITC labeling sultion and 5μl PI,and then mix gently.Set the blank control without Annexin V and PI,the control only with Annexin V and the control only with PI.Incubate away from light and at temperature for 15 min,then add 400μl binding buffer and detect by the flow cytometer within 1 h.See chapterⅠfor the methods of the EPC double staining for EPC number and the determinations of the EPC abilities of adherence, migration,proliferation and in vitro angiogenesis.Results:1.The results of Western blot:the p-p38 expression increased in the EPC with time dependence when treated by 100μg/ml oxLDL and the expression level reached peak at 25 min,the ratios between the expression levels of p-p38 in the oxLDL group at 5,15,25,25 and 35 min and those of the control group were 1.4±0.15,2.1±0.22,3.2±0.34 and 1.5±0.13,respectively,meanwhile,the p38 inhibitor SB203580 could inhibit the expression and the ratio to the control group is 1.2±0.13,and there were significant differences at different times(P＜0.05).The observations at 25 min after the treatment of different concentrations indicated that the p-p38 expressions increased with dose dependence,and the ratios between the expression levels of p-p38 in the groups treated by 25μg /ml,50μg /ml,100μg /ml,200μg/ml oxLDL were 1.2±0.13, 1.7±0.18,3.2±0.35 and 3.4±0.35,respectively.LDL had no effect to p-p38 expression.The p38 expression in the EPC was not affected by the time and concentrations.2.The treatment of 100μg/ml oxLDL led to the decrease of EPC amount,reduce of proliferation,increase of apoptosis,the ability decreases of adherence,migration and in vitro angiogenesis.However, the SB203580 could decrease the effect of 100μg/ml oxLDL to EPC. The EPC numbers in the control group,oxLDL group and SB+oxLDL group were 71.8±7.52,32.5±3.40 and 52.8±5.85,respectively;and the adherent cell numbers were 31.7±3.32,16.3±1.71 and 24.5±2.57, respectively;and the migrated cell numbers were 24.2±2.53,10.5±1.10 and 18.2±1.91,respectively;and the numbers of the cells forming in vitro blood vessels were 25.2±2.64,12.3±1.29 and 17.8±1.87,respectively; and the proliferations were 1.32±0.139,0.45±0.048 and 1.133±0.119, respectively;and the apoptosis rates were 10.2±1.07%,21.3±2.23%and 13.2±1.4%respectively.The comparison between the SB+oxLDL group and oxLDL group indicated that there were significant differences on the cell numbers,proliferation,apoptosis and the abilities of adherence, migration and in vitro angiogenesis between these two groups(P＜0.05).Conclusions:the effect of oxLDL to EPC is related to the expression and activity of the intracellular phosphorylated p38.The p38 inhibitor SB203580 can inhibit the toxic actions of oxLDL to EPC.These results suggested that the effect of oxLDL to EPC is transduced by the p38 signal.