P38信号通路在氧化性低密度脂蛋白导致内皮祖细胞数量及功能改变方面的作用
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摘要
内皮祖细胞(EPC)是一类能循环、增殖并分化为血管内皮细胞,但尚未表达成熟血管内皮细胞表型特征的前体细胞。研究发现,EPC不仅参与人胚胎血管生成,同时也参与出生后血管新生和内皮损伤后的修复过程。干细胞因子(SCF)、血管内皮细胞生长因子(VEGF),HMG-coA还原酶抑制剂、粒系巨噬系集落刺激因子(GM-CSF)等都能白骨髓动员EPC,并促进其增殖、分化、粘附和迁移能力.氧化性低密度脂蛋白是冠心病独立危险因素,其生物学作用主要是影响内皮细胞功能,并进一步导致动脉粥样硬化。研究证明EPC对维持内皮结构和功能的完整性具有重要作用,并且参与了机体多种生理、病理性血管重建过程,在新形成的血管中,EPC来源的内皮细胞约占总内皮细胞的25%。EPC促进血管生成和内皮再生是因为EPC能够从骨髓动员到外周循环中,并归巢到血管损伤和生成的部位。EPC归巢需要多步骤协调作用,包括趋化、粘附、跨内皮迁移及最终分化为内皮细胞。最近,临床试验表明冠状动脉疾病患者培养的EPC数量及迁移能力明显下降。氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)为心血管疾病的重要独立危险因素,而且冠状动脉疾病及糖尿病患者血浆oxLDL生成明显增加。以前的研究表明oxLDL能够诱导内皮细胞凋亡、增加内皮细胞粘附分子的表达、抑制内皮细胞迁移而抑制血管新生。基于以上研究背景,我们推测。oxLDL可能为影响EPC数量和功能的因素。本研究的目的是观察oxLDL是否影响外周血EPC的数量;是否改变了EPC的增殖功能、迁移功能及粘附能力;oxLDL对EPC的体外血管生成能力的影响程度;同时观察oxLDL是否影响EPC的P38信号表达,来影响EPC的数量、功能。本研究分为二个部分,主要研究方法和结果如下:
第一章氧化型低密度脂蛋白对内皮祖细胞存活和功能的影响
目的:
研究氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)对EPC存活及功能的影响
方法:
1.EPC的分离、培养:取健康成人空腹外周静脉血,用密度梯度离心法获取单个核细胞,培养7天,收集贴壁细胞。贴壁细胞随机分成5组:①对照组;②oxLDL各浓度组(共3组):在培养液中分别加入25,50,100,200μg/ml后培养24小时;③LDL组:含100μg/ml的条件培养液培养24h。
2.细胞染色与鉴定:分离获得的单个核细胞培养7d后形成了梭形的内皮样细胞。用acLDL-Dil和FITC-UEA-I对细胞染色后,通过荧光显微镜鉴定,FITC-UEA-I和Dil-acLDL双染色阳性细胞为正在分化的EPCs,在荧光显微镜下(~*200)对每孔细胞进行计数
3.细胞表型检测:将2×10~5贴壁细胞分别与FITC标记的VEGFR-2,CD31和CD34单克隆杭体以及PE标记的KDR,在4℃孵育30 min后,用300μl PBS悬浮细胞后上机检测。
4.EPC粘附能力检测:收集贴壁细胞,悬浮在500μl培养液并计数,然后将等量EPC接种到包被有人纤维连接蛋白培养板,在37℃培养30 min,计数贴壁细胞。
5.EPC迁移能力检测:收集贴壁细胞并计数。将600μl培养液和VEGF(50 ng/mL)加入改良的Boyden室的下室,将2×10~4 EPC悬浮在100μl培养液注入上室,培养24 h,刮去滤膜上面的未移动细胞,计数迁移到低层的细胞。
6.EPC增殖能力检测:采用promega公司的Non-Radioactive CellProliferation Assay试剂盒MTS/PMS比色法检测细胞增殖率,按操作程序执行,简化如下:将消化的各组EPC细胞溶于含0.5%BSA的EBM-2中(含50ng/ml的VEGF)其中细胞浓度是1×10~5/ml,取各组细胞液50μl加入96孔,设三孔对照,培养72小时,各孔加入配备的染液15μl,再培养4小时,加停止液,过夜。置酶标仪于波长570nm记录各孔值,三孔对照均值作为各组本次试验值,
7.体外血管生成能力检测:采用体外血管生成试剂盒检测EPC的血管生成能力.将ECM成胶。胰蛋白酶消化贴壁细胞获取EPC,并重新悬浮于培养液,调整细胞数为5×10~4/ml。将EPC接种于ECM胶上。37℃培养24h,在200倍倒置显微镜下观察小(血)管生成情况,随机选择6个显微镜视野(x200),计数小管数。
结果:
1.EPC的鉴定:分离获得的单个核细胞培养3天即可看到贴壁生长,7天后形成梭形的内皮样细胞。用acLDL-DiI和FITC-UEA-I对细胞染色后,通过荧光显微镜鉴定UEA-I和DiLDL双染色阳性细胞被认为是正在分化的EPC占贴壁细胞的90%。流式细胞仪检测贴壁细胞表面标志,结果显示:表达KDR(VECFR-2)(68.8±7.5)%,CD34(25.4±9.1%),CD31(71.2±7.2%)和CD144:(73.9±6.3)%,进一步明确为EPC。
2.oxLDL对外周血EPC数量的影响:
不同浓度的OxLDL干预EPC后24小时,结果显示:oxLDL显著减少EPC数量,并且EPC数量随着oxLDL的浓度的增加而减少。而LDL对EPC数量无影响。50μg/ml 100μg/ml,200μg/ml的oxLDL组导致EPC数量分别为46.3±4.85,34.2±3.59,22.7±2.38都较对照组EPC的70.7±7.41低,差异有显著性,P＜0.05。而oxLDL25μg/ml及LDL导致EPC数量为63.8±6.69,68.3±7.16,差异无显著性,P＞0.05
3.OxLDL对外周血EPC粘附能力的影响:oxLDL显著减少EPC的贴壁数,并且贴壁数随着其浓度的增加而减少,而LDL对EPC的贴壁无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC粘附数分别为21.7±2.28,16.3±1.71,10.2±1.07都较对照组EPC的30.2±3.17低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC粘附数为25.8±2.71,28.3±2.97,差异无显著性,P＞0.05。
4.OxLDL对外周血EPC迁移功能的影响:OxLDL各浓度组明显减少EPC的迁移能力,而LDL对EPC的迁移无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC迁移数分别为16.5±1.73,10.8±1.13,6.2±0.65都较对照组EPC的25.5±2.67低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC迁移数为22.3±2.34,23.8±2.50,差异无显著性,P＞0.05。
5.OxLDL对外周血EPC增殖功能的影响:EPC的增殖能力在OxLDL各浓度组抑制了EPC的增殖,并且随着其浓度的增加而加剧。LDL对EPC的增殖无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC增值率分别为0.73±0.762,0.45±0.048,0.31±0.033都较对照组EPC的1.29±0.135低,差异有显著性,P＜0.05。而oxLDL25μg/ml及LDL导致EPC增值率为1.12±0.118,1.198±0.126,差异无显著性,P＞0.05。
6.OxLDL对外周血EPC体外血管生成能力的影响:体外血管生成实验模拟体内血管生成,检测EPC参与血管新生的能力.结果显示,OxLDL各浓度组显著减弱EPC的体外血管生成能力,在浓度为200μg/ml时最为显著。而LDL对EPC的成血管能力无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC成血管数数分别为19.2±2.01,13.2±1.38,9.5±1.00都较对照组EPC的26.2±2.75低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC迁移数为23.0±2.41,25.8±2.70,差异无显著性,P＞0.05。
结论:
1.OxLDL在体外能减少EPC数量,作用呈浓度依赖性;
2.OxLDL在体外能抑制EPC的增殖、迁移、粘附能力,作用呈浓度和依赖性;
3.OxLDL能降低EPC的体外血管生成能力:
提示OxLDL是导致EPC数量及功能减弱的因素之一。
第二章P38信号通路参与oxLDL对内皮祖细胞数量和功能的影响
目的:
研究oxLDL是否导致EPC细胞内P38及P-P38的表达异常,以及oxLDL对EPC的作用是否与细胞内P38的信号有关。
方法:
1.EPC分离,培养,及分组:EPC的分离及培养见第一章,收集贴壁细胞。贴壁细胞随机三组:①对照组:②oxLDL组:在培养液中加入100μg/ml的oxLDL③SB203580组:在培养液中加入100μg/ml的oxLDL前,预先半小时加入0.5μM的SB203580。
2.Western检测p38及p-p38的表达:oxLDL(100μg/ml)处理EPC后0,5,15,25,35分钟及SB203580预处理后细胞内p38,及p-p38蛋白的表达;以及oxLDL 25,50,100,200μg/ml以及LDL100μg/ml处理30分钟后细胞内p38,及p-p38蛋白的表达。
3.EPC凋亡分析:
按BD Biosciences公司的说明进行操作:各组EPC处理结束后,用0.1%胰酶消化,4°C PBS洗两次后,重悬于结合缓冲液中,调整细胞浓度为1 X 106/ml。取100μl细胞悬液至5 ml试管中,加5μlAnnexin V-FITC标记液和5μl PI,轻轻混匀。并设立无Annexin V及PI的空白对照、仅Annexin V的对照、仅PI的对照。室温避光孵育15min后,加400μl结合缓冲液,1小时内上流式细胞仪检测EPC双染色鉴定EPC数量,EPC粘附能力检测,迁移能力检测,EPC增殖能力检测,体外血管生成能力检测实验方法见第一章
结果:
1.western检测结果:100μg/ml的oxLDL处理EPC后,细胞内p-p38成时间依赖性表达增强,大约25min时达顶峰,5,15,25,25,35分钟细胞内p-p38的表达是对照组的1.4±0.15倍,2.1±0.22倍,3.2±0.34倍,1.5±0.13倍,且p38拮抗剂sb203580可抑制其表达,是对照组的1.2±0.13倍,与各时间点比较有显著性差异,P＜0.05。不同浓度的OxLDL处理25min后观察,细胞内p-p38成剂量依赖性表达增强,25μg/ml,50μg/ml,100μg/ml,200μg/ml处理EPC后,其细胞内p-p38的表达是分别对照组的1.2±0.13,1.7±0.18,3.2±0.35,3.4±0.35。而LDL对其没影响。细胞内p38表达不受时间和浓度的影响。
2.100μg/ml的oxLDL处理EPC后,导致EPC数量减少,增殖减低,凋亡增加,粘附及迁移能力减低,体外成血管能力减弱。而sb203580能减弱100μg/ml的oxLDL对EPC的作用。其中对照组,oxLDL组和SB+oxLDL组的EPC数量分别为71.8±7.52,32.5±3.40,52.8±5.85;粘附细胞数分别为31.7±3.32,16.3±1.71,24.5±2.57;迁移细胞数分别为24.2±2.53,10.5±1.10,18.2±1.91;体外成血管细胞数分别为25.2±2.64,12.3±1.29,17.8±1.87。各组增殖率分别为1.32±0.139,0.45±0.048,1.133±0.119。各组的凋亡率分别10.2±1.07%,21.3±2.23%,13.2±1.4%。SB+oxLDL与oxLDL组比较,在细胞数量,增殖,凋亡及黏附,迁移,体外成血管能力方面有差异显著性,P＜0.05。
结论:
oxLDL对EPC的作用与细胞内磷酸化的p38表达及活性有关。P38的抑制剂sb203580能抑制oxLDL对EPC的毒性作用。提示oxLDL对EPC的作用是通过细胞p38信号作用的。
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 cell
Object:
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 oxLDL
Objective:
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.
第一章氧化型低密度脂蛋白对内皮祖细胞存活和功能的影响
目的:
研究氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)对EPC存活及功能的影响
方法:
1.EPC的分离、培养:取健康成人空腹外周静脉血,用密度梯度离心法获取单个核细胞,培养7天,收集贴壁细胞。贴壁细胞随机分成5组:①对照组;②oxLDL各浓度组(共3组):在培养液中分别加入25,50,100,200μg/ml后培养24小时;③LDL组:含100μg/ml的条件培养液培养24h。
2.细胞染色与鉴定:分离获得的单个核细胞培养7d后形成了梭形的内皮样细胞。用acLDL-Dil和FITC-UEA-I对细胞染色后,通过荧光显微镜鉴定,FITC-UEA-I和Dil-acLDL双染色阳性细胞为正在分化的EPCs,在荧光显微镜下(~*200)对每孔细胞进行计数
3.细胞表型检测:将2×10~5贴壁细胞分别与FITC标记的VEGFR-2,CD31和CD34单克隆杭体以及PE标记的KDR,在4℃孵育30 min后,用300μl PBS悬浮细胞后上机检测。
4.EPC粘附能力检测:收集贴壁细胞,悬浮在500μl培养液并计数,然后将等量EPC接种到包被有人纤维连接蛋白培养板,在37℃培养30 min,计数贴壁细胞。
5.EPC迁移能力检测:收集贴壁细胞并计数。将600μl培养液和VEGF(50 ng/mL)加入改良的Boyden室的下室,将2×10~4 EPC悬浮在100μl培养液注入上室,培养24 h,刮去滤膜上面的未移动细胞,计数迁移到低层的细胞。
6.EPC增殖能力检测:采用promega公司的Non-Radioactive CellProliferation Assay试剂盒MTS/PMS比色法检测细胞增殖率,按操作程序执行,简化如下:将消化的各组EPC细胞溶于含0.5%BSA的EBM-2中(含50ng/ml的VEGF)其中细胞浓度是1×10~5/ml,取各组细胞液50μl加入96孔,设三孔对照,培养72小时,各孔加入配备的染液15μl,再培养4小时,加停止液,过夜。置酶标仪于波长570nm记录各孔值,三孔对照均值作为各组本次试验值,
7.体外血管生成能力检测:采用体外血管生成试剂盒检测EPC的血管生成能力.将ECM成胶。胰蛋白酶消化贴壁细胞获取EPC,并重新悬浮于培养液,调整细胞数为5×10~4/ml。将EPC接种于ECM胶上。37℃培养24h,在200倍倒置显微镜下观察小(血)管生成情况,随机选择6个显微镜视野(x200),计数小管数。
结果:
1.EPC的鉴定:分离获得的单个核细胞培养3天即可看到贴壁生长,7天后形成梭形的内皮样细胞。用acLDL-DiI和FITC-UEA-I对细胞染色后,通过荧光显微镜鉴定UEA-I和DiLDL双染色阳性细胞被认为是正在分化的EPC占贴壁细胞的90%。流式细胞仪检测贴壁细胞表面标志,结果显示:表达KDR(VECFR-2)(68.8±7.5)%,CD34(25.4±9.1%),CD31(71.2±7.2%)和CD144:(73.9±6.3)%,进一步明确为EPC。
2.oxLDL对外周血EPC数量的影响:
不同浓度的OxLDL干预EPC后24小时,结果显示:oxLDL显著减少EPC数量,并且EPC数量随着oxLDL的浓度的增加而减少。而LDL对EPC数量无影响。50μg/ml 100μg/ml,200μg/ml的oxLDL组导致EPC数量分别为46.3±4.85,34.2±3.59,22.7±2.38都较对照组EPC的70.7±7.41低,差异有显著性,P＜0.05。而oxLDL25μg/ml及LDL导致EPC数量为63.8±6.69,68.3±7.16,差异无显著性,P＞0.05
3.OxLDL对外周血EPC粘附能力的影响:oxLDL显著减少EPC的贴壁数,并且贴壁数随着其浓度的增加而减少,而LDL对EPC的贴壁无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC粘附数分别为21.7±2.28,16.3±1.71,10.2±1.07都较对照组EPC的30.2±3.17低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC粘附数为25.8±2.71,28.3±2.97,差异无显著性,P＞0.05。
4.OxLDL对外周血EPC迁移功能的影响:OxLDL各浓度组明显减少EPC的迁移能力,而LDL对EPC的迁移无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC迁移数分别为16.5±1.73,10.8±1.13,6.2±0.65都较对照组EPC的25.5±2.67低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC迁移数为22.3±2.34,23.8±2.50,差异无显著性,P＞0.05。
5.OxLDL对外周血EPC增殖功能的影响:EPC的增殖能力在OxLDL各浓度组抑制了EPC的增殖,并且随着其浓度的增加而加剧。LDL对EPC的增殖无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC增值率分别为0.73±0.762,0.45±0.048,0.31±0.033都较对照组EPC的1.29±0.135低,差异有显著性,P＜0.05。而oxLDL25μg/ml及LDL导致EPC增值率为1.12±0.118,1.198±0.126,差异无显著性,P＞0.05。
6.OxLDL对外周血EPC体外血管生成能力的影响:体外血管生成实验模拟体内血管生成,检测EPC参与血管新生的能力.结果显示,OxLDL各浓度组显著减弱EPC的体外血管生成能力,在浓度为200μg/ml时最为显著。而LDL对EPC的成血管能力无影响。50μg/ml,100μg/ml,200μg/ml的oxLDL组导致EPC成血管数数分别为19.2±2.01,13.2±1.38,9.5±1.00都较对照组EPC的26.2±2.75低,差异有显著性,P＜0.05。而oxLDL 25μg/ml及LDL导致EPC迁移数为23.0±2.41,25.8±2.70,差异无显著性,P＞0.05。
结论:
1.OxLDL在体外能减少EPC数量,作用呈浓度依赖性;
2.OxLDL在体外能抑制EPC的增殖、迁移、粘附能力,作用呈浓度和依赖性;
3.OxLDL能降低EPC的体外血管生成能力:
提示OxLDL是导致EPC数量及功能减弱的因素之一。
第二章P38信号通路参与oxLDL对内皮祖细胞数量和功能的影响
目的:
研究oxLDL是否导致EPC细胞内P38及P-P38的表达异常,以及oxLDL对EPC的作用是否与细胞内P38的信号有关。
方法:
1.EPC分离,培养,及分组:EPC的分离及培养见第一章,收集贴壁细胞。贴壁细胞随机三组:①对照组:②oxLDL组:在培养液中加入100μg/ml的oxLDL③SB203580组:在培养液中加入100μg/ml的oxLDL前,预先半小时加入0.5μM的SB203580。
2.Western检测p38及p-p38的表达:oxLDL(100μg/ml)处理EPC后0,5,15,25,35分钟及SB203580预处理后细胞内p38,及p-p38蛋白的表达;以及oxLDL 25,50,100,200μg/ml以及LDL100μg/ml处理30分钟后细胞内p38,及p-p38蛋白的表达。
3.EPC凋亡分析:
按BD Biosciences公司的说明进行操作:各组EPC处理结束后,用0.1%胰酶消化,4°C PBS洗两次后,重悬于结合缓冲液中,调整细胞浓度为1 X 106/ml。取100μl细胞悬液至5 ml试管中,加5μlAnnexin V-FITC标记液和5μl PI,轻轻混匀。并设立无Annexin V及PI的空白对照、仅Annexin V的对照、仅PI的对照。室温避光孵育15min后,加400μl结合缓冲液,1小时内上流式细胞仪检测EPC双染色鉴定EPC数量,EPC粘附能力检测,迁移能力检测,EPC增殖能力检测,体外血管生成能力检测实验方法见第一章
结果:
1.western检测结果:100μg/ml的oxLDL处理EPC后,细胞内p-p38成时间依赖性表达增强,大约25min时达顶峰,5,15,25,25,35分钟细胞内p-p38的表达是对照组的1.4±0.15倍,2.1±0.22倍,3.2±0.34倍,1.5±0.13倍,且p38拮抗剂sb203580可抑制其表达,是对照组的1.2±0.13倍,与各时间点比较有显著性差异,P＜0.05。不同浓度的OxLDL处理25min后观察,细胞内p-p38成剂量依赖性表达增强,25μg/ml,50μg/ml,100μg/ml,200μg/ml处理EPC后,其细胞内p-p38的表达是分别对照组的1.2±0.13,1.7±0.18,3.2±0.35,3.4±0.35。而LDL对其没影响。细胞内p38表达不受时间和浓度的影响。
2.100μg/ml的oxLDL处理EPC后,导致EPC数量减少,增殖减低,凋亡增加,粘附及迁移能力减低,体外成血管能力减弱。而sb203580能减弱100μg/ml的oxLDL对EPC的作用。其中对照组,oxLDL组和SB+oxLDL组的EPC数量分别为71.8±7.52,32.5±3.40,52.8±5.85;粘附细胞数分别为31.7±3.32,16.3±1.71,24.5±2.57;迁移细胞数分别为24.2±2.53,10.5±1.10,18.2±1.91;体外成血管细胞数分别为25.2±2.64,12.3±1.29,17.8±1.87。各组增殖率分别为1.32±0.139,0.45±0.048,1.133±0.119。各组的凋亡率分别10.2±1.07%,21.3±2.23%,13.2±1.4%。SB+oxLDL与oxLDL组比较,在细胞数量,增殖,凋亡及黏附,迁移,体外成血管能力方面有差异显著性,P＜0.05。
结论:
oxLDL对EPC的作用与细胞内磷酸化的p38表达及活性有关。P38的抑制剂sb203580能抑制oxLDL对EPC的毒性作用。提示oxLDL对EPC的作用是通过细胞p38信号作用的。
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 cell
Object:
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 oxLDL
Objective:
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.
引文
[1] Stary HC. Evolution and progression of atherosclerotic lesions in coronary arteries of children and young adults. Arteriosclerosis. 1989;9(1 Suppl):I19-32.
[2] Buschmann I, Schaper W. The pathophysiology of the collateral circulation (arteriogenesis). J Pathol. 2000;190:338-342.
[3] RisauW. Mechanisms of angiogenesis.Nature. 1997;386:671-674
[4] Risau W, Flamme I. Vasculogenesis. Annu Rev Cell Dev Biol. 1995; 11:73-91.
[5] Satoshi S,Toyoaki,Hisao 1, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation, 2001, 103:2776-2779.
[6] Gill M, Dias S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2+AC 133+ endothelial precursor cells.Circ Res, 2001,88:167-174.
[7] Isner J,Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest, 1999, 103:1231- 1236.
[8] Kawamoto A, Gwon H C,Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia.Circulation,2001, 103:634-637.54
[9] Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001;89:E1-7.30
[10] Loomans CJ, de Koning EJ, Staal J, et al. Endothelial progenitor cell dysfunction:a novel concept in the pathogenesis of vascular complications of type 1 diabetes.2004; Diabetes. 53:195-199.
[11] Tepper OM, Galiano RD, Capla JM, et al. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002.106:2781-2786.
[12] Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 2001; 103:2885-90.
[13] Yla-Herttuala S, Palinski W Rosenfeld ME, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989; 84:1086-1095.
[14] Witztum JL. Role of modified lipoproteins in diabetic macroangiopathy.Diabetes. 1997; 46:S112-114.
[15] Khan BV, Parthasarathy SS, Alexander RWet al. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest. 1995; 95:1262-70.
[16] Harada-Shiba M, Kinoshita M, Kamido H,et al. Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms. J Biol Chem. 1998 ;273:9681-7.
[17] Plane F, Bruckdorfer KR, Kerr P, et al .Oxiditive modification of low- density lipoproteins and the inhibition of relaxations mediated endothelial derived nitric oxide in rabbit aorta. Br J Pharmacol. 1992; 105:216- 222
[18] Oren M. Tepper, Robert D. Galiano, Jennifer M. Capla,et al.Human Endothelial Progenitor Cells From Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation Into Vascular Structures. Circulation,2002,106:2781-2786
[19] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature,1993,362:801-809.
[20] Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med. 2005;353:999 -1007.
[21] Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events:proof of concept for the clinical importance of endogenous vascular repair.Circulation. 2005; 111:2981-2987.
[22] Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerate reendothelialization. Circulation, 2002, 105:3017-3024.
[23] Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med, 2003 348:593-600.
[24] Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation, 2001, 104:1046-1052.
[25] Rehman J, Li J, Orschell CM, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation, 2003, 107:1164-1169.
[26] Vajkoczy P, Blum S, Lamparter M, et al. Multistep nature of microvascular recruitment of ex vivo-expanded embryonic endothelial progenitor cells during tumor angiogenesis. J Exp Med.2003;197:1755-1765.
[27] Adams GB, Chabner KT, Foxall RB, et al. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment. Blood 2003; 101:45-51.
[28] Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases. J Pharmacol Sci.2003;91:172-176.
[29] Seeger FH, Haendeler J, Walter DH, Rochwalsky U, Reinhold J, Urbich C, et al.p38 mitogen-activated protein kinase downregulates endothelial progenitor cells.Circulation 2005; 111: 1184- 1191.
[30] Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events:proof of concept for the clinical importance of endogenous vascular repair.Circulation. 2005; 111:2981 -2987.
[31] Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerate reendothelialization. Circulation, 2002, 105:3017-3024.
[32] Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells,vascular function, and cardiovascular risk. N Engl J Med, 2003 348:593-600.
[33] Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation, 2001, 104:1046-1052.
[34] Rehman J, Li J, Orschell CM, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation, 2003,107:1164-1169.
[35] Adams RH, Porras A, Alonso G, Jones M, Vinterstewn K, Panelli S,et al.Essential role of p38aMAP kinase in placental but not embryonic cardiovascular development. Mol Cell 2000; 6: 106-116.
[36] Matsumoto T, Turesson I, Book M, Gerwins P, Claesson-Welsh L. p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2-stimulated angiogenesis. J Cell Biol 2002; 156:149-160.
[37] Gratton JP, Morales-Ruiz M, Kureishi Y, Fulton D, Walsh K, Sessa WC. Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells. J Biol Chem 2001; 276: 30359-30365.
[38] Taro M,Ingela T,Majlis B,et al.P38MAP Kinase negatively regulates endothelial cell survival,proliferation and differentiation in FGF-2-stimulated angiogenesis.J Cell Biology,2002,156( 1): 149-160.
[1]Jiang Y,Li Z,Schwarz EM,et al.Structure-function studies of p38mitogen-activated protein kinase.Loop 12 influences substrate specificity and autophosphorylation,but not upstream kinase selection.J Biol Chem,1997,272(7):11096-11102.
[2]Kan W,Zhao KS,Jiang Yet al.Role of p38 mitogen-activated protein kinase in signal transduction of inducible of nitric oxide synthase expression.Shock,2004,21(3) 281-287.
[3]Jiang YChen C,Li Z,et al.Characterizadon of the structure and function of a new mitogen-activated protein kinase(p38b).J Biol Chem,1996,271(30):17920-17926.
[4]Serger R,Krebs EGI.The MAPK signaling cascade.FASEB J,1995,9(9):726-735.
[5]Brewster JL,De Valoir T,DwyND,et al.An osmosensing signal transduction pathway inyeast.J.Science,1993,259(5099):1760-1763.
[6]Han J,Lee JD,Bibbs L,et ol.AMAP kinase targeted by endotoxin and hyperosmolarityin mammalian cells.Science,1994,265(5173):808-811.
[7]Bodero AJ,Ye R,Lees-Miller SPo UV-light induces p38 MAPK-dependent phosphorylation of Bcll0.Biomchem Biophys Res Commun.2003,301(4):923-926.
[8]Taro M,Ingela T,Majlis B,et al.P38MAP Kinase negatively regulates endothelial cell survival,proliferation and differentiation in FGF-2-stimulated angiogenesis.J Cell Biology,2002,156(1):149-160.
[9] Mi-Sun K,Hyun J, Jae-Young U,et al .Agonists of proteinase-activated receptor 2 induce TNF-alpha secretion from astrocytoma cells.Cell Biomchem Funct, 2002,20 (4): 339-345.
[10] Huang CD,Tliba O;Panettieri RA,et al.Bradykinin induces interleukin-8 production in human aiyway smooth muscle cells:modulation by TV cytokines and dexamethasone.Am J Respir Cell Mol Biol,2003,28(3):330-338.
[11] Raingeaud J, Whitmarsh AJ, Barrett T, et al . MKK3 and MKK6 regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathwayl. Mol Cell Biol, 1996, 16(3): 1247. 1255.
[12] Bagrodia S, Derijard B, Davis RJ, Cerione RA. Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J Biol Chem. 1995 Nov 24;270(47):27995-8.
[13] Zhang S,Huan J,Sells MA,et al. Rho Family GTPases Regulate p38 Mitogen-activated Protein Kinase through the Downstream Mediator Pak1. J Biol Chem, 1995,270:23934-23936.
[14] Knaus UG,Morris S, Dong HJ,et al. Regulation of Human Leukocyte p21-Activated Kinases Through G Protein-Coupled Receptors. Science,1995,269:221-223.
[15] Manner E,Leung T,Salihuddin H,et al. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature, 1994,367:40-46.
[16] Chan-Hui PY,Weaver R. Biochem J, Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades 。 Biochem J. 1998 Dec 15;336 ( Pt 3):599-609 .
[17] Tibbies LA,Ing YL,Kiefer F,et al. MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6. EMBO J, 1996,15:7026-7035.
[18] Cuenda A ,Dorow DS. Biochem J, Differential activation of stress-activated protein kinase kinases SKK4/MKK7 and SKK1/MKK4 by the mixed-lineage kinase-2 and mitogen-activated protein kinase kinase (MKK) kinase-1 1998,333(Pt 1): 11-15.
[19] Terada Y,Nakashima O,Inoshita S,et al. Mitogen-activated protein kinase cascade and transcription factors: the opposite role of MKK3/6-p38K and MKK1-MAPK. Nephrol Dial Transplant, 1999,1:45-47.
[20] Moriguchi T,Kuroyanagi N,Yamaguchi K,et al. A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3. J Biol Chem,1996,271:13675-13679.
[21 ] Stein B,Brady H,Yang MX,et al. Cloning and characterization of MEK6, a novel member of the mitogen-activated protein kinase kinase cascade. J Biol Chem,1996,271:11427-11433.
[22] Nick JA,Avdi NJ,Young SK,et al. Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils . J Clin Invest, 1999,103: 851-858.
[23] Lu HT,Yang DD,Wysk M,et al. Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice . EMBO J,1999,18:1845-1857.
[24] Han J,Wang X,Jiang Y,et al. Identification and characterization of a predominant isoform of human MKK3. FEBS Lett, 1997,403:19-22.
[25] Jiang Y,Li Z,Schwarz EM,et al. Structure-function studies of p38 mitogen-activated protein kinase. J Biol Chem, 1997,272:11096-11102.
[26] Zhao M,New L,Kravchenko VV,et al. Regulation of the MEF2 family of transcription factors by p38. Mol Cell Biol, 1999,19:21-30.
[27] Ornatsky OI, Cox DM, Tangirala P, Andreucci JJ, Quinn ZA, Wrana JL, Prywes R, Yu YT, McDermott JC. Post-translational control of the MEF2A transcriptional regulatory protein. Nucleic Acids Res. 1999 Jul 1;27(13):2646-54.
[28] Yang SH,Galanis A,Sharrocks AD. Targeting of p38 mitogen-activated protein kinases to MEF2 transcription factors. .Mol Cell Biol, 1999,19: 4028-4038.
[29] Han J,Jiang Y,Li Z,et al. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature, 1997,386: 296-299.
[30] Hai T,Wolfgang CD,Marsee DK,et al. ATF3 and stress responses. Gene Expr, 1999,7:321-335
[31] Thuerauf DJ,Arnold ND,Zechner D,et al. p38 Mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6. J Biol Chem, 1998,273:20636-20643.
[32] Casillas AM,Amaral K,Chegini-Farahani S,et al. Okadaic acid activates p42 mitogen-activated protein kinase (MAP kinase; ERK-2) in B-lymphocytes but inhibits rather than augments cellular proliferation: contrast with phorbol 12-myristate 13-acetate. Biochem J, 1993,290:545-550.
[33] Raingeaud J,Gupta S,Rogers JS,et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem, 1995,270:7420-7426.
[34] Raingeaud J,Whitmarsh AJ,Barrett T,et al. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol, 1996,16:1247-1255.
[35] Baldassare JJ,Bi Y,Bellone CJ. The role of p38 mitogen-activated protein kinase in IL-1 beta transcription. J Immunol, 1999,162:5367-5373.
[36] Wang XZ,Ron D. Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science, 1996,272:1347-1349.
[37] Rouse J,Cohen P,Trigon S,et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins Cell, 1994,78:1027-1037.
[38] Engel K,Kotlyarov A,Gaestel M. Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J, 1998,17:3363-3371.
[39] Ben-Levy R,Hooper S,Wilson R,et al. Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol,1998,8: 1049-1057.
[40] Schafer PH,Wang L,Wadsworth SA,et al. T cell activation signals up-regulate p38 mitogen-activated protein kinase activity and induce TNF-alpha production in a manner distinct from LPS activation of monocytes. J Immunol, 1999,162:659-668.
[41] Pierrat B,Correia JS,Mary JL,et al. RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38alpha mitogen-activated protein kinase (p38alphaMAPK). J Biol Chem, 1998, 273:29661-29671.
[42] New L,Jiang Y,Zhao M,et al. PRAK, a novel protein kinase regulated by the p38 MAP kinase .EMBO J, 1998,17: 3372-3384
[43] Parker CG,Hunt J,Diener K,et al. Identification of stathmin as a novel substrate for p38 delta. Biochem Biophys Res Commun, 1998,249:791-796.
[44] Hedges JC,Dechert MA,Yamboliev IA,et al. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem, 1999,274:24211-24219.
[45] Guay J,Lambert H,Gingras-Breton G,et al. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.J Cell Sci, 1997, 110: 357-368.
[46] Jeffrey RJ,Brian B,Leonard H,et al.Pharmacological effects of SB203580,a selective inhibitor of p38 mitogen-activated protein kinase,in angiogenesis and chronic inflammatory disease models.J Pharmacol Exp Ther,1998,284(2):687-692.
[47] Lee JC,Kacsis S,Kumar S,et al. P38 mitogen-activated protein kinase inhibitors:mechanisms and therapeutic potential.J Pharmac Ther,1999,82:389-397.
[48] Frantz B,Klatt T,Pang Met al.The activation state of p38 mitogen-activated protein kinase determines the efficiency of ATP competition for pyridinylimidazole inhibitor binding.J Biochemistry, 1998,37(39):13846-13853.
[49] ShethK, Friel J, Nolan B, et al. Inhibition of p38 mitogen activated protein kinase increases LPS induced inhibition of apoptosis in neutrophils by activating extracellular signal regulated kinase. Surgery, 2001, 130(2):242. 248.
[50] Staneley M, Chappell SA, Jopling CL, et al. c-Myc protein synthesis is initiated from the intemal ribosome entrv segment during apoptosis. . Mol Cell Biol, 2000, 20(4): 1162-1169.
[51] Bulavin DV, Saito S, Hollander MC, et al . Phosphorylation of human p53 by p38 kinase coordinates N—terminal phosphorylation and apoptosis in responsetoUV radiation. EMBO, 1999, 18(23): 6845-6854
[52] Kommann M, Ishiwam T, Kleef J, et 01. Fas and Fas. ligand expression in human pancreatic cancer . Ann Surg 2000, 231(3): 368-379
[53] Han J, Jiang Y, Li z, et al . Activation of the transcription factor MEF2C bytheMAP kinase p38ininflammation . Nature, 1997, 386(6622): 296—299.
[54] Ghatan S, Lanier S, Kinoshita Y, et al . p38 MAP kinase mediates bax translocation in nitric oxide, induced apoptosis in neurons . Cereb Blood new Metab, 2000, 150(2): 335—347
[55] Nagata Y. Todokom K. Requirement of activation of JNK and p38 for environmental stress . induced erythroid diferentiation and apoptosisand inhibition ofERK for apoptosis. Blood, 1999, 94(3): 853-863
[56] Dong X, Liu Y, Du M, et al . Related Articles, links-Abstract, p38 mitogen. activated protein kinase inhibition attenuates pulmonary inflammatory responsein a rat cardiopulmonary by pass model. Eur J Cardiotholac Surg,2006, 30(1): 77-84
[57] Chen BC, Chen YH, An WW, etal. Involvement of p38 mitogen-activated protein kinase in lipopolysaecharide-induced iNOS and COX2 expression in J774 macmphages . Immunology, 1999, 97(1): 124-129
[58] TamuraDY, MooreEE, Johnson JIJ, et al . p38mitogen-activated protein kinase inhibition attenuates intercellar adhesian molecule-1 up-regulation on human pulmonary microvascular endothelial cells . Surgery, 1998. 124(2):403-408.
[59]王玉,李晓玫,王海燕.白介素-1β通过JNK,p38信号转导通路调控肾系膜细胞表达a平滑肌肌动蛋白.生理学报,2002,54(3):244.250
[60]张梅,唐嘉薇,李晓玫.IL-I通过p38MAPK信号途径诱导肾小管细胞转分化并影响其移行功能.中华医学杂志,2003,83(13):1161.1165)
[61]Hoffmeyer A,Grosse-Wilde A,Flory E,et al.Different mitogen-activated protein kinase signaling pathways cooperate to regulate tumor necrosis factor alpha gene expression in T lymphocytes.J Biol Chem,1999,274:4319-4327.
[62]Shibayama H,Anzai N,Braun SE,et al.H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation.Blood,1999,93:1540-1548.
[63]Xing J,Kornhauser JM,Xia Z,et al.Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation.Mol Cell Biol,1998,18:1946-1955.
[64]Yang SH,Yates PR,Whitmarsh AJ,et al.The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif.Mol Cell Biol,1998,18:710-720.
[65]Yang SH,Whitmarsh AJ,Davis RJ,et al.Differential targeting of MAP kinases to the ETS-domain transcription factor Elk-1.EMBO J,1998,17:1740-1749.
[66]Singh RP,Dhawan P,Golden C,et al.One-way cross-talk between p38(MAPK)and p42/44(MAPK).Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.J Biol Chem,1999,274:19593-19600.
[67]Rosenberger SF,Gupta A,Bowden GT,et al.Inhibition of p38 MAP kinase increases okadaic acid mediated AP-1 expression and DNA binding but has no effect on TRE dependent transcription.Oncogene,1999,18:3626-3632.
[68]Ju H,Behm DJ,Nerurkar S,et al.p38 MAPK Inhibitors Ameliorate Target Organ Damage in Hypertension:Part 1,p38 MAPK-Dependent Endothelial Dysfunction and Hypertension.J Pharmacol Exp Ther2003,307(3):9322938.
[69]McGinn S,Saad S,Poronnik P,Pollock CA.High glucose-mediated ef2 fects on endothelial cell proliferation occur via p38 MAP kinase . Am J Physiol Endocrinol Metab ,2003 ,285 (4) :E7082717.
[70] Niwa K,Inanami O ,Ohta T , et al . p38 MAPK and Ca2 + contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERKdoes not . Free Radic Res, 2001,35 (5) :5192527.
[71] Kevil CG, Oshima T , Alexander JS. The role of p38 MAP kinase in hydrogen peroxide mediated endothelial solute permeability.Endotheli2um, 2001,8(2) :1072116.
[72] Kiemer AK, Weber NC , Furst R , et al. Inhibition of p38 MAPK activation via induction of MKP21 : atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability . Circ Res ,2002 ,90(8) :8742881.
[73] Jersmann HP , Hii CS , Ferrante JV , et al . Bacterial lipopolysaccharide and tumor necrosis factor alpha synergistically increase expression of human endothelial adhesion molecules through activation of NF-kappaB and p38 mitogen2activated protein kinase signaling pathways . Infect Immun ,2001 ,69(3): 127321279.
[74] van Gorp RM, Heeneman S , Broers JL , et al . Glutathione oxidation in calcium2 and p38 MAPK-dependent membrane blebbing of endothelial cells .Biochim Biophys Acta ,2002 ,1591 (123) :1292138.
[75] Clermont F , Adam E , Dumont JE , et al . Survival pathways regulating the apoptosis induced by tumour necrosis factor-alpha in primary cultured bovine endothelial cells . Cell Signal ,2003 ,15(5) :5392546.
[76] Nakagami H , Morishita R , Yamamoto K, et al . Phosphorylation of p38 mitogen2activated protein kinase downstream of bax-caspase23 pathway leads to cell death induced by high D2glucose in human endothelial cells . Diabetes.2001 ,50(6): 147221481.
[77] Zhang X, Hu K, Li CY. Protection against oxidized low-density lipoprotein induced vascular endothelial cell death by integrin-linked kinase .Circulation ,2001 ,104(23) :276222766.
[78]Hyman KM,Seghezzi G,Pintucci G,et al.Transforming growth factor betal induces apoptosis in vascular endothelial cells by activation of mito2gen2activated protein kinase.Surgery,2002,132(2):1732179.
[79]Zhang X,Shan P,Otterbein LE,et al.Carbon monoxide inhibition of apoptosis during ischemia2reperfusion lung injury is dependent on the p38mitogen2activated protein kinase pathway and involves caspase 3.J Biol Chem,2003,278(2):124821258.
[80]Zhang X,Shan P,AlamJ,et al.Carbon monoxide modulates Fas/Fas ligand,caspases,and Bc122 family proteins via the p38alpha mitogen activated protein kinase pathway during ischemia-reperfusion lung injury.J Biol Chem,2003,278(24):22061222070.
[81]Razandi M,PedramA,Levin ER.Estrogen signals to the preservation of endothelial cell form and function.J Biol Chem,2000,275(49):38540-38546.
[82]蔡海江编.动脉粥样硬化基础与临床,南京:江苏科学技术出版社,1996,第1版:110-125.
[83]杨向东,黎明健.氧化型低密度脂蛋白诱导U937细胞凋亡及其对p53,p21和Bcl-2表达的影响,中国动脉硬化杂志,2000;8(1):21.
[84]Cybulsky MI,Liyama K,Li H,et al.A major role for VCAM-1,but not ICAM-1in early atherosclerosis.J ClinInvest.2001.107:1255-1262.
[85]梁萍,孙雷,唐建武,等.细胞间粘附分子1、血管细胞粘附分子1和肿瘤坏死因子在人动脉粥样硬化病灶中的表达及意义.中国动脉硬化杂志,2004;12(4):427-429.
[86]VanderA,Vander PT.Intercellular adhesion molecule-1.J Mo Med,1996;74(1):13.
[87]Peter K,Nawroth P,Conradt C,et al.circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating Intercellular adhesion molecule-1,E-selectin,P-selectin and thrombomodulin.ArtherosclerThromb Vas Biol,1997;17(3):55.
[88]Amberger A,Maczek C,Jurgens Qet al.Co-expression of ICAM-1,VCAM-1 and Hsp60 in human arterial and venous endothelial cell in response to cytokins and oxidized low-density lipoproteins. cell Stress Chaperones,1997;2(2):94.
[89] Gu L,Okada Y,Clinton S,et al.Absence of monocyte chemoattactant protein-1 reduces atherosclerosis in low-density lipoprotein-deficient mice.Mol Cell.1998;2:275-281.
[90] Ikeda U,Matsui K,Murakmi Y. Monocyte chemoattractant protein-I and coronary disease .Clin Cardiol,2000;25(4):143 — 147.
[91] Barter PJ. Inhibition of endothelial cell adhesion molecule expression by high density liporoteins. Clin Exp Pharmaco Physiol ,1997;24(4):286.
[92] Porrca E,Di Febbo C,Reale M, et al .Monocyte chemoatractant protein —l(MCP-l) is a mitogen for cultured rat vascular smooth muscle cell . Vasc Res,1997;34:58-56.
[93] Blandberg FQ Wen P,Dai M,et al.Detection of early atherosclerosis with radiolabeled monocyte chemoatractant protein-1 in prediabetic Zucker rats.Pediatr Radiol,2002;31:827-835.
[94] Rahman A; Anwar KN; Minhajuddin M;et. cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L1017-24
[95] Li D, Williams V, Liu L, Chen H, Sawamura T, Antakli T, Mehta JL.LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation.Am J Physiol Heart Circ Physiol. 2002 Nov;283(5):H1795-801
[96] Van Wetering S; Van den Berk N; van Buul JD;et al. VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration.Am J Physiol Cell Physiol. 2003 Aug;285(2):C343-52
[97] Lei ZB, Zhang Z, Jing Q ,et al. oxLDL upregulates CXCR2 expression in monocytes via scavenger receptors and activation of p38 mitogen-activated protein kinase. Cardiovasc Res. 2002 Feb 1;53(2):524-32.
[98] Chen XL, Grey JY, Thomas S, Qiu FH, Medford RM, Wasserman MA, Kunsch C. Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells:upregulation by oscillatory flow.Am J Physiol Heart Circ Physiol.2004 Oct;287(4):H1452-8.
[99]Khreiss T,J6zsef L,Potempa LA,Filep JG Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells.Circulation.2004 Apr 27;109(16):2016-22
[100]Domoto K,Taniguchi T,Takaishi H,Takahashi T,Fujioka Y,Takahashi A,Ishikawa Y,Yokoyama M.Chylomicron remnants induce monocyte chemoattractant protein-1 expression via p38MAPK activation in vascular smooth muscle cells.Atherosclerosis.2003 Dec;171(2):193-200
[101]Li Yanbo;Deng Huacong;Zheng Dan;et.Role of p38 Mitogen-activated Protein Kinase in Mediating Monocyte Chemoattractant Protein-1 in Human Umbilical Vein Endothelial Cells.Chinese Medical Sciences Journal,2004;19(1):71.
[102]Guo ZJ;Hou FF;Liang M;et.Advanced glycation end products stimulate human endothelial cells to produce monocyte chemoattractant protein-1.Zhonghua yi xue za zhi.2003;83(12):1075-9.
[103]Werle,Martina;Schmal,Ulrike;Hanna,Katharina;et.MCP-I induces activation of MAP-kinases ERK,JNK and p38 MAPK in human endothelial cells.Cardiovascular Research,2002;56(2):284.
[104]Ashida N;Arai H;Yamasaki M;rt al.Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis.The Journal of biological chemistry.J Biol Chem 2001;276(19):16555-60.
[105]李艳波,邓华聪,郑丹,等.P38信号通路对人脐静脉内皮细胞单核细胞趋化蛋白-1表达的影响.中华糖尿病杂志,2004;12(4):287-289.
[106]Zhao M,Liu Y,Wang X,New L,Han J,Brunk UT.Activation of the p38 MAP kinase pathway is required for foam cell formation from macrophages exposed to oxidized LDL.APMIS.2002 Jun;110(6):458-68.
[107]Jing Q;Xin SM;Cheng ZJ;et.Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells:mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity.Circulation research.1999;84(7):831-9.
[108]Ohashi N;Matsumori A;Furukawa Y;et.Role of p38 mitogen-activated protein kinase in neointimal hyperplasia after vascular injury.Arteriosclerosis,thrombosis,and vascular biology.2000;20(12):2521-6.
[109]Qing Jing,Sun-Mei Xin.Lysophosphatidylcholine Activates p38 and p42144Mitogen-Activated Protein Kinases in Monocytic THP-1 Cells,but Only p38Activation Is Involved in Its Stimulated Chemotaxis Circulation Research 2000;87:52-59.
[110]Ju H;Behm DJ;Nerurkar S;et al.p38 MAPK inhibitors ameliorate target organ damage in hypertension:Part 1.p38 MAPK-dependent endothelial dysfunction and hypertension.The Journal of pharmacology and experimental therapeutics.Pharmacol Exp Therl 2003;307(3):932-8.
[111]林蓉,甘伟杰.药物治疗动脉粥样硬化的新途径一抗炎治疗,中国药理学通报,,2004;20(5):499-503.
[1]Risau W,et al.Vasculogenesis.Annu Rev Cell Dev Biol,1995,11:73-91.
[2]Risau W.Mechanisms ofangiogenesis.Nature,1997,386:671-674
[3]Choi K.The hemangioblast:a common progenitor of hematopoietic and endothelial cells.J Hematother Stem Cell Res.2002 Feb;11(1):91-101
[4]Mikkola HK,Orkin SH.The search for the hemangioblast.J Hematother Stem Cell Res.2002 Feb;11(1):9-17.
[5]Asahara T,Murohara T,Sullivan A,Silver M,van der Zee R,Li T,Witzenbichler B,Schatteman G;Isner JM.Isolation of putative progenitor endothelial cells for angiogenesis.Science.1997 Feb 14;275(5302):964-7
[6]Suda T,Takakura N,Oike Y.Hematopoiesis and angiogenesis.Int J Hematol.2000 Feb;71(2):99-107
[7]Heilmann C,Beyersdorf F,Lutter G.Collateral growth:cells arrive at the construction site.Cardiovasc Surg.2002 Dec;10(6):570-8
[8]Choi K,Kennedy M,Kazarov A,Papadimitriou JC,Keller G.A common precursor for hematopoietic and endothelial cells.Development.1998Feb;125(4):725-32.
[9]Kalka C,Tehrani H,Laudenberg B,et al.VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease.Ann Thorac Surg,2000,70:829-834.
[10]Murasawa S,Llevadot J,Silver M,et al.Constitutive human telomerase reverse transcriptase expression enhances regenerativeproperties of endothelial progenitor cells.Circulation,2002,106:1133-1139
[11] Akashi M ,Hisashi I, Yoshimichi S ,et al. Platelet — derived growth factor BB( PDGF-BB) induces differentiation of bone marrow endothelial progenitor cellderived cell line TRBME2 into mural cells ,and changes the phenotype .Cell Physiol, 2005, 04:948-955.
[12] Friedrich EB, Walenta K, Scharlau J, et al. CD342/ CD133+NEGFR22+ endothelial progenitor cell subpopulation with potent vasoregenerative capacities .Circ Res ,2006 ,98 (3):e20-25.
[13] Kalka C, Tehrani H, Laudenberg B, et al.VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease. Ann Thorac Surg, 2000,70:829 - 834.
[14] Peichev M,Naiyer AJ, Pereira D ,et al.Expression of VEGFR-2 and AC133 by circulating human CD34+cells identifies a population of functional endothelial precursors. Blood ,2000,95 :952-958.
[15] ASAHARA T, MASUDA H, TAKAHASHI T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization .Circ Res ,1999, 85:221-228.
[16] TAKAHASHI T, KALKA C, MASUDA H, et al.Ischemia and cytokine-induced mobilization of bone derived endothelial progenitor cells for neovascularization.Nat Med, 1999, 5:434-438.
[17] YAMAGUCHI J, KUSANO K F, MASUO 0, et al. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization .Circulation, 2003,107:1322-1328
[18] GILL M, DIA S, HATTORI K, et al.Vascular trauma mobilization of VEGFR2+AC133+endothelial precursor induces rapid but transient cells。Circ Res, 2001, 8:167-174.
[19] Shibuya M. Vascular endothelial growth factor receptor family genes :when did the three genes phylogenetically segregate. Biol Chem, 2002 ,383 :1573-1579.
[20] Wang C, Jiao C, Hanlon HD, et al. Mechanical, cellular, and molecular factors interact to modulate circulating endothelial cell progenitors. AmJ Physiol Heart Circ Physiol, 2004;8 :8-12
[21] Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor 1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation, 2003,107:1322-1328.
[22] Amano K, Okigaki M, Adachi Y, et al. Mechanismfor IL21b2mediated neovascularization unmasked by IL21b knock-out mice. J Mol Cell Cardiol,2004,36 :469-480.
[23] Imanishi T, Hano T, Moriwaki C, et al.Angiotensin II potentiates vegf2 induced proliferation and network formation of endothelial progenitor cells. AJH 2004,17:1555-7.
[24] Sarukawa M, Yamamoto K, Ueda N. Kinetics of circulating endothelial progenitor cells in congestive heart failure. JACC, 2004,3:202A.
[25] Miller HI, George J, Herzl, The number and adhesive properties of circulating endothelial progenitor cells in patients with in - stent restenosis. JACC 2004,3:353A.
[26] Kunz GA, LiangqCuculoski F. David gregg circulating endothelialprogenitor cells predict coronary artery disease severity. JACC, 2004,3:496A.
[27] Moriwaki C, Imanishi T, Hano T, et al.Endothelial progenitor cell senescence is related to telomerase activity in patients with hypertension. AJH ,2004,17:202A.
[28] Dimmeler S, Aicher A, Vasa M, et al.HMG2CoA reductase inhibitors (statins)increase endothelial progenitor cells via the PI 32kinase/ Akt pathway. J Clin Invest, 2001,108:391-397.
[29] Murayama T, Tepper OM, Silver M, Ma H, Losordo DW, Isner JM,Asahara T,Kalka C. Determination of bone marrow-derived endothelial progenitor cell significance in anglogemc factor-induced neovascularization in Vivo. Exp Hematol. 104 2002;30(8):967-72
[30] Fadini GP, Sartore S, Schiavon Avogaro M, Albiero M, Baesso I, Cabrelle A,Agostini C, Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats Diabetologia. 2006;49(12):3075-8
[31 ] Hiasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M, Ichiki T,Takeshita A, Egashira K. Gene transfer of stromal cell-derived factor-1 alpha enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization. Circulation. 2004 May 25;109(20):2454-61.
[32] Shi Q,Rafii S,Wu M,et al.Evidence for eireulatiag bone marow-derived endothelial cells .Blood, 1998;92(2):362-367
[33] Lin Y, Weisdorf DJ, Solovey A, et al. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest, 2000,105:71-77.
[34] Tiwari A, Hamilton G, Seifalian AM, et al. Regarding "Isolation of endothelial cells and their progenitor cells from human peripheral blood".J Vasc Surg. 2002 Apr;35(4):827)
[35] Kalka C,IAasuda H,Takahashi T,et al.Vascular endothelial growth factor 165 geng transfer augments circulating endothelial progenitor cells in human subjects. Circ Res,2000;86(12):1198-1202.
[36] Burger PE,Coetzee S,Mckeehan WL,et,al. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells .Blood,2002;100(10):3527-353
[37] Boyer M, Townsend LE, Vogel LM, Falk J, Reitz-Vick D, Trevor KT, Villalba M, Bendick PJ, Glover JL. Isolation of endothelial cells and their progenitor cells from human peripheral blood J Vasc Surg. 2000 Jan;31(1 Pt 1):181-9
[38] Harraz M, Jiao C, Hanlon HD, Hartley RS, Schatteman GC. CD34- blood-derived human endothelial cell progenitors.Stem Cells. 2001;19(4):304-12.
[39] Kalka C,IAasuda H,Takahashi T,et al.Vascular endothelial growth factor 165 geng transfer augments circulating endothelial progenitor cells in human subjects.CircRes,2000;86(12):1198-1202.
[40] Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T,Isner JM, Asahara T. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration.Circulation. 2002 Feb 12;105(6):732-8.
[41] Kawamoto A,Gwon HC,Iwaguro H,et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia .Circulation, 2001; 103(5):634-637.
[42] Assmus B, Sch(?)chinger V, Teupe C, Britten M, Lehmann R, Dobert N, Griinwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI).Circulation. 2002 Dec 10;106(24):3009-17
[43] He H, Shirota T, Yasui H, Matsuda T. Canine endothelial progenitor cell-lined hybrid vascular graft with nonthrombogenic potential. J Thorac Cardiovasc Surg. 2003 Aug;126(2):455-64.
[44] Kaushal S, Amiel GE, Guleserian KJ, Shapira OM, Perry T, Sutherland FW,Rabkin E, Moran AM, Schoen FJ, Atala A, Soker S, Bischoff J, Mayer JE Jr.Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001 Sep;7(9):1035-40
[45] Vasa M,et al.Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res:El-E7,2001
[46] Tepper OM,et al. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation ,adhesion,and incorporation into vascular struction.Circulation 2002, 106:2781-2786
[47] Verma S , et al.C-reactive protein attenuates endothelial progenoitor cell survival ,differentiation, and function: Further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation 2004,109:2058-2067
[48] Refii S, et al. Therapeutic Sterm and pregenitor cell transplantation for organ vascularization and regenetation. Nat Med 2003; 9: 702
[49] Gulati R. Autologous cuhure modified mononuclear cells confer vascular protection after artery injuny. Circulation 2003; 108: 1520
[50] Hill JM, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003; 348: 593
[51] Takahashi T, et al .Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med1999,5:1999.
[52] Dimmerler S ,et al.HMG-CoA reductase inhibitors(statins)increase endothelial progenitor cells via the PI-kinase/Akt pathway.J Clin Invest 2001 108:391-397.
[53] Asahara A ,et al .VEGF contributes to postnastal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. Embol J 1999,18:3964.
[54] Sterhlow K ,et al. Estrogen increase bone marrow-derived endothelial progenitor cell product and diminishes neointima formation .Circulation 2003,107:3059
[55] Heeschen C, et al .Erytheropoietin is a potent physiological stimulus for endothelial progenitor cell mobilization. Blood 2003,17:17
[56] Laufs U ,et al .Physical training increases endothelial progenitor cells, inhibits neointima formation.and enhances angiogenesis.Circulation 2003,22:22
[57] Seeger FH, et al .P38 Mitogen-activated protein kinase Downregulates endothelial progenitor cells.circulation 2005,8:1184.
[58] Walter DH ,et al. Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease .Cir Res 2005 ,25:1142.
[59] Urbish C,et al. Cathepsin L is required for endothelial progenitor cell-induced neovascularization .Nat Med 2005 ,11:206
[2] Buschmann I, Schaper W. The pathophysiology of the collateral circulation (arteriogenesis). J Pathol. 2000;190:338-342.
[3] RisauW. Mechanisms of angiogenesis.Nature. 1997;386:671-674
[4] Risau W, Flamme I. Vasculogenesis. Annu Rev Cell Dev Biol. 1995; 11:73-91.
[5] Satoshi S,Toyoaki,Hisao 1, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation, 2001, 103:2776-2779.
[6] Gill M, Dias S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2+AC 133+ endothelial precursor cells.Circ Res, 2001,88:167-174.
[7] Isner J,Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest, 1999, 103:1231- 1236.
[8] Kawamoto A, Gwon H C,Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia.Circulation,2001, 103:634-637.54
[9] Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001;89:E1-7.30
[10] Loomans CJ, de Koning EJ, Staal J, et al. Endothelial progenitor cell dysfunction:a novel concept in the pathogenesis of vascular complications of type 1 diabetes.2004; Diabetes. 53:195-199.
[11] Tepper OM, Galiano RD, Capla JM, et al. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002.106:2781-2786.
[12] Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 2001; 103:2885-90.
[13] Yla-Herttuala S, Palinski W Rosenfeld ME, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989; 84:1086-1095.
[14] Witztum JL. Role of modified lipoproteins in diabetic macroangiopathy.Diabetes. 1997; 46:S112-114.
[15] Khan BV, Parthasarathy SS, Alexander RWet al. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest. 1995; 95:1262-70.
[16] Harada-Shiba M, Kinoshita M, Kamido H,et al. Oxidized low density lipoprotein induces apoptosis in cultured human umbilical vein endothelial cells by common and unique mechanisms. J Biol Chem. 1998 ;273:9681-7.
[17] Plane F, Bruckdorfer KR, Kerr P, et al .Oxiditive modification of low- density lipoproteins and the inhibition of relaxations mediated endothelial derived nitric oxide in rabbit aorta. Br J Pharmacol. 1992; 105:216- 222
[18] Oren M. Tepper, Robert D. Galiano, Jennifer M. Capla,et al.Human Endothelial Progenitor Cells From Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation Into Vascular Structures. Circulation,2002,106:2781-2786
[19] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature,1993,362:801-809.
[20] Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med. 2005;353:999 -1007.
[21] Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events:proof of concept for the clinical importance of endogenous vascular repair.Circulation. 2005; 111:2981-2987.
[22] Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerate reendothelialization. Circulation, 2002, 105:3017-3024.
[23] Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med, 2003 348:593-600.
[24] Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation, 2001, 104:1046-1052.
[25] Rehman J, Li J, Orschell CM, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation, 2003, 107:1164-1169.
[26] Vajkoczy P, Blum S, Lamparter M, et al. Multistep nature of microvascular recruitment of ex vivo-expanded embryonic endothelial progenitor cells during tumor angiogenesis. J Exp Med.2003;197:1755-1765.
[27] Adams GB, Chabner KT, Foxall RB, et al. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment. Blood 2003; 101:45-51.
[28] Yoshizumi M, Abe J, Tsuchiya K, Berk BC, Tamaki T. Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases. J Pharmacol Sci.2003;91:172-176.
[29] Seeger FH, Haendeler J, Walter DH, Rochwalsky U, Reinhold J, Urbich C, et al.p38 mitogen-activated protein kinase downregulates endothelial progenitor cells.Circulation 2005; 111: 1184- 1191.
[30] Schmidt-Lucke C, Rossig L, Fichtlscherer S, et al. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events:proof of concept for the clinical importance of endogenous vascular repair.Circulation. 2005; 111:2981 -2987.
[31] Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerate reendothelialization. Circulation, 2002, 105:3017-3024.
[32] Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells,vascular function, and cardiovascular risk. N Engl J Med, 2003 348:593-600.
[33] Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation, 2001, 104:1046-1052.
[34] Rehman J, Li J, Orschell CM, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation, 2003,107:1164-1169.
[35] Adams RH, Porras A, Alonso G, Jones M, Vinterstewn K, Panelli S,et al.Essential role of p38aMAP kinase in placental but not embryonic cardiovascular development. Mol Cell 2000; 6: 106-116.
[36] Matsumoto T, Turesson I, Book M, Gerwins P, Claesson-Welsh L. p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2-stimulated angiogenesis. J Cell Biol 2002; 156:149-160.
[37] Gratton JP, Morales-Ruiz M, Kureishi Y, Fulton D, Walsh K, Sessa WC. Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells. J Biol Chem 2001; 276: 30359-30365.
[38] Taro M,Ingela T,Majlis B,et al.P38MAP Kinase negatively regulates endothelial cell survival,proliferation and differentiation in FGF-2-stimulated angiogenesis.J Cell Biology,2002,156( 1): 149-160.
[1]Jiang Y,Li Z,Schwarz EM,et al.Structure-function studies of p38mitogen-activated protein kinase.Loop 12 influences substrate specificity and autophosphorylation,but not upstream kinase selection.J Biol Chem,1997,272(7):11096-11102.
[2]Kan W,Zhao KS,Jiang Yet al.Role of p38 mitogen-activated protein kinase in signal transduction of inducible of nitric oxide synthase expression.Shock,2004,21(3) 281-287.
[3]Jiang YChen C,Li Z,et al.Characterizadon of the structure and function of a new mitogen-activated protein kinase(p38b).J Biol Chem,1996,271(30):17920-17926.
[4]Serger R,Krebs EGI.The MAPK signaling cascade.FASEB J,1995,9(9):726-735.
[5]Brewster JL,De Valoir T,DwyND,et al.An osmosensing signal transduction pathway inyeast.J.Science,1993,259(5099):1760-1763.
[6]Han J,Lee JD,Bibbs L,et ol.AMAP kinase targeted by endotoxin and hyperosmolarityin mammalian cells.Science,1994,265(5173):808-811.
[7]Bodero AJ,Ye R,Lees-Miller SPo UV-light induces p38 MAPK-dependent phosphorylation of Bcll0.Biomchem Biophys Res Commun.2003,301(4):923-926.
[8]Taro M,Ingela T,Majlis B,et al.P38MAP Kinase negatively regulates endothelial cell survival,proliferation and differentiation in FGF-2-stimulated angiogenesis.J Cell Biology,2002,156(1):149-160.
[9] Mi-Sun K,Hyun J, Jae-Young U,et al .Agonists of proteinase-activated receptor 2 induce TNF-alpha secretion from astrocytoma cells.Cell Biomchem Funct, 2002,20 (4): 339-345.
[10] Huang CD,Tliba O;Panettieri RA,et al.Bradykinin induces interleukin-8 production in human aiyway smooth muscle cells:modulation by TV cytokines and dexamethasone.Am J Respir Cell Mol Biol,2003,28(3):330-338.
[11] Raingeaud J, Whitmarsh AJ, Barrett T, et al . MKK3 and MKK6 regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathwayl. Mol Cell Biol, 1996, 16(3): 1247. 1255.
[12] Bagrodia S, Derijard B, Davis RJ, Cerione RA. Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J Biol Chem. 1995 Nov 24;270(47):27995-8.
[13] Zhang S,Huan J,Sells MA,et al. Rho Family GTPases Regulate p38 Mitogen-activated Protein Kinase through the Downstream Mediator Pak1. J Biol Chem, 1995,270:23934-23936.
[14] Knaus UG,Morris S, Dong HJ,et al. Regulation of Human Leukocyte p21-Activated Kinases Through G Protein-Coupled Receptors. Science,1995,269:221-223.
[15] Manner E,Leung T,Salihuddin H,et al. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature, 1994,367:40-46.
[16] Chan-Hui PY,Weaver R. Biochem J, Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades 。 Biochem J. 1998 Dec 15;336 ( Pt 3):599-609 .
[17] Tibbies LA,Ing YL,Kiefer F,et al. MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6. EMBO J, 1996,15:7026-7035.
[18] Cuenda A ,Dorow DS. Biochem J, Differential activation of stress-activated protein kinase kinases SKK4/MKK7 and SKK1/MKK4 by the mixed-lineage kinase-2 and mitogen-activated protein kinase kinase (MKK) kinase-1 1998,333(Pt 1): 11-15.
[19] Terada Y,Nakashima O,Inoshita S,et al. Mitogen-activated protein kinase cascade and transcription factors: the opposite role of MKK3/6-p38K and MKK1-MAPK. Nephrol Dial Transplant, 1999,1:45-47.
[20] Moriguchi T,Kuroyanagi N,Yamaguchi K,et al. A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3. J Biol Chem,1996,271:13675-13679.
[21 ] Stein B,Brady H,Yang MX,et al. Cloning and characterization of MEK6, a novel member of the mitogen-activated protein kinase kinase cascade. J Biol Chem,1996,271:11427-11433.
[22] Nick JA,Avdi NJ,Young SK,et al. Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils . J Clin Invest, 1999,103: 851-858.
[23] Lu HT,Yang DD,Wysk M,et al. Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice . EMBO J,1999,18:1845-1857.
[24] Han J,Wang X,Jiang Y,et al. Identification and characterization of a predominant isoform of human MKK3. FEBS Lett, 1997,403:19-22.
[25] Jiang Y,Li Z,Schwarz EM,et al. Structure-function studies of p38 mitogen-activated protein kinase. J Biol Chem, 1997,272:11096-11102.
[26] Zhao M,New L,Kravchenko VV,et al. Regulation of the MEF2 family of transcription factors by p38. Mol Cell Biol, 1999,19:21-30.
[27] Ornatsky OI, Cox DM, Tangirala P, Andreucci JJ, Quinn ZA, Wrana JL, Prywes R, Yu YT, McDermott JC. Post-translational control of the MEF2A transcriptional regulatory protein. Nucleic Acids Res. 1999 Jul 1;27(13):2646-54.
[28] Yang SH,Galanis A,Sharrocks AD. Targeting of p38 mitogen-activated protein kinases to MEF2 transcription factors. .Mol Cell Biol, 1999,19: 4028-4038.
[29] Han J,Jiang Y,Li Z,et al. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature, 1997,386: 296-299.
[30] Hai T,Wolfgang CD,Marsee DK,et al. ATF3 and stress responses. Gene Expr, 1999,7:321-335
[31] Thuerauf DJ,Arnold ND,Zechner D,et al. p38 Mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6. J Biol Chem, 1998,273:20636-20643.
[32] Casillas AM,Amaral K,Chegini-Farahani S,et al. Okadaic acid activates p42 mitogen-activated protein kinase (MAP kinase; ERK-2) in B-lymphocytes but inhibits rather than augments cellular proliferation: contrast with phorbol 12-myristate 13-acetate. Biochem J, 1993,290:545-550.
[33] Raingeaud J,Gupta S,Rogers JS,et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem, 1995,270:7420-7426.
[34] Raingeaud J,Whitmarsh AJ,Barrett T,et al. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol, 1996,16:1247-1255.
[35] Baldassare JJ,Bi Y,Bellone CJ. The role of p38 mitogen-activated protein kinase in IL-1 beta transcription. J Immunol, 1999,162:5367-5373.
[36] Wang XZ,Ron D. Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science, 1996,272:1347-1349.
[37] Rouse J,Cohen P,Trigon S,et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins Cell, 1994,78:1027-1037.
[38] Engel K,Kotlyarov A,Gaestel M. Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J, 1998,17:3363-3371.
[39] Ben-Levy R,Hooper S,Wilson R,et al. Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol,1998,8: 1049-1057.
[40] Schafer PH,Wang L,Wadsworth SA,et al. T cell activation signals up-regulate p38 mitogen-activated protein kinase activity and induce TNF-alpha production in a manner distinct from LPS activation of monocytes. J Immunol, 1999,162:659-668.
[41] Pierrat B,Correia JS,Mary JL,et al. RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38alpha mitogen-activated protein kinase (p38alphaMAPK). J Biol Chem, 1998, 273:29661-29671.
[42] New L,Jiang Y,Zhao M,et al. PRAK, a novel protein kinase regulated by the p38 MAP kinase .EMBO J, 1998,17: 3372-3384
[43] Parker CG,Hunt J,Diener K,et al. Identification of stathmin as a novel substrate for p38 delta. Biochem Biophys Res Commun, 1998,249:791-796.
[44] Hedges JC,Dechert MA,Yamboliev IA,et al. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem, 1999,274:24211-24219.
[45] Guay J,Lambert H,Gingras-Breton G,et al. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.J Cell Sci, 1997, 110: 357-368.
[46] Jeffrey RJ,Brian B,Leonard H,et al.Pharmacological effects of SB203580,a selective inhibitor of p38 mitogen-activated protein kinase,in angiogenesis and chronic inflammatory disease models.J Pharmacol Exp Ther,1998,284(2):687-692.
[47] Lee JC,Kacsis S,Kumar S,et al. P38 mitogen-activated protein kinase inhibitors:mechanisms and therapeutic potential.J Pharmac Ther,1999,82:389-397.
[48] Frantz B,Klatt T,Pang Met al.The activation state of p38 mitogen-activated protein kinase determines the efficiency of ATP competition for pyridinylimidazole inhibitor binding.J Biochemistry, 1998,37(39):13846-13853.
[49] ShethK, Friel J, Nolan B, et al. Inhibition of p38 mitogen activated protein kinase increases LPS induced inhibition of apoptosis in neutrophils by activating extracellular signal regulated kinase. Surgery, 2001, 130(2):242. 248.
[50] Staneley M, Chappell SA, Jopling CL, et al. c-Myc protein synthesis is initiated from the intemal ribosome entrv segment during apoptosis. . Mol Cell Biol, 2000, 20(4): 1162-1169.
[51] Bulavin DV, Saito S, Hollander MC, et al . Phosphorylation of human p53 by p38 kinase coordinates N—terminal phosphorylation and apoptosis in responsetoUV radiation. EMBO, 1999, 18(23): 6845-6854
[52] Kommann M, Ishiwam T, Kleef J, et 01. Fas and Fas. ligand expression in human pancreatic cancer . Ann Surg 2000, 231(3): 368-379
[53] Han J, Jiang Y, Li z, et al . Activation of the transcription factor MEF2C bytheMAP kinase p38ininflammation . Nature, 1997, 386(6622): 296—299.
[54] Ghatan S, Lanier S, Kinoshita Y, et al . p38 MAP kinase mediates bax translocation in nitric oxide, induced apoptosis in neurons . Cereb Blood new Metab, 2000, 150(2): 335—347
[55] Nagata Y. Todokom K. Requirement of activation of JNK and p38 for environmental stress . induced erythroid diferentiation and apoptosisand inhibition ofERK for apoptosis. Blood, 1999, 94(3): 853-863
[56] Dong X, Liu Y, Du M, et al . Related Articles, links-Abstract, p38 mitogen. activated protein kinase inhibition attenuates pulmonary inflammatory responsein a rat cardiopulmonary by pass model. Eur J Cardiotholac Surg,2006, 30(1): 77-84
[57] Chen BC, Chen YH, An WW, etal. Involvement of p38 mitogen-activated protein kinase in lipopolysaecharide-induced iNOS and COX2 expression in J774 macmphages . Immunology, 1999, 97(1): 124-129
[58] TamuraDY, MooreEE, Johnson JIJ, et al . p38mitogen-activated protein kinase inhibition attenuates intercellar adhesian molecule-1 up-regulation on human pulmonary microvascular endothelial cells . Surgery, 1998. 124(2):403-408.
[59]王玉,李晓玫,王海燕.白介素-1β通过JNK,p38信号转导通路调控肾系膜细胞表达a平滑肌肌动蛋白.生理学报,2002,54(3):244.250
[60]张梅,唐嘉薇,李晓玫.IL-I通过p38MAPK信号途径诱导肾小管细胞转分化并影响其移行功能.中华医学杂志,2003,83(13):1161.1165)
[61]Hoffmeyer A,Grosse-Wilde A,Flory E,et al.Different mitogen-activated protein kinase signaling pathways cooperate to regulate tumor necrosis factor alpha gene expression in T lymphocytes.J Biol Chem,1999,274:4319-4327.
[62]Shibayama H,Anzai N,Braun SE,et al.H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation.Blood,1999,93:1540-1548.
[63]Xing J,Kornhauser JM,Xia Z,et al.Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation.Mol Cell Biol,1998,18:1946-1955.
[64]Yang SH,Yates PR,Whitmarsh AJ,et al.The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif.Mol Cell Biol,1998,18:710-720.
[65]Yang SH,Whitmarsh AJ,Davis RJ,et al.Differential targeting of MAP kinases to the ETS-domain transcription factor Elk-1.EMBO J,1998,17:1740-1749.
[66]Singh RP,Dhawan P,Golden C,et al.One-way cross-talk between p38(MAPK)and p42/44(MAPK).Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.J Biol Chem,1999,274:19593-19600.
[67]Rosenberger SF,Gupta A,Bowden GT,et al.Inhibition of p38 MAP kinase increases okadaic acid mediated AP-1 expression and DNA binding but has no effect on TRE dependent transcription.Oncogene,1999,18:3626-3632.
[68]Ju H,Behm DJ,Nerurkar S,et al.p38 MAPK Inhibitors Ameliorate Target Organ Damage in Hypertension:Part 1,p38 MAPK-Dependent Endothelial Dysfunction and Hypertension.J Pharmacol Exp Ther2003,307(3):9322938.
[69]McGinn S,Saad S,Poronnik P,Pollock CA.High glucose-mediated ef2 fects on endothelial cell proliferation occur via p38 MAP kinase . Am J Physiol Endocrinol Metab ,2003 ,285 (4) :E7082717.
[70] Niwa K,Inanami O ,Ohta T , et al . p38 MAPK and Ca2 + contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERKdoes not . Free Radic Res, 2001,35 (5) :5192527.
[71] Kevil CG, Oshima T , Alexander JS. The role of p38 MAP kinase in hydrogen peroxide mediated endothelial solute permeability.Endotheli2um, 2001,8(2) :1072116.
[72] Kiemer AK, Weber NC , Furst R , et al. Inhibition of p38 MAPK activation via induction of MKP21 : atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability . Circ Res ,2002 ,90(8) :8742881.
[73] Jersmann HP , Hii CS , Ferrante JV , et al . Bacterial lipopolysaccharide and tumor necrosis factor alpha synergistically increase expression of human endothelial adhesion molecules through activation of NF-kappaB and p38 mitogen2activated protein kinase signaling pathways . Infect Immun ,2001 ,69(3): 127321279.
[74] van Gorp RM, Heeneman S , Broers JL , et al . Glutathione oxidation in calcium2 and p38 MAPK-dependent membrane blebbing of endothelial cells .Biochim Biophys Acta ,2002 ,1591 (123) :1292138.
[75] Clermont F , Adam E , Dumont JE , et al . Survival pathways regulating the apoptosis induced by tumour necrosis factor-alpha in primary cultured bovine endothelial cells . Cell Signal ,2003 ,15(5) :5392546.
[76] Nakagami H , Morishita R , Yamamoto K, et al . Phosphorylation of p38 mitogen2activated protein kinase downstream of bax-caspase23 pathway leads to cell death induced by high D2glucose in human endothelial cells . Diabetes.2001 ,50(6): 147221481.
[77] Zhang X, Hu K, Li CY. Protection against oxidized low-density lipoprotein induced vascular endothelial cell death by integrin-linked kinase .Circulation ,2001 ,104(23) :276222766.
[78]Hyman KM,Seghezzi G,Pintucci G,et al.Transforming growth factor betal induces apoptosis in vascular endothelial cells by activation of mito2gen2activated protein kinase.Surgery,2002,132(2):1732179.
[79]Zhang X,Shan P,Otterbein LE,et al.Carbon monoxide inhibition of apoptosis during ischemia2reperfusion lung injury is dependent on the p38mitogen2activated protein kinase pathway and involves caspase 3.J Biol Chem,2003,278(2):124821258.
[80]Zhang X,Shan P,AlamJ,et al.Carbon monoxide modulates Fas/Fas ligand,caspases,and Bc122 family proteins via the p38alpha mitogen activated protein kinase pathway during ischemia-reperfusion lung injury.J Biol Chem,2003,278(24):22061222070.
[81]Razandi M,PedramA,Levin ER.Estrogen signals to the preservation of endothelial cell form and function.J Biol Chem,2000,275(49):38540-38546.
[82]蔡海江编.动脉粥样硬化基础与临床,南京:江苏科学技术出版社,1996,第1版:110-125.
[83]杨向东,黎明健.氧化型低密度脂蛋白诱导U937细胞凋亡及其对p53,p21和Bcl-2表达的影响,中国动脉硬化杂志,2000;8(1):21.
[84]Cybulsky MI,Liyama K,Li H,et al.A major role for VCAM-1,but not ICAM-1in early atherosclerosis.J ClinInvest.2001.107:1255-1262.
[85]梁萍,孙雷,唐建武,等.细胞间粘附分子1、血管细胞粘附分子1和肿瘤坏死因子在人动脉粥样硬化病灶中的表达及意义.中国动脉硬化杂志,2004;12(4):427-429.
[86]VanderA,Vander PT.Intercellular adhesion molecule-1.J Mo Med,1996;74(1):13.
[87]Peter K,Nawroth P,Conradt C,et al.circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating Intercellular adhesion molecule-1,E-selectin,P-selectin and thrombomodulin.ArtherosclerThromb Vas Biol,1997;17(3):55.
[88]Amberger A,Maczek C,Jurgens Qet al.Co-expression of ICAM-1,VCAM-1 and Hsp60 in human arterial and venous endothelial cell in response to cytokins and oxidized low-density lipoproteins. cell Stress Chaperones,1997;2(2):94.
[89] Gu L,Okada Y,Clinton S,et al.Absence of monocyte chemoattactant protein-1 reduces atherosclerosis in low-density lipoprotein-deficient mice.Mol Cell.1998;2:275-281.
[90] Ikeda U,Matsui K,Murakmi Y. Monocyte chemoattractant protein-I and coronary disease .Clin Cardiol,2000;25(4):143 — 147.
[91] Barter PJ. Inhibition of endothelial cell adhesion molecule expression by high density liporoteins. Clin Exp Pharmaco Physiol ,1997;24(4):286.
[92] Porrca E,Di Febbo C,Reale M, et al .Monocyte chemoatractant protein —l(MCP-l) is a mitogen for cultured rat vascular smooth muscle cell . Vasc Res,1997;34:58-56.
[93] Blandberg FQ Wen P,Dai M,et al.Detection of early atherosclerosis with radiolabeled monocyte chemoatractant protein-1 in prediabetic Zucker rats.Pediatr Radiol,2002;31:827-835.
[94] Rahman A; Anwar KN; Minhajuddin M;et. cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Am J Physiol Lung Cell Mol Physiol. 2004 Nov;287(5):L1017-24
[95] Li D, Williams V, Liu L, Chen H, Sawamura T, Antakli T, Mehta JL.LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation.Am J Physiol Heart Circ Physiol. 2002 Nov;283(5):H1795-801
[96] Van Wetering S; Van den Berk N; van Buul JD;et al. VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration.Am J Physiol Cell Physiol. 2003 Aug;285(2):C343-52
[97] Lei ZB, Zhang Z, Jing Q ,et al. oxLDL upregulates CXCR2 expression in monocytes via scavenger receptors and activation of p38 mitogen-activated protein kinase. Cardiovasc Res. 2002 Feb 1;53(2):524-32.
[98] Chen XL, Grey JY, Thomas S, Qiu FH, Medford RM, Wasserman MA, Kunsch C. Sphingosine kinase-1 mediates TNF-alpha-induced MCP-1 gene expression in endothelial cells:upregulation by oscillatory flow.Am J Physiol Heart Circ Physiol.2004 Oct;287(4):H1452-8.
[99]Khreiss T,J6zsef L,Potempa LA,Filep JG Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells.Circulation.2004 Apr 27;109(16):2016-22
[100]Domoto K,Taniguchi T,Takaishi H,Takahashi T,Fujioka Y,Takahashi A,Ishikawa Y,Yokoyama M.Chylomicron remnants induce monocyte chemoattractant protein-1 expression via p38MAPK activation in vascular smooth muscle cells.Atherosclerosis.2003 Dec;171(2):193-200
[101]Li Yanbo;Deng Huacong;Zheng Dan;et.Role of p38 Mitogen-activated Protein Kinase in Mediating Monocyte Chemoattractant Protein-1 in Human Umbilical Vein Endothelial Cells.Chinese Medical Sciences Journal,2004;19(1):71.
[102]Guo ZJ;Hou FF;Liang M;et.Advanced glycation end products stimulate human endothelial cells to produce monocyte chemoattractant protein-1.Zhonghua yi xue za zhi.2003;83(12):1075-9.
[103]Werle,Martina;Schmal,Ulrike;Hanna,Katharina;et.MCP-I induces activation of MAP-kinases ERK,JNK and p38 MAPK in human endothelial cells.Cardiovascular Research,2002;56(2):284.
[104]Ashida N;Arai H;Yamasaki M;rt al.Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis.The Journal of biological chemistry.J Biol Chem 2001;276(19):16555-60.
[105]李艳波,邓华聪,郑丹,等.P38信号通路对人脐静脉内皮细胞单核细胞趋化蛋白-1表达的影响.中华糖尿病杂志,2004;12(4):287-289.
[106]Zhao M,Liu Y,Wang X,New L,Han J,Brunk UT.Activation of the p38 MAP kinase pathway is required for foam cell formation from macrophages exposed to oxidized LDL.APMIS.2002 Jun;110(6):458-68.
[107]Jing Q;Xin SM;Cheng ZJ;et.Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells:mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity.Circulation research.1999;84(7):831-9.
[108]Ohashi N;Matsumori A;Furukawa Y;et.Role of p38 mitogen-activated protein kinase in neointimal hyperplasia after vascular injury.Arteriosclerosis,thrombosis,and vascular biology.2000;20(12):2521-6.
[109]Qing Jing,Sun-Mei Xin.Lysophosphatidylcholine Activates p38 and p42144Mitogen-Activated Protein Kinases in Monocytic THP-1 Cells,but Only p38Activation Is Involved in Its Stimulated Chemotaxis Circulation Research 2000;87:52-59.
[110]Ju H;Behm DJ;Nerurkar S;et al.p38 MAPK inhibitors ameliorate target organ damage in hypertension:Part 1.p38 MAPK-dependent endothelial dysfunction and hypertension.The Journal of pharmacology and experimental therapeutics.Pharmacol Exp Therl 2003;307(3):932-8.
[111]林蓉,甘伟杰.药物治疗动脉粥样硬化的新途径一抗炎治疗,中国药理学通报,,2004;20(5):499-503.
[1]Risau W,et al.Vasculogenesis.Annu Rev Cell Dev Biol,1995,11:73-91.
[2]Risau W.Mechanisms ofangiogenesis.Nature,1997,386:671-674
[3]Choi K.The hemangioblast:a common progenitor of hematopoietic and endothelial cells.J Hematother Stem Cell Res.2002 Feb;11(1):91-101
[4]Mikkola HK,Orkin SH.The search for the hemangioblast.J Hematother Stem Cell Res.2002 Feb;11(1):9-17.
[5]Asahara T,Murohara T,Sullivan A,Silver M,van der Zee R,Li T,Witzenbichler B,Schatteman G;Isner JM.Isolation of putative progenitor endothelial cells for angiogenesis.Science.1997 Feb 14;275(5302):964-7
[6]Suda T,Takakura N,Oike Y.Hematopoiesis and angiogenesis.Int J Hematol.2000 Feb;71(2):99-107
[7]Heilmann C,Beyersdorf F,Lutter G.Collateral growth:cells arrive at the construction site.Cardiovasc Surg.2002 Dec;10(6):570-8
[8]Choi K,Kennedy M,Kazarov A,Papadimitriou JC,Keller G.A common precursor for hematopoietic and endothelial cells.Development.1998Feb;125(4):725-32.
[9]Kalka C,Tehrani H,Laudenberg B,et al.VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease.Ann Thorac Surg,2000,70:829-834.
[10]Murasawa S,Llevadot J,Silver M,et al.Constitutive human telomerase reverse transcriptase expression enhances regenerativeproperties of endothelial progenitor cells.Circulation,2002,106:1133-1139
[11] Akashi M ,Hisashi I, Yoshimichi S ,et al. Platelet — derived growth factor BB( PDGF-BB) induces differentiation of bone marrow endothelial progenitor cellderived cell line TRBME2 into mural cells ,and changes the phenotype .Cell Physiol, 2005, 04:948-955.
[12] Friedrich EB, Walenta K, Scharlau J, et al. CD342/ CD133+NEGFR22+ endothelial progenitor cell subpopulation with potent vasoregenerative capacities .Circ Res ,2006 ,98 (3):e20-25.
[13] Kalka C, Tehrani H, Laudenberg B, et al.VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease. Ann Thorac Surg, 2000,70:829 - 834.
[14] Peichev M,Naiyer AJ, Pereira D ,et al.Expression of VEGFR-2 and AC133 by circulating human CD34+cells identifies a population of functional endothelial precursors. Blood ,2000,95 :952-958.
[15] ASAHARA T, MASUDA H, TAKAHASHI T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization .Circ Res ,1999, 85:221-228.
[16] TAKAHASHI T, KALKA C, MASUDA H, et al.Ischemia and cytokine-induced mobilization of bone derived endothelial progenitor cells for neovascularization.Nat Med, 1999, 5:434-438.
[17] YAMAGUCHI J, KUSANO K F, MASUO 0, et al. Stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization .Circulation, 2003,107:1322-1328
[18] GILL M, DIA S, HATTORI K, et al.Vascular trauma mobilization of VEGFR2+AC133+endothelial precursor induces rapid but transient cells。Circ Res, 2001, 8:167-174.
[19] Shibuya M. Vascular endothelial growth factor receptor family genes :when did the three genes phylogenetically segregate. Biol Chem, 2002 ,383 :1573-1579.
[20] Wang C, Jiao C, Hanlon HD, et al. Mechanical, cellular, and molecular factors interact to modulate circulating endothelial cell progenitors. AmJ Physiol Heart Circ Physiol, 2004;8 :8-12
[21] Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor 1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation, 2003,107:1322-1328.
[22] Amano K, Okigaki M, Adachi Y, et al. Mechanismfor IL21b2mediated neovascularization unmasked by IL21b knock-out mice. J Mol Cell Cardiol,2004,36 :469-480.
[23] Imanishi T, Hano T, Moriwaki C, et al.Angiotensin II potentiates vegf2 induced proliferation and network formation of endothelial progenitor cells. AJH 2004,17:1555-7.
[24] Sarukawa M, Yamamoto K, Ueda N. Kinetics of circulating endothelial progenitor cells in congestive heart failure. JACC, 2004,3:202A.
[25] Miller HI, George J, Herzl, The number and adhesive properties of circulating endothelial progenitor cells in patients with in - stent restenosis. JACC 2004,3:353A.
[26] Kunz GA, LiangqCuculoski F. David gregg circulating endothelialprogenitor cells predict coronary artery disease severity. JACC, 2004,3:496A.
[27] Moriwaki C, Imanishi T, Hano T, et al.Endothelial progenitor cell senescence is related to telomerase activity in patients with hypertension. AJH ,2004,17:202A.
[28] Dimmeler S, Aicher A, Vasa M, et al.HMG2CoA reductase inhibitors (statins)increase endothelial progenitor cells via the PI 32kinase/ Akt pathway. J Clin Invest, 2001,108:391-397.
[29] Murayama T, Tepper OM, Silver M, Ma H, Losordo DW, Isner JM,Asahara T,Kalka C. Determination of bone marrow-derived endothelial progenitor cell significance in anglogemc factor-induced neovascularization in Vivo. Exp Hematol. 104 2002;30(8):967-72
[30] Fadini GP, Sartore S, Schiavon Avogaro M, Albiero M, Baesso I, Cabrelle A,Agostini C, Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats Diabetologia. 2006;49(12):3075-8
[31 ] Hiasa K, Ishibashi M, Ohtani K, Inoue S, Zhao Q, Kitamoto S, Sata M, Ichiki T,Takeshita A, Egashira K. Gene transfer of stromal cell-derived factor-1 alpha enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization. Circulation. 2004 May 25;109(20):2454-61.
[32] Shi Q,Rafii S,Wu M,et al.Evidence for eireulatiag bone marow-derived endothelial cells .Blood, 1998;92(2):362-367
[33] Lin Y, Weisdorf DJ, Solovey A, et al. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest, 2000,105:71-77.
[34] Tiwari A, Hamilton G, Seifalian AM, et al. Regarding "Isolation of endothelial cells and their progenitor cells from human peripheral blood".J Vasc Surg. 2002 Apr;35(4):827)
[35] Kalka C,IAasuda H,Takahashi T,et al.Vascular endothelial growth factor 165 geng transfer augments circulating endothelial progenitor cells in human subjects. Circ Res,2000;86(12):1198-1202.
[36] Burger PE,Coetzee S,Mckeehan WL,et,al. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells .Blood,2002;100(10):3527-353
[37] Boyer M, Townsend LE, Vogel LM, Falk J, Reitz-Vick D, Trevor KT, Villalba M, Bendick PJ, Glover JL. Isolation of endothelial cells and their progenitor cells from human peripheral blood J Vasc Surg. 2000 Jan;31(1 Pt 1):181-9
[38] Harraz M, Jiao C, Hanlon HD, Hartley RS, Schatteman GC. CD34- blood-derived human endothelial cell progenitors.Stem Cells. 2001;19(4):304-12.
[39] Kalka C,IAasuda H,Takahashi T,et al.Vascular endothelial growth factor 165 geng transfer augments circulating endothelial progenitor cells in human subjects.CircRes,2000;86(12):1198-1202.
[40] Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T,Isner JM, Asahara T. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration.Circulation. 2002 Feb 12;105(6):732-8.
[41] Kawamoto A,Gwon HC,Iwaguro H,et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia .Circulation, 2001; 103(5):634-637.
[42] Assmus B, Sch(?)chinger V, Teupe C, Britten M, Lehmann R, Dobert N, Griinwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI).Circulation. 2002 Dec 10;106(24):3009-17
[43] He H, Shirota T, Yasui H, Matsuda T. Canine endothelial progenitor cell-lined hybrid vascular graft with nonthrombogenic potential. J Thorac Cardiovasc Surg. 2003 Aug;126(2):455-64.
[44] Kaushal S, Amiel GE, Guleserian KJ, Shapira OM, Perry T, Sutherland FW,Rabkin E, Moran AM, Schoen FJ, Atala A, Soker S, Bischoff J, Mayer JE Jr.Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001 Sep;7(9):1035-40
[45] Vasa M,et al.Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res:El-E7,2001
[46] Tepper OM,et al. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation ,adhesion,and incorporation into vascular struction.Circulation 2002, 106:2781-2786
[47] Verma S , et al.C-reactive protein attenuates endothelial progenoitor cell survival ,differentiation, and function: Further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation 2004,109:2058-2067
[48] Refii S, et al. Therapeutic Sterm and pregenitor cell transplantation for organ vascularization and regenetation. Nat Med 2003; 9: 702
[49] Gulati R. Autologous cuhure modified mononuclear cells confer vascular protection after artery injuny. Circulation 2003; 108: 1520
[50] Hill JM, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003; 348: 593
[51] Takahashi T, et al .Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med1999,5:1999.
[52] Dimmerler S ,et al.HMG-CoA reductase inhibitors(statins)increase endothelial progenitor cells via the PI-kinase/Akt pathway.J Clin Invest 2001 108:391-397.
[53] Asahara A ,et al .VEGF contributes to postnastal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. Embol J 1999,18:3964.
[54] Sterhlow K ,et al. Estrogen increase bone marrow-derived endothelial progenitor cell product and diminishes neointima formation .Circulation 2003,107:3059
[55] Heeschen C, et al .Erytheropoietin is a potent physiological stimulus for endothelial progenitor cell mobilization. Blood 2003,17:17
[56] Laufs U ,et al .Physical training increases endothelial progenitor cells, inhibits neointima formation.and enhances angiogenesis.Circulation 2003,22:22
[57] Seeger FH, et al .P38 Mitogen-activated protein kinase Downregulates endothelial progenitor cells.circulation 2005,8:1184.
[58] Walter DH ,et al. Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease .Cir Res 2005 ,25:1142.
[59] Urbish C,et al. Cathepsin L is required for endothelial progenitor cell-induced neovascularization .Nat Med 2005 ,11:206
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