高糖诱导内皮祖细胞凋亡的内质网应激途径及其在糖尿病冠脉病变中的作用
|
摘要
研究背景
内皮祖细胞是1997年发现的在成人体内能分化为内皮细胞的前体细胞,存在于骨髓及外周血中。内皮祖细胞主要参与内皮细胞修复即血管的再内皮化以及参与血管新生,由此近年来内皮祖细胞在血管病变中的作用越来越受到重视。糖尿病血管病变的重要发病机制是内皮细胞功能受损及凋亡。血管内皮层完整性遭到破坏,炎性细胞浸润至内皮下层促进动脉粥样硬化的发生发展,因此内皮功能受损被视为糖尿病血管病变的始动因素。正常状态下,受损的内皮由周边内皮及循环中的内皮祖细胞分化为内皮来修复,保持内皮层的完整性,一旦内皮祖细胞受损,内皮损害和修复之间的平衡被打破,内皮层的完整性遭到破坏,由此发生动脉粥样病变,因此内皮祖细胞在糖尿病血管病变中具有重要作用。已有研究证实糖尿病时循环内皮祖细胞数量和功能均受损害,而循环内皮祖细胞来源于骨髓,由此我们推测糖尿病时骨髓内皮祖细胞可能也受损。我们已知内质网应激介导的β细胞凋亡是糖尿病发病的重要机制,由此我们推测内质网应激可能介导内皮祖细胞的凋亡参与糖尿病血管病变的发病。迄今尚无观察糖尿病对骨髓内皮祖细胞的凋亡有无影响及其机制的研究,本研究的目的旨在:
目的
1.建立糖尿病大鼠模型。
2.研究大鼠骨髓内皮祖细胞体外分离、诱导、培养及鉴定的方法。
3.检测糖尿病大鼠和正常大鼠骨髓内皮祖细胞凋亡率,明确糖尿病时骨髓内皮祖细胞是否受损。
4.检测糖尿病大鼠和正常大鼠骨髓内皮祖细胞内质网应激标志物GRP78及内质网应激特异性凋亡因子—caspase-12活性,以了解内质网应激是否参与介导糖尿病时骨髓内皮祖细胞的凋亡。
方法
1.糖尿病(diabetes mellitus,DM)大鼠模型的制备
雄性Wistar大鼠27只,随机分为2组:对照组12只,DM组15只。标准大鼠饲料喂养1周后,禁食12h,DM组大鼠给以腹腔内注射链脲佐菌素(STZ)65mg/kg,对照组大鼠注射等量柠檬酸钠/柠檬酸缓冲液。糖尿病大鼠成模标准为:连续2次空腹血糖＞16.7mmol/L。未达成模标准者剔除。DM组大鼠血糖升高后,饲以高脂高糖饮食,对照组则给予标准饮食喂养。
2.大鼠骨髓源内皮祖细胞的培养、鉴定。
2.1大鼠骨髓内皮祖细胞的培养
大鼠拉颈处死后无菌状态下分离股骨胫骨,将骨髓细胞以无菌PBS液完全冲洗至离心管,应用大鼠淋巴细胞分离液(LTS-1083)以密度梯度离心法(2 000rpm,30 min)分离单个核细胞。洗涤后以含20%胎牛血清的M199培养液重悬,接种于培养瓶中,37℃、5%CO_2条件下培养。细胞培养24 h(早期贴壁)后收集未贴壁细胞离心后以选择性培养基(M199培养基加入20%胎牛血清,100μg/mL链霉素,100U/ml青霉素,40 ng/mL大鼠血管内皮生长因子(VEGF,Peprotech,USA),2 ng/mL人成纤维细胞生长因子(FGF,Peprotech,USA),10 ng/mL人表皮细胞生长因子(EGF,Peprotech,USA)以及10U/mL肝素)重悬计数,1×10~6/mL转入包被0.1%明胶(Sigma,USA)的24孔板和培养瓶中二次贴壁,4天后换液,培养至第7天鉴定。
2.2大鼠骨髓EPCs的鉴定
细胞在含DiI-ac-LDL(2.4μg/mL)的培养液中37℃避光孵育1h,然后以2%的多聚甲醛固定10min,PBS液漂洗后再加FITC-UEA-1(10μg/mL)避光孵育1h。PBS液漂洗后在荧光显微镜下观察,显示红色荧光的为ac-LDL阳性细胞,显示绿色荧光的为UEA-1阳性细胞,显示双荧光阳性(黄色)的细胞被认为是EPCs。
3.流式细胞仪检测大鼠骨髓源内皮祖细胞凋亡率
ANEXIN V/PI凋亡检测试剂盒流式细胞仪检测凋亡。
4.RT-PCR检测GRP78、CASPASE-12基因表达
结果
1.大鼠基本情况及血糖检测
实验过程中,对照组大鼠精神状态良好,体重增加明显,反应敏捷,毛色白而光泽。DM组大鼠出现多食、多饮、多尿和消瘦等症状,体重增加迟缓,精神萎靡,皮毛无光泽,部分出现烂尾、白内障等。最终共23只完成实验,其中对照组12只,DM组11只。
注射STZ之前,对照组与DM组血糖无明显差异(P＞0.05)。注射STZ 1周后,DM组血糖明显高于对照组(P＜0.01),并持续至实验末。
2.大鼠骨髓源内皮祖细胞培养、鉴定
早期贴壁细胞呈毛发样生长,多为骨髓基质细胞。二次贴壁的细胞大多在3天左右伸展成梭形或纺锤形。7～10天增殖速度加快可见内皮细胞特异性条索状结构。2周左右细胞可达80～90%融合,大部分细胞呈多角形。80%以上细胞可同时吸收DiI-ac-LDL结合FITC-UEA-1即鉴定为内皮祖细胞。
3.流式细胞仪检测大鼠骨髓源内皮祖细胞凋亡率
DM大鼠骨髓源内皮祖细胞凋亡率明显高于对照组(20.3±4.64%vs.11.2±2.85,P＜0.05)。
4.RT-PCR检测GRP78、CASPASE-12 mRNA表达
1)GRP78mRNA表达:DM组内皮祖细胞GRP78mRNA表达均较对照组明显增高(0.64±0.07%vs.0.43±0.05%,P＜0.05)。
2)CASPASE-12 mRNA表达:DM组内皮祖细胞CASPASE-12 mRNA表达均较对照组明显增高(0.56±0.09%vs.0.36±0.04,P＜0.05)。
结论
1)糖尿病大鼠骨髓源内皮祖细胞凋亡率明显增加。
2)DM大鼠骨髓源内皮祖细胞GRP78及CASPASE-12 mRNA表达增加,提示内质网应激参与介导糖尿病大鼠骨髓源内皮祖细胞凋亡,可能在糖尿病血管病变的发生发展中起重要作用。
研究背景
内质网应激(endoplasmic reticulum stress,ER stress)是内质网功能紊乱时出现错误折叠与未折叠蛋白在腔内聚集以及Ca~(2+)平衡紊乱的状态,对决定应激细胞的结局如抵抗、适应、损伤或凋亡有重要作用。一定程度的内质网应激因能激活保护机制例如分子伴侣表达而有细胞保护作用,应激过强时,保护机制不能与损伤抗衡,内质网应激可通过caspase-12独立地介导细胞凋亡。内质网应激使caspase-12活化,继而激活下游caspase,导致细胞凋亡,内质网应激反应性细胞凋亡是不同于死亡受体介导或经线粒体介导的一种新的细胞凋亡途径,caspase-12作为凋亡起始因子发挥了关键作用。有研究已证实内质网应激介导的凋亡参与胰岛β细胞的凋亡,是糖尿病发病的重要机制。内皮祖细胞在糖尿病血管病变中有重要作用,研究表明糖尿病时循环内皮祖细胞数量及功能均受损,我们在体内实验也发现其在糖尿病大鼠骨髓中凋亡率明显增加,同时内皮祖细胞内质网应激激活,结合内皮祖细胞具有内皮细胞修复以及参与血管新生的作用,我们推测内皮祖细胞凋亡增加很可能在以动脉粥样硬化为重要特征的糖尿病血管病变中发挥重要作用。那么内质网应激介导的凋亡是否参与了高糖诱导的大鼠骨髓源内皮祖细胞的凋亡呢?本工作拟通过高糖刺激体外培养的大鼠骨髓源内皮祖细胞,观察高糖对其凋亡的影响及内质网应激特异凋亡因子caspase-12是否参与介导其凋亡,以期为糖尿病血管病变的预防和治疗提供新的机制和靶点。
目的
1.观察高糖培养对大鼠骨髓EPCs凋亡的影响。
2.观察高糖培养是否激活大鼠骨髓EPCs内质网应激反应。
3.观察CASPASE-12介导的内质网应激途径是否在高糖诱导的大鼠骨髓EPCs凋亡过程中发挥重要作用。
方法
1.大鼠骨髓内皮祖细胞(Bone Marrow-derived Endothelial Progenitor Cells,BM-EPCs)的培养、鉴定
1.1骨髓EPCs的分离、培养
6周龄雄性体重250—300克Wistar大鼠购自山东大学动物实验中心,拉颈处死后无菌条件下取出四肢长骨,将骨髓细胞以无菌PBS液完全冲洗至离心管用大鼠淋巴细胞分离液(LTS-1083)以密度梯度离心法(2 000 rpm,30 min)分离单个核细胞。洗涤后以含20%胎牛血清的M199培养液重悬,接种于培养瓶中,37℃、5%CO_2条件下培养。细胞培养24 h(早期贴壁)后收集未贴壁细胞离心后以选择性培养基(M199培养基加入20%胎牛血清,100μg/mL链霉素,100U/ml青霉素,40 ng/mL大鼠血管内皮生长因子(VEGF,Peprotech,USA),2 ng/mL人成纤维细胞生长因子(FGF,Peprotech,USA),10 ng/mL人表皮细胞生长因子(EGF,Peprotech,USA)以及10U/mL肝素)重悬计数,1×10~6/mL转入包被0.1%明胶(Sigma,USA)的24孔板和培养瓶中二次贴壁,4天后换液,培养至第7天鉴定。
1.2 EPCs的鉴定
细胞在含DiI-ac-LDL(2.4μg/mL)的培养液中37℃避光孵育1h,然后以2%的多聚甲醛固定10min,PBS液漂洗后再加FITC-UEA-1(10μg/mL)避光孵育1h。PBS液漂洗后在荧光显微镜下观察,显示红色荧光的为ac-LDL阳性细胞,显示绿色荧光的为UEA-1阳性细胞,显示双荧光阳性(黄色)的细胞被认为是EPCs。
2.流式细胞仪测定不同浓度糖培养BM-EPCs凋亡率
骨髓EPCs给予不同浓度高糖(5mmol/L、12.5mmol/L及25mmol/L)刺激24h,流式细胞仪检测细胞凋亡率
3.Western-blot测定不同浓度糖培养骨髓EPCs GRP78、CASPASE-12、CASPASE-3蛋白表达改变
4.流式细胞仪测定给予或未给予caspase-12抑制剂(Z-ATAD-FMK 5uM)预处理后高糖(25mmol/L)培养骨髓EPCs凋亡率
5.Western-blot测定给予或未给予caspase-12抑制剂(Z-ATAD-FMK 5uM)预处理后高糖(25mmol/L)培养骨髓EPCs CSAPASE-12、CASPASE-3蛋白表达改变
结果
1.大鼠骨髓内皮祖细胞的培养、鉴定
早期贴壁细胞呈毛发样生长,多为骨髓基质细胞。二次贴壁的细胞大多在3天左右伸展成梭形或纺锤形。7～10天增殖速度加快可见内皮细胞特异性条索状结构。2周左右细胞可达80～90%融合,大部分细胞呈多角形,可呈铺路石样排列。80%以上细胞可同时吸收DiI-ac-LDL结合FITC-UEA-1即鉴定为内皮祖细胞。
2.高糖对骨髓EPCs凋亡的影响
不同浓度糖(5mmol/L、12.5mmol/L及25mmol/L)孵育EPCs 24h,结果显示高糖呈浓度依赖性诱导EPCs凋亡(7.34±0.44%,11.59±1.22%,36.82±2.16%),与正常对照组(6.18±0.33%)比较差异均有统计学意义(P＜0.05),其中以糖浓度为25mmol/L时凋亡率最高。高渗对照25mmol/L甘露醇组凋亡率与对照组比较无显著性差异(5.95±0.81%vs.6.18±0.33%,P＞0.05)。
3.高糖激活骨髓EPCs内质网应激
不同浓度糖(5mmol/L、12.5mmol/L及25mmol/L)孵育EPCs 24h,westernblot法测定GRP78表达,结果显示高糖激活内质网应激特异蛋白GRP78表达,并呈浓度依赖性(127.33±9.71,152.33±7.09,183.33±4.51),与正常对照组(39±6.56)比较差异均有统计学意义(P＜0.05)。
4.高糖对CASPASE-12和CASPASE-3蛋白表达的影响
不同浓度(5mmol/L、12.5mmol/L及25mmol/L)孵育EPCs 24h,western blot法测定CASPASE-12和CASPASE-3蛋白水平表达,结果显示高糖激活CASPASE-12和CASPASE-3,并呈浓度依赖性(cleaved-CASPASE-12:34.67±9.07,71.33±4.16,125.33±11.68;cleaved-CASPASE-3:64.67±7.37,131.67±9.02,165.33±5.69),与正常对照组比较差异有统计学意义(cleaved-CASPASE-12;cleaved-CASPASE-3均无表达,P＜0.01)。
5.CASPASE-12抑制剂(Z-ATAD-FMK)对高糖诱导的骨髓EPCs凋亡的影响
25mmol/L高糖培养组予5uM Z-ATAD-FMK预处理后,与未预处理组比较高糖诱导的凋亡率明显下降(17.91±0.94%vs.36.82±2.16%,P＜0.05),而对照组应用Z-ATAD-FMK预处理与未预处理组凋亡率无明显差异(5.62±0.92%vs.6.18±0.33%,P＞0.05);
6.CASPASE-12抑制剂(Z-ATAD-FMK)对高糖诱导的骨髓EPCs CASPASE-12和CASPASE-3蛋白表达的影响
与未加抑制剂预处理组比较高糖加预处理组CASPASE-12和CASPASE-3活性均被明显抑制(cleaved-CASPASE-12:0 vs.58±7.21,P＜0.01;cleaved-CASPASE-3:155±10.58 vs.185.33±12.01,P＜0.05)。对照组预处理组与未预处理组cleaved-CASPASE-12、cleaved-CASPASE-3均无表达)。
结论
1)高糖培养可以浓度依赖性诱导大鼠骨髓EPCs凋亡。
2)高糖可以激活大鼠骨髓EPCs内质网应激。
3)CASPASE-12介导的内质网应激途径可能在高糖诱导的大鼠骨髓EPCs凋亡过程中发挥重要作用。
研究背景
2型糖尿病(type 2 diabetes mellitus,T2DM)的发病率逐年升高,已成为威胁人类健康的常见疾病,糖尿病是冠心病的独立危险因素之一,近年来更加认为糖尿病是冠心病的等危症。T2DM常伴有多种心血管疾病的危险因素,易合并以动脉粥样硬化和血栓形成为特征的大血管病变,冠心病是引起T2DM患者死亡的主要原因,其中最为严重的是急性冠状动脉综合征(acute coronary syndroms,ACS)。近年的研究显示,动脉粥样硬化斑块破裂和血栓形成是ACS的主要发病机制,而斑块不稳定性是这些环节中的始动因素。急性冠脉综合征的发生与否取决于斑块的稳定程度,因此易损斑块的临床识别以及探索稳定斑块的方法具有十分重要的临床意义。由于存在血管重构,冠脉造影(coronary angiography,CAG)并不能真实地反映出冠脉病变程度—常低估冠脉病变。血管内超声(Intravascularultrosound,IVUS)能提供管腔、管壁横截面图像,能够分辨出斑块的大小、组成成分、分布以及观察斑块处血管的重构情况,在斑块稳定性、靶病变严重程度的评估等方面具有冠脉造影无法比拟的优势。
内皮祖细胞(endothelial progenitor cells,EPCs)是一类能增殖并分化为血管内皮细胞的前体细胞。对于调节内皮细胞凋亡/再生平衡及保持内皮层的完整性有着重要作用。另外EPCs还参与新生血管的生成,这对于缺血组织侧枝循环的形成至关重要。因此,EPCs在糖尿病冠脉病变的发生发展中有重要的作用。冠脉造影研究显示与不伴有糖尿病的冠心病患者相比糖尿病患者冠状动脉病变严重,病变范围广并且冠状动脉侧支血管形成能力明显下降,应用血管内超声评价糖尿病对冠脉病变影响的研究尚少见报道;以往研究表明糖尿病患者循环EPCs数量和功能均受影响,但此种改变与急性冠脉综合征靶病变斑块稳定性及病变程度有何关系尚不明了。由此构成本课题的设计思路和研究目的。本课题应用血管内超声技术分析急性冠脉综合征患者靶病变严重程度、斑块负荷与外周血EPCs数量的关系,研究糖尿病合并ACS时对外周血EPCs数量的影响以及与易损斑块特征的关系。
目的
1.应用血管内超声方法研究糖尿病合并急性冠脉综合征患者靶病变斑块特征。
2.观察糖尿病合并急性冠脉综合征患者循环内皮祖细胞数量变化。
3.研究糖尿病合并急性冠脉综合征患者循环内皮祖细胞数量变化与靶病变严重程度和斑块负荷有无相关性。
方法
1.研究对象
选自2007年7月至2008年1月间在我院接受冠状动脉造影(CAG)检查确诊为急性冠脉综合征(ACS)40例患者。包括急性心肌梗死(AMI)16例和不稳定型心绞痛(UAP)24例。AMI的临床诊断为伴有心肌坏死的血清心肌标志物浓度的动态演变的持续性静息状态胸痛的临床病史。靶血管病变经心电图、CAG和超声心动室壁运动资料确定。根据是否患有糖尿病将患者分为两组:糖尿病组(n=20),非糖尿病组(n=20)。糖尿病诊断标准符合2005年ADA修订的诊断及分型标准,均为2型糖尿病。入院时收集患者的病史包括冠心病高危因素如高血压、高脂血症、吸烟史等临床资料。
2.检查方法
2.1冠状动脉造影检查所有患者按常规进行CAG检查,冠脉造影诊断标准按照美国Coronary Artery Surgery Study(CASS)研究:冠状动脉左主干狭窄≥50%,左前降支、左旋支及右冠状动脉狭窄≥70%为阳性。
2.2血管内超声检查在常规冠状动脉血管造影结束后,采用标准的冠状动脉内介入导管操作技术进行IVUS检查,追加3000IU肝素,然后把0.014英寸的导丝送至靶血管,常规冠脉内注入200μg硝酸甘油。将IVUS导管沿导引钢丝越过靶病变,至靶病变远端1cm以上,利用自动回撤仪以0.5mm/s的速度自动匀速回撤至靶血管开口,所有获取的IVUS图像实时连续采集并存入硬盘,供以后脱机分析。
2.3 IVUS图像的定量测量斑块形态由二位研究者采用盲法分别进行分析和处理。
2.3.1 EEM-CSA:即血管外弹力膜的横断面积,手工描记外膜内边缘,由仪器自身的测量系统测得的。
2.3.2 L-CSA(lumen-CSA):是指血管腔的横断面积,手工描记内膜的内边缘,由仪器自身的测量系统测得的。当斑块包绕探头时,推测管腔的大小等于探头的大小。
2.3.3 P-CSA(plaque-CSA):经由外弹力膜面积减去血管管腔面积计算获得。
2.3.4 PB(plaque burden):即斑块负荷,为斑块面积除以外弹力膜面积乘以100%,它不同于管腔狭窄率。
2.4循环内皮祖细胞培养、鉴定、计数
2.4.1内皮祖细胞培养
无菌采集外周血20mL,肝素抗凝,用PBS液等倍稀释血液。将稀释血液按1:2的比例置于人淋巴细胞分层液(LTS-1077)上以2 000 rpm离心30min。分离出单个核细胞后用5倍体积的PBS洗涤2次计数,调整细胞浓度以1×10~6/mL接种在包被0.1%明胶的24孔培养板。选择性M199培养基(含20%胎牛血清,青霉素100U/mL,链霉素100μg/mL,人血管内皮生长因子(VEGF)40 ng/mL,人成纤维细胞生长因子(FGF)2 ng/mL,人表皮生长因子(EGF)10 ng/mL,10U/mL肝素)诱导培养,4d后换液,继续培养至7d,PBS洗掉未贴壁细胞,贴壁细胞进行细胞鉴定。
2.4.2内皮祖细胞鉴定
内皮祖细胞可结合FITC标记的植物凝集素(FITC-UEA-1)并且能吸收DiI标记的乙酰化低密度脂蛋白(DiI-ac-LDL),能结合两者的细胞可鉴定为内皮祖细胞。首先在37℃细胞与DiI-ac-LDL(2.4μg/ml)共同孵育1小时,然后用2%多聚甲醛固定10分钟,轻轻冲洗后与FITC-UEA-1(10μg/mL)共同孵育1小时,PBS冲洗后在倒置荧光显微镜下观察,双染阳性者为内皮祖细胞。
2.4.3内皮祖细胞计数
由两位研究者采用盲法分别计数双染细胞,每孔随机选取3个高倍镜视野计数,取平均值。
结果
靶病变分布情况:左前降支22例(55%),左回旋支8例(20%),右冠10例(25%)。
血管内超声结果
血管内超声分析示,糖尿病组管腔面积,外弹力膜面积,斑块面积和斑块负荷分别为3.3±1.6,14.3±3.0 and 11.1±2.7 mm~2,76.5%±11.7%;非糖尿病组管腔面积,外弹力膜面积,斑块面积和斑块负荷分别为4.9±2.2,13.8±3.4 and 9.0±2.6mm~2,66.2%±13.2%。与非糖尿病组比较糖尿病组斑块负荷明显增大。
循环内皮祖细胞计数
糖尿病组循环内皮祖细胞数量明显低于非糖尿病组(30.4±6.99 vs.51.7±11.1EPCs/×200 field,P＜0.05)。
循环内皮祖细胞数量与斑块负荷的相关性
糖尿病组循环内皮祖细胞数量与斑块负荷呈负相关(r=-0.427,P＜0.05)。
结论
1.糖尿病组病变部位的斑块面积和斑块负荷均大于非糖尿病组,管腔面积小于非糖尿病组;
2.糖尿病组患者循环内皮祖细胞数量明显低于非糖尿病组;
3.糖尿病组患者斑块负荷、靶病变严重程度与循环内皮祖细胞数量负相关,提示靶病变严重程度与循环内皮祖细胞数量相关;
4.糖尿病急性冠脉综合征患者冠脉病变程度严重可能与内皮祖细胞减少导致的斑块迅速增长及血管内皮的再内皮化降低有关。
Background
The endothelial progenitors cells(EPCs)were reported in 1997 to differentiate in vitro into endothelial cell(EC)and seemed to be incorporated at sites of active angiogenesisin various animal models of ischaemia,which led to the new concept that vasculogenesis and angiogenesis may occur simultaneously in postnatal.These cells are derived from the bone marrow and can circulate in the blood.The majority of circulating EPCs reside in the bone marrow where exist an optimal microenviroment. EPCs are capable of differentiating into endothelial cells and homing to sites of ischaemia and play a important role in vascular repair and reendothelialisation of injured blood vessels and collateral vessels formation.Moreover,diabetic macro -angiopathy develop as the result of an atherogenesis process initiated by endothelial damage and dysfunction.And recent insights show that the injured endothelial monolayer is regenerated by circulating EPCs,which accelerate reendothelialization and limits atherosclerotic lesion formation.There is a balance between endothelial cell damage and endothelial cell regeneration.Apoptotic and necrotic endothelial cells are regenerated by circulating EPCs.However,the system becomes imbalanced in conditions of enhanced endothelial cell damage and exhausted EPCs-mediated endothelial cell repair.The resulting the disequilibrium between damage and regeneration,results in the atherogenesis progression.So the EPCs would play a vital role in the diabetic macroangiopathy.It has been reported that the number of circulating EPCs which origin from bone marrow were impaired in patients with diabetes mellitus(DM).Moreover,recent studies have revealed that ER stress mediiated apoptosis inβcells could be a cause of DM.Based on these results,this present study hypothesized that the EPCs might be impaired in bone marrow in diabetes mellitus and that ER stress mediated apoptosis in bone marrow derived EPCs (BM-derived EPCs)might be a cause of diabetic macroangiopathy.The objectives of this study are as follow:
Objectives
1.To establish a DM animal model.
2.To investigate the methods of isolating,culturing and identifying BM-derived EPCs
3.To examine the occurrence of apoptosis of BM-derived EPCs in DM rat and control group to determine whether or not the EPCs is impaired in bone marrow in DM.
4.To assess the mRNA express of GRP78 and CASPASE-12 of BM-derived EPCs in diabetic or non-diabetic rats to investigate whether or not the ER stress mediated apoptosis in bone marrow derived EPCs.
Methods
1.Establishment of DM animal model
All protocols were approved by the Shandong University experimental animal center. Twenty-seven male age-matched Wistar rats(Experimental Animal Center of Shandong University,Ji'nan,China)were randomly divided into 2 groups:control group(n=12)and DM group(n=15).Diabetes was induced by a single intraperitoneal injection of streptozotocin(STZ,65 mg/kg body wt and dissolved in 0.1 mol/l citrate buffer,pH 4.2)in DM group.The rats in control group received citrate buffer alone. One week after injection of STZ,fasting plasma glucose levels was measured,and rats with plasma glucose at least two times higher than 16.7mmol/L were used.DM group rats were fed high fat and glucose diet and the rats in control group were fed standard rat diet.
2.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and Characterization
2.1 EPCs culture
BM cells were obtained by flushing the tibias and femurs of rat.BM mononuclear cells were isolated by density centrifugation over rat lymphocyte separating medium(LTS-1083)at 2000 rpm for 30 min.Then mononuclear cells were isolated and plated culture flasks in M199 medium(Gibco,USA)supplemented with 20% fatal calf serum,100μg/mL streptomycin,100U/ml penicillin.After 24 h in culture, the nonadherent cells were harvested and resuspended,then 1×10~6 cells were plated on 24-well culture dishes or T25 flasks pre-coated with 0.1%gelatin(Sigma,USA) and maintained in M199 medium supplemented with 20%fatal calf serum,100μg/mL streptomycin,100U/ml penicillin,40 ng/mL vascular endothelial growth factor (VEGF,Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA), 10 ng/mL epidermal growth factor(EGF,Peprotech,USA),and 10U/mL heparin. After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS,new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.
2.2 Characterization of EPCs
Fluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FIYC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,St.Louis,USA)and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo -carbocyanine(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular ProbeCo,Eugene,USA).To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%parafor -maldehyde for 10 minutes.After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..
3.EPCs apoptosis detection by flow cytometer.
The BM-derived EPCs apoptosis of the rats in DM and control groups was determined by flow cytometric detection according to the manufacturer's instructions of the annexin V-FITC apoptosis detection kit(Jingmei,China).Briefly,EPCs were washed once with PBS and resuspended in binding buffer were stained with annexin Ⅴ(0.5μg/ml)and propidium iodide(0.6μg/ml)for 5 min at room temperature.After staining,cells were immediately analyzed using a FACScan flow cytometer(Becton Dickinson,CA)with simultaneous monitoring of green fluorescence(530 nm,30 nm band-pass filter)for annexin V-FITC and red fluorescence(long-pass emission filter that transmits light＞650 nm)associated with propidium iodide.10,000 events were collected and analyzed to determine the percentage of annexin-ⅤFITC and PI-positive cells.The cells were analyzed using Cell Quest analysis software (Becton-Dickinson,CA).
4.The mRNA expression of GRP78 and CASPASE-12 by RT-PCR
Total RNA from DM and control rat BM-defived EPCs was isolated using the TRIzol Reagent(Invitrogen)as described by the manufacturer.For RT-PCR,lug total RNA and oligo(dT)(Promega)was reverse-transcribed into cDNA with 1 unit/ml M-MLV reverse transcriptase(Promega)at 42℃for 1b.Primers sequences for rat grp78, caspase-12 andβ-actin are available with Taq Polymerase(Takara)for PCR.
Results
1.General features of the experimental rats
At the end of the experiment,11 diabetic rats induced by STZ and 12 control rats survived.Glucose levels were significantly elevated in diabetic rats compared with control rats after STZ injection.Other symptoms,such as lower body weights, polyuria and polyphagia,which are normally associated with diabetic state were also observed in the diabetic rats.
2.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and characterization
The secondarily attached cells stretched after 3 days.The cells proliferated faster after 7-10 days' incubation when cord-like structure was observed.After 2 weeks' induction, most of the cells exhibited multangular morphology.More than 80%attached cells took up DiI-ac-LDL,bound FITC-UEA-1(double positive fluorescence).
3.EPCs apoptosis detection by flow cytometer.
BM-derived EPCs significantly increased apoptosis in DM rat compared with control group(20.3±4.64%vs.11.2±2.85,P＜0.05).
4.The mRNA expression of GRP78 and CASPASE-12 by RT-PCR
The mRNA expression of GRP78 and CASPASE-12 significantly increased in DM rat when compared with control group(GRP78:0.64±0.07%vs.0.43±0.05%,P＜0.05; CASPASE-12:0.56±0.09%vs.0.36±0.04,P＜0.05).
Conclusions
1.BM-derived EPCs significantly increased apoptosis in DM rat.
2.The mRNA expression of GRP78 and CASPASE-12 significantly increased in DM rat which suggest that ER stress mediated apoptosis in bone marrow derived EPCs might be one of causes of diabetic macroangiopathy.
Background
Endoplasmic reticulum(ER)stress is a conditions interfering with endoplasmic reticulum function will perturb the folding of newly synthesized membrane or secreted proteins.The accumulation of unfolded proteins in ER causes cells ER stress response,which reduces the amount and aggregation of unfolded proteins in ER.In case the response fails to solve ER stress,the apoptosis pathway will be activated.ER stress can lead to apoptosis by various pathways,among them caspase-12 has been proposed as a specific mediator of ER-stress-induced apoptosis.It has been suggested that ER stress might induce cell apoptosis via caspase-12 independently.The involvement of apoptosis via ER stress is a relatively new pathway other than the death receptor or mitochondrial death pathways.
It has been shown that type 2 diabetic patients have reduced pancreatic isletsβcell mass and increasedβcell apoptosis rates.Recent in vitro studies suggest that elevated glucose levels induced apoptosis in culturedβcells possibly through ER stress.And we observed the occurrence of apoptosis of EPCs elevated in DM rat bone marrow as well as ER stress was actived in vivo study.Moreover,the endothelial progenitor cells (EPCs)play a important role in vascular repair and reendothelialisation of injured blood vessels,which means the EPCs play a key factor in diabetic vascular disease. Based on these results,we hypothesized that elevated glucose levels impair the bone marrow-derived EPCs(BM-derived EPCs)survival and induce the cells apoptosis. Then whether the ER stress-mediated apoptotic pathway is a component of high concentration of glucose induced the cells apoptois is unknown.In order to test this hypothesis,we cultured rat BM-derived EPCs under different concentration of glucose to investigate that the effect of high concentration of glucose on EPCs and a possible role of ER stress in the apoptosis.
Objectives
1.To assess the effect of apoptosis indued by high concentration of glucose on rat BM-derived EPCs.
2.To assess whether the ER stress was actived in rat BM-derived EPCs by high concentration of glucose.
3.To assess whether the ER stress-mediated apoptotic pathway is a component of high concentration of glucose induced rat BM-derived EPCs apoptois.
Methods
1.rat BM-derived EPCs culture and characterization
1.1 EPCs culture
Adult male Wistar age-matched(6 weeks)rats(Experimental Animal Center of Shandong University,Ji'nan,China)weighing 250-300 g were used.All protocols were approved by the shandong university experimental animal center.BM cells were obtained by flushing the tibias and femurs of rat.BM mononuclear cells were isolated by density centrifugation over LTS-1083 at 2000 rpm for 30 min.Then mononuclear cells were isolated and plated culture flasks in M199 medium(Gibco,USA) supplemented with 20%fatal calf serum,100μg/mL streptomycin,100U/ml penicillin.After 24 h in culture,the nonadherent cells were harvested and resuspended, then 1×10~6 cells were plated on 24-well culture dishes or T25 flasks pre-coated with 0.1%gelatin(Sigma,USA)and maintained in M199 medium supplemented with 20% fatal calf serum,100μg/mL streptomycin,100U/ml penicillin,40 ng/mL vascular endothelial growth factor(VEGF,Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA),10 ng/mL epidermal growth factor(EGF,Peprotech, USA),and 10U/mL heparin.After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS,new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.
1.2 Characterization of EPCs
Fluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FITC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,St.Louis,USA)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo -carbocyanine(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular Probe,Eugene,USA).To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%parafor -maldehyde for 10 minutes.After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..
1.3 Passage of EPCs
The cells should be subcultured when they reach 80-85%confluence.Aspirate the medium and wash the cells with PBS,then add trypsin-EDTA.Incubate 37℃for a few minutes meanwhile should be monitored using an inverted phase-contrast microscope.Once the cells have begun to detach inactivate the trypsin with medium and centrifuge at 1000 rpm for 8 min.Aspirate the supernatants and resuspend cells with medium then split the cells at 1:3 ratio to expand and be ready to use.
2.EPCs apoptosis detection by flow cytometer.
2.1 Flow cytometer was performed to characterize the occurrence of apoptosis of various concentration of glucose(control,5mmol/L,12.Smmol/L,25mmol/L and 25mmol/L manitol)treatment for 24 h on EPCs.
2.2 Flow cytometer was performed to characterize the occurrence of apoptosis of pre-treating or no pretreating the EPCs with Z-ATAD-FMK(caspase-12 specific inhibitor)in the presence or absence of high glucose(25mmol/L).
3.Western blot analysis
3.1 The protein expression of the effect of various concentration of glucose(control, 5mmol/L,12.Smmol/L and 25mmol/L)treatment for 24h on EPCs were investigated by western blot analysis for the GRP78,CASPASE-12,CASPASE-3,respectively.
3.2 The protein expression of the effect of pre-treating or no pretreating the EPCs with Z-ATAD-FMK in the presence or absence of high concentration of glucose(25mmol/L) by western blot analysis for the CASPASE-12,CASPASE-3,respectively.
Results
1.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and characterization
The BM mononuclear cells were isolated and the secondarily attached cells stretched after 3 days.The cells proliferated faster after 7-10 days resulted in a spindle-shaped, EC-like morphology.After 2 weeks' induction,most of the cells exhibited multangular morphology.The cord-like structures and cobblestone appearance was observed.Fluorescent chemical detection of EPCs was performed on second attached cells after 7 days in culture.Uptake of ac-LDL was detected red-stained cells,binding UEA-1 was detected green-stained cells and dual-stained cells for both UEA-1 and ac-LDL were judged to be EPCs.More than 80%attached cells were double positive fluorescence.
2.The effect of high concentration of glucose on BM-derived EPCs apoptosis FAC were performed to characterize the effect of high concentration of glucose treatment on EPCs.Treatment with high concentration of glucose(Smmol/L, 12.5mmol/L and 25mmol/L)for 24h significantly increased apoptosis in cultured rat BM-EPCs compared with control group in a dose-dependent manner(7.34±0.44%vs 6.18±0.33%,P＜0.05;11.59±1.22%vs 6.18±0.33%,P＜0.05;36.82±2.16%vs. 6.18±0.33%,P＜0.05;n=3).Treatment of the cells with 25mmol/L mannitol,used as an osmotic control,had no significant effect on apoptosis compared with control group in rat BM-EPCs(5.95±0.81%vs.6.18±0.33%,P＜0.05;n=3).
3.The ER stress by high concentration of glucose
Treatment with various concentration of glucose(5mmol/L,12.5mmol/L and 25mmol/L)for 24h significantly increased the protein expression of the GRP78 in EPCs compared with control in a dose-dependent manner(127.33±9.71 vs 39±6.56, P＜0.05;152.33±7.09 vs 39±6.56,P＜0.05;183.33±4.51 vs 39±6.56,P＜0.05;n=3).
4.Protein expression of high concentration of glucose on EPCs for CASPASE-12 and CASPASE-3
Treatment of BM-EPCs with various concentration of glucose(Smmol/L,12.5mmol/L and 25mmol/L)for 24h markedly increased protein expression of cleaved-caspase-12 and cleaved-caspase-3 in a dose-dependent manner(cleaved-caspase-12:34.67±9.07, 71.33±4.16,125.33±11.68;cleaved-caspase-3:64.67±7.37,131.67±9.02,165.33±5.69)compared with control group(cleaved-caspase-12:0,P＜0.01;cleaved-caspase-3: 0,P＜0.01;n=3).
5.The effect of caspase-12 specific inhibitor(Z-ATAD-FMK)on apoptosis induced by high concentration of glucose in rat BM-derived EPCs
Pretreatment with Z-ATAD-FMK significantly decreased apoptosis in high glucose (25mmol/L)cultured rat BM-EPCs compared with those of no pretreating group(17.91±0.94%vs.36.82±2.16%,P＜0.05;n=3).
In contrast,pretreatment or no with Z-ATAD-FMK had no significant effect on apoptosis of rat BM-EPCs in normal glucose(5.62±0.92%vs.6.18±0.33%,P＞0.05;n =3).
6.The effect of caspase-12 specific inhibitor(Z-ATAD-FMK)on protein expression of caspase-12 and caspase-3 induced by high concentration of glucose in rat BM-derived EPCs
Pretreatment with Z-ATAD-FMK significantly decreased protein expression of cleaved-caspase-12 and cleaved-caspase-3 compared with those of no pretreatment (cleaved-caspase-12:0 vs.58±7.21,P＜0.01;cleaved-caspase-3.155±10.58 vs. 185.33±12.01,P＜0.05;n=3)in group with high concentration of glucose.
In contrast,pretreatment or no with Z-ATAD-FMK had no significant effect on protein expression of cleaved-caspase-12 and cleaved-caspase-3(both had no expression of cleaved-caspase-12 and cleaved-caspase-3;n=3).
Conclusions
1.High concentration of glucose may induce the rat BM-derived EPCs apoptosis in a dose-dependent manner.
2.ER stress in rat BM-derived EPCs was actived by high concentration of glucose.
3.ER stress pathway mediated by caspase-12 may play a important role in rat BM-derived EPCs apoptosis induced by high concentration of glucose.
Background
The prevalence of type 2 diabetes is increasing year by year.As an independent risk factor for cardiovascular disease,type 2 diabetes has been implicated as a coronary heart disease equivalent and it is an important component of the complex of common cardiovascular risk factors.Cardiovascular complications are the leading causes of morbidity and mortality in patients with diabetes mellitus(DM).Among them the acute coronary syndroms(ACS)is the extremely severe outcome.Recent studies have shown that the key pathobiological processes are responsible for transforming stable atherosclerotic plaques into active rupture-prone plaques in ACS.Moreover,the processes may be enhanced in diabetes.So it is important for clinician to detect the vulnerable plaque and seek the method to stabilize the plaques.As a method to diagnose the coronary disese,the coronary angiography(CAG)has several inherent limitations because it is unable to take account of arterial remodeling.Incontrast with angiography,Intravascular ultrasound(IVUS)is unique in its ability to assess both the intraluminal plaque and the surrounding arterial wall morphology.IVUS provides cross-sectional morphometric detail and quantifies atherosclerotic plaque area and plaque burden.
Endothelial progenitor cells(EPCs)are capable of differentiating into endothelial cells and homing to sites of ischaemia and play a important role in vascular repair and reendothelialisation of injured blood vessels and collateral vessels formation. Moreover,the endothelial dysfunction and damage are common starting points for diabetic macroangiopathy.And recent insights show that the injured endothelial monolayer is regenerated by circulating EPCs,which accelerate reendothelialization and limits atherosclerotic lesion formation.So the EPCs would play a vital role in the diabetic macroangiopathy.Then it would be of significance to assess the correlation between EPCs levels and extent of lesion in diabetic coronary disease.There was analysis of the CAG show that the lesion is more extensive and severe,the ability to form collateral circulation is descent in patients with DM.And it was reported that the number of circulating EPCs has been impaired in patients with diabetes mellitus and been increased in patients with ACS.However,few data are available on the relationship between the number of circulating EPCs and the instability of plaque as well as the extension of lesion assessed by intravascular ultrasound in diabetic patients with ACS.The objectives of this study are as follow:
objectives
1.To investigate the coronary culprit plaque in diabetic patients with ACS by IVUS.
2.To investigate the number of circulating EPCs in diabetic patients with ACS.
3.To investigate the potential relation between the number of circulating EPCs and plaque burden and severity of coronary culprit lesions using IVUS in diabetic patients with ACS.
Methods
1.Subjects
From July 2007 to January 2008,40 patients with diagnosis of ACS with de novo culprit lesions underwent preintervention IVUS were enrolled in this study.Among the subjects,there were 24 patients with unstable angina,16 patients with acute myocardial infarction.Acute myocardial infarction was defined as continuous chest pain at rest with abnormal levels of cardiac enzymes(creatininekinase-MB or troponin T).The culprit lesion in ACS was identified by the combination of left ventricular wall motion abnormalities,electrocardiographic findings,angiographic -lesion morphology.The lesion with the worst diameter stenosis was selected as the target lesion for IVUS analysis.According to the American Diabetes Association 2005 criteria for insulin-independent diabetes mellitus,the patients were classified to diabetic group and non-diabetic group:diabetic group(n=20),nondiabetic group (n=20).The medical history,including the pre-existing coronary riskfactors such as hypertension,hypercholesterolemia,and smoking,was recorded for each patient.
2.Examinational Methods
2.1 Coronary angiography:Coronary angiography was performed according to standard angiographic techniques.Angiographic scoring was done by interventional cardiologists who were blinded to the study protocol.Angiographic diagnosed criteria fulfilling the Coronary Artery Surgery Study:The left main coronary arterywas defined as a lumen diameter decrease of at least 50%,the anterior descending,left circumflex,and right coronary arteries were at least 70%,respectively.
2.2 intravascular ultrasound
After completion of the diagnostic coronary angiography,the culprit lesion was localized according to the angiographic signs of vessel morphology and electrocardiogrampatterns.Before the IVUS procedure,200μg intracoronary nitroglycerine was administered to prevent vasospasm,and a 2.9F IVUS imaging catheter(Eagle Eye;Volcano Corporation,USA)was advanced distally to the culprit lesion and pulled back at a pull-back speed of 0.5 mm/sec.In parallel,the images were recorded on videotapes for subsequent off-line analysis.IVUS images were analyzed independently by 2 experienced observers.Conventional gray-scale quantitative IVUS analyses were performed according to criteria of the clinical expert consensus document on IVUS to include external elastic membrane(EEM),luminal, and plaque areas.The external elastic membrane cross-sectional area(defined as the area encompassed by the media-adventitia border)were measured in diastolic frames. The plaque cross-sectional area,defined as the intimamedia area,was calculated from the difference between the external elastic membrane and lumen cross-sectional areas. The plaque burden on each cross section was calculated as 100×(external elastic membrane cross-sectional area-lumen cross-sectional area)/external elastic membrane cross-sectional area).
2.3 EPCs Culture,Characterization and Count
2.3.1 EPCs Culture Assay
After placement of an arterial sheath,3 to 5 ml of blood was wasted and then 20 ml of whole blood obtained for cell culture.The heparinized specimen was diluted with phosphate buffered saline(PBS),and the specimen was centrifuged across a 1.077 density gradient over human lymphocyte separating medium(LTS-1077)at 2000 rpm for 30 min.Then mononuclear cells were isolated from the diluted peripheral blood. Immediately after isolation,1×10~6 mononuclear cells were plated on 24-well culture dishes pre-coated with 0.1%gelatin(Sigma,USA)and maintained in M199 medium (Gibco,USA)supplemented with 20%fatal calf serum,100μg/mL streptomycin, 100U/ml penicillin,40 ng/mL human vascular endothelial growth factor(VEGF, Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA),10 ng/mL epidermal growth factor(EGF,Peprotech,USA),and 10U/mL heparin.After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS, new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.
2.3.2 Characterization of EPCs
Fluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FITC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,USA)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyani -ne(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular Probe,USA). To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%paraformaldehyde for 10 minutes. After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..
2.3.3 Count of EPCs
Dual-stained positive cells were counted per well.Two independent investigators who was blinded to the clinical profile of the patients evaluated the number of EPCs per well by counting 3 randomly selected high-power fields.
Results
The culprit lesion was localized in the left anterior escending,left circumflex,or right coronary artery in 22(55%),8(20%)and 10(25%)patients,respectively.
IVUS data
IVUS findings of culprit lesions,comparing diabetic patients and without diabetes. The minimum luminal area site lumen,external elastic membrane,and plaque plus media cross-sectional areas were 3.3±1.6,14.3±3.0 and 11.1±2.7 mm~2 respectively, whereas the plaque burden was 76.5%±11.7%in diabetes group.Another group,the minimum luminal area site lumen,external elastic membrane,and plaque plus media cross-sectional areas were 4.9±2.2,13.8±3.4 and 9.0±2.6 mm~2 respectively,and the plaque burden was 66.2%±13.2%.DM group had greater pique burden compared with the nondiabetes.
EPCs Number
When patients were classified according to DM or nondiabete group,the significantly lower levels of EPCs were found in the patients with DM,compared with EPCs levels in the patients without DM(30.4±6.99 vs.51.7±11.1 EPCs/×200 field,P＜0.05).
Correlation between EPCs number and plaque burden determined with IVUS
The lower levels of circulating EPCs were measured in association with lesion with greater plaque burden.In addition,the plaque burden of the lesion was associated with significantly decreased levels of EPCs(r=-0.427,P＜0.05)in diabetes group.
Conclusions
1.Coronary culprit plaque in ACS patients with DM showed a higher extent of coronary stenosis,a greater plaque burden and a smaller lumen cross-sectional area.
2.The number of circulating EPCs was significantly lower in patients with DM compared to those without DM.
3.The number of EPCs was negatively associated with the plaque burden.Decreased number of circulating EPCs may reflect the severity of coronary culprit lesion in diabetic patients with ACS.
4.Given the important role of EPCs in process of atherogenesis,a decrease in the number of EPCs may contribute to the accelerated atherogenesis and impaired endothelial regeneration in diabetic patients with ACS.
内皮祖细胞是1997年发现的在成人体内能分化为内皮细胞的前体细胞,存在于骨髓及外周血中。内皮祖细胞主要参与内皮细胞修复即血管的再内皮化以及参与血管新生,由此近年来内皮祖细胞在血管病变中的作用越来越受到重视。糖尿病血管病变的重要发病机制是内皮细胞功能受损及凋亡。血管内皮层完整性遭到破坏,炎性细胞浸润至内皮下层促进动脉粥样硬化的发生发展,因此内皮功能受损被视为糖尿病血管病变的始动因素。正常状态下,受损的内皮由周边内皮及循环中的内皮祖细胞分化为内皮来修复,保持内皮层的完整性,一旦内皮祖细胞受损,内皮损害和修复之间的平衡被打破,内皮层的完整性遭到破坏,由此发生动脉粥样病变,因此内皮祖细胞在糖尿病血管病变中具有重要作用。已有研究证实糖尿病时循环内皮祖细胞数量和功能均受损害,而循环内皮祖细胞来源于骨髓,由此我们推测糖尿病时骨髓内皮祖细胞可能也受损。我们已知内质网应激介导的β细胞凋亡是糖尿病发病的重要机制,由此我们推测内质网应激可能介导内皮祖细胞的凋亡参与糖尿病血管病变的发病。迄今尚无观察糖尿病对骨髓内皮祖细胞的凋亡有无影响及其机制的研究,本研究的目的旨在:
目的
1.建立糖尿病大鼠模型。
2.研究大鼠骨髓内皮祖细胞体外分离、诱导、培养及鉴定的方法。
3.检测糖尿病大鼠和正常大鼠骨髓内皮祖细胞凋亡率,明确糖尿病时骨髓内皮祖细胞是否受损。
4.检测糖尿病大鼠和正常大鼠骨髓内皮祖细胞内质网应激标志物GRP78及内质网应激特异性凋亡因子—caspase-12活性,以了解内质网应激是否参与介导糖尿病时骨髓内皮祖细胞的凋亡。
方法
1.糖尿病(diabetes mellitus,DM)大鼠模型的制备
雄性Wistar大鼠27只,随机分为2组:对照组12只,DM组15只。标准大鼠饲料喂养1周后,禁食12h,DM组大鼠给以腹腔内注射链脲佐菌素(STZ)65mg/kg,对照组大鼠注射等量柠檬酸钠/柠檬酸缓冲液。糖尿病大鼠成模标准为:连续2次空腹血糖＞16.7mmol/L。未达成模标准者剔除。DM组大鼠血糖升高后,饲以高脂高糖饮食,对照组则给予标准饮食喂养。
2.大鼠骨髓源内皮祖细胞的培养、鉴定。
2.1大鼠骨髓内皮祖细胞的培养
大鼠拉颈处死后无菌状态下分离股骨胫骨,将骨髓细胞以无菌PBS液完全冲洗至离心管,应用大鼠淋巴细胞分离液(LTS-1083)以密度梯度离心法(2 000rpm,30 min)分离单个核细胞。洗涤后以含20%胎牛血清的M199培养液重悬,接种于培养瓶中,37℃、5%CO_2条件下培养。细胞培养24 h(早期贴壁)后收集未贴壁细胞离心后以选择性培养基(M199培养基加入20%胎牛血清,100μg/mL链霉素,100U/ml青霉素,40 ng/mL大鼠血管内皮生长因子(VEGF,Peprotech,USA),2 ng/mL人成纤维细胞生长因子(FGF,Peprotech,USA),10 ng/mL人表皮细胞生长因子(EGF,Peprotech,USA)以及10U/mL肝素)重悬计数,1×10~6/mL转入包被0.1%明胶(Sigma,USA)的24孔板和培养瓶中二次贴壁,4天后换液,培养至第7天鉴定。
2.2大鼠骨髓EPCs的鉴定
细胞在含DiI-ac-LDL(2.4μg/mL)的培养液中37℃避光孵育1h,然后以2%的多聚甲醛固定10min,PBS液漂洗后再加FITC-UEA-1(10μg/mL)避光孵育1h。PBS液漂洗后在荧光显微镜下观察,显示红色荧光的为ac-LDL阳性细胞,显示绿色荧光的为UEA-1阳性细胞,显示双荧光阳性(黄色)的细胞被认为是EPCs。
3.流式细胞仪检测大鼠骨髓源内皮祖细胞凋亡率
ANEXIN V/PI凋亡检测试剂盒流式细胞仪检测凋亡。
4.RT-PCR检测GRP78、CASPASE-12基因表达
结果
1.大鼠基本情况及血糖检测
实验过程中,对照组大鼠精神状态良好,体重增加明显,反应敏捷,毛色白而光泽。DM组大鼠出现多食、多饮、多尿和消瘦等症状,体重增加迟缓,精神萎靡,皮毛无光泽,部分出现烂尾、白内障等。最终共23只完成实验,其中对照组12只,DM组11只。
注射STZ之前,对照组与DM组血糖无明显差异(P＞0.05)。注射STZ 1周后,DM组血糖明显高于对照组(P＜0.01),并持续至实验末。
2.大鼠骨髓源内皮祖细胞培养、鉴定
早期贴壁细胞呈毛发样生长,多为骨髓基质细胞。二次贴壁的细胞大多在3天左右伸展成梭形或纺锤形。7～10天增殖速度加快可见内皮细胞特异性条索状结构。2周左右细胞可达80～90%融合,大部分细胞呈多角形。80%以上细胞可同时吸收DiI-ac-LDL结合FITC-UEA-1即鉴定为内皮祖细胞。
3.流式细胞仪检测大鼠骨髓源内皮祖细胞凋亡率
DM大鼠骨髓源内皮祖细胞凋亡率明显高于对照组(20.3±4.64%vs.11.2±2.85,P＜0.05)。
4.RT-PCR检测GRP78、CASPASE-12 mRNA表达
1)GRP78mRNA表达:DM组内皮祖细胞GRP78mRNA表达均较对照组明显增高(0.64±0.07%vs.0.43±0.05%,P＜0.05)。
2)CASPASE-12 mRNA表达:DM组内皮祖细胞CASPASE-12 mRNA表达均较对照组明显增高(0.56±0.09%vs.0.36±0.04,P＜0.05)。
结论
1)糖尿病大鼠骨髓源内皮祖细胞凋亡率明显增加。
2)DM大鼠骨髓源内皮祖细胞GRP78及CASPASE-12 mRNA表达增加,提示内质网应激参与介导糖尿病大鼠骨髓源内皮祖细胞凋亡,可能在糖尿病血管病变的发生发展中起重要作用。
研究背景
内质网应激(endoplasmic reticulum stress,ER stress)是内质网功能紊乱时出现错误折叠与未折叠蛋白在腔内聚集以及Ca~(2+)平衡紊乱的状态,对决定应激细胞的结局如抵抗、适应、损伤或凋亡有重要作用。一定程度的内质网应激因能激活保护机制例如分子伴侣表达而有细胞保护作用,应激过强时,保护机制不能与损伤抗衡,内质网应激可通过caspase-12独立地介导细胞凋亡。内质网应激使caspase-12活化,继而激活下游caspase,导致细胞凋亡,内质网应激反应性细胞凋亡是不同于死亡受体介导或经线粒体介导的一种新的细胞凋亡途径,caspase-12作为凋亡起始因子发挥了关键作用。有研究已证实内质网应激介导的凋亡参与胰岛β细胞的凋亡,是糖尿病发病的重要机制。内皮祖细胞在糖尿病血管病变中有重要作用,研究表明糖尿病时循环内皮祖细胞数量及功能均受损,我们在体内实验也发现其在糖尿病大鼠骨髓中凋亡率明显增加,同时内皮祖细胞内质网应激激活,结合内皮祖细胞具有内皮细胞修复以及参与血管新生的作用,我们推测内皮祖细胞凋亡增加很可能在以动脉粥样硬化为重要特征的糖尿病血管病变中发挥重要作用。那么内质网应激介导的凋亡是否参与了高糖诱导的大鼠骨髓源内皮祖细胞的凋亡呢?本工作拟通过高糖刺激体外培养的大鼠骨髓源内皮祖细胞,观察高糖对其凋亡的影响及内质网应激特异凋亡因子caspase-12是否参与介导其凋亡,以期为糖尿病血管病变的预防和治疗提供新的机制和靶点。
目的
1.观察高糖培养对大鼠骨髓EPCs凋亡的影响。
2.观察高糖培养是否激活大鼠骨髓EPCs内质网应激反应。
3.观察CASPASE-12介导的内质网应激途径是否在高糖诱导的大鼠骨髓EPCs凋亡过程中发挥重要作用。
方法
1.大鼠骨髓内皮祖细胞(Bone Marrow-derived Endothelial Progenitor Cells,BM-EPCs)的培养、鉴定
1.1骨髓EPCs的分离、培养
6周龄雄性体重250—300克Wistar大鼠购自山东大学动物实验中心,拉颈处死后无菌条件下取出四肢长骨,将骨髓细胞以无菌PBS液完全冲洗至离心管用大鼠淋巴细胞分离液(LTS-1083)以密度梯度离心法(2 000 rpm,30 min)分离单个核细胞。洗涤后以含20%胎牛血清的M199培养液重悬,接种于培养瓶中,37℃、5%CO_2条件下培养。细胞培养24 h(早期贴壁)后收集未贴壁细胞离心后以选择性培养基(M199培养基加入20%胎牛血清,100μg/mL链霉素,100U/ml青霉素,40 ng/mL大鼠血管内皮生长因子(VEGF,Peprotech,USA),2 ng/mL人成纤维细胞生长因子(FGF,Peprotech,USA),10 ng/mL人表皮细胞生长因子(EGF,Peprotech,USA)以及10U/mL肝素)重悬计数,1×10~6/mL转入包被0.1%明胶(Sigma,USA)的24孔板和培养瓶中二次贴壁,4天后换液,培养至第7天鉴定。
1.2 EPCs的鉴定
细胞在含DiI-ac-LDL(2.4μg/mL)的培养液中37℃避光孵育1h,然后以2%的多聚甲醛固定10min,PBS液漂洗后再加FITC-UEA-1(10μg/mL)避光孵育1h。PBS液漂洗后在荧光显微镜下观察,显示红色荧光的为ac-LDL阳性细胞,显示绿色荧光的为UEA-1阳性细胞,显示双荧光阳性(黄色)的细胞被认为是EPCs。
2.流式细胞仪测定不同浓度糖培养BM-EPCs凋亡率
骨髓EPCs给予不同浓度高糖(5mmol/L、12.5mmol/L及25mmol/L)刺激24h,流式细胞仪检测细胞凋亡率
3.Western-blot测定不同浓度糖培养骨髓EPCs GRP78、CASPASE-12、CASPASE-3蛋白表达改变
4.流式细胞仪测定给予或未给予caspase-12抑制剂(Z-ATAD-FMK 5uM)预处理后高糖(25mmol/L)培养骨髓EPCs凋亡率
5.Western-blot测定给予或未给予caspase-12抑制剂(Z-ATAD-FMK 5uM)预处理后高糖(25mmol/L)培养骨髓EPCs CSAPASE-12、CASPASE-3蛋白表达改变
结果
1.大鼠骨髓内皮祖细胞的培养、鉴定
早期贴壁细胞呈毛发样生长,多为骨髓基质细胞。二次贴壁的细胞大多在3天左右伸展成梭形或纺锤形。7～10天增殖速度加快可见内皮细胞特异性条索状结构。2周左右细胞可达80～90%融合,大部分细胞呈多角形,可呈铺路石样排列。80%以上细胞可同时吸收DiI-ac-LDL结合FITC-UEA-1即鉴定为内皮祖细胞。
2.高糖对骨髓EPCs凋亡的影响
不同浓度糖(5mmol/L、12.5mmol/L及25mmol/L)孵育EPCs 24h,结果显示高糖呈浓度依赖性诱导EPCs凋亡(7.34±0.44%,11.59±1.22%,36.82±2.16%),与正常对照组(6.18±0.33%)比较差异均有统计学意义(P＜0.05),其中以糖浓度为25mmol/L时凋亡率最高。高渗对照25mmol/L甘露醇组凋亡率与对照组比较无显著性差异(5.95±0.81%vs.6.18±0.33%,P＞0.05)。
3.高糖激活骨髓EPCs内质网应激
不同浓度糖(5mmol/L、12.5mmol/L及25mmol/L)孵育EPCs 24h,westernblot法测定GRP78表达,结果显示高糖激活内质网应激特异蛋白GRP78表达,并呈浓度依赖性(127.33±9.71,152.33±7.09,183.33±4.51),与正常对照组(39±6.56)比较差异均有统计学意义(P＜0.05)。
4.高糖对CASPASE-12和CASPASE-3蛋白表达的影响
不同浓度(5mmol/L、12.5mmol/L及25mmol/L)孵育EPCs 24h,western blot法测定CASPASE-12和CASPASE-3蛋白水平表达,结果显示高糖激活CASPASE-12和CASPASE-3,并呈浓度依赖性(cleaved-CASPASE-12:34.67±9.07,71.33±4.16,125.33±11.68;cleaved-CASPASE-3:64.67±7.37,131.67±9.02,165.33±5.69),与正常对照组比较差异有统计学意义(cleaved-CASPASE-12;cleaved-CASPASE-3均无表达,P＜0.01)。
5.CASPASE-12抑制剂(Z-ATAD-FMK)对高糖诱导的骨髓EPCs凋亡的影响
25mmol/L高糖培养组予5uM Z-ATAD-FMK预处理后,与未预处理组比较高糖诱导的凋亡率明显下降(17.91±0.94%vs.36.82±2.16%,P＜0.05),而对照组应用Z-ATAD-FMK预处理与未预处理组凋亡率无明显差异(5.62±0.92%vs.6.18±0.33%,P＞0.05);
6.CASPASE-12抑制剂(Z-ATAD-FMK)对高糖诱导的骨髓EPCs CASPASE-12和CASPASE-3蛋白表达的影响
与未加抑制剂预处理组比较高糖加预处理组CASPASE-12和CASPASE-3活性均被明显抑制(cleaved-CASPASE-12:0 vs.58±7.21,P＜0.01;cleaved-CASPASE-3:155±10.58 vs.185.33±12.01,P＜0.05)。对照组预处理组与未预处理组cleaved-CASPASE-12、cleaved-CASPASE-3均无表达)。
结论
1)高糖培养可以浓度依赖性诱导大鼠骨髓EPCs凋亡。
2)高糖可以激活大鼠骨髓EPCs内质网应激。
3)CASPASE-12介导的内质网应激途径可能在高糖诱导的大鼠骨髓EPCs凋亡过程中发挥重要作用。
研究背景
2型糖尿病(type 2 diabetes mellitus,T2DM)的发病率逐年升高,已成为威胁人类健康的常见疾病,糖尿病是冠心病的独立危险因素之一,近年来更加认为糖尿病是冠心病的等危症。T2DM常伴有多种心血管疾病的危险因素,易合并以动脉粥样硬化和血栓形成为特征的大血管病变,冠心病是引起T2DM患者死亡的主要原因,其中最为严重的是急性冠状动脉综合征(acute coronary syndroms,ACS)。近年的研究显示,动脉粥样硬化斑块破裂和血栓形成是ACS的主要发病机制,而斑块不稳定性是这些环节中的始动因素。急性冠脉综合征的发生与否取决于斑块的稳定程度,因此易损斑块的临床识别以及探索稳定斑块的方法具有十分重要的临床意义。由于存在血管重构,冠脉造影(coronary angiography,CAG)并不能真实地反映出冠脉病变程度—常低估冠脉病变。血管内超声(Intravascularultrosound,IVUS)能提供管腔、管壁横截面图像,能够分辨出斑块的大小、组成成分、分布以及观察斑块处血管的重构情况,在斑块稳定性、靶病变严重程度的评估等方面具有冠脉造影无法比拟的优势。
内皮祖细胞(endothelial progenitor cells,EPCs)是一类能增殖并分化为血管内皮细胞的前体细胞。对于调节内皮细胞凋亡/再生平衡及保持内皮层的完整性有着重要作用。另外EPCs还参与新生血管的生成,这对于缺血组织侧枝循环的形成至关重要。因此,EPCs在糖尿病冠脉病变的发生发展中有重要的作用。冠脉造影研究显示与不伴有糖尿病的冠心病患者相比糖尿病患者冠状动脉病变严重,病变范围广并且冠状动脉侧支血管形成能力明显下降,应用血管内超声评价糖尿病对冠脉病变影响的研究尚少见报道;以往研究表明糖尿病患者循环EPCs数量和功能均受影响,但此种改变与急性冠脉综合征靶病变斑块稳定性及病变程度有何关系尚不明了。由此构成本课题的设计思路和研究目的。本课题应用血管内超声技术分析急性冠脉综合征患者靶病变严重程度、斑块负荷与外周血EPCs数量的关系,研究糖尿病合并ACS时对外周血EPCs数量的影响以及与易损斑块特征的关系。
目的
1.应用血管内超声方法研究糖尿病合并急性冠脉综合征患者靶病变斑块特征。
2.观察糖尿病合并急性冠脉综合征患者循环内皮祖细胞数量变化。
3.研究糖尿病合并急性冠脉综合征患者循环内皮祖细胞数量变化与靶病变严重程度和斑块负荷有无相关性。
方法
1.研究对象
选自2007年7月至2008年1月间在我院接受冠状动脉造影(CAG)检查确诊为急性冠脉综合征(ACS)40例患者。包括急性心肌梗死(AMI)16例和不稳定型心绞痛(UAP)24例。AMI的临床诊断为伴有心肌坏死的血清心肌标志物浓度的动态演变的持续性静息状态胸痛的临床病史。靶血管病变经心电图、CAG和超声心动室壁运动资料确定。根据是否患有糖尿病将患者分为两组:糖尿病组(n=20),非糖尿病组(n=20)。糖尿病诊断标准符合2005年ADA修订的诊断及分型标准,均为2型糖尿病。入院时收集患者的病史包括冠心病高危因素如高血压、高脂血症、吸烟史等临床资料。
2.检查方法
2.1冠状动脉造影检查所有患者按常规进行CAG检查,冠脉造影诊断标准按照美国Coronary Artery Surgery Study(CASS)研究:冠状动脉左主干狭窄≥50%,左前降支、左旋支及右冠状动脉狭窄≥70%为阳性。
2.2血管内超声检查在常规冠状动脉血管造影结束后,采用标准的冠状动脉内介入导管操作技术进行IVUS检查,追加3000IU肝素,然后把0.014英寸的导丝送至靶血管,常规冠脉内注入200μg硝酸甘油。将IVUS导管沿导引钢丝越过靶病变,至靶病变远端1cm以上,利用自动回撤仪以0.5mm/s的速度自动匀速回撤至靶血管开口,所有获取的IVUS图像实时连续采集并存入硬盘,供以后脱机分析。
2.3 IVUS图像的定量测量斑块形态由二位研究者采用盲法分别进行分析和处理。
2.3.1 EEM-CSA:即血管外弹力膜的横断面积,手工描记外膜内边缘,由仪器自身的测量系统测得的。
2.3.2 L-CSA(lumen-CSA):是指血管腔的横断面积,手工描记内膜的内边缘,由仪器自身的测量系统测得的。当斑块包绕探头时,推测管腔的大小等于探头的大小。
2.3.3 P-CSA(plaque-CSA):经由外弹力膜面积减去血管管腔面积计算获得。
2.3.4 PB(plaque burden):即斑块负荷,为斑块面积除以外弹力膜面积乘以100%,它不同于管腔狭窄率。
2.4循环内皮祖细胞培养、鉴定、计数
2.4.1内皮祖细胞培养
无菌采集外周血20mL,肝素抗凝,用PBS液等倍稀释血液。将稀释血液按1:2的比例置于人淋巴细胞分层液(LTS-1077)上以2 000 rpm离心30min。分离出单个核细胞后用5倍体积的PBS洗涤2次计数,调整细胞浓度以1×10~6/mL接种在包被0.1%明胶的24孔培养板。选择性M199培养基(含20%胎牛血清,青霉素100U/mL,链霉素100μg/mL,人血管内皮生长因子(VEGF)40 ng/mL,人成纤维细胞生长因子(FGF)2 ng/mL,人表皮生长因子(EGF)10 ng/mL,10U/mL肝素)诱导培养,4d后换液,继续培养至7d,PBS洗掉未贴壁细胞,贴壁细胞进行细胞鉴定。
2.4.2内皮祖细胞鉴定
内皮祖细胞可结合FITC标记的植物凝集素(FITC-UEA-1)并且能吸收DiI标记的乙酰化低密度脂蛋白(DiI-ac-LDL),能结合两者的细胞可鉴定为内皮祖细胞。首先在37℃细胞与DiI-ac-LDL(2.4μg/ml)共同孵育1小时,然后用2%多聚甲醛固定10分钟,轻轻冲洗后与FITC-UEA-1(10μg/mL)共同孵育1小时,PBS冲洗后在倒置荧光显微镜下观察,双染阳性者为内皮祖细胞。
2.4.3内皮祖细胞计数
由两位研究者采用盲法分别计数双染细胞,每孔随机选取3个高倍镜视野计数,取平均值。
结果
靶病变分布情况:左前降支22例(55%),左回旋支8例(20%),右冠10例(25%)。
血管内超声结果
血管内超声分析示,糖尿病组管腔面积,外弹力膜面积,斑块面积和斑块负荷分别为3.3±1.6,14.3±3.0 and 11.1±2.7 mm~2,76.5%±11.7%;非糖尿病组管腔面积,外弹力膜面积,斑块面积和斑块负荷分别为4.9±2.2,13.8±3.4 and 9.0±2.6mm~2,66.2%±13.2%。与非糖尿病组比较糖尿病组斑块负荷明显增大。
循环内皮祖细胞计数
糖尿病组循环内皮祖细胞数量明显低于非糖尿病组(30.4±6.99 vs.51.7±11.1EPCs/×200 field,P＜0.05)。
循环内皮祖细胞数量与斑块负荷的相关性
糖尿病组循环内皮祖细胞数量与斑块负荷呈负相关(r=-0.427,P＜0.05)。
结论
1.糖尿病组病变部位的斑块面积和斑块负荷均大于非糖尿病组,管腔面积小于非糖尿病组;
2.糖尿病组患者循环内皮祖细胞数量明显低于非糖尿病组;
3.糖尿病组患者斑块负荷、靶病变严重程度与循环内皮祖细胞数量负相关,提示靶病变严重程度与循环内皮祖细胞数量相关;
4.糖尿病急性冠脉综合征患者冠脉病变程度严重可能与内皮祖细胞减少导致的斑块迅速增长及血管内皮的再内皮化降低有关。
Background
The endothelial progenitors cells(EPCs)were reported in 1997 to differentiate in vitro into endothelial cell(EC)and seemed to be incorporated at sites of active angiogenesisin various animal models of ischaemia,which led to the new concept that vasculogenesis and angiogenesis may occur simultaneously in postnatal.These cells are derived from the bone marrow and can circulate in the blood.The majority of circulating EPCs reside in the bone marrow where exist an optimal microenviroment. EPCs are capable of differentiating into endothelial cells and homing to sites of ischaemia and play a important role in vascular repair and reendothelialisation of injured blood vessels and collateral vessels formation.Moreover,diabetic macro -angiopathy develop as the result of an atherogenesis process initiated by endothelial damage and dysfunction.And recent insights show that the injured endothelial monolayer is regenerated by circulating EPCs,which accelerate reendothelialization and limits atherosclerotic lesion formation.There is a balance between endothelial cell damage and endothelial cell regeneration.Apoptotic and necrotic endothelial cells are regenerated by circulating EPCs.However,the system becomes imbalanced in conditions of enhanced endothelial cell damage and exhausted EPCs-mediated endothelial cell repair.The resulting the disequilibrium between damage and regeneration,results in the atherogenesis progression.So the EPCs would play a vital role in the diabetic macroangiopathy.It has been reported that the number of circulating EPCs which origin from bone marrow were impaired in patients with diabetes mellitus(DM).Moreover,recent studies have revealed that ER stress mediiated apoptosis inβcells could be a cause of DM.Based on these results,this present study hypothesized that the EPCs might be impaired in bone marrow in diabetes mellitus and that ER stress mediated apoptosis in bone marrow derived EPCs (BM-derived EPCs)might be a cause of diabetic macroangiopathy.The objectives of this study are as follow:
Objectives
1.To establish a DM animal model.
2.To investigate the methods of isolating,culturing and identifying BM-derived EPCs
3.To examine the occurrence of apoptosis of BM-derived EPCs in DM rat and control group to determine whether or not the EPCs is impaired in bone marrow in DM.
4.To assess the mRNA express of GRP78 and CASPASE-12 of BM-derived EPCs in diabetic or non-diabetic rats to investigate whether or not the ER stress mediated apoptosis in bone marrow derived EPCs.
Methods
1.Establishment of DM animal model
All protocols were approved by the Shandong University experimental animal center. Twenty-seven male age-matched Wistar rats(Experimental Animal Center of Shandong University,Ji'nan,China)were randomly divided into 2 groups:control group(n=12)and DM group(n=15).Diabetes was induced by a single intraperitoneal injection of streptozotocin(STZ,65 mg/kg body wt and dissolved in 0.1 mol/l citrate buffer,pH 4.2)in DM group.The rats in control group received citrate buffer alone. One week after injection of STZ,fasting plasma glucose levels was measured,and rats with plasma glucose at least two times higher than 16.7mmol/L were used.DM group rats were fed high fat and glucose diet and the rats in control group were fed standard rat diet.
2.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and Characterization
2.1 EPCs culture
BM cells were obtained by flushing the tibias and femurs of rat.BM mononuclear cells were isolated by density centrifugation over rat lymphocyte separating medium(LTS-1083)at 2000 rpm for 30 min.Then mononuclear cells were isolated and plated culture flasks in M199 medium(Gibco,USA)supplemented with 20% fatal calf serum,100μg/mL streptomycin,100U/ml penicillin.After 24 h in culture, the nonadherent cells were harvested and resuspended,then 1×10~6 cells were plated on 24-well culture dishes or T25 flasks pre-coated with 0.1%gelatin(Sigma,USA) and maintained in M199 medium supplemented with 20%fatal calf serum,100μg/mL streptomycin,100U/ml penicillin,40 ng/mL vascular endothelial growth factor (VEGF,Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA), 10 ng/mL epidermal growth factor(EGF,Peprotech,USA),and 10U/mL heparin. After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS,new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.
2.2 Characterization of EPCs
Fluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FIYC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,St.Louis,USA)and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo -carbocyanine(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular ProbeCo,Eugene,USA).To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%parafor -maldehyde for 10 minutes.After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..
3.EPCs apoptosis detection by flow cytometer.
The BM-derived EPCs apoptosis of the rats in DM and control groups was determined by flow cytometric detection according to the manufacturer's instructions of the annexin V-FITC apoptosis detection kit(Jingmei,China).Briefly,EPCs were washed once with PBS and resuspended in binding buffer were stained with annexin Ⅴ(0.5μg/ml)and propidium iodide(0.6μg/ml)for 5 min at room temperature.After staining,cells were immediately analyzed using a FACScan flow cytometer(Becton Dickinson,CA)with simultaneous monitoring of green fluorescence(530 nm,30 nm band-pass filter)for annexin V-FITC and red fluorescence(long-pass emission filter that transmits light＞650 nm)associated with propidium iodide.10,000 events were collected and analyzed to determine the percentage of annexin-ⅤFITC and PI-positive cells.The cells were analyzed using Cell Quest analysis software (Becton-Dickinson,CA).
4.The mRNA expression of GRP78 and CASPASE-12 by RT-PCR
Total RNA from DM and control rat BM-defived EPCs was isolated using the TRIzol Reagent(Invitrogen)as described by the manufacturer.For RT-PCR,lug total RNA and oligo(dT)(Promega)was reverse-transcribed into cDNA with 1 unit/ml M-MLV reverse transcriptase(Promega)at 42℃for 1b.Primers sequences for rat grp78, caspase-12 andβ-actin are available with Taq Polymerase(Takara)for PCR.
Results
1.General features of the experimental rats
At the end of the experiment,11 diabetic rats induced by STZ and 12 control rats survived.Glucose levels were significantly elevated in diabetic rats compared with control rats after STZ injection.Other symptoms,such as lower body weights, polyuria and polyphagia,which are normally associated with diabetic state were also observed in the diabetic rats.
2.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and characterization
The secondarily attached cells stretched after 3 days.The cells proliferated faster after 7-10 days' incubation when cord-like structure was observed.After 2 weeks' induction, most of the cells exhibited multangular morphology.More than 80%attached cells took up DiI-ac-LDL,bound FITC-UEA-1(double positive fluorescence).
3.EPCs apoptosis detection by flow cytometer.
BM-derived EPCs significantly increased apoptosis in DM rat compared with control group(20.3±4.64%vs.11.2±2.85,P＜0.05).
4.The mRNA expression of GRP78 and CASPASE-12 by RT-PCR
The mRNA expression of GRP78 and CASPASE-12 significantly increased in DM rat when compared with control group(GRP78:0.64±0.07%vs.0.43±0.05%,P＜0.05; CASPASE-12:0.56±0.09%vs.0.36±0.04,P＜0.05).
Conclusions
1.BM-derived EPCs significantly increased apoptosis in DM rat.
2.The mRNA expression of GRP78 and CASPASE-12 significantly increased in DM rat which suggest that ER stress mediated apoptosis in bone marrow derived EPCs might be one of causes of diabetic macroangiopathy.
Background
Endoplasmic reticulum(ER)stress is a conditions interfering with endoplasmic reticulum function will perturb the folding of newly synthesized membrane or secreted proteins.The accumulation of unfolded proteins in ER causes cells ER stress response,which reduces the amount and aggregation of unfolded proteins in ER.In case the response fails to solve ER stress,the apoptosis pathway will be activated.ER stress can lead to apoptosis by various pathways,among them caspase-12 has been proposed as a specific mediator of ER-stress-induced apoptosis.It has been suggested that ER stress might induce cell apoptosis via caspase-12 independently.The involvement of apoptosis via ER stress is a relatively new pathway other than the death receptor or mitochondrial death pathways.
It has been shown that type 2 diabetic patients have reduced pancreatic isletsβcell mass and increasedβcell apoptosis rates.Recent in vitro studies suggest that elevated glucose levels induced apoptosis in culturedβcells possibly through ER stress.And we observed the occurrence of apoptosis of EPCs elevated in DM rat bone marrow as well as ER stress was actived in vivo study.Moreover,the endothelial progenitor cells (EPCs)play a important role in vascular repair and reendothelialisation of injured blood vessels,which means the EPCs play a key factor in diabetic vascular disease. Based on these results,we hypothesized that elevated glucose levels impair the bone marrow-derived EPCs(BM-derived EPCs)survival and induce the cells apoptosis. Then whether the ER stress-mediated apoptotic pathway is a component of high concentration of glucose induced the cells apoptois is unknown.In order to test this hypothesis,we cultured rat BM-derived EPCs under different concentration of glucose to investigate that the effect of high concentration of glucose on EPCs and a possible role of ER stress in the apoptosis.
Objectives
1.To assess the effect of apoptosis indued by high concentration of glucose on rat BM-derived EPCs.
2.To assess whether the ER stress was actived in rat BM-derived EPCs by high concentration of glucose.
3.To assess whether the ER stress-mediated apoptotic pathway is a component of high concentration of glucose induced rat BM-derived EPCs apoptois.
Methods
1.rat BM-derived EPCs culture and characterization
1.1 EPCs culture
Adult male Wistar age-matched(6 weeks)rats(Experimental Animal Center of Shandong University,Ji'nan,China)weighing 250-300 g were used.All protocols were approved by the shandong university experimental animal center.BM cells were obtained by flushing the tibias and femurs of rat.BM mononuclear cells were isolated by density centrifugation over LTS-1083 at 2000 rpm for 30 min.Then mononuclear cells were isolated and plated culture flasks in M199 medium(Gibco,USA) supplemented with 20%fatal calf serum,100μg/mL streptomycin,100U/ml penicillin.After 24 h in culture,the nonadherent cells were harvested and resuspended, then 1×10~6 cells were plated on 24-well culture dishes or T25 flasks pre-coated with 0.1%gelatin(Sigma,USA)and maintained in M199 medium supplemented with 20% fatal calf serum,100μg/mL streptomycin,100U/ml penicillin,40 ng/mL vascular endothelial growth factor(VEGF,Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA),10 ng/mL epidermal growth factor(EGF,Peprotech, USA),and 10U/mL heparin.After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS,new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.
1.2 Characterization of EPCs
Fluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FITC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,St.Louis,USA)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo -carbocyanine(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular Probe,Eugene,USA).To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%parafor -maldehyde for 10 minutes.After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..
1.3 Passage of EPCs
The cells should be subcultured when they reach 80-85%confluence.Aspirate the medium and wash the cells with PBS,then add trypsin-EDTA.Incubate 37℃for a few minutes meanwhile should be monitored using an inverted phase-contrast microscope.Once the cells have begun to detach inactivate the trypsin with medium and centrifuge at 1000 rpm for 8 min.Aspirate the supernatants and resuspend cells with medium then split the cells at 1:3 ratio to expand and be ready to use.
2.EPCs apoptosis detection by flow cytometer.
2.1 Flow cytometer was performed to characterize the occurrence of apoptosis of various concentration of glucose(control,5mmol/L,12.Smmol/L,25mmol/L and 25mmol/L manitol)treatment for 24 h on EPCs.
2.2 Flow cytometer was performed to characterize the occurrence of apoptosis of pre-treating or no pretreating the EPCs with Z-ATAD-FMK(caspase-12 specific inhibitor)in the presence or absence of high glucose(25mmol/L).
3.Western blot analysis
3.1 The protein expression of the effect of various concentration of glucose(control, 5mmol/L,12.Smmol/L and 25mmol/L)treatment for 24h on EPCs were investigated by western blot analysis for the GRP78,CASPASE-12,CASPASE-3,respectively.
3.2 The protein expression of the effect of pre-treating or no pretreating the EPCs with Z-ATAD-FMK in the presence or absence of high concentration of glucose(25mmol/L) by western blot analysis for the CASPASE-12,CASPASE-3,respectively.
Results
1.Bone Marrow-derived Endothelial Progenitor Cells(BM-derived EPCs)culture and characterization
The BM mononuclear cells were isolated and the secondarily attached cells stretched after 3 days.The cells proliferated faster after 7-10 days resulted in a spindle-shaped, EC-like morphology.After 2 weeks' induction,most of the cells exhibited multangular morphology.The cord-like structures and cobblestone appearance was observed.Fluorescent chemical detection of EPCs was performed on second attached cells after 7 days in culture.Uptake of ac-LDL was detected red-stained cells,binding UEA-1 was detected green-stained cells and dual-stained cells for both UEA-1 and ac-LDL were judged to be EPCs.More than 80%attached cells were double positive fluorescence.
2.The effect of high concentration of glucose on BM-derived EPCs apoptosis FAC were performed to characterize the effect of high concentration of glucose treatment on EPCs.Treatment with high concentration of glucose(Smmol/L, 12.5mmol/L and 25mmol/L)for 24h significantly increased apoptosis in cultured rat BM-EPCs compared with control group in a dose-dependent manner(7.34±0.44%vs 6.18±0.33%,P＜0.05;11.59±1.22%vs 6.18±0.33%,P＜0.05;36.82±2.16%vs. 6.18±0.33%,P＜0.05;n=3).Treatment of the cells with 25mmol/L mannitol,used as an osmotic control,had no significant effect on apoptosis compared with control group in rat BM-EPCs(5.95±0.81%vs.6.18±0.33%,P＜0.05;n=3).
3.The ER stress by high concentration of glucose
Treatment with various concentration of glucose(5mmol/L,12.5mmol/L and 25mmol/L)for 24h significantly increased the protein expression of the GRP78 in EPCs compared with control in a dose-dependent manner(127.33±9.71 vs 39±6.56, P＜0.05;152.33±7.09 vs 39±6.56,P＜0.05;183.33±4.51 vs 39±6.56,P＜0.05;n=3).
4.Protein expression of high concentration of glucose on EPCs for CASPASE-12 and CASPASE-3
Treatment of BM-EPCs with various concentration of glucose(Smmol/L,12.5mmol/L and 25mmol/L)for 24h markedly increased protein expression of cleaved-caspase-12 and cleaved-caspase-3 in a dose-dependent manner(cleaved-caspase-12:34.67±9.07, 71.33±4.16,125.33±11.68;cleaved-caspase-3:64.67±7.37,131.67±9.02,165.33±5.69)compared with control group(cleaved-caspase-12:0,P＜0.01;cleaved-caspase-3: 0,P＜0.01;n=3).
5.The effect of caspase-12 specific inhibitor(Z-ATAD-FMK)on apoptosis induced by high concentration of glucose in rat BM-derived EPCs
Pretreatment with Z-ATAD-FMK significantly decreased apoptosis in high glucose (25mmol/L)cultured rat BM-EPCs compared with those of no pretreating group(17.91±0.94%vs.36.82±2.16%,P＜0.05;n=3).
In contrast,pretreatment or no with Z-ATAD-FMK had no significant effect on apoptosis of rat BM-EPCs in normal glucose(5.62±0.92%vs.6.18±0.33%,P＞0.05;n =3).
6.The effect of caspase-12 specific inhibitor(Z-ATAD-FMK)on protein expression of caspase-12 and caspase-3 induced by high concentration of glucose in rat BM-derived EPCs
Pretreatment with Z-ATAD-FMK significantly decreased protein expression of cleaved-caspase-12 and cleaved-caspase-3 compared with those of no pretreatment (cleaved-caspase-12:0 vs.58±7.21,P＜0.01;cleaved-caspase-3.155±10.58 vs. 185.33±12.01,P＜0.05;n=3)in group with high concentration of glucose.
In contrast,pretreatment or no with Z-ATAD-FMK had no significant effect on protein expression of cleaved-caspase-12 and cleaved-caspase-3(both had no expression of cleaved-caspase-12 and cleaved-caspase-3;n=3).
Conclusions
1.High concentration of glucose may induce the rat BM-derived EPCs apoptosis in a dose-dependent manner.
2.ER stress in rat BM-derived EPCs was actived by high concentration of glucose.
3.ER stress pathway mediated by caspase-12 may play a important role in rat BM-derived EPCs apoptosis induced by high concentration of glucose.
Background
The prevalence of type 2 diabetes is increasing year by year.As an independent risk factor for cardiovascular disease,type 2 diabetes has been implicated as a coronary heart disease equivalent and it is an important component of the complex of common cardiovascular risk factors.Cardiovascular complications are the leading causes of morbidity and mortality in patients with diabetes mellitus(DM).Among them the acute coronary syndroms(ACS)is the extremely severe outcome.Recent studies have shown that the key pathobiological processes are responsible for transforming stable atherosclerotic plaques into active rupture-prone plaques in ACS.Moreover,the processes may be enhanced in diabetes.So it is important for clinician to detect the vulnerable plaque and seek the method to stabilize the plaques.As a method to diagnose the coronary disese,the coronary angiography(CAG)has several inherent limitations because it is unable to take account of arterial remodeling.Incontrast with angiography,Intravascular ultrasound(IVUS)is unique in its ability to assess both the intraluminal plaque and the surrounding arterial wall morphology.IVUS provides cross-sectional morphometric detail and quantifies atherosclerotic plaque area and plaque burden.
Endothelial progenitor cells(EPCs)are capable of differentiating into endothelial cells and homing to sites of ischaemia and play a important role in vascular repair and reendothelialisation of injured blood vessels and collateral vessels formation. Moreover,the endothelial dysfunction and damage are common starting points for diabetic macroangiopathy.And recent insights show that the injured endothelial monolayer is regenerated by circulating EPCs,which accelerate reendothelialization and limits atherosclerotic lesion formation.So the EPCs would play a vital role in the diabetic macroangiopathy.Then it would be of significance to assess the correlation between EPCs levels and extent of lesion in diabetic coronary disease.There was analysis of the CAG show that the lesion is more extensive and severe,the ability to form collateral circulation is descent in patients with DM.And it was reported that the number of circulating EPCs has been impaired in patients with diabetes mellitus and been increased in patients with ACS.However,few data are available on the relationship between the number of circulating EPCs and the instability of plaque as well as the extension of lesion assessed by intravascular ultrasound in diabetic patients with ACS.The objectives of this study are as follow:
objectives
1.To investigate the coronary culprit plaque in diabetic patients with ACS by IVUS.
2.To investigate the number of circulating EPCs in diabetic patients with ACS.
3.To investigate the potential relation between the number of circulating EPCs and plaque burden and severity of coronary culprit lesions using IVUS in diabetic patients with ACS.
Methods
1.Subjects
From July 2007 to January 2008,40 patients with diagnosis of ACS with de novo culprit lesions underwent preintervention IVUS were enrolled in this study.Among the subjects,there were 24 patients with unstable angina,16 patients with acute myocardial infarction.Acute myocardial infarction was defined as continuous chest pain at rest with abnormal levels of cardiac enzymes(creatininekinase-MB or troponin T).The culprit lesion in ACS was identified by the combination of left ventricular wall motion abnormalities,electrocardiographic findings,angiographic -lesion morphology.The lesion with the worst diameter stenosis was selected as the target lesion for IVUS analysis.According to the American Diabetes Association 2005 criteria for insulin-independent diabetes mellitus,the patients were classified to diabetic group and non-diabetic group:diabetic group(n=20),nondiabetic group (n=20).The medical history,including the pre-existing coronary riskfactors such as hypertension,hypercholesterolemia,and smoking,was recorded for each patient.
2.Examinational Methods
2.1 Coronary angiography:Coronary angiography was performed according to standard angiographic techniques.Angiographic scoring was done by interventional cardiologists who were blinded to the study protocol.Angiographic diagnosed criteria fulfilling the Coronary Artery Surgery Study:The left main coronary arterywas defined as a lumen diameter decrease of at least 50%,the anterior descending,left circumflex,and right coronary arteries were at least 70%,respectively.
2.2 intravascular ultrasound
After completion of the diagnostic coronary angiography,the culprit lesion was localized according to the angiographic signs of vessel morphology and electrocardiogrampatterns.Before the IVUS procedure,200μg intracoronary nitroglycerine was administered to prevent vasospasm,and a 2.9F IVUS imaging catheter(Eagle Eye;Volcano Corporation,USA)was advanced distally to the culprit lesion and pulled back at a pull-back speed of 0.5 mm/sec.In parallel,the images were recorded on videotapes for subsequent off-line analysis.IVUS images were analyzed independently by 2 experienced observers.Conventional gray-scale quantitative IVUS analyses were performed according to criteria of the clinical expert consensus document on IVUS to include external elastic membrane(EEM),luminal, and plaque areas.The external elastic membrane cross-sectional area(defined as the area encompassed by the media-adventitia border)were measured in diastolic frames. The plaque cross-sectional area,defined as the intimamedia area,was calculated from the difference between the external elastic membrane and lumen cross-sectional areas. The plaque burden on each cross section was calculated as 100×(external elastic membrane cross-sectional area-lumen cross-sectional area)/external elastic membrane cross-sectional area).
2.3 EPCs Culture,Characterization and Count
2.3.1 EPCs Culture Assay
After placement of an arterial sheath,3 to 5 ml of blood was wasted and then 20 ml of whole blood obtained for cell culture.The heparinized specimen was diluted with phosphate buffered saline(PBS),and the specimen was centrifuged across a 1.077 density gradient over human lymphocyte separating medium(LTS-1077)at 2000 rpm for 30 min.Then mononuclear cells were isolated from the diluted peripheral blood. Immediately after isolation,1×10~6 mononuclear cells were plated on 24-well culture dishes pre-coated with 0.1%gelatin(Sigma,USA)and maintained in M199 medium (Gibco,USA)supplemented with 20%fatal calf serum,100μg/mL streptomycin, 100U/ml penicillin,40 ng/mL human vascular endothelial growth factor(VEGF, Peprotech,USA),2 ng/mL fibroblast growth factor-2(FGF,Peprotech,USA),10 ng/mL epidermal growth factor(EGF,Peprotech,USA),and 10U/mL heparin.After 4 days in culture,nonadherent cells were removed by a thorough washing with PBS, new media was applied,and adherent cells were maintained through day 7 then underwent cytochemical analysis.
2.3.2 Characterization of EPCs
Fluorescent chemical detection of EPCs was performed by using to detect dual binding of fluorescein isothiocyanate(FITC)-labeled Ulex europaeus agglutinin (UEA)-1(lectin;Sigma,USA)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyani -ne(DiI)-labeled acetylated low-density lipoprotein(ac-LDL;Molecular Probe,USA). To detect the uptake of ac-LDL,cells were incubated with ac-LDL(2.4μg/mL)at 37℃for 1 hour.Then cells were fixed with 2%paraformaldehyde for 10 minutes. After being washed,cells were incubated with lectin(10μg/mL)for 1 hour.After being washed with PBS,samples were viewed using an inverted fluorescent microscope(Olympus,Japan).Dual-stained cells positive for both lectin and ac-LDL were judged to be EPCs..
2.3.3 Count of EPCs
Dual-stained positive cells were counted per well.Two independent investigators who was blinded to the clinical profile of the patients evaluated the number of EPCs per well by counting 3 randomly selected high-power fields.
Results
The culprit lesion was localized in the left anterior escending,left circumflex,or right coronary artery in 22(55%),8(20%)and 10(25%)patients,respectively.
IVUS data
IVUS findings of culprit lesions,comparing diabetic patients and without diabetes. The minimum luminal area site lumen,external elastic membrane,and plaque plus media cross-sectional areas were 3.3±1.6,14.3±3.0 and 11.1±2.7 mm~2 respectively, whereas the plaque burden was 76.5%±11.7%in diabetes group.Another group,the minimum luminal area site lumen,external elastic membrane,and plaque plus media cross-sectional areas were 4.9±2.2,13.8±3.4 and 9.0±2.6 mm~2 respectively,and the plaque burden was 66.2%±13.2%.DM group had greater pique burden compared with the nondiabetes.
EPCs Number
When patients were classified according to DM or nondiabete group,the significantly lower levels of EPCs were found in the patients with DM,compared with EPCs levels in the patients without DM(30.4±6.99 vs.51.7±11.1 EPCs/×200 field,P＜0.05).
Correlation between EPCs number and plaque burden determined with IVUS
The lower levels of circulating EPCs were measured in association with lesion with greater plaque burden.In addition,the plaque burden of the lesion was associated with significantly decreased levels of EPCs(r=-0.427,P＜0.05)in diabetes group.
Conclusions
1.Coronary culprit plaque in ACS patients with DM showed a higher extent of coronary stenosis,a greater plaque burden and a smaller lumen cross-sectional area.
2.The number of circulating EPCs was significantly lower in patients with DM compared to those without DM.
3.The number of EPCs was negatively associated with the plaque burden.Decreased number of circulating EPCs may reflect the severity of coronary culprit lesion in diabetic patients with ACS.
4.Given the important role of EPCs in process of atherogenesis,a decrease in the number of EPCs may contribute to the accelerated atherogenesis and impaired endothelial regeneration in diabetic patients with ACS.
引文
1. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelia angiogenesis. Science, 1997;275:964-967.
2. Ziegler D. Type 2 diabetes as an inflammatory cardiovascular disorder. Curr Mol Med,2005;5:309-322.
3. Grundy SM, Benjamin IJ, Burke GL, et al. Diabetes and cardiovascular disease a statement for healthcare professionals from the American Heart Association. Circulation, 1999; 100:1134-1146.
4. Hink U, Li H, Mollnau H, et al. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res,2001;88:el4-e22.
5. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med, 2003;348:593-600.
6. 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:El-E7.
7. Krankel N, Adams V, Linke A, et al. Hyperglycemia reduces surrival and impairs function of circulating blood-derived progenitor cell. Arterioscler Thromb Vasc Biol,2005;25:698-703.
8. Fadini GP, Miorin M, Facco M, et al. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Colle Cardiol, 2005;45:1449-1457.
9. Yoon YS, Uchida S, Masuo O, et al. Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation,2005;111:2073-2085.
10. Ayalasomayajula SP, Komepella UB. Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozotocin-induced diabetic rat model. Eur J Pharmacol, 2003;458:283-289.
11.Sasso FC,Carbonara O,Persico E,et al.Increased vascular endothelial growth factor mRNA expression in the heart of streptozotocin-induced diabetic rats.Metabolism,2003;52:675-678.
12.魏来,赵强,付晗等.大鼠骨髓内皮祖细胞的分离、培养和鉴定.复旦学报(医学版),2006.33:404-407.
13.Kalka C,Masuda H,Takahashi T,et al.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.Proc Natl Acad Sci USA,2000;97:3422-3427.
14.Suda T,Yakakura N,Oike Y,et al.Hematopoiesis and angiogenesis.Int J Hematol,2000;71:99-107.
15.Tavian M,Cortes F,Robin C,et al.The hemangioblast,common precursor of endothelial and hematopoietic cells.Transfus Clin Biol,2000;7:238-241.
16.Reyes M,Dudek A,Jahagirdar B,et al.Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002;109:337-346.
17.Peichev M,Naiyer AJ,Pereira D,et al.Expression of VEGF2 and CD_(133)by circulation human CD_(34)~+ cells identifies a population of functional endothelial precursors.Blood,2000;95:952-958.
18.Wang C,Jiao C,Hanlon HD,et al.Mechanical,cellular,and molecular factors interact to modulate circulating endothelial cell progenitors.Am J Physiol Heart Circ Physiol,2004;286:1985-1993.
19.Mihail H,Wolfgang Erl,Peter CW,et al.Endothelial progenitor cells mobilization,differentiation,and homing.Arterioscler Thromb Vasc Biol,2003,23:1185-1189.
20.Asahara T,Takahashi T,Masuda H,et al.VEGF contributes to postnatal neovascu -larization by mobilizing bone marrow-derived endothelial progenitor cells.EMBO J,1999;18:3964-3972.
21.Yakahashi T,Kalka C,Masuda H,et al.Ischemiaand cytokine-induced mobili -zation of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med,1999;5:434-438.
22.Shintani S,Murohara T,Ikeda H,et al.Augmentation of postnatal neovasculariza -tion with autologous bone marrow transplantation. Circulation, 2001; 103: 2776 -2779.
23. Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res,2000;86:1198-1202.
24. Moore MA, Hattori K, Heissig B, et al. Mobilization ofendothelial and hemotopoietic stem and progenitor cells by adenovector-mediated elevation of serum level of SDF-1, VEGF, and angiopoientin-1. Ann NY Acad Sci, 2001; 938: 36-45.
25. Schuch G, Heymach JV, Nomi M, et al. Endostatin inhibits the vascular endothelial growth factor-induced mobilization of endothelial progenitor cells. Cancer Res,2003;63:8345-8350.
26. Heesschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood, 2003; 102; 1340 -1346.
27. Edelberg JM, Tang L, Hattonk, et al. Young adult bone marrow-derived endothelial progenitor cells restore aging impaired angiogenesis function. Circ Res, 2002; 90: e89-e93.
28. Adams V, Lenk K, Linke A, et al. Increase of circulating endothelial progenitor cells in patients with coronary artery disease after exercise-induced ischemia. Arterioscler Thromb Vasc Biol, 2004;24: 684-690.
29. Strehlow K, Werner N, Berweiler J, et al. Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation. Circulation,2003; 107:3059-3065.
30. Urbich C, Derbach E, Zeiher AM, et al. Double-edged role of statins in angiogenesis signaling. Cir Res,2002;90:737-744.
31. Stamm C, Westphal B, Kleine HD, et al. Autologous bone marrow stem cell transplantation for myocardial regeneration. Lancet,2003;361:45-46.
32. Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerates reendothelia-lization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation,2002; 105:3017 -3024.
33. Abaci A, Oguzhan A, Kahraman S, et al. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation, 1999;99:2239-2242.
34. Fallow GD, Singh J. The prevalence, type and severity of cardiovascular disease in diabetic and non-diabetic patients: Amatched-paired retrospective analysis using coronary angiography as the diagnostic tool. Mol Cell Biochem,2004;261: 263-269.
35. Nassar T, Kadery B, Lotan C, et al. Effects of the superoxide dismutase-mimetic compound tempol on endothelial dysfunction in streptozotocin-induced diabetic rats. Eur J Pharmacol,2002;436:111 -118.
36. Bansilal S, Farkouh ME, Fuster V. Role of insulin resistance and hyperglycemia in the development of atherosclerosis. Am J Cardiol,2007;99:6B-14B.
37. Xu Y, He Z, King GL. Introduction of hyperglycemia and dyslipidemia in the pathogenesis of diabetic vascular complications. Curr Diab Rep,2005;5:91-97.
38. 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.
39. Waltenberger J. Impaired collateral vessel development in diabetes: potential cellular mechanisms and therapeutic implications. Cardiovasc Res,2001; 49:554 -560.
40. Loomans CJ, de Koning EJ, Staal FJ, et al. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes, 2004;53:195-199.
41. Tepper OM, Galiano RD, Capla JM, et al. Human endothelial progenitor cells from type 2 diabetics exhibit impaired proliferation,adhesion,and incorporation intovascularstructures. Circulation,2002;106:2781-2786.
42. Fadini GP, Sartore S, Schiavon M, et al. Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats. Diabetologia, 2006;49:3075-3084.
43. He T, Smith LA, Harrington S, et al. Transplantation of circulating endothelial progenitor cells restores endothelial function of denuded rabbit carotid arteries. Stroke,2004;35:2378-2384.
44. Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res,2004;95:343-353.
45. He T, Peterson TE, Holmuhamedov EL, et al. Human endothelial progenitor cells tolerate oxidative stress due to intrinsically high expression of manganese superoxide dismutase. Arterioscler Thromb Vasc Biol,2004;24:2021-2027.
46. Laybutt DR, Preston AM, Akerfeldt MC, et al. Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia, 2007;50:752 -763.
47. Donath MY, Ehses JA, Maedler K, et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes,2005;54 Suppl 2:S108-S113.
48. Lipson KL, Fonseca SG, Urano F. Endoplasmic reticulum stress-induced apoptosis and autoimmunity in diabetes. Curr Mol Med,2006;6:71-77.
49. Araki E, Oyadomari S, Mori M. Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus. Exp Biol Med, 2003;228:1213-1217.
50. Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis,2002;7:335-345.
1.Quirici N,Soligo D,Caneva L,et al.Differentiation and expansion of endothelial cells from human bone marrow CD133(+)cells.Br J Haematol,2001;115:186-194.
2.Asahara T,Murohara T,Sullivan A,et al.Isolation of putative progenitor endothe -lia angiogenesis.Science,1997;275:964-967.
3.Kalka C,Masuda H,Takahashi T,et al.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.Proc Natl Acad Sci USA,2000;97:3422-3427.
4.魏来,赵强,付晗等.大鼠骨髓内皮祖细胞的分离、培养和鉴定。复旦学报(医学版),2006:33:404-407.
5.Suda T,Takakura N,Oike Y,et al.Hematopoiesis and angiogenesis.Int J Hematol,2000;71:99-107.
6.Tavian M,Cortes F,Robin C,et al.The hemangioblast,common precursor of endothelial and hematopoietic cells.Transfus Clin Biol,2000;7:238-241.
7.Shi Q,Rafii S,Wu MH,et al.Evidence for circulating bone marrow-derived endothelial progenitor cells.Blood,1998;92:362-367.
8.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.
9.Reyes M,Dudek A,Jahagirdar B,et al.Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002;109:337-346.
10.Peichev M,Naiyer AJ,Pereira D,et al.Expression of VEGF2 and CD_(133)by circulation human CD_(34)~+ cells identifies a population of functional endothelial precursors.Blood,2000;95:952-958.
11.Hristov M,Weber C.Endothelial progenitor cells:characterization,pathophy -siology,and possible clinical relevance.J Cell Mol Med,2004;8:498-508.
12.Rumpold H,Wolf D,Koeck R,et al.Endothelial progenitor cells:a source for therapeutic vasculogenesis? J Cell Mol Med.2004;8:509-518.
13. Khakoo AY, Finkel T. Endothelial progenitor cells. Annu Rev Med,2005;56:79 -101.
14. Isner JM, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair. Ann N Y Acad Sci,2001;953:75-84.
15. Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem,1999;274:20049-20052.
16. Chang HY, Yang X. Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev,2000;64:821-846.
17. Takahashi A. Caspase: executioner and undertaker of apoptosis. Int J Hematol, 1999;70:226-232.
18. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, et al. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell, 1998; 1:949-957.
19. Zhou P,Chou J,Olea RS,et al.Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific adaptor/caspase interaction. Proc Natl Acad Sci USA, 1999;96:11265-11270.
20. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science, 1998; 281:1305-1308.
21. Kaufman RJ. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Gene Dev,1999;13: 1211-1233.
22. Mori K. Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell,2000; 101:451-454.
23. Michalak M, Robert Parker JM, et al. Ca~(2+) signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium,2002;32:269-278.
24. Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic reticulum resident kinase. Nature,1999;397:271-274.
25. Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis,2002;7:335-345.
26. Liu J, Bhalgat M, Zhang C, et al. Fluorescent molecular probes V: a sensitive caspase-3 substrate for fluorometric assays. Bioorg Med Chem Lett,1999;9:3231 vascular biology. Circ Res,2004;95:343-353.
38. He T, Peterson TE, Holmuhamedov EL, et al. Human endothelial progenitor cells tolerate oxidative stress due to intrinsically high expression of manganese superoxide dismutase. Arterioscler Thromb Vasc Biol,2004;24:2021-2027.
39. Dernbach E, Urbich C, Brandes RP ,et al. Antioxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress. Blood,2004;104:3591-3597.
40. Hoetzer GL, Irmiger HM, Keith RS, et al. Endothelial nitric oxide synthase inhibition does not alter endothelial progenitor cell colony forming capacity or migratory activity. J Cardiovasc Pharmacol,2005;46:387-389.
41. Chen YH, Lin SJ, Lin FY, et al. High glucose impairs early and late endothelial progenitor cells by modifying nitric oxide-related but not oxidative stress -mediated mechanisms. Diabetes,2007;56:1559-1568.
42. Laybutt DR, Preston AM, Akerfeldt MC, et al. Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia, 2007;50:752 -763.
43. Donath MY, Ehses JA, Maedler K, et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes,2005;54 Suppl 2:S108-S113.
44. Lipson KL, Fonseca SG, Urano F. Endoplasmic reticulum stress-induced apoptosis and autoimmunity in diabetes. Curr Mol Med,2006;6:71-77.
45. Araki E, Oyadomari S, Mori M. Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus. Exp Biol Med,2003;228:1213 -1217.
1. Vinik A, Flemmer M. Diabetes and macrovascular disease. J Diabetes Complications,2002;16:235-245.
2. Coccheri S. Approaches to prevention of cardiovascular complications and events in diabetes mellitus. Drugs,2007;67:997-1026.
3. Karra R, Vemullapalli S, Dong C, et al. Molecular evidence for arterial repair in atherosclerosis. Proc Natl Acad Sci USA, 2005;102:16789-16794.
4. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: part Ⅰ. Circulation, 2003;108:1664-1672.
5. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part Ⅱ. Circulation, 2003;108:1772-1778.
6. Hristov M, Erl W, Weber PC. Endothelial progenitor cells mobilization, differentiation, and homing. Arterioscler Thromb Vasc Biol,2003;23:1185-1189.
7. Wojakowski W, Tendera M. Mobilization of bone marrow-derived progenitor cells in acute coronary syndromes. Folia Histochem Cytobiol,2005;43:229-232.
8. Shintani S, Murohara T, Ikeda H, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation,2001; 103:2776-2779.
9. George J, Goldstein E, Abashidze S, et al. Circulating endothelial progenitor cells in patients with unstable angina association with systemic inflammation. Eur Heart J, 2004;25:1003-1008.
10. Leone AM, Rutella S, Bonanno G, et al. Mobilization of bone marrow-derived stem cells after myocardial infarction and left ventricular function. Eur Heart J, 2005;26:1196-1204.
11. Thum T, Fraccarollo D, Galuppo P, et al. Bone marrow molecular alterations after myocardial infarction: Impact on endothelial progenitor cells. Cardiovasc Res, 2006; 70:50-60.
12.Loomans CJ,de Koning EJ,Staal FJ,et al.Endothelial progenitor cell dysfunction:a novel concept in the pathogenesis of vascular complications of type 1 diabetes.Diabetes,2004;53:195-199.
13.Krankel N,Adams V,Linke A,et al.Hyperglycemia reduces survival and impairs function of circulating blood-derived progenitor cells.Arterioscler Thromb Vasc Biol,2005;25:698-703.
14.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-e7.
15.Fallow GD,Singh J.The prevalence,type and severity of cardiovascular disease in diabetic and non-diabetic patients:Amatched-paired retrospective analysis using coronary angiography as the diagnostic toot.Mol Cell Biochem,2004;261:263-269.
16.中华医学会心血管病学分会,中华心血管病杂志编辑委员会,中国循环杂志编辑委员会.急性心肌梗死诊断和治疗指南.中华心血管病杂志,2001,29:710-725.
17.CASS Principal investingators and their associates:Coronary Artery Surgery Study(CASS):a randomized trial of coronary artery,bypasss Surgery.Circulation,1983;28:939-950.
18.St Goar FG,Pinto FJ,Alderman EL,et al.Intravascular ultrasound imaging of angiographically normal coronary arteries:an in vivo comparison with quantita -tive angiography.J Am Coll Cardiol.1991;18:952-958.
19.Fitzgerald PJ,St Goar FG,Connolly AJ,et al.Intravascular ultrasound imaging of coronary arteries.Is three layers the norm? Circulation,1992;86:154-158.
20.Mintz GS,Nissen SE,Anderson WD,et al.ACC Clinical Expert Consensus Document on Standards for the acquisition,measurement and reporting of intravascular ultrasound studies:a report of the American College of Cardiology Task Force on Clinical Expert Consensus Documents(Committee to Develop a Clinical Expert Consensus Document on Standards for Acquisition,Measurement and Reporting of Intravascular Ultrasound Studies[IVUS]).J Am Coll Cardiol, 2001 ;37:1478-1492.
21.Mintz GS, Maehara A, Bui AB, et al. Multiple versus single coronary plaque ruptures detected by intravascular ultrasound in stable and unstable angina pectoris and in acute myocardial infarction. Am J Cardiol,2003;91:1333-1335.
22. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelia angiogenesis. Science, 1997;275:964-967.
23. Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA,2000;97:3422-3427.
24. Ziegler D. Type 2 diabetes as an inflammatory cardiovascular disorder. Curr Mol Med,2005;5:309-322.
25. Grundy SM, Benjamin IJ, Burke GL, et al. Diabetes and cardiovascular disease a statement for healthcare professionals from the American Heart Association. Circulation, 1999; 100:1134-1146.
26. Clark CM, Clark PR. Type 2 diabetes and macrovascular disease. Epidemiology and etiology. Am Heart J, 1999; 138:5330-5333.
27. Amos AF, McCarty DJ, Zimmet P. The rising global burden of diabetes and its complications: Estimates and projections to the year 2010. Diabetes Med,1997; 14:S7-S15.
28. Vinik AI, Vinik E. Prevention of the complications of diabetes. Am J Manag Care,2003;9(3 suppl):S63-S80.
29. Abaci A, Oguzhan A, Kahraman S, et al. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation, 1999;99:2239-2242.
30. Lindvall B, Brorsson B, Herlitz J, et al. Comparison of diabetic and non-diabetic patients referred for coronary angiography. Int J Cardiol,1999;70:33-42.
31. Schaar JA, Muller JE, Falk E, et al. Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque, June 17 and 18, 2003, Santorini, Greece. Eur Heart J, 2004;25:1077-1082.
32. Schoenhagen P, Nissen S. Understanding coronary artery disease: tomographic imaging with intravascular ultrasound. Heart,2002;88:91-96.
33. Swallow RA, Court IA, Calver AL, et al. The limitations of coronary angiography: identification of a critical coronary stenosis using intravascular ultrasound. Int J Cardiol,2006;106:123-125.
34. Nissen SE, Yock P. Intravascular ultrasound: novel pathophysiological insights and current clinical applications. Circulation,2001; 103:604-616.
35. Gussenhoven EJ, Essed CE, Lancee CT, et al. Arterial wall characteristics determined by intravascular ultrasound imaging: an in vitro study. J Am Coll Cardiol, 1989;14:947-952.
36. Hodgson JM, Reddy KG, Suneja R, et al. Intracoronary ultrasound imaging: correlation of plaque morphology with angiography, clinical syndrome and procedural results in patients undergoing coronary angioplasty. J Am Coll Cardiol, 1993; 21:35-44.
37. Virmani R, Kolodgie FD, Burke AP, et al. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol,2000;20:1262-1275.
38. Libby P. Changing concepts of atherogenesis. J Intern Med,2000,247(3): 349-358.
39. Lusis AJ. Atherosclerosis. Nature,2000;407:233-241.
40. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 1993;362: 801-809.
41. Walter D, Rittig K, Bahlmann F, et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation,2002; 105:3017-3024.
42. Griese D, Ehsan A, Melo L, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy. Circulation,2003; 108:2710-2715.
43. Fujiyama S, Amano K, Uehira K, et al. Bone marrow monocyte lineage cells adhere on injured endothelium in a monocyte chemoattractant protein-1-dependent manner and accelerate reendothelialization as endothelial progenitor cells. Circ Res,2003;10:980-989.
44. Gotlieb AI. Atherosclerosis and acute coronary syndromes. Cardiovasc Pathol, 2005;14:181-184.
45. Zoll J, Fontaine V, Gourdy P, et al. Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition. Cardiovasc Res,2008;77: 471-480.
46. Dickson BC, Gotlieb AI. Towards understanding acute destabilization of vulnerable atherosclerotic plaques. Cardiovasc Pathol,2003; 12:237-248.
47. Virmani R, Burke AP, Farb A, et al. Pathology of the vulnerable plaque. J Am Coll Cardiol,2006;18;47(8 Suppl):C13-C18.
48. Shan PK. Mechanisms of plaque vulnerability and rupture. J Am Coll Cardiol, 2003;41(4 suppl S):15S-22S.
49. Zhao Y, Glesne D, Huberman E, et al. A human peripheral blood monocyte derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA,2003;100: 2426-2431.
50. Asahara T, Takahashi T, Masuda H, et al. VEGF contributes to postnatal neovascu-larization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J,1999;18:3964-3972.
51. Takahashi T, Kalka C, Masuda H, et al. Ischemiaand cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med,1999;5:434-438.
52. Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor165 gene transfer augments circulationendothelial progenitor cells in human subjects. CircRes,2000;86:1198-1202.
53. Moore MA, Hattori K, Heissig B, et al. Mobilization of endothelial and hemotopoietic stem and progenitor cells by adenovector-mediated elevation of serum level of SDF-1,VEGF, and angiopoientin-1. Ann NY Acad Sci,2001,938:36-45.
54. Urbich C, Derbach E, Zeiher AM, et al. Double-edged role of statins in angiogenesis signaling. Cir Res,2002;90:737-744.
55. Tepper OM, Galiano RD, Capla JM, et al. Human endothelial progenitor cells from type 2 diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation,2002;106:2781-2786.
56. Kunz GA, Liang G, Cuculi F, et al. Circulating endothelial progenitor cells predict coronary artery disease severity. Am Heart J,2006; 152:190-195.
57. Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med, 2005;353:999-1007.
58. 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.
59. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med, 2003;348:593-600.
60. Rosenzweig A. Circulating endothelial progenitors-cells as biomarkers. N Engl J Med,2005;353:1055-1057.
61. Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mono -nuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation,2001; 104:1046-1052.
62. Kawamoto A, Gwon HC, Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation,2001; 103:634-637.
63. Orlic D, Kajstura J, Chimenti S, et al. Transplanted adult bone marrow cells repair myocardial infarcts in mice. Ann NY Acad Sci,2001;938:221-229.
64. Hamano K, Nishida M, Hirata K, et al. Local implantation of autologous bone marrow cells for therapeutic angiogenesis in patients with ischemic heart disease: clinical trial and preliminary results. Jpn Circ J,2001;65:845-847.
65. Badorff C, Brands RP, Popp R, et al. Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation,2003; 107:1024-1032.
66. Szmiko PE, Fedak PW, Weisel RD, et al. Endothelial progenitor cells: new hope for a broken heart. Circulation,2003; 107:3093-3100.
2. Ziegler D. Type 2 diabetes as an inflammatory cardiovascular disorder. Curr Mol Med,2005;5:309-322.
3. Grundy SM, Benjamin IJ, Burke GL, et al. Diabetes and cardiovascular disease a statement for healthcare professionals from the American Heart Association. Circulation, 1999; 100:1134-1146.
4. Hink U, Li H, Mollnau H, et al. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res,2001;88:el4-e22.
5. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med, 2003;348:593-600.
6. 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:El-E7.
7. Krankel N, Adams V, Linke A, et al. Hyperglycemia reduces surrival and impairs function of circulating blood-derived progenitor cell. Arterioscler Thromb Vasc Biol,2005;25:698-703.
8. Fadini GP, Miorin M, Facco M, et al. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Colle Cardiol, 2005;45:1449-1457.
9. Yoon YS, Uchida S, Masuo O, et al. Progressive attenuation of myocardial vascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy: restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardiomyopathy after replenishment of local vascular endothelial growth factor. Circulation,2005;111:2073-2085.
10. Ayalasomayajula SP, Komepella UB. Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozotocin-induced diabetic rat model. Eur J Pharmacol, 2003;458:283-289.
11.Sasso FC,Carbonara O,Persico E,et al.Increased vascular endothelial growth factor mRNA expression in the heart of streptozotocin-induced diabetic rats.Metabolism,2003;52:675-678.
12.魏来,赵强,付晗等.大鼠骨髓内皮祖细胞的分离、培养和鉴定.复旦学报(医学版),2006.33:404-407.
13.Kalka C,Masuda H,Takahashi T,et al.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.Proc Natl Acad Sci USA,2000;97:3422-3427.
14.Suda T,Yakakura N,Oike Y,et al.Hematopoiesis and angiogenesis.Int J Hematol,2000;71:99-107.
15.Tavian M,Cortes F,Robin C,et al.The hemangioblast,common precursor of endothelial and hematopoietic cells.Transfus Clin Biol,2000;7:238-241.
16.Reyes M,Dudek A,Jahagirdar B,et al.Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002;109:337-346.
17.Peichev M,Naiyer AJ,Pereira D,et al.Expression of VEGF2 and CD_(133)by circulation human CD_(34)~+ cells identifies a population of functional endothelial precursors.Blood,2000;95:952-958.
18.Wang C,Jiao C,Hanlon HD,et al.Mechanical,cellular,and molecular factors interact to modulate circulating endothelial cell progenitors.Am J Physiol Heart Circ Physiol,2004;286:1985-1993.
19.Mihail H,Wolfgang Erl,Peter CW,et al.Endothelial progenitor cells mobilization,differentiation,and homing.Arterioscler Thromb Vasc Biol,2003,23:1185-1189.
20.Asahara T,Takahashi T,Masuda H,et al.VEGF contributes to postnatal neovascu -larization by mobilizing bone marrow-derived endothelial progenitor cells.EMBO J,1999;18:3964-3972.
21.Yakahashi T,Kalka C,Masuda H,et al.Ischemiaand cytokine-induced mobili -zation of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med,1999;5:434-438.
22.Shintani S,Murohara T,Ikeda H,et al.Augmentation of postnatal neovasculariza -tion with autologous bone marrow transplantation. Circulation, 2001; 103: 2776 -2779.
23. Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res,2000;86:1198-1202.
24. Moore MA, Hattori K, Heissig B, et al. Mobilization ofendothelial and hemotopoietic stem and progenitor cells by adenovector-mediated elevation of serum level of SDF-1, VEGF, and angiopoientin-1. Ann NY Acad Sci, 2001; 938: 36-45.
25. Schuch G, Heymach JV, Nomi M, et al. Endostatin inhibits the vascular endothelial growth factor-induced mobilization of endothelial progenitor cells. Cancer Res,2003;63:8345-8350.
26. Heesschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood, 2003; 102; 1340 -1346.
27. Edelberg JM, Tang L, Hattonk, et al. Young adult bone marrow-derived endothelial progenitor cells restore aging impaired angiogenesis function. Circ Res, 2002; 90: e89-e93.
28. Adams V, Lenk K, Linke A, et al. Increase of circulating endothelial progenitor cells in patients with coronary artery disease after exercise-induced ischemia. Arterioscler Thromb Vasc Biol, 2004;24: 684-690.
29. Strehlow K, Werner N, Berweiler J, et al. Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation. Circulation,2003; 107:3059-3065.
30. Urbich C, Derbach E, Zeiher AM, et al. Double-edged role of statins in angiogenesis signaling. Cir Res,2002;90:737-744.
31. Stamm C, Westphal B, Kleine HD, et al. Autologous bone marrow stem cell transplantation for myocardial regeneration. Lancet,2003;361:45-46.
32. Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerates reendothelia-lization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation,2002; 105:3017 -3024.
33. Abaci A, Oguzhan A, Kahraman S, et al. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation, 1999;99:2239-2242.
34. Fallow GD, Singh J. The prevalence, type and severity of cardiovascular disease in diabetic and non-diabetic patients: Amatched-paired retrospective analysis using coronary angiography as the diagnostic tool. Mol Cell Biochem,2004;261: 263-269.
35. Nassar T, Kadery B, Lotan C, et al. Effects of the superoxide dismutase-mimetic compound tempol on endothelial dysfunction in streptozotocin-induced diabetic rats. Eur J Pharmacol,2002;436:111 -118.
36. Bansilal S, Farkouh ME, Fuster V. Role of insulin resistance and hyperglycemia in the development of atherosclerosis. Am J Cardiol,2007;99:6B-14B.
37. Xu Y, He Z, King GL. Introduction of hyperglycemia and dyslipidemia in the pathogenesis of diabetic vascular complications. Curr Diab Rep,2005;5:91-97.
38. 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.
39. Waltenberger J. Impaired collateral vessel development in diabetes: potential cellular mechanisms and therapeutic implications. Cardiovasc Res,2001; 49:554 -560.
40. Loomans CJ, de Koning EJ, Staal FJ, et al. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes, 2004;53:195-199.
41. Tepper OM, Galiano RD, Capla JM, et al. Human endothelial progenitor cells from type 2 diabetics exhibit impaired proliferation,adhesion,and incorporation intovascularstructures. Circulation,2002;106:2781-2786.
42. Fadini GP, Sartore S, Schiavon M, et al. Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats. Diabetologia, 2006;49:3075-3084.
43. He T, Smith LA, Harrington S, et al. Transplantation of circulating endothelial progenitor cells restores endothelial function of denuded rabbit carotid arteries. Stroke,2004;35:2378-2384.
44. Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res,2004;95:343-353.
45. He T, Peterson TE, Holmuhamedov EL, et al. Human endothelial progenitor cells tolerate oxidative stress due to intrinsically high expression of manganese superoxide dismutase. Arterioscler Thromb Vasc Biol,2004;24:2021-2027.
46. Laybutt DR, Preston AM, Akerfeldt MC, et al. Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia, 2007;50:752 -763.
47. Donath MY, Ehses JA, Maedler K, et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes,2005;54 Suppl 2:S108-S113.
48. Lipson KL, Fonseca SG, Urano F. Endoplasmic reticulum stress-induced apoptosis and autoimmunity in diabetes. Curr Mol Med,2006;6:71-77.
49. Araki E, Oyadomari S, Mori M. Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus. Exp Biol Med, 2003;228:1213-1217.
50. Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis,2002;7:335-345.
1.Quirici N,Soligo D,Caneva L,et al.Differentiation and expansion of endothelial cells from human bone marrow CD133(+)cells.Br J Haematol,2001;115:186-194.
2.Asahara T,Murohara T,Sullivan A,et al.Isolation of putative progenitor endothe -lia angiogenesis.Science,1997;275:964-967.
3.Kalka C,Masuda H,Takahashi T,et al.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.Proc Natl Acad Sci USA,2000;97:3422-3427.
4.魏来,赵强,付晗等.大鼠骨髓内皮祖细胞的分离、培养和鉴定。复旦学报(医学版),2006:33:404-407.
5.Suda T,Takakura N,Oike Y,et al.Hematopoiesis and angiogenesis.Int J Hematol,2000;71:99-107.
6.Tavian M,Cortes F,Robin C,et al.The hemangioblast,common precursor of endothelial and hematopoietic cells.Transfus Clin Biol,2000;7:238-241.
7.Shi Q,Rafii S,Wu MH,et al.Evidence for circulating bone marrow-derived endothelial progenitor cells.Blood,1998;92:362-367.
8.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.
9.Reyes M,Dudek A,Jahagirdar B,et al.Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002;109:337-346.
10.Peichev M,Naiyer AJ,Pereira D,et al.Expression of VEGF2 and CD_(133)by circulation human CD_(34)~+ cells identifies a population of functional endothelial precursors.Blood,2000;95:952-958.
11.Hristov M,Weber C.Endothelial progenitor cells:characterization,pathophy -siology,and possible clinical relevance.J Cell Mol Med,2004;8:498-508.
12.Rumpold H,Wolf D,Koeck R,et al.Endothelial progenitor cells:a source for therapeutic vasculogenesis? J Cell Mol Med.2004;8:509-518.
13. Khakoo AY, Finkel T. Endothelial progenitor cells. Annu Rev Med,2005;56:79 -101.
14. Isner JM, Kalka C, Kawamoto A, et al. Bone marrow as a source of endothelial cells for natural and iatrogenic vascular repair. Ann N Y Acad Sci,2001;953:75-84.
15. Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem,1999;274:20049-20052.
16. Chang HY, Yang X. Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev,2000;64:821-846.
17. Takahashi A. Caspase: executioner and undertaker of apoptosis. Int J Hematol, 1999;70:226-232.
18. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, et al. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell, 1998; 1:949-957.
19. Zhou P,Chou J,Olea RS,et al.Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific adaptor/caspase interaction. Proc Natl Acad Sci USA, 1999;96:11265-11270.
20. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science, 1998; 281:1305-1308.
21. Kaufman RJ. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Gene Dev,1999;13: 1211-1233.
22. Mori K. Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell,2000; 101:451-454.
23. Michalak M, Robert Parker JM, et al. Ca~(2+) signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium,2002;32:269-278.
24. Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic reticulum resident kinase. Nature,1999;397:271-274.
25. Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis,2002;7:335-345.
26. Liu J, Bhalgat M, Zhang C, et al. Fluorescent molecular probes V: a sensitive caspase-3 substrate for fluorometric assays. Bioorg Med Chem Lett,1999;9:3231 vascular biology. Circ Res,2004;95:343-353.
38. He T, Peterson TE, Holmuhamedov EL, et al. Human endothelial progenitor cells tolerate oxidative stress due to intrinsically high expression of manganese superoxide dismutase. Arterioscler Thromb Vasc Biol,2004;24:2021-2027.
39. Dernbach E, Urbich C, Brandes RP ,et al. Antioxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress. Blood,2004;104:3591-3597.
40. Hoetzer GL, Irmiger HM, Keith RS, et al. Endothelial nitric oxide synthase inhibition does not alter endothelial progenitor cell colony forming capacity or migratory activity. J Cardiovasc Pharmacol,2005;46:387-389.
41. Chen YH, Lin SJ, Lin FY, et al. High glucose impairs early and late endothelial progenitor cells by modifying nitric oxide-related but not oxidative stress -mediated mechanisms. Diabetes,2007;56:1559-1568.
42. Laybutt DR, Preston AM, Akerfeldt MC, et al. Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia, 2007;50:752 -763.
43. Donath MY, Ehses JA, Maedler K, et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes,2005;54 Suppl 2:S108-S113.
44. Lipson KL, Fonseca SG, Urano F. Endoplasmic reticulum stress-induced apoptosis and autoimmunity in diabetes. Curr Mol Med,2006;6:71-77.
45. Araki E, Oyadomari S, Mori M. Impact of endoplasmic reticulum stress pathway on pancreatic beta-cells and diabetes mellitus. Exp Biol Med,2003;228:1213 -1217.
1. Vinik A, Flemmer M. Diabetes and macrovascular disease. J Diabetes Complications,2002;16:235-245.
2. Coccheri S. Approaches to prevention of cardiovascular complications and events in diabetes mellitus. Drugs,2007;67:997-1026.
3. Karra R, Vemullapalli S, Dong C, et al. Molecular evidence for arterial repair in atherosclerosis. Proc Natl Acad Sci USA, 2005;102:16789-16794.
4. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: part Ⅰ. Circulation, 2003;108:1664-1672.
5. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part Ⅱ. Circulation, 2003;108:1772-1778.
6. Hristov M, Erl W, Weber PC. Endothelial progenitor cells mobilization, differentiation, and homing. Arterioscler Thromb Vasc Biol,2003;23:1185-1189.
7. Wojakowski W, Tendera M. Mobilization of bone marrow-derived progenitor cells in acute coronary syndromes. Folia Histochem Cytobiol,2005;43:229-232.
8. Shintani S, Murohara T, Ikeda H, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation,2001; 103:2776-2779.
9. George J, Goldstein E, Abashidze S, et al. Circulating endothelial progenitor cells in patients with unstable angina association with systemic inflammation. Eur Heart J, 2004;25:1003-1008.
10. Leone AM, Rutella S, Bonanno G, et al. Mobilization of bone marrow-derived stem cells after myocardial infarction and left ventricular function. Eur Heart J, 2005;26:1196-1204.
11. Thum T, Fraccarollo D, Galuppo P, et al. Bone marrow molecular alterations after myocardial infarction: Impact on endothelial progenitor cells. Cardiovasc Res, 2006; 70:50-60.
12.Loomans CJ,de Koning EJ,Staal FJ,et al.Endothelial progenitor cell dysfunction:a novel concept in the pathogenesis of vascular complications of type 1 diabetes.Diabetes,2004;53:195-199.
13.Krankel N,Adams V,Linke A,et al.Hyperglycemia reduces survival and impairs function of circulating blood-derived progenitor cells.Arterioscler Thromb Vasc Biol,2005;25:698-703.
14.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-e7.
15.Fallow GD,Singh J.The prevalence,type and severity of cardiovascular disease in diabetic and non-diabetic patients:Amatched-paired retrospective analysis using coronary angiography as the diagnostic toot.Mol Cell Biochem,2004;261:263-269.
16.中华医学会心血管病学分会,中华心血管病杂志编辑委员会,中国循环杂志编辑委员会.急性心肌梗死诊断和治疗指南.中华心血管病杂志,2001,29:710-725.
17.CASS Principal investingators and their associates:Coronary Artery Surgery Study(CASS):a randomized trial of coronary artery,bypasss Surgery.Circulation,1983;28:939-950.
18.St Goar FG,Pinto FJ,Alderman EL,et al.Intravascular ultrasound imaging of angiographically normal coronary arteries:an in vivo comparison with quantita -tive angiography.J Am Coll Cardiol.1991;18:952-958.
19.Fitzgerald PJ,St Goar FG,Connolly AJ,et al.Intravascular ultrasound imaging of coronary arteries.Is three layers the norm? Circulation,1992;86:154-158.
20.Mintz GS,Nissen SE,Anderson WD,et al.ACC Clinical Expert Consensus Document on Standards for the acquisition,measurement and reporting of intravascular ultrasound studies:a report of the American College of Cardiology Task Force on Clinical Expert Consensus Documents(Committee to Develop a Clinical Expert Consensus Document on Standards for Acquisition,Measurement and Reporting of Intravascular Ultrasound Studies[IVUS]).J Am Coll Cardiol, 2001 ;37:1478-1492.
21.Mintz GS, Maehara A, Bui AB, et al. Multiple versus single coronary plaque ruptures detected by intravascular ultrasound in stable and unstable angina pectoris and in acute myocardial infarction. Am J Cardiol,2003;91:1333-1335.
22. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelia angiogenesis. Science, 1997;275:964-967.
23. Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA,2000;97:3422-3427.
24. Ziegler D. Type 2 diabetes as an inflammatory cardiovascular disorder. Curr Mol Med,2005;5:309-322.
25. Grundy SM, Benjamin IJ, Burke GL, et al. Diabetes and cardiovascular disease a statement for healthcare professionals from the American Heart Association. Circulation, 1999; 100:1134-1146.
26. Clark CM, Clark PR. Type 2 diabetes and macrovascular disease. Epidemiology and etiology. Am Heart J, 1999; 138:5330-5333.
27. Amos AF, McCarty DJ, Zimmet P. The rising global burden of diabetes and its complications: Estimates and projections to the year 2010. Diabetes Med,1997; 14:S7-S15.
28. Vinik AI, Vinik E. Prevention of the complications of diabetes. Am J Manag Care,2003;9(3 suppl):S63-S80.
29. Abaci A, Oguzhan A, Kahraman S, et al. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation, 1999;99:2239-2242.
30. Lindvall B, Brorsson B, Herlitz J, et al. Comparison of diabetic and non-diabetic patients referred for coronary angiography. Int J Cardiol,1999;70:33-42.
31. Schaar JA, Muller JE, Falk E, et al. Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque, June 17 and 18, 2003, Santorini, Greece. Eur Heart J, 2004;25:1077-1082.
32. Schoenhagen P, Nissen S. Understanding coronary artery disease: tomographic imaging with intravascular ultrasound. Heart,2002;88:91-96.
33. Swallow RA, Court IA, Calver AL, et al. The limitations of coronary angiography: identification of a critical coronary stenosis using intravascular ultrasound. Int J Cardiol,2006;106:123-125.
34. Nissen SE, Yock P. Intravascular ultrasound: novel pathophysiological insights and current clinical applications. Circulation,2001; 103:604-616.
35. Gussenhoven EJ, Essed CE, Lancee CT, et al. Arterial wall characteristics determined by intravascular ultrasound imaging: an in vitro study. J Am Coll Cardiol, 1989;14:947-952.
36. Hodgson JM, Reddy KG, Suneja R, et al. Intracoronary ultrasound imaging: correlation of plaque morphology with angiography, clinical syndrome and procedural results in patients undergoing coronary angioplasty. J Am Coll Cardiol, 1993; 21:35-44.
37. Virmani R, Kolodgie FD, Burke AP, et al. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol,2000;20:1262-1275.
38. Libby P. Changing concepts of atherogenesis. J Intern Med,2000,247(3): 349-358.
39. Lusis AJ. Atherosclerosis. Nature,2000;407:233-241.
40. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 1993;362: 801-809.
41. Walter D, Rittig K, Bahlmann F, et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation,2002; 105:3017-3024.
42. Griese D, Ehsan A, Melo L, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy. Circulation,2003; 108:2710-2715.
43. Fujiyama S, Amano K, Uehira K, et al. Bone marrow monocyte lineage cells adhere on injured endothelium in a monocyte chemoattractant protein-1-dependent manner and accelerate reendothelialization as endothelial progenitor cells. Circ Res,2003;10:980-989.
44. Gotlieb AI. Atherosclerosis and acute coronary syndromes. Cardiovasc Pathol, 2005;14:181-184.
45. Zoll J, Fontaine V, Gourdy P, et al. Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition. Cardiovasc Res,2008;77: 471-480.
46. Dickson BC, Gotlieb AI. Towards understanding acute destabilization of vulnerable atherosclerotic plaques. Cardiovasc Pathol,2003; 12:237-248.
47. Virmani R, Burke AP, Farb A, et al. Pathology of the vulnerable plaque. J Am Coll Cardiol,2006;18;47(8 Suppl):C13-C18.
48. Shan PK. Mechanisms of plaque vulnerability and rupture. J Am Coll Cardiol, 2003;41(4 suppl S):15S-22S.
49. Zhao Y, Glesne D, Huberman E, et al. A human peripheral blood monocyte derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA,2003;100: 2426-2431.
50. Asahara T, Takahashi T, Masuda H, et al. VEGF contributes to postnatal neovascu-larization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J,1999;18:3964-3972.
51. Takahashi T, Kalka C, Masuda H, et al. Ischemiaand cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med,1999;5:434-438.
52. Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor165 gene transfer augments circulationendothelial progenitor cells in human subjects. CircRes,2000;86:1198-1202.
53. Moore MA, Hattori K, Heissig B, et al. Mobilization of endothelial and hemotopoietic stem and progenitor cells by adenovector-mediated elevation of serum level of SDF-1,VEGF, and angiopoientin-1. Ann NY Acad Sci,2001,938:36-45.
54. Urbich C, Derbach E, Zeiher AM, et al. Double-edged role of statins in angiogenesis signaling. Cir Res,2002;90:737-744.
55. Tepper OM, Galiano RD, Capla JM, et al. Human endothelial progenitor cells from type 2 diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation,2002;106:2781-2786.
56. Kunz GA, Liang G, Cuculi F, et al. Circulating endothelial progenitor cells predict coronary artery disease severity. Am Heart J,2006; 152:190-195.
57. Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med, 2005;353:999-1007.
58. 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.
59. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med, 2003;348:593-600.
60. Rosenzweig A. Circulating endothelial progenitors-cells as biomarkers. N Engl J Med,2005;353:1055-1057.
61. Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mono -nuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation,2001; 104:1046-1052.
62. Kawamoto A, Gwon HC, Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation,2001; 103:634-637.
63. Orlic D, Kajstura J, Chimenti S, et al. Transplanted adult bone marrow cells repair myocardial infarcts in mice. Ann NY Acad Sci,2001;938:221-229.
64. Hamano K, Nishida M, Hirata K, et al. Local implantation of autologous bone marrow cells for therapeutic angiogenesis in patients with ischemic heart disease: clinical trial and preliminary results. Jpn Circ J,2001;65:845-847.
65. Badorff C, Brands RP, Popp R, et al. Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation,2003; 107:1024-1032.
66. Szmiko PE, Fedak PW, Weisel RD, et al. Endothelial progenitor cells: new hope for a broken heart. Circulation,2003; 107:3093-3100.