单核细胞趋化蛋白1对人脐静脉平滑肌细胞增殖的影响

目的 研究单核细胞趋化蛋白1对人脐静脉平滑肌细胞增殖的影响。方法用不同浓度单核细胞趋化蛋1（0．1、1．0、10及100μg/L）作用于人脐静脉平滑肌细胞，采用细胞计数、MTT、流式细胞术检测细胞增殖情况；采用逆转录聚合酶链反应检测细胞中c-fos基因的表达。结果单核细胞趋化蛋白1能诱导人脐静脉平滑肌细胞的增殖，呈剂量依赖关系，100μg/L单核细胞趋化蛋白1对人脐静脉血管平滑肌细胞的增殖作用强于平滑肌细胞增殖因子血小板源生长因子的作用（P〈0．001），单核细胞趋化蛋白l在诱导血管平滑肌细胞增殖的同时引起c-fos基因的高表达（P〈0．001）。结论单核细胞趋化蛋白1不仅是趋化激活剂，而且是人脐静脉平滑肌细胞的促增殖因子，并且其促增殖的功能可能与增强c-fos基因的表达有关。     

普伐他汀对血管平滑肌细胞CX3CR1表达及其介导的趋化作用的影响

目的探讨普伐他汀对体外培养大鼠血管平滑肌细胞趋化因子受体CX3CR1表达及其介导的趋化作用的影响。方法分别以γ干扰素（IFN-γ）和氧化低密度脂蛋白（ox-LDL）作用于体外培养的大鼠血管平滑肌细胞,以免疫组化法观察CX3CR1表达。用普伐他汀进行干预,通过免疫组织化学法、ELISA法观察其是否会抑制IFN-γ诱导的CX3CR1表达。利用Transwell细胞趋化试验观察Fractalkine对体外培养平滑肌细胞的趋化作用,及普伐他汀对趋化作用的影响。结果 IFN-γ和ox-LDL均可诱导血管平滑肌细胞CX3CR1表达,与对照组比较有统计学差异。以普伐他汀对血管平滑肌细胞预作用一段时间后I,FN-γ诱导的CX3CR1表达受到显著抑制。Fractalkine对血管平滑肌细胞具有趋化作用,普伐他汀可显著抑制Fractalkine对血管平滑肌细胞的趋化作用。结论普伐他汀可抑制血管平滑肌细胞CX3CR1表达及其介导的趋化作用。     

趋化因子促进冠状动脉粥样硬化斑块不稳定的分子机制

目的 研究单核细胞趋化因子在动脉粥样硬化斑块不稳定中作用的分子机制。方法 对50例稳定性心绞痛(SAP)和50例不稳定性心绞痛(UAP)患者分别进行冠状动脉造影(CAG)和血管内超声(IVUS)检查。应用Transwell小室检测2组患者血单核细胞的趋化活性,ELISA法检测血清中高敏C反应蛋白(hs-CRP)、单核细胞趋化蛋白-1(MCP-1)、T细胞表达和分泌因子(RANTES)和Fractalkine的水平,用实时PCR检测单核细胞中MCP-1、RANTES和Fractalkine的mRNA表达水平。结果 IVUS共检出SAP组85处斑块,UAP组90处斑块,其中,UAP组主要为脂质性斑块48％ (43/90),而SAP组主要为纤维性斑块54％ (46/85),脂质斑块仅占16％( 14/85)。与SAP组比较,UAP组斑块负荷和血管重构指数明显大于前者（P均＜0.01）。UAP组单核细胞趋化活性明显增强,单核细胞移动数量明显多于SAP组,两组比较差异有统计学意义(P＜0.01)。UAP组血清中hs-CRP、MCP-1、RANTES和Fractalkine水平明显高于SAP组（P＜0.05或P＜0.01）。UAP组MCP-1、RANTES和Fractalkine的mRNA表达水平明显高于SAP组(P＜0.05)。结论 单核细胞趋化因子MCP-1、RANTES和Fractalkine可能促进了冠状动脉粥样硬化斑块的不稳定。     

γ-干扰素对大鼠血管平滑肌细胞增殖及增殖细胞核抗原表达的影响

为观察γ 干扰素对血管平滑肌细胞增殖的影响及探讨其作用机制 ,培养大鼠主动脉血管平滑肌细胞 ,以γ 干扰素 (5 0 0ku L)刺激血管平滑肌细胞 2 4h后 ,通过氚标记胸腺嘧啶脱氧核苷掺入法及Western杂交印迹法观察γ 干扰素作用 2 4h对大鼠血管平滑肌细胞DNA合成及增殖细胞核抗原表达的影响。结果发现γ 干扰素明显抑制 10 %胎牛血清刺激血管平滑肌细胞引起的增殖细胞核抗原表达增多及氚标记胸腺嘧啶脱氧核苷掺入增高 (P <0 .0 1) ,表明γ 干扰素具有较强的抑制大鼠血管平滑肌细胞增殖的作用。     

阿司匹林对血管平滑肌细胞增殖及增殖细胞核抗原表达的影响

目的探讨阿司匹林对大鼠血管平滑肌细胞增殖的影响及相关机制。方法体外培养大鼠主动脉平滑肌细胞,用不同浓度的阿司匹林处理,并且设置对照组,采用MTT法检测阿司匹林对血管平滑肌细胞增殖活性的影响,用免疫细胞化学和逆转录聚合酶链反应检测阿司匹林作用后血管平滑肌细胞中增殖细胞核抗原的表达。结果较高浓度(5×10-3mol/L)的阿司匹林对血管平滑肌细胞的增殖有明显抑制作用(P<0.05),相对抑制率为31.5%。与对照组相比,该浓度组血管平滑肌细胞中增殖细胞核抗原的蛋白和mRNA表达量均显著降低(P<0.05)。结论阿司匹林可能通过降低增殖细胞核抗原的表达来抑制血管平滑肌的增殖,对心血管系统具有抗血小板聚集之外的保护作用。     

玉葵清对糖基化终产物诱导的人肾系膜细胞趋化因子表达的影响

目的 探讨玉葵清对糖基化终产物（AGEs）诱导的人肾系膜细胞（HRMC）趋化因子表达及其趋化效应的影响. 方法 糖基化牛血清白蛋白（AGE-BSA）和玉葵清干预HRMC. 结果 AGE-BSA组较BSA组HRMC趋化单核细胞数增加,单核细胞趋化蛋白-1（MCP-1）、Fractalkine（FKN）中和抗体组HRMC趋化单核细胞数较AGE-BSA组减少;玉葵清组MCP-1、Fractalkine基因表达、上清中的蛋白含量和趋化单核细胞较BSA组降低（P＜0.05）. 结论 玉葵清减弱AGE-BSA诱导的HRMC趋化因子表达及其趋化效应,可能改善DN肾脏炎症反应.     

川芎嗪对血管壁细胞血管细胞粘附分子1和单核细胞趋化蛋白1表达的作用及可能机制

目的观察川芎嗪对血管内皮细胞和平滑肌细胞表达血管细胞粘附分子1、单核细胞趋化蛋白1和核因子κB的影响,探讨川芎嗪抗动脉粥样硬化分子机制。方法体外培养人脐静脉内皮细胞和大鼠胸主动脉平滑肌细胞。用氧化型低密度脂蛋白、氧化型极低密度脂蛋白、血管紧张素Ⅱ和(或)川芎嗪作用于细胞后,采用免疫细胞化学和原位分子杂交检测各组细胞血管细胞粘附分子1、单核细胞趋化蛋白1和核因子κB的表达情况。用单核细胞粘附试验检测川芎嗪对单核细胞粘附于内皮细胞的影响。结果氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导内皮细胞血管细胞粘附分子1蛋白相对表达量分别为0.552±0.008、0.460±0.006和0.486±0.025,明显高于对照组的0.365±0.019(P<0.01);诱导平滑肌细胞血管细胞粘附分子1蛋白相对表达量分别为0.564±0.007、0.513±0.021和0.524±0.008,明显高于对照组(0.416±0.013,P<0.01)。氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导内皮细胞单核细胞趋化蛋白1蛋白相对表达量分别为0.962±0.051、0.878±0.014和0.824±0.006,明显高于对照组的0.303±0.008(P<0.01);诱导平滑肌细胞单核细胞趋化蛋白1蛋白相对表达量分别为0.877±0.011、0.845±0.023和0.881±0.009,明显高于对照组的0.362±0.018(P<0.01)。氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导组血管细胞粘附分子1和单核细胞趋化蛋白1的mRNA表达明显高于对照组(P<0.01)。同时加入川芎嗪后血管细胞粘附分子1和单核细胞趋化蛋白1蛋白和mRNA表达明显低于相应诱导组(P<0.01)。氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导核因子κB在核内表达,同时加入川芎嗪后核因子κB表达于胞浆,核内阴性。与氧化型低密度脂蛋白或氧化型极低密度脂蛋白诱导组(2.047±0.011和1.936±0.014)比较,加入川芎嗪后,粘附于内皮细胞的单核细胞明显减少(1.282±0.020和1.265±0.016,P<0.01)。结论川芎嗪通过抑制或阻断动脉粥硬化危险因素氧化型低密度脂蛋白、氧化型极低密度脂蛋白和血管紧张素Ⅱ诱导的核因子κB活化及核内移位,抑制血管壁细胞血管细胞粘附分子1和单核细胞趋化蛋白1表达,抑制单核细胞粘附于内皮,而发挥其抗动脉粥样硬化作用。     

趋化素促进小鼠血管平滑肌细胞增殖的实验研究

目的:利用慢病毒构建趋化素基因缺陷性小鼠血管平滑肌细胞株,观察沉默趋化素基因后血管平滑肌细胞的增殖情况。方法:将正常血管平滑肌细胞、趋化素基因干扰对照血管平滑肌细胞株、趋化素基因缺陷性血管平滑肌细胞株分成正常组、增殖组、对照组和沉默组,增殖组、沉默组加入血小板源性生长因子-BB促进增殖,采用细胞计数和BrdU法检测血管平滑肌细胞增殖。结果:增殖组血管平滑肌细胞的细胞数目和BrdUOD值显著高于正常组(P0.05);与正常组相比,沉默组的血管平滑肌细胞数目和BrdUOD值显著降低(P0.05);与此同时,对照组与正常组之间的血管平滑肌细胞增殖情况无明显差异(P0.05)。结论:趋化素具有促进血管平滑肌细胞增殖的作用。     

缬沙坦和卡托普利对兔血管平滑肌细胞组织因子和组织因子途径抑制物表达及活性的影响

目的观察氧化修饰低密度脂蛋白（ox-LDL）、血管紧张素转换酶抑制剂和血管紧张素受体拮抗剂对血管平滑肌细胞组织因子（TF）和组织因子途径抑制物（TFPI）表达和活性的影响。方法采用组织贴块法培养兔主动脉平滑肌细胞,进行形态学和α-肌动蛋白（actin）免疫组化鉴定。在细胞水平,采用免疫组化、免疫荧光法检测ox-LDL和缬沙坦对TF、TFPI蛋白表达的影响,激光共聚焦法检测ox-LDL和缬沙坦对TF蛋白表达的影响,ELISA法检测ox-LDL、缬沙坦和卡托普利对TF、TFPI抗原表达的影响,发色底物法检测ox-LDL、缬沙坦和卡托普利对TF活性的影响,RT—PCR检测ox-LDL和缬沙坦对TF mRNA表达的影响。结果ox-LDL可增加兔血管平滑肌细胞TF抗原活性和mRNA的表达,在mRNA水平调节TF的表达。ox-LDL可降低兔血管平滑肌细胞TFPI抗原的表达。不同浓度的缬沙坦和卡托普利可明显减少ox-LDL刺激的平滑肌细胞TF抗原表达及活性,缬沙坦对TF表达的影响呈浓度依赖性,同时缬沙坦还可明显减少ox-LDL刺激的TF mRNA表达,证明缬沙坦在mRNA水平上调节TF的表达。不同浓度的缬沙坦和卡托普利可增加ox-LDL刺激的血管平滑肌细胞TFPI抗原的表达,缬沙坦对TFPI表达的影响呈浓度依赖性,该结果由ELISA法得出。结论本实验在细胞水平观察到ox-LDL对血管平滑肌细胞TF表达和活性有促进作用,血管紧张素转换酶抑制剂和血管紧张素受体拮抗剂对其有抑制作用,但这两种药对TFPI的表达有促进作用。从一个新的角度说明这两种药对动脉粥样硬化斑块特别是斑块促凝性的影响,它们可能是通过部分调节TF和TFPI的表达水平而发挥作用的。     

氯沙坦对血管平滑肌细胞内单核细胞趋化因子-1表达的影响

为了探讨血管紧张素Ⅱ受体拮抗剂氯沙坦对血管平滑肌细胞内单核细胞趋化蛋白 1表达的影响 ,以培养幼兔主动脉平滑肌细胞为研究对象 ,分别给予不同浓度血管紧张素Ⅱ和 或血管紧张素Ⅱ受体拮抗剂氯沙坦 ,采用免疫组织化学、原位杂交与酶联免疫吸附技术检测不同处理组平滑肌细胞内单核细胞趋化蛋白 1蛋白及其mR NA表达和平滑肌细胞培养介质中单核细胞趋化蛋白 1含量的变化。结果发现 ,10 - 6 ～ 10 - 1 0 mol L血管紧张素Ⅱ呈剂量依赖性地增加平滑肌细胞内单核细胞趋化蛋白 1蛋白及其mRNA表达水平 ,增高培养介质中单核细胞趋化蛋白 1蛋白含量 (P均 <0 .0 0 1)。 10 - 5 ～ 10 - 7mol L氯沙坦预处理使血管紧张素Ⅱ刺激的平滑肌细胞内单核细胞趋化蛋白 1蛋白及其mRNA表达水平及培养介质中单核细胞趋化蛋白 1蛋白含量明显减低 (P均 <0 .0 0 1)。以上结果提示 ,氯沙坦可拮抗血管紧张素Ⅱ所致的平滑肌细胞内单核细胞趋化蛋白 1的表达与分泌 ,这可能有助于减轻或防治某些病理状态下血管平滑肌细胞的增殖与迁移。     

经活化SAPK/JNK和ERK1/2激酶途径上调单核细胞趋化蛋白1表达在介导血管紧张素Ⅱ促进大鼠血管平滑肌细胞增殖中的作用

目的探讨血管紧张素Ⅱ(AngⅡ)诱导大鼠主动脉平滑肌细胞(VSMC)单核细胞趋化蛋白1(MCP-1)表达的分子机制及生物学功能。方法按指定时间,给予不同浓度AngⅡ处理原代培养大鼠VSMC,运用细胞增殖毒性检测试剂盒(CCK-8)研究AngⅡ对平滑肌细胞增殖的影响,并用MCP-1受体抑制剂(RS102895)和血管紧张素Ⅱ1型受体(AT1R)拮抗剂氯沙坦预处理细胞,观察MCP-1是否参与AngⅡ的促增殖作用;逆转录聚合酶链反应(RT-PCR)方法和酶联免疫吸附实验(ELISA)检测MCP-1mRNA和培养基中的MCP-1蛋白含量,并用氯沙坦干预,观察氯沙坦是否具有抗炎作用;免疫印迹法观察不同时间细胞内丝裂原活化蛋白激酶(MAPK)信号转导途径对MCP-1表达的影响。结果 AngⅡ(1μmol/L)作用48h明显促进平滑肌细胞增殖,MCP-1受体抑制剂(RS102895)能抵消该作用。在一定时间内,不同浓度的AngⅡ均上调MCP-1mRNA和蛋白表达水平(P<0.05或P<0.01),氯沙坦能抵消AngⅡ的上调作用(均P<0.05)。与AngⅡ(1μmol/L)单独孵育12h比较,AngⅡ(1μmol/L)+氯沙坦(10μmol/L)明显抑制SAPK/JNK(0.581±0.037比1.143±0.012)、ERK1/2激酶(0.062±0.020比0.342±0.098,均P<0.05)活化,差异均有统计学意义。此外,SAPK/JNK激酶抑制剂(SP600125)和ERK1/2激酶抑制剂(U0126)分别抑制SAPK/JNK和ERK1/2激酶活性并下调MCP-1表达(P<0.05或P<0.01)。结论 AngⅡ通过AT1R,活化SAPK/JNK和ERK1/2激酶途径上调大鼠VSMCMCP-1的表达,并且MCP-1在一定程度上介导AngⅡ的促平滑肌细胞增殖。     

Treatment with sulfasalazine or sulfapyridine, but not 5-aminosalicyclic acid, inhibits basic fibroblast growth factor-induced endothelial cell chemotaxis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression. METHODS: SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites. RESULTS: HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78. CONCLUSION: These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.     

大鼠胸主动脉球囊损伤后平滑肌细胞PCNA和P27表达的变化

目的探讨大鼠胸主动脉球囊损伤后增殖细胞核抗原(PCNA)、细胞周期蛋白依赖性激酶抑制蛋白P27表达变化规律。方法30只400～500g的雄性SD大鼠随机分为2组。手术组(n=24)行球囊损伤大鼠胸主动脉术;对照组(n=6)不行球囊损伤,作为正常对照。于术后2d、7d、14d、28d取胸主动脉应用HE染色、免疫组化和计算机图像分析法进行形态学、PCNA和P27表达水平检测。结果(1)正常动脉壁不表达PCNA,球囊损伤后开始表达,术后2d时中膜表达显著(24.08±2.35);术后7d出现新生内膜,PCNA在内膜高度表达(35.32±3.46)而中膜表达已明显下降(9.47±1.56);后内膜、中膜表达均逐渐下降,28d时形成显著的新生内膜(0.173±0.030)mm2,管腔狭窄;(2)正常动脉壁显著表达P27(19.29±1.54),球囊损伤后中膜表达迅速下降,2d达最低水平(2.93±0.55),后逐渐回升;14d、28d新生内膜中可见P27表达,并逐渐增多;(3)P27表达与PCNA表达呈显著负相关(r=0.868P<0.001)。结论球囊损伤大鼠主动脉后管壁P27表达下调伴随PCNA表达上调,血管平滑肌细胞恢复增殖能力并向内膜下迁移并过度增殖,从而形成显著的新生内膜、管腔狭窄。     

Inhibition of restenosis by tissue factor pathway inhibitor: in vivo and in vitro evidence for suppressed monocyte chemoattraction and reduced gelatinolytic activity

Activation of inflammatory and procoagulant mechanisms is thought to contribute significantly to the initiation of restenosis, a common complication after balloon angioplasty of obstructed arteries. During this process, expression of tissue factor (TF) represents one of the major physiologic triggers of coagulation that results in thrombus formation and the generation of additional signals leading to vascular smooth muscle cell (VSMC) proliferation and migration. In this study, we have investigated the mechanisms by which inhibition of coagulation at an early stage through overexpression of tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of TF, might reduce restenosis. In a rabbit femoral artery model, percutaneous delivery of TFPI using a recombinant adenoviral vector resulted in a significant reduction of the intimamedia ratio 21 days after injury. Investigating several markers of inflammation and coagulation, we found reduced neointimal expression of monocyte chemoattractant protein-1 (MCP-1), lesional monocyte infiltration, and expression of vascular TF, matrix metalloproteinase-2 (MMP-2), and MMP-9. Moreover, overexpression of TFPI suppressed the autocrine release of platelet-derived growth factor BB (PDGF-BB), MCP-1, and MMP-2 in response to factors VIIa and Xa from VSMCs in vitro and inhibited monocyte TF activity. These results suggest that TFPI exerts its action in vivo through not only thrombotic, but also nonthrombotic mechanisms.     

Tissue factor induction by aggregated LDL depends on LDL receptor-related protein expression (LRP1) and Rho A translocation in human vascular smooth muscle cells

OBJECTIVE: Low density lipoprotein (LDL) internalized in the vascular wall and modified by binding to extracellular matrix-proteoglycans (ECM) becomes aggregated (agLDL). AgLDL induces tissue factor (TF) expression and activity in human vascular smooth muscle cells (VSMC). TF expression in vascular cells promotes the prothrombotic transformation of the vascular wall. However, the mechanisms by which agLDL induces TF are not known. The aim of this study was to investigate the mechanisms involved in TF activation by extracellular matrix-modified LDL in human VSMC. METHODS AND RESULTS: AgLDL significantly induces TF expression (real time PCR and Western blot analysis) and procoagulant activity (factor Xa generation test) in human VSMC. HMG-CoA reductase inhibition completely prevents agLDL-induced TF expression and partially inhibits agLDL-TF activation. These effects are reverted by geranylgeranyl pyrophosphate (GGPP) but not by farnesyl pyrophosphate (FPP), suggesting the involvement of a geranylated protein in agLDL-TF induction. AgLDL increases Rho A translocation (2-fold) from the cytoplasm to the cell membrane in control but not in simvastatin-treated VSMC. Exoenzyme C3, a specific Rho A inhibitor, completely prevents agLDL-induced TF overexpression and partially agLDL-TF activation. Blocking LRP1, the receptor of agLDL, with anti-LRP1 antibodies or inhibiting LRP1 expression by small interference RNA treatment (siRNA-LRP1) impairs agLDL-induced TF overexpression and activation. CONCLUSIONS: These results demonstrate that TF induction by agLDL depends on LRP1 expression and requires Rho A translocation to the cellular membrane.     

纤维蛋白原诱导大鼠主动脉平滑肌细胞增殖及趋化

目的观察不同浓度纤维蛋白原对体外培养的血管平滑肌细胞增殖、趋化和随机迁移的影响,探讨其致动脉粥样硬化的可能作用及其机制。方法采用胶原酶消化法培养大鼠主动脉平滑肌细胞,用BrdU掺入法和细胞计数法观察其增殖能力,刮伤实验及慢速显微摄像技术检测其随机迁移能力,微孔滤膜法观察其趋化性,Western印迹法检测其明胶酶A的表达,明胶酶谱分析其分泌的明胶酶活性。结果细胞计数(r=0.860,P<0.05)和BrdU掺入实验(r=0.880,P<0.05)发现0.2gL～3.2gL纤维蛋白原呈剂量依赖性地促进血管平滑肌细胞增殖,其中0.4、0.8、1.6和3.2gL纤维蛋白原作用48h,细胞计数依次为(6.32±0.39)×107L、(10.63±0.25)×107L、(12.31±0.44)×107L和(13.59±0.43)×107L,BrdU掺入率依次为5.2%±2.2%、17.5%±2.0%、21.5%±2.3%和25.5%±2.5%,与对照组[分别为(5.63±0.34)×107L和2.0%±0.8%]比较差异均有显著性(P<0.01)。微孔滤膜法趋化实验发现0.2gL～3.2gL纤维蛋白原呈剂量依赖性地促进血管平滑肌细胞趋化(r=0.957,P<0.01)。刮伤实验中细胞迁移速度及慢速显微摄像单细胞随机迁移速度在纤维蛋白原组及对照组细胞均无明显差别(P>0.05)。Western印迹及明胶酶谱发现纤维蛋白原促使血管平滑肌细胞明胶酶A的表达及活性上调。结论纤维蛋白原可能通过促进血管平滑肌细胞增殖和趋化在动脉粥样硬化发生过程中起重要作用。     

Chemokine receptor-8 (CCR8) mediates human vascular smooth muscle cell chemotaxis and metalloproteinase-2 secretion

The response of the arterial vascular wall to injury is characterized by vascular smooth muscle cell (VSMC) migration, a process requiring metalloproteinase production. This migration is induced by cytokines, however the agonists involved are not fully defined. The CC chemokine receptor 8 (CCR8) is expressed on monocytes and T lymphocytes and is the sole receptor for the human CC chemokine 1 (CCL1, I-309) and for the viral chemokine, vCCL1 (viral macrophage inflammatory protein 1 [vMIP-1]). We have reported that CCR8 is expressed on human umbilical vein endothelial cells (HUVECs) and mediates chemotaxis induced by CCL1. The conditioned medium from incubation mixtures of lipoprotein(a) (Lp(a)) and HUVECs (LCM) contained CCL1 and stimulated both monocyte and HUVEC chemotaxis, providing novel mechanisms for the atherogenicity of Lp(a). We now report that CCL1, vCCL1, and LCM stimulate chemotaxis of human VSMCs that is blocked by murine monoclonal antibody against CCR8 and by the G-protein inhibitor pertussis toxin. The effect of anti-CCR8 was specific, as this antibody failed to effect the chemotaxis of VSMCs in response to CCL3 or by platelet-derived growth factor BB (PDGF-BB). VSMCs contained CCR8 mRNA and CCR8 antigen coprecipitated with VSMC membranes. Antibodies against metalloproteinase-2 (MMP-2) inhibited the CCL1-induced chemotaxis of VSMCs, whereas anti-MMP-9 was less effective. CCL1 induced VSMC pro-MMP-2 mRNA and protein secretion. Poxvirus MC148 inhibited the increase in MMP-2 induced by CCL1, documenting that CCR8 was the receptor responsible. In mouse femoral arteries, CCR8 and TCA3 antigen colocalized with VSMCs and were up-regulated after injury. The induction of CCR8 and CCL1/TCA3 under conditions associated with VSMC proliferation and migration raises the possibility that CCR8 may play an important role in vessel wall pathology.