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丹参冻干粉剂对非ST段抬高心肌梗死病人血浆PAI-1的影响

【摘要】  目的观察非ST段抬高心肌梗死(NSTEMI)病人血浆组织型纤溶酶原激活物抑制物1(PAI-1)变化和丹参冻干粉剂对其影响。方法将110例病人随机分为治疗组和对照组,两组均给予西医常规治疗,治疗组加用丹参冻干粉剂。两组分别于治疗前、治疗后14 d测定血浆PAI-1浓度,观察心绞痛、严重心律失常、心力衰竭的发生情况。结果治疗组和对照组治疗前PAI-1浓度接近,治疗后14 d治疗组PAI-1水平低于对照组(t=2.434,P<0.05),心绞痛、严重心律失常、心力衰竭的发生率均显著低于对照组(χ2=3.891~4.385,P<0.05)。结论丹参冻干粉剂治疗NSTEMI 的效果确切,并能降低心绞痛、严重心律失常、心力衰竭的发生率。

【关键词】  心肌梗死;丹参;纤溶酶原激活物抑制物1

 [ABSTRACT]ObjectiveTo observe the changes of plasminogen activator inhibitor-1(PAI-1) in patients with non-ST-segment-elevation myocardial infarction (MI) and evaluate the efficacy of Danshen freeze-dried powder injection on it.MethodsThe present study included 110 MI patients who were randomly divided into treatment group and control group. Both groups were given conventional Western medicine, while those in the treatment group were given extra Danshen freeze-dried powder injection. The plasma PAI-1 levels were detected before and after 14 days of therapy. The occurrence of angina, serious cardiac arrhythmia (SCA) and cardiac failure (CF) were observed.ResultsBefore treatment, the plasma PAI-1 levels were similar between the two groups; after 14 days of treatment, the levels of PAI-1 in the treatment group were lower than that in the control (t=2.434,P<0.05), and the incidence of angina, SCA and CF were also dramatically lower than that in the control (χ2=3.891-4.385,P<0.05).ConclusionThe effectiveness of Danshen freeze-dried powder injection for patients with non-ST-segment-elevation MI is certain, the medicine can reduce the occurrence of angina, SCA and CF.

  [KEY WORDS]myocardial infarction; salvia miltiorrhiza; plasminogen activator inhibitor 1

  丹参是我国传统医学中常用药物之一,有悠久的临床应用历史。近几十年来, 采用现代医学方法,结合化学、分子生物学和细胞生物学等多学科的技术手段, 对丹参进行了较系统的研究, 取得了大量新的认识和实验结果,并开发出一系列疗效良好的临床应用药物。本文在西医常规治疗非ST段抬高心肌梗死(NSTEMI)的基础上,加用丹参冻干粉剂,以观察病人治疗前后血浆组织型纤溶酶原激活物抑制物1(PAI-1)变化及其心绞痛、严重心律失常、心力衰竭的发生情况,现报告如下。

  1资料与方法

  1.1一般资料

  2008年10月—2009年5月,选择本院收治的NSTEMI病人110例,诊断参照文献[1]有关标准。将病人随机分为2组:治疗组58例,男33例,女25例,年龄42~76岁,平均58.64岁;对照组52例,男29例,女23例,年龄40~75岁,平均58.52岁。2组病人的性别、年龄、病情等差异均无显著性,均排除并发严重心律失常、心力衰竭、糖尿病、出血性疾病、高血压、脑血管病和出血倾向者,既往无心肌梗死史。

  1.2治疗方法

  所有病人入院后均收入CCU,按急性心肌梗死一般常规护理,包括绝对卧床,持续心电图、血压和呼吸监护,密切观察心律、心率、血压和心功能的变化,持续吸氧,阿司匹林、氯吡格雷抗凝,保持大便通畅等,并给予硝酸异山梨酯注射液(商品名异舒吉,珠海许瓦兹制药有限公司生产)加50 g/L葡萄糖注射液250 mL 以20 mg/d静滴,开始剂量4 μg/min,每4~5 min增加10~20 μg/min,一般2~10 mg/h。根据个体的血压、心率和其他血流动力学参数来调整用量。治疗组同时给予丹参冻干粉针(哈药集团中药二厂生产,批准文号Z10970093,产品批号080526)800 mg加50 g/L葡萄糖注射液250 mL静脉滴注,每日1次。2组均以14 d为1个疗程。

  1.3观察指标

  所有病人在入院后治疗前检测外周血常规、大便常规、尿常规、生化全套、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血时间(TT)、胸部X线片、心电图、心肌酶学及心脏彩超等。定期查心肌酶谱,每日行常规18导联心电图检查。于治疗前、治疗后14 d测定血浆PAI-1浓度,观察病人心绞痛、严重心律失常(心室颤动、多形性室速、Ⅱ度或Ⅲ度房室传导阻滞伴有血流动力学功能障碍、室上性快速性心律失常)、心力衰竭(LVEF≤40%)的发生情况。用发色底物法测定血浆PAI-1浓度,试剂盒由上海亚都生物科技有限公司提供,操作按说明书进行。

  1.4统计学处理

  应用SSPS 17.0软件进行统计学处理,组间比较采用χ2检验和t检验。

  2结果

  治疗组和对照组病人治疗前的PAI-1浓度分别为(13.718±2.027)、(13.703±1.654)mg/L,两组比较差异无显著性(P>0.05);治疗14 d后治疗组和对照组病人PAI-1浓度分别为(11.505±1.634)、(12.313±1.879)mg/L,治疗组PAI-1水平显著低于对照组(t=2.434,P<0.05)。治疗14 d后治疗组病人心绞痛、严重心律失常、心力衰竭的发生率均显著低于对照组(χ2=3.891~4.385,P<0.05)。见表1。表1两组治疗14 d后严重心律失常、心绞痛、心力衰竭的发生率比较

  3讨论

  凝血与纤溶系统的动态平衡对维持生理性纤维蛋白溶解和预防病理性血栓形成起着重要的作用,组织型纤溶酶原激活物t-PA是纤溶系统的主要物质,能使纤溶酶原转化为纤溶酶而降解纤维蛋白凝块,使纤溶活性增加。而组织型纤溶酶原的抑制物PAI是t-PA的抑制物,能促使血栓形成,对抑制纤溶有重要作用。近年来的研究表明,血浆 PAI-1活性变化所致纤溶活性下降在冠心病发生、发展及急性缺血事件发生过程中起重要作用[2]。另有研究表明,纤溶活性降低在急性冠状动脉综合征(ACS)的发病中具有重要作用,与再发性缺血事件密切相关,并且具有独立预测价值[3]。

  丹参冻干粉剂含有丹参酮、丹参素、琥珀酸、维生素E、黄芩苷、胡萝卜苷等化学成分,可扩张冠状动脉,增加冠状动脉血流量,改善缺血心肌的血循环,减轻缺血心肌的损伤程度,加速心肌缺血和损伤的恢复。同时,本品具有钙通道阻滞剂样作用,可以抑制心肌细胞复极时钙离子的缓慢内流,干扰电机械收缩耦联,抑制血管平滑肌收缩,使主动脉阻抗及左心室后负荷降低,从而减轻心脏负荷,降低心肌耗氧量。本品还有抗血小板聚集,促进纤溶,降低血液黏滞度,提高低氧耐受力,抗脂质过氧化,清除自由基等作用[4]。

  丹参具有明显抑制PAI-1的活性和使t-PA的活性增强的作用,并具有调节t-PA/PAI-1的比例而维持血管内皮细胞的抗凝作用。使用丹参粉针治疗冠心病心肌梗死能明显改善病人的全血黏度、红细胞聚集指数,改善血液流变性和心肌微循环,从而改善心肌的缺血低氧状况,增强心肌对低氧的耐受力,因而能解除胸闷、心悸和心绞痛等症状及改善心电图的缺血波形。同时, 硝酸异山梨酯注射液可扩张冠状动脉,降低阻力,增加冠状动脉的血流量,通过扩张周围血管,减少静脉回心血量,降低心室容量、心腔内压,减轻心脏前后负荷,降低心肌的需氧量。

  本研究结果表明,注射用丹参冻干粉剂可调节NSTEMI病人纤溶系统的平衡,降低心绞痛、严重心律失常的发生率,改善心功能。

【参考文献】
   \[1\]陆再英,钟南山,谢毅,等. 内科学\[M\]. 7版.北京:人民卫生出版社, 2008:286-291.

  \[2\]韩勃,李红云,张社华,等. 冠心病病人血浆PAI-1活性的变化\[J\]. 青岛大学医学院学报, 2000,36(2):93-94.

  \[3\]史卫国,王志辉,王修卫,等. 急性冠状动脉综合征病人血清CRP与纤溶活性变化\[J\]. 齐鲁医学杂志, 2008,23(2):128-130.

  \[4\]董振行,关淑敏,孙仁俊,等. 丹参防治动脉粥样硬化的机制\[J\]. 心肺血管病杂志, 1993,12(2):121-123.

  

日期:2011年6月30日 - 来自[2010年第25卷第6期]栏目
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PAI-1基因多态性与脓毒血症的研究进展

【关键词】  PAI-1 腕毒血症

  血浆纤溶酶原激活物抑制剂-1(Plasminogen activator inhibitor-1,PAI-1)是纤溶系统的主要调节因子,与纤溶酶原激活物结合后,迅速失活而发挥抗纤溶作用。故可减少纤维蛋白降解,引起纤维蛋白聚集,保持人体正常血液中纤溶系统与凝血系统的动态平衡。

  1  PAI-1的生物学特点和影响因素

  PAI-1属于丝氨酸蛋白酶抑制剂超家族,是一种单链糖蛋白,分子量52000, 成熟的蛋白含379个氨基酸,其中含有一个由23个氨基酸残基组成的信号肽,反应中心在346位的Arg和347位的Met残基处,Arg-Met肽键被称为“诱饵肽键”。PAI-1特异的抑制纤溶酶原激活物(PA),调节纤溶活性。组织型PA (tPA)或尿激酶型PA (uPA)与PAI-1形成1:1复合物,它们攻击PAI-1的Arg346-Met347肽键,此时tPA或uPA被脂酰化而失活[1,2]。PAI主要包括3种类型: PAI-1、PAI-2、PAI-3。PAI-1存在于人类所有组织,内皮细胞、血小板、单核细胞等均可表达PAI-1。PAI-1是纤溶系统的主要抑制物。PAI-2主要存在于孕妇血浆及非孕妇的单核细胞中, PAI-3主要存在于尿中,二者在血浆中浓度及活性很低,故PAI:A主要反应PAI-1的活性功能变化[3]。PAI-1主要由血管内皮细胞产生,再进入细胞间质和血液循环。初释放时有活性,为活化型,很快衰减为无活性的潜在型。循环中的PAI-1与一种黏附蛋白-玻璃连接蛋白(Vitronectin,VN)结合,后者可稳定并保持其活性。PAI-1的生理功能主要为:①在结缔组织演变、凝血、纤溶、补体激活和炎症反应等过程中具有抑制蛋白降解作用。②保护基底膜不被血浆来源的酶所降解。③保护细胞间的接触面而维持组织结构的完整性。④通过对tPA和uPA的特异性抑制作用,影响细胞的多种生理功能,如胶原酶的激活、组织修复、神经生长等。⑤在细胞周期中,PAI-1转录水平的变化及其在细胞内的积聚,对细胞形态的维持、细胞与其间质的粘附、细胞增殖、信号转导及基因表达等都有重要意义[1]。体内的PAI-1主要被活化蛋白C或凝血酶中和而破坏。过多的PAI-1导致血浆中纤溶酶原(PA)活性下降,血浆纤溶活性降低,而易发生血栓。影响PAI-1的因素很多,如细胞因子(IL-1,TNF-α,INF-α,IFN-γ)、生长因子(TGF-β,PDGF,IGF-1)、激素(胰岛素、胰岛素原、糖皮质激素)、脂蛋白、血管紧张素Ⅱ、细菌内毒素和脂多糖等均可增加内皮细胞PAI-1的合成和分泌。这些因素多直接作用于PAI-1基因上的DNA调控顺序(如启动子区的糖皮质区激素反应元件),刺激基因转录,使mRNA水平增加或稳定性增强,从而通过增加PAI-1基因表达使PAI-1活性升高。

  2  脓毒血症

  脓毒血症(Sepsis),是指微生物入侵机体感染后所致的全身性炎症反应综合征(SIRS),严重全身性感染(Severe sepsis)及多器官功能障碍综合征(MODS)是脓毒血症渐进性恶化的后果。PAI-1在脓毒血症的发生和发展过程中起着关键性的作用[4]。脓毒血症(Sepsis)是当今危重病医学所面临的棘手难题,美国疾病控制中心的统计调查结果显示:在美国,每年大约有20万人死于疾病及其后续症,已成为老年人十大死因之一。在我国,随着经济发展和科技进步,尤其是先进医疗技术的采用(如有创监测)及免疫抑制剂使用、抗生素的误用、人口老龄化等,多发伤、严重创伤、外科大手术后sepsis、严重感染、感染性休克及多器官功能障碍综合征(MODS)的患病率及死亡率与国外报道一致。虽然,国内外对sepsis的基础研究和临床研究十分重视,但迄今其死亡率仍高达30%~60%。本文就PAI-1与Sepsis的研究进展作一介绍。

  3  PAI-1的基因结构和多态性

  PAI-1基因定位于7号染色体长臂2区1. 3-2带(7q21.3-22),由15867bp组成,全长12.3kb,包含9个外显子和8个内含子。其mRNA有2.4kb和3.2kb两种。PAI-1基因主要有以下几种多态性:(1)HindⅢ限制性片段长度多态性(RFLP),即不同个体PAI-1DNA经HindⅢ酶切后得到不同长度的片段,有3种基因型1/1、1/2、2/2;(2)启动子区-675bp处4G/5G多态性,即含4个鸟甘酸(G)的一段序列中插入或缺失第5个G,位于启动子区域,转录起始点上游-675bp处,有3种基因型4G/4G、5G/4G、5G/5G;(3)第四内含子(CA)n二核甘酸重复序列多态性,即一个位点上有8个等位基因,其间差异仅在双核甘酸(CA)的重复数不同;(4)启动子区转录起始点上游-172bp~-153bp处(CA)n二核甘酸重复序列多态性;(5)启动子区-844bp处G→A突变,与-675bp处4G/5G多态性明显的连锁不平衡,该突变位点位于Ets核蛋白结合部位,而Ets蛋白参与生长调控、细胞分化、T细胞激活和器官发育等许多生物过程中基因表达的调节;(6)第7内含子+9785bp处G→A突变,与3′端非编码区+11319bp~+11345bpCGCGCCCCC插入/缺失多态性有明显锁不平衡,与+11053bp处T→G突变无连锁不平衡;(7)3′端+12078bp处A→G突变,与A-844G和-675 4G/5G基因型有显著的连锁不平衡。

  4  PAI-1基因多态性与脓毒血症

  PAI-1和组织型纤溶酶原激活物(t-PA)相互作用,构成纤溶系统的动态平衡,PAI-1活性过强或浓度过高会引起血管内凝血、微血栓形成,反之则引起凝血障碍。而PAI-1基因多态性则可影响血浆PAI-1浓度[5]。PAI-1基因中与脓毒血症有关的多态性出现在启动子区域-675位点G的缺失(4G)或插入(5G),前者称为PAI-14G,后者称为PAI-15G。近年研究证实,PAI-1基因4G/5G多态性与转录水平有关。PAI-1基因5G位点既可以结合一个转录因子,也可以结合一个抑制因子,而4G位点只结合转录因子,且4G/4G纯合子型者的细胞有较高的PAI-1分泌功能和血浆较高的PAI-1水平。升高的PAI-1主要通过抑制局部纤溶酶的产生而导致纤溶活性下降。目前研究发现PAI-1基因启动子区675bp处单个核苷酸的缺失/插入(4G/5G)的多态性与血浆PAI-1活性有密切关系,由此推断PAI-1基因4G/5G的多态性可能与脓毒血症的发生有关。PAI-1基因多态性与冠心病[3,6]、心肌梗死[7]、非胰岛素依赖性糖尿病[2]、周围动脉闭塞性疾病[8]等的关系已有报道,但和脓毒血症的关系则报道甚少。感染脑膜炎双球菌的病人有些发展为脓毒血症休克,而有些则只发展到菌血症或脑膜炎。Westendorp等[9]通过研究PAI-1基因多态性来分析其中是否有遗传上的差异。为此他们检测了50例感染脑膜炎双球菌的病人、另183名感染此菌的病人第一直系亲属和131名正常对照组PAI-1的4G、5G基因型分布和等位基因频率,结果发现,病人和对照组基因型分布和等位基因频率基本相同,但发现脓毒血症休克病人其基因型为4G/4G的频率(44%)是只发展到脑膜炎的病人该基因型频率(14%)的3.14倍。通过分析计算,他们认为携带4G/4G基因型的感染病人发展成脓毒血症休克的风险概率是携带其他基因型病人的5倍,而第一直系亲属携带该基因型的感染病人发展成脓毒血症休克的风险概率是第一直系亲属携带其他基因型的感染病人的6倍。Menges等[10]的研究结果表明了PAI-1基因多态性与严重创伤病人脓毒血症易感性的关系。他们发现84%的基因型为4G/4G的严重创伤病人并发脓毒血症,而基因型为4G/5G和5G/5G的严重创伤病人并发脓毒血症的比例分别是38%和15%。其结果还表明,PAI-14G等位基因和血浆中高PAI-1浓度有关,而且基因型为4G/4G的严重创伤病人其死亡率(58%)显著高于基因型为4G/5G(28%)和5G/5G(15%)的严重创伤病人。总之,PAI-1基因多态性是脓毒血症的危险因素。深入了解PAI-1基因的结构和功能,改善各种危险因素,对脓毒血症的防治意义重大。

【参考文献】
    [1] 焦秀敏, 刘宽芝. PAI-1与糖尿病肾病[J].国外医学内分泌学分册, 2002, 22(5): 338~340.

  [2] 楚新梅,何秉贤. 纤溶酶原激活物抑制剂-1基因多态性与代谢综合征[J]. 中华心血管病杂志, 2004, 32(2):184~186.

  [3] 翟艳苓, 付 研, 王旭东,等. 冠心病发病与血浆纤溶酶原激活物抑制剂-1及其基因4G/5G多态性的关系[J]. 临床荟萃, 2003, 18(5):246~248.

  [4] American college of chest physicians/society of critical care medicine consensus conference: definitions for sesis and organ failure and guidelines for the use of innovative therapies in sepsis[J]. Crit Care Med, 1992, 20:864.

  [5] 张道杰, 蒋建新. 基因多态性与脓毒症的关系研究进展[J]. 国外医学外科学分册, 2002, 29(3):129~132.

  [6] Margaglione M, Cappucci G, Colaizzo D, et al. The PAI-1 Gene Locus 4G/5G Polymorphism Is Associated With a Family History of Coronary Artery Disease[J]. Arteriosclerosis, Thrombosis, and Vascular Biology, 1998, 18:152~156.

  [7] 富路, 孔一慧, 李佳,等. 纤溶酶原激活剂抑制物-1基因启动子区4G/5G多态性与血栓性疾病的相关性研究[J]. 中华心血管病杂志, 2001, 29(3): 144~147.

  [8] 康兰,李小鹰,李金生,等. 血浆纤溶酶原激活物抑制剂-1基因启动子区4G/5G多态性与周围动脉闭塞性疾病关系的研究[J]. 中华老年心脑血管病杂志, 2003,5(2): 92~95.

  [9] Westendorp RG, Hottenga JJ, Slagboom PE. Variation in plasminogen activtor inhibitor-1 gene and risk of meningococcal septic shock[J]. Lancet, 1999, 354(9178):561~563.

  [10] Menges T, Hermans PW, Little SG, et al. Plasminogen activator inhibitor-1 4G/5G promoter polymorphism and prognosis of severely injured patients[J]. Lancet, 2001, 357(9262):1096~1097.


作者单位:台州学院医学院, 浙江 台州 318000.

日期:2010年1月13日 - 来自[2007年第7卷第8期]栏目

PAI-1在糖尿病肾病患者中的变化

【摘要】    目的 探讨2型糖尿病(T2DM)患者血浆纤溶酶原激活物抑制物(PAI-1)和组织型纤溶酶原激活物(t-PA)活性水平的变化及其与糖尿病肾病(DN)的关系。 方法 选择133例T2DM患者,根据尿蛋白排泄率(UAER)分为无DN组67例、DN微量白蛋白尿组51例、DN临床蛋白尿组15例,正常对照组27例。测定其血浆t-PA、PAI-1的活性水平。 结果 T2DM各组患者血浆PAI-1水平均显著高于正常对照组,且随着UAER的增高而递增(P<0.05, P<0.01 );DN患者血浆PAI-1活性与UAER 、胰岛素抵抗指数(HOMA-IR)、空腹胰岛素(FINS)、胆固醇(TG)、低密度脂蛋白(LDL)、甘油三酯(TC)、体重指数(BMI)呈显著正相关;t-PA活性在各组间差异无显著性。 结论 DN患者血浆PAI-1活性增高;胰岛素抵抗、高TG、高LDL、高TC血症及肥胖等因素可能与PAI-1活性增高有关。

【关键词】  组织型纤溶酶原激活物 纤溶酶原激活物抑制物 糖尿病肾病

  The variation of levels of PAI-1 in patients with diabetic nepropathy.

  XIE Fang-qiu, LI Wei, GUAN Pin.

  (Hainan Provincial People’s Hospital, Haikou 570311, Hainan, P. R. China)
   
  Abstract:Objective  To invetigate the variations of plasminogen activator inhibitor-1(PAI-1) and tissue-type plsminogen activator(t-PA) in patients with diabetic nephropathy.  Methods  The Plasmic levels of PAI-1 and t-PA in diabetic patients with or without diabetic nephropathy and normal controls were tested by ELISA.  Results  The concentrations of of PAI-1 in diabetic patients were higher than that in normal controls, and that in diabetic patients with diabetic nephropathy wer higher than that of diabetic patients without diabetic nephropathy (P<0.05, P<0.01).  However, there no significant differences were observed in the levels of t-PA among these groups.  Conclusion  The procoagulant activity is associated with diabetic nephropathy in type-2 diabetes mellitus.
   
  Key words:Plasminogen activator inhibitor-1;Tissue-type plsminogen activator;Diabetic nephropathy

  糖尿病肾病(Diabetic nephropathy, DN)是糖尿病的常见并发症,也是糖尿病死亡的主要原因之一。DN肾脏损害主要表现为微血管病变。有研究表明,凝血和纤溶活性的异常在微血管病变中具有重要的作用[1]。本文旨在探讨组织型纤溶酶原激活物(Tissue-type plsminogen activator, t-PA)和其抑制物(Plasminogen activator inhibitor-1, PAI-1)在DN患者中的变化及其影响因素。

  1  资料和方法

  1.1  一般资料  133例2型糖尿病(T2DM)病人均为我院住院患者,均符合1999年WHO糖尿病诊断标准。按尿蛋白排泄率(UAER)将T2DM患者分为3组:无DN组67例,UAER<30mg/24h,年龄34~77岁;DN微量白蛋白尿组51例,UAER30~300mg/24h,年龄42~77岁;DN临床蛋白尿组15例,UAER>300mg/24h,年龄46~80岁。正常对照组27例,均为健康体检者,年龄34~74岁,无高血压及明显心、脑、肝、肾疾病。所有研究对象近期均无感染、外伤及出血性疾病,亦未使用过溶栓、抗凝药物。

  1.2  方法  受试者空腹12h取静脉血,检测血浆t-PA、PAI-1、空腹血糖(FBG)、空腹胰岛素(FINS)、糖化血红蛋白(HbA1c)、胆固醇(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、甘油三酯(TC)浓度。t-PA、PAI-1采用发色底物法,试剂由上海太阳生物技术公司提供 ;FBG采用葡萄糖酶氧化法;FINS采用放免法;HbA1c采用免疫凝集抑制法;TG、HDL、 LDL 、TC采用酶比色法。取受试者24h尿液测UAER,采用免疫法,试剂由中国原子能科学研究院提供;胰岛素抵抗指数(HOMA-IR)=FBG×FINS/22.5,体重指数(BMI)=体重(kg)/身高2(m2)。

  1.3  统计学分析  数据处理采用SPSS10.0软件,所有数据采用x±s表示;各组间均数比较用方差分析,各组间差异采用q检验,PAI-1与有关因素的相关性用Person直线相关分析。P<0.05为差异有显著性。

  2  结果

  2.1  各组间t-PA、PAI-1比较  对照组、无DN组、DN微量白蛋白尿组、DN临床蛋白尿组血浆PAI-1水平呈递增性增高,组间有显著性差异; 而t-PA水平在各组间比较无显著性差异。见表1。

  表1  各组t-PA、PAI-1比较(略)

  注:与对照组比较, P<0.01;与糖尿病无DN组比较,△P<0.01,△△P<0.05;与DN微量白蛋白尿组比较,▲P<0.05。

  2.2  PAI-1与有关因素的相关性分析  UAER、HOMA-IR、FINS、TG、BMI分别与PAI-1呈正相关,FBG、HbA1c与PAI-1水平无相关。见表2。

  表2  PAI-1与有关因素的相关性分析(略)

  3  讨论
   
  t-PA、PAI-1是调节纤溶系统生理功能的一对重要因子[2]。t-PA、PAI-1由内皮细胞合成和释放 ,t-PA为纤溶系统活化始动因子,PAI-1能快速作用于t-PA,与之形成复合物,使之灭活[3]。PAI-1作用增强或t-PA作用降低,或两者均存在时,局部纤溶减弱,降低了排除血管内纤维蛋白的功能,易造成血管狭窄闭塞,使血流灌注不足,血管壁出现小的缺血域,导致继发性血栓形成,引起并加重血管内皮损伤。
   
  DN的主要病理学特征是肾小球基底膜(GBM)和肾小管基底膜(TBM)的增厚,肾小球系膜区细胞外基质(ECM)进行性积聚及小管间质纤维化,从而导致肾小球硬化,并出现蛋白尿、肾衰竭等[4]。已知ECM积聚是肾小球硬化的病理基础,纤溶系统在ECM的降解中起重要作用。实验证实[5],通过基因转染技术使PAI-1基因在肾脏中定位表达,结果显示,随PAI-1表达水平增加,局部出现ECM过度积聚,在肾小球纤维化区域也可检测出PAI-1表达增高;PAI-1表达水平增加使t-PA活性下降,基质降解障碍,ECM进行性积聚;同时ECM积聚可刺激成纤维细胞侵入增加,产生大量纤维蛋白原,使纤维蛋白基质更加难以分解。
   
  本组资料结果显示,T2DM患者血浆PAI-1水平明显高于正常对照组,随着UAER 升高, PAI-1逐渐升高,提示糖尿病早期存在血管内皮细胞功能受损,平滑肌细胞增殖并向内膜移行,以及血小板被激活, PAI-1合成、释放增加;过量PAI-1与t-PA结合使其灭活,致纤溶活性减低,促进血栓形成;肾病时伴有的微血管内皮结构和功能受损有可导致PAI-1升高,形成恶性循环。有文献报道[6],某些血栓性疾病的发生并非由于t-PA含量减少,而可能是由于PAI-1活性增强所致。本组资料亦未看到DN患者t-PA水平降低。以上表明PAI-1是肾脏损害的重要物质。
   
  影响PAI-1的因素很多,本组资料相关分析显示T2DM患者PAI-1水平与HOMA-IR、FINS、TG、LDL-C、TC、BMI呈显著正相关,而与FBG、HbA1c、HDL-C无相关性。文献报道[7],在IR状态下,胰岛素及胰岛素原升高使内皮细胞、肝细胞合成PAI-1增多;肥胖患者内脏脂肪堆积,脂肪组织产生PAI-1增多;动物实验[8]证实,高TG可诱导内皮细胞PAI-1mRNA的表达。这表明PAI-1可能是胰岛素抵抗(IR)、高TG、高LDL、高TC、肥胖和DN相互联系的纽带,PAI-1活性升高可能是IR与DN之间的桥梁,PAI-1是一个心血管病变的独立危险因素。
   
  DN起病隐匿,综上所述,血浆PAI-1水平是早期诊断DN较敏感指标,并可监测DN的发生、发展。T2DM患者在控制代谢紊乱的同时,及早监测、纠正纤溶功能异常,积极降低血浆PAI-1活性对预防DN的发生及延缓其进展有着重要的意义。

【参考文献】
    [1] 陶少平. 糖尿病肾病早期诊治进展[J]. 实用全科医学,2003,1(1):61~65.

  [2] Galajda P,Martinda E,Mokan M,et al. Endothelial markers in diabetes Mellitus[J].Thromb Res,1997,85:63~65.

  [3] 陈高翔,屈燧林. 纤溶酶原激活物抑制剂-1与肾脏疾病[J]. 肾脏病与透析肾移植杂志,1999,8(2):155~158.

  [4] 苏进,田浩明.纤维连接蛋白与糖尿病肾病[J].医学综述,2001,7(11):654~655.

  [5] 秦蓉,张农,陈广平,等.肝素对大鼠系膜细胞增殖和PAI-1表达的影响[J].复旦学报·医学版,2002,29:247~250.

  [6] Estelles A,Tormo G,Aznar J,et al.Reduced fibrinolytic octivity in cornary heart disease in dasal condition and ajter exercise[J]. Thomb Res, 1985,40:373~378.

  [7] 张悦,苏胜偶.纤溶活性与2型糖尿病及其大血管病变[J]. 国外医学内分泌学分册,2005,25:42~43.

  [8] 陈良华,龚兰生.降低胆固醇在冠心病防治中的作用[J].国外医学内科分册,1997,24:291.


作者单位:海南省人民医院医疗康复中心,海南 海口 570311.

日期:2010年1月13日 - 来自[2007年第7卷第7期]栏目
循环ads

Neutralization of Plasminogen Activator Inhibitor I (PAI-1) by the Synthetic Antagonist PAI-749 via a Dual Mechanism of Action

【关键词】  Neutralization

    PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC50 values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent Kd (254 nM) was similar to the IC50 (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.

    Plasminogen activator inhibitor 1 (PAI-1) is a rapidly acting inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (Dellas and Loskutoff, 2005). PAI-1 is a member of the serpin class of serine protease inhibitors that characteristically produce SDS-stable complexes with their cognate protease targets (Silverman et al., 2001). Formation of the acyl-enzyme adduct between PAI-1 and the protease involves initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational changes that trap the protease in a covalent complex (Olson et al., 2001). Two other conformation states of PAI-1 are known. First, the acyl-enzyme adduct between PAI-1 and tPA (or uPA) can be hydrolyzed to form cleaved (inactive) PAI-1 and regenerate active plasminogen activator (PA) (Declerck et al., 1992). Second, active PAI-1 can undergo a spontaneous large conformation change that gives rise to an inactive (latent) state of the inhibitor (Levin and Santell, 1987; Mottonen et al., 1992).

    PAI-1 plays a pivotal role in a myriad of physiological processes that involve activation of plasminogen (Dellas and Loskutoff, 2005). High levels of PAI-1 activity are associated with a broad spectrum of pathophysiological states, including thrombosis, cancer, inflammation, neurodegenerative diseases, and possibly metabolic diseases (Dellas and Loskutoff, 2005). Thus, neutralization of PAI-1 has been championed as a promising strategy to intervene in a number of diverse disease states involving suppressed extracellular proteolysis.

    Small-molecule synthetic antagonists of PAI-1 have been described previously (Charlton et al., 1996; Friederich et al., 1997; Neve et al., 1999; Gils et al., 2002; Einholm et al., 2003; Elokdah et al., 2004; Liang et al., 2005; Gorlatova et al., 2007). The metastable conformation of PAI-1 along with the intricacies of the pathway by which PAI-1 inhibits tPA affords numerous potential opportunities whereby a PAI-1 inhibitor could block PAI-1 activity. In this report, we probe the mechanism of action of a novel synthetic PAI-1 antagonist, PAI-749 (Fig. 1, inset; H. Elokdah, G. R. McFarlane, S. C. Mayer, J. A. Krueger, J. Hennan, S. J. Gardell, D. L. Crandall, J. A. Butera, R. Magolda, G. P. Vlasuk, et al., manuscript in preparation) using purified components. Our investigation reveals that PAI-749 can neutralize PAI-1 activity via a dual mechanism of action: 1) direct inhibitory impact on PAI-1 activity and 2) rendering PAI-1 vulnerable to plasmin-mediated degradation.

    Fig. 1. PAI-749 preserved plasminogen activator activity in the presence of PAI-1. PAI-1 was treated with the indicated amount of PAI-749 for 5 min and then mixed with tPA or uPA. Ten min later, plasminogen activator activity was assayed with the respective amidolytic substrate for tPA or uPA. Residual tPA (or uPA) activity (%) is shown. , tPA + PAI-1; , uPA + PAI-1. Each data point depicts the mean (S.E.M.), n = 4. The inset shows the structure of PAI-749.

    Materials.PAI-749,1-benzyl-3-pentyl-2-[6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-1H-indole; compound B, 1-benzyl-2-[5-methyl-6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-3-pentyl-1H-indole; compound C, 1-benzyl-2-[5-chloro-6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-3-pentyl-1H-indole; compound D, 1-benzyl-2-[5-bromo-6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-3-pentyl-1H-indole; compound E, 2-[5-chloro-6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-1-methyl-3-pentyl-1H-indole; compound F, 1-methyl-2-[5-methyl-6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-3-pentyl-1H-indole; compound G, 2-[5-bromo-6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-3-pentyl-1-[2-(trifluoromethoxy)benzyl]-1H-indole; compound H, 1-(4-tert-butylbenzyl)-3-pentyl-2-[6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-1H-indole were synthesized as described elsewhere (H. Elokdah, G. R. McFarlane, S. C. Mayer, J. A. Krueger, J. Hennan, S. J. Gardell, D. L. Crandall, J. A. Butera, R. Magolda, G. P. Vlasuk, et al., manuscript in preparation). Compounds were prepared as 1 mM stocks in dimethylsulfoxide (DMSO) and diluted into aqueous buffer (maintaining a final 1% DMSO concentration in all assays). Human PAI-1 (both active and latent), human PAI-1 variant (Ser119 to Cys) tagged with N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD), Lys-plasmin, Glu-plasminogen, two-chain tPA (2c-tPA), high molecular weight uPA, human 2-antiplasmin, monomeric vitronectin, and human antithrombin III were from Molecular Innovations (Southfield, MI). Human recombinant single-chain tPA (tPA) (Activase) was produced by Genentech (South San Francisco, CA). Spectrozyme tPA, Spectrozyme uPA, Spectrozyme FXa, and plasminogen activator inhibitor type 2 (PAI-2) were from American Diagnostica (Greenwich, CT). HEPES-free acid, aprotinin, p-aminobenzamidine (PAB), D-Val-Leu-Lys-pNA (plasmin substrate) and SAR-Pro-Arg-pNA (trypsin substrate) were from Sigma-Aldrich (St. Louis, MO). PEG-8000 was from U.S. Biochemical Corp. (Cleveland, OH). Dimethyldecyl-phosphine oxide (apo-10) was from Calbiochem (San Diego, CA). Human 1-antitrypsin and human pancreatic trypsin were from Athens Research and Technology (Athens, GA). Human factor Xa was from Enzyme Research Laboratories (South Bend, IN). Fondaparinux sodium (Arixtra) was manufactured by GlaxoSmithKline (Uxbridge, Middlesex, UK). Novex precast gels (4–12% Bis-Tris gels), SilverXpress Staining kit, 4 x NuPAGE LDS sample prep buffer, Native PAGE Novex Bis-Tris gel system (4–16% Bis-Tris gels), and SeeBlue Plus2 molecular weight standard were from Invitrogen (Carlsbad, CA). For molar concentration determinations, the following relative molecular weights were used: Mr of 43,000 for PAI-1, Mrof 66,000 for tPA, and Mr of 83,000 for plasmin.

    Assay of Functional PAI-1 Activity. PAI-1 (12 or 24 nM) was mixed with varying amounts of PAI-749 (or DMSO control) for 5 min at room temperature (final volume = 480 µl) in 0.1 M HEPES, 0.1 M NaCl, pH 7.4, 1 mM EDTA, 0.1% PEG-8000, 2 mM apo-10 (HNEPA buffer). The nonionic detergent apo-10 was included in the assay buffer to stabilize PA activity. Apo-10 at 2 mM (which is below the critical micelle concentration of 4.6 mM) does not interfere with PAI-1 activity or the ability of PAI-749 to neutralize PAI-1 (in contrast to nonionic detergents such as Tween 80 and Triton X-100; data not shown). Twenty-microliter aliquots of tPA, 2c-tPA, or uPA (final concentration, 10 or 20 nM) was added, and the samples were placed at room temperature for 10 min. Seventy-five microliters of each sample (in triplicate) was transferred to individual wells of a 96-well microtiter dish containing 25 µl of Spectrozyme tPA (final concentration, 500 µM) or Spectrozyme uPA (final concentration, 250 µM). Reactions were continuously monitored at A405 in kinetic mode using a SPECTRAmax 340PC plate spectrophotometer (Molecular Devices). The IC50 values for PAI-749 were obtained using nonlinear regression (sigmoidal) curve fit analysis (Prism software; GraphPad Software Inc., San Diego, CA).

    Serpin Selectivity Assays. Selectivity assays for other serpins were performed essentially according to the PAI-1 and tPA assay protocol (described above) with the following assay-specific conditions. PAI-2 selectivity assay: PAI-2, 25 nM; 2c-tPA, 10 nM; Spectrozyme tPA, 500 µM. 2-antiplasmin selectivity assay: 2-antiplasmin, 7.5 nM; plasmin, 5 nM; D-Val-Leu-Lys-pNA, 200 µM. 1-antitrypsin selectivity assay: 1-antitrypsin, 50 nM; trypsin, 2.5 nM; SAR-Pro-Arg-pNA, 50 µM; HNEPA buffer was spiked with 10 mM CaCl2. Antithrombin III selectivity assay: antithrombin III, 10 nM; low-molecular-weight heparin (fondaparinux), 10 nM, factor Xa, 1 nM; Spectrozyme FXa, 100 µM.

    SDS-PAGE Analysis of PAI-1 and tPA Reaction Products. Samples (generated as described above) were mixed with 1/3 volume of 4 x NuPAGE LDS sample buffer without reducing agents, heated to 70°C for 10 min, and fractionated by SDS-PAGE using NuPAGE (4–12%) Novex Bis-Tris gels. Proteins were visualized with the SilverXpress Staining Kit according to the manufacturer's instructions. Gels were analyzed with a flatbed scanner at 600 dpi, and the resultant images were analyzed using Quantity One software (Bio-Rad Laboratories, Hercules, CA). The intensity of all bands was below pixel intensity saturation plateau. Local background correction for each band was calculated based on the average intensity of pixels immediately surrounding the defined area of analysis. Global background correction for each band was calculated based on the average pixel intensity of a defined section across the entire gel judged to free of protein staining. No significant difference in EC50 was apparent using either method of background correction.

    Blue Native PAGE Analysis of PAI-1. PAI-1 (20 nM) was mixed with DMSO control (1% final) or PAI-749 for 60 min at room temperature. Samples (60 µl) were mixed with 1/3 volume of 4 x native gel sample buffer or 4 x LDS sample buffer. The former were subjected to BN-PAGE performed essentially as described previously (Schägger et al., 1991) using commercially available reagents. The latter were fractionated by SDS-PAGE as described above. Proteins were visualized by silver staining.

    Fig. 2. Impact of PAI-749 on the Fl signal of PAI-NBD119, the Fl-tagged PAI-1 variant with NBD at the amino acid 119 position. A, spectral scans (excitation, 480 nm; emission, 500–600 nm) of PAI-NBD119 in the absence and presence of increasing concentrations of PAI-749. The PAI-749 concentrations (nanomolar) and associated color-coded scans are shown. B, determination of the apparent Kd for PAI-749 binding to PAI-NBD119. The Fl emission signal at 525 nm was used to derive this value. C, time-dependent effects of PAI-749 on the Fl signal of PAI-NBD119. 1, PAI-NBD119 (50 nM); 2, PAI-NBD119 (50 nM) + PAI-749 (1 µM). Samples were mixed, and the Fl signal was immediately monitored for 300 s (excitation, 480 nm; emission, 525 nm).

    Binding of Compounds to PAI-1 Using NBD-Labeled PAI-1 Variants. Fluorescence (Fl) measurements with NBD-labeled PAI-1 variants were performed using a LS50B fluorimeter (PerkinElmer Life and Analytical Sciences, Waltham, MA). Excitation was at 480 nm; emission was at 500 to 600 nm (scan mode) or 525 nm (kinetic mode). Slit widths were 10 nm for both the excitation and emission settings. Experiments were performed in HNEPA buffer at room temperature. Experiments depicted in Figs. 2A, 3, and 4 involved exposure of PAI-NBD119 or latent PAI-NBD119 (50 nM) to DMSO vehicle (1% final) or various concentrations of PAI-749 (or PAI-749 analogs) for 5 min before assessment of Fl spectra. The latent PAI-NBD119 was prepared by incubation of active PAI-NBD119 at 37°C in the dark for 16 h. "Kinetic mode" experiments were also performed to probe the inhibitory mechanism of PAI-749. In the experiments shown in Figure 2C, PAI-NBD119 (50 nM) was mixed with vehicle or PAI-749 (1 µM) and assayed immediately (over a 5-min interval).

    Fig. 3. Scatter plot of IC50 values (neutralization of PAI-1 activity toward tPA) versus apparent Kd values (perturbation of the Fl signal of PAI-NBD119) for PAI-749 and its closely related structural analogs. Compounds (PAI-749, B–H) are depicted as filled labeled circles. Linear regression analysis of all data points is shown (r2 = 0.65). The structures of the compounds are identified in Table 1.

    Fig. 4. PAI-749 does not induce formation of the latent form of PAI-1. Active PAI-NBD119 or latent PAI-NBD119 (50 nM) was mixed with DMSO control or PAI-749 (1 µM). After 5 min, the samples were scanned for the Fl signal (excitation, 480 nm; emission, 500–600 nm). The identities of the samples are indicated in the side legend.

    PAB Displacement Experiments to Assess Occupancy of the PA Active Site by PAI-1. PAI-1 (1 µM) or PAI-1 pretreated (for 2 min) with 2 µM compound B (closely related analog of PAI-749 with lesser Fl signal interference; see Table 1 for its identity) was mixed with 0.5 µM uPA (or 2c-tPA) that was pretreated with 200 µM PAB. After 5 min, samples (final volume, 600 µl) were scanned using the fluorometer (excitation at 325 nm; emission scan from 340–400 nm). The emission signal at 355 nm was used to calculate active site occupancy of uPA (or 2c-tPA) by PAB.

    TABLE 1 Assays of PAI-749 and its closely-related analogs The basic template structure for the PAI-749 chemical series with the 2 variable positions (R1 and R2) is depicted. The identity of the substituents in the R1 and R2 positions for PAI-749 and compounds B to H are shown. For each compound, the IC50 value using unmodified human PAI-1 (and tPA) and the apparent Kd value using PAI-NBD119 are shown.

    Assaying for Residual Inhibition of PAI-1 after Transient Exposure to PAI-749. Human PAI-1 (20 nM) was mixed with PAI-749 (1 µM) or DMSO in HNEP buffer. The final DMSO concentration in all samples was 1%. Samples were prepared in duplicate. After 5 or 60 min at room temperature, 0.1% Tween 80 was added to one set of samples (which sequesters PAI-749 in ensuing detergent micelles). tPA (20 nM) was added to the appropriate samples. After 10 min, 75 µl of each sample was mixed with 25 µl of Spectrozyme tPA (500 µM), and residual tPA activity was monitored at A405 nm for 10 min using a SPECTRAmax 340PC plate spectrophotometer.

    Assaying for the Impact of Vitronectin on the Ability of PAI-749 to Inactivate PAI-1. Human PAI-1 (24 nM) was mixed with PAI-749 (2 µM), vitronectin (VN) (250 nM), or BSA (250 nM) in HNEPA buffer. The final DMSO concentration in all samples was 1%. Samples were prepared in duplicate. After 5 min at room temperature, VN, BSA, or PAI-749 was added to certain samples as shown in Table 4. After 5 min, tPA (20 nM) was added to all samples. After 10 min, 75 µl of each sample was mixed with 25 µl of Spectrozyme tPA (500 µM), and residual tPA activity was monitored at A405 nm for 10 min using a SPECTRAmax 340PC plate spectrophotometer.

    TABLE 4 Impact of VN on PAI-749 mediated inactivation of PAI-1 Stage I: HNEPA buffer alone or containing human PAI-1 (24 nM), VN (250 nM), BSA (250 nM), and/or PAI-749 (1 µM) was placed at room temperature for 5 min. Stage II: VN, BSA, or PAI-749 was added as indicated; samples were placed at room temperature for 5 min. Stage III: tPA (20 nM) was added to all samples. After 10 min at room temperature, all samples were mixed with Spectrozyme tPA and residual tPA activity (mOD/min) was assayed. Each value shows the mean values (SD); n = 3.

    Neutralization of PAI-1 by Plasmin. Mixtures of active or latent PAI-1 (100 nM) and PAI-749 (1 µM) (or DMSO control) were equilibrated for 5 min at room temperature. Reactions were initiated by the addition of plasmin (10 nM) (with and without aprotinin), incubated at 25°C for 30 min, quenched with 4 x LDS sample prep buffer, and heated at 70°C for 10 min. Samples were fractionated by SDS-PAGE (4–12% Bis-Tris NOVEX precast gels) and visualized by silver staining as described above.

    PAI-749 Preserved tPA and uPA Activity in the Presence of PAI-1. PAI-749 (Fig. 1, inset) dose-dependently blocked PAI-1-mediated inactivation of tPA activity toward its low-molecular-weight amidolytic substrate, Spectrozyme tPA (Fig. 1). The IC50 value of PAI-749 for preservation of tPA activity was 157 ± 9 nM. Likewise, PAI-749 dose dependently prevented PAI-1 mediated inactivation of uPA activity toward its low-molecular-weight substrate, Spectrozyme uPA (Fig. 1). The IC50 value of PAI-749 for blocking PAI-1 mediated inhibition of uPA was 87 ± 3 nM. The ability of PAI-749 to preserve tPA and uPA activities toward Glu-plasminogen in the presence of PAI-1 was also demonstrated (data not shown).

    PAI-749 Displayed Selectivity for PAI-1 Compared with Other Serpin Class Inhibitors. The selectivity of PAI-749 for PAI-1 was evaluated against a panel of other serpins and their target proteases. Pretreatment of PAI-2 (25 nM) with vehicle control or PAI-749 (5 µM) and subsequent addition to tPA (10 nM) yielded 62 and 48% inhibition of tPA activity, respectively. Pretreatment of 2-antiplasmin (7.5 nM) with vehicle or PAI-749 (5 µM) and subsequent addition to plasmin (5 nM) caused 88 and 58% inhibition of plasmin activity, respectively. Pretreatment of antithrombin III (10 nM) with vehicle or PAI-749 (5 µM) and subsequent addition to factor Xa (1 nM) in the presence of fondaparinux (10 nM) produced 72 and 46% inhibition of Factor Xa activity, respectively. Finally, pretreatment of 1-antitrypsin (50 nM) with vehicle or PAI-749 (5 µM) and subsequent addition to trypsin (2.5 nM) caused 54 and 52% inhibition of trypsin activity, respectively. Hence, only slight neutralization of antithrombin III, 2-antiplasmin, and PAI-2 was observed in the presence of 5 µM PAI-749, whereas 1-antitrypsin was fully refractory to the effects of PAI-749 (at 5 µM). Control experiments established that PAI-749 (5 µM) did not directly affect the activity of the proteases used for these selectivity assays. Potential solubility problems at higher concentrations of PAI-749 in assay buffer precluded a more rigorous evaluation of the potency of the compound toward these other serpins. Nevertheless, these data clearly show that PAI-749 displayed marked selectivity for PAI-1 relative to other serpins.

    Direct, Rapid, and Reversible Inhibition of PAI-1 by PAI-749. Treatment of a PAI-1 variant tagged with the NBD fluorophore at position 119 (PAI-NBD119) with PAI-749 for 5 min caused concentration-dependent quenching of the Fl signal (Fig. 2A). An apparent Kd value of 254 nM was deduced from the Fl signal perturbation at 525 nm (Fig. 2B). This value is less than 2-fold different from the IC50 (142 nM) of the PAI-NBD119 variant for tPA (data not shown). The change in the Fl spectrum was no different when the treatment interval was extended to 1 h (data not shown). Moreover, similar results were obtained with a different Fl-tagged PAI-1 species (PAI-NBDP1') in which the NBD moiety was present at the P1' position of the reactive loop region of PAI-1 (data not shown).

    PAI-NBD119 was used to assess the rapidity of PAI-749 binding to PAI-1 (Fig. 2C). The Fl signal of PAI-NBD119 was stable over the course of the 5-min observation period (progress curve 1). Addition of PAI-749 to PAI-NBD119 produced a rapid decline in the Fl signal (progress curve 2). The bulk of the Fl change occurred within the brief time interval (15 s) between reagent addition, sample mixing, and cuvette placement. Progress curve 2 best fit to a single exponential curve fit. The limiting Fl quench signal was asymptotically reached by 5 min.

    We aimed to firmly establish that PAI-749 binding deduced from the compound-triggered Fl change of PAI-NBD119 was linked to inactivation of PAI-1. Although the close similarity between the IC50 and apparent Kd values of PAI-749 for PAI-NBD119 is consistent with this notion, we evaluated the IC50 and apparent Kd values of several closely related compounds in the PAI-749 chemical series to more fully assess whether these two estimates for binding affinity reflect the same binding event (Table 1). All compounds differed from PAI-749 at the R1/R2 positions depicted in the structural template. Remarkably, the same rank order of potency was observed for this set of compounds with respect to both the IC50 and apparent Kd determinations (Fig. 3). The apparent Kd values are consistently higher than the corresponding IC50 values of all tested compounds for reasons that have yet to be defined but are likely to be inherent to the different assay methods. Nevertheless, these data establish that the binding events that perturb the Fl signal and neutralize PAI-1 activity are tightly linked.

    A novel experimental strategy was used to test whether inhibition of PAI-1 by PAI-749 was sustained after limited exposure to the compound. This approach exploited the fortuitous finding that PAI-749 was sequestered by Tween 80 detergent micelles (data not shown). PAI-1 was treated with PAI-749 for either 5 or 60 min; subsequently, the samples were exposed to buffer without or with 0.1% Tween 80 for the remainder of the experimental protocol. Tween 80 itself did not affect the ability of PAI-1 to inhibit tPA (Table 2; compare samples 1 and 2, 4 and 5, 7 and 8, and 10 and 11). The 2-fold greater tPA activity in the presence of Tween 80 reflected the ability of the nonionic detergent to stabilize tPA activity (perhaps by minimizing nonspecific absorption of tPA to the multiwell assay plate). In the absence of Tween 80, pretreatment of PAI-1 with PAI-749 (for 5 min) and subsequent addition of tPA (sample 3) is associated with marked preservation of tPA activity (i.e., indicative of PAI-1 neutralization). However, pretreatment of PAI-1 with PAI-749 for 5 min (in the absence of Tween 80), followed by exposure to Tween 80 and subsequent addition to tPA (sample 6), yielded virtually complete suppression of tPA activity (i.e., failure of PAI-749 to inhibit PAI-1). Hence, limited exposure of PAI-1 to PAI-749 is insufficient to elicit a sustained inhibitory effect. It is noteworthy that when PAI-1 was pretreated with PAI-749 for 60 min, the subsequent addition of Tween 80 was largely ineffective at reversing the inhibitory effect of PAI-749 (Table 2; compare samples 7 and 9, 10 and 12). Data shown below may provide the explanation why Tween 80-mediated reversibility is affected by the exposure time between PAI-1 and PAI-749.

    TABLE 2 Assaying for residual PAI-1 inhibition after limited exposure to PAI-749 Stage I: HNEP buffer alone (samples 1, 4, 7, 10) or HNEP buffer containing 20 nM human PAI-1 (all other samples) was mixed with 1 µM PAI-749 (samples 3, 6, 9, 12) or DMSO vehicle control (all other samples) in HNEP buffer for 5 (samples 1–6) or 60 min (samples 7–12). Stage II: 0.1% Tween-80 was added to samples 4 to 6 and 10 to 12 (which sequesters/neutralizes PAI-749 in detergent micelles). Stage III: tPA (20 nM) was added to all samples. After 10 min at room temperature, all samples were mixed with Spectrozyme tPA and residual tPA activity (mOD/min) was assayed. Each value shows the mean values (sd); n = 3.

    Elucidating the Mechanism of Action of PAI-749. The latent form of PAI-NBD119 was produced by incubation of active PAI-NBD119 at 37°C for 16 h. A time-dependent change of the Fl signal and concomitant loss of PAI-1 inhibitory activity was observed (data not shown). Analysis of the samples by reverse fibrin zymography (which reactivates latent PAI-1) showed minimal loss of PAI-1 inhibitory activity (data not shown). These observations established that the latent form of PAI-NBD119 was generated.

    The Fl signal displayed by latent PAI-NBD119 in the presence of PAI-749 was markedly different from that of active PAI-NBD119 treated with PAI-749 (Fig. 4, gray dashed scan versus black dashed scan, respectively). Moreover, the Fl signal of latent PAI-NBD119 was only slightly perturbed by PAI-749 (compare gray solid and gray dashed scans) in contrast to that of active PAI-NBD119 (compare black solid and black dashed scans). These results showed that PAI-749 did not induce formation of the latent form of PAI-1 (a conclusion corroborated by additional findings described below). Moreover, the very slight perturbation of the Fl signal of latent PAI-1 in the presence of PAI-749 suggests that the affinity of PAI-749 for latent PAI-1 is low. However, we cannot exclude the less likely possibility that PAI-749 bound to latent PAI-NBD119 but failed to perturb the Fl signal of the NBD-tag (unlike with active PAI-NBD119).

    The canonical product of the reaction between PAI-1 and tPA is an SDS-stable complex. PAI-749 caused concentration-dependent inhibition of the formation of the SDS-stable tPA/PAI-1 complex (Fig. 5A). The decrease in the abundance of the tPA/PAI-1 complex with increasing amounts of PAI-749 was accompanied by concomitant increases in both the tPA and (uncleaved) PAI-1 bands. Densitometry analysis revealed that the EC50 for blockade of the SDS-stable tPA/PAI-1 complex was 190 nM (Fig. 5B), a value that is very similar to the IC50 deduced from the activity assay described above. PAI-749 also interfered with formation of the SDS-stable complex between uPA and PAI-1; the concentration dependence of the PAI-749 effect matched the impact on preservation of uPA activity (data not shown).

    Fig. 5. Impact of PAI-749 on the fate of PAI-1 when added to tPA as assessed by SDS-PAGE. A, PAI-1 (24 nM) was preincubated with PAI-749 for 5 min before the addition of tPA (20 nM). After 10 min, the reactions were quenched by the addition of SDS-containing sample prep buffer, fractionated by SDS-PAGE, and proteins were visualized by silver staining. The PAI-749 concentrations in each sample were 0, 43, 54, 67, 84, 105, 131, 164, 205, 256, 320, 400, and 500 nM (lanes 1–13, respectively). A mock reaction containing 24 nM PAI-1 without PAI-749 or tPA is shown in lane 14. B, densitometry analysis of the formation of covalent complex between tPA and PAI-1 as a function of PAI-749 concentration. For each concentration of PAI-749, pixel intensity of the band corresponding to the serpin-protease complex was normalized as percentage total complex observed in absence of PAI-749 (see Materials and Methods).

    The effect of PAI-749 on the production of the cleaved form of PAI-1 was very slight if at all (Fig. 5A). A subtle biphasic concentration dependence of PAI-749 on the generation of the cleaved form of PAI-1 might be evident. At intermediate concentrations of PAI-749, there was a modestly enhanced formation of the cleaved species; however, with steadily increasing amounts of PAI-749, the generation of the cleaved species was abolished. In any event, PA-mediated cleavage of PAI-1 in the presence of PAI-749 does not seem to be a major contributor to neutralization of PAI-1 by PAI-749.

    The marked ability of PAI-749 to block formation of the SDS-stable complex between PAI-1 and tPA could very likely reflect interference with an upstream event in the reaction pathway governing the multistep interaction between tPA and PAI-1 (Olson et al., 2001). To shed light on the actual step affected by PAI-749, we examined the effect of a PAI-749 analog on the ability of PAI-1 to displace p-aminobenzamidine (PAB) from the PA active site. For this study, we used compound B (a closely related analog of PAI-749; see Table 1 for its identity) instead of PAI-749 because the former posed less interference with the Fl signal of PAB (data not shown). In addition, uPA was used because it produced a more robust signal than tPA or 2c-tPA; however, similar conclusions were drawn from studies with 2c-tPA (data not shown). Binding of PAB to the uPA active site increased the intrinsic Fl signal of PAB (Table 3, sample 1; value depicts signal augmentation normalized to PAB control). As expected, addition of PAB to PAI-1 had no effect on the PAB Fl signal (sample 2). Treatment of the uPA/PAB complex with PAI-1 (sample 3) reduced the PAB Fl signal to yield a value that was indistinguishable from "no uPA" (sample 2). This result agreed with published data showing that PAI-1 displaced PAB from the active site of 2c-tPA (Olson et al., 2001). Compound B had little impact on the enhanced Fl signal of PAB when added to uPA (sample 4). Likewise, compound B had little effect on the minor Fl signal of PAB in the presence of PAI-1 (sample 5). It is noteworthy that pretreatment of PAI-1 with compound B negated the ability of PAI-1 to suppress the augmented Fl signal of PAB in the presence of uPA (sample 6). This result revealed that compound B (and by extrapolation, PAI-749, its closely related analog) blocked the ability of PAI-1 to occupy the primary specificity pocket of uPA. Binding of the P1 residue in PAI-1 to the primary specificity pocket of the PA is proposed to occur concomitantly with formation of the putative Michaelis-like complex (Ibarra et al., 2004). Consequently, this experiment reveals that the effect of PAI-749 on PAI-1 occurs at the earliest step of the postulated pathway describing the interaction between PAI-1 and the PA (i.e., PAI-749 seems to block formation of the reversible Michaelis-like complex between PAI-1 and the PA).

    TABLE 3 Compound B-treated PAI-1 does not displace PAB from uPA active site PAB added to buffer or buffer containing compound B served as the background measurements for samples 1 to 3 and 4 to 6, respectively. Pretreatments at room temperature (5 min) are shown by grouping of reagents within parentheses. All other additions were placed at room temperature for 5 min before analysis of the Fl signal (excitation, 325 nm; emission, 355 nm). Each value shows mean (S.E.M.), n = 3.

    It was reported previously that negatively charged organochemical inactivators of PAI-1 convert PAI-1 to inactive polymers (Pedersen et al., 2003). We thus employed (nondenaturing) BN-PAGE (Schägger et al., 1991) to test whether PAI-749 similarly elicited PAI-1 polymerization. PAI-1 was mixed with increasing concentrations of PAI-749 for 60 min and was then subjected to both SDS-denaturing PAGE and BN-PAGE. Treatment of PAI-1 with PAI-749 had no effect on the mobility of PAI-1 as assessed by SDS-PAGE (Fig. 6A). In the absence of PAI-749 treatment, PAI-1 exhibited a tendency toward polymerization when analyzed by BN-PAGE (Fig. 6B, lane 1). With increasing concentrations of PAI-749, there was a dramatic shift in the mobility of PAI-1 to higher molecular species when analyzed by BN-PAGE. The virtual disappearance of PAI-1 in the presence of elevated PAI-749 as assessed by BN-PAGE reflects diffuse migration of the heterogeneous PAI-1 polymer. This assertion is amply supported by cross-reference to Fig. 6A showing no diminution of signal when the same samples where analyzed by SDS-PAGE. The concentration dependence of the PAI-749 effect on PAI-1 mobility during BN-PAGE (Fig. 6B) was strikingly similar to that of neutralization of PAI-1 activity (Fig. 1) and perturbation of the Fl signal of PAI-NBD119 (Fig. 2B). Hence, all of these effects of PAI-749 on PAI-1 seem to be linked. It is noteworthy that the mobility of latent PAI-1 during BN-PAGE was unaltered by pretreatment with PAI-749 (data not shown).

    Fig. 6. PAI-749 promoted PAI-1 polymerization as assessed by BN-PAGE. PAI-1 (20 nM final) was mixed with the indicated concentrations of PAI-749 in HNEPA buffer. After 60 min at room temperature, samples (from the same experiment) were mixed with LDS sample buffer and fractionated by SDS-PAGE (A) or mixed with native gel sample buffer and fractionated by BN-PAGE (B). Samples were detected by protein silver staining.

    When the treatment interval between PAI-1 and PAI-749 was only 5 min (actual exposure time is approximate because there was no quench step before BN-PAGE), PAI-1 polymerization was evident but was less extensive (data not shown). The apparent dependence of PAI-1 polymerization on exposure time to PAI-749 (5 versus 60 min) is reminiscent of the aforementioned impact of exposure time between PAI-1 and PAI-749 on the ability of Tween 80 to "reverse" inactivation of PAI-1 activity (Table 2).

    Impact of Vitronectin on the Ability of PAI-749 to Neutralize PAI-1. VN was shown to bind tightly to PAI-1 (Declerck et al., 1988; Wiman et al., 1988). Indeed, PAI-1 exists in plasma largely as a complex with VN. We thus examined the possible impact of VN on the ability of PAI-749 to inhibit PAI-1 activity (Table 4). The addition of VN or BSA (control) to tPA did not alter its activity toward Spectrozyme tPA. As expected, addition of PAI-1 to tPA virtually abolished the catalytic activity of tPA. Pretreatment of PAI-1 with VN or BSA did not suppress its ability to inactivate tPA. Again, as expected, pretreatment of PAI-1 with PAI-749 neutralized the ability of PAI-1 to inhibit tPA. Addition of VN to PAI-1 before the addition of PAI-749 largely blocked the ability of PAI-749 to neutralize PAI-1 inhibitory activity. BSA failed to protect PAI-1 from inactivation by PAI-749. On the other hand, pretreatment of PAI-1 with PAI-749 followed by the addition of VN yielded inactive PAI-1. PAI-1 treated sequentially with PAI-749 and BSA was also largely incapable of inactivating tPA. We conclude that PAI-1, when complexed with VN is shielded from the inhibitory effects of PAI-749. Nonetheless, if PAI-749 neutralized PAI-1 first, then the subsequent encounter with VN did not reverse the inhibitory effect of the compound.

    Impact of PAI-749 on the Vulnerability of PAI-1 to Plasmin-Mediated Proteolysis. It was shown previously that plasmin combines with PAI-1 to produce an SDS-stable complex (Reilly et al., 1993). The robust plasmin-generating potential near a thrombus prompted us to explore the effects of PAI-749 on the interaction between plasmin and PAI-1. Addition of plasmin to PAI-1 resulted in formation of small but discernible amounts of the SDS-stable plasmin/PAI-1 complex as shown by SDS-PAGE and protein silver staining (Fig. 7, lane 3). As expected, formation of the plasmin/PAI-1 complex was blocked by the presence of PAI-749 (Fig. 7, lane 4). We were surprised to find that the presence of PAI-749 also triggered the virtual disappearance of the band corresponding to PAI-1 (Fig. 7, lane 4). Aprotinin, a potent plasmin inhibitor, blocked the PAI-749 induced disappearance of the PAI-1 protein band (as well as the appearance of the plasmin/PAI-1 covalent complex) (Fig. 7, lane 5). PAI-749 did not increase the activity of plasmin toward a chromogenic plasmin substrate (data not shown); hence, the virtual disappearance of PAI-1 in the presence of PAI-749 and plasmin does not seem to be due to a generalized stimulation of plasmin proteolytic activity. The ability of PAI-749 to promote plasmin-mediated degradation of PAI-1 was also evident from data showing a concomitant decrease of residual PAI-1 inhibitory activity (data not shown). It is noteworthy that latent PAI-1 was refractory to the ability of PAI-749 to promote plasmin-mediated degradation (Fig. 7, lanes 7–9). This result corroborated the aforementioned conclusion that PAI-749 neither interacts with latent PAI-1 nor induces its formation.

    Fig. 7. PAI-749 promotes plasmin-mediated degradation of active PAI-1 but not latent PAI-1. Plasmin, PAI-1 (both active and latent), PAI-749, and aprotinin concentrations were 10 nM, 100 nM, 1 µM, and 200 nM, respectively. All reactions were performed at room temperature for 30 min before quenching and fractionation by SDS-PAGE. Lane 1, plasmin; lane 2; active PAI-1; lane 3, active PAI-1 + plasmin; lane 4, active PAI-1 + PAI-749 + plasmin; lane 5, active PAI-1 + PAI-749 + plasmin + aprotinin; lane 6, latent PAI-1; lane 7, latent PAI-1 + plasmin; lane 8, latent PAI-1 + PAI-749 + plasmin; and lane 9, latent PAI-1 + PAI-749 + plasmin + aprotinin. Proteins were visualized by silver staining.

    PAI-749 preserved tPA and uPA activity in the presence of neutralizing amounts of PAI-1. This outcome could arise if 1) PAI-749 bound to PAI-1 and neutralized its PA-inhibitory activity or 2) PAI-749 bound to the PA and blocked its subsequent interaction with PAI-1. The data presented herein strongly support the former hypothesis, in which PAI-749 binds directly to PAI-1 and this binding event mediates neutralization of PA inhibitory activity (i.e., PAI-749 is a bona fide PAI-1 antagonist). The fact that structurally related PAI-749 analogs displayed the same rank order of potency for altering the Fl signal of PAI-NBD119 and inhibiting PAI-1 activity firmly established that binding to PAI-1 and antagonism of PAI-1 activity are inextricably linked. Although the preponderance of data points to PAI-749 being a direct inhibitor of PAI-1, it is unresolved why the apparent IC50 values of PAI-749 for tPA and uPA are not identical (albeit less than 2-fold different). The apparent ability of PAI-749 to induce polymerization of PAI-1 could possibly exert disparate effects with respect to neutralization of uPA and tPA.

    Elegant studies by Olson et al. (2001) helped to elucidate the reaction pathway for PAI-1 mediated inhibition of tPA activity. Insight gleaned from these investigations reveals a number of potential mechanisms by which PAI-749 might neutralize PAI-1. First, PAI-749 could promote the active-to-latent conformational change in PAI-1. Production of the latent state of PAI-1 occurs spontaneously; hence, a compound could bind to PAI-1 and, in turn, decrease the activation energy barrier to formation of the thermodynamically more favorable latent state. Second, PAI-749 could block formation of the initial reversible Michaelis complex between PAI-1 and its target protease. Third, PAI-749 could impede nucleophilic attack of the active site serine of the PA on the reactive site of PAI-1 thereby abrogating formation of the acyl-enzyme intermediate. Fourth, PAI-749 could convert PAI-1 from a suicide inhibitor to a substrate for tPA by facilitating hydrolytic attack on the acyl-enzyme intermediate. The goal of this investigation was to determine which of these proposed inhibitory mechanisms is indeed responsible for the ability of PAI-749 to antagonize PAI-1 inhibitory activity.

    PAI-749 did not neutralize PAI-1 by inducing formation of latent PAI-1. Support for this conclusion is derived from several different lines of evidence. First, PAI-1 activity was preserved despite limited exposure to inhibitory amounts of PAI-749 (using "Tween 80 sequestration" to reduce the effective PAI-749 exposure before subsequent addition of the PA). This result is incompatible with production of the latent form of PAI-1, a stable conformational state that is not reversed upon removal of the triggering agent/condition. Second, the Fl signal of the latent form of PAI-NBD119 either in the presence or absence of PAI-749 differed from that observed when PAI-749 was added to active PAI-NBD119. Third, latent PAI-1 was not susceptible to plasmin-mediated proteolytic degradation in the presence of PAI-749 in stark contrast to active PAI-1 that was treated with PAI-749. Fourth, polymerization of PAI-1 by PAI-749 as shown by BN-PAGE was not seen with latent PAI-1. The results, in sum, clearly established that PAI-749 did not neutralize PAI-1 by triggering formation of latent PAI-1.

    PAI-749 seemed to exert an inhibitory effect on PAI-1 activity by interfering with the earliest step in the proposed reaction pathway: formation of the Michaelis-like complex between PAI-1 and the PA. The argument rests on two basic premises: 1) compound B (closely related analog of PAI-749) blocked the ability of PAI-1 to displace PAB from the active site of uPA and 2) the model structure of the Michaelis complex between PAI-1 and tPA based on the crystal structure of the noncovalent Manduca sexta serpin 1B-trypsin complex showed that the side chain and amide backbone nitrogen of Arg-346 (PAI-1 P1 residue) are optimally situated in the active site pocket (Ibarra et al., 2004). Hence, displacement of PAB from the PA active site by PAI-1 is predicted to accompany formation of the Michaelis complex. Based on the aforementioned arguments, failure of PAI-749-treated PAI-1 to displace PAB from the PA signifies that the Michaelis complex between PAI-1 and the PA is not produced in the presence of PAI-749. This conclusion is consistent with other experimental observations showing that more distal events in the reaction pathway (e.g., formation of the SDS stable complex) have been abolished as well. The most parsimonious explanation for at least one component of the mechanism of action of PAI-749 is that the compound binds directly to PAI-1 and interferes with the ability of PAI-1 to engage in a Michaelis-like complex with tPA.

    The ability of low-molecular-weight PAI-1 antagonists to elicit serpin multimerization was reported previously (Pedersen et al., 2003). PAI-1 polymerization in the presence of PAI-749 was thus was not surprising. However, the impressive potency of PAI-749 at producing this effect is particularly noteworthy. In any event, the PAI-1 polymerization outcome created uncertainty with regard to the mechanism of inhibition of PAI-1 by PAI-749. Was it due to the formation of the binary complex between PAI-1 and PAI-749 or to PAI-749–triggered PAI-1 polymerization? The reversibility studies with Tween 80 shed light on the answer to this key question. Tween 80 reversed inactivation of PAI-1 by PAI-749 when PAI-1 and PAI-749 were allowed to interact for 5 min. However, the Tween 80-induced reversibility was not apparent after 60-min treatment of PAI-1 with PAI-749. We hypothesize that the rapid inhibition of PAI-1 activity by PAI-749 reflects formation of the binary complex (reversed by Tween 80). The subsequent PAI-1 polymerization (not reversed by Tween 80) is not imperative for PAI-1 inactivation but does commit PAI-1 to a pseudoirreversible state. This interpretation of the data further implies that the rapid change of the Fl signal of PAI-NBD119 by PAI-749 is also probably due to the formation of the binary complex and not the ensuing PAI-1 polymerization. A high likelihood exists that the proximal molecular events after the initial interaction between PAI-1 and PAI-749 display greater complexity with respect to discrete states/conformations of PAI-1. For instance, this possibility is suggested by the apparent biphasic concentration-dependent impact of PAI-749 on the substrate-like behavior of PAI-1 within the bounds of a short (5-min) treatment interval (depicted in Fig. 5). Although our investigation has provided key mechanistic insight into the action of PAI-749 on PAI-1, there are certain aspects of this interaction that have yet to be elucidated and will require further investigation.

    Fig. 8. PAI-749 exerts a dual mechanism of action for neutralizing PAI-1 activity. This scheme shows the proposed reaction pathway for the interaction between plasminogen activator inhibitor type I (PAI) and a plasminogen activator (PA). PAIlatent, latent PAI; PA · PAI, Michaelis-type complex; PAPAI, acyl-enzyme intermediate; PAI*, PAI-1 clipped at the reactive site; PA–PAI, SDS-stable covalent complex. The data from this study show that PAI-749 binds directly to PAI-1 and interferes with formation of the Michaelis complex between PAI-1 and the PA. Moreover, PAI-749 upon binding to PAI-1 promotes plasmin-mediated degradation of PAI-1.

    The ability of PAI-749 to inhibit formation of the plasmin/PAI-1 complex represents another potential profibrinolytic effect of PAI-749; however, the importance of PAI-1 to plasmin neutralization in vivo is uncertain because of the vast potential excess of plasmin over PAI-1. An unexpected and potentially more significant finding is that PAI-749 promotes plasmin-mediated degradation of PAI-1. The putative conformational change in PAI-1 (perhaps coincident with formation of serpin multimers) induced by the binding of PAI-749 seemed to expose regions in PAI-1 that are cleaved by plasmin. This PAI-749-mediated effect represents a potential "feed forward" profibrinolytic mechanism. Accordingly, direct PAI-1 antagonism by PAI-749 promotes plasmin formation that, in turn, can lead to greater plasmin production as a result of plasmin-mediated degradation of the PAI-1/PAI-749 complex. Other PAI-1 antagonists were shown previously to increase the susceptibility of PAI-1 to proteolytic degradation by a variety of proteases such as papain and subtilisin (Einholm et al., 2003). However, plasmin (which was not previously examined) is a particularly relevant protease in light of its high abundance near the thrombus.

    The ability of VN to shield PAI-1 from the effects of PAI-749 coupled with the fact that PAI-1 exists predominantly in plasma as a complex with VN might prompt speculation that PAI-749 would not inhibit PAI-1 activity in vivo. However, whereas PAI-1 is expressed widely, VN seems to be expressed predominantly by the liver (Seiffert et al., 1994). Hence, "naked" PAI-1 will exist at least transiently after secretion by the source cell and should thus be a target for PAI-749 until it combines with VN. The fact that inactivation of PAI-1 activity by PAI-749 is not reversed by subsequent addition of VN is consistent with this hypothesis. It is noteworthy that this proposal is also supported by preclinical in vivo experiments in which PAI-749 exerts an impressive antithrombotic effect (J. Hennan, G. A. Morgan, R. E. Swillo, A. J. Ji, L. Guan, S. J. Gardell, and D. L. Crandall, manuscript in preparation).

    In conclusion, this investigation has uncovered a dual mechanism by which PAI-749 might neutralize PAI-1 activity (Fig. 8). Binding of PAI-749 to PAI-1 blocks the ability of PAI-1 to engage in a complex with the PA. The ensuing increase of PA-mediated plasmin production may lead, in turn, to further PAI-749-dependent neutralization of PAI-1 because of proteolytic degradation of the PAI-1/PAI-749 complex by plasmin. These two PAI-749-mediated effects on PAI-1 should work in concert to elevate tPA and plasmin activities at regions of vascular injury, thereby preserving blood vessel patency and contributing to antithrombotic efficacy. Detailed studies of the impact of PAI-749 on PAI-1 activity in blood as well as in the setting of active thrombolysis in vivo will be necessary to further explore the clinical antithrombotic potential of this PAI-1 antagonist.

    Acknowledgements

    We thank Bruce Malcolm for helpful discussions during the preparation of the manuscript.

    ABBREVIATIONS: PAI, plasminogen activator inhibitor; PAI-749, 1-benzyl-3-pentyl-2-[6-(1H-tetrazol-5-ylmethoxy)naphthalen-2-yl]-1H-indole; PA, plasminogen activator; tPA, single-chain tissue-type plasminogen activator; 2c-tPA, two-chain tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; NBD, N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); PAI-NBD119, PAI-1 tagged with NBD at amino acid position 119; Fl, fluorescence; PAB, p-aminobenzamidine; Apo-10, dimethyldecylphosphine oxide; HNEP, HEPES/NaCl/EDTA/PEG-8000; HNEPA, HEPES/NaCl/EDTA/PEG-8000/apo-10; DMSO, dimethylsulfoxide; PAGE, polyacrylamide gel electrophoresis; BN-PAGE, blue native polyacrylamide gel electrophoresis; VN, vitronectin; pNA, p-nitroanilide; BSA, bovine serum albumin; LDS, lithium dodecyl sulfate.

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作者单位:Departments of Cardiovascular and Metabolic Diseases (S.J.G., J.A.K., T.A.A., S.J.O., D.L.C., G.P.V.) and Chemical & Screening Sciences (H.E., S.M.), Wyeth Research, Collegeville, Pennsylvania

日期:2009年8月25日 - 来自[2007年第69卷第10期]栏目

PAI-1对心脏的纤维化作用

佛蒙特大学心血管研究院的研究人员发现,当心脏表达过高的纤维蛋白溶酶原活化抑制剂第一型(PAI-1)  时是前纤维化,他们的结果发表在《实验生物学与医学》2009年3月份上。PAI-1的过度表达已知和胰岛素抗性及第二类型糖尿病有关,现在更进一步发现可能是增加糖尿病患心肌梗塞后造成心脏衰竭的一个重要因子。  研究人员以10  周大小鼠进行冠状动脉闲塞实验,正常对照组及相同C57BL/6背景但过度表达PAI-1的转殖小鼠在左心室心肌细胞有相同量的PAI-1,虽然后者的血液中有较多的PAI-1,但在进行冠状状动脉闲塞后六个礼拜,过度表达PAI-1的转殖小鼠左心室心肌有二倍量PAI-1的增加,组织化学的分析也发现左心室多33%的纤维化,PAI-1增加伴随的纤维化带来功能上的障碍包括,舒张及收缩时减少的左心室壁厚度、收缩时左心室后期容量增加、低下的部分缩短,左心室结节功能的更大的伤害,以及更大的传导E-波幅度。
总结,心脏过度表达PAI-1会改变心肌梗塞后左心室的反应,冠状动脉闲塞后PAI-1的增加会伴随着纤维化的增加以及收缩和舒张的机能的失调。Sobel  博士说,结合我们以前的报告转殖小鼠PAI-1在心脏表达的增加会使胰岛素抗性增加,  这次的研究结果建议那些有胰岛素抗性及第二类型糖尿病的病人在心肌梗塞后有较高机会会发生严重心脏衰竭的原因,可能部份和心脏增加PAI-1的表达,因而加速纤维化以及带来的左心室功能受损有关。
《实验生物学与医学》期刊总编辑Steven  R.  Goodman博士说,这个由Sobel博士及他同事提供优雅的研究,对于我们了解第二类型糖尿病人因为增加PAI-1在心脏的表达会增加心肌梗塞后,有较大的机会发生严重心脏衰竭提供了一个很重要的机制,这对于我们了解糖尿病和心血管疾病间的相关联过程是很重要的一步。
日期:2009年3月7日 - 来自[心脑血管相关]栏目
循环ads

Association of Plasminogen Activator Inhibitor (PAI)-1 (SERPINE1) SNPs With Myocardial Infarction, Plasma PAI-1, and Metabolic Parameters

【摘要】  Objective— The purpose of this study was to investigate the effects of plasminogen activator inhibitor-1 (PAI-1) gene (SERPINE1) single nucleotide polymorphisms (SNPs) on the risk of myocardial infarction (MI), on PAI-1 levels, and factors related to the metabolic syndrome.

Methods and Results— Eleven SNPs capturing the common genetic variation of the SERPINE1 gene were genotyped in the HIFMECH study. In the 510 male cases and their 543 age-matched controls, a significant gene-smoking interaction was observed. In nonsmokers, the rs7242-G allele was more frequent in cases than in controls (0.486 versus 0.382, P =0.013) whereas the haplotype G)-G and rs2227683-A alleles was 3-fold lower in cases than in controls (0.042 versus 0.115, P =0.006). SERPINE1 haplotypes explained 3.5% ( P =0.007) of the variability of PAI-1 levels, which was attributable to G, rs2227666, and rs2227694. The rs6092 (Ala15Thr) and rs7242 SNPs acted additively to explain 4.4% of the variability of plasma insulin levels and 1.6% of the variability of BMI ( P <10 –3 and P =0.023, respectively).

Conclusions— SERPINE1 haplotypes are mildly associated with plasma levels of PAI-1 and with the risk of MI in nonsmokers. They are also associated with insulin levels and BMI.

HIFMECH is a European case-control study for myocardial infarction (MI). In the 510 male cases and their 543 age-matched controls, SERPINE1 haplotypes were mildly associated with plasma levels of PAI-1 and with the risk of MI in nonsmokers. They were also associated with insulin levels and BMI.

【关键词】  metabolic syndrome myocardial infarction PAI SERPINE


Introduction


In blood, fibrinolysis breaks down fibrin and maintains vessel patency, and in tissues it breaks down the extracellular matrix and controls cell adhesion and migration and thus participates in tissue remodeling. Fibrinolysis is primarily regulated by plasminogen activator inhibitor type-1 (PAI-1), which controls the extent of this potentially destructive protease system. 1–3


Increased PAI-1 levels may predispose patients to the formation of atherosclerosis plaque prone to rupture, with a high lipid-to-vascular smooth muscle cells ratio as a result of decreased cell migration. 4 In humans, there is clinical evidence that increased PAI-1 levels are associated with atherothrombosis. 5,6 In large epidemiological studies, elevated plasma PAI-1 levels have been identified as a predictor of myocardial infarction (MI). 7–11 Remarkably, the predictive ability of PAI-1 disappears after adjustment for markers of the metabolic syndrome (MetS), 8,12–16 suggesting that the MetS is a prerequisite to high plasma PAI-1 levels in patients prone to atherothrombosis. Moreover, it has been hypothesized that PAI-1 participates in the development of key features of the MetS. Indeed, several studies 17–20 showed that high plasma PAI-1 levels independently predict the development of type II diabetes. Whether PAI-1 plays a direct role in MI, MetS, or diabetes, or is only a bystander, is difficult to assess in humans. One way to verify this hypothesis is to look for a relation between single nucleotide polymorphisms (SNPs) influencing PAI-1 expression or activity, MI, and parameters belonging to the MetS. Several SERPINE1 (formerly PAI-1) gene SNPs have been identified, 21–23 among which the polymorphism 4G/5G (rs1799889) located in position –675 of the promoter region has been quite extensively studied. The 4G allele has been shown to be associated with increased SERPINE1 transcription compared with the 5G allele in in vitro studies 21,24 and with increased plasma PAI-1 levels in vivo. 22 A large systematic review found that the 4G/4G genotype was associated with a modest 1.2-fold increased risk of MI. 25 We have shown, in a previous report of the HIFMECH study, that the –675 4G/5G polymorphism is associated with the risk of MI but that this effect is considerably influenced by the presence of underlying MetS. 26 Results about the relation between this polymorphism and variables related to the MetS were however contradictory, carriers of the 4G allele being more prone to obesity and MetS in some studies 22,23,26–28 but not in others. 29–32 One possible explanation for these observed discrepancies could be that other SERPINE1 SNPs in linkage disequilibrium (LD) with the 4G/5G polymorphism are associated with MetS, suggesting that haplotype analysis of SERPINE1 SNPs could be of great interest. For example, another potentially functional polymorphism of the promoter, 33 G (rs2227631), in strong LD with the 4G/5G polymorphism, could be responsible, instead of the 4G/5G, for the association between PAI-1, MetS, and MI. Three haplotype association studies have recently been performed to address the influence of SERPINE1 SNPs on PAI-1 plasma levels and on the risk of cardiovascular disease. 34–36 Kathiresan et al 34 have shown that SERPINE1 SNPs explained about 5% of the variability of PAI-1 plasma levels and this association could be attributable to 3 SNPs, rs6465787, G. This haplotype analysis could not completely exclude the possibility that the effect of the G SNP was the consequence of its strong LD with the –675 4G/5G. On the contrary, Ding et al, 35 using a similar approach, showed that the SERPINE1 effect on PAI-1 plasma levels seems to be restricted to the –675 4G/5G polymorphism, G. Neither of these studies found any relation between common haplotypes of the SERPINE1 and the overall risk of cardiovascular disease. However, Su et al, 36 in a group of Chinese subjects, detected a SERPINE1-smoking interaction on CHD risk, such as the main haplotype carrying the –844A and –6754G allele significantly increased the risk of CHD in nonsmokers only.


The aim of our study was to simultaneously evaluate, in a case-control study of White individuals, the association of SERPINE1 SNPs with MI, plasma PAI-1 levels, and metabolic parameters using a haplotype-based approach. We aimed to test whether the association already described between the SERPINE1 variants and myocardial infarction could be first modulated by smoking and secondly be partly the result of the relationship between these variants and some features of the metabolic syndrome.


Materials and Methods


Study Subjects


Full details of the study design and recruitment criteria are presented elsewhere. 26 Male survivors of a first MI aged <60 years (excluding patients with familial hypercholesterolemia and insulin dependent diabetes mellitus) and population-based individuals of the same age were recruited from the 4 centers as part of the HIFMECH study: Stockholm (Sweden), London (UK), Marseille (France), and San Giovanni Retondo (Italy). Consecutive patients were invited to participate, along with randomly selected healthy individuals from the same catchment areas. In all, a total of 510 postinfarction patients and 543 controls were included in the present study. Postinfarction patients were investigated 3 to 6 months after the acute event. Patients and control subjects were examined in parallel in the early morning after an overnight fast. Height and weight were recorded and the body mass index (BMI) was calculated as kg m –2. Smokers were considered as current or ex-smokers at the time of the MI onset.


Determination of PAI-1 antigen was centrally performed with a commercially available kit (Asserachrom PAI-1; Stago). Each plasma sample was run in duplicate. Interassay variation coefficient of pooled plasma from 30 healthy volunteers was 8%. Assay method for insulin has been described. 37


Choice of PAI-1 Tag Polymorphisms


SERPINE1 has been sequenced by the Seattle SNPs program for Genomics Application project in 23 individuals of European ancestry (http://pga.gs.washington.edu/). From the identified SNPs spanning 13 kb of the SERPINE1 gene, the minimum number of SNPs (tag SNP) required to characterize 100% of the haplotypic diversity of the SERPINE1 gene was determined. 10 tag SNP with minor allele 0.04 were found to be enough to fully characterize this gene and were further genotyped in the HIFMECH study. These G), rs6092 (Ala15Thr), rs7242, rs2227708, rs2227662, rs2227666, rs2227667, rs2227672, rs2227683, rs2227694. The rs1799889 (–675 4G/5G) was also genotyped as it is widely used in SERPINE1 genetic studies, has been shown to be functional, 24 and is associated with PAI-1 plasma levels.


Genotyping were performed under contract by Kbioscience, Cambridge, UK (http://www.kbioscience.co.uk), except for 4G-675 5G, which was genotyped by allele specific polymerase chain reaction (PCR), 3'): forward: TCAGCCAGACAAGGTTGTTG, reverse: TTTTCCCCCAGGGCTGTCCA, 4G: GTCTGGACACGTGGGGA, 5G: GTCTGGACACGTGGGGG. PCR conditions were an initial denaturation step of 1?30 at 95°C, followed by 35 cycles of these 3 steps: 95°C: 30", 62°C: 45", 72°C: 1? and then a final extension step of 5? at 72°C. PCR were then kept at 15°C for immediate use or frozen for later use.


Statistical Analysis


Allele frequencies were estimated by gene counting, and departure from Hardy-Weinberg (HW) equilibrium was testing using a 2 with 1 degree of freedom. Allele frequencies were compared between cases and controls by use of a 2 with 1 degree of freedom. Conditional logistic regression analysis for matched case-control study was used to investigate the association between MI and explanatory variables. Genotypic association of SERPINE1 polymorphisms with plasma PAI-1 and insulin levels was first investigated by use of a classical linear model. Plasma PAI-1 and insulin were square-root and log-transformed to remove positive skewness, respectively.


LD analysis was carried by the THESIAS software 38 (www. genecanvas.org) based on the SEM algorithm. 39 The extent of LD was expressed in terms of D'. 40 THESIAS was also used for haplotype analyses. For the haplotype analyses, systematic analyses of all possible combinations of 1 to 9 polymorphisms were carried out to reduce the haplotype dimension and to search for the most informative and parsimonious haplotype configuration in terms of prediction of the phenotypes variability using the previously described Akaike?s Information Criterion-based strategy. 41,42 The homogeneity of allelic and haplotypic effects across North and South or across cases and controls was assessed by the Mantel-Haenszel statistics. 43 All analyses were adjusted for age, gender, smoking, center, and case-control status when appropriate. A probability value of <0.05 was taken as statistically significant.


Results


Baseline Characteristics of Cases and Controls


Cases were more likely to be smokers and suffer from diabetes. Parameters of the MetS such as BMI, plasma insulin, and triglycerides (TG) levels were significantly higher in cases than in controls (supplemental Table I, available online at http://atvb.ahajournals.org). PAI-1 levels were significantly higher in cases than in controls (40.10±28.46 versus 29.48±22.50 ng/mL, P <10 –4 ), an effect seen in both the North (42.64 versus 31.85 ng/mL) and South (38.28 versus 27.82 ng/mL). As expected, plasma PAI-1 levels were highly correlated with insulin levels, =0.44 and =0.37 in controls and cases, respectively (both P <10 –4 ), but also to BMI ( =0.43 and =0.24, P <10 –4, respectively) and with TG ( =0.38 and =0.30, P <10 –4, respectively).


Description of Studied SERPINE1 SNPs


Among the 11 genotyped polymorphisms, 1 was found to be nonpolymorphic (rs2227662), whereas the rs2227708 was relatively rare (frequency of 0.012 in the whole HIFMECH study). Therefore, as shown in Figure 1, the present analysis focused on 9 polymorphisms, rs2227631 G), rs1799889 (–675 4G/5G), rs6092 (Ala15Thr), rs2227666, rs2227667, rs2227672, rs2227683, rs2227694, and rs7242 G). The genotype distribution of all the SNPs were in HW equilibrium and their allele frequencies were very similar in North and South (supplemental Table II). Pairwise LD was relatively strong between all polymorphisms, except for the G and –675 4G/5G SNPs. As a consequence, 9 haplotypes with frequency greater than 3% were inferred and accounted for about 93% of the whole chromosomes (see below). As the pattern of LD and the resulting haplotypic structure were very similar in North and South (supplemental Table III), the following analyses were performed on the whole HIFMECH sample, while checking for this homogeneity of the associations across regions.


Figure 1. Location of the 11 polymorphisms analyzed in the SERPINE1 gene. Usual names, when existing, are given with the rs numbers. The exons are denoted as squares (white: untranslated, black: coding region). The 9th exon contains the end of the coding sequence and the 3'UTR.


Association of SERPINE1 SNPs With MI


In the whole sample, none of the polymorphisms was significantly associated with MI either using single-locus (supplemental Table IV) or haplotype ( Table 1 ) analyses. The rs6092-Thr allele was carried by only 1 haplotype and tended to be less frequent in cases than in controls (0.099 versus 0.125), but this difference failed to reach significance ( P =0.07). However, the association of SERPINE1 SNPs with MI was found to be modulated by smoking. Two SNPs were associated with MI only in nonsmokers (supplemental Table V). Although SERPINE1 haplotypes were not associated with MI in smokers ( P =0.42), they were highly associated with MI in nonsmokers ( P =0.004). The best model in terms of predicting G, rs2227683, and rs7242 ( Table 2 ). Table 2 also includes the information on the –675 4G/5G, to examine in more detail its contribution on the risk of MI. Consistent with univariate analysis, the rs7242-G allele carried by 1 frequent haplotype (H2) was more frequent in cases than in controls (0.486 versus 0.383, P =0.013). Because the effect of this haplotype that also carries the –844A allele was not significantly different from the 3 other haplotypes carrying the –844A allele (H1, H3, H4; P =0.27), it cannot be completely excluded that the effect of the rs7242 SNP was G. In addition, the frequency of the haplotype carrying the –844G and rs2227683-A alleles (H6) was 3-fold lower in nonsmoker cases than in nonsmoker controls (0.042 versus 0.115, P =0.006). It is important to note that the –6754G/5G does not participate in this gene x smoking interaction.


Table 1. Association of Main SERPINE1 Haplotypes With MI in the HIFMECH Study


Table 2. Association Between MI and Main SERPINE1 Haplotypes Derived From the rs2227631, rs1799889, rs2227683, and rs7242 Polymorphisms According to Smoking


Association of SERPINE1 SNPs With PAI-1 Levels


Although effects were generally larger in cases than control (supplemental Table VI), there was no significant evidence for genetic effect heterogeneity (all P 0.15) nor across smokers and non-smokers (data not shown). Therefore, Table 3 provides a full description of the single-SNP association analyses in G, –675 4G/5G, rs2227667, and rs2227672, were significantly associated with PAI-1 levels. Overall, the percentage of variance explained by these SNPs were 1.25% ( P =0.002), 1.12% ( P =0.004), 0.77% ( P =0.02), and 0.72% ( P =0.03), respectively. SERPINE1 haplotypes were significantly associated with PAI-1 levels ( P =0.007) and explained 3.8% ( P =0.05) and 3.1% ( P =0.11) of the variability of PAI-1 levels in controls and cases, respectively (supplemental Table VII). The best model found in the systematic exploration of haplotype effects in G (already found to be associated with the risk of MI in nonsmokers), rs2227666, and rs2227694 polymorphisms. Detailed haplotype analysis of these 3 polymorphisms is summarized in Figure 2. The information on the –6754G/5G SNP is also provided to get better insight into its contribution on PAI-1 levels variability. Firstly, the –844G allele was carried by 2 haplotypes that differed only at position rs2227694 and were both associated with similar PAI-1 levels (2.33 versus 2.14, P =0.51). By comparison to the most frequent A[4G]GG haplotype, these 2 haplotypes were associated with lower PAI-1 levels (2.55 versus 2.30, P =0.02), a result compatible with an increasing effect on PAI-1 levels of the –844A allele. The observation that the unique haplotype pair A[4G]GG and A[5G]GG that differed only at the –6754G/5G locus did not show difference in PAI-1 levels (2.55 versus 2.59, P =0.87) would additionally suggest that the effect of the –675 to 4G/5G polymorphism observed in univariate analysis was the consequence of its LD with other SERPINE1 SNPs. In addition, 2 haplotypes, A[4G]GA and A[4G]AG, were associated with higher mean PAI-1 levels than the most frequent haplotype (3.02 versus 2.55 and 3.01 versus 2.55, respectively; P =0.03 for both). Because the A[4G]AG haplotype is the only one carrying the rs2227666 A allele, these results would suggest an increasing effect on PA-I levels of the rs2227666A allele in addition to an increasing effect of the rs222794 A allele when associated with the –844A allele on the same haplotype. These effects were similar in controls and cases, in North and South, and in smokers and nonsmokers (data not shown).


Table 3. Association Between PAI-1 Gene Polymorphisms and PAI-1 Levels in the HIFMECH Study (n=948)


Figure 2. Association between plasma PAI-1 levels and SERPINE1 haplotypes derived from the rs2227631 (A/G), rs1799889 (4G/5G), rs2227666 (G/A), and rs2227694 (G/A) polymorphisms. Polymorphisms are ordered according to their position on the genomic sequence. Each bar and its 95% CI brackets corresponds to the expected mean of PAI-1 levels (square rooted) associated with 1 dose of haplotype under the assumption of additive haplotype effects.


Association of SERPINE1 SNPs With Metabolic Parameters


In univariate analysis, the rs7242 was found to be significantly associated with insulin levels in cases only (supplemental Table VIII). No single locus nor haplotype effects were observed in controls (test for homogeneity between cases and controls for the rs7242 P =0.039, supplemental data). Conversely, haplotype analysis revealed that the Ala15Thr and the rs7242 SNPs were strongly associated with insulin levels in cases. These 2 SNPs defined 3 haplotypes ( Table 4 ) that were highly associated with insulin ( R 2 =4.4%, 2 =15.89 with 2 df, P <10 –3 ). By comparison to the most frequent Ala-T haplotype, both Ala-G and Thr-T haplotypes were associated with higher insulin levels ( Table 4 ). These results were compatible with independent and increasing effects of the Thr15 (+0.24 [0.05 to 0.43], P =0.015) and rs7242 G (+0.17 [0.08 to 0.27], P <10 –3 ) alleles. These effects were similar in smokers and nonsmokers (data not shown), and were G, –675 4G/5G, and rs227683 (supplemental Table IX). The same pattern of association was observed with BMI, the rs6092 and rs7242 SNPs explaining 1.6% of the variability of BMI ( P =0.023) in cases only (supplemental Table X). No SERPINE1 genotype or haplotype was associated with a significant effect on TG levels.


Table 4. SERPINE1 Haplotype Analysis of the rs6092 and rs7242 Polymorphisms in Relation to Insulin Levels According to Case–Control Status


Discussion


The main findings of the study are that different SERPINE1 haplotypes are associated with the risk of MI in nonsmokers and with plasma levels of PAI-1 in both cases and controls and with insulin and BMI in cases. In all, 6 SNPs were associated with these different clinical and biological phenotypes ( Figure 1 ). The G is particularly of interest as it is both related with MI in nonsmokers and with plasma PAI-1 levels. As previously reported in Chinese Han subjects, 36 a highly significant gene x smoking interaction was detected, characterized by a strong association of SERPINE1 haplotypes with MI in nonsmokers only. In nonsmokers, the rs7242 SNP was associated with MI, as the rs7242-G allele was more frequent in cases than in controls (0.51 versus 0.41). This allele was carried out by only 1 haplotype, a haplotype that also carries the –844A and –675 to 4G alleles, and that was found to be associated with higher risk of MI in nonsmoking Chinese. 36 Because of the strong LD, G is responsible for the observed association. However, haplotype analysis revealed that the –675 4G/5G is unlikely to explain this association. In addition, we observed that the frequency of the haplotype defined by the –844-G and rs2227683-A alleles was 3-fold lower in cases than in controls. Thus, the G polymorphism is more relevant than the –675 4G/5G for the association with MI in nonsmokers.


The fact that the impact of the SERPINE1 polymorphisms on MI was only observed in nonsmokers remains puzzling. We can hypothesize that SERPINE1 SNPs effect is relatively modest so that it is overwhelmed by the strong effect of smoking on risk. However, only 84 cases and 201 controls were nonsmokers, and this result must be replicated in a larger study conducted in nonsmokers. In HIFMECH, besides its effect on the risk of MI in nonsmokers, G SNP was also associated with PAI-1 plasma levels. It must be underlined that this SNP explained only a small amount of PAI-1 levels variability (1.25%) and that this association may not be of clinical relevance. As it has been also implicated in the regulation of the SERPINE1 gene, as a part of an Ets nuclear protein consensus sequence binding site, 33 G could be attributable to modifications of SERPINE1 expression. In the present study, the observation that adjustment for PAI-1 plasma levels did not modify the relation between SERPINE1 haplotypes and the MI risk in nonsmokers suggests that PAI-1 plasma levels might not reflect the local expression of PAI-1. This latter hypothesis is supported by the fact that PAI-1 mRNA expression is increased in the endothelial cells located in the vicinity of thrombi, and that this overexpression is not correlated with plasma PAI-1 antigen levels. 44


Besides a direct effect via modification of PAI-1 expression in the arterial wall, SERPINE1 polymorphisms could influence the risk of MI by influencing well-known cardiovascular risk factors such as the MetS. Indeed, several studies conducted in vitro and in vivo support a role of SERPINE1 in the development MetS (reviewed in 45 ). This led us to study the association between SERPINE1 SNPs and several variables of the MetS such as BMI, insulin, and TG. Haplotype analysis revealed that 2 polymorphisms, rs6092 and rs7242, independently affect BMI and plasma insulin levels in cases. The rs6092 could be of functional importance as it is an Ala15Thr located in the central hydrophobic core of the PAI-1 signal peptide. 23 The Ala15 allele increases hydrophobicity and -helix propensity, indicating that it could stabilize the -helix confirmation of the signal peptide. These 2 properties are known to be important in signal peptide function and, therefore, the mutations might modulate the secreted PAI-1 level. Lopes et al 23 have shown that the Ala15 allele tended to be associated with a higher risk of CHD in diabetic subjects. In the present study, in univariate analysis, the Ala15 allele tended to be more frequent in individuals with MI as compared with those without, however this difference did not reach significance ( P =0.06). As regards the rs7242 polymorphism located in the 3' untranslated region of the SERPINE1 gene, no specific information on a functional role is available; however, studies to investigate its functional impact could be useful because this polymorphism was also found associated with MI in nonsmokers.


It is of note that these associations were not modified by adjustment for circulating PAI-1 levels. The reason for the restriction of the relation between insulin and SERPINE1 polymorphisms to cases with MI is not understood. It could be attributable to the effect of another factor, overrepresented in MI and more generally in a stressed inflammatory situation that reinforces the link between SERPINE1 and the MetS, but such a factor has yet to be identified.


In the first report of the HIFMECH study, 26 only the –675 4G/5G polymorphism was studied in relation with the risk of MI and was found to interact with insulin to modulate the risk of MI, such that the risk mediated by higher insulin levels was only observed in 4G carriers. The haplotype analysis performed in this report suggested that this interaction was in fact the consequence of the joint effects of the rs6092 and rs7242 on insulin observed in cases only.


SERPINE1 haplotypes explained about 3.5% of the variability of PAI-1 levels, which is similar to that observed in the Framingham Heart Study. 34 The present study confirmed results from this latter study by demonstrating that other polymorphisms located outside the SERPINE1 promoter influence PAI-1 levels, but this is in disagreement with results from the study of Ding et al 35 which exclusively observed an effect of the –675 4G/5G polymorphism. The analysis here suggested that the observed haplotype effects were attributable to the combined effects G, rs2227666, and rs2227694. In the Framingham Heart Study, it was impossible to distinguish which of the 2 polymorphisms (A–844G and –675 4G/5G) was responsible for the association with PAI-1 levels. In the present study, the data suggest that the effect of the –675 4G/5G polymorphism observed in univariate analysis was the consequence G. In view of the results of the Framingham Heart Study, 34 2 others polymorphisms, rs6465787 and rs2227692, were also found associated with PAI-1 plasma levels. The rs6465787 SNP was not genotyped in our study because of its minor allele frequency (2%, 34) and its complete LD with G, which meant it would be impossible to distinguish its potential effect on PAI-1 levels from that of G SNP. The rs2227692 was not genotyped in our study as it is completely tagged by the rs2227667 and rs2227672 SNPs, the rs2227692-T allele being equivalent to the haplotype defined by the rs2227667-G and rs2227672-G alleles (see HapMap database at www. hapmap.org).


It is to note that, because of the LD among the 9 SNPs studied, a standard Bonferroni correction for multiple testing would not be appropriate because it would have been too conservative. Using the method proposed by Li and Ji, 46 the number of independent components underlying the LD structure of the set of SNPs was estimated to be 7. Correcting for this number, which corresponds to consider significant any probability value 0.007 should be considered as significant, would not have altered the main conclusions of our analyses.


Conclusions


SERPINE1 haplotypes are not associated with the risk of MI in the overall cohort. However, they are mildly associated with plasma levels of PAI-1 and with the risk of MI in nonsmokers. They are also mildly associated with insulin levels and BMI. Six SNPs are associated with these different clinical and biological phenotypes, suggesting that modifications of SERPINE1 expression involved in these processes are regulated via different pathways. Besides, our haplotype analysis suggested that among the 2 promoter G) and rs1799889 (–675 4G/5G) which are in tight LD, the former associated with MI, is more likely to be associated with PAI-1 levels.


Appendix


HIFMECH Investigators


Stockholm: A. Hamsten (coordinator), S. Boquist, C.G. Ericsson, P. Lundman, A. Samnegard, A. Silveira, P. Tornvall; London: J.S. Yudkin, V. Mohamed-Ali, A. Holmes; Marseille: I. Juhan-Vague, M.F. Aillaud, P.E. Morange, M.C. Alessi, P. Ambrosi, I. Canavy. F. Paganelli, R. Didelot, J. Ansaldi, M. Billerey; San Giovanni Rotondo: G. Di Minno, M. Margaglione, D. Cimino, N. Dello Iacono, A. Cimino, G. Gaeta, C. Blasich, G. Pucciarelli; London: S.E. Humphries; Leiden: V. van Hinsbergh, T. Kooistra; Milan: E. Tremoli, C. Banfi, L. Mussoni.


Acknowledgments


Sources of Funding


The HIFMECH Study was also supported by the European Commission (BMH4-CT96–0272), the Swedish Medical Research Council, the Swedish Heart-Lung Foundation, INSERM, and Université de la Méditerranée (INSERM U626), Fondation pour la Recherche Médicale (FRM) and Programme Hospitalier de Recherche Clinique (PHRC 1996). Steve Humphries is supported by the British Heart Foundation (RG2005/014).


Disclosures


None.

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作者单位:From INSERM, U626 (P.E.M., N.S., M.C.A., I.J.V.), Université de la Méditerranée, Marseille, France; the Diabetes and Cardiovascular Disease Academic Unit (J.S.Y.), Archway Campus, Royal Free and University College Medical School, London, UK; Instituto di Ricovero e Cura a Caratt

日期:2008年12月28日 - 来自[2007年第27卷第10期]栏目

PAI-1 and the Metabolic Syndrome

【摘要】  The link between plasminogen activator inhibitor (PAI)-1 and the metabolic syndrome with obesity was established many years ago. Increased PAI-1 level can be now considered a true component of the syndrome. The metabolic syndrome is associated with an increased risk of developing cardiovascular disease, and PAI-1 overexpression may participate in this process. The mechanisms of PAI-1 overexpression during obesity are complex, and it is conceivable that several inducers are involved at the same time at several sites of synthesis. Interestingly, recent in vitro and in vivo studies showed that besides its role in atherothrombosis, PAI-1 is also implicated in adipose tissue development and in the control of insulin signaling in adipocytes. These findings suggest PAI-1 inhibitors serve in the control of atherothrombosis and insulin resistance.

The metabolic syndrome is associated with overexpression of PAI-1; the mechanisms involved are complex. Besides its role in atherothrombosis, PAI-1 is also implicated in obesity and insulin resistance. The development of PAI-1 inhibitors is a challenge.

【关键词】  adipose tissue atherothrombosis metabolic syndrome obesity PAI


Introduction


In blood, fibrinolysis breaks down fibrin and maintains vessel patency, and in tissues it breaks down the extracellular matrix and controls cell adhesion and migration and thus participates in tissue remodeling. Fibrinolysis is primarily regulated by plasminogen activator inhibitor type-1 (PAI-1), which prevents the escape of this potentially destructive protease system. 1-3


Increased PAI-1 levels may predispose patients to the formation of atherosclerotic plaques prone to rupture with a high lipid-to-vascular smooth muscle cells ratio as a result of decreased cell migration. 4 In humans, there is clinical evidence that increased PAI-1 levels are associated with atherothrombosis. 5,6 In large epidemiological studies, elevated plasma PAI-1 levels have been identified as a predictor of myocardial infarction. 7-11 Remarkably, the predictive ability of PAI-1 disappears after adjustment for markers of the metabolic syndrome (MetS) such as body mass index (BMI), triglycerides, and high-density lipoprotein cholesterol, 8,12-16 suggesting that the MetS is a prerequisite to high plasma PAI-1 levels in patients prone to atherothrombosis.


The purpose of this review is to update our understanding of the connections between PAI-1 and the MetS. We discuss the possible mechanisms linking increased circulating PAI-1 levels to the MetS and the recent findings implicating PAI-1 in adipose tissue development and insulin signaling, making PAI-1 more an actor rather than a simple marker of the MetS ( Figure ).


Interaction between metabolic syndrome and PAI-1 overexpression.


Contribution of MetS to PAI-1 Synthesis


Description of the MetS


The MetS consists of a cluster of metabolic abnormalities that cooccur in an individual more often than by chance. These abnormalities include obesity with a distribution of the fat in the central part of the body (visceral or android obesity), impaired glucose tolerance, hyperinsulinemia, dyslipidemia with elevated triglyceride level, low high-density lipoprotein cholesterol concentration, increased proportion of small dense lipoparticles, and hypertension, all well-documented risk factors for cardiovascular disease. 17 Individuals with the MetS are at increased risk for diabetes mellitus and cardiovascular disease. 18,19 Some investigators called into question the existence of this syndrome in the sense of its having a single underlying cause 20 and argued that the MetS does not add to global cardiovascular risk as assessed by current algorithms, which already include some of the MetS features. It is clear that the MetS has more than one cause. 21 The extent to which the MetS adds to the global risk of cardiovascular disease has to be defined, given its growing prevalence worldwide. Ectopic fat depots such as the visceral one may play a central part in linking the MetS to cardiovascular disease. Ectopic fat could be secondary to a defect of peripheral fat cell proliferation, as observed in severe insulin resistance associated with hereditary lipodystrophy, or to the failure of fat cells to increase their size and therefore to accommodate an increased energy influx, leading to a reorientation of fat storage. 22 The resulting ectopic fat depots may be a source of toxicity toward the surrounding tissues by releasing active substances such as free fatty acid 23 and deleterious adipokines such as inflammatory cytokines and PAI-1. 24


PAI-1 Is a True Component of the MetS


The link between PAI-1 and the MetS was first described by our group in 1986 and is now well-established. 25,26 Circulating PAI-1 is increased in obese subjects with the MetS, as well as in patients with type II diabetes. The more severe the MetS, the higher the plasma level of PAI-1. 27 The MetS explains a major part of plasma PAI-1 level variability, with this relationship being stronger in men than in women (45% versus 26%). 28 Interventional studies reported that if insulin resistance is improved, plasma PAI-1 levels decrease. Decreased plasma PAI-1 concentrations were observed after weight reduction by a hypocaloric diet and were associated with decreased body fat. 29 In addition, treatment with insulin-sensitizing drugs like metformin or troglitazone decrease plasma PAI-1 levels in subjects with type II diabetes and to some extent in normal obese subjects. 30,31 It has been proposed to consider increased PAI-1 levels as a true component of the MetS. 26,32


Possible Mechanisms Linking PAI-1 Overexpression to the MetS


Obviously, induction of PAI-1 overexpression is a complex process and several inducers may be involved at the same time at several sites of synthesis, including vessel walls. 33,34 Factorial analysis showed 35 that elevated circulating PAI-1 levels during obesity are not associated with the interleukin (IL)-6 driven inflammation (C-reactive protein , fibrinogen), as one would expect because PAI-1 is considered an acute phase protein whose synthesis is induced by IL6. 36,37 PAI-1 levels are also not associated with dyslipidemia but rather with the fat redistribution phenotype assessed by the measure of waist circumference and the insulin resistance state. 35,38 In this context, the role of ectopic fat depots as sites of PAI-1 synthesis may be relevant.


Adipose Tissue and Ectopic Fat Depots as Sites of PAI-1 Overexpression


Several groups have described the ability of adipocyte cell lines 39-41 and murine adipose tissue 42 to synthesize PAI-1. Subsequent reverse-transcription polymerase chain reaction (RT-PCR) and in situ hybridization studies suggested that the increased plasma PAI-1 originates primarily from the adipocyte in response to chronically elevated levels of tumor necrosis factor (TNF), insulin, and transforming growth factor (TGF)-beta. 43 PAI-1 is also produced by human adipose tissue explants, 44,45 but it is mainly localized in the stromal compartment of the adipose tissue. PAI-1 antigen was detected in purely stromal area and in small cells in direct contact with adipocytes of macrophage origin. 46,47 During human preadipocyte differentiation, PAI-1 secretion appears to be produced by contaminant macrophages. 46,48,49 These results suggest that macrophages infiltrating adipose tissues may be one of the main source of PAI-1 in patients with a MetS.


Several groups have stressed the exclusive association between high plasma PAI-1 levels and visceral obesity. 50-52 For example, changes in plasma PAI-1 levels during a weight-reducing program correlated with changes in visceral fat depot but not in subcutaneous fat depot. 53 In obese rats PAI-1 mRNA is found in both types of fat tissue but its level increased only in visceral fat during the development of obesity. 52 In obese patients, abdominal visceral fat expressed 5-fold more PAI-1 than subcutaneous tissue. 46


Ectopic fat accumulation in human liver was also associated with a strong expression of PAI-1 close to fat cells. 54 This could be related to the strong relationship between circulating PAI-1 antigen and hepatic but not peripheral insulin resistance in Pima Indians. 55 All these findings suggest that circulating PAI-1 levels are not closely dependent on fat mass but rather that they reflect fat redistribution and may be considered as a biomarker of ectopic fat storage. However, several questions remain unanswered. Is there a direct ectopic fat mass effect, an indirect connection between ectopic fat and PAI-1 through a mediator, or a common ground with a parallel evolution of PAI-1 and ectopic fat without a real connection?


Arguments for the Contribution of TNF and TGF-beta to PAI-1 Overexpression


TNF is involved in insulin resistance. 56 The group of Loskutoff was the first to emphasize the contribution of TNF in PAI-1 regulation during obesity. In ob/ob mice, deletion of both TNF receptors (TNF RI and RII) significantly reduced the plasma PAI-1 levels as well as the adipose tissue PAI-1 mRNA levels. TNF-neutralizing antibodies decreased plasma PAI-1 level, proving a direct link between TNF and PAI-1 during obesity. 57,58 Moreover, the invalidation of both TNF receptors decreased TGF-beta expression in the adipose tissue, 57 and in humans, TNF receptors, TGF-beta, and PAI-1 levels were strongly correlated within adipose tissue. 59,60 These results suggest that the TNF and TGF-beta pathways are connected within adipose tissue and may both control PAI-1 expression. The possible connection between insulin resistance, TGF-beta, and PAI-1 is further supported by the lowered expression of PAI-1 in FOXC2 +/- mice in response to TGF-beta1 treatment 61 because FOXC2 has been implicated in insulin resistance. 62,63 Thiazolidinediones decrease plasma PAI-1 levels 30,31,64 and inhibit PAI-1 synthesis through anti-TNF properties in the absence of inducible peroxisome proliferator activated receptor (PPAR ) activation, 65 indicating that the TNF pathway is a potential PAI-1 inducer during the MetS and underscoring the ability of thiazolinedione to exert anti-inflammatory properties independent of PPAR activation in several cell types. 66


In summary, the parallelism between circulating PAI-1 levels and the features of the MetS may be the reflection of an active TNF/TGF-beta pathway that modulates both insulin resistance and PAI-1 synthesis.


Possible Contribution of Local Cortisol Production


Clinical observations have highlighted the link between glucocorticoids and visceral obesity. Dexamethasone and cortisol are potent inducers of PAI-1 synthesis by cultured adipocytes and human adipose tissue, 67,68 making cortisol a possible inducer of PAI-1 during visceral obesity. Excess of circulating cortisol has failed to be demonstrated during obesity but a local cortisol production may occur within adipose tissue. 11-beta-hydroxysteroid dehydrogenase (11ß-HSD1) is expressed in most tissues. It potentiates the action of endogenous glucocorticoids by converting inactive cortisone into cortisol. Several recent experiments suggested that MetS may result from elevated 11ß-HSD1 activity. 69,70 Interestingly, in adipose tissue, 11ß-HSD1 levels paralleled those of PAI-1. Using human adipose tissue explants we observed that inactive cortisone stimulated PAI-1 secretion in an 11 ß-HSD1-dependent manner. 71 11-beta-HSD1 may be a central player in linking PAI-1, the MetS, and atherosclerosis because beside its improvement of MetS 11-beta-HSD1 inhibition prevents progression of atherosclerosis in mice. 72


The Culprit May Be Glucidolipidic Disturbances Associated With the MetS


Several groups as well as ours have suggested hyperinsulinemia and hypertriglyceridemia contribute to PAI-1 synthesis. Most cell culture experiments confirm that excesses of insulin or proinsulin, 73-75 free fatty acid, or very-low-density lipoprotein (VLDL), 76-78 directly increase PAI-1 synthesis. The apparent contradiction between the inability of insulin to induce glucose uptake and its capacity to stimulate PAI-1 synthesis led some investigators to demonstrate that some genes became insulin-resistant, whereas others, including PAI-1, continued to respond normally to insulin although insulin resistance was established. 79 Moreover the signaling pathway of PAI-1 synthesis differs in normal and insulin-resistant adipocytes. 80 This difference supports the hypothesis that the signaling pathways that remain insulin-sensitive may contribute to vascular disease associated with obesity and type II diabetes.


Such a direct effect of insulin or lipoprotein is not always supported by clinical observations. Postprandial hyperinsulinemia and hypertriglyceridemia as well as hyperinsulinemia induced during a hyperinsulinemic euglycemic clamp are not associated with increased circulating level of PAI-1 in healthy or obese subjects. 55,81-83 Even more, low-dose insulin infusion was shown to decrease Egr-1, a pro-inflammatory transcription factor, in mononuclear cells as well as some prothrombotic factors such as tissue factor and PAI-1 plasma levels in obese individuals. 84


Possible Contribution of the Renin Angiotensin System


The renin-angiotensin system mainly controls blood pressure. Angiotensin-converting enzyme inhibition significantly reduces plasma PAI-1 in obese subjects. 85 The renin angiotensin system is completely expressed in human adipose tissue and several reports suggested it plays a role on PAI-1 synthesis. Angiotensin II promotes PAI-1 production and release in cultured human adipocytes via the angiotensin II type 1 receptor (AT1 receptor), and AT1 receptor blockade reduces basal PAI-1 release and abolishes angiotensin II-stimulated PAI-1 release from adipocytes. 86 It is not known how angiotensin II acts on PAI-1 synthesis. Angiotensin II may promote PAI-1 secretion in many ways because it has been involved in local uptake, synthesis, and oxidation of lipids, inflammation, as well as cellular migration and proliferation mechanisms, but a more direct pathway cannot be ruled out. 87,88


Oxidative Stress as a Central Player


In nondiabetic humans, fat accumulation as well as PAI-1 level closely positively correlates with the markers of systemic oxidative stress. 89 Production of reactive oxygen species (ROS) increased selectively in adipose tissue of obese mice, partly because of augmented expression of NADPH oxidase and lowered expression of antioxidative enzymes induced by fat accumulation. This locally increased oxidative stress dysregulates the production of adipokines by adipose tissue such as TNF, MCP-1, and PAI-1. 90 Macrophages infiltrated in adipose tissue 91 may be involved in this process by elevating ROS production in the obese adipose tissue. Remarkably, obese mice treated with an NADPH oxidase inhibitor, adipocynin, showed reduced ROS production, improved diabetes and hyperlipidemia, and attenuated dysregulation of adipokines. 90 Thus, oxidative stress in adipose tissue and probably in other tissues may play a central role in linking most of the features that characterize the MetS and plasma PAI-1 levels.


There has been a long-lasting debate about the role of ROS in oxygen sensing. Interestingly, hypoxia and ROS increase PAI-1 expression in adipocytes via distinct signaling pathways, suggesting that both may participate in PAI-1 overexpression during obesity. 92


Place of the Circadian Rhythm of PAI-1


Finally, one could wonder whether the dysregulation of PAI-1 synthesis observed during the MetS may not be driven by dysregulation of the circadian clock, an endogenous self-sustained machinery of rhythmically acting transcriptional loops. Both clock:bmal1 and clock:bmal2 heterodimers activate the PAI-1 promoter. 93 Deletion of the Clock and Bmal1 genes results not only in circadian disturbances but also in metabolic abnormalities of lipid and glucose homeostasis, a phenotype resembling the MetS. 94,95 It could thus be suspected that PAI-1 synthesis dysregulation in obese patients is secondary to alteration of the self-regulated circadian clock, but this dysregulation needs to be elucidated.


In conclusion, the causes of PAI-1 overexpression in the MetS are complex, with much interference between biological systems. Establishment of inflammation or oxidative stress at the macrophage level as fundamental precursors is tempting and may reveal interesting avenues for a better understanding of the link between atherosclerosis and the MetS.


Contribution of PAI-1 to the Development of Adipose Tissue and Insulin Resistance


Clinical Evidence that PAI-1 Is Diabetogenic


High PAI-1 levels may help to identify a high-risk population with the potential of developing atherosclerotic disease and type II diabetes. Indeed, Festa et al showed that high plasma PAI-1 levels predict the development of diabetes. In their study the association of CRP and fibrinogen with incident diabetes was significantly attenuated after adjustment for body fat, waist circumference, or insulin sensitivity. In a logistic regression model that included age, sex, ethnicity, clinical center, smoking, BMI, insulin sensitivity, physical activity, and family history of diabetes, PAI-1 still remained significantly related to incident type II diabetes. 96 Furthermore, the same group has recently shown that progression of PAI-1 levels over time, in addition to high baseline PAI-1 levels, is associated with incident diabetes. 97 Similar findings were obtained in 2 other populations. 98,99 Based on these results it has been hypothesized that PAI-1 participates in the development of key features of the MetS. This hypothesis is also sustained by the relationship between PAI-1 gene polymorphisms, obesity, and insulin resistance in population studies. The PAI-1 gene polymorphism 4G/5G in the promoter region in position -675 has been especially studied; the 4G allele is associated with increased PAI-1 transcription compared with the 5G allele in in vitro studies and with increased plasma PAI-1 levels in vivo. 100 In some studies, 4G allele carriers were more prone to obesity and MetS but not in others. 101-104 We have recently shown there is an interaction between insulin and pro-insulin levels and the -675 4G/5G PAI-1 gene polymorphism for the risk of myocardial infarction. Patients with the highest pro-insulin levels were at risk for myocardial infarction only if they were homozygous for the 4G allele, suggesting that PAI-1 genotype may influence the vascular risk associated with hyperinsulinemia. 105 All together, these results are in favor of a role of PAI-1 gene variability in the modulation of obesity-associated phenotypes.


In Vitro Support of the Role of PAI-1 in Insulin Signaling and Adipocyte Differentiation


Studies on cultured fibroblasts showed that PAI-1 interferes with insulin signaling by preventing binding of vitronectin to integrin v ß 3 and can inhibit insulin-induced protein kinase B phosphorylation. 106,107 PAI-1 also binds IGF-5 binding protein and thus impairs insulin action. 108 Studies with adipocytes revealed interesting results. Overexpression of PAI-1 by adenovirus-mediated gene transfer inhibited differentiation. Conversely, preadipocytes from PAI-1 -/- mice showed greater differentiation than those issued from wild type mice and exhibited enhanced basal as well as insulin-stimulated glucose uptake. Inhibition of PAI-1 with a neutralizing antibody promoted 3T3 adipocyte differentiation. Remarkably, PAI-1 deficiency was able to blunt the deleterious effect induced by TNF on glucose uptake and on adipocyte differentiation marker expression levels. 109 Intriguingly, using a synthetic PAI-1 inhibitor we recently observed not an increase but a decrease in human adipocyte differentiation. 110 This effect could be attributed to the human origin of the cells or to unknown properties of the inhibitor.


In Vivo Support of the Role of PAI-1 in Obesity and the Related Glucidolipidic Disturbances


The effect of PAI-1 excess has been investigated in vivo. Mice overexpressing murine PAI-1 under the control of the aP2 promoter develop high PAI-1 expression within adipose tissue. 111 These mice exhibited adipocyte hypotrophy and a higher mRNA level of a preadipocyte marker in adipose tissue, suggesting adipocyte differentiation potential is decreased. These differences were exacerbated under high-fat diet with a significant lower body weight and smaller adipocytes associated with a lower feeding efficiency in transgenic mice. These findings suggest that PAI-1 overexpression induces impaired adipose tissue growth, which is in line with the in vitro effect of PAI-1 on murine adipocyte differentiation described earlier. 110 When looking at the metabolic parameters, it appears that old transgenic mice maintained on standard fat diet exhibit significantly higher insulinemia and a tendency to higher triglyceride levels despite lower body fat. 112 These data indicate that PAI-1 overexpression may worsen the metabolic profile; these differences, however, were not found when younger transgenic mice were subjected to high-fat diet. 111 One could wonder whether local and/or systemic PAI-1 contributes to this phenotype and whether PAI-1 is directly or indirectly involved through the modulation of TNF or TGF-beta actions. PAI-1 deficiency protects against TNF effects on adipocytes 109 and it has been recently hypothesized that PAI-1 could exert its action through inhibition of the proprotein convertase, furine, involved in TGF-beta activation and insulin receptor shedding. 113


Because of the improved insulin-stimulated glucose uptake and increased differentiation induced by PAI-1 inhibition in murine cultured adipocytes, one could expect PAI-1 deficiency to lead to higher subcutaneous fat accumulation in vivo under high-fat diet. Whereas 2 studies did not demonstrate any effect of PAI- deficiency on weight gain, 114,115 2 groups found that fat accumulation was prevented with a concomitant improvement in insulin sensitivity in mice lacking PAI-1 in 2 kinds of models, a nutritionally induced 116 and a genetic 117 murine model of obesity. The protection against obesity was linked to an increase in metabolic rate, total energy expenditure, and thermogenesis. These findings suggest the plasminogen activation system may be implicated in the control of fat accumulation in a more systemic way than that initially proposed. In the adult central nervous system, tissue-type plasminogen activator (tPA) is expressed at the mRNA and protein levels in many sites. 118,119 tPA is considered a major relay to the hypothalamic paraventricular nucleus controlling satiety and conveying both excitatory and inhibitory information to the hypothalamic-pituitary-adrenal axis. 120 It could reasonably be proposed that inhibition of tPA in the central nervous system by excess systemic or local PAI-1 may affect the control of body weight by the central nervous system; this aspect needs to be investigated.


Interestingly, we recently observed that inhibition of PAI-1 with the same synthetic inhibitor previously cited, 110 may improve insulin sensitivity in mice. This synthetic, low-molecular-weight PAI-1 inhibitor was added to the normal chow of wild-type mice for 4 weeks. After insulin injection, glycemia was lower in treated animals as insulin levels after glucose injection, suggesting higher insulin sensitivity in treated mice. 112 During high-fat diet, mice treated with the same PAI-1 inhibitor had lower body weight, glycemia, and triglyceride level than nontreated mice. 110 Overall, these data support the concept that PAI inhibition has the potential to reduce obesity and improve insulin sensitivity and may represent a new therapeutic target. This needs to be confirmed in different experimental models and the mechanisms involved should be precisely defined.


Conclusion


Several new features have been added to the MetS over time because they were frequently found associated with the metabolic syndrome. PAI-1, the main inhibitor of the fibrinolytic system, belongs to this cluster and could be considered a true component of the MetS. The mechanisms linking PAI-1 to the MetS are complex and probably interrelated, and several inducers may act jointly and at several sites of synthesis. In vitro and in vivo studies have indicated that PAI-1 might be involved in the development of obesity. Thus PAI-1 may serve as a feedback loop to limit adipose tissue expansion. Further efforts with experimental and clinical studies are needed to better understand this complex interplay. In any case, these findings support the rationale to develop PAI-1 inhibitors as they may serve to control atherothrombosis and insulin resistance.


Acknowledgments


Disclosures


None.

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作者单位:Marie-Christine Alessi; Irène Juhan-VagueFrom the Laboratory of Hematology, INSERM UMR 62 Faculty of Medicine, Marseilles, France.

日期:2008年12月28日 - 来自[2006年第26卷第10期]栏目
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Augmentation of Proliferation of Vascular Smooth Muscle Cells by Plasminogen Activator Inhibitor Type

【摘要】  Objective- Proliferation of vascular smooth muscle cells (VSMCs) contributes to restenosis after coronary intervention. We have shown previously that increased expression of plasminogen activator inhibitor type 1 (PAI-1) limits VSMC apoptosis. Because apoptosis and proliferation appear to be linked, we sought to determine whether increased PAI-1 would affect VSMC proliferation.

Methods and Results- VSMCs were explanted from control and transgenic mice (SM22-PAI + ) in which VSMC expression of PAI-1 was increased. Increased growth of SM22-PAI + -VSMCs (2.3±0.4-fold) reflected, at least partially, increased proliferation. Greater expression of FLICE-like inhibitory protein (FLIP; 2.7-fold) and its cleaved active form were seen in SM22-PAI + -VSMCs. The balance between caspase-8 and FLIP favored proliferation in SM22-PAI + -VSMCs. Increased expression of NF- B and activation of extracellular signal-regulated kinase (ERK) were demonstrated in SM22-PAI + -VSMCs (fold=NF- B=2.2±0.1, fold=phosphorylated-ERK=1.6±0.1). Results were confirmed when expression of PAI-1 was increased by transfection. Inhibition of NF- B and ERK attenuated proliferation in SM22-PAI + -VSMCs. Increased expression of PAI-1 promoted proliferation when VSMCs were exposed to tumor necrosis factor (TNF).

Conclusions- Increased expression of PAI-1 is associated with greater activity of FLIP that promotes VSMC proliferation through NF- B and ERK. Thus, when vascular wall expression of PAI-1 is increased, restenosis after coronary intervention is likely to be potentiated by greater proliferation of VSMC and resistance to apoptosis.

We determined that increased expression of PAI-1 increased proliferation of VSMCs. The balance between caspase-8 and FLIP favored proliferation when PAI-1 was increased. Thus, when vascular wall PAI-1 is increased, restenosis after coronary intervention is likely to be potentiated by greater proliferation of VSMCs and resistance to apoptosis.

【关键词】  proliferation VSMC plasminogen activator inhibitor type FLIP restenosis


Introduction


Despite technological advances, restenosis remains an important limitation of coronary intervention, particularly in patients with diabetes. 1,2 Proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in restenosis after vessel injury associated with coronary intervention. 3,4 Consistent with this observation, inhibition of VSMC proliferation decreases neointimal cellularity after balloon injury. 5


In contrast to the role of VSMCs in restenosis, migration of VSMCs into the neointima is a determinant of plaque vulnerability. 6,7 We have shown that increased expression of plasminogen activator inhibitor type 1 (PAI-1) limits migration of VSMCs, a phenomenon that may result in promoting generation of plaques more prone to rupture. 8 The present study was designed to determine whether increased PAI-1 influences the proliferation of VSMCs.


Increased expression of PAI-1 has been associated with cellular proliferation and restenosis; however, the mechanism(s) responsible have not been elucidated. Increased expression of PAI-1 is associated with greater proliferation of neoplastic cells. 9,10 Expression of PAI-1 is increased in the vessel wall in patients with diabetes, a group particularly prone to exhibit restenosis. 11,12 In mice and other laboratory animals, increased arterial wall expression of PAI-1 has been found to promote increased neointimal cellularity after vascular injury. 13-15 By contrast, the cellular response to exogenous injury was significantly decreased after arterial injury in PAI-1-deficient mice. 14,16


Results of recent studies have suggested that the balance between the activity of FLICE-like inhibitory protein (FLIP) and caspase-8 determines whether selected signals lead to apoptosis or proliferation. FLIP diverts Fas-mediated signals from death to proliferation in lymphocytes. 17,18 In addition, increased expression of FLIP decreases apoptosis of pancreatic ß cells and increases their proliferation. 19 We have shown previously that increased expression of PAI-1 inhibits apoptosis of VSMCs by directly inhibiting caspase-3. 20 Because inhibition of caspase activity was found to increase expression of FLIP, 21,22 we hypothesized that inhibition of caspase-3 by PAI-1 would increase expression or activation of FLIP. FLIP has been demonstrated to lead to activation of nuclear factor B (NF- B) and extracellular signal-regulated kinase (ERK) that promote proliferation. 18 NF- B is a key regulator of genes involved in cell activation, survival, and proliferation. Activation of NF- B induces VSMC proliferation, 23 and inhibition of NF- B inhibits smooth muscle cell proliferation and promotes apoptosis. 24,25 ERK signaling influences cellular processes such as proliferation, differentiation, and cell cycle progression. 26 Inhibition of ERK decreases the growth and proliferation of smooth muscle cells. 27 Accordingly, we sought to determine whether increased expression of PAI-1 affected the expression and activation of FLIP and its downstream mediators NF- B and ERK to promote cell survival/proliferation.


Materials and Methods


Cell Culture


VSMCs were obtained by explantation from the aortas of SM22-PAI + mice that exhibit a 3-fold increased expression of PAI-1 8,20 and negative control littermates, and grown in Dulbecco Modified Eagle Medium (DMEM; Gibco-BRL) supplemented with 20% fetal bovine serum as we described previously. 20 The identity of smooth muscle cells was confirmed by Western blot and flow cytometry with smooth muscle cell specific -actin antibody. 20 Experiments were performed with cells in DMEM with Hams nutrient mixture F12 (DME/F12; Gibco-BRL). Tumor necrosis factor (TNF) was purchased from Sigma. All experiments were performed with VSMCs maintained in culture for 2 to 8 passages.


Growth of VSMC was determined by cell counts performed in triplicate daily for 6 days with the use of flow cytometry (Beckman Coulter, Epics XL). Each VSMC line was characterized twice.


Inhibitors of ERK and NF- B


VSMCs at 80% confluence were exposed to an inhibitor of: MAPK (PD98059, 10 µmol/L; Calbiochem) 27 and an inhibitor of NF- B: aminium pyrrolidithiocarbamate (APDC, 50 µmol/L; Calbiochem) 28 or control media. The accumulation and the proliferation of cells were determined 24 hours after the cells were exposed to inhibitors or control conditions.


Adenovirus-Mediated PAI-1 Gene Transfection


VSMCs at 50% confluence were infected with adenovirus containing PAI-1(AdPAI-1) or control (AdRR5 without PAI-1) adenovirus (kindly provided by Dr P Carmeliet, University of Leuven, Belgium) 29 at 200 virus particle per cell in serum-free media. After 5 hours, serum-containing growth medium was added. Media containing the adenovirus were removed after 12 hours and replaced with DME/F12 media. The proliferation of AdPAI-1-infected VSMCs was determined after 72 hours and compared with that of VSMC infected with AdRR5.


Determination of Proliferation of VSMCs


DNA synthesis was assayed by the incorporation of 5-bromo-2-deoxyuridine (BrdU) 10 (ABSULUTE-S T proliferation kit; Phoenix Flow Systems). Cells that were 30% to 50% confluent were pulse labeled 2 hours with BrdU and fixed in ice cold 70% ethanol overnight at -20°C. Photolysis of DNA at sites of BrdU incorporation was induced with ultraviolet light. Subsequent labeling with deoxynucleotide triphosphate was catalyzed with terminal deoxynucleotidyl transferase. BrdU incorporation was identified with a fluorescein-labeled anti-BrdU antibody by flow cytometry.


Cell proliferation was determined also by the dye dilution method with carboxyfluorescein diacetate succinimidyl ester (CFSE) as described. 30 VSMCs (1 x 10 7 ) were washed with PBS, exposed to 2 µmol/L CFSE (Sigma) for 10 minutes, and then diluted with DMEM plus 10% FBS before being washed 3 times. After incubation for 1 hour, VSMC were divided equally into 3 groups. The first group was analyzed immediately with the use of flow cytometry to delineate fluorescence associated with the parent cells. The other 2 groups were incubated for 48 hours. After dissociation from the culture plate, cells were analyzed with the use of flow cytometry (Beckman Coulter) and MODFIT software.


Western Blot


VSMCs that had been exposed to serum-free DME/F12 media or DME/F12 with TNF (10 ng/mL) were lysed in lysis buffer containing 20 mmol/L Tris-HCl, pH 7.4, 0.4 mol/L KCl, 2 mmol/L dithiothreitol, and 10% glycerol. Concentrations of protein were determined with Bicinchoninic acid kit (Sigma). Extracted proteins were separated through a sodium dodecyl sulfate polyacrylamide gel, transferred to polyvinylidene difluoride membrane (Bio-Rad), and incubated with anti-FLIP, anti-Caspase-8 (Alexis Biotechnology Inc), anti-NF- B (Saint Cruz Biotech Inc), and anti-ERK, anti-pERK, anti-MAPK, anti-p-MAPK, anti-Raf-1, and anti-p-Raf-1 (Cell Signaling Technology) or anti-GAPDH monoclonal antibody (Research Diagnostics Inc, to confirm equal loading). The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence detection reagents (Roche Applied Science). The density of bands was analyzed with the use of densitometry and Kodak software (Eastman Kodak).


Statistical Analysis


Results are mean±SD. Differences between groups were identified with the use of Student t tests. Significance was defined as P <0.05.


Results


Growth and Proliferation of VSMCs From SM22-PAI + Mice


We have previously generated PAI-1 transgenic mice with increased expression of PAI-1 in VSMCs and demonstrated that increased expression of PAI-1 renders VSMCs more resistant to apoptosis. 8,20 In the present studies, greater proliferation was demonstrated in VSMCs from PAI-1 transgenic mice (SM22-PAI + ). After 6 days in culture, the average number of SM22-PAI + VSMCs was 2.3±0.4-fold greater than control VSMCs ( Figure 1 A, n=6, P <0.001). The greater accumulation of cells reflected greater proliferation of VSMCs from SM22-PAI + mice when assessed by BrdU incorporation ( Figure 1 B, control=17±2% and SM22-PAI + =31±6%, n=4, P =0.006) or with the use of cell tracking dye. SM22-PAI + VSMCs exhibited a 45±8% greater (n=4, P <0.001) rate of proliferation ( Figure 1 C).


Figure 1. Growth and proliferation of VSMCs from SM22-PAI + and control mice. A, VSMCs from 3 pairs of littermates were cultured in growth media. VSMCs from the littermate pairs were characterized at the same passage. Cells were counted in triplicate daily for 6 days with flow cytometry. Two independent determinations were performed for each VSMC pair. Results are mean±SD. B, Incorporation of BrdU was determined in 30% to 50% confluent cells as described in Material and Methods. The mean±SD of the percentage of cells with BrdU incorporation from 4 independent experiments is shown. C, Representative CFSE fluorescence histograms. VSMCs (1 x 10 7 cells) were labeled with 2 µmol/L CFSE for 1 hour and then divided into 3 equal groups. One group was analyzed immediately with the use of a flow cytometer to detect the fluorescence of parent cells. The remaining aliquots were incubated for 48 hours before flow cytometric analysis. Results were compared with the use of MODFIT software. Two independent experiments were performed for each cell line.


Expression and Activation of FLIP, Caspase-8, NF- B, and ERK


Increased intracellular expression and activity of FLIP were seen in SM22-PAI + -VSMCs compared control VSMCs ( Figure 2A and 2 B). The expression of FLIP in SM22-PAI + -VSMCs was 2.7-fold greater than that in control cells ( Figure 2 B, n=4, P <0.001). In addition, the cleaved active form of FLIP (p43), known to promote proliferation, was the predominant species of FLIP seen in VSMC from SM22-PAI + mice ( Figure 2 A). By contrast, the full-length form was the predominant species in cells from controls.


Figure 2. Expression of FLIP, caspase-8, and NF- B in VSMCs from SM22-PAI + and control mice. A and B, Western blot analysis of the expression of FLIP and NF- B in VSMCs from SM22-PAI + mice and negative control littermates. A representative blot from 4 (FLIP) or 6 (NF- B) independent experiments is shown in A. Density of bands was analyzed with the use of densitometry. After results were normalized for expression of GAPDH, the relative density of FLIP (total and cleaved forms) and NF- B (p65) in VSMCs from control was defined as 1. The mean±SD of the fold increases of relative density of FLIP and NF- B in VSMCs from SM22-PAI + mice is shown in B. C and D, Western blot analysis of the expression of Caspase-8 in VSMCs from SM22-PAI + mice and negative control littermates. A representative blot from 4 independent experiments is shown in C. The density of the cleaved forms of caspase-8 was compared with that of the pro-caspase-8 in each cell lines to determine the ratio of cleaved caspase-8 to pro-caspase-8. The mean±SD of this ratio in VSMCs from SM22-PAI + mice and their control littermates is shown.


Caspase-8 can activate caspase-3 to induce apoptosis or cleave FLIP to generate p43 that induces proliferation. The protein expression of caspase-8 by VSMCs from SM22-PAI + mice and their control littermates was similar ( Figure 2 C). Cleavage of procaspase-8 was apparent in protein extracts from SM22-PAI + VSMCs and from VSMCs explanted from control littermates. However, the ratio of the cleaved form to procaspase-8 was greater in VSMCs from SM22-PAI + mice ( Figure 2C and 2 D, n=4, P <0.001).


NF- B mediates the proliferative effect of FLIP. 18 Expression of NF- B was greater in SM22-PAI + -VSMCs under control condition ( Figure 2A and 2 B, fold induction=2.2±0.1, n=6, P <0.001). Thus, increased protein expression of NF- B in SM22-PAI + VSMC may contribute to the increased proliferation of these cells associated with increased expression and activation of FLIP.


A second mechanism by which FLIP may induce proliferation is through ERK signaling. 18 ERK signaling appears to be critical in diverting FLIP-mediated death receptor-induced apoptosis signals such as TNF from apoptosis to cell survival or proliferation. 18 The intracellular protein expression of nonphosphorylated Raf-1, ERK, and p38MAPK was similar in VSMCs from SM22-PAI + mice and control littermates. Addition of TNF to culture media did not affect the expression of the nonphosphorylated molecules ( Figure 3 A). By contrast, activation (phosphorylation) of Raf-1 and ERK was increased in VSMCs from SM22-PAI + mice compared with that from control littermates ( Figure 3A through 3 C, n=3, P <0.05). Moreover, activation of ERK (p-ERK) and Raf-1 (p-Raf-1) was increased by TNF in SM22-PAI + VSMCs but not control VSMCs ( Figure 3A through 3 C, n=3, P <0.05). Activation of p38MAPK was similar in VSMCs from SM22-PAI + mice and control littermates, and it was not affected by TNF. These results demonstrate that activation of ERK and Raf-1 is increased in VSMCs with increased expression of PAI-1 and that increased PAI-1 by VSMCs diverts the death signal of TNF to that for survival/proliferation.


Figure 3. Expression of ERK, Raf-1, and p38MAPK in VSMCs from SM22-PAI + and control mice. A, Western blot analyses were performed with proteins extracted from SM22-PAI + and control VSMCs exposed to control media or TNF (10 ng/mL in DME/F12). Antibodies against Raf-1, p-Raf-1, ERK, p-ERK, p38MAPK, and p-p38MAPK were used. Representative blot of 3 independent experiments is shown. B and C, The density of bands was analyzed with the use of densitometry and normalized to the density of GAPDH. The relative density of pRaf-1 and pERK in control VSMCs exposed to DMEM/F12 was defined as 1, respectively. The relative density of the pRaf-1 and pERK in control VSMC exposed to TNFs or in SM22-PAI + VSMCs exposed to DMEM/F12 and TNF was compared with that in control VSMCs exposed to DMEM/F12. The mean±SD of 3 independent experiments is shown.


Adenoviral Transfection of PAI-1


Consistent with our observation with SM22-PAI + -VSMCs, VSMCs in which expression of PAI-1 was increased by adenovirus transfection (AdPAI-1) exhibited increased growth and proliferation compared with VSMCs infected with control virus ( Figure 4A and 4 B, n=3, P <0.05). The expression and cleavage of FLIP was increased in VSMC infected with AdPAI-1 compared with control virus ( Figure 4C and 4 D, n=3, P <0.05). Further, increased NF- B and p-ERK was identified in AdPAI-1-infected cells ( Figure 4C and 4 D, n=3, P <0.05).


Figure 4. A and B, Growth (A) and proliferation (B) of VSMCs infected with AdPAI-1 or control virus. VSMCs from control mice were grown to 50% confluence and infected with adenovirus expressing PAI-1 (AdPAI-1) or control (AdRR5 without PAI-1) adenovirus. Growth and proliferation were determined after 72 hours. The mean±SD of three independent experiments is shown. C and D, Western blot analyses of FLIP, p-ERK, and NF- B in VSMCs infected with AdPAI-1 or control virus were performed with specific antibodies. Representative blots of 3 independent experiments are shown in C. The density of bands was analyzed with densitometry and normalized to the density of GAPDH. The relative density of FLIP, p-ERK, and NF- B in VSMCs infected with control virus was defined as 1, respectively. The mean±SD of 3 independent experiments is shown.


Inhibition of NF- B and ERK


Inhibition of NF- B decreased proliferation of VSMCs from control and SM22-PAI + mice ( Figure 5, n=3, P <0.001). Inhibition of ERK did not affect proliferation of control-VSMCs ( Figure 5, n=3, P =NS) but did inhibit proliferation of SM22-PAI + -VSMCs ( Figure 5, n=3, P <0.05). Accordingly, these results demonstrate that NF- B is a key mediator of VSMC proliferation in vitro and are consistent with our observation that increased expression of PAI-1 increases proliferation through both ERK and NF- B.


Figure 5. Effect of ERK and NF- B antagonists on VSMC proliferation. VSMCs explanted from control and SM22-PAI + mice were grown in culture media with and without APDC (50 µmol/L) or PD98059 (10 µmol/L) for 24 hours. Proliferation was determined by the incorporation of BrdU. The percentage of control VSMCs that incorporated BrdU in the absence of antagonist was defined as 100%. Results are mean±SD of 3 independent experiments and are the relative rates of incorporation compared with control VSMCs.


Discussion


In the present study we demonstrated that increased expression of PAI-1 by VSMCs increases their proliferation. Previously we have reported that increased expression of PAI-1 renders VSMCs resistant to apoptosis. 20 The resistance to apoptosis and the induction of increased proliferation appear to be linked. PAI-1 inhibits directly the activity of caspase-3 but not caspase-8. 20 This inhibition of caspase-3 parallels increased expression and cleavage of FLIP and downstream regulators of proliferation regulators such as NF- B and ERK.


Our observation that increased expression of PAI-1 increases proliferation of VSMCs is consistent with those made with cells from rats, 13,15 mice, 14 and humans. 31 Increased expression of PAI-1 increases neointimal formation after balloon injury of carotid arteries in rats 13 and mice. 14 Conversely, decreased expression of PAI-1 is associated with attenuation of VSMC proliferation. 32 By contrast, Carmeliet and colleagues found that the proliferation of VSMCs after electrical injury was similar in wild-type compared with PAI-1 knockout mice. 29 Notably, PAI-1 was not detected in uninjured arteries from the wild-type mice. 29 Thus, the similar response to arterial injury in wild-type and PAI-1 knockout mice may reflect the lack of a substantial difference in the expression of PAI-1 in VSMCs in the two groups. Their observation of decreased neointimal formation after injury when PAI-1 expression was restored with adenoviral gene transfer 29 is consistent with our previous observation that increased expression of PAI-1 inhibits VSMC contribution to neointimal formation presumably by inhibiting migration. 8 Differing effects of PAI-1 on the in vitro proliferation of aortic endothelial cells 33 and VSMCs 34 are likely to be a reflection of differences in cell type, culture conditions, genotypes, and experimental design. Nevertheless, these results are consistent with the observation that increased intracellular expression PAI-1 promotes proliferation of VSMCs.


Interaction between signals that initiate apoptosis and proliferation have been observed in studies of lymphocytes 19 and VSMCs. 35 FLIP renders many types of cells resistant to death receptor-mediated apoptosis. 36,37 The expression of both PAI-1 and FLIP are increased in highly proliferative cells 9,38 consistent with a link between these two proteins and cell proliferation. Because FLIP is an enzymatically inactive homologue of caspase-8, 36 the relative expression of FLIP and caspase-8 determines whether cells undergo apoptosis or proliferation. 18,39 In TNF-induced apoptosis, procaspase-8 binds to the death effector domain and undergoes autocatalytic activation that generates an active heterotetramer consisting of two large (p20) and two small subunits (p10). 40 Caspase-8 initiates apoptosis through cleavage of downstream substrates such as procaspase-3. 41 FLIP can limit apoptosis by binding to death receptors thereby blocking activation of caspase-8. 36


We found that the expression of caspase-8 was similar in VSMCs from SM22-PAI + mice and negative control littermates. This observation is consistent with previous work suggesting that expression of caspase-8 is quite stable. 42 Minimal or no variation in the expression of caspase-8 has been seen in response to diverse stimuli. Modest changes in expression of FLIP appear to determine whether a cell proliferates or dies in response to selected stimuli. 42 Thus, our finding of increased expression of FLIP in VSMCs with increased expression of PAI-1 is consistent with a pivotal role of FLIP in cell proliferation.


Decreased apoptosis and increased proliferation have been seen in lymphocytes from human subjects who were homozygous for deficiency of caspase-8. 43 Similar results have been obtained in studies of mice lacking caspase-8 in their T-cell lineage. 44 Thus, the absence of caspase-8 is associated with increased proliferation in lymphocytes, an observation consistent with our findings in VSMCs with constitutively increased expression of PAI-1.


Caspase-8 cleaves FLIP at Asp-376 to generate N-terminal FLIP (p43) and C-terminal FLIP(p12). 36,40,45 FLIP in turn permits the cleavage of procaspase-8. 36 The cleaved fragment of FLIP (p43) binds preferentially compared with caspase-8 to the death effector domain. 39,40 Further, cleaved (p43) FLIP binds TNF-receptor-associate factor 2 (TRAF2) more effectively than full-length FLIP. 45 The binding of TNF to TRAF2 promotes activation of NF- B. 45 We observed increased expression of the cleaved form of FLIP (p43) associated with increased intracellular expression of PAI-1, a phenomenon expected to promote proliferation.


Increased proliferation of VSMCs from SM22-PAI + mice appears to be mediated, at least in part, by increased expression of NF- B. Activation of NF- B induces proliferation of VSMCs. 23 Inhibition of NF- B decreases proliferation in a variety of cells including hepatocytes, epithelial cells and VSMCs. 24,25 In addition, activation of ERK promotes smooth muscle cell growth and proliferation. 27 Our results demonstrate that increased expression of PAI-1 is associated with increased activation of ERK and Raf-1, but not p38MAPK. Further, increased expression of PAI-1 appears to divert the death signal of TNF to a signal that promotes proliferation (phosphorylation of ERK and Raf-1). Accordingly, we hypothesized that increased intracellular expression of PAI-1 that inhibits apoptosis and promotes cell survival/proliferation in VSMC through FLIP and downstream mediators NF- B and ERK ( Figure 6 ).


Figure 6. Proposed model for the influence of PAI-1 on the interaction between signals for apoptosis and proliferation. Activation of caspase-3 induces apoptosis. Inhibition of caspase-3 by PAI-1 decreases apoptosis in VSMCs. Increased expression and cleavage of FLIP induces NF- B and ERK signaling that induces proliferation. Accordingly, increased expression of PAI-1 in VSMCs may increase expression and activation of FLIP and its downstream NF- B and ERK signaling pathways and there by promote proliferation.


In summary, we have demonstrated that increased expression of PAI-1 increases proliferation of VSMCs as well as rendering them more resistant to apoptosis. We have shown previously that PAI-1 attenuates apoptosis by inhibiting caspase-3. Inhibition of caspase-3 appears to promote caspase-8-mediated cleavage and activation of FLIP that promotes proliferation through induction of NF- B and activation of ERK signaling. These results are consistent with a direct effect of PAI-1 on VSMC proliferation likely to contribute to restenosis in patients with conditions such as diabetes mellitus that are associated with increased expression of PAI-1 in vessel walls.


Acknowledgments


The authors thank Heidi Taatjes for her excellent technical support and the Vermont Cancer Center Flow Cytometry Facility for technical assistance.


Source of Funding


This work was supported by a Scientist Development Grant from American Heart Associate to Y. Chen (National Center).


Disclosures


None.

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作者单位:Department of Medicine and the Cardiovascular Research Institute (Y.C., R.C.B., R.J.K., B.E.S., D.J.S.), University of Vermont, Burlington; and the Department of Pathology (Y.C.), University of Alabama at Birmingham.

日期:2008年12月28日 - 来自[2006年第26卷第8期]栏目
共 2 页,当前第 1 页 9 1 2 :

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