游离脂肪酸与代谢综合征中促凝血状态的关系研究
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摘要
背景
代谢综合征患者表现为显著的促凝血状态,其中凝血功能增强,纤维溶解活性降低,血小板的活性和功能增强。促凝血状态的存在造成患者病理性血栓形成的风险增加,进而导致心肌梗死、脑卒中、外周血管疾病等临床事件的发生。虽然众多的临床研究证实了促凝血状态是代谢综合征的重要特征之一,但其病理生理学特点及具体的发生机制尚不明确。
活性蛋白C(activated protein C, APC)是体内重要的抗凝分子。凝血酶(thrombin)与血栓调节蛋白(thrombomodulin, TM)结合后,能高效地促使蛋白C转化为活性蛋白C。APC能和辅助因子蛋白S结合,降解凝血因子Ⅷa和Va,从而发挥抗凝作用。另外,APC能抑制组织型纤溶酶原激活物抑制剂(PAl-1)的活性,进而促进纤维蛋白溶解。此外,APC还具有抗炎和抑制凋亡的功能。TM和内皮表面蛋白C受体(endothelial protein C receptor, EPCR)能够调控蛋白C的激活过程。以往的研究表明,TM表达减少能抑制蛋白C的激活,造成血栓形成。局部TM表达减少与动脉粥样硬化病变有关。因此,TM可能发挥血管保护作用从而发挥预防心血管疾病的作用。TM-蛋白C系统的调控异常可能与代谢综合征中促凝血状态的发生有关。
代谢综合征中促凝血状态的发生是一个非常复杂的病理生理过程。循环中游离脂肪酸(free fatty acids, FFAs)水平升高是代谢综合征的重要特征。因此,代谢综合征患者的血管系统长期暴露于高FFAs状态中。临床研究表明,循环中FFAs水平升高能直接损害血管功能,与心肌梗死、脑卒中和猝死的发生密切相关。长期的高FFAs可能导致异常的血栓形成。但是游离脂肪酸能否调节TM的表达以及具体的机制如何,目前尚缺乏相关研究。
针对以上问题,我们提出以下假设：高FFAs能导致TM-蛋白C系统表达失调,进而促使代谢综合征中促凝血状态的发生。通过本研究有助于进一步了解代谢综合征中促凝血状态的发生机制,为急性心脑血管疾病的预防和治疗提供新的思路和治疗靶点。
研究目的
1.观察高游离脂肪酸水平与代谢综合征中促凝血状态的关系。
2.探讨游离脂肪酸对TM-蛋白C抗凝系统的作用以及可能的分子生物学机制。
材料与方法
1.动物模型
8-10周龄的雄性小鼠30只,分为两组,每组15只。一组给与普通饮食(含10%脂肪),一组给与高脂饮食(含50%脂肪)20周。实验结束后,留取主动脉,用于后续的病理学检查和分子生物学检查。
2.鼠尾静脉出血时间测定
小鼠麻醉后,在鼠尾直径约为1mm左右处,用锐利的刀片横断鼠尾。然后迅速将鼠尾置于37摄氏度的PBS溶液中,记录从出血到血流终止的时间,记为出血时间。
3.三氯化铁诱导的颈动脉血栓模型
利用FeCl3损伤方法造成血栓形成。小鼠腹腔麻醉后,行颈部正中切口,钝性分离,暴露左侧颈总动脉,把超声探头置于颈动脉表面,监测基础状态下血流情况。把滤纸切成1.5 mm X 2 mm的细条,在10%的FeCl3溶液中充分浸泡,将滤纸片置于颈动脉表面,放置3分钟,然后去除滤纸片。迅速用超声探头持续监测颈动脉血流情况,监测时间为30分钟。记录从放置滤纸片到血流完全中断的时间。
4.代谢指标检测
实验结束前,抽取小鼠静脉血。利用常规生物学方法,检测血液中游离脂肪酸(FFA)、血糖、胰岛素,血脂TQ VLDL, LDL, HDL水平。
5.病理学检测
实验结束,留取小鼠主动脉,多聚甲醛固定,包埋,制作组织切片。免疫组织化学方法观察血管壁TM,PAI-1,TF的表达。
6.内皮细胞培养
人主动脉内皮细胞株(human aortic endothelial cells, HAECs),使用内皮细胞生长培养基-2(endothelial cell growth medium-2, EGM-2),在37℃和5%CO2孵育箱中进培养。每2-3天更换培养液。第5-9代细胞用于实验。
7.脂肪酸制备
首先将游离脂肪酸(FFAs)溶解在酒精中,并与10%的不含FFAs,低内毒素的BSA混合,使其终浓度在1-5 mM。所有溶液的PH值调整在7.5左右,使用滤器消毒,-200C储存。将不含有PA的BSA溶液作为对照。在处理细胞之前,将储存的PA按照1：10的比例,使用培养基进行稀释。
8.蛋白C活性检测
内皮细胞被种植在96孔板中,给与不同浓度、不同种类的FFAs刺激24小时,然后根据试剂盒说明,检测蛋白C的活性。
9.siRNA诱导的基因沉默
使用特异性的siRNA沉默相关基因,应用LipofectAMINETM 2000脂质体包裹,并转染HAECs.
10.蛋白印迹分析
使用细胞裂解液或组织裂解液提取细胞和小鼠主动脉的总蛋白。以SDS-PAGE电泳、转膜、一抗孵育、二抗孵育,最后采用化学发光法观察结果。
11.实时定量PCR
使用Trizol提取细胞的总RNA。应用iScript cDNA合成酶将mRNA逆转录成cDNA。使用iCycler iQ荧光探针系统进行实时PCR。
12.质粒DNA转染
分别使用野生型表达质粒(wide type plasmid)、持续激活型表达质粒(constitutively active plasmid)、显性负向表达质粒(dominant negative plasmid),首先使用LipofectAMINETM 2000脂质体包裹,并转染内皮细胞。然后再给与PA处理。
结果
1.动物的基本特征
与普通饮食的小鼠相比,高脂饮食导致小鼠代谢指标明显异常。体重明显增加,血液中游离脂肪酸、血糖、胰岛素、血脂水平明显增加(P均<0.05)。
2.高脂饮食导致小鼠体内出现明显的促凝血状态
与对照组小鼠相比,高脂饮食导致小鼠尾静脉出血时间明显缩短,三氯化铁损伤后颈总动脉血栓形成时间明显缩短(P0.01),提示这些小鼠体内存在明显的凝血异常。
3.高脂饮食导致小鼠体内TM表达明显减少,PAI-1和TF表达增加
我们检测了小鼠主动脉中TM,PAI-1和TF等凝血指标的表达。免疫组化结果显示,与对照组比较,高脂喂养的小鼠血管壁TM表达明显减少,PAI-1和TF的表达增加。此外,Western blot结果表明,高脂喂养的小鼠主动脉TM表达明显减少(P<0.001),PAI-1和TF的表达增加(P<0.01)。
4.高脂饮食能激活体内JNK和p38 MAPK应激信号通路
Western blot结果表明,高脂饮食导致小鼠主动脉内JNK和p38 MAPK磷酸化蛋白／总蛋白的比值增加(P<0.01)。
5.游离脂肪酸抑制内皮细胞中TM和EPCR的表达,并增加TF的表达
内皮细胞给与不同浓度的棕榈酸(palmitic acid, PA)、亚油酸(linoleic acid,LA)、油酸(oleic acid, OA)刺激24小时。Western blot结果显示,PA和LA能显著抑制TM和EPCR的表达(P均<0.01),并增加TF的表达(P<0.05)。但是OA对TM,EPCR和TF的蛋白表达没有明显作用(P>0.05)。RT-PCR结果显示PA能剂量依赖性地抑制TM mRNA的表达(P<0.01)。
6.游离脂肪酸抑制内皮细胞中蛋白c的激活
用不同浓度的PA,LA和OA处理内皮细胞24小时,检测蛋白C的活性。PA和LA呈剂量依赖性的抑制蛋白C的活性(P<0.01),但是OA对蛋白C的活性没有明显作用(P>0.05)。
7.JNK和p38信号通路参与PA抑制TM基因表达的过程
使用特异性的siRNA分别敲除JNK和p38通路,PA导致的TM表达降低的作用减弱(P<0.01)。野生型JNK质粒DNA转染能加强PA对TM的抑制作用(P<0.05),而显性负向的JNK质粒DNA转染没有这一作用。
8.转录因子Foxol参与PA抑制TM基因表达的过程
使用特异性的Foxo1 siRNA沉默Foxol的表达,PA抑制的TM蛋白表达的作用减低(P<0.001)。野生型和持续激活型Foxol质粒DNA转染能加强PA对TM的抑制作用(P<0.05),而显性负向的Foxol质粒DNA转染能阻止这一作用(P>0.05)。
结论
1.高脂饮食导致小鼠血液中游离脂肪酸水平升高,出血时间缩短,血栓形成时间缩短,提示代谢综合征模型小鼠体内存在促凝血状态。
2.高脂饮食小鼠主动脉中抗凝因子TM表达减少,促凝因子PAI-1和TF表达增加,提示这些小鼠体内凝血和抗凝调控异常。
3.PA和LA抑制内皮细胞TM和EPCR的表达,并增加PAI-1的表达,降低蛋白C的活性,说明游离脂肪酸能导致细胞中凝血和抗凝因子表达失调。
4.在PA抑制TM表达的过程中,应激信号通路JNK和p38MAPK参与了对TM表达的调节。
5.在PA抑制TM表达的过程中,Foxol作为重要的转录因子参与了抑制TM表达的过程。
总之,代谢综合征模型小鼠血液中游离脂肪酸水平升高,体内凝血和抗凝平衡失调,表现为明显的促凝血状态。PA能减少内皮细胞中TM的表达,降低蛋白C的活性。PA可以通过JNK,p38/Foxol信号传导通路抑制内皮细胞中TM的表达。
背景
代谢综合征患者表现为显著的促凝血状态,这些患者发生心肌梗死、脑卒中、外周血管疾病等血栓性并发症的风险增加。临床上抗血栓治疗能够改善急性心脑血管并发症患者的生存率。虽然既往的研究已经发现代谢综合征患者凝血和纤维溶解功能异常,但是有关促凝血状态的发生机制目前尚未完全明确。
血管内皮能分泌多种活性物质,例如：vWF,血栓调节蛋白(thrombomodulin,TM),组织型纤溶酶原激活剂(tissue plasminogen activator, t-PA),组织型纤溶酶原激活物抑制剂(plasminogen activator inhibitor, PAI-1),在调控促凝和抗凝平衡过程发挥着重要作用。在这些活性分子中,TM-蛋白C系统是内皮组织重要的生理性抗凝系统。内皮细胞功能失调可导致凝血功能异常,并促使血栓形成。
TM是一种定位于内皮细胞表面的糖蛋白,是激活蛋白C的一种重要的活性物质。TM与凝血酶(thrombin)结合后,促使蛋白C转化为有活性的蛋白C。活性蛋白C一方面通过降解凝血因子Ⅷa和Va发挥抑制凝血过程；一方面可使PAI-1失去活性从而增强纤维溶解过程。TM在抗凝血过程中发挥着重要作用。TM基因突变或表达减少能促使动脉血栓的形成；而局部TM基因过度表达能避免兔动脉血栓形成。另外,TM基因还具有抗炎和抑制凋亡的作用。多项研究发现,TM能抑制炎症反应,阻止细胞凋亡。在动脉粥样硬化等血管性疾病发生过程中,TM基因表达降低。多种刺激,诸如：炎症因子、管壁张力、氧化的脂质等能抑制TM基因的表达。虽然众多的研究发现TM表达降低与病理性血栓形成有关,但是导致TM表达降低的具体机制尚不完全明确。
MAPKs作为体内广泛存在的丝氨酸／苏氨酸蛋白调节激酶,在细胞的增殖、分化、转化和凋亡过程中发挥着重要作用。现有研究表明,包括动脉粥样硬化在内的多种心血管疾病过程,伴随着JNK和p38 MAPK应激信号通路的异常激活。多种代谢刺激和炎症因子能激活JNK和p38 MAPK应激信号通路。游离脂肪酸(free fatty acids,FFAs)也能够激活JNK和p38 MAPK应激信号通路,并参与游离脂肪酸导致胰岛素抵抗的过程。我们已经发现JNK和p38 MAPK应激信号通路参与调控TM基因的表达,但是具体的分子生物学机制目前尚不清楚。
ATF-2是位于JNK和p38 MAPK应激信号通路下游的转录因子。以往的研究显示ATF-2参与调控LPS诱导的组织因子(tissue factor,TF)表达的过程,因此转录因子ATF-2可能与血栓形成失调有关。但是,转录因子ATF-2能否参与调控TM基因的表达目前尚不明确。
因此,在这项研究中,我们检测了转录因子ATF-2作为JNK的下游转录因子,通过与HDAC4结合形成转录抑制复合物从而抑制TM基因的表达的具体机制。这将为病理性血栓及心血管疾病的治疗干预提供新的干预靶点。
研究目的
1.明确转录因子ATF-2在调控TM基因表达过程中的作用。
2.探讨ATF-2调控TM基因表达的可能的分子生物学机制。
材科与方法
1.细胞培养
人主动脉内皮细胞株(human aortic endothelial cells, HAECs),使用含有2%的胎牛血清,FGF-2, VEGF, IGF-1, EGF等生长因子的内皮细胞生长培养基-2(endothelial cell growth medium-2, EGM-2),在37℃和5%CO2孵育箱中进培养。每2-3天更换培养液。第5-9代细胞用于实验。内皮细胞被种植在6孔板中,然后给与不同浓度的棕榈酸(palmitic acid,PA)或siRNA刺激。
2.棕榈酸制备
根据文献方法制备棕榈酸。首先用酒精溶解棕榈酸至终浓度为200mM,然后加入10%的不含游离脂肪酸的牛血清稀释至终浓度为1-5mM。调节pH值在7.5左右,放入-20℃冰箱中备用。
3. siRNA转染方法
使用特异的siRNA诱导基因沉默,应用LipofectAMINETM 2000脂质体包裹,并转染HAECs。被转染的HAECs再给与棕榈酸刺激24小时。
4. Western blot分析
收集细胞后,使用细胞裂解液提取总蛋白,煮沸5分钟。将15μg左右蛋白的标本和蛋白标记物一起加样,以SDS-PAGE电泳,然后用一湿电转移电转到聚偏氟乙烯膜(PVDF)上。封闭液封闭,一抗孵育过夜。洗膜后,用二抗孵育。用显色液显色。目的蛋白和β-actin条带积分光密度的比值表示蛋白表达的多少。
5.细胞RNA提取和实时定量PCR
根据试剂盒说明,使用Trizol提取细胞的总RNA。用iScript cDNA合成酶将mRNA逆转录成cDNA。应用iCycler iQ荧光探针进行实时定量PCR。TM基因mPCR扩增引物：上游序列5'-CCGATGTCATTTCCTTGCTA-3',下游序列5'-GTTGTCTCCCGTAACCCACT-3'。反应结束得到循环阈值(Ct值),用TM与β-actin的循环阈值的比值表示mRNA的相对水平。
6.染色质免疫沉淀分析
根据试剂盒说明,使用Chip试剂盒进行染色质免疫沉淀分析。首先将处理过的内皮细胞用1%的甲醛在37℃中孵育15分钟,以形成DNA-蛋白质复合体。裂解细胞,离心,应用特异抗体-蛋白A-琼脂糖浆对DNA-蛋白质复合体进行免疫沉淀(IgG作为阴性对照)。将这些免疫复合物珠子进行清洗,洗脱和解链。应用苯酚／氯仿／易戊酯酒精混合物萃取DNA片段。然后,将免疫沉淀的DNA片段作PCR,使用1.5%的琼脂糖胶分离PCR产物。
7.免疫沉淀分析
将处理过的细胞放置冰预冷的裂解液中裂解60分钟。然后与抗体和蛋白A／G-琼脂糖珠子在4℃中孵育过夜以进行免疫沉淀。使用提取液清洗两遍,然后再使用含有0.5 M氯化锂的提取液清洗两遍。使用SDS样本试剂直接洗脱蛋白质,然后进行蛋白印迹分析。
8.统计分析
实验数据以均数±标准差表示。应用SPSS13.0软件进行分析,多组之间采用单因素方差分析,P<0.05认为差异具有统计学意义。
结果
1.游离脂肪酸抑制内皮细胞中TM基因的表达
人主动脉内皮细胞(HAECs)给与不同浓度的PA刺激24小时,Western blot结果显示PA能明显抑制TM蛋白的表达,差异有统计学意义(P<0.001)。
2.游离脂肪酸能激活转录因子ATF-2
人主动脉内皮细胞(HAECs)给与不同浓度的PA刺激24小时,Western blot方法检测ATF-2通路磷酸化蛋白和总蛋白的表达水平,用磷酸化蛋白／总蛋白的比值表示蛋白活性。PA能够增加转录因子ATF-2的活性,差异有统计学意义(P<0.05)。
3.转录因子ATF-2能直接与TM基因启动子结合
TM基因启动子区域包含多个ATF-2结合位点(agTGACGgatt at-1288/-1277, gcTGACTcgct at-1026/-1016, and ccTGACAgtgt at-939/-929)。首先用ChiP分析检测ATF-2能否与TM启动子区域结合。ChiP结果显示,ATF-2能与TM启动子gcTGACTcgct (-1026/-1016)和ccTGACAgtgt (-939/929)位点结合。并且,PA能增强ATF-2与TM启动子的结合。
4.转录因子ATF-2参与PA抑制TM基因表达的过程
检测转录因子ATF-2能否调控TM表达。使用特异性的ATF-2 siRNA沉默ATF-2表达,能阻止PA抑制的TM mRNA的表达(P<0.05)。
5.ATF-2与HDAC4结合在TM基因启动子区域形成转录抑制复合物
ChiP结果显示,PA能明显增强转录抑制分子HDAC4与TM基因启动子相结合。双ChiP结果表明ATF-2能与HDAC4结合形成转录抑制复合物从而抑制TM基因的表达。
结论
1.在PA抑制内皮细胞中TM表达的过程中,转录因子ATF-2参与了调控TM基因的表达过程。
2.ATF-2通过与HDAC4结合,在TM基因启动子区域形成转录抑制复合物从而抑制TM的表达。这将有助于进一步明确TM表达降低的分子生物学机制,从而为基因治疗提供新的靶点。
Backgroud
Metabolic syndrome displays a significant prothrombotic state with increased coagulation, inhibited fibronolysis and increased platelet activation. The prothrombotic state induces the increased risk of pathological thrombosis, and renders patients highly susceptible to myocardial infarction, ischemic stroke and peripheral vascular diseases. Although many clinical studies showed that the prothrombotic state is a key feature of metabolic syndrome, the pathogenesis and mechanisms of the state is not completely understood.
Protein C, a key endogenous anticoagulant molecule, is efficiently activated on the surface of endothelial cells by the binding of thrombin to the endothelial transmembrane glycoprotein, Thrombomodulin. Once activated, protein C, together with its cofactor protein S, inhibits coagulation by proteolytic degradation of factor Villa and factor Va. In addition, activated protein C (APC) promotes fibrin degradation by inhibiting the activity of plasminogen activator inhibitor—1 (PAI-1). Moreover, APC has significant anti-inflammatory and anti-apoptotic functions. Thus, APC not only regulates hemostasis and inflammation, but also provides additional levels of cellular protection. Thrombomodulin and endothelial protein C receptor (EPCR) control the protein C anticoagulation pathway. Maximal rates of protein C activation require thrombin binding to thrombomodulin and protein C binding to EPCR. Loss of thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis. Localized loss of thrombomodulin from endothelium has been associated with arteriosclerotic lesions, therefore, thrombomodulin may act in a vasoprotective manner to prevent cardiovascular diseases. Deregulation of this system may be responsible for the prothrombotic state in metabolic syndrome.
The prothrombotic state in metabolic syndrome has a complex pathogenesis. Metabolic syndrome is often characterized by high circulating concentrations of FFAs. Thus, the vasculature of patients with metabolic syndrome is constantly exposed to high levels of FFAs. Clinical studies have shown that high FFA levels directly impair vascular functions are associated with myocardial infarction, stroke, and sudden death. Long-term increases in FFA levels may have an adverse effect on the regulation of thrombosis. But it is not understood whether FFAs can regulate TM expression and the mechanisms involved.
In this study, we hypothesize that the metabolic stress of high concentrations of FFAs may deregulate the thrombomodulin-APC system, which in turn may be responsible for the development of the prothrombotic state in metabolic syndrome. Our study may be helpful to understand the mechanisms of the prothrombotic state in metabolic syndrome, and supply a new therapeutic target in treating and preventing cardiovascular diseases in metabolic syndrome.
Objectives
1. Elucidate the effects of high serum FFAs level on the prothrombotic state in metabolic syndrome.
2. Examine the effects of FFAs on the expression of TM-protion C system and the possible mechanisms involved.
Methods
1. Animal model
8 to 10 weeks of male wide-type mice were divided into two groups. One group of mice were fed a chow diet containing 10% fat, while the other group of mice were fed a high-fat diet containing 50% fat for 20 weeks. The aortas were rapidly excised and rinsed in ice-cold saline, and then stored for histology and further use.
2. Tail-bleeding time
Mice were anesthetized and placed on a 37℃heating pad. The tail was transected with a sterile scalpel at a point where the tail diameter was approximately 1 mm wide. After transection, the tail was immediately placed in a 50 mL tube containing 0.9% NaCl warmed to 37℃. The time between bleeding to stop was recorded.
3. FeCl3 induced carotid artery thrombosis model
FeCl3 induced thrombosis was used in this study. In brief, mice were anesthetized. An incision was made, and a segment of the right common carotid artery was exposed with blunt dissection. A small piece of filter paper(2 mm X 3 mm) was saturated with 10% FeCl3 was deposited on the isolated artery for 3 min, followed by washing with saline. Blood flow was monitored using a Doppler flow probe for 30 min after FeCl3 application. The occlusion time was recorded as the first image that showed 0 flow.
4. Metabolic factors measurements
Blood samples were collected from retio-orbital sinus. Circulating free fatty acids, glocuse, insulin, TG, VLDL, LDL, HDL were measured.
5. Histological and morphology analyses
Sections were stained with hematoxylin and eosin(H&E). TM, PAI-1, TF were detected with immunostaining.
6. Cell culture
Primary human aortic endothelial cells (HAECs) were cultured at 37℃in 5% CO2 in endothelial cell growth medium-2 (EGM-2). Cells between passages 5 to 9 were used for all of the experiments. The medium was changed every two or three days.
7. Preparation of fatty acid-albumin complexes.
Saturated PA, polyunsaturated LA, and monounsaturated OA were used in this study. Lipid-containing media were prepared by conjugation of FFAs with BSA using a modification of the method described previously. Briefly, FFAs were first dissolved in ethanol at 200mM, and then combined with 10% FFA-free low endotoxin BSA to final concentrations of 1-5mM. The pH of all solutions was adjusted to approximately 7.5, and the stock solutions were filter-sterilized and stored at-20℃until use. Control solution containing ethanol and BSA was prepared similarly. Working solutions were prepared fresh by diluting stock solution (1:10) in 2% FCS-EBM.
8. Protein C activation.
HAECs in 96-well plates were treated with different kind and different dose of FFAs for 24 hours.Then the activation of protein C was detected according to the manufacturer's instructions.
9. siRNA-induced gene sliencing
Sliencing gene expression wsas achieved by using specific siRNAs. HAECs were transfected with siRNAs using Lipofectamine2000 according to the manufacture's instructions.
10. Western blot analysis
Cell extracts and mice aortas were prepared with lysis buffer. Equal amounts of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Following blocking with 5% non-fat milk, the blots were incubated with a primary antibody at 4℃overnight. Then the blots were washed with TBST and incubated with HRP-conjugated secondary antibody. The blots were then visualized by use of enhanced chemiluminescence.
11. RT-PCR
Total RNA was extracted from HAECs with Trizol according to the manufacturer's instructions. Signal-strand cDNA was synthesized with iScript cDNA synthesis kit. Semi-quantitative real-time PCR was performed with iCycler iQ real-time PCR detection system.
12. Plasmid DNA transfection
HAECs were transfected with different types of plasmids including wide type, constitutively active and dominant-negative by using LipofectAMINETM 2000 according to the manufacturer's instructions. Transfected cells were then treated with PA.
Results
1. Mice model
Mice fed a high-fat diet showed notable characteristics of metabolic syndrome with higher body weight and elevated blood levels of FFAs, TG, VLDLs, glucose and insulin(P<0.05).
2. Prothrombotic state in mice fed a high-fat diet
The tail bleeding time was significantly lower in mice fed a high-fat diet than that in chow diet. Furthermore, the occlusion time following FeCl3 injury of the carotid artery was significantly shorter in mice fed a high-fat diet compared to that in control mice, indicating that the development of prothrombotic state in mice fed a high-fat diet(P<0.01).
3. Reduced expression of thrombomodulin and increased expression of PAI-1 and TF in mice fed a high-fat diet
We examined the expression of TM, PAI-1 and TF in the aorta of obese mice. Immunostaining of the vascular wall showed significantly decreased expression of thrombomodulin(P<0.001), and increased expression of PAI-1 and TF(P<0.01) on the surface of the endothelium in the aorta of mice fed a high-fat diet. This finding is consistent with the results of Western blot. These results show that thrombomodulin is down-regulated in obese mice, which may contribute to the hypercoagulable state in obesity and metabolic syndrome.
4. JNK and p38 MAPK stress signaling pathways were activated in mice fed a high-fat diet
Western blot showed that JNK and p38 MAPK stress pathways were activated in mice fed a high-fat diet(P<0.01), indicating that the activation of stree pathways may contribute to the prothrombotic state in these mice.
5. Suppression of thrombomodulin and EPCR expression and increased expression of TF by FFAs.
To determine the potential role of FFAs in thrombosis dysregulation, we examined the effect of FFAs on the expression of prothrombotic factors and antithrombotic factors in endothelial cells. Saturated fatty acid PA, polyunsaturated fatty acid LA, and monounsaturated fatty acid oleic acid (OA) were used for the study. PA and LA significantly decreased the expression of thrombomodulin in a dose-dependent fashion(P<0.01), whereas OA had a minimal effect on thrombomodulin(P>0.05). Similarly, treatment with PA and LA substantially inhibited EPCR expression and increased to varying degrees the expression of the prothrombotic factor, PAI-1(P<0.01). PA significantly decreased mRNA for thrombomodulin in a dose-dependent manner.
6. FFAs inhibited endothelial-mediated protein C activation
We compared the activation of protein C in FFA-treated and untreated HAECs. PA and LA caused a significant dose-dependent decrease in protein C activation(P<0.01), whereas OA had no effect(P>0.05).
7. JNK and p38 stress pathways were involved in PA's inhibitory effect on the expression of TM.
Silencing JNK and p38 with specific siRNAs decreased PA-induced thrombomodulin suppression(P<0.01), indicating that JNK and p38 pathways mediated PA-induced inhibition of thrombomodulin expression. Furthermore, wide type of JNK plasmid can enhance PA's inhibitory effect on TM expression, while dominant-negative of JNK plasmid can abrogate the effect.
8. Foxol was involved in PA-inhibited TM expression
When HAECs cells were transiently transfected with Foxol-specific siRNAs before treated with PA, PA-induced inhibition of TM expression was significantly decreased(P<0.001). Wide type and constitutively active of Foxol plasmids enhanced PA's inhibitory effect of TM expression(P<0.05), while dominant-negative of Foxol plasmid can abrogate the effect(P>0.05).
Conclusions
1. A high-fat diet induced high elevated circulating FFAs level, the shortened bleeding time, indicating that the prothrombotic state in metabotic syndrome mice.
2. The aortas from mice fed a high-fat diet showed decreased TM expression, and increased expression of PAI-1 and TF, indicating that the imbalance of coagulation and anticoagulation.
3. PA and LA inhibited the expression of TM and EPCR, and increased PAI-1 expression in endothelial cells, indicating that FFAs can induce the imbalance of coagulation and anticoagulation in endothelial cells.
4. JNK and p38 stress pathways mediated PA-inhibited TM expression in HAECs.
5. Foxol was involved in PA-inhibited TM expression in HAECs.
6. JNK, p38/Foxol pathway mediated free fat acid's inhibitory effect of thrombomodulin expression, which may be a new therapeutic target in treating cardiovascular and cerebrovascular complications.
Backgroud
Metabolic syndrome is associated with a prothrombotic state, which contributes to the atherothrombotic complications, such as myocardial infarction, stroke, and peripheral vascular complications. Antithrombotic therapy improves survival in acute cardiac and cerebrovascular complications patients. Although the dysregulation in coagulation and fibrinolysis has been reported, the development of prothrombotic state is not well understood.
Endothelium plays an active role in regulating pro-coagulation and anti-coagulation balance by generating several active regulatory molecules, such as von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Among these factors, thrombomodulin-protein C pathway is a major physiological anticoagulation system of the endothelium. Endothelial dysfunction can cause coagulation dysregulation and promote vascular thrombosis.
Endothelium plays an active role in regulating pro-coagulation and anti-coagulation balance by generating several active regulatory molecules, such as von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Among these factors, thrombomodulin-protein C pathway is a major physiological anticoagulation system of the endothelium. Endothelial dysfunction can cause coagulation dysregulation and promote vascular thrombosis.
Thrombomodulin, a glycoprotein on the surface of endothelial cells, is a key factor in protein C activation3. When bound to thrombin, TM triggers the activation of protein C by facilitating the conversion of circulating protein C to activated protein C (APC). Activated protein C can inhibit coagulation by degradingⅧa and factor Va and enhance fibrinolysis by inactivating PAI-1. TM plays a key role in anticoagulation, and mutation or down-regulation of TM promotes while overexpression of TM prevents arterial thrombosis. In addition, TM functions as an anti-inflammatory and anti-apoptotic molecule. It has been shown that TM inhibited inflammatory response and blocked cell apoptosis. TM is down-regulated in vascular diseases including atherosclerotic lesions, and TM is negatively regulated by inflammatory factors, wall tension and oxidized lipids. However, many reports showed that the down-regulated expression of TM was associated with the pathologic thrombosis, the mechanisms involved in the down-regulation of TM expression are not completely understood.
Mitogen-activated protein kinases (MAPKs) are serine/threonine-specific protein kinases that contribute to extracellular stimuli and regulate various cellular activities, such as gene expression, mitosis, transformation, apoptosis. Stress signaling JNK and p38 pathways are activated in many cardiovascular diseases including atherosclerosis and are involved in pathphysiological changes in these conditions. JNK and p38 signaling pathways are activated by metabolic stress and inflammatory factors. Previous study showed that JNK and p38 can be activated by free fatty acids (FFA) and were involved in vascular insulin resistance. We have found that JNK and p38 pathways are involved in the dowm-regulation of TM, but the detailed mechanisms involved are not well understood.
How stress signaling down-regulates TM expression? ATF-2 is a downstream target transcription factor of JNK and p38 pathways. Previous study has reported that ATF-2 mediated LPS-induced TF expression and may be involved in thrombosis dysregulation. But whether transcriptional factor ATF-2 is involved in the regulation of TM expression is still unclear.
So in this study, we examined the effects of transcriptional factor ATF-2 on the regulation of TM expression, which may be helpful for the development of therapeutic intervention for pathologic thrombosis and cardiovascular complications.
Objectives
1. Identify the effects of transcriptional factor ATF-2 in the regulation of TM expression in HAECs.
2. Examine the possible mechanisms how ATF-2 mediated TM expression.
Metarials and Methods
1. Cell culture
Primary human aortic endothelial cells (HAECs) were cultured at 37℃in 5% CO2 in endothelial cell growth medium-2 (EGM-2) containing 2% FBS, FGF-2, VEGF, IGF-1, EGF. Cells between passages 5 to 9 were used for all of the experiments. The medium was changed every two or three days. The endothelial cells were plated on 6-well plates, and then treated with different doses of palmitic acid or transfected with siRNAs.
2. Preparation of palmitic acids:
Preparation of PA was carried out as previously. Briefly, PA was dissolved in ethanol at 200mM, and then combined with 10% FFA-free low endotoxin BSA to final concentrations of 1-5mM. The pH of all solutions was adjusted to approximately 7.5, and the stock solutions were stored at-20℃.
3. siRNA transfection
Silencing gene expression was achieved using specific siRNA. HAECs were transfected with siRNAs using Lipofectamine2000 according to the manufacturer's instructions. Transfected cells were then treated with palmitic acid for 24h.
4. Western blot analysis
Cell extracts were prepared with lysis buffer, and boiled with 5 min. Protein samples(15μg per lane) and protein marker were seperated by SDS-PAGE gel electropohresis and transferred to PVDF membranes. The membranes were blocked with blocking buffer, and then incubated with the primary antibody overnight. The membranse were washed with PBST, and then incubated with the HRP-conjugated secondary antibody. The blots were then visualized with enhanced chemiluminescene. The expression of cytokine was demonstrated by the ration of integral optical density between cytokine and p-actin
5. RNA extration and real-time quantitative PCR
Total RNA was extracted from HAECs with Trizol according to the manufacturer's instructions. Signal-strand cDNA was synthesized with iScript cDNA synthesis kit. Semi-quantitative real-time PCR was performed with iCycler iQ real-time PCR detection system. The primers for human thrombomodulin mRNA were as follows:forward:5'-CCGATGTCATTTCCTTGCTA-3'; reverse:5'-GTTGTCTCCCGTAACCCACT-3'. The mRNA levels were estimated from the value of the threshold cycle (Ct) of the real-time PCR adjusted by that ofβ-actin.
6. Chromatin immunoprecipitation assay
The ChIP assay kit (Upstate) was used according to the manufacturer's instructions. Treated HAECs were first incubated with 1% formaldehyde at 37℃for 15 min to cross-link DNA-protein complexes. Cells were then rinsed and lysed. Cell lysates were sonicated and centrifuged to produce chromatin fragments. The supernatants were pre-cleared with a mixture of salmon sperm DNA/protein A/protein G, followed by immunoprecipitation with antibody-protein A-agarose slurry. (IgG served as the negative control.) The DNA was recovered by extraction with the phenol/chloroform/isoamyl alcohol mixture. The immunoprecipitated DNA was used as a template for PCR. The PCR products were separated by 1.5% agarose gel.
7. Immunoprecipitation
Immunoprecipitation was conducted as described previously.Treated cells were lysed for 60 min in ice-cold extraction buffer. For immunoprecipitation, cleared cell lysates were incubated with the appropriate antibody precoupled to protein A/G-agarose beads (Santa Cruz Biotechnology) at 4℃overnight. The beads were washed twice with extraction buffer and twice with extraction buffer containing 0.5 M LiCl. Proteins were eluted directly in SDS sample buffer for Western blot analysis.
8. Statistical analysis
Data are presented as mean±SEM. One-way ANOVA was used to analyze the differences among groups. P values< 0.05 were considered statistically significant.
Results:
1. Free fatty acids suppressed thrombomodulin expression in HAECs.
HAECs were incubated with different concentrations of PA for 24h. Western blotting showed that PA significantly suppressed the expression of thrombomodulin in a dose-dependent manner. Furthermore, PA significantly inhibited the expression of thrombomodulin mRNA, indicating that PA suppressed thrombomodulin expression at the transcriptional level.
2. Free fatty acids activated ATF-2 pathway.
HAECs were treated with different doses of PA for 24h. The phosphorylated protein and total protein of ATF-2 were examined by Western blot. The activation of pathways was demonstrated by the ratio of phosphorylated and total protein. Consistent with previous observation, ATF-2 was activated by PA in a dose-dependent manner.
3. Transcriptional factor ATF-2 binded directly to the TM promoter.
The promoter region in the TM gene contained many ATF-2 binding sites(agTGACGgatt at-1288/-1277, gcTGACTcgct at-1026/-1016, and ccTGACAgtgt at-939/-929). ChiP assay was used to examine whether ATF-2 can bind to the TM promoter. The results of ChiP assay showed that ATF-2 can bind to the TM promoter at the gcTGACTcgct (-1026/-1016) and ccTGACAgtgt (-939/-929) sites. Importantly, the binding was significantly increased by PA treatment, indicating that binding of ATF-2 to TM promoter may be involved in PA-induced suppression of thrombomodulin transcription.
4. ATF-2 was involved in PA-inhibited TM expression.
We then examined whether ATF-2 can regulate TM expression. When HAECs cells were transiently transfected with ATF-2-specific siRNAs before treated with PA, PA-induced inhibition of TM expression was significantly prevented, which showed that a critical role for ATF-2 transcription factor in PA-induced down-regulation of thrombomodulin transcription (P<0.05).
5. Recruitment of HDAC4 and formation of ATF-2/HDAC4 transcription repressor complex in the TM promoter.
We finally investigated how the transcription factor ATF-2 can bind to the TM promoter and inhibit gene expression. ChiP assay showed that PA treatment significantly increased transcription repressor HDAC4 binding to the TM promoter. The double-chip assay showed that ATF-2 and HDAC4 were in the same transcription repression complex in the TM promoter.
Conclusions
1. PA significantly inhibited thrombomodulin expression in human aortic endothelial cells(HAECs).
2. ATF-2 was involved in the down-regulation of thrombomodulin expression.
3. ATF-2 was mediated TM suppression by recruitment of HDAC4 and formation of ATF-2/HDAC4 transcription repressor complex in the TM promoter.
代谢综合征患者表现为显著的促凝血状态,其中凝血功能增强,纤维溶解活性降低,血小板的活性和功能增强。促凝血状态的存在造成患者病理性血栓形成的风险增加,进而导致心肌梗死、脑卒中、外周血管疾病等临床事件的发生。虽然众多的临床研究证实了促凝血状态是代谢综合征的重要特征之一,但其病理生理学特点及具体的发生机制尚不明确。
活性蛋白C(activated protein C, APC)是体内重要的抗凝分子。凝血酶(thrombin)与血栓调节蛋白(thrombomodulin, TM)结合后,能高效地促使蛋白C转化为活性蛋白C。APC能和辅助因子蛋白S结合,降解凝血因子Ⅷa和Va,从而发挥抗凝作用。另外,APC能抑制组织型纤溶酶原激活物抑制剂(PAl-1)的活性,进而促进纤维蛋白溶解。此外,APC还具有抗炎和抑制凋亡的功能。TM和内皮表面蛋白C受体(endothelial protein C receptor, EPCR)能够调控蛋白C的激活过程。以往的研究表明,TM表达减少能抑制蛋白C的激活,造成血栓形成。局部TM表达减少与动脉粥样硬化病变有关。因此,TM可能发挥血管保护作用从而发挥预防心血管疾病的作用。TM-蛋白C系统的调控异常可能与代谢综合征中促凝血状态的发生有关。
代谢综合征中促凝血状态的发生是一个非常复杂的病理生理过程。循环中游离脂肪酸(free fatty acids, FFAs)水平升高是代谢综合征的重要特征。因此,代谢综合征患者的血管系统长期暴露于高FFAs状态中。临床研究表明,循环中FFAs水平升高能直接损害血管功能,与心肌梗死、脑卒中和猝死的发生密切相关。长期的高FFAs可能导致异常的血栓形成。但是游离脂肪酸能否调节TM的表达以及具体的机制如何,目前尚缺乏相关研究。
针对以上问题,我们提出以下假设：高FFAs能导致TM-蛋白C系统表达失调,进而促使代谢综合征中促凝血状态的发生。通过本研究有助于进一步了解代谢综合征中促凝血状态的发生机制,为急性心脑血管疾病的预防和治疗提供新的思路和治疗靶点。
研究目的
1.观察高游离脂肪酸水平与代谢综合征中促凝血状态的关系。
2.探讨游离脂肪酸对TM-蛋白C抗凝系统的作用以及可能的分子生物学机制。
材料与方法
1.动物模型
8-10周龄的雄性小鼠30只,分为两组,每组15只。一组给与普通饮食(含10%脂肪),一组给与高脂饮食(含50%脂肪)20周。实验结束后,留取主动脉,用于后续的病理学检查和分子生物学检查。
2.鼠尾静脉出血时间测定
小鼠麻醉后,在鼠尾直径约为1mm左右处,用锐利的刀片横断鼠尾。然后迅速将鼠尾置于37摄氏度的PBS溶液中,记录从出血到血流终止的时间,记为出血时间。
3.三氯化铁诱导的颈动脉血栓模型
利用FeCl3损伤方法造成血栓形成。小鼠腹腔麻醉后,行颈部正中切口,钝性分离,暴露左侧颈总动脉,把超声探头置于颈动脉表面,监测基础状态下血流情况。把滤纸切成1.5 mm X 2 mm的细条,在10%的FeCl3溶液中充分浸泡,将滤纸片置于颈动脉表面,放置3分钟,然后去除滤纸片。迅速用超声探头持续监测颈动脉血流情况,监测时间为30分钟。记录从放置滤纸片到血流完全中断的时间。
4.代谢指标检测
实验结束前,抽取小鼠静脉血。利用常规生物学方法,检测血液中游离脂肪酸(FFA)、血糖、胰岛素,血脂TQ VLDL, LDL, HDL水平。
5.病理学检测
实验结束,留取小鼠主动脉,多聚甲醛固定,包埋,制作组织切片。免疫组织化学方法观察血管壁TM,PAI-1,TF的表达。
6.内皮细胞培养
人主动脉内皮细胞株(human aortic endothelial cells, HAECs),使用内皮细胞生长培养基-2(endothelial cell growth medium-2, EGM-2),在37℃和5%CO2孵育箱中进培养。每2-3天更换培养液。第5-9代细胞用于实验。
7.脂肪酸制备
首先将游离脂肪酸(FFAs)溶解在酒精中,并与10%的不含FFAs,低内毒素的BSA混合,使其终浓度在1-5 mM。所有溶液的PH值调整在7.5左右,使用滤器消毒,-200C储存。将不含有PA的BSA溶液作为对照。在处理细胞之前,将储存的PA按照1：10的比例,使用培养基进行稀释。
8.蛋白C活性检测
内皮细胞被种植在96孔板中,给与不同浓度、不同种类的FFAs刺激24小时,然后根据试剂盒说明,检测蛋白C的活性。
9.siRNA诱导的基因沉默
使用特异性的siRNA沉默相关基因,应用LipofectAMINETM 2000脂质体包裹,并转染HAECs.
10.蛋白印迹分析
使用细胞裂解液或组织裂解液提取细胞和小鼠主动脉的总蛋白。以SDS-PAGE电泳、转膜、一抗孵育、二抗孵育,最后采用化学发光法观察结果。
11.实时定量PCR
使用Trizol提取细胞的总RNA。应用iScript cDNA合成酶将mRNA逆转录成cDNA。使用iCycler iQ荧光探针系统进行实时PCR。
12.质粒DNA转染
分别使用野生型表达质粒(wide type plasmid)、持续激活型表达质粒(constitutively active plasmid)、显性负向表达质粒(dominant negative plasmid),首先使用LipofectAMINETM 2000脂质体包裹,并转染内皮细胞。然后再给与PA处理。
结果
1.动物的基本特征
与普通饮食的小鼠相比,高脂饮食导致小鼠代谢指标明显异常。体重明显增加,血液中游离脂肪酸、血糖、胰岛素、血脂水平明显增加(P均<0.05)。
2.高脂饮食导致小鼠体内出现明显的促凝血状态
与对照组小鼠相比,高脂饮食导致小鼠尾静脉出血时间明显缩短,三氯化铁损伤后颈总动脉血栓形成时间明显缩短(P0.01),提示这些小鼠体内存在明显的凝血异常。
3.高脂饮食导致小鼠体内TM表达明显减少,PAI-1和TF表达增加
我们检测了小鼠主动脉中TM,PAI-1和TF等凝血指标的表达。免疫组化结果显示,与对照组比较,高脂喂养的小鼠血管壁TM表达明显减少,PAI-1和TF的表达增加。此外,Western blot结果表明,高脂喂养的小鼠主动脉TM表达明显减少(P<0.001),PAI-1和TF的表达增加(P<0.01)。
4.高脂饮食能激活体内JNK和p38 MAPK应激信号通路
Western blot结果表明,高脂饮食导致小鼠主动脉内JNK和p38 MAPK磷酸化蛋白／总蛋白的比值增加(P<0.01)。
5.游离脂肪酸抑制内皮细胞中TM和EPCR的表达,并增加TF的表达
内皮细胞给与不同浓度的棕榈酸(palmitic acid, PA)、亚油酸(linoleic acid,LA)、油酸(oleic acid, OA)刺激24小时。Western blot结果显示,PA和LA能显著抑制TM和EPCR的表达(P均<0.01),并增加TF的表达(P<0.05)。但是OA对TM,EPCR和TF的蛋白表达没有明显作用(P>0.05)。RT-PCR结果显示PA能剂量依赖性地抑制TM mRNA的表达(P<0.01)。
6.游离脂肪酸抑制内皮细胞中蛋白c的激活
用不同浓度的PA,LA和OA处理内皮细胞24小时,检测蛋白C的活性。PA和LA呈剂量依赖性的抑制蛋白C的活性(P<0.01),但是OA对蛋白C的活性没有明显作用(P>0.05)。
7.JNK和p38信号通路参与PA抑制TM基因表达的过程
使用特异性的siRNA分别敲除JNK和p38通路,PA导致的TM表达降低的作用减弱(P<0.01)。野生型JNK质粒DNA转染能加强PA对TM的抑制作用(P<0.05),而显性负向的JNK质粒DNA转染没有这一作用。
8.转录因子Foxol参与PA抑制TM基因表达的过程
使用特异性的Foxo1 siRNA沉默Foxol的表达,PA抑制的TM蛋白表达的作用减低(P<0.001)。野生型和持续激活型Foxol质粒DNA转染能加强PA对TM的抑制作用(P<0.05),而显性负向的Foxol质粒DNA转染能阻止这一作用(P>0.05)。
结论
1.高脂饮食导致小鼠血液中游离脂肪酸水平升高,出血时间缩短,血栓形成时间缩短,提示代谢综合征模型小鼠体内存在促凝血状态。
2.高脂饮食小鼠主动脉中抗凝因子TM表达减少,促凝因子PAI-1和TF表达增加,提示这些小鼠体内凝血和抗凝调控异常。
3.PA和LA抑制内皮细胞TM和EPCR的表达,并增加PAI-1的表达,降低蛋白C的活性,说明游离脂肪酸能导致细胞中凝血和抗凝因子表达失调。
4.在PA抑制TM表达的过程中,应激信号通路JNK和p38MAPK参与了对TM表达的调节。
5.在PA抑制TM表达的过程中,Foxol作为重要的转录因子参与了抑制TM表达的过程。
总之,代谢综合征模型小鼠血液中游离脂肪酸水平升高,体内凝血和抗凝平衡失调,表现为明显的促凝血状态。PA能减少内皮细胞中TM的表达,降低蛋白C的活性。PA可以通过JNK,p38/Foxol信号传导通路抑制内皮细胞中TM的表达。
背景
代谢综合征患者表现为显著的促凝血状态,这些患者发生心肌梗死、脑卒中、外周血管疾病等血栓性并发症的风险增加。临床上抗血栓治疗能够改善急性心脑血管并发症患者的生存率。虽然既往的研究已经发现代谢综合征患者凝血和纤维溶解功能异常,但是有关促凝血状态的发生机制目前尚未完全明确。
血管内皮能分泌多种活性物质,例如：vWF,血栓调节蛋白(thrombomodulin,TM),组织型纤溶酶原激活剂(tissue plasminogen activator, t-PA),组织型纤溶酶原激活物抑制剂(plasminogen activator inhibitor, PAI-1),在调控促凝和抗凝平衡过程发挥着重要作用。在这些活性分子中,TM-蛋白C系统是内皮组织重要的生理性抗凝系统。内皮细胞功能失调可导致凝血功能异常,并促使血栓形成。
TM是一种定位于内皮细胞表面的糖蛋白,是激活蛋白C的一种重要的活性物质。TM与凝血酶(thrombin)结合后,促使蛋白C转化为有活性的蛋白C。活性蛋白C一方面通过降解凝血因子Ⅷa和Va发挥抑制凝血过程；一方面可使PAI-1失去活性从而增强纤维溶解过程。TM在抗凝血过程中发挥着重要作用。TM基因突变或表达减少能促使动脉血栓的形成；而局部TM基因过度表达能避免兔动脉血栓形成。另外,TM基因还具有抗炎和抑制凋亡的作用。多项研究发现,TM能抑制炎症反应,阻止细胞凋亡。在动脉粥样硬化等血管性疾病发生过程中,TM基因表达降低。多种刺激,诸如：炎症因子、管壁张力、氧化的脂质等能抑制TM基因的表达。虽然众多的研究发现TM表达降低与病理性血栓形成有关,但是导致TM表达降低的具体机制尚不完全明确。
MAPKs作为体内广泛存在的丝氨酸／苏氨酸蛋白调节激酶,在细胞的增殖、分化、转化和凋亡过程中发挥着重要作用。现有研究表明,包括动脉粥样硬化在内的多种心血管疾病过程,伴随着JNK和p38 MAPK应激信号通路的异常激活。多种代谢刺激和炎症因子能激活JNK和p38 MAPK应激信号通路。游离脂肪酸(free fatty acids,FFAs)也能够激活JNK和p38 MAPK应激信号通路,并参与游离脂肪酸导致胰岛素抵抗的过程。我们已经发现JNK和p38 MAPK应激信号通路参与调控TM基因的表达,但是具体的分子生物学机制目前尚不清楚。
ATF-2是位于JNK和p38 MAPK应激信号通路下游的转录因子。以往的研究显示ATF-2参与调控LPS诱导的组织因子(tissue factor,TF)表达的过程,因此转录因子ATF-2可能与血栓形成失调有关。但是,转录因子ATF-2能否参与调控TM基因的表达目前尚不明确。
因此,在这项研究中,我们检测了转录因子ATF-2作为JNK的下游转录因子,通过与HDAC4结合形成转录抑制复合物从而抑制TM基因的表达的具体机制。这将为病理性血栓及心血管疾病的治疗干预提供新的干预靶点。
研究目的
1.明确转录因子ATF-2在调控TM基因表达过程中的作用。
2.探讨ATF-2调控TM基因表达的可能的分子生物学机制。
材科与方法
1.细胞培养
人主动脉内皮细胞株(human aortic endothelial cells, HAECs),使用含有2%的胎牛血清,FGF-2, VEGF, IGF-1, EGF等生长因子的内皮细胞生长培养基-2(endothelial cell growth medium-2, EGM-2),在37℃和5%CO2孵育箱中进培养。每2-3天更换培养液。第5-9代细胞用于实验。内皮细胞被种植在6孔板中,然后给与不同浓度的棕榈酸(palmitic acid,PA)或siRNA刺激。
2.棕榈酸制备
根据文献方法制备棕榈酸。首先用酒精溶解棕榈酸至终浓度为200mM,然后加入10%的不含游离脂肪酸的牛血清稀释至终浓度为1-5mM。调节pH值在7.5左右,放入-20℃冰箱中备用。
3. siRNA转染方法
使用特异的siRNA诱导基因沉默,应用LipofectAMINETM 2000脂质体包裹,并转染HAECs。被转染的HAECs再给与棕榈酸刺激24小时。
4. Western blot分析
收集细胞后,使用细胞裂解液提取总蛋白,煮沸5分钟。将15μg左右蛋白的标本和蛋白标记物一起加样,以SDS-PAGE电泳,然后用一湿电转移电转到聚偏氟乙烯膜(PVDF)上。封闭液封闭,一抗孵育过夜。洗膜后,用二抗孵育。用显色液显色。目的蛋白和β-actin条带积分光密度的比值表示蛋白表达的多少。
5.细胞RNA提取和实时定量PCR
根据试剂盒说明,使用Trizol提取细胞的总RNA。用iScript cDNA合成酶将mRNA逆转录成cDNA。应用iCycler iQ荧光探针进行实时定量PCR。TM基因mPCR扩增引物：上游序列5'-CCGATGTCATTTCCTTGCTA-3',下游序列5'-GTTGTCTCCCGTAACCCACT-3'。反应结束得到循环阈值(Ct值),用TM与β-actin的循环阈值的比值表示mRNA的相对水平。
6.染色质免疫沉淀分析
根据试剂盒说明,使用Chip试剂盒进行染色质免疫沉淀分析。首先将处理过的内皮细胞用1%的甲醛在37℃中孵育15分钟,以形成DNA-蛋白质复合体。裂解细胞,离心,应用特异抗体-蛋白A-琼脂糖浆对DNA-蛋白质复合体进行免疫沉淀(IgG作为阴性对照)。将这些免疫复合物珠子进行清洗,洗脱和解链。应用苯酚／氯仿／易戊酯酒精混合物萃取DNA片段。然后,将免疫沉淀的DNA片段作PCR,使用1.5%的琼脂糖胶分离PCR产物。
7.免疫沉淀分析
将处理过的细胞放置冰预冷的裂解液中裂解60分钟。然后与抗体和蛋白A／G-琼脂糖珠子在4℃中孵育过夜以进行免疫沉淀。使用提取液清洗两遍,然后再使用含有0.5 M氯化锂的提取液清洗两遍。使用SDS样本试剂直接洗脱蛋白质,然后进行蛋白印迹分析。
8.统计分析
实验数据以均数±标准差表示。应用SPSS13.0软件进行分析,多组之间采用单因素方差分析,P<0.05认为差异具有统计学意义。
结果
1.游离脂肪酸抑制内皮细胞中TM基因的表达
人主动脉内皮细胞(HAECs)给与不同浓度的PA刺激24小时,Western blot结果显示PA能明显抑制TM蛋白的表达,差异有统计学意义(P<0.001)。
2.游离脂肪酸能激活转录因子ATF-2
人主动脉内皮细胞(HAECs)给与不同浓度的PA刺激24小时,Western blot方法检测ATF-2通路磷酸化蛋白和总蛋白的表达水平,用磷酸化蛋白／总蛋白的比值表示蛋白活性。PA能够增加转录因子ATF-2的活性,差异有统计学意义(P<0.05)。
3.转录因子ATF-2能直接与TM基因启动子结合
TM基因启动子区域包含多个ATF-2结合位点(agTGACGgatt at-1288/-1277, gcTGACTcgct at-1026/-1016, and ccTGACAgtgt at-939/-929)。首先用ChiP分析检测ATF-2能否与TM启动子区域结合。ChiP结果显示,ATF-2能与TM启动子gcTGACTcgct (-1026/-1016)和ccTGACAgtgt (-939/929)位点结合。并且,PA能增强ATF-2与TM启动子的结合。
4.转录因子ATF-2参与PA抑制TM基因表达的过程
检测转录因子ATF-2能否调控TM表达。使用特异性的ATF-2 siRNA沉默ATF-2表达,能阻止PA抑制的TM mRNA的表达(P<0.05)。
5.ATF-2与HDAC4结合在TM基因启动子区域形成转录抑制复合物
ChiP结果显示,PA能明显增强转录抑制分子HDAC4与TM基因启动子相结合。双ChiP结果表明ATF-2能与HDAC4结合形成转录抑制复合物从而抑制TM基因的表达。
结论
1.在PA抑制内皮细胞中TM表达的过程中,转录因子ATF-2参与了调控TM基因的表达过程。
2.ATF-2通过与HDAC4结合,在TM基因启动子区域形成转录抑制复合物从而抑制TM的表达。这将有助于进一步明确TM表达降低的分子生物学机制,从而为基因治疗提供新的靶点。
Backgroud
Metabolic syndrome displays a significant prothrombotic state with increased coagulation, inhibited fibronolysis and increased platelet activation. The prothrombotic state induces the increased risk of pathological thrombosis, and renders patients highly susceptible to myocardial infarction, ischemic stroke and peripheral vascular diseases. Although many clinical studies showed that the prothrombotic state is a key feature of metabolic syndrome, the pathogenesis and mechanisms of the state is not completely understood.
Protein C, a key endogenous anticoagulant molecule, is efficiently activated on the surface of endothelial cells by the binding of thrombin to the endothelial transmembrane glycoprotein, Thrombomodulin. Once activated, protein C, together with its cofactor protein S, inhibits coagulation by proteolytic degradation of factor Villa and factor Va. In addition, activated protein C (APC) promotes fibrin degradation by inhibiting the activity of plasminogen activator inhibitor—1 (PAI-1). Moreover, APC has significant anti-inflammatory and anti-apoptotic functions. Thus, APC not only regulates hemostasis and inflammation, but also provides additional levels of cellular protection. Thrombomodulin and endothelial protein C receptor (EPCR) control the protein C anticoagulation pathway. Maximal rates of protein C activation require thrombin binding to thrombomodulin and protein C binding to EPCR. Loss of thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis. Localized loss of thrombomodulin from endothelium has been associated with arteriosclerotic lesions, therefore, thrombomodulin may act in a vasoprotective manner to prevent cardiovascular diseases. Deregulation of this system may be responsible for the prothrombotic state in metabolic syndrome.
The prothrombotic state in metabolic syndrome has a complex pathogenesis. Metabolic syndrome is often characterized by high circulating concentrations of FFAs. Thus, the vasculature of patients with metabolic syndrome is constantly exposed to high levels of FFAs. Clinical studies have shown that high FFA levels directly impair vascular functions are associated with myocardial infarction, stroke, and sudden death. Long-term increases in FFA levels may have an adverse effect on the regulation of thrombosis. But it is not understood whether FFAs can regulate TM expression and the mechanisms involved.
In this study, we hypothesize that the metabolic stress of high concentrations of FFAs may deregulate the thrombomodulin-APC system, which in turn may be responsible for the development of the prothrombotic state in metabolic syndrome. Our study may be helpful to understand the mechanisms of the prothrombotic state in metabolic syndrome, and supply a new therapeutic target in treating and preventing cardiovascular diseases in metabolic syndrome.
Objectives
1. Elucidate the effects of high serum FFAs level on the prothrombotic state in metabolic syndrome.
2. Examine the effects of FFAs on the expression of TM-protion C system and the possible mechanisms involved.
Methods
1. Animal model
8 to 10 weeks of male wide-type mice were divided into two groups. One group of mice were fed a chow diet containing 10% fat, while the other group of mice were fed a high-fat diet containing 50% fat for 20 weeks. The aortas were rapidly excised and rinsed in ice-cold saline, and then stored for histology and further use.
2. Tail-bleeding time
Mice were anesthetized and placed on a 37℃heating pad. The tail was transected with a sterile scalpel at a point where the tail diameter was approximately 1 mm wide. After transection, the tail was immediately placed in a 50 mL tube containing 0.9% NaCl warmed to 37℃. The time between bleeding to stop was recorded.
3. FeCl3 induced carotid artery thrombosis model
FeCl3 induced thrombosis was used in this study. In brief, mice were anesthetized. An incision was made, and a segment of the right common carotid artery was exposed with blunt dissection. A small piece of filter paper(2 mm X 3 mm) was saturated with 10% FeCl3 was deposited on the isolated artery for 3 min, followed by washing with saline. Blood flow was monitored using a Doppler flow probe for 30 min after FeCl3 application. The occlusion time was recorded as the first image that showed 0 flow.
4. Metabolic factors measurements
Blood samples were collected from retio-orbital sinus. Circulating free fatty acids, glocuse, insulin, TG, VLDL, LDL, HDL were measured.
5. Histological and morphology analyses
Sections were stained with hematoxylin and eosin(H&E). TM, PAI-1, TF were detected with immunostaining.
6. Cell culture
Primary human aortic endothelial cells (HAECs) were cultured at 37℃in 5% CO2 in endothelial cell growth medium-2 (EGM-2). Cells between passages 5 to 9 were used for all of the experiments. The medium was changed every two or three days.
7. Preparation of fatty acid-albumin complexes.
Saturated PA, polyunsaturated LA, and monounsaturated OA were used in this study. Lipid-containing media were prepared by conjugation of FFAs with BSA using a modification of the method described previously. Briefly, FFAs were first dissolved in ethanol at 200mM, and then combined with 10% FFA-free low endotoxin BSA to final concentrations of 1-5mM. The pH of all solutions was adjusted to approximately 7.5, and the stock solutions were filter-sterilized and stored at-20℃until use. Control solution containing ethanol and BSA was prepared similarly. Working solutions were prepared fresh by diluting stock solution (1:10) in 2% FCS-EBM.
8. Protein C activation.
HAECs in 96-well plates were treated with different kind and different dose of FFAs for 24 hours.Then the activation of protein C was detected according to the manufacturer's instructions.
9. siRNA-induced gene sliencing
Sliencing gene expression wsas achieved by using specific siRNAs. HAECs were transfected with siRNAs using Lipofectamine2000 according to the manufacture's instructions.
10. Western blot analysis
Cell extracts and mice aortas were prepared with lysis buffer. Equal amounts of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Following blocking with 5% non-fat milk, the blots were incubated with a primary antibody at 4℃overnight. Then the blots were washed with TBST and incubated with HRP-conjugated secondary antibody. The blots were then visualized by use of enhanced chemiluminescence.
11. RT-PCR
Total RNA was extracted from HAECs with Trizol according to the manufacturer's instructions. Signal-strand cDNA was synthesized with iScript cDNA synthesis kit. Semi-quantitative real-time PCR was performed with iCycler iQ real-time PCR detection system.
12. Plasmid DNA transfection
HAECs were transfected with different types of plasmids including wide type, constitutively active and dominant-negative by using LipofectAMINETM 2000 according to the manufacturer's instructions. Transfected cells were then treated with PA.
Results
1. Mice model
Mice fed a high-fat diet showed notable characteristics of metabolic syndrome with higher body weight and elevated blood levels of FFAs, TG, VLDLs, glucose and insulin(P<0.05).
2. Prothrombotic state in mice fed a high-fat diet
The tail bleeding time was significantly lower in mice fed a high-fat diet than that in chow diet. Furthermore, the occlusion time following FeCl3 injury of the carotid artery was significantly shorter in mice fed a high-fat diet compared to that in control mice, indicating that the development of prothrombotic state in mice fed a high-fat diet(P<0.01).
3. Reduced expression of thrombomodulin and increased expression of PAI-1 and TF in mice fed a high-fat diet
We examined the expression of TM, PAI-1 and TF in the aorta of obese mice. Immunostaining of the vascular wall showed significantly decreased expression of thrombomodulin(P<0.001), and increased expression of PAI-1 and TF(P<0.01) on the surface of the endothelium in the aorta of mice fed a high-fat diet. This finding is consistent with the results of Western blot. These results show that thrombomodulin is down-regulated in obese mice, which may contribute to the hypercoagulable state in obesity and metabolic syndrome.
4. JNK and p38 MAPK stress signaling pathways were activated in mice fed a high-fat diet
Western blot showed that JNK and p38 MAPK stress pathways were activated in mice fed a high-fat diet(P<0.01), indicating that the activation of stree pathways may contribute to the prothrombotic state in these mice.
5. Suppression of thrombomodulin and EPCR expression and increased expression of TF by FFAs.
To determine the potential role of FFAs in thrombosis dysregulation, we examined the effect of FFAs on the expression of prothrombotic factors and antithrombotic factors in endothelial cells. Saturated fatty acid PA, polyunsaturated fatty acid LA, and monounsaturated fatty acid oleic acid (OA) were used for the study. PA and LA significantly decreased the expression of thrombomodulin in a dose-dependent fashion(P<0.01), whereas OA had a minimal effect on thrombomodulin(P>0.05). Similarly, treatment with PA and LA substantially inhibited EPCR expression and increased to varying degrees the expression of the prothrombotic factor, PAI-1(P<0.01). PA significantly decreased mRNA for thrombomodulin in a dose-dependent manner.
6. FFAs inhibited endothelial-mediated protein C activation
We compared the activation of protein C in FFA-treated and untreated HAECs. PA and LA caused a significant dose-dependent decrease in protein C activation(P<0.01), whereas OA had no effect(P>0.05).
7. JNK and p38 stress pathways were involved in PA's inhibitory effect on the expression of TM.
Silencing JNK and p38 with specific siRNAs decreased PA-induced thrombomodulin suppression(P<0.01), indicating that JNK and p38 pathways mediated PA-induced inhibition of thrombomodulin expression. Furthermore, wide type of JNK plasmid can enhance PA's inhibitory effect on TM expression, while dominant-negative of JNK plasmid can abrogate the effect.
8. Foxol was involved in PA-inhibited TM expression
When HAECs cells were transiently transfected with Foxol-specific siRNAs before treated with PA, PA-induced inhibition of TM expression was significantly decreased(P<0.001). Wide type and constitutively active of Foxol plasmids enhanced PA's inhibitory effect of TM expression(P<0.05), while dominant-negative of Foxol plasmid can abrogate the effect(P>0.05).
Conclusions
1. A high-fat diet induced high elevated circulating FFAs level, the shortened bleeding time, indicating that the prothrombotic state in metabotic syndrome mice.
2. The aortas from mice fed a high-fat diet showed decreased TM expression, and increased expression of PAI-1 and TF, indicating that the imbalance of coagulation and anticoagulation.
3. PA and LA inhibited the expression of TM and EPCR, and increased PAI-1 expression in endothelial cells, indicating that FFAs can induce the imbalance of coagulation and anticoagulation in endothelial cells.
4. JNK and p38 stress pathways mediated PA-inhibited TM expression in HAECs.
5. Foxol was involved in PA-inhibited TM expression in HAECs.
6. JNK, p38/Foxol pathway mediated free fat acid's inhibitory effect of thrombomodulin expression, which may be a new therapeutic target in treating cardiovascular and cerebrovascular complications.
Backgroud
Metabolic syndrome is associated with a prothrombotic state, which contributes to the atherothrombotic complications, such as myocardial infarction, stroke, and peripheral vascular complications. Antithrombotic therapy improves survival in acute cardiac and cerebrovascular complications patients. Although the dysregulation in coagulation and fibrinolysis has been reported, the development of prothrombotic state is not well understood.
Endothelium plays an active role in regulating pro-coagulation and anti-coagulation balance by generating several active regulatory molecules, such as von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Among these factors, thrombomodulin-protein C pathway is a major physiological anticoagulation system of the endothelium. Endothelial dysfunction can cause coagulation dysregulation and promote vascular thrombosis.
Endothelium plays an active role in regulating pro-coagulation and anti-coagulation balance by generating several active regulatory molecules, such as von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Among these factors, thrombomodulin-protein C pathway is a major physiological anticoagulation system of the endothelium. Endothelial dysfunction can cause coagulation dysregulation and promote vascular thrombosis.
Thrombomodulin, a glycoprotein on the surface of endothelial cells, is a key factor in protein C activation3. When bound to thrombin, TM triggers the activation of protein C by facilitating the conversion of circulating protein C to activated protein C (APC). Activated protein C can inhibit coagulation by degradingⅧa and factor Va and enhance fibrinolysis by inactivating PAI-1. TM plays a key role in anticoagulation, and mutation or down-regulation of TM promotes while overexpression of TM prevents arterial thrombosis. In addition, TM functions as an anti-inflammatory and anti-apoptotic molecule. It has been shown that TM inhibited inflammatory response and blocked cell apoptosis. TM is down-regulated in vascular diseases including atherosclerotic lesions, and TM is negatively regulated by inflammatory factors, wall tension and oxidized lipids. However, many reports showed that the down-regulated expression of TM was associated with the pathologic thrombosis, the mechanisms involved in the down-regulation of TM expression are not completely understood.
Mitogen-activated protein kinases (MAPKs) are serine/threonine-specific protein kinases that contribute to extracellular stimuli and regulate various cellular activities, such as gene expression, mitosis, transformation, apoptosis. Stress signaling JNK and p38 pathways are activated in many cardiovascular diseases including atherosclerosis and are involved in pathphysiological changes in these conditions. JNK and p38 signaling pathways are activated by metabolic stress and inflammatory factors. Previous study showed that JNK and p38 can be activated by free fatty acids (FFA) and were involved in vascular insulin resistance. We have found that JNK and p38 pathways are involved in the dowm-regulation of TM, but the detailed mechanisms involved are not well understood.
How stress signaling down-regulates TM expression? ATF-2 is a downstream target transcription factor of JNK and p38 pathways. Previous study has reported that ATF-2 mediated LPS-induced TF expression and may be involved in thrombosis dysregulation. But whether transcriptional factor ATF-2 is involved in the regulation of TM expression is still unclear.
So in this study, we examined the effects of transcriptional factor ATF-2 on the regulation of TM expression, which may be helpful for the development of therapeutic intervention for pathologic thrombosis and cardiovascular complications.
Objectives
1. Identify the effects of transcriptional factor ATF-2 in the regulation of TM expression in HAECs.
2. Examine the possible mechanisms how ATF-2 mediated TM expression.
Metarials and Methods
1. Cell culture
Primary human aortic endothelial cells (HAECs) were cultured at 37℃in 5% CO2 in endothelial cell growth medium-2 (EGM-2) containing 2% FBS, FGF-2, VEGF, IGF-1, EGF. Cells between passages 5 to 9 were used for all of the experiments. The medium was changed every two or three days. The endothelial cells were plated on 6-well plates, and then treated with different doses of palmitic acid or transfected with siRNAs.
2. Preparation of palmitic acids:
Preparation of PA was carried out as previously. Briefly, PA was dissolved in ethanol at 200mM, and then combined with 10% FFA-free low endotoxin BSA to final concentrations of 1-5mM. The pH of all solutions was adjusted to approximately 7.5, and the stock solutions were stored at-20℃.
3. siRNA transfection
Silencing gene expression was achieved using specific siRNA. HAECs were transfected with siRNAs using Lipofectamine2000 according to the manufacturer's instructions. Transfected cells were then treated with palmitic acid for 24h.
4. Western blot analysis
Cell extracts were prepared with lysis buffer, and boiled with 5 min. Protein samples(15μg per lane) and protein marker were seperated by SDS-PAGE gel electropohresis and transferred to PVDF membranes. The membranes were blocked with blocking buffer, and then incubated with the primary antibody overnight. The membranse were washed with PBST, and then incubated with the HRP-conjugated secondary antibody. The blots were then visualized with enhanced chemiluminescene. The expression of cytokine was demonstrated by the ration of integral optical density between cytokine and p-actin
5. RNA extration and real-time quantitative PCR
Total RNA was extracted from HAECs with Trizol according to the manufacturer's instructions. Signal-strand cDNA was synthesized with iScript cDNA synthesis kit. Semi-quantitative real-time PCR was performed with iCycler iQ real-time PCR detection system. The primers for human thrombomodulin mRNA were as follows:forward:5'-CCGATGTCATTTCCTTGCTA-3'; reverse:5'-GTTGTCTCCCGTAACCCACT-3'. The mRNA levels were estimated from the value of the threshold cycle (Ct) of the real-time PCR adjusted by that ofβ-actin.
6. Chromatin immunoprecipitation assay
The ChIP assay kit (Upstate) was used according to the manufacturer's instructions. Treated HAECs were first incubated with 1% formaldehyde at 37℃for 15 min to cross-link DNA-protein complexes. Cells were then rinsed and lysed. Cell lysates were sonicated and centrifuged to produce chromatin fragments. The supernatants were pre-cleared with a mixture of salmon sperm DNA/protein A/protein G, followed by immunoprecipitation with antibody-protein A-agarose slurry. (IgG served as the negative control.) The DNA was recovered by extraction with the phenol/chloroform/isoamyl alcohol mixture. The immunoprecipitated DNA was used as a template for PCR. The PCR products were separated by 1.5% agarose gel.
7. Immunoprecipitation
Immunoprecipitation was conducted as described previously.Treated cells were lysed for 60 min in ice-cold extraction buffer. For immunoprecipitation, cleared cell lysates were incubated with the appropriate antibody precoupled to protein A/G-agarose beads (Santa Cruz Biotechnology) at 4℃overnight. The beads were washed twice with extraction buffer and twice with extraction buffer containing 0.5 M LiCl. Proteins were eluted directly in SDS sample buffer for Western blot analysis.
8. Statistical analysis
Data are presented as mean±SEM. One-way ANOVA was used to analyze the differences among groups. P values< 0.05 were considered statistically significant.
Results:
1. Free fatty acids suppressed thrombomodulin expression in HAECs.
HAECs were incubated with different concentrations of PA for 24h. Western blotting showed that PA significantly suppressed the expression of thrombomodulin in a dose-dependent manner. Furthermore, PA significantly inhibited the expression of thrombomodulin mRNA, indicating that PA suppressed thrombomodulin expression at the transcriptional level.
2. Free fatty acids activated ATF-2 pathway.
HAECs were treated with different doses of PA for 24h. The phosphorylated protein and total protein of ATF-2 were examined by Western blot. The activation of pathways was demonstrated by the ratio of phosphorylated and total protein. Consistent with previous observation, ATF-2 was activated by PA in a dose-dependent manner.
3. Transcriptional factor ATF-2 binded directly to the TM promoter.
The promoter region in the TM gene contained many ATF-2 binding sites(agTGACGgatt at-1288/-1277, gcTGACTcgct at-1026/-1016, and ccTGACAgtgt at-939/-929). ChiP assay was used to examine whether ATF-2 can bind to the TM promoter. The results of ChiP assay showed that ATF-2 can bind to the TM promoter at the gcTGACTcgct (-1026/-1016) and ccTGACAgtgt (-939/-929) sites. Importantly, the binding was significantly increased by PA treatment, indicating that binding of ATF-2 to TM promoter may be involved in PA-induced suppression of thrombomodulin transcription.
4. ATF-2 was involved in PA-inhibited TM expression.
We then examined whether ATF-2 can regulate TM expression. When HAECs cells were transiently transfected with ATF-2-specific siRNAs before treated with PA, PA-induced inhibition of TM expression was significantly prevented, which showed that a critical role for ATF-2 transcription factor in PA-induced down-regulation of thrombomodulin transcription (P<0.05).
5. Recruitment of HDAC4 and formation of ATF-2/HDAC4 transcription repressor complex in the TM promoter.
We finally investigated how the transcription factor ATF-2 can bind to the TM promoter and inhibit gene expression. ChiP assay showed that PA treatment significantly increased transcription repressor HDAC4 binding to the TM promoter. The double-chip assay showed that ATF-2 and HDAC4 were in the same transcription repression complex in the TM promoter.
Conclusions
1. PA significantly inhibited thrombomodulin expression in human aortic endothelial cells(HAECs).
2. ATF-2 was involved in the down-regulation of thrombomodulin expression.
3. ATF-2 was mediated TM suppression by recruitment of HDAC4 and formation of ATF-2/HDAC4 transcription repressor complex in the TM promoter.
引文
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