模式识别受体在血管内膜增生及血管平滑肌细胞激活中的作用
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摘要
背景和目的:血管内膜增生是动脉血管对各种损伤的一种反应,也是动脉粥样硬化和经皮冠状动脉介入治疗(percutaneous coronary intervention, PCI)术后再狭窄的重要标志。血管平滑肌细胞( vascular smooth muscle cell,VSMC)被各种损伤刺激激活后,由静息状态转变为增殖表型并移行到内膜下,参与合成各种细胞外基质,在内膜增生形成过程中具有重要作用。越来越多的证据表明感染和各种组织损伤能够通过诱导先天免疫反应,促进动脉壁的炎症,在VSMC激活和血管内膜增生过程中具有重要作用。但这些免疫反应调节和相互作用的分子生物学机制还不是很清楚。
模式识别受体(pattern-recognition receptors,PRRs)是一种经过广泛选择的种系编码的受体,它们通过识别病原体关联的分子模式(pathogen-associated molecular patterns,PAMPs)来感知侵入机体的危险信号。它们与其它免疫组份一起构成了机体的第一道防线。Toll样受体(toll-like receptors,TLRs)和核苷酸结合的寡聚结构域(nucleotide- binding oligomerization domain,NOD)受体是近期确定的参与炎症疾病的两个PRRs家族。TLRs主要位于细胞表面,识别来源于细胞外部的危险信号。细菌为了避免被清除已经发展出一些侵入和通过上皮层的对策。在侵入上皮下间隙后,细菌还可以遭遇到吞噬细胞的局部抵抗,但是细菌能够利用各种对策避免被吞噬细胞的溶酶体吞食和降解而侵入细胞内。NOD受体是一种功能上与TLR相似,但位于细胞胞质溶胶的蛋白家族,它们能识别侵入细胞内的细菌产物,负责识别侵入细胞内的危险信号,启动免疫反应,代表了细胞内的一种防御监测手段。TLRs和NOD受体都具有一个富含亮氨酸的重复结构域(leucine–rich repeat domain,LRR),负责与特异的PAMPs结合。TLRs和NOD受体被激活后能启动下游衔接分子的级联反应,激活NF-κB信号传导通路,启动炎症细胞因子和其它先天免疫反应成分的转录,在清除病原感染的炎症反应中起着重要作用。NF-κB信号传导通路的激活介导了许多炎症因子的表达,这些炎症因子参与血管损伤炎症反应,在VSMC增殖、分化和凋亡的过程中起到重要的调节作用,由此推测TLRs和NOD受体介导的信号传导通路可能在血管内膜增生和VSMC激活过程中起到重要作用。
本研究利用大鼠颈总动脉狭窄这一经典动物模型,观察血管内膜增生过程中TLR4/NF-κB信号传导通路的变化,以及NOD1受体在动脉损伤后增生的血管内膜中的表达和功能。比较NOD1受体在增生的内膜平滑肌细胞(iSMC)和血管中层平滑肌细胞(mSMC)中的功能。同时还对NOD1和NOD2受体在人冠状动脉VSMC中的表达及其介导的先天免疫信号传导通路在VSMC的激活过程中的作用进行研究,并探讨NOD2与TLR2、4介导的先天免疫信号传导通路在此过程中的相互关系,探讨先天免疫模式识别受体在血管内膜增生和VSMC激活过程中的作用,为防治动脉粥样硬化形成和PCI术后再狭窄提供新的思路。
方法和结果:第一部分:建立大鼠颈动脉血管损伤模型,将大鼠分为正常血管组、损伤7天组和损伤14天组,用免疫组织化学染色、Western blotting和半定量反转录多聚酶联聚合反应(RT-PCR)的方法,检测了TLR4和NF-κB在损伤的大鼠颈动脉中的表达。结果发现大鼠颈动脉新生内膜在血管损伤后7天增加,增厚的内膜中以VSMC为主,损伤14天后血管内膜增生更加明显。新生的内膜平滑肌中TLR4和NF-κB染色阳性信号损伤14天组(TLR4:37.20±2.10;NF-κB:23.2±0.68)高于损伤7天组(TLR4:15.58±0.91;NF-κB:9.8±0.5),损伤7天组要高于正常组(TLR4:1.50±0.36;NF-κB:1.0±0.3)。与此一致,增生的内膜平滑肌中TLR4蛋白和mRNA表达水平也随损伤时间增加,损伤14天组(TLR4蛋白:57.1±2.2;TLR4 mRNA:0.49±0.019)高于损伤7天组(TLR4蛋白:26.2±0.9;TLR4 mRNA:0.39±0.014),损伤7天组要高于正常组(TLR4蛋白:12.0±0.5;TLR4 mRNA:0.18±0.013)。第二部分:我们用计算机比对分析的方法在大鼠基因组中确定了人类NOD1基因的类似物,并发现它们在保守的结构域上高度同源。我们应用大鼠颈动脉血管损伤模型,用免疫组织化学染色和半定量RT-PCR的方法,检测了大鼠NOD1在损伤血管中的表达。我们发现在大鼠NOD1 mRNA在颈动脉损伤后表达增加,通过免疫组化染色的方法确定了NOD1主要位于增生的内膜中。我们将增生的iSMC分离培养,并用NOD1受体特异的配体diaminopimalic acid(DAP)刺激iSMC,用实时定量RT-PCR法检测了iSMC中诱生型一氧化氮合酶(inducible nitric oxide synthase ,iNOS)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的表达,结果发现DAP能以时间和剂量依赖的方式诱导iSMC表达iNOS。刺激NOD1还能够诱导TNF-α在iSMC中短暂的表达上调。DAP还能以时间和剂量依赖的方式诱导iSMC分泌一氧化氮(nitric oxide ,NO)。我们分别用DAP和干扰素-γ(interferon-γ,IFN-γ)单独或联合刺激iSMC和mSMC 24小时,用Western blot的方法分析NOD1、受体相互作用蛋白2(receptor-interacting protein 2,Rip2)和iNOS蛋白的表达,并检测细胞培养物上清液中NO的浓度。结果发现DAP只能诱导iSMC表达RIP2和iNOS。mSMC中没有RIP2蛋白表达。DAP和IFN-γ还能协同诱导iSMC产生NO,而在mSMC中没有发现这种协同现象。第三部分:(一)体外培养人冠状动脉VSMC,用NOD1受体的激动剂PGN(10μg/ml)刺激0、3、6和24h,用实时定量RT- PCR检测VSMC中NOD1和成纤维生长因子-2(fibroblast growth factor-2,FGF-2) mRNA的表达,用ELISA法测定细胞培养物上清夜中白细胞介素8(interleukin-8,IL-8)和TNF-α的浓度。发现VSMC在静息状态下表达低水平的NOD1。PGN刺激能够引起VSMC中NOD1 mRNA表达上调(从0.164±0.005增加到0.231±0.027,p<0.05)。伴随NOD1表达上调,PGN刺激能引起VSMC中FGF-2 mRNA快速、短暂的表达上调,在6h(22.72±0.58)达到高峰,随即下降(24h:8.85±0.96;3h:13.74±0.77; 0h:8.05±0.26)。PGN刺激能引起细胞培养物上清液中IL-8和TNF-α的分泌显著增加(IL-8从0.12±0.01 ng/ml增加到2.31±0.11ng/ml;TNF-α从4.22±0.50 pg/ml增加到143.11±12.58 pg/ml)。(二)体外培养人冠状动脉VSMC,用NOD2激动剂胞壁酰二肽(Muramyl dipeptide,MDP)、TLR2激动剂(Pam3CSK4,PAM3)和TLR4激动剂脂多糖(lipopolysaccharides,LPS)单独或联合进行刺激,用实时定量RT- PCR检测VSMC中NOD2和FGF-2 mRNA的表达,用ELISA法测定细胞培养物上清液中IL-8和TNF-α的浓度,用MTT检测VSMC增殖活性。MDP能使VSMC中NOD2 mRNA呈时间依赖性表达上调(0h:0.028±0. 001;3h:0.045±0.002;6h:0.053±0.002;24h:0.162±0.013),并能引起VSMC中FGF-2 mRNA的表达增加(9.32±0.37 vs对照组7.44±0.25;p<0.05),细胞培养物上清液中IL-8(2.41±0.22 ng/ml vs对照组1.02±0.13 ng/ml;p<0.05)和TNF-α(51.9±4.73 pg/ml vs对照组29.4±3.73 pg/ml;p<0.05)的分泌增多和细胞增殖活性增加(0.87±0.05 vs对照组0.72±0.02;p<0.05)。MDP还能协同LPS、PAM3诱导VSMC活性增加,细胞培养物上清液中IL-8和TNF-α的分泌增加。
结论:1.在大鼠颈总动脉球囊损伤后再狭窄模型中,TLR4的mRNA及蛋白水平均表达增高,NF-κB活性增加,表明TLR4/NF-κB通路有可能参与了再狭窄形成的过程。
2. NOD1在大鼠组织中也有表达并与人NOD1高度同源。NOD1在血管损伤修复过程中表达上调,并主要集中于增生的iSMC。iSMC中的NOD1激活后能强烈诱导iNOS和TNF-α这两个NF- B依赖的基因的表达,表明NOD1是一个特异的血管内膜增生先天免疫反应介质。iSMC与mSMC胞浆内的NOD1受体功能的不同,这可能是由两者下游的信号通路不同所致,在mSMC中缺乏NOD1信号传导通路中关键的下游衔接分子RIP2蛋白表达。
3. VSMC中NOD1介导的先天免疫信号传导通路被激活后能引起下游的促炎症因子如IL-8和TNF-α分泌增加,诱导VSMC表达FGF-2。这可能代表一个与血管炎症和内膜形成有关的新的先天免疫反应激活通路。
4. NOD2介导的先天免疫信号传导通路的激活能导致VSMC增殖活性增加,诱导其分泌促炎症细胞因子。该信号通路与TLR2、4介导的先天免疫信号传导通路在促进VSMC增殖活性增加,诱导VSMC分泌促炎症细胞因子的过程中具有协同作用。
Background and Objective: Vascular intimal hyperplasia is a response of the arterial wall upon a wide range of stimuli. It is also a prominent hallmark of atherosclerosis and restenosis after percutaneous coronary intervention (PCI). Activation of vascular smooth muscle cells (VSMC), which switch from quiescent state to proliferative phenotype, migrates into the intima to proliferate and produce extracellular matrix proteins, is a key event in intimal hyperplasia in response to inflammation or mechanical injury. More and more evidences show that infections and injurious stimuli play an important role in the activation of VSMC and intimal hyperplasia by triggering innate immune responses and promoting inflammation in the vessel wall. Yet, underlying molecular bases remain undefined.
Pattern recognition receptors (PRRs) represent a diverse collection of germline-encoded receptors responsible for detecting danger signals by sensing pathogen-associated molecular patterns (PAMPs), and together with other immune components they are involved in the first line of defence. Two families of PRRs, toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) receptors have recently been identified and implicated in inflammatory diseases. Among them, TLRs are expressed on the surface of cells and responsible for detecting extracelluar PAMPs. To avoid recognition and escape from defense, bacteria have developed strategies for invading and crossing the epithelium. Upon arrival at the subepithelial space, bacteria encounter locally resident, as well as newly infiltrated, professional phagocytic cells. However, bacteria use a variety of strategies to avoid engulfment and degradation by the endolysosomal compartment by phagocytes. NOD receptors have the similar function of TLRs, which are cytoplasmic proteins, involved in intracellular bacterial detection, representing a means of cytosolic surveillance.
Both TLRs and NOD receptors have a leucine rich repeat domain, which is involved in specific PAMPs recognition. Activation of TLRs and NOD receptors can initiate downstream cascade reaction of adapter molecules, which eventually lead the activation of nuclear factor-κB (NF-κB) and initiate the transcription of inflammatory cytokines and other host response elements, play a key role in the clearance of infecting pathogens. NF-κB signal transduction pathway play a central role in regulating VSMC proliferation, differentiation and apoptosis by inducing the expression of many kinds of proinflammatory cytokines. Quibus we suppose the signal pathway mediated by TLR or NOD receptors may play an important role in the VSMC activation and intimal hyperplasia.
To explore the role of PRRs in the VSMC activation and intimal hyperplasia, and provide a novel insight for preventing atherosclerosis and restenosis, we explore the expression of TLR4/NF-κB and NOD1 signal pathway during the progression of intimal hyperplasia by a well established animal model of restenosis of rat balloon injury of the common carotid artery. We also observe the expression and function of NOD1 in the rat intimal smooth muscle cells (iSMC), and the NOD1 functions difference in iSMC and medial SMC (mSMC). We further investigate the expression of NOD1 and NOD2 in human VSMC, and the role of NOD1 and NOD2 mediated innate immune signaling pathway in the activation VSMC. We also explore the possible interaction of NOD2 signal pathways with TLR2 and 4 signal pathways in the induction of proinflammatory cytokines in VSMC.
Methods and Results: Part one: We use immunohistochemistry, Western blotting and semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) to detect the expression of TLR4 and NF-κB on rat carotid arteries following angioplastic injury. We found the level of TLR4 both in mRNA and protein was increase following the neointimal formation. The expression of NF-κB was also up-regulated with intima hyperplasia.
Part two: Using the methods of immunohistochemistry and semiquantitative RT-PCR, we found that expression of human homologue of NOD1 gene was highly up-regulated with the development of intima after angioplastic injury to rat carotid artery, and predominantly localized in intimal lesion. Studies using isolated iSMC showed that stimulation of NOD1 by its ligand diaminopimalic acid (DAP) led to marked induction of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in time-depend and dose-depend manner. Stimulation of NOD1 also led to a transient up-regulation of tumor necrosis factor-α?TNF-α??in iSMC. Rat iSMCs and mSMC were stimulated with DAP and/or IFN- ?for 24 hours. Expression of NOD1 and receptor-interacting protein 2(RIP2) and iNOS were assessed by Western blot analysis. In spite of comparable levels of NOD1 protein in iSMCs and mSMC under basal condition, activation of RIP2 and expression of iNOS were elicited merely in iSMC subsequent to the stimulation. In addition, a synergistic effect of DAP and IFN- ?was shown in the induction of NO production by iSMC whereas such effect could not be found in mSMC.
Part three: (1) Human coronary artery SMCs were in vitro stimulated with NOD1 receptor agonist PGN(10μg/ml)for 0, 3, 6 and 24 hours. The mRNA expression of NOD1 and fibroblast growth factor-2(FGF-2) were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of interleukin-8(IL-8) and TNF-αwas determined by ELISA. We found human SMC constitutively expressed a low level of NOD1 under resting condition. Upon PGN stimulation, the expression of NOD1 mRNA was up-regulated in SMC, from 0.164±0.005 to 0.231±0.027 (p< 0.05). Stimulation of NOD1 also led to a transient up-regulation of FGF-2, which reaches the peak at 6 hour(22.72±0.58), then decrease quickly(24h:8.85±0.96;3h:13.74±0.77; 0h:8.05±0.26)。ELISA showed the concentration in the culture supernatants of IL-8 and TNF-αincreased from 0.12±0.01ng/ml to 2.31±0.11 ng/ml and from 4.22±0.50 pg/ml to 143.11±12.58 pg/ml, respectively (p< 0.01).
(2) Human coronary artery SMCs were in vitro stimulated with NOD2 agonist Muramyl dipeptide (MDP), TLR2 agonist Pam3CSK4(PAM3)and TLR4 agonist lipopolysaccharides (LPS) alone or MDP in synergy with either PAM3 or LPS. The mRNA expression of NOD2 and FGF-2 were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of IL-8 and TNF-αwas determined by ELISA. VSMC proliferation viability was analyzed by the MTT assay. MDP can up-regulate the expression of NOD2 mRNA as a time-dependent manner (0h: 0.028±0. 001; 3h: 0.045±0.002; 6h: 0.053±0.002; 24h: 0.162±0.013) in VSMC. It can also up-regulate the expression of FGF-2 mRNA (9.32±0.37 vs control 7.44±0.25; p<0.05) in VSMC, induce the production of IL-8 and TNF-α(2.41±0.22 ng/ml vs control 1.02±0.13 ng/ml; 51.9±4.73 pg/ml vs control 29.4±3.73 pg/ml; respectively, p < 0.05) and increase the proliferation viability of VSMC (0.87±0.05 vs control 0.72±0.02; p<0.05). Additional, MDP can synergy with LPS and PAM3 to increase the proliferation viability of VSMC and induce the production of IL-8 and TNF-α.
Conclusion: 1.In the model of vascular restenosis by balloon injury, the levels of TLR4 mRNA and protein of carotid arteries were significantly increased and companied with activation of NF-κB. It implied that TLR4/ NF-κB pathway may play an important role in the developing of restenosis. 2. NOD1 is also expressed in rat tissue and exhibits highly homology with the human NOD1. Rat NOD1 has an increased expression during intimal hyperplasia in a rat carotid artery injury model and preferentially expressed by iSMCs. Activation of NOD1 can robustly induce the expression of iNOS and TNF-α, which regulated by NF-κB signal pathway. It implied that NOD1 is an intimal specific innate immune response mediator. The functional difference of NOD1 in iSMC and mSMC maybe due to the difference of their downstream mediator. The mSMC lacks RIP2 protein, a key component of NOD1 signal pathway.
3. The activation of NOD1 mediated innate immune signaling pathway can induce the production of proinflammatory cytokines such as IL-8 and TNF-αin VSMC. It can also induce the expression of FGF-2. This may represent a novel innate immune activation pathway in vascular inflammation and intimal formation.
4. The activation of NOD2 mediated innate immune signaling pathway can increase the proliferation viability of VSMC and induce the production of proinflammatory cytokines in VSMC. It had a synergistic effect with those of TLR2 and TLR4 mediated signaling pathways in this process.
模式识别受体(pattern-recognition receptors,PRRs)是一种经过广泛选择的种系编码的受体,它们通过识别病原体关联的分子模式(pathogen-associated molecular patterns,PAMPs)来感知侵入机体的危险信号。它们与其它免疫组份一起构成了机体的第一道防线。Toll样受体(toll-like receptors,TLRs)和核苷酸结合的寡聚结构域(nucleotide- binding oligomerization domain,NOD)受体是近期确定的参与炎症疾病的两个PRRs家族。TLRs主要位于细胞表面,识别来源于细胞外部的危险信号。细菌为了避免被清除已经发展出一些侵入和通过上皮层的对策。在侵入上皮下间隙后,细菌还可以遭遇到吞噬细胞的局部抵抗,但是细菌能够利用各种对策避免被吞噬细胞的溶酶体吞食和降解而侵入细胞内。NOD受体是一种功能上与TLR相似,但位于细胞胞质溶胶的蛋白家族,它们能识别侵入细胞内的细菌产物,负责识别侵入细胞内的危险信号,启动免疫反应,代表了细胞内的一种防御监测手段。TLRs和NOD受体都具有一个富含亮氨酸的重复结构域(leucine–rich repeat domain,LRR),负责与特异的PAMPs结合。TLRs和NOD受体被激活后能启动下游衔接分子的级联反应,激活NF-κB信号传导通路,启动炎症细胞因子和其它先天免疫反应成分的转录,在清除病原感染的炎症反应中起着重要作用。NF-κB信号传导通路的激活介导了许多炎症因子的表达,这些炎症因子参与血管损伤炎症反应,在VSMC增殖、分化和凋亡的过程中起到重要的调节作用,由此推测TLRs和NOD受体介导的信号传导通路可能在血管内膜增生和VSMC激活过程中起到重要作用。
本研究利用大鼠颈总动脉狭窄这一经典动物模型,观察血管内膜增生过程中TLR4/NF-κB信号传导通路的变化,以及NOD1受体在动脉损伤后增生的血管内膜中的表达和功能。比较NOD1受体在增生的内膜平滑肌细胞(iSMC)和血管中层平滑肌细胞(mSMC)中的功能。同时还对NOD1和NOD2受体在人冠状动脉VSMC中的表达及其介导的先天免疫信号传导通路在VSMC的激活过程中的作用进行研究,并探讨NOD2与TLR2、4介导的先天免疫信号传导通路在此过程中的相互关系,探讨先天免疫模式识别受体在血管内膜增生和VSMC激活过程中的作用,为防治动脉粥样硬化形成和PCI术后再狭窄提供新的思路。
方法和结果:第一部分:建立大鼠颈动脉血管损伤模型,将大鼠分为正常血管组、损伤7天组和损伤14天组,用免疫组织化学染色、Western blotting和半定量反转录多聚酶联聚合反应(RT-PCR)的方法,检测了TLR4和NF-κB在损伤的大鼠颈动脉中的表达。结果发现大鼠颈动脉新生内膜在血管损伤后7天增加,增厚的内膜中以VSMC为主,损伤14天后血管内膜增生更加明显。新生的内膜平滑肌中TLR4和NF-κB染色阳性信号损伤14天组(TLR4:37.20±2.10;NF-κB:23.2±0.68)高于损伤7天组(TLR4:15.58±0.91;NF-κB:9.8±0.5),损伤7天组要高于正常组(TLR4:1.50±0.36;NF-κB:1.0±0.3)。与此一致,增生的内膜平滑肌中TLR4蛋白和mRNA表达水平也随损伤时间增加,损伤14天组(TLR4蛋白:57.1±2.2;TLR4 mRNA:0.49±0.019)高于损伤7天组(TLR4蛋白:26.2±0.9;TLR4 mRNA:0.39±0.014),损伤7天组要高于正常组(TLR4蛋白:12.0±0.5;TLR4 mRNA:0.18±0.013)。第二部分:我们用计算机比对分析的方法在大鼠基因组中确定了人类NOD1基因的类似物,并发现它们在保守的结构域上高度同源。我们应用大鼠颈动脉血管损伤模型,用免疫组织化学染色和半定量RT-PCR的方法,检测了大鼠NOD1在损伤血管中的表达。我们发现在大鼠NOD1 mRNA在颈动脉损伤后表达增加,通过免疫组化染色的方法确定了NOD1主要位于增生的内膜中。我们将增生的iSMC分离培养,并用NOD1受体特异的配体diaminopimalic acid(DAP)刺激iSMC,用实时定量RT-PCR法检测了iSMC中诱生型一氧化氮合酶(inducible nitric oxide synthase ,iNOS)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的表达,结果发现DAP能以时间和剂量依赖的方式诱导iSMC表达iNOS。刺激NOD1还能够诱导TNF-α在iSMC中短暂的表达上调。DAP还能以时间和剂量依赖的方式诱导iSMC分泌一氧化氮(nitric oxide ,NO)。我们分别用DAP和干扰素-γ(interferon-γ,IFN-γ)单独或联合刺激iSMC和mSMC 24小时,用Western blot的方法分析NOD1、受体相互作用蛋白2(receptor-interacting protein 2,Rip2)和iNOS蛋白的表达,并检测细胞培养物上清液中NO的浓度。结果发现DAP只能诱导iSMC表达RIP2和iNOS。mSMC中没有RIP2蛋白表达。DAP和IFN-γ还能协同诱导iSMC产生NO,而在mSMC中没有发现这种协同现象。第三部分:(一)体外培养人冠状动脉VSMC,用NOD1受体的激动剂PGN(10μg/ml)刺激0、3、6和24h,用实时定量RT- PCR检测VSMC中NOD1和成纤维生长因子-2(fibroblast growth factor-2,FGF-2) mRNA的表达,用ELISA法测定细胞培养物上清夜中白细胞介素8(interleukin-8,IL-8)和TNF-α的浓度。发现VSMC在静息状态下表达低水平的NOD1。PGN刺激能够引起VSMC中NOD1 mRNA表达上调(从0.164±0.005增加到0.231±0.027,p<0.05)。伴随NOD1表达上调,PGN刺激能引起VSMC中FGF-2 mRNA快速、短暂的表达上调,在6h(22.72±0.58)达到高峰,随即下降(24h:8.85±0.96;3h:13.74±0.77; 0h:8.05±0.26)。PGN刺激能引起细胞培养物上清液中IL-8和TNF-α的分泌显著增加(IL-8从0.12±0.01 ng/ml增加到2.31±0.11ng/ml;TNF-α从4.22±0.50 pg/ml增加到143.11±12.58 pg/ml)。(二)体外培养人冠状动脉VSMC,用NOD2激动剂胞壁酰二肽(Muramyl dipeptide,MDP)、TLR2激动剂(Pam3CSK4,PAM3)和TLR4激动剂脂多糖(lipopolysaccharides,LPS)单独或联合进行刺激,用实时定量RT- PCR检测VSMC中NOD2和FGF-2 mRNA的表达,用ELISA法测定细胞培养物上清液中IL-8和TNF-α的浓度,用MTT检测VSMC增殖活性。MDP能使VSMC中NOD2 mRNA呈时间依赖性表达上调(0h:0.028±0. 001;3h:0.045±0.002;6h:0.053±0.002;24h:0.162±0.013),并能引起VSMC中FGF-2 mRNA的表达增加(9.32±0.37 vs对照组7.44±0.25;p<0.05),细胞培养物上清液中IL-8(2.41±0.22 ng/ml vs对照组1.02±0.13 ng/ml;p<0.05)和TNF-α(51.9±4.73 pg/ml vs对照组29.4±3.73 pg/ml;p<0.05)的分泌增多和细胞增殖活性增加(0.87±0.05 vs对照组0.72±0.02;p<0.05)。MDP还能协同LPS、PAM3诱导VSMC活性增加,细胞培养物上清液中IL-8和TNF-α的分泌增加。
结论:1.在大鼠颈总动脉球囊损伤后再狭窄模型中,TLR4的mRNA及蛋白水平均表达增高,NF-κB活性增加,表明TLR4/NF-κB通路有可能参与了再狭窄形成的过程。
2. NOD1在大鼠组织中也有表达并与人NOD1高度同源。NOD1在血管损伤修复过程中表达上调,并主要集中于增生的iSMC。iSMC中的NOD1激活后能强烈诱导iNOS和TNF-α这两个NF- B依赖的基因的表达,表明NOD1是一个特异的血管内膜增生先天免疫反应介质。iSMC与mSMC胞浆内的NOD1受体功能的不同,这可能是由两者下游的信号通路不同所致,在mSMC中缺乏NOD1信号传导通路中关键的下游衔接分子RIP2蛋白表达。
3. VSMC中NOD1介导的先天免疫信号传导通路被激活后能引起下游的促炎症因子如IL-8和TNF-α分泌增加,诱导VSMC表达FGF-2。这可能代表一个与血管炎症和内膜形成有关的新的先天免疫反应激活通路。
4. NOD2介导的先天免疫信号传导通路的激活能导致VSMC增殖活性增加,诱导其分泌促炎症细胞因子。该信号通路与TLR2、4介导的先天免疫信号传导通路在促进VSMC增殖活性增加,诱导VSMC分泌促炎症细胞因子的过程中具有协同作用。
Background and Objective: Vascular intimal hyperplasia is a response of the arterial wall upon a wide range of stimuli. It is also a prominent hallmark of atherosclerosis and restenosis after percutaneous coronary intervention (PCI). Activation of vascular smooth muscle cells (VSMC), which switch from quiescent state to proliferative phenotype, migrates into the intima to proliferate and produce extracellular matrix proteins, is a key event in intimal hyperplasia in response to inflammation or mechanical injury. More and more evidences show that infections and injurious stimuli play an important role in the activation of VSMC and intimal hyperplasia by triggering innate immune responses and promoting inflammation in the vessel wall. Yet, underlying molecular bases remain undefined.
Pattern recognition receptors (PRRs) represent a diverse collection of germline-encoded receptors responsible for detecting danger signals by sensing pathogen-associated molecular patterns (PAMPs), and together with other immune components they are involved in the first line of defence. Two families of PRRs, toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) receptors have recently been identified and implicated in inflammatory diseases. Among them, TLRs are expressed on the surface of cells and responsible for detecting extracelluar PAMPs. To avoid recognition and escape from defense, bacteria have developed strategies for invading and crossing the epithelium. Upon arrival at the subepithelial space, bacteria encounter locally resident, as well as newly infiltrated, professional phagocytic cells. However, bacteria use a variety of strategies to avoid engulfment and degradation by the endolysosomal compartment by phagocytes. NOD receptors have the similar function of TLRs, which are cytoplasmic proteins, involved in intracellular bacterial detection, representing a means of cytosolic surveillance.
Both TLRs and NOD receptors have a leucine rich repeat domain, which is involved in specific PAMPs recognition. Activation of TLRs and NOD receptors can initiate downstream cascade reaction of adapter molecules, which eventually lead the activation of nuclear factor-κB (NF-κB) and initiate the transcription of inflammatory cytokines and other host response elements, play a key role in the clearance of infecting pathogens. NF-κB signal transduction pathway play a central role in regulating VSMC proliferation, differentiation and apoptosis by inducing the expression of many kinds of proinflammatory cytokines. Quibus we suppose the signal pathway mediated by TLR or NOD receptors may play an important role in the VSMC activation and intimal hyperplasia.
To explore the role of PRRs in the VSMC activation and intimal hyperplasia, and provide a novel insight for preventing atherosclerosis and restenosis, we explore the expression of TLR4/NF-κB and NOD1 signal pathway during the progression of intimal hyperplasia by a well established animal model of restenosis of rat balloon injury of the common carotid artery. We also observe the expression and function of NOD1 in the rat intimal smooth muscle cells (iSMC), and the NOD1 functions difference in iSMC and medial SMC (mSMC). We further investigate the expression of NOD1 and NOD2 in human VSMC, and the role of NOD1 and NOD2 mediated innate immune signaling pathway in the activation VSMC. We also explore the possible interaction of NOD2 signal pathways with TLR2 and 4 signal pathways in the induction of proinflammatory cytokines in VSMC.
Methods and Results: Part one: We use immunohistochemistry, Western blotting and semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) to detect the expression of TLR4 and NF-κB on rat carotid arteries following angioplastic injury. We found the level of TLR4 both in mRNA and protein was increase following the neointimal formation. The expression of NF-κB was also up-regulated with intima hyperplasia.
Part two: Using the methods of immunohistochemistry and semiquantitative RT-PCR, we found that expression of human homologue of NOD1 gene was highly up-regulated with the development of intima after angioplastic injury to rat carotid artery, and predominantly localized in intimal lesion. Studies using isolated iSMC showed that stimulation of NOD1 by its ligand diaminopimalic acid (DAP) led to marked induction of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in time-depend and dose-depend manner. Stimulation of NOD1 also led to a transient up-regulation of tumor necrosis factor-α?TNF-α??in iSMC. Rat iSMCs and mSMC were stimulated with DAP and/or IFN- ?for 24 hours. Expression of NOD1 and receptor-interacting protein 2(RIP2) and iNOS were assessed by Western blot analysis. In spite of comparable levels of NOD1 protein in iSMCs and mSMC under basal condition, activation of RIP2 and expression of iNOS were elicited merely in iSMC subsequent to the stimulation. In addition, a synergistic effect of DAP and IFN- ?was shown in the induction of NO production by iSMC whereas such effect could not be found in mSMC.
Part three: (1) Human coronary artery SMCs were in vitro stimulated with NOD1 receptor agonist PGN(10μg/ml)for 0, 3, 6 and 24 hours. The mRNA expression of NOD1 and fibroblast growth factor-2(FGF-2) were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of interleukin-8(IL-8) and TNF-αwas determined by ELISA. We found human SMC constitutively expressed a low level of NOD1 under resting condition. Upon PGN stimulation, the expression of NOD1 mRNA was up-regulated in SMC, from 0.164±0.005 to 0.231±0.027 (p< 0.05). Stimulation of NOD1 also led to a transient up-regulation of FGF-2, which reaches the peak at 6 hour(22.72±0.58), then decrease quickly(24h:8.85±0.96;3h:13.74±0.77; 0h:8.05±0.26)。ELISA showed the concentration in the culture supernatants of IL-8 and TNF-αincreased from 0.12±0.01ng/ml to 2.31±0.11 ng/ml and from 4.22±0.50 pg/ml to 143.11±12.58 pg/ml, respectively (p< 0.01).
(2) Human coronary artery SMCs were in vitro stimulated with NOD2 agonist Muramyl dipeptide (MDP), TLR2 agonist Pam3CSK4(PAM3)and TLR4 agonist lipopolysaccharides (LPS) alone or MDP in synergy with either PAM3 or LPS. The mRNA expression of NOD2 and FGF-2 were measured by real time quantitative RT-PCR. The concentration in the culture supernatants of IL-8 and TNF-αwas determined by ELISA. VSMC proliferation viability was analyzed by the MTT assay. MDP can up-regulate the expression of NOD2 mRNA as a time-dependent manner (0h: 0.028±0. 001; 3h: 0.045±0.002; 6h: 0.053±0.002; 24h: 0.162±0.013) in VSMC. It can also up-regulate the expression of FGF-2 mRNA (9.32±0.37 vs control 7.44±0.25; p<0.05) in VSMC, induce the production of IL-8 and TNF-α(2.41±0.22 ng/ml vs control 1.02±0.13 ng/ml; 51.9±4.73 pg/ml vs control 29.4±3.73 pg/ml; respectively, p < 0.05) and increase the proliferation viability of VSMC (0.87±0.05 vs control 0.72±0.02; p<0.05). Additional, MDP can synergy with LPS and PAM3 to increase the proliferation viability of VSMC and induce the production of IL-8 and TNF-α.
Conclusion: 1.In the model of vascular restenosis by balloon injury, the levels of TLR4 mRNA and protein of carotid arteries were significantly increased and companied with activation of NF-κB. It implied that TLR4/ NF-κB pathway may play an important role in the developing of restenosis. 2. NOD1 is also expressed in rat tissue and exhibits highly homology with the human NOD1. Rat NOD1 has an increased expression during intimal hyperplasia in a rat carotid artery injury model and preferentially expressed by iSMCs. Activation of NOD1 can robustly induce the expression of iNOS and TNF-α, which regulated by NF-κB signal pathway. It implied that NOD1 is an intimal specific innate immune response mediator. The functional difference of NOD1 in iSMC and mSMC maybe due to the difference of their downstream mediator. The mSMC lacks RIP2 protein, a key component of NOD1 signal pathway.
3. The activation of NOD1 mediated innate immune signaling pathway can induce the production of proinflammatory cytokines such as IL-8 and TNF-αin VSMC. It can also induce the expression of FGF-2. This may represent a novel innate immune activation pathway in vascular inflammation and intimal formation.
4. The activation of NOD2 mediated innate immune signaling pathway can increase the proliferation viability of VSMC and induce the production of proinflammatory cytokines in VSMC. It had a synergistic effect with those of TLR2 and TLR4 mediated signaling pathways in this process.
引文
1. Ross R. Atherosclerosis-An inflammatory disease. N Engl J Med.1999; 340(2):115-26.
2. Mulvihill ER, Jaeger J, Sengupta R, et al. Atherosclerotic plague smooth muscle cells have a distinct phenotype. Arterioscl Thromb Vasc Biol.2004; 24(7): 1283-9.
3. Janeway Jr, Medzhitov R. Innate Immune Recognition. Annu Rev Immunol. 2002; 20: 197–216.
4. Opal SM, Huber Ce.Bench-to-bedside review: Toll-like receptors and their role septic shock. Crit Care. 2002; 6(2): 125-136.
5. Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol.2004; 4(7):499-511.
6. Faure E,Thomas L, Xu H, et al. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial: role of NF-kappa B activation. J Immunol.2001; 166(3):2018-24.
7. Xu XH, Shah PK, Faure E, et al. Toll-like receptor 4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation. 2001; 104(25):3103-8.
8. Sen R, Baltimore D Multiple nuclear factors interact with the immunogobulin enhancer sequences .Cell.1986; 46(5):705-16.
9. Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004; 25(6): 280-8.
10. Ialenti A, Innaro A, Maffia P, et al. Role of nuclear factor kappa B in a rat model of vascular injures. Naunyn Schmicdebergs Arch Pharmacol.2001; 364(4):343-50.
11. Autieri MV.Expression of anaphase promoting complex 5 in balloon angioplasty injured rat carotid arteries and mitogen-stimulated human vascular smooth muscle cells.Biochem Biophys Res Commun.2001; 282(3):723-28.
12. Medzhitov R, Preston-Hulbert P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997; 388(6640): 394-7.
13. Sasu S, LaVerda D, Qureshi N, et al. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-likereceptor4 and p44/p42 mitogen- activated protein kinase activation. Circ Res. 2001; 89(3):244–50.
14. Hollestelle SC, De Vries MR, Van Keulen JK, et al.Toll-Like Receptor 4 is involved in outward arterial remodeling. Circulation.2004; 109(3):393-8.
15. Okamura Y, Watari M, Jerud ES, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem. 2001; 276(13):10229–33.
1. Laberge MA, Moore KJ, Freeman MW. Atherosclerosis and innate immune signaling. Ann Med. 2005; 37(2):130-40.
2. Fritz JH, Ferrero RL, Philpott DJ, et al. Nod-like proteins in immunity, inflammation and disease. Nat Immunol. 2006; 7(12):1250-7.
3. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs. 2006; 66(1):15-29.
4. de Graaf R, Kloppenburg G, Kitslaar PJ, et al. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4. Microbes Infect.2006; 8(7):1859-65.
5. Schoneveld AH, Oude Nijhuis MM, van Middelaar B, et al. Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development. Cardiovasc Res. 2005; 66(1):162-9.
6. Strober W, Murray PJ, Kitani A, et al. Signaling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol. 2006; 6(1):9-20.
7. Dziarski R, Gupta D. Peptidoglycan recognition in innate immunity. J Endotoxin Res. 2005; 11(5):304-10.
8. Girardin SE, Boneca IG, Carneiro LA, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science. 2003; 300 (5625):1584-7.
9. Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003; 278(11): 8869-72.
10. Chamaillard M, Girardin SE, Viala J, et al. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol. 2003; 5(9): 581-92.
11. Inohara N, Nunez G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol. 2003; 3(5):371-82.
12. Inohara N, Koseki T, del Peso L, et al. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem. 1999; 274(21):14560-7.
13. Park JH, Kim YG, McDonald C, et al. RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs. J Immunol. 2007; 178(4):2380-6.
14. Ogura Y, Inohara N, Benito A, et al. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J Biol Chem. 2001; 276 (7):4812-8.
15. Laman JD, Schoneveld AH, Moll FL, et al. Significance of peptidoglycan, a proinflammatory bacterial antigen in atherosclerotic arteries and its association with vulnerable plaques. Am J Cardiol. 2002; 90(2):119-23.
16. Yan ZQ, Yokota T, Zhang W, et al. Expression of inducible nitric oxide synthase inhibits platelet adhesion and restores blood flow in the injured artery. Circ Res. 1996; 79(1):38-44.
17. Yan Z, Hansson GK. Overexpression of inducible nitric oxide synthase by neointimal smooth muscle cells. Circ Res. 1998; 82(1):21-9.
18. Opitz B, Forster S, Hocke AC, et al. Nod1-mediated endothelial cell activation by Chlamydophila pneumoniae. Circ Res. 2005; 96(3):319-26.
19. Niemann-Jonsson A, Ares MP, Yan ZQ, et al. Increased rate of apoptosis in intimal arterial smooth muscle cells through endogenous activation of TNF receptors. Arterioscler Thromb Vasc Biol. 2001; 21(12):1909-14.
20. Dichtl W, Stocker EM, Mistlberger K, et al. Countervailing effects of rapamycin (sirolimus) on nuclear factor-kappa B activities in neointimal and medial smooth muscle cells. Atherosclerosis. 2006; 186(2):321-30.
21. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352(16):1685-95.
22. de Winther MP, Kanters E, Kraal G, et al. Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005; 25(5):904-14.
23. Kufer TA, Banks DJ, Philpott DJ. Innate immune sensing of microbes by Nod proteins. Ann N Y Acad Sci. 2006; 1072:19-27.
24. Lowe HC, Oesterle SN, Khachigian LM. Coronary in-stent restenosis: current status and future strategies. J Am Coll Cardiol. Jan 16 2002; 39(2):183-193.
25. Barbato JE, Tzeng E. Nitric oxide and arterial disease. J Vasc Surg. 2004; 40 (1):187-93.
26. Holifield B, Helgason T, Jemelka S, et al. Differentiated vascular myocytes: are they involved in neointimal formation? J Clin Invest. 1996; 97(3):814-25.
27. Bochaton-Piallat ML, Ropraz P, Gabbiani F, et al. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol. 1996; 16 (6): 815-20.
1. Mulvihill ER, Jaeger J, Sengupta R, et al. Atherosclerotic plague smooth muscle cells have a distinct phenotype. Arterioscl Thromb Vasc Biol. 2004; 24(7): 1283-9.
2. Hansson GK. Inflammation, atherosclerosis and coronary artery disease. N Eng J Med. 2005; 352(16):1685-95.
3. Sirard JC, Vignal C, Dessein R et al. Nod-like receptors: cytosolic watchdogs for immunity against pathogens. PloS Pathog. 2007; 3(12):1829-36.
4. Kanneganti TD, Lamkanfi M, Nunez G. Intracellular NOD-like receptors in host defense and disease. Immunity. 2007; 27(4):549-59.
5. Kufer TA, Banks DJ, Philpott DJ. Innate immune sensing of microbes by Nod proteins. Ann N Y Acad Sci. 2006; 1072: 19-27.
6. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell.2006; 124(4): 783-801.
7. Uematsu S, Akira S. Toll-like receptors and innate immunity. J Mol Med.2006; 84(9): 712-25.
8. Hornef MW, Wick MJ, Rhen M, et al. Bacterial strategies for overcoming host innate and adaptive immune responses. Nat Immunol.2002; 3(11): 1033-40.
9. Yan ZQ, Hansson GK. Innate immunity, macrophage activation, and atherosclerosis. Immunol Rev. 2007; 219(1):187-203.
10. de Graaf R, Kloppenburg G, Kitslaar PJ, et al. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4. Microbes Infect. 2006; 8(7): 1859-65.
11.马林,丁彦春,崔晓琼,等.Toll样受体4-核因子κB信号通路在血管成形术后再狭窄过程中的作用.中国动脉硬化杂志. 2006; 14(7):596-600.
12. Cao F, Castrillo A, Tontonoz P, et al. Chlamydia pneumoniae-induced macrophage foam cell formation is mediated by Toll-like receptor 2. Infect Immun. 2007; 75(2):753–9.
13. Inohara, Chamaillard, McDonald C, et al. NOD-LRR proteins: role in host- microbial interactions and inflammatory disease. Annu Rev Biochem. 2005; 74:355–83.
14. Opitz B, Forster S, Hocke AC, et al. Nod1-mediated endothelial cell activation by Chlamydophila pneumoniae. Circ Res. 2005; 96(3):319–26.
15. Fong IW, Chiu B, Jang D, et al. De Novo induction of atherosclerosis by Chlamydia pneumoniae in a rabbit model. Infect Immun. 1999; 67(11):6048-55.
16. Laman JD, Schoneveld AH, Moll FL, et al. Significance of peptidoglycan, a proinflammatory bacterial antigen in atherosclerotic arteries and its association with vulnerable plaques. Am J Cardiol. 2002; 90(2): 119-23.
17. Gupta S, Tripathi CD.Current status of TNF blocking therapy in heart failure. Indian J Med Sci. 2005; 59(8):363-6.
18. Wang Z, Rao PJ, Castresana MR, et al. TNF-αinduces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype. Am J Physiol Heart Circ Physiol. 2005; 288(1):293-301.
19. Kobayashi Y.The role of chemokines in neutrophil biology. Front Biosci. 2008; 13(1):2400-07.
20. Qi X, Li S, Li J. The prognostic value of IL-8 for cardiac events and restenosis in patients with coronary heart diseases after percutaneous coronary intervention. Jpn Heart J. 2003; 44(5):623-32.
21. Braunersreuther V, Mach F, Steffens S. The specific role of chemokines in atherosclerosis. Thromb Haemost. 2007; 97(5):714-21.
22. Millette E, Rauch BN, Kenagy RD, et al. Platelet-derived growth factor-BB transactivates the fibroblast growth factor receptor to induce proliferation in human smooth muscle cells. Trends Cardiovasc Med. 2006; 16(1): 25-8.
23. Grayston JT, Kronmal RA, Jackson LA, et al. Azithromycin for the secondary prevention of coronary events. N Engl J Med. 2005; 352(16):1637-45.
24. Cannon CP, Braunwald E, McCabe CH,et al. Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome. N Engl J Med. 2005; 352(16): 1646–54.
1. Kufer, T.A., D.J. Banks, D.J. Philpott. Innate immune sensing of microbes by Nod proteins. Ann N Y Acad Sci. 2006; 1072: 19-27.
2. Uematsu S, Akira S. Toll-like receptors and innate immunity. J Mol Med. 2006; 84(9): 712-25.
3.和亚萍,曲鹏,李桂华.Toll样受体在Goldblatt鼠左室心肌中的表达-炎症与左心室肥厚相关的一个证据.高血压杂志. 2005; 13(8):483-7.
4.王纪文,曲鹏,姜华,等.抑制NF-κB对Toll样受体4在Goldblatt鼠左室心肌中表达的影响.高血压杂志. 2005; 13(7):427-31.
5.王虹艳,曲鹏,富晶,等.TLR4激动上调内皮细胞氧化低密度脂蛋白受体LOX-1表达.高血压杂志. 2005; 13(7):422-6.
6.许柳青,叶琼,吴可贵.P38MAPK与血管重构.高血压杂志. 2005; 13 (5):262-5.
7. Inohara, Chamaillard, McDonald C, et al. NOD-LRR proteins: role in host- microbial interactions and inflammatory disease. Annu Rev Biochem. 2005; 74: 355-83.
8. Watanabe T, Kitani A, Murray PJ, et al. NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nature Immunology. 2004; 5(8): 800-8.
9. Uehara A, Yang S, Fujimoto Y, et al. Muramyldipeptide and diaminopimelic acid-containing desmuramylpeptides in combination with chemically synthesized Toll-like receptor agonists synergistically induced production of interleukin-8 in a NOD2-and NOD1- dependent manner, respectively, in human monocytic cells in culture. Cell Microbiology. 2005; 7 (1): 53-61.
10. Fritz J, Girardin SE, Fitting G, et al. Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor and NOD-1 and NOD-2 activating agonists. Eur J Immunol. 2005; 35 (8): 2459-70.
11. Morris GE, Whyte MK, Martin GF, et al. Agonists of Toll-like receptors 2 and 4 activate airway smooth muscle via mononuclear leukocytes. Am J Respir Crit Care Med.2005; 171(8): 814-22.
12. de Graaf R, Kloppenburg G, Kitslaar PJ, et al. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4. Microbes Infect. 2006; 8(7): 1859-65.
13. Millette E, Rauch BN, Kenagy RD, et al. Platelet-derived growth factor-BBtransactivates the fibroblast growth factor receptor to induce proliferation in human smooth muscle cells. Trends Cardiovasc Med. 2006; 16 (1): 25-8.
14. Wang Z-B, Rao PJ, Castresana MR, et al. TNF-αinduces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype. Am J Physiol Heart Circ Physiol. 2005; 288(1): 293-301.
15. Qi X, Li S, Li J. The prognostic value of IL-8 for cardiac events and restenosis in patients with coronary heart diseases after percutaneous coronary intervention. Jpn Heart J. 2003; 44 (5): 623-32.
1. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell.2006; 124(4):783-801.
2. Libby P, Ridker PM. Inflammation and atherothrombosis from population biology and bench research to clinical practice. J Am Coll Cardiol. 2006; 48(9 Suppl):A33-46.
3. Epstein SE, Zhou YF, Zhu J. Infection and atherosclerosis: emerging mechanistic paradigms. Circulation. 1999; 100(4):e20-28.
4. Maass M, Bartels C, Kruger S, et al. Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease. Atherosclerosis. 1998; 140( Suppl) 1:S25-30.
5. Dechend R, Maass M, Gieffers J, et al. Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappaB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circulation. 1999;100(13):1369-73.
6. Gibson FC, 3rd, Hong C, Chou HH, et al. Innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice. Circulation. 2004;109(22):2801-6.
7. Gelfand EV, Cannon CP. Antibiotics for secondary prevention of coronary artery disease: an ACES hypothesis but we need to PROVE IT. Am Heart J. 2004;147(2):202-9.
8. Harats D, George J. Beta2-glycoprotein I and atherosclerosis. Curr Opin Lipidol. 2001;12(5):543-6.
9. Wick G, Knoflach M, Xu Q. Autoimmune and inflammatory mechanisms in atherosclerosis. Annu Rev Immunol. 2004;22:361-403.
10. Binder CJ, Shaw PX, Chang MK, et al. The role of natural antibodies in atherogenesis. J Lipid Res. 2005;46(7):1353-63.
11. Binder CJ, Silverman GJ. Natural antibodies and the autoimmunity of atherosclerosis. Springer Semin Immunopathol. Mar 2005;26(4):385-404.
12. Shaw PX, Horkko S, Chang MK, et al. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest. 2000;105(12):1731-40.
13. Montero I, Orbe J, Varo N, et al. C-reactive protein induces matrixmetalloproteinase-1 and -10 in human endothelial cells: implications for clinical and subclinical atherosclerosis. J Am Coll Cardiol. 2006;47(7): 1369-78.
14. Mukhopadhyay S, Gordon S. The role of scavenger receptors in pathogen recognition and innate immunity. Immunobiology. 2004;209(1-2):39-49.
15. Miller YI, Chang MK, Binder CJ, et al. Oxidized low density lipoprotein and innate immune receptors. Curr Opin Lipidol. 2003;14(5):437-45.
16. Kunjathoor VV, Febbraio M, Podrez EA, et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem. 2002;277(51):49982-8.
17. Medeiros LA, Khan T, El Khoury JB, et al. Fibrillar amyloid protein present in atheroma activates CD36 signal transduction. J Biol Chem. 2004; 279 (11): 10643-8.
18. Hoebe K, Georgel P, Rutschmann S, et al. CD36 is a sensor of diacylglycerides. Nature. 2005;433(7025):523-7.
19. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs. 2006;66(1):15-29.
20. de Winther MP, Kanters E, Kraal G, et al. Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005;25(5):904-14.
21. Edfeldt K, Swedenborg J, Hansson GK, et al. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation. 2002;105(10):1158-61.
22. Kol A, Lichtman AH, Finberg RW, et al. Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immunol. 2000;164(1):13-7.
23. Miller YI, Viriyakosol S, Worrall DS, et al. Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages. Arterioscler Thromb Vasc Biol. Jun 2005;25(6):1213-1219.
24. Janeway CA, Jr., Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216.
25. Bjorkbacka H, Kunjathoor VV, Moore KJ, et al. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation ofinnate immunity signaling pathways. Nat Med.2004;10(4):416-21.
26. Isoda K, Sawada S, Ayaori M, et al. Deficiency of interleukin-1 receptor antagonist deteriorates fatty liver and cholesterol metabolism in hyper- cholesterolemia mice. J Biol Chem.2005;280(8):7002-9.
27. Elhage R, Jawien J, Rudling M, et al. Reduced atherosclerosis in interleukin-
18 deficient apolipoprotein E-knockout mice. Cardiovasc Res. 2003;59(1): 234-40.
28. Strober W, Murray PJ, Kitani A, et al. Signaling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol.2006;6(1):9-20.
29. Laman JD, Schoneveld AH, Moll FL, et al. Significance of peptidoglycan, a proinflammatory bacterial antigen in atherosclerotic arteries and its association with vulnerable plaques. Am J Cardiol.2002;90(2):119-23.
1. Janeway CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216.
2. Fritz JH, Girardin SE. How Toll-like receptors and Nod-like receptors contribute to innate immunity in mammals. J Endotoxin Res. 2005;11(6): 390-4.
3. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs. 2006;66(1):15-29.
4. Hornef MW, Wick MJ, Rhen M, et al. Bacterial strategies for overcoming host innate and adaptive immune responses. Nat Immunol. 2002;3(11):1033-40.
5. Inohara, Chamaillard, McDonald C, et al. NOD-LRR proteins: role in host- microbial interactions and inflammatory disease. Annu Rev Biochem. 2005; 74:355-83.
6. Ting JP, Davis BK. CATERPILLER: a novel gene family important in immunity, cell death, and diseases. Annu Rev Immunol. 2005;23:387-414.
7. Chamaillard M, Girardin SE, Viala J, et al. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol. 2003;5(9): 581- 92.
8. Boneca IG. The role of peptidoglycan in pathogenesis. Curr Opin Microbiol. 2005;8(1):46-53.
9. Girardin SE, Boneca IG, Carneiro LA, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science. 2003; 300(5625):1584-7.
10. Chamaillard M, Hashimoto M, Horie Y, et al. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol. 2003;4(7):702-7.
11. Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003;278(11): 8869-72.
12. Inohara N, Ogura Y, Fontalba A, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J Biol Chem. 2003;278(8):5509-12.
13. Strober W, Murray PJ, Kitani A, et al. Signaling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol. 2006;6(1):9-20.
14. Vavricka SR, Musch MW, Chang JE, et al. hPepT1 transports muramyl dipeptide,activating NF-kappaB and stimulating IL-8 secretion in human colonic Caco2/ bbe cells. Gastroenterology. 2004;127(5):1401-9.
15. Kufer TA, Fritz JH, Philpott DJ. NACHT-LRR proteins (NLRs) in bacterial infection and immunity. Trends Microbiol. 2005;13(8):381-8.
16. Abbott DW, Wilkins A, Asara JM, et al. The Crohn's disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO. Curr Biol. 2004;14(24):2217-27.
17. Pauleau AL, Murray PJ. Role of nod2 in the response of macrophages to toll-like receptor agonists. Mol Cell Biol. 2003;23(21):7531-9.
18. Kobayashi KS, Chamaillard M, Ogura Y, et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science. 2005;307(5710): 731-4.
19. Mariathasan S, Newton K, Monack DM, et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature. 2004;430(6996): 213-8.
20. Hysi P, Kabesch M, Moffatt MF, et al. NOD1 variation, immunoglobulin E and asthma. Hum Mol Genet. 2005;14(7):935-41.
21. Nau GJ, Richmond JF, Schlesinger A, et al. Human macrophage activation programs induced by bacterial pathogens. Proc Natl Acad Sci U S A. 2002; 99(3):1503-8.
22. McGovern DP, Hysi P, Ahmad T, et al. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease. Hum Mol Genet. 2005;14(10):1245-50.
23. Kanazawa N, Okafuji I, Kambe N, et al. Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome. Blood. 2005;105(3):1195-7.
2. Mulvihill ER, Jaeger J, Sengupta R, et al. Atherosclerotic plague smooth muscle cells have a distinct phenotype. Arterioscl Thromb Vasc Biol.2004; 24(7): 1283-9.
3. Janeway Jr, Medzhitov R. Innate Immune Recognition. Annu Rev Immunol. 2002; 20: 197–216.
4. Opal SM, Huber Ce.Bench-to-bedside review: Toll-like receptors and their role septic shock. Crit Care. 2002; 6(2): 125-136.
5. Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol.2004; 4(7):499-511.
6. Faure E,Thomas L, Xu H, et al. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial: role of NF-kappa B activation. J Immunol.2001; 166(3):2018-24.
7. Xu XH, Shah PK, Faure E, et al. Toll-like receptor 4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation. 2001; 104(25):3103-8.
8. Sen R, Baltimore D Multiple nuclear factors interact with the immunogobulin enhancer sequences .Cell.1986; 46(5):705-16.
9. Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004; 25(6): 280-8.
10. Ialenti A, Innaro A, Maffia P, et al. Role of nuclear factor kappa B in a rat model of vascular injures. Naunyn Schmicdebergs Arch Pharmacol.2001; 364(4):343-50.
11. Autieri MV.Expression of anaphase promoting complex 5 in balloon angioplasty injured rat carotid arteries and mitogen-stimulated human vascular smooth muscle cells.Biochem Biophys Res Commun.2001; 282(3):723-28.
12. Medzhitov R, Preston-Hulbert P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997; 388(6640): 394-7.
13. Sasu S, LaVerda D, Qureshi N, et al. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-likereceptor4 and p44/p42 mitogen- activated protein kinase activation. Circ Res. 2001; 89(3):244–50.
14. Hollestelle SC, De Vries MR, Van Keulen JK, et al.Toll-Like Receptor 4 is involved in outward arterial remodeling. Circulation.2004; 109(3):393-8.
15. Okamura Y, Watari M, Jerud ES, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem. 2001; 276(13):10229–33.
1. Laberge MA, Moore KJ, Freeman MW. Atherosclerosis and innate immune signaling. Ann Med. 2005; 37(2):130-40.
2. Fritz JH, Ferrero RL, Philpott DJ, et al. Nod-like proteins in immunity, inflammation and disease. Nat Immunol. 2006; 7(12):1250-7.
3. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs. 2006; 66(1):15-29.
4. de Graaf R, Kloppenburg G, Kitslaar PJ, et al. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4. Microbes Infect.2006; 8(7):1859-65.
5. Schoneveld AH, Oude Nijhuis MM, van Middelaar B, et al. Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development. Cardiovasc Res. 2005; 66(1):162-9.
6. Strober W, Murray PJ, Kitani A, et al. Signaling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol. 2006; 6(1):9-20.
7. Dziarski R, Gupta D. Peptidoglycan recognition in innate immunity. J Endotoxin Res. 2005; 11(5):304-10.
8. Girardin SE, Boneca IG, Carneiro LA, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science. 2003; 300 (5625):1584-7.
9. Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003; 278(11): 8869-72.
10. Chamaillard M, Girardin SE, Viala J, et al. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol. 2003; 5(9): 581-92.
11. Inohara N, Nunez G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol. 2003; 3(5):371-82.
12. Inohara N, Koseki T, del Peso L, et al. Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem. 1999; 274(21):14560-7.
13. Park JH, Kim YG, McDonald C, et al. RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs. J Immunol. 2007; 178(4):2380-6.
14. Ogura Y, Inohara N, Benito A, et al. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB. J Biol Chem. 2001; 276 (7):4812-8.
15. Laman JD, Schoneveld AH, Moll FL, et al. Significance of peptidoglycan, a proinflammatory bacterial antigen in atherosclerotic arteries and its association with vulnerable plaques. Am J Cardiol. 2002; 90(2):119-23.
16. Yan ZQ, Yokota T, Zhang W, et al. Expression of inducible nitric oxide synthase inhibits platelet adhesion and restores blood flow in the injured artery. Circ Res. 1996; 79(1):38-44.
17. Yan Z, Hansson GK. Overexpression of inducible nitric oxide synthase by neointimal smooth muscle cells. Circ Res. 1998; 82(1):21-9.
18. Opitz B, Forster S, Hocke AC, et al. Nod1-mediated endothelial cell activation by Chlamydophila pneumoniae. Circ Res. 2005; 96(3):319-26.
19. Niemann-Jonsson A, Ares MP, Yan ZQ, et al. Increased rate of apoptosis in intimal arterial smooth muscle cells through endogenous activation of TNF receptors. Arterioscler Thromb Vasc Biol. 2001; 21(12):1909-14.
20. Dichtl W, Stocker EM, Mistlberger K, et al. Countervailing effects of rapamycin (sirolimus) on nuclear factor-kappa B activities in neointimal and medial smooth muscle cells. Atherosclerosis. 2006; 186(2):321-30.
21. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352(16):1685-95.
22. de Winther MP, Kanters E, Kraal G, et al. Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005; 25(5):904-14.
23. Kufer TA, Banks DJ, Philpott DJ. Innate immune sensing of microbes by Nod proteins. Ann N Y Acad Sci. 2006; 1072:19-27.
24. Lowe HC, Oesterle SN, Khachigian LM. Coronary in-stent restenosis: current status and future strategies. J Am Coll Cardiol. Jan 16 2002; 39(2):183-193.
25. Barbato JE, Tzeng E. Nitric oxide and arterial disease. J Vasc Surg. 2004; 40 (1):187-93.
26. Holifield B, Helgason T, Jemelka S, et al. Differentiated vascular myocytes: are they involved in neointimal formation? J Clin Invest. 1996; 97(3):814-25.
27. Bochaton-Piallat ML, Ropraz P, Gabbiani F, et al. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol. 1996; 16 (6): 815-20.
1. Mulvihill ER, Jaeger J, Sengupta R, et al. Atherosclerotic plague smooth muscle cells have a distinct phenotype. Arterioscl Thromb Vasc Biol. 2004; 24(7): 1283-9.
2. Hansson GK. Inflammation, atherosclerosis and coronary artery disease. N Eng J Med. 2005; 352(16):1685-95.
3. Sirard JC, Vignal C, Dessein R et al. Nod-like receptors: cytosolic watchdogs for immunity against pathogens. PloS Pathog. 2007; 3(12):1829-36.
4. Kanneganti TD, Lamkanfi M, Nunez G. Intracellular NOD-like receptors in host defense and disease. Immunity. 2007; 27(4):549-59.
5. Kufer TA, Banks DJ, Philpott DJ. Innate immune sensing of microbes by Nod proteins. Ann N Y Acad Sci. 2006; 1072: 19-27.
6. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell.2006; 124(4): 783-801.
7. Uematsu S, Akira S. Toll-like receptors and innate immunity. J Mol Med.2006; 84(9): 712-25.
8. Hornef MW, Wick MJ, Rhen M, et al. Bacterial strategies for overcoming host innate and adaptive immune responses. Nat Immunol.2002; 3(11): 1033-40.
9. Yan ZQ, Hansson GK. Innate immunity, macrophage activation, and atherosclerosis. Immunol Rev. 2007; 219(1):187-203.
10. de Graaf R, Kloppenburg G, Kitslaar PJ, et al. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4. Microbes Infect. 2006; 8(7): 1859-65.
11.马林,丁彦春,崔晓琼,等.Toll样受体4-核因子κB信号通路在血管成形术后再狭窄过程中的作用.中国动脉硬化杂志. 2006; 14(7):596-600.
12. Cao F, Castrillo A, Tontonoz P, et al. Chlamydia pneumoniae-induced macrophage foam cell formation is mediated by Toll-like receptor 2. Infect Immun. 2007; 75(2):753–9.
13. Inohara, Chamaillard, McDonald C, et al. NOD-LRR proteins: role in host- microbial interactions and inflammatory disease. Annu Rev Biochem. 2005; 74:355–83.
14. Opitz B, Forster S, Hocke AC, et al. Nod1-mediated endothelial cell activation by Chlamydophila pneumoniae. Circ Res. 2005; 96(3):319–26.
15. Fong IW, Chiu B, Jang D, et al. De Novo induction of atherosclerosis by Chlamydia pneumoniae in a rabbit model. Infect Immun. 1999; 67(11):6048-55.
16. Laman JD, Schoneveld AH, Moll FL, et al. Significance of peptidoglycan, a proinflammatory bacterial antigen in atherosclerotic arteries and its association with vulnerable plaques. Am J Cardiol. 2002; 90(2): 119-23.
17. Gupta S, Tripathi CD.Current status of TNF blocking therapy in heart failure. Indian J Med Sci. 2005; 59(8):363-6.
18. Wang Z, Rao PJ, Castresana MR, et al. TNF-αinduces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype. Am J Physiol Heart Circ Physiol. 2005; 288(1):293-301.
19. Kobayashi Y.The role of chemokines in neutrophil biology. Front Biosci. 2008; 13(1):2400-07.
20. Qi X, Li S, Li J. The prognostic value of IL-8 for cardiac events and restenosis in patients with coronary heart diseases after percutaneous coronary intervention. Jpn Heart J. 2003; 44(5):623-32.
21. Braunersreuther V, Mach F, Steffens S. The specific role of chemokines in atherosclerosis. Thromb Haemost. 2007; 97(5):714-21.
22. Millette E, Rauch BN, Kenagy RD, et al. Platelet-derived growth factor-BB transactivates the fibroblast growth factor receptor to induce proliferation in human smooth muscle cells. Trends Cardiovasc Med. 2006; 16(1): 25-8.
23. Grayston JT, Kronmal RA, Jackson LA, et al. Azithromycin for the secondary prevention of coronary events. N Engl J Med. 2005; 352(16):1637-45.
24. Cannon CP, Braunwald E, McCabe CH,et al. Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome. N Engl J Med. 2005; 352(16): 1646–54.
1. Kufer, T.A., D.J. Banks, D.J. Philpott. Innate immune sensing of microbes by Nod proteins. Ann N Y Acad Sci. 2006; 1072: 19-27.
2. Uematsu S, Akira S. Toll-like receptors and innate immunity. J Mol Med. 2006; 84(9): 712-25.
3.和亚萍,曲鹏,李桂华.Toll样受体在Goldblatt鼠左室心肌中的表达-炎症与左心室肥厚相关的一个证据.高血压杂志. 2005; 13(8):483-7.
4.王纪文,曲鹏,姜华,等.抑制NF-κB对Toll样受体4在Goldblatt鼠左室心肌中表达的影响.高血压杂志. 2005; 13(7):427-31.
5.王虹艳,曲鹏,富晶,等.TLR4激动上调内皮细胞氧化低密度脂蛋白受体LOX-1表达.高血压杂志. 2005; 13(7):422-6.
6.许柳青,叶琼,吴可贵.P38MAPK与血管重构.高血压杂志. 2005; 13 (5):262-5.
7. Inohara, Chamaillard, McDonald C, et al. NOD-LRR proteins: role in host- microbial interactions and inflammatory disease. Annu Rev Biochem. 2005; 74: 355-83.
8. Watanabe T, Kitani A, Murray PJ, et al. NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nature Immunology. 2004; 5(8): 800-8.
9. Uehara A, Yang S, Fujimoto Y, et al. Muramyldipeptide and diaminopimelic acid-containing desmuramylpeptides in combination with chemically synthesized Toll-like receptor agonists synergistically induced production of interleukin-8 in a NOD2-and NOD1- dependent manner, respectively, in human monocytic cells in culture. Cell Microbiology. 2005; 7 (1): 53-61.
10. Fritz J, Girardin SE, Fitting G, et al. Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor and NOD-1 and NOD-2 activating agonists. Eur J Immunol. 2005; 35 (8): 2459-70.
11. Morris GE, Whyte MK, Martin GF, et al. Agonists of Toll-like receptors 2 and 4 activate airway smooth muscle via mononuclear leukocytes. Am J Respir Crit Care Med.2005; 171(8): 814-22.
12. de Graaf R, Kloppenburg G, Kitslaar PJ, et al. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4. Microbes Infect. 2006; 8(7): 1859-65.
13. Millette E, Rauch BN, Kenagy RD, et al. Platelet-derived growth factor-BBtransactivates the fibroblast growth factor receptor to induce proliferation in human smooth muscle cells. Trends Cardiovasc Med. 2006; 16 (1): 25-8.
14. Wang Z-B, Rao PJ, Castresana MR, et al. TNF-αinduces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype. Am J Physiol Heart Circ Physiol. 2005; 288(1): 293-301.
15. Qi X, Li S, Li J. The prognostic value of IL-8 for cardiac events and restenosis in patients with coronary heart diseases after percutaneous coronary intervention. Jpn Heart J. 2003; 44 (5): 623-32.
1. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell.2006; 124(4):783-801.
2. Libby P, Ridker PM. Inflammation and atherothrombosis from population biology and bench research to clinical practice. J Am Coll Cardiol. 2006; 48(9 Suppl):A33-46.
3. Epstein SE, Zhou YF, Zhu J. Infection and atherosclerosis: emerging mechanistic paradigms. Circulation. 1999; 100(4):e20-28.
4. Maass M, Bartels C, Kruger S, et al. Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease. Atherosclerosis. 1998; 140( Suppl) 1:S25-30.
5. Dechend R, Maass M, Gieffers J, et al. Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappaB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circulation. 1999;100(13):1369-73.
6. Gibson FC, 3rd, Hong C, Chou HH, et al. Innate immune recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice. Circulation. 2004;109(22):2801-6.
7. Gelfand EV, Cannon CP. Antibiotics for secondary prevention of coronary artery disease: an ACES hypothesis but we need to PROVE IT. Am Heart J. 2004;147(2):202-9.
8. Harats D, George J. Beta2-glycoprotein I and atherosclerosis. Curr Opin Lipidol. 2001;12(5):543-6.
9. Wick G, Knoflach M, Xu Q. Autoimmune and inflammatory mechanisms in atherosclerosis. Annu Rev Immunol. 2004;22:361-403.
10. Binder CJ, Shaw PX, Chang MK, et al. The role of natural antibodies in atherogenesis. J Lipid Res. 2005;46(7):1353-63.
11. Binder CJ, Silverman GJ. Natural antibodies and the autoimmunity of atherosclerosis. Springer Semin Immunopathol. Mar 2005;26(4):385-404.
12. Shaw PX, Horkko S, Chang MK, et al. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest. 2000;105(12):1731-40.
13. Montero I, Orbe J, Varo N, et al. C-reactive protein induces matrixmetalloproteinase-1 and -10 in human endothelial cells: implications for clinical and subclinical atherosclerosis. J Am Coll Cardiol. 2006;47(7): 1369-78.
14. Mukhopadhyay S, Gordon S. The role of scavenger receptors in pathogen recognition and innate immunity. Immunobiology. 2004;209(1-2):39-49.
15. Miller YI, Chang MK, Binder CJ, et al. Oxidized low density lipoprotein and innate immune receptors. Curr Opin Lipidol. 2003;14(5):437-45.
16. Kunjathoor VV, Febbraio M, Podrez EA, et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem. 2002;277(51):49982-8.
17. Medeiros LA, Khan T, El Khoury JB, et al. Fibrillar amyloid protein present in atheroma activates CD36 signal transduction. J Biol Chem. 2004; 279 (11): 10643-8.
18. Hoebe K, Georgel P, Rutschmann S, et al. CD36 is a sensor of diacylglycerides. Nature. 2005;433(7025):523-7.
19. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs. 2006;66(1):15-29.
20. de Winther MP, Kanters E, Kraal G, et al. Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005;25(5):904-14.
21. Edfeldt K, Swedenborg J, Hansson GK, et al. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation. 2002;105(10):1158-61.
22. Kol A, Lichtman AH, Finberg RW, et al. Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immunol. 2000;164(1):13-7.
23. Miller YI, Viriyakosol S, Worrall DS, et al. Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages. Arterioscler Thromb Vasc Biol. Jun 2005;25(6):1213-1219.
24. Janeway CA, Jr., Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216.
25. Bjorkbacka H, Kunjathoor VV, Moore KJ, et al. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation ofinnate immunity signaling pathways. Nat Med.2004;10(4):416-21.
26. Isoda K, Sawada S, Ayaori M, et al. Deficiency of interleukin-1 receptor antagonist deteriorates fatty liver and cholesterol metabolism in hyper- cholesterolemia mice. J Biol Chem.2005;280(8):7002-9.
27. Elhage R, Jawien J, Rudling M, et al. Reduced atherosclerosis in interleukin-
18 deficient apolipoprotein E-knockout mice. Cardiovasc Res. 2003;59(1): 234-40.
28. Strober W, Murray PJ, Kitani A, et al. Signaling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol.2006;6(1):9-20.
29. Laman JD, Schoneveld AH, Moll FL, et al. Significance of peptidoglycan, a proinflammatory bacterial antigen in atherosclerotic arteries and its association with vulnerable plaques. Am J Cardiol.2002;90(2):119-23.
1. Janeway CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216.
2. Fritz JH, Girardin SE. How Toll-like receptors and Nod-like receptors contribute to innate immunity in mammals. J Endotoxin Res. 2005;11(6): 390-4.
3. Cristofaro P, Opal SM. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs. 2006;66(1):15-29.
4. Hornef MW, Wick MJ, Rhen M, et al. Bacterial strategies for overcoming host innate and adaptive immune responses. Nat Immunol. 2002;3(11):1033-40.
5. Inohara, Chamaillard, McDonald C, et al. NOD-LRR proteins: role in host- microbial interactions and inflammatory disease. Annu Rev Biochem. 2005; 74:355-83.
6. Ting JP, Davis BK. CATERPILLER: a novel gene family important in immunity, cell death, and diseases. Annu Rev Immunol. 2005;23:387-414.
7. Chamaillard M, Girardin SE, Viala J, et al. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol. 2003;5(9): 581- 92.
8. Boneca IG. The role of peptidoglycan in pathogenesis. Curr Opin Microbiol. 2005;8(1):46-53.
9. Girardin SE, Boneca IG, Carneiro LA, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science. 2003; 300(5625):1584-7.
10. Chamaillard M, Hashimoto M, Horie Y, et al. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol. 2003;4(7):702-7.
11. Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003;278(11): 8869-72.
12. Inohara N, Ogura Y, Fontalba A, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J Biol Chem. 2003;278(8):5509-12.
13. Strober W, Murray PJ, Kitani A, et al. Signaling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol. 2006;6(1):9-20.
14. Vavricka SR, Musch MW, Chang JE, et al. hPepT1 transports muramyl dipeptide,activating NF-kappaB and stimulating IL-8 secretion in human colonic Caco2/ bbe cells. Gastroenterology. 2004;127(5):1401-9.
15. Kufer TA, Fritz JH, Philpott DJ. NACHT-LRR proteins (NLRs) in bacterial infection and immunity. Trends Microbiol. 2005;13(8):381-8.
16. Abbott DW, Wilkins A, Asara JM, et al. The Crohn's disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO. Curr Biol. 2004;14(24):2217-27.
17. Pauleau AL, Murray PJ. Role of nod2 in the response of macrophages to toll-like receptor agonists. Mol Cell Biol. 2003;23(21):7531-9.
18. Kobayashi KS, Chamaillard M, Ogura Y, et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science. 2005;307(5710): 731-4.
19. Mariathasan S, Newton K, Monack DM, et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature. 2004;430(6996): 213-8.
20. Hysi P, Kabesch M, Moffatt MF, et al. NOD1 variation, immunoglobulin E and asthma. Hum Mol Genet. 2005;14(7):935-41.
21. Nau GJ, Richmond JF, Schlesinger A, et al. Human macrophage activation programs induced by bacterial pathogens. Proc Natl Acad Sci U S A. 2002; 99(3):1503-8.
22. McGovern DP, Hysi P, Ahmad T, et al. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease. Hum Mol Genet. 2005;14(10):1245-50.
23. Kanazawa N, Okafuji I, Kambe N, et al. Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome. Blood. 2005;105(3):1195-7.