Notch1通路在人主动脉瓣间质细胞中对Toll样受体4介导的炎症反应的调节作用
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摘要
研究背景
钙化性主动脉瓣疾病是老年人群中一种常见的疾病。随着社会人口老龄化的进展,钙化性主动脉瓣疾病发病率正逐年上升,目前已经发展成为仅次于冠心病和高血压病的第三大心血管疾病。传统观念认为钙化性主动脉瓣疾病是“退行性”改变,是机体老化的一种体现,是退行性钙磷沉积不可修复的过程。最近的研究发现钙化性主动脉瓣疾病的发展可能是高度调节的自主过程,可能存在着一个复杂的多步骤的内在机制在发挥作用,而并非年龄导致的不可避免的结果,病变有可能通过药物逆转。由于发病机制尚未完全明确,目前临床上还没有针对该病的治疗性药物,因此对该病发病机制的研究显得尤其重要。越来越多研究证明了炎症在钙化性主动脉瓣疾病发生发展过程中起重要作用。首先,通过对瓣膜置换手术中被置换出来的主动脉瓣膜进行组织病理分析发现有炎症的证据。其次,由于在发生主动脉瓣钙化并狭窄的瓣膜里发现有与口腔感染一致的细菌证据,并且在实验兔子接种口腔细菌可以诱导出主动脉瓣病变,推测慢性口腔感染可能是主动脉瓣钙化和狭窄发病的重要机制之一。
人主动脉瓣间质细胞是主动脉瓣膜最主要的细胞成分,而主动脉瓣钙化往往发生在瓣膜的间质部分,因此人主动脉瓣间质细胞在钙化性主动脉瓣膜疾病的发生发展过程中起关键作用。Toll样受体(Toll-like receptors,TLRs)是识别病原微生物的跨膜受体家族,主要通过识别保守的病原相关分子模式(PAMPs)感知病原微生物的存在,对之产生免疫炎症反应,在人类天然免疫系统中起到重要作用。目前已经发现哺乳动物中存在有13种TLRs,而TLR4是最早被发现且被研究最多的TLR,广泛存在于内皮细胞、巨噬细胞、中性粒细胞和树突状细胞等多种细胞中。TLR4被生物配体识别及结合后,可以活化细胞内的信号传导通路,促进炎症介质的产生和释放。因此,TLR4在细胞的免疫与炎症反应中起着关键作用。近来研究已经发现人主动脉瓣间质细胞能够表达有功能的TLR4,刺激这种受体后能够引起心脏瓣膜间质细胞的炎症和成骨反应,这在钙化性主动脉瓣疾病发生发展的病理过程中起重要作用。间质细胞的炎症激活可以诱导单核细胞和巨噬细胞聚集和浸润,而炎症介质促进主动脉瓣膜钙化的作用已被多个研究证实,因此我们推测炎症是主动脉瓣疾病初始阶段的促发因素,继而促进主动脉瓣膜成骨反应和钙沉积,从而导致钙化性主动脉瓣疾病的发生和发展。
核转录因子κB (NF-κB)广泛存在于真核生物中,是一个由复杂的多肽亚单位组成的蛋白家族。它作为信号传导途径中的枢纽,与免疫,肿瘤的发生、发展,细胞凋亡的调节以及胚胎发育等有着密切联系,是细胞内最重要的核转录因子。NF-κB在许多细胞刺激后介导的细胞内信息的转录调控中起核心作用,参与多种基因的表达和调控,它调控的基因编码急性期反应蛋白、细胞因子、细胞粘附分子、免疫调节分子、病毒瘤基因、生长因子、转录和生长调控因子等。在静息细胞中,NF-κB二聚体通过非共价键的形式与其抑制蛋白IKB结合而分散在细胞质内,当细胞受到肿瘤坏死因子(TNF)、脂多糖(LPS)等NF-κB激活剂刺激时,其抑制蛋白IKB解离和降解,NF-κB发生磷酸化,激活后的NF-κB进入细胞核,与DNA模块上的特异蛋白结合,诱导目标mRNA的产生,最后转录、生成和释放各种目标产物。
Notch蛋白是一组高度保守的细胞表面跨膜受体,在哺乳动物细胞中含有四种亚型,Notchl,Notch2, Notch3以及Notch4,Notch受体的配体有Jagged1, Jagged2,Delta like ligand (D11)l、D113和D114。Notch被γ分泌酶裂解,释放出其胞内域NICD,而NICD能够调控细胞命运及调节细胞功能。近来有研究表明细菌产物和脂多糖能够激活单核巨噬细胞的Notchl受体,从而调节细胞功能。抑制细胞γ分泌酶,就可以抑制NICD1的产生,同时也减少了巨噬细胞中脂多糖诱导的炎症因子的产生。然而Notchl信号通路在主动脉瓣间质细胞中对TLR4介导的炎症反应所起的作用目前尚不清楚。
白介素37(Interleukine37,IL-37)属于白介素1家族,而白介素1家族目前拥有11个成员。至今已有五种亚型的人类IL-37被报道,分别是IL-37、. IL-37b、IL-37c、IL-37d和IL-37e。目前尚未发现小鼠表达IL-37,但人IL-37能在小鼠体内发挥作用。IL-37普遍存在于扁桃体、皮肤、食道、胎盘、黑色素瘤、乳腺癌、前列腺癌、结肠癌和肺癌等组织及THP-1巨噬细胞,A549表皮细胞和外周血单核细胞等细胞中。IL-37能以低亲和力的方式与白介素18受体(IL-18R)结合但却不发挥激动或拮抗作用。除了胞外作用,IL-37也能在细胞内起作用,在细胞受到刺激后IL-37能转入核内并可能充当转录调节因子调节细胞功能。体内及体外研究均证明了人IL-37具有抗炎作用。体内实验,人IL-37转基因小鼠对结肠炎模型有保护作用。体外实验,巨噬细胞及表皮细胞转染IL-37后能显著降低LPS及IL-1诱导的炎症介质的表达。
本研究中,我们假想TLR4激活能诱导人主动脉瓣间质细胞的炎症反应,而这种炎症反应在来自主动脉瓣狭窄病人瓣膜组织的主动脉瓣间质细胞比来自正常瓣膜组织的细胞更明显,TLR4信号通路与Notch1信号通路发生交互作用,从而调节TLR4诱导的炎症反应水平,而人IL-37能通过Notch1/NF-KB轴抑制TLR4诱导的炎症反应,因此本研究可能对寻找钙化性主动脉瓣疾病的潜在防治靶点提供理论依据。本研究中我们利用TLR4受体激活剂LPS刺激人主动脉瓣间质细胞的TLR4受体,探讨TLR4介导的炎症反应,以及Notchl信号通路和人IL-37对其的调节作用及可能机制,本研究将分三部分进行。
第一部分TLR4与Notch1通路间的交互作用对人主动脉瓣间质细胞炎症反应的调节作用
目的
研究TLR4与Notch1信号通路间的交互作用对人主动脉瓣间质细胞炎症反应的调节作用。
方法
分别从正常主动脉瓣膜组织和主动脉瓣狭窄病人主动脉瓣膜组织分离出人主动脉瓣间质细胞,用TLR4激活物脂多糖(LPS)进行干预1到24小时。用免疫印迹方法分析细胞间粘附分子(ICAM-1)蛋白表达。用免疫印迹和免疫荧光染色方法分析Notch1信号通路的活化水平。用酶联免疫吸附(ELISA)方法分析单核细胞趋化蛋白(MCP-1),白介素8(IL-8)及Notch1特异性配体Jagged1的释放。应用γ分泌酶抑制剂DAPT抑制Notch1的活化,应用基因沉默的方法减少Notch1蛋白表达量,减少Notch1活化分子NICD1的产生,用Notch1特异性配体Jagged1激活Notch1,然后分析TLR4介导的ICAM-1表达量及MCP-1和IL-8的分泌水平。
结果
LPS诱导人主动脉瓣间质细胞ICAM-1蛋白的表达,MCP-1和IL-8的释放,以及Notch1信号通路的活化。来自主动脉瓣狭窄病人主动脉瓣膜组织的主动脉瓣间质细胞比来自正常瓣膜组织的主动脉瓣间质细胞显示出更强的炎症反应和Notch1信号通路的活化以及更多的Notch1特异性配体Jagged1释放。γ分泌酶抑制剂DAPT能抑制Notch1信号通路的活化,同时抑制TLR4介导的ICAM-1蛋白表达,IL-8和MCP-1释放。用基因沉默的方法降低Notch1表达水平,能减少NICD1的生成量,同时也减少TLR4介导的ICAM-1蛋白表达量。Notch1受体的特异性配体Jagged1能增强TLR4介导的ICAM-1蛋白表达,IL-8和MCP-1的释放。
结论
LPS能诱导人主动脉瓣间质细胞的炎症反应及Notch1信号通路的活化,而且这些反应在来自主动脉瓣狭窄病人瓣膜组织的主动脉瓣间质细胞中更明显。Notch1通路的活化能调控TLR4介导的炎症反应,Notch1过度活化可能是主动脉瓣狭窄病人瓣膜组织来源的主动脉瓣间质细胞炎症反应增强的重要机制。
第二部分Notch1通路在人主动脉瓣间质细胞中调节TLR4介导的NF-κB活化的机制研究
目的
探讨Notch1信号通路在人主动脉瓣间质细胞调节TLR4介导的炎症反应的机制。
方法
分别从正常瓣膜组织和主动脉瓣狭窄病人瓣膜组织分离出人主动脉瓣间质细胞,用TLR4激活物LPS处理细胞1到24小时。用免疫印迹和免疫组化方法分析Notch1蛋白基础表达水平,用免疫印迹和免疫荧光染色方法分析核因子κB(NF-κB)的活化水平,用免疫共沉淀方法分析NF-κB上游IKB激酶(IKK)和Notch1信号通路活化分子NICD1间的直接相互作用。用γ分泌酶抑制齐(?)DAPT抑制Notch1的活化,用基因沉默的方法减少Notch1蛋白表达量,用Notch1特异性配体Jagged1激活Notch1,然后分析TLR4介导的NF-κB的活化水平。
结果
LPS诱导入主动脉瓣间质细胞NF-κB的活化,其活化水平均在来自主动脉瓣狭窄病人瓣膜组织的主动脉瓣间质细胞中增高。来自主动脉瓣狭窄病人的主动脉瓣膜组织和问质细胞的Notch1蛋白基础表达水平比来自正常瓣膜的组织和细胞高。γ分泌酶抑制剂(DAPT)能抑制TLR4介导的NF-κB的磷酸化。用基因沉默的方法降低Notch1表达水平,可减少TLR4介导的NF-κB的磷酸化。Notch1受体的特异性配体Jagged1能增强TLR4介导的NF-κB的活化。免疫共沉淀结果显示NICD1与NF-κB上游的IκB激酶IKK有直接相互作用。
结论
LPS诱导人主动脉瓣膜间质细胞NF-κB信号通路的活化,其活化水平在来自主动脉瓣狭窄病人的细胞中增高。Notch1的活化分子NICD1能通过与IKK直接相互作用调节TLR4介导的NF-κB的活化水平,从而调控TLR4介导的炎症反应。
第三部分IL-37对人主动脉瓣间质细胞中对TLR4诱导的炎症反应的调节作用及其机制探讨
目的
探讨IL-37在人主动脉瓣间质细胞中对TLR4诱导的炎症反应的调节作用及其相关机制。
方法
分别从正常瓣膜和主动脉瓣狭窄病人瓣膜分离出人主动脉瓣间质细胞,用IL-37蛋白多肽与TLR4激活物LPS进行干预。用免疫印迹方法分析IL-37、ICAM-1蛋白表达水平,NF-κB和Notchl信号通路的活化水平,用RT-PCR方法分析IL-37的mRNA表达水平,用免疫荧光方法分析NF-κB核内转位。用基因沉默的方法减少IL-37的表达,然后分析TLR4介导的ICAM-1蛋白表达量。
结果
来自主动脉瓣狭窄病人瓣膜组织和来自正常瓣膜组织的主动脉瓣膜间质细胞均有IL-37mRNA和蛋白的表达,其水平在来自病变瓣膜组织的细胞较低。IL-37能抑制TLR4介导的ICAM-1表达水平,此作用在来自病变瓣膜组织的细胞中更明显。用基因沉默的方法减少IL-37的表达水平,可以增加TLR4介导的ICAM-1表达量。IL-37干预能减少Notchl的活化水平,并降低TLR4介导的NF-κB的活化水平。
结论
IL-37在人主动脉瓣膜间质细胞中表达,相对于来自正常瓣膜组织的主动脉瓣间质细胞,其表达水平在来自病变瓣膜组织的细胞较低。IL-37能通过抑制Notch1/NF-κB信号转导通路从而调节TLR4介导的炎症反应。
全文总结
1.Toll样受体4(TLR4)与Notch1通路的交互作用在人主动脉瓣间质细胞中能调节TLR4介导的炎症反应。
2.Notch1通路通过其活化分子NICD1与NF-κB上游Iκ3激酶IKK直接相互作用调节NF-κB通路的活性。
3.TLR4与Notch1通路间过多的交互作用可能是钙化性主动脉瓣疾病发生发展的重要机制。
4.白介素37(IL-37)可通过抑(?)(?)Notch1/NF-κB信号通路轴抑制TLR4介导的炎症反应,具有防治钙化性主动脉瓣疾病进展的潜力。
Background
Calcific aortic valve disease is a common disease in old people. Given the progressive aging of the general population, the incidence of calcific aortic valve disease is increasing every year. Currently, calcific aortic valve disease has become the third leading cardiovascular disease. Calcific aortic valve disease was traditionly regarded as a degenerative change which was irreversible process of calcium deposit in the aging people. Recent studies have found that calcific aortic valve disease is an active process, and multi-step complecated mechanisms are involved in the progression of this disease. Calcific aortic valve disease might be treated by drug. However, pharmacological intervention for the suppression of progression of calcific aortic valve disease is unavailable due to the limited knowledge of the underlying mechanism. Therefore, it is particularly important to study the pathogenesis of the disease. Increasing evidence suggest that inflammation plays an important role in the mechanism of calcific aortic valve disease. Firstly, evidence of inflammation had been found in the pathological analysis of replaced stenotic aortic valves. Secondly, the bacterial products found in the replaced stenotic aortic valve were consistent with the oral bacteria. Inoculation of experimental rabbits with oral bacteria induced aortic valve disease. We speculate that chronic oral infection plays an important role in the pathogenesis and progression of aortic valve calcification and stenosis.
Human aortic valve interstitial cell is the dominent cell component of human aortic valve. Aortic valve calcification nodules are usualy located in the interstitial portion of the valve. Therefore, human aortic valve interstitial cells play a critical role in the progression of calcific aortic valve disease. Toll-like receptors (TLRs) are the transmembrane receptors which recognize pathogens from bacterials, virus and other sources. TLRs play a pivotal role in the innate immune system. Currently,13TLRs have been reported and presented in endothelial cells, macrophages, neutrophils, dendritic cells and other cells. TLR4was the first TLR being discoveried and was one of the well-studied TLRs. TLR4activates intracellular signaling transduction pathways and induces the production and release of inflammatory mediators after stimulation by its biological ligands. Thus TLR4plays a key role in the immune and inflammatory responses. Previous studies have found that human aortic valve interstitial cells express the functional TLR4. Stimulation of TLR4induces inflammatory and osteogenic responses that play an important role in the pathologenesis and progression of calcific aortic valve disease. Inflammation of interstitial cells induces monocyte and macrophage accumulation and infiltration in aortic valve tissue. Studies have found that inflammatory mediators promote calcific aortic valve disease. Therefore we hypothesize that inflammation is an initial factor in the early stage of aortic valve diseases which promotes aortic valve osteogenic response and calcium deposition, thereby causes calcific aortic valve diseases.
Nuclear factor κB (NF-κB) is a protein family which composed of polypeptide subunits and prerented in eukaryotic organisms. NF-κB is the most important transcription factor in cells and plays an important role in the signal transduction pathways. It is closely related to important events, such as immunization, cancer development, cell apoptosis and the embryonic development. In addition, NF-κB plays a central role in the transcriptional regulation in cells after stimulation, and is involved in various expression and regulation of genes which encode acute phase response proteins, cytokines, cell adhesion molecules, immune regulation molecules, virus cancer genes, growth factors, transcription and growth regulators and so on. NF-κB is involved in the immune response, inflammatory response, apoptosis, tumorigenesis and other biological processes by regulating the expression of multiple genes. In the resting cells, a noncovalent bond links NF-κB dimer and its inhibitor IκB in the cytoplasm. When the cells are stimulated by NF-κB activators such as TNF and LPS, its inhibitor IκB is separated and degraded, and NF-κB is phosphorylated. After activated NF-κB entered the cell nucleus, it binds to the specific protein in the DNA module and induced the production of specific mRNA, then transcribed, produced and released various final targeted products.
Notch proteins are the highly conserved transmembrane receptors expressed on cell surface. Notch1, Notch2, Notch3and Notch4are the isoforms of Notch receptors in mammalian cells. The ligands that bind to Notch receptors are composed of Delta like group and Jagged group. The former includes Delta like ligand (D11)1, D113and D114. The latter includes Jaggedl and Jagged2. Upon ligand binding, Notch receptors undergo proteolytic cleavage, leading to the release of their intracellular domains (NICDs) that control cell fate and modulate cell functions. Bacterial lipopeptide and lipopolysaccharide (LPS) have been found to induce Notchl activation in macrophages. Inhibition of y-secretase, which processes Notchl to release NICD1, reduces LPS-induced expression of pro-inflammatory cytokines in macrophages. Currently, the role of Notchl in TLR4-mediated inflammatory response in human aortic valve interstitial cells has not been determined, and it is unknown whether human aortic valve interstitial cells of stenotic valves have exaggerated Notch1activation in response to TLR4stimulation.
Interleukine-37(IL-37) belongs to the IL-1family, which currently has11members. Five alternatively spliced transcript variants encoding distinct isoforms of human IL-37have been reported (IL-37a, IL-37b, IL-37c, IL-37d and IL-37e), whereas mouse IL-37has not been identified currently. However, human IL-37is active in mouse cells. Human IL-37has been identified presented in tonsils, skin, esophagus, placenta, melanoma, carcinomas of the breast, prostate, colon, lung and albeit, as well as human THP-1macrophages, A549epithelial cells and peripheral blood mononuclear cells. IL-37binds to IL-18Receptor a chain (IL-18Rα) with low affinity but does not exert any IL-18agonistic or antagonistic effect. Besides an extracellular role for IL-37, endogenous IL-37had been reported to translocate to the nucleus after cell stimulation and might also act as transcriptional modulators. Human IL-37has been demonstrated anti-inflammatory properties in vitro and in vivo. In vivo, expression of IL-37in mice protects mice from colitis,. In vitro, transfected with IL-37markedly reduced levels of cytokines induced by LPS and IL-1stimulation. However, it is unknown currently whether human IL-37presents in human aortic valve interstitial cells, what role it might play, and how IL-37exerts an effect in human aortic valve interstitial cells.
In the present study, we hypothesized that TLR4stimulation induces the expression of inflammatory mediators in human aortic valve interstitial cells. Human aortic valve interstitial cells from stenotic valves exhibit exaggerated inflammatory response in comparison to the cells of normal aortic valves. Crosstalk between the TLR4and Notchl pathways augments the inflammatory response in the interstitial cells of stenotic human aortic valves. Human IL-37can suppress TLR4-induced inflammatory response in human aortic valve interstitial cells and might be a therapeutic potential for prevention of progression of calcified aortic valve disease. We applied the TLR4agonist LPS to stimulate TLR4in human aortic valve interstitial cell, and evaluated the levels of TLR4-mediated inflammatory responses,. We also investigated the effects of Notchl pathway and human IL-37on TLR4-mediated inflammatory responses human aortic valve interstitial cells and possible underlying mechanisms. This study will be divided into3parts.
Part1Crosstalk between the TLR4and Notchl pathways modulates the inflammatory response in the interstitial cells of stenotic human aortic valves. Objective
To investigate the effect of crosstalk between the TLR4and Notchl pathways on the inflammatory response in the interstitial cells of stenotic human aortic valves.
Method
Human aortic valve interstitial cells were isolated from the normal aortic valves and stenotic aortic valves and treated with TLR4agonist lipopolysaccharide (LPS) for1to24hours. The levels of intercellular adhesion molecule1(ICAM-1) expression were analyzed with immunoblotting. The levels of Notchl signaling pathway activation were analyzed with immunoblotting and immunofluoresence staining. The release of monocyte chemotactic protein (MCP-1), interleukin8(IL-8) and Notchl specific ligand Jaggedl were examined with Enzyme-linked immuno sorbent assay (ELISA).γ-secretase inhibitor DAPT was applied to inhibit activation of Notchl. Gene silencing was applied to the knockdown the Notchl and reduce the production of Notchl activation molecular NICD1. Notch1specific ligand Jagged1was applied to activate Notchl. The levels of ICAM-1,MCP-1and IL-8were examined after treatements.
Results
LPS induced the protein expression of ICAM-1, the releases of MCP-1and IL-8, as well as the activation of Notchl signaling pathway in human aortic valve interstitial cells. Aortic valve interstitial cells of stenotic valves exhibited greater inflammatory response and activation of the Notchl signaling pathway than normal cells, γ-secretase inhibitor (DAPT) inhibited TLR4-mediated expression of ICAM-1and release of IL-8and MCP-1through inhibition of Notchl activation. Knockdown of Notchl inhibited the production of NICD1, and reduced TLR4-mediated expression of ICAM-1protein. Activation of Notchl with specific ligand Jagged1enhanced the TLR4-mediated protein expression of ICAM-1and the release of IL-8and MCP-1.
Conclusion
LPS induces inflammatory response and activation of Notchl signaling pathway in human aortic valve interstitial cells. The inflammatory response and Notchl activation in cells of stenotic aortic valves are greater than that in cells of normal aortic valves. Notchl activation regulates TLR4-mediated inflammatory response in human aortic valve interstitial cells, and the excessive cross-talk between the TLR4and Notchl pathways might be one of the mechanisms underlying exaggareted inflammatory response in aortic valve interstitial cells of stenotic arotic valves.
Part2The mechanistic study of modulaiotn of TLR4-induced NF-κB activation by Notchl pathway in human aortic valve interstitial cells Objective
To investigate the mechanism by which Notchl signaling pathway modulates TLR4-mediated inflammatory response in human aortic valve interstitial cells.
Methods
Human aortic valve interstitial cells were isolated from the normal aortic valves and stenotic aortic valves and treated with TLR4agonist lipopolysaccharide (LPS) for1to24hours. The levels of Notch1expression were analyzed with immunohistochemistry and immunoblotting. NF-κB signaling pathways were analyzed with immunoblotting and immunofluorescence. Physical interaction between Notch1and NF-κB signaling pathways were examined with co-immunoprecipitation. γ-secretase inhibitor DAPT was applied to inhibit activation of Notch1. Knockdown of Notchl was applied to reduce the protein expression of Notchl and production of NICD1. Notchl specific ligand Jagged1was applied to activate Notchl. The levels of NF-κB phosphorylation were analyzed after treatments.
Results
LPS induced the activation of NF-κB signaling pathways in human aortic valve interstitial cell. Activation of NF-κB signaling pathways were greater in human aortic valve interstitial cells of stenotic aortic valve than cells of normal aortic valves. The levels of Notchl protein expression in stenotic aortic valve tissues and interstitial cells were higher than that in normal aortic valve, y-secretase inhibitor (DAPT) inhibited TLR4-mediated NF-κB phosphorylation. Knockdown of Notchl inhibited TLR4-mediated NF-κB phosphorylation and reduced the production of NICD1. Notchl receptor specific ligand Jagged1enhanced TLR4-mediated NF-κB phosphorylation. Co-immunoprecipitation results showed that NICD1interacted with NF-κB upstream IκB kinase IKK physically.
Conclusion
LPS induces the activation of NF-κB signaling pathway in human aortic heart valve interstitial cell and the activations of these two signaling pathways are greater in human aortic valve interstitial cells from stenotic valves that those in normal cells. Activation of Notch1modulates TLR4-mediated NF-κB phosphorylation through interaction of NICD1with IKK physically, and subsequently regulates TLR4-mediated inflammatory responses in human aortic valve interstitial cells.
Part3The study of effect and mechanism of IL-37on TLR4-mediated inflammatory response in human aortic valve interstitial cells
Objective
To investigate the effect of IL-37on the modulation of TLR4-mediated inflammatory response in human aortic valve interstitial cells and the underlying mechanisms.
Methods
Human aortic valve interstitial cells were isolated from the normal aortic valves and stenotic aortic valves and treated with TLR4agonist lipopolysaccharide (LPS). The mRNA levels of human IL-37were examined with RT-PCR. The levels of protein expression of IL-37, ICAM-1, activation of NF-κB and Notchl signaling pathways were analyzed with immunoblotting. Nuclear localization of NF-κB was examined with immunofluorescence. Knockdown was applied to reduce the expression of IL-37. The levels of ICAM-1protein expression were analyzed with immunoblotting after treatments.
Results
Human aortic valve interstitial cells expressed IL-37and the levels of IL-37expression were lower in the interstitial cells of human stenotic aortic valves in comparison to that in normal cells. IL-37inhibited TLR4-mediated ICAM-1expression in human aortic valve interstitial cells.knockdown of IL-37markly decreased IL-37expression, and increase the TLR4-mediated ICAM-1expression. IL-37treatment reduces the levels of TLR4-mediated Notchl and NF-κB activation.
Conclusions
Human aortic valve interstitial cells expressed IL-37and the levels of IL-37expression were lower in the interstitial cells of human stenotic aortic valves in comparison to that in normal cells. IL-37regulates TLR4-mediated inflammatory response through inhibition of Notch1/NF-κB axis. Thus, IL-37might have therapeutic potential for the progression of calcific aortic valve disease.
Summary
1. Cross-talk between the TLR4and Notchl pathways modulates TLR4-mediated inflammatory response in human aortic valve interstitial cells.
2. Notchl pathway modulates TLR4-mediated NF-kB activation in human aortic valve interstitial cells through interaction of NICD1with IKK directly.
3. Excessive cross-talk between TLR4and Notch1pathways in human aortic valve interstitial cells may contribute to the mechanism underlying pathogenesis and progression of calcific aortic valve disease.
4. IL-37suppressed TLR4-mediated inflammatory response through inhibition of Notch1/NF-κB axis in human aortic valve interstitial cells. Thus, IL-37may have therapeutic potential for prevention of progression of calcific aortic valve disease.
钙化性主动脉瓣疾病是老年人群中一种常见的疾病。随着社会人口老龄化的进展,钙化性主动脉瓣疾病发病率正逐年上升,目前已经发展成为仅次于冠心病和高血压病的第三大心血管疾病。传统观念认为钙化性主动脉瓣疾病是“退行性”改变,是机体老化的一种体现,是退行性钙磷沉积不可修复的过程。最近的研究发现钙化性主动脉瓣疾病的发展可能是高度调节的自主过程,可能存在着一个复杂的多步骤的内在机制在发挥作用,而并非年龄导致的不可避免的结果,病变有可能通过药物逆转。由于发病机制尚未完全明确,目前临床上还没有针对该病的治疗性药物,因此对该病发病机制的研究显得尤其重要。越来越多研究证明了炎症在钙化性主动脉瓣疾病发生发展过程中起重要作用。首先,通过对瓣膜置换手术中被置换出来的主动脉瓣膜进行组织病理分析发现有炎症的证据。其次,由于在发生主动脉瓣钙化并狭窄的瓣膜里发现有与口腔感染一致的细菌证据,并且在实验兔子接种口腔细菌可以诱导出主动脉瓣病变,推测慢性口腔感染可能是主动脉瓣钙化和狭窄发病的重要机制之一。
人主动脉瓣间质细胞是主动脉瓣膜最主要的细胞成分,而主动脉瓣钙化往往发生在瓣膜的间质部分,因此人主动脉瓣间质细胞在钙化性主动脉瓣膜疾病的发生发展过程中起关键作用。Toll样受体(Toll-like receptors,TLRs)是识别病原微生物的跨膜受体家族,主要通过识别保守的病原相关分子模式(PAMPs)感知病原微生物的存在,对之产生免疫炎症反应,在人类天然免疫系统中起到重要作用。目前已经发现哺乳动物中存在有13种TLRs,而TLR4是最早被发现且被研究最多的TLR,广泛存在于内皮细胞、巨噬细胞、中性粒细胞和树突状细胞等多种细胞中。TLR4被生物配体识别及结合后,可以活化细胞内的信号传导通路,促进炎症介质的产生和释放。因此,TLR4在细胞的免疫与炎症反应中起着关键作用。近来研究已经发现人主动脉瓣间质细胞能够表达有功能的TLR4,刺激这种受体后能够引起心脏瓣膜间质细胞的炎症和成骨反应,这在钙化性主动脉瓣疾病发生发展的病理过程中起重要作用。间质细胞的炎症激活可以诱导单核细胞和巨噬细胞聚集和浸润,而炎症介质促进主动脉瓣膜钙化的作用已被多个研究证实,因此我们推测炎症是主动脉瓣疾病初始阶段的促发因素,继而促进主动脉瓣膜成骨反应和钙沉积,从而导致钙化性主动脉瓣疾病的发生和发展。
核转录因子κB (NF-κB)广泛存在于真核生物中,是一个由复杂的多肽亚单位组成的蛋白家族。它作为信号传导途径中的枢纽,与免疫,肿瘤的发生、发展,细胞凋亡的调节以及胚胎发育等有着密切联系,是细胞内最重要的核转录因子。NF-κB在许多细胞刺激后介导的细胞内信息的转录调控中起核心作用,参与多种基因的表达和调控,它调控的基因编码急性期反应蛋白、细胞因子、细胞粘附分子、免疫调节分子、病毒瘤基因、生长因子、转录和生长调控因子等。在静息细胞中,NF-κB二聚体通过非共价键的形式与其抑制蛋白IKB结合而分散在细胞质内,当细胞受到肿瘤坏死因子(TNF)、脂多糖(LPS)等NF-κB激活剂刺激时,其抑制蛋白IKB解离和降解,NF-κB发生磷酸化,激活后的NF-κB进入细胞核,与DNA模块上的特异蛋白结合,诱导目标mRNA的产生,最后转录、生成和释放各种目标产物。
Notch蛋白是一组高度保守的细胞表面跨膜受体,在哺乳动物细胞中含有四种亚型,Notchl,Notch2, Notch3以及Notch4,Notch受体的配体有Jagged1, Jagged2,Delta like ligand (D11)l、D113和D114。Notch被γ分泌酶裂解,释放出其胞内域NICD,而NICD能够调控细胞命运及调节细胞功能。近来有研究表明细菌产物和脂多糖能够激活单核巨噬细胞的Notchl受体,从而调节细胞功能。抑制细胞γ分泌酶,就可以抑制NICD1的产生,同时也减少了巨噬细胞中脂多糖诱导的炎症因子的产生。然而Notchl信号通路在主动脉瓣间质细胞中对TLR4介导的炎症反应所起的作用目前尚不清楚。
白介素37(Interleukine37,IL-37)属于白介素1家族,而白介素1家族目前拥有11个成员。至今已有五种亚型的人类IL-37被报道,分别是IL-37、. IL-37b、IL-37c、IL-37d和IL-37e。目前尚未发现小鼠表达IL-37,但人IL-37能在小鼠体内发挥作用。IL-37普遍存在于扁桃体、皮肤、食道、胎盘、黑色素瘤、乳腺癌、前列腺癌、结肠癌和肺癌等组织及THP-1巨噬细胞,A549表皮细胞和外周血单核细胞等细胞中。IL-37能以低亲和力的方式与白介素18受体(IL-18R)结合但却不发挥激动或拮抗作用。除了胞外作用,IL-37也能在细胞内起作用,在细胞受到刺激后IL-37能转入核内并可能充当转录调节因子调节细胞功能。体内及体外研究均证明了人IL-37具有抗炎作用。体内实验,人IL-37转基因小鼠对结肠炎模型有保护作用。体外实验,巨噬细胞及表皮细胞转染IL-37后能显著降低LPS及IL-1诱导的炎症介质的表达。
本研究中,我们假想TLR4激活能诱导人主动脉瓣间质细胞的炎症反应,而这种炎症反应在来自主动脉瓣狭窄病人瓣膜组织的主动脉瓣间质细胞比来自正常瓣膜组织的细胞更明显,TLR4信号通路与Notch1信号通路发生交互作用,从而调节TLR4诱导的炎症反应水平,而人IL-37能通过Notch1/NF-KB轴抑制TLR4诱导的炎症反应,因此本研究可能对寻找钙化性主动脉瓣疾病的潜在防治靶点提供理论依据。本研究中我们利用TLR4受体激活剂LPS刺激人主动脉瓣间质细胞的TLR4受体,探讨TLR4介导的炎症反应,以及Notchl信号通路和人IL-37对其的调节作用及可能机制,本研究将分三部分进行。
第一部分TLR4与Notch1通路间的交互作用对人主动脉瓣间质细胞炎症反应的调节作用
目的
研究TLR4与Notch1信号通路间的交互作用对人主动脉瓣间质细胞炎症反应的调节作用。
方法
分别从正常主动脉瓣膜组织和主动脉瓣狭窄病人主动脉瓣膜组织分离出人主动脉瓣间质细胞,用TLR4激活物脂多糖(LPS)进行干预1到24小时。用免疫印迹方法分析细胞间粘附分子(ICAM-1)蛋白表达。用免疫印迹和免疫荧光染色方法分析Notch1信号通路的活化水平。用酶联免疫吸附(ELISA)方法分析单核细胞趋化蛋白(MCP-1),白介素8(IL-8)及Notch1特异性配体Jagged1的释放。应用γ分泌酶抑制剂DAPT抑制Notch1的活化,应用基因沉默的方法减少Notch1蛋白表达量,减少Notch1活化分子NICD1的产生,用Notch1特异性配体Jagged1激活Notch1,然后分析TLR4介导的ICAM-1表达量及MCP-1和IL-8的分泌水平。
结果
LPS诱导人主动脉瓣间质细胞ICAM-1蛋白的表达,MCP-1和IL-8的释放,以及Notch1信号通路的活化。来自主动脉瓣狭窄病人主动脉瓣膜组织的主动脉瓣间质细胞比来自正常瓣膜组织的主动脉瓣间质细胞显示出更强的炎症反应和Notch1信号通路的活化以及更多的Notch1特异性配体Jagged1释放。γ分泌酶抑制剂DAPT能抑制Notch1信号通路的活化,同时抑制TLR4介导的ICAM-1蛋白表达,IL-8和MCP-1释放。用基因沉默的方法降低Notch1表达水平,能减少NICD1的生成量,同时也减少TLR4介导的ICAM-1蛋白表达量。Notch1受体的特异性配体Jagged1能增强TLR4介导的ICAM-1蛋白表达,IL-8和MCP-1的释放。
结论
LPS能诱导人主动脉瓣间质细胞的炎症反应及Notch1信号通路的活化,而且这些反应在来自主动脉瓣狭窄病人瓣膜组织的主动脉瓣间质细胞中更明显。Notch1通路的活化能调控TLR4介导的炎症反应,Notch1过度活化可能是主动脉瓣狭窄病人瓣膜组织来源的主动脉瓣间质细胞炎症反应增强的重要机制。
第二部分Notch1通路在人主动脉瓣间质细胞中调节TLR4介导的NF-κB活化的机制研究
目的
探讨Notch1信号通路在人主动脉瓣间质细胞调节TLR4介导的炎症反应的机制。
方法
分别从正常瓣膜组织和主动脉瓣狭窄病人瓣膜组织分离出人主动脉瓣间质细胞,用TLR4激活物LPS处理细胞1到24小时。用免疫印迹和免疫组化方法分析Notch1蛋白基础表达水平,用免疫印迹和免疫荧光染色方法分析核因子κB(NF-κB)的活化水平,用免疫共沉淀方法分析NF-κB上游IKB激酶(IKK)和Notch1信号通路活化分子NICD1间的直接相互作用。用γ分泌酶抑制齐(?)DAPT抑制Notch1的活化,用基因沉默的方法减少Notch1蛋白表达量,用Notch1特异性配体Jagged1激活Notch1,然后分析TLR4介导的NF-κB的活化水平。
结果
LPS诱导入主动脉瓣间质细胞NF-κB的活化,其活化水平均在来自主动脉瓣狭窄病人瓣膜组织的主动脉瓣间质细胞中增高。来自主动脉瓣狭窄病人的主动脉瓣膜组织和问质细胞的Notch1蛋白基础表达水平比来自正常瓣膜的组织和细胞高。γ分泌酶抑制剂(DAPT)能抑制TLR4介导的NF-κB的磷酸化。用基因沉默的方法降低Notch1表达水平,可减少TLR4介导的NF-κB的磷酸化。Notch1受体的特异性配体Jagged1能增强TLR4介导的NF-κB的活化。免疫共沉淀结果显示NICD1与NF-κB上游的IκB激酶IKK有直接相互作用。
结论
LPS诱导人主动脉瓣膜间质细胞NF-κB信号通路的活化,其活化水平在来自主动脉瓣狭窄病人的细胞中增高。Notch1的活化分子NICD1能通过与IKK直接相互作用调节TLR4介导的NF-κB的活化水平,从而调控TLR4介导的炎症反应。
第三部分IL-37对人主动脉瓣间质细胞中对TLR4诱导的炎症反应的调节作用及其机制探讨
目的
探讨IL-37在人主动脉瓣间质细胞中对TLR4诱导的炎症反应的调节作用及其相关机制。
方法
分别从正常瓣膜和主动脉瓣狭窄病人瓣膜分离出人主动脉瓣间质细胞,用IL-37蛋白多肽与TLR4激活物LPS进行干预。用免疫印迹方法分析IL-37、ICAM-1蛋白表达水平,NF-κB和Notchl信号通路的活化水平,用RT-PCR方法分析IL-37的mRNA表达水平,用免疫荧光方法分析NF-κB核内转位。用基因沉默的方法减少IL-37的表达,然后分析TLR4介导的ICAM-1蛋白表达量。
结果
来自主动脉瓣狭窄病人瓣膜组织和来自正常瓣膜组织的主动脉瓣膜间质细胞均有IL-37mRNA和蛋白的表达,其水平在来自病变瓣膜组织的细胞较低。IL-37能抑制TLR4介导的ICAM-1表达水平,此作用在来自病变瓣膜组织的细胞中更明显。用基因沉默的方法减少IL-37的表达水平,可以增加TLR4介导的ICAM-1表达量。IL-37干预能减少Notchl的活化水平,并降低TLR4介导的NF-κB的活化水平。
结论
IL-37在人主动脉瓣膜间质细胞中表达,相对于来自正常瓣膜组织的主动脉瓣间质细胞,其表达水平在来自病变瓣膜组织的细胞较低。IL-37能通过抑制Notch1/NF-κB信号转导通路从而调节TLR4介导的炎症反应。
全文总结
1.Toll样受体4(TLR4)与Notch1通路的交互作用在人主动脉瓣间质细胞中能调节TLR4介导的炎症反应。
2.Notch1通路通过其活化分子NICD1与NF-κB上游Iκ3激酶IKK直接相互作用调节NF-κB通路的活性。
3.TLR4与Notch1通路间过多的交互作用可能是钙化性主动脉瓣疾病发生发展的重要机制。
4.白介素37(IL-37)可通过抑(?)(?)Notch1/NF-κB信号通路轴抑制TLR4介导的炎症反应,具有防治钙化性主动脉瓣疾病进展的潜力。
Background
Calcific aortic valve disease is a common disease in old people. Given the progressive aging of the general population, the incidence of calcific aortic valve disease is increasing every year. Currently, calcific aortic valve disease has become the third leading cardiovascular disease. Calcific aortic valve disease was traditionly regarded as a degenerative change which was irreversible process of calcium deposit in the aging people. Recent studies have found that calcific aortic valve disease is an active process, and multi-step complecated mechanisms are involved in the progression of this disease. Calcific aortic valve disease might be treated by drug. However, pharmacological intervention for the suppression of progression of calcific aortic valve disease is unavailable due to the limited knowledge of the underlying mechanism. Therefore, it is particularly important to study the pathogenesis of the disease. Increasing evidence suggest that inflammation plays an important role in the mechanism of calcific aortic valve disease. Firstly, evidence of inflammation had been found in the pathological analysis of replaced stenotic aortic valves. Secondly, the bacterial products found in the replaced stenotic aortic valve were consistent with the oral bacteria. Inoculation of experimental rabbits with oral bacteria induced aortic valve disease. We speculate that chronic oral infection plays an important role in the pathogenesis and progression of aortic valve calcification and stenosis.
Human aortic valve interstitial cell is the dominent cell component of human aortic valve. Aortic valve calcification nodules are usualy located in the interstitial portion of the valve. Therefore, human aortic valve interstitial cells play a critical role in the progression of calcific aortic valve disease. Toll-like receptors (TLRs) are the transmembrane receptors which recognize pathogens from bacterials, virus and other sources. TLRs play a pivotal role in the innate immune system. Currently,13TLRs have been reported and presented in endothelial cells, macrophages, neutrophils, dendritic cells and other cells. TLR4was the first TLR being discoveried and was one of the well-studied TLRs. TLR4activates intracellular signaling transduction pathways and induces the production and release of inflammatory mediators after stimulation by its biological ligands. Thus TLR4plays a key role in the immune and inflammatory responses. Previous studies have found that human aortic valve interstitial cells express the functional TLR4. Stimulation of TLR4induces inflammatory and osteogenic responses that play an important role in the pathologenesis and progression of calcific aortic valve disease. Inflammation of interstitial cells induces monocyte and macrophage accumulation and infiltration in aortic valve tissue. Studies have found that inflammatory mediators promote calcific aortic valve disease. Therefore we hypothesize that inflammation is an initial factor in the early stage of aortic valve diseases which promotes aortic valve osteogenic response and calcium deposition, thereby causes calcific aortic valve diseases.
Nuclear factor κB (NF-κB) is a protein family which composed of polypeptide subunits and prerented in eukaryotic organisms. NF-κB is the most important transcription factor in cells and plays an important role in the signal transduction pathways. It is closely related to important events, such as immunization, cancer development, cell apoptosis and the embryonic development. In addition, NF-κB plays a central role in the transcriptional regulation in cells after stimulation, and is involved in various expression and regulation of genes which encode acute phase response proteins, cytokines, cell adhesion molecules, immune regulation molecules, virus cancer genes, growth factors, transcription and growth regulators and so on. NF-κB is involved in the immune response, inflammatory response, apoptosis, tumorigenesis and other biological processes by regulating the expression of multiple genes. In the resting cells, a noncovalent bond links NF-κB dimer and its inhibitor IκB in the cytoplasm. When the cells are stimulated by NF-κB activators such as TNF and LPS, its inhibitor IκB is separated and degraded, and NF-κB is phosphorylated. After activated NF-κB entered the cell nucleus, it binds to the specific protein in the DNA module and induced the production of specific mRNA, then transcribed, produced and released various final targeted products.
Notch proteins are the highly conserved transmembrane receptors expressed on cell surface. Notch1, Notch2, Notch3and Notch4are the isoforms of Notch receptors in mammalian cells. The ligands that bind to Notch receptors are composed of Delta like group and Jagged group. The former includes Delta like ligand (D11)1, D113and D114. The latter includes Jaggedl and Jagged2. Upon ligand binding, Notch receptors undergo proteolytic cleavage, leading to the release of their intracellular domains (NICDs) that control cell fate and modulate cell functions. Bacterial lipopeptide and lipopolysaccharide (LPS) have been found to induce Notchl activation in macrophages. Inhibition of y-secretase, which processes Notchl to release NICD1, reduces LPS-induced expression of pro-inflammatory cytokines in macrophages. Currently, the role of Notchl in TLR4-mediated inflammatory response in human aortic valve interstitial cells has not been determined, and it is unknown whether human aortic valve interstitial cells of stenotic valves have exaggerated Notch1activation in response to TLR4stimulation.
Interleukine-37(IL-37) belongs to the IL-1family, which currently has11members. Five alternatively spliced transcript variants encoding distinct isoforms of human IL-37have been reported (IL-37a, IL-37b, IL-37c, IL-37d and IL-37e), whereas mouse IL-37has not been identified currently. However, human IL-37is active in mouse cells. Human IL-37has been identified presented in tonsils, skin, esophagus, placenta, melanoma, carcinomas of the breast, prostate, colon, lung and albeit, as well as human THP-1macrophages, A549epithelial cells and peripheral blood mononuclear cells. IL-37binds to IL-18Receptor a chain (IL-18Rα) with low affinity but does not exert any IL-18agonistic or antagonistic effect. Besides an extracellular role for IL-37, endogenous IL-37had been reported to translocate to the nucleus after cell stimulation and might also act as transcriptional modulators. Human IL-37has been demonstrated anti-inflammatory properties in vitro and in vivo. In vivo, expression of IL-37in mice protects mice from colitis,. In vitro, transfected with IL-37markedly reduced levels of cytokines induced by LPS and IL-1stimulation. However, it is unknown currently whether human IL-37presents in human aortic valve interstitial cells, what role it might play, and how IL-37exerts an effect in human aortic valve interstitial cells.
In the present study, we hypothesized that TLR4stimulation induces the expression of inflammatory mediators in human aortic valve interstitial cells. Human aortic valve interstitial cells from stenotic valves exhibit exaggerated inflammatory response in comparison to the cells of normal aortic valves. Crosstalk between the TLR4and Notchl pathways augments the inflammatory response in the interstitial cells of stenotic human aortic valves. Human IL-37can suppress TLR4-induced inflammatory response in human aortic valve interstitial cells and might be a therapeutic potential for prevention of progression of calcified aortic valve disease. We applied the TLR4agonist LPS to stimulate TLR4in human aortic valve interstitial cell, and evaluated the levels of TLR4-mediated inflammatory responses,. We also investigated the effects of Notchl pathway and human IL-37on TLR4-mediated inflammatory responses human aortic valve interstitial cells and possible underlying mechanisms. This study will be divided into3parts.
Part1Crosstalk between the TLR4and Notchl pathways modulates the inflammatory response in the interstitial cells of stenotic human aortic valves. Objective
To investigate the effect of crosstalk between the TLR4and Notchl pathways on the inflammatory response in the interstitial cells of stenotic human aortic valves.
Method
Human aortic valve interstitial cells were isolated from the normal aortic valves and stenotic aortic valves and treated with TLR4agonist lipopolysaccharide (LPS) for1to24hours. The levels of intercellular adhesion molecule1(ICAM-1) expression were analyzed with immunoblotting. The levels of Notchl signaling pathway activation were analyzed with immunoblotting and immunofluoresence staining. The release of monocyte chemotactic protein (MCP-1), interleukin8(IL-8) and Notchl specific ligand Jaggedl were examined with Enzyme-linked immuno sorbent assay (ELISA).γ-secretase inhibitor DAPT was applied to inhibit activation of Notchl. Gene silencing was applied to the knockdown the Notchl and reduce the production of Notchl activation molecular NICD1. Notch1specific ligand Jagged1was applied to activate Notchl. The levels of ICAM-1,MCP-1and IL-8were examined after treatements.
Results
LPS induced the protein expression of ICAM-1, the releases of MCP-1and IL-8, as well as the activation of Notchl signaling pathway in human aortic valve interstitial cells. Aortic valve interstitial cells of stenotic valves exhibited greater inflammatory response and activation of the Notchl signaling pathway than normal cells, γ-secretase inhibitor (DAPT) inhibited TLR4-mediated expression of ICAM-1and release of IL-8and MCP-1through inhibition of Notchl activation. Knockdown of Notchl inhibited the production of NICD1, and reduced TLR4-mediated expression of ICAM-1protein. Activation of Notchl with specific ligand Jagged1enhanced the TLR4-mediated protein expression of ICAM-1and the release of IL-8and MCP-1.
Conclusion
LPS induces inflammatory response and activation of Notchl signaling pathway in human aortic valve interstitial cells. The inflammatory response and Notchl activation in cells of stenotic aortic valves are greater than that in cells of normal aortic valves. Notchl activation regulates TLR4-mediated inflammatory response in human aortic valve interstitial cells, and the excessive cross-talk between the TLR4and Notchl pathways might be one of the mechanisms underlying exaggareted inflammatory response in aortic valve interstitial cells of stenotic arotic valves.
Part2The mechanistic study of modulaiotn of TLR4-induced NF-κB activation by Notchl pathway in human aortic valve interstitial cells Objective
To investigate the mechanism by which Notchl signaling pathway modulates TLR4-mediated inflammatory response in human aortic valve interstitial cells.
Methods
Human aortic valve interstitial cells were isolated from the normal aortic valves and stenotic aortic valves and treated with TLR4agonist lipopolysaccharide (LPS) for1to24hours. The levels of Notch1expression were analyzed with immunohistochemistry and immunoblotting. NF-κB signaling pathways were analyzed with immunoblotting and immunofluorescence. Physical interaction between Notch1and NF-κB signaling pathways were examined with co-immunoprecipitation. γ-secretase inhibitor DAPT was applied to inhibit activation of Notch1. Knockdown of Notchl was applied to reduce the protein expression of Notchl and production of NICD1. Notchl specific ligand Jagged1was applied to activate Notchl. The levels of NF-κB phosphorylation were analyzed after treatments.
Results
LPS induced the activation of NF-κB signaling pathways in human aortic valve interstitial cell. Activation of NF-κB signaling pathways were greater in human aortic valve interstitial cells of stenotic aortic valve than cells of normal aortic valves. The levels of Notchl protein expression in stenotic aortic valve tissues and interstitial cells were higher than that in normal aortic valve, y-secretase inhibitor (DAPT) inhibited TLR4-mediated NF-κB phosphorylation. Knockdown of Notchl inhibited TLR4-mediated NF-κB phosphorylation and reduced the production of NICD1. Notchl receptor specific ligand Jagged1enhanced TLR4-mediated NF-κB phosphorylation. Co-immunoprecipitation results showed that NICD1interacted with NF-κB upstream IκB kinase IKK physically.
Conclusion
LPS induces the activation of NF-κB signaling pathway in human aortic heart valve interstitial cell and the activations of these two signaling pathways are greater in human aortic valve interstitial cells from stenotic valves that those in normal cells. Activation of Notch1modulates TLR4-mediated NF-κB phosphorylation through interaction of NICD1with IKK physically, and subsequently regulates TLR4-mediated inflammatory responses in human aortic valve interstitial cells.
Part3The study of effect and mechanism of IL-37on TLR4-mediated inflammatory response in human aortic valve interstitial cells
Objective
To investigate the effect of IL-37on the modulation of TLR4-mediated inflammatory response in human aortic valve interstitial cells and the underlying mechanisms.
Methods
Human aortic valve interstitial cells were isolated from the normal aortic valves and stenotic aortic valves and treated with TLR4agonist lipopolysaccharide (LPS). The mRNA levels of human IL-37were examined with RT-PCR. The levels of protein expression of IL-37, ICAM-1, activation of NF-κB and Notchl signaling pathways were analyzed with immunoblotting. Nuclear localization of NF-κB was examined with immunofluorescence. Knockdown was applied to reduce the expression of IL-37. The levels of ICAM-1protein expression were analyzed with immunoblotting after treatments.
Results
Human aortic valve interstitial cells expressed IL-37and the levels of IL-37expression were lower in the interstitial cells of human stenotic aortic valves in comparison to that in normal cells. IL-37inhibited TLR4-mediated ICAM-1expression in human aortic valve interstitial cells.knockdown of IL-37markly decreased IL-37expression, and increase the TLR4-mediated ICAM-1expression. IL-37treatment reduces the levels of TLR4-mediated Notchl and NF-κB activation.
Conclusions
Human aortic valve interstitial cells expressed IL-37and the levels of IL-37expression were lower in the interstitial cells of human stenotic aortic valves in comparison to that in normal cells. IL-37regulates TLR4-mediated inflammatory response through inhibition of Notch1/NF-κB axis. Thus, IL-37might have therapeutic potential for the progression of calcific aortic valve disease.
Summary
1. Cross-talk between the TLR4and Notchl pathways modulates TLR4-mediated inflammatory response in human aortic valve interstitial cells.
2. Notchl pathway modulates TLR4-mediated NF-kB activation in human aortic valve interstitial cells through interaction of NICD1with IKK directly.
3. Excessive cross-talk between TLR4and Notch1pathways in human aortic valve interstitial cells may contribute to the mechanism underlying pathogenesis and progression of calcific aortic valve disease.
4. IL-37suppressed TLR4-mediated inflammatory response through inhibition of Notch1/NF-κB axis in human aortic valve interstitial cells. Thus, IL-37may have therapeutic potential for prevention of progression of calcific aortic valve disease.
引文
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1. Meng, X., et al., Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells:potential roles in aortic valve inflammation and stenosis. Am J Physiol Cell Physiol,2008.294(1):p. C29-C35.
2. Shin, H.M., et al., Notchl augments NF-kappaB activity by facilitating its nuclear retention. EMBO J,2006.25(1):p.129-38.
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6. Su, X., et al., Oxidized low density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4. J Biol Chem,2011.286(14):p.12213-20.
7. Mohler, E.R.,3rd, et al., Bone formation and inflammation in cardiac valves. Circulation,2001.103(11):p.1522-8.
8. Rajamannan, N.M., B. Gersh, and R.O. Bonow, Calcific aortic stenosis:from bench to the bedside--emerging clinical and cellular concepts. Heart,2003. 89(7):p.801-5.
9. Skowasch, D., et al., Pathogen burden in degenerative aortic valves is associated with inflammatory and immune reactions. J Heart Valve Dis,2009. 18(4):p.411-7.
10. Cote, N., et al., Inflammation Is Associated with the Remodeling of Calcific Aortic Valve Disease. Inflammation,2012.
11. Mohler, E.R.,3rd, et al., Identification and characterization of calcifying valve cells from human and canine aortic valves. J Heart Valve Dis,1999.8(3):p. 254-60.
12. Zhu, C, et al., Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-kappaB pathway in microglia cells. PLoS One,2012.7(4):p. e35125.
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22. Foldi, J.. et al., Autoamplification of Notch signaling in macrophages by TLR-induced and RBP-J-dependent induction of Jaggedl. J Immunol,2010. 185(9):p.5023-31.
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1. Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998.391(6669):p.806-11.
2. Bernstein, E., et al., Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001.409(6818):p.363-6.
3. Su, X., et al., Oxidized low density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4.J Biol Chem,2011.286(14):p.12213-20.
4. Skowasch, D., et al., Pathogen burden in degenerative aortic valves is associated with inflammatory and immune reactions. J Heart Valve Dis,2009. 18(4):p.411-7.
5. Cote, N., et al., Inflammation Is Associated with the Remodeling of Calcific Aortic Valve Disease. Inflammation,2012.
6. Steiner, I., et al., Calcific aortic valve stenosis:Immunohistochemical analysis of inflammatory infiltrate. Pathol Res Pract,2012.208(4):p.231-4.
7. Miller, J.D., R.M. Weiss, and D.D. Heistad, Calcific aortic valve stenosis: methods, models, and mechanisms. Circ Res,2011.108(11):p.1392-412.
8. Meng, X., et al., Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells:potential roles in aortic valve inflammation and stenosis. Am J Physiol Cell Physiol,2008.294(1):p. C29-C35.
9. Yang, X., et al., Pro-osteogenic phenotype of human aortic valve interstitial cells is associated with higher levels of Toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2. J Am Coll Cardiol,2009.53:p. 491-500.
10. Paquette, D.W., N. Brodala, and T.C. Nichols, Cardiovascular disease, inflammation, and periodontal infection. Periodontol 2000,2007.44:p.113-26.
11. Niedzielska, I., et al., The effect of chronic periodontitis on the development of atherosclerosis:review of the literature. Med Sci Monit,2008.14(7):p. RA103-6.
12. Erridge, C., The roles of pathogen-associated molecular patterns in atherosclerosis. Trends Cardiovasc Med,2008.18(2):p.52-6.
13. Mohler, E.R.,3rd, et al., Bone formation and inflammation in cardiac valves. Circulation,2001.103(11):p.1522-8.
14. Rajamannan, N.M., B. Gersh, and R.O. Bonow, Calcific aortic stenosis:from bench to the bedside--emerging clinical and cellular concepts. Heart,2003. 89(7):p.801-5.
15. Babu, A.N., et al., Lipopolysaccharide stimulation of human aortic valve interstitial cells activates inflammation and osteogenesis. Ann Thorac Surg, 2008.86(1):p.71-6.
16. Charo, I.F. and R.M. Ransohoff, The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med,2006.354(6):p.610-21.
17. Gerard, C. and B.J. Rollins, Chemokines and disease. Nat Immunol,2001.2(2): p.108-15.
18. Springer, T.A., Traffic signals for lymphocyte recirculation and leukocyte emigration:the multistep paradigm. Cell,1994.76(2):p.301-14.
19. Liu, G., et al., Src phosphorylation of endothelial cell surface intercellular adhesion molecule-1 mediates neutrophil adhesion and contributes to the mechanism of lung inflammation. Arterioscler Thromb Vasc Biol,2011.31(6): p.1342-50.
20. Lawson, C., et al., Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells:induction of VCAM-1 but no proliferation. Cardiovasc Res,2001.50(3):p.547-55.
21. Lawson, C. and S. Wolf, ICAM-1 signaling in endothelial cells. Pharmacol Rep, 2009.61(1):p.22-32.
22. Fortini, M.E., Notch signaling:the core pathway and its posttranslational regulation. Dev Cell,2009.16(5):p.633-47.
23. Palaga, T., et al., Notch signaling is activated by TLR stimulation and regulates macrophage functions. Eur J Immunol,2008.38(1):p.174-83.
24. Hu, X., et al., Integrated regulation of Toll-like receptor responses by Notch and interferon-gamma pathways. Immunity,2008.29(5):p.691-703.
25. Outtz, H.H., et al., Notch 1 deficiency results in decreased inflammation during wound healing and regulates vascular endothelial growth factor receptor-1 and inflammatory cytokine expression in macrophages. J Immunol,2010.185(7):p. 4363-73.
26. Foldi, J., et al., Autoamplification of Notch signaling in macrophages by TLR-induced and RBP-J-dependent induction of Jaggedl. J Immunol,2010. 185(9):p.5023-31.
27. Cao, Q., et al., Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells. Glia,2008.56(11):p.1224-37.
28. Lopez, J., et al., Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells. Int J Cardiol, 2012.158(1):p.18-25.
29. Mohler, E.R.,3rd, et al., Identification and characterization of calcifying valve cells from human and canine aortic valves. J Heart Valve Dis,1999.8(3):p. 254-60.
1. Meng, X., et al., Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells:potential roles in aortic valve inflammation and stenosis. Am J Physiol Cell Physiol,2008.294(1):p. C29-C35.
2. Shin, H.M., et al., Notchl augments NF-kappaB activity by facilitating its nuclear retention. EMBO J,2006.25(1):p.129-38.
3. Monsalve, E., et al., Notchl upregulates LPS-induced macrophage activation by increasing NF-kappaB activity. Eur J Immunol,2009.39(9):p.2556-70.
4. Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998.391(6669):p.806-11.
5. Bernstein, E., et al., Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001.409(6818):p.363-6.
6. Su, X., et al., Oxidized low density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4. J Biol Chem,2011.286(14):p.12213-20.
7. Mohler, E.R.,3rd, et al., Bone formation and inflammation in cardiac valves. Circulation,2001.103(11):p.1522-8.
8. Rajamannan, N.M., B. Gersh, and R.O. Bonow, Calcific aortic stenosis:from bench to the bedside--emerging clinical and cellular concepts. Heart,2003. 89(7):p.801-5.
9. Skowasch, D., et al., Pathogen burden in degenerative aortic valves is associated with inflammatory and immune reactions. J Heart Valve Dis,2009. 18(4):p.411-7.
10. Cote, N., et al., Inflammation Is Associated with the Remodeling of Calcific Aortic Valve Disease. Inflammation,2012.
11. Mohler, E.R.,3rd, et al., Identification and characterization of calcifying valve cells from human and canine aortic valves. J Heart Valve Dis,1999.8(3):p. 254-60.
12. Zhu, C, et al., Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-kappaB pathway in microglia cells. PLoS One,2012.7(4):p. e35125.
13. Barnes, P.J. and M. Karin, Nuclear factor-kappaB:a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med,1997.336(15):p.1066-71.
14. Granet, C., W. Maslinski, and P. Miossec, Increased AP-1 and NF-kappaB activation and recruitment with the combination of the proinflammatory cytokines IL-lbeta, tumor necrosis factor alpha and IL-17 in rheumatoid synoviocytes. Arthritis Res Ther,2004.6(3):p. R190-8.
15. Zhu, Y.M., et al., Transcriptional regulation of interleukin (IL)-8 by bradykinin in human airway smooth muscle cells involves prostanoid-dependent activation of AP-1 and nuclear factor (NF)-IL-6 and prostanoid-independent activation of NF-kappaB. J Biol Chem,2003.278(31):p.29366-75.
16. Artavanis-Tsakonas, S., M.D. Rand, and R.J. Lake, Notch signaling:cell fate control and signal integration in development. Science,1999.284(5415):p. 770-6.
17. Brou, C, et al., A novel proteolytic cleavage involved in Notch signaling:the role of the disintegrin-metalloprotease TACE. Mol Cell,2000.5(2):p.207-16.
18. Oswald, F., et al., SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. EMBO J,2002.21(20):p.5417-26.
19. Fortini, M.E., Notch signaling:the core pathway and its posttranslational regulation. Dev Cell,2009.16(5):p.633-47.
20. Palaga, T., et al., Notch signaling is activated by TLR stimulation and regulates macrophage functions. Eur J Immunol,2008.38(1):p.174-83.
21. Hu, X., et al., Integrated regulation of Toll-like receptor responses by Notch and interferon-gamma pathways. Immunity,2008.29(5):p.691-703.
22. Foldi, J.. et al., Autoamplification of Notch signaling in macrophages by TLR-induced and RBP-J-dependent induction of Jaggedl. J Immunol,2010. 185(9):p.5023-31.
23. Nickoloff, B.J., et al., Jagged-1 mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma. Cell Death Differ,2002.9(8):p.842-55.
24. Vilimas, T., et al., Targeting the NF-kappaB signaling pathway in Notch1-induced T-cell leukemia. Nat Med,2007.13(1):p.70-7.
25. Song, L.L., et al., Notch-1 associates with IKKalpha and regulates IKK activity in cervical cancer cells. Oncogene,2008.27(44):p.5833-44.
26. Oswald, F., et al., NF-kappaB2 is a putative target gene of activated Notch-1 via RBP-Jkappa. Mol Cell Biol,1998.18(4):p.2077-88.
27. Espinosa, L., et al., The Notch/Hesl pathway sustains NF-kappaB activation through CYLD repression in T cell leukemia. Cancer Cell,2010.18(3):p. 268-81.
1. Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998.391(6669):p.806-11.
2. Bernstein, E., et al., Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001.409(6818):p.363-6.
3. Su, X., et al., Oxidized low density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4. J Biol Chem,2011.286(14):p.12213-20.
4. Skowasch, D., et al., Pathogen burden in degenerative aortic valves is associated with inflammatory and immune reactions. J Heart Valve Dis,2009. 18(4):p.411-7.
5. New, S.E. and E. Aikawa, Cardiovascular calcification:an inflammatory disease. Circ J,2011.75(6):p.1305-13.
6. Rajamannan, N.M., et al., Calcific aortic valve disease:not simply a degenerative process:A review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group. Executive summary:Calcific aortic valve disease-2011 update. Circulation,2011.124(16): p.1783-91.
7. Kaden, J.J., et al., Inflammatory regulation of extracellular matrix remodeling in calcific aortic valve stenosis. Cardiovasc Pathol,2005.14(2):p.80-7.
8. Mohler, E.R.,3rd, et al., Bone formation and inflammation in cardiac valves. Circulation,2001.103(11):p.1522-8.
9. Rajamannan, N.M., B. Gersh, and R.O. Bonow, Calcific aortic stenosis:from bench to the bedside--emerging clinical and cellular concepts. Heart,2003. 89(7):p.801-5.
10. New, S.E. and E. Aikawa, Molecular imaging insights into early inflammatory stages of arterial and aortic valve calcification. Circ Res,2011.108(11):p. 1381-91.
11. Otto, C.M., et al., Characterization of the early lesion of 'degenerative' valvular aortic stenosis. Histological and immunohistochemical studies. Circulation, 1994.90(2):p.844-53.
12. Nakano, K., et al., Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens. J Clin Microbiol,2006.44(9): p.3313-7.
13. Nakano, K., et al., Detection of oral bacteria in cardiovascular specimens. Oral Microbiol Immunol,2009.24(1):p.64-8.
14. Cohen, D.J., et al., Role of oral bacterial flora in calcific aortic stenosis:an animal model. Ann Thorac Surg,2004.77(2):p.537-43.
15. Meng, X., et al., Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells:potential roles in aortic valve inflammation and stenosis. Am J Physiol Cell Physiol,2008.294(1):p. C29-C35.
16. Babu, A.N., et al., Lipopolysaccharide stimulation of human aortic valve interstitial cells activates inflammation and osteogenesis. Ann Thorac Surg, 2008.86(1):p.71-6.
17. Yang, X., et al., Pro-osteogenic phenotype of human aortic valve interstitial cells is associated with higher levels of Toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2. J Am Coll Cardiol,2009.53:p. 491-500.
18. Kaden, J.J., et al., Tumor necrosis factor alpha promotes an osteoblast-like phenotype in human aortic valve myofibroblasts:a potential regulatory mechanism of valvular calcification. Int J Mol Med,2005.16(5):p.869-72.
19. Kaden, J.J., et al., Interleukin-1 beta promotes matrix metalloproteinase expression and cell proliferation in calcific aortic valve stenosis. Atherosclerosis,2003.170(2):p.205-11.
20. Lee, J.H., et al., Stenotic aortic valves have dysfunctional mechanisms of anti-inflammation:implications for aortic stenosis. J Thorac Cardiovasc Surg, 2011.141(2):p.481-6.
21. McNamee, E.N., et al., Interleukin 37 expression protects mice from colitis. Proc Natl Acad Sci U S A,2011.108(40):p.16711-6.
22. Bulau, A.M., et al., In vivo expression of interleukin-37 reduces local and systemic inflammation in concanavalin A-induced hepatitis. Scientific WorldJournal,2011.11:p.2480-90.
23. Sharma, S., et al., The IL-1 family member 7b translocates to the nucleus and down-regulates proinflammatory cytokines. J Immunol,2008.180(8):p. 5477-82.
24. Nold, M.F., et al., IL-37 is a fundamental inhibitor of innate immunity. Nat Immunol,2011.11(11):p.1014-22.
25. Lopez, J., et al., Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells. Int J Cardiol, 2012.158(1):p.18-25.
26. Yang, L., et al., ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow. Blood, 2005.106(2):p.584-92.
27. Lawson, C. and S. Wolf, ICAM-1 signaling in endothelial cells. Pharmacol Rep, 2009.61(1):p.22-32.
28. Lawson, C., et al., Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells:induction of VCAM-1 but no proliferation. Cardiovasc Res,2001.50(3):p.547-55.
29. Lawson, C., et al., Ligation of ICAM-1 on endothelial cells leads to expression of VCAM-1 via a nuclear factor-kappaB-independent mechanism. J Immunol, 1999.162(5):p.2990-6.
30. Liu, G., et al., Src phosphorylation of endothelial cell surface intercellular adhesion molecule-1 mediates neutrophil adhesion and contributes to the mechanism of lung inflammation. Arterioscler Thromb Vasc Biol,2011.31(6): p.1342-50.
31. Kumar, S., et al., Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma production. Cytokine,2002.18(2):p.61-71.
32. Bufler, P., et al., A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity. Proc Natl Acad Sci U S A,2002.99(21):p. 13723-8.
33. Bufler, P., et al., Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide. Biochem J,2004.381(Pt 2):p.503-10.
1. Skowasch, D., et al., Pathogen burden in degenerative aortic valves is associated with inflammatory and immune reactions. J Heart Valve Dis,2009. 18(4):p.411-7.
2. Cote, N., et al., Inflammation Is Associated with the Remodeling of Calcific Aortic Valve Disease. Inflammation,2012.
3. Steiner, I., et al., Calcific aortic valve stenosis:Immunohistochemical analysis of inflammatory infiltrate. Pathol Res Pract,2012.208(4):p.231-4.
4. Miller, J.D., R.M. Weiss, and D.D. Heistad, Calcific aortic valve stenosis: methods, models, and mechanisms. Circ Res,2011108(11):p.1392-412.
5. New, S.E. and E. Aikawa, Cardiovascular calcification:an inflammatory disease. Circ J,2011.75(6):p.1305-13.
6. Rajamannan, N.M., et al., Calcific aortic valve disease:not simply a degenerative process:A review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group. Executive summary:Calcific aortic valve disease-2011 update. Circulation,2011.124(16): p.1783-91.
7. Kaden, J.J., et al., Inflammatory regulation of extracellular matrix remodeling in calcific aortic valve stenosis. Cardiovasc Pathol,2005.14(2):p.80-7.
8. Meng, X., et al., Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells:potential roles in aortic valve inflammation and stenosis. Am J Physiol Cell Physiol,2008.294(1):p. C29-C35.
9. Yang, X., et al., Pro-osteogenic phenotype of human aortic valve interstitial cells is associated with higher levels of Toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2. J Am Coll Cardiol,2009.53:p. 491-500.
10. Paquette, D.W., N. Brodala, and T.C. Nichols, Cardiovascular disease, inflammation, and periodontal infection. Periodontol 2000,2007.44:p.113-26.
11. Niedzielska, I., et al., The effect of chronic periodontitis on the development of atherosclerosis:review of the literature. Med Sci Monit,2008.14(7):p. RA103-6.
12. Erridge, C., The roles of pathogen-associated molecular patterns in atherosclerosis. Trends Cardiovasc Med,2008.18(2):p.52-6.
13. Mohler, E.R.,3rd, et al., Bone formation and inflammation in cardiac valves. Circulation,2001.103(11):p.1522-8.
14. Rajamannan, N.M., B. Gersh, and R.O. Bonow, Calcific aortic stenosis:from bench to the bedside--emerging clinical and cellular concepts. Heart,2003. 89(7):p.801-5.
15. Babu, A.N., et al., Lipopolysaccharide stimulation of human aortic valve interstitial cells activates inflammation and osteogenesis. Ann Thorac Surg, 2008.86(1):p.71-6.
16. Charo, I.F. and R.M. Ransohoff, The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med,2006.354(6):p.610-21.
17. Gerard, C. and B.J. Rollins, Chemokines and disease. Nat Immunol,2001.2(2): p.108-15.
18. Springer, T.A., Traffic signals for lymphocyte recirculation and leukocyte emigration:the multistep paradigm. Cell,1994.76(2):p.301-14.
19. Artavanis-Tsakonas, S., M.D. Rand, and R.J. Lake, Notch signaling:cell fate control and signal integration in development. Science,1999.284(5415):p. 770-6.
20. Brou, C., et al., A novel proteolytic cleavage involved in Notch signaling:the role of the disintegrin-metalloprotease TACE. Mol Cell,2000.5(2):p.207-16.
21. Oswald, F., et al., SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. EMBO J,2002.21(20):p.5417-26.
22. Fortini, M.E., Notch signaling:the core pathway and its posttranslational regulation. Dev Cell,2009.16(5):p.633-47.
23. Monsalve, E., et al., Notchl upregulates LPS-induced macrophage activation by increasing NF-kappaB activity. Eur J Immunol,2009.39(9):p.2556-70.
24. Palaga, T., et al., Notch signaling is activated by TLR stimulation and regulates macrophage functions. Eur J Immunol,2008.38(1):p.174-83.
25. Hu, X., et al., Integrated regulation of Toll-like receptor responses by Notch and interferon-gamma pathways. Immunity,2008.29(5):p.691-703.
26. Foldi, J., et al., Autoamplification of Notch signaling in macrophages by TLR-induced and RBP-J-dependent induction of Jaggedl.J Immunol,2010. 185(9):p.5023-31.
27. Cao, Q., et al., Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells. Glia,2008.56(11):p.1224-37.
28. Lopez, J., et al., Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells.Int J Cardiol, 2012.158(1):p.18-25.
29. Mohler, E.R.,3rd, et al., Identification and characterization of calcifying valve cells from human and canine aortic valves. J Heart Valve Dis,1999.8(3):p. 254-60.
30. Zhu, C., et al., Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-kappaB pathway in microglia cells. PLoS One,2012.7(4):p. e35125.
31. Barnes, P.J. and M. Karin, Nuclear factor-kappaB:a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med,1997.336(15):p.1066-71.
32. Granet, C., W. Maslinski, and P. Miossec, Increased AP-1 and NF-kappaB activation and recruitment with the combination of the proinflammatory cytokines IL-lbeta, tumor necrosis factor alpha and IL-17 in rheumatoid synoviocytes. Arthritis Res Ther,2004.6(3):p. R190-8.
33. Zhu, Y.M., et al., Transcription al regulation of interleukin (IL)-8 by bradykinin in human airway smooth muscle cells involves prostanoid-dependent activation of AP-1 and nuclear factor (NF)-IL-6 and prostanoid-independent activation of NF-kappaB. J Biol Chem,2003.278(31):p.29366-75.
34. Nickoloff, B.J., et al., Jagged-1 mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma. Cell Death Differ,2002.9(8):p.842-55.
35. Vilimas, T., et al., Targeting the NF-kappaB signaling pathway in Notch1-induced T-cell leukemia. Nat Med,2007.13(1):p.70-7.
36. Song, L.L., et al., Notch-1 associates with IKKalpha and regulates IKK activity in cervical cancer cells. Oncogene,2008.27(44):p.5833-44.
37. Oswald, F., et al., NF-kappaB2 is a putative target gene of activated Notch-1 via RBP-Jkappa. Mol Cell Biol,1998.18(4):p.2077-88.
38. Shin, H.M., et al., Notchl augments NF-kappaB activity by facilitating its nuclear retention. EMBO J,2006.25(1):p.129-38.
39. Espinosa, L., et al., The Notch/Hes1 pathway sustains NF-kappaB activation through CYLD repression in T cell leukemia. Cancer Cell,2010.18(3):p. 268-81.
40. Shimizu, T., et al., Notch signaling induces osteogenic differentiation and mineralization of vascular smooth muscle cells:role of Msx2 gene induction via Notch-RBP-Jk signaling. Arterioscler Thromb Vasc Biol,2009.29(7):p. 1104-11.
41. Shimizu, T., et al., Notch signaling pathway enhances bone morphogenetic protein 2 (BMP2) responsiveness of Msx2 gene to induce osteogenic differentiation and mineralization of vascular smooth muscle cells. J Biol Chem, 2011.286(21):p.19138-48.
42. Nobta, M., et al., Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notchl signaling. J Biol Chem,2005.280(16):p.15842-8.
43. Tezuka, K., et al., Stimulation of osteoblastic cell differentiation by Notch. J Bone Miner Res,2002.17(2):p.231-9.
44. Fukuda, D., et al., Notch ligand Delta-like 4 blockade attenuates atherosclerosis and metabolic disorders. Proc Natl Acad Sci U S A,2012.109(27):p. E1868-77.
45. Garg, V., et al., Mutations in NOTCH1 cause aortic valve disease. Nature,2005. 437(7056):p.270-4.
46. Lee, J.H., et al., Stenotic aortic valves have dysfunctional mechanisms of anti-inflammation:implications for aortic stenosis. J Thorac Cardiovasc Surg, 2011.141(2):p.481-6.
47. McNamee, E.N., et al., Interleukin 37 expression protects mice from colitis. Proc Natl Acad Sci U S A,2011.108(40):p.16711-6.
48. Bulau, A.M., et al., In vivo expression of interleukin-37 reduces local and systemic inflammation in concanavalin A-induced hepatitis. Scientific WorldJournal,2011.11:p.2480-90.
49. Sharma, S., et al., The IL-1 family member 7b translocates to the nucleus and down-regulates proinflammatory cytokines. J Immunol,2008.180(8):p. 5477-82.
50. Nold, M.F., et al., IL-37 is a fundamental inhibitor of innate immunity. Nat Immunol,2011.11(11):p.1014-22.
51. Yang, L., et al., ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow. Blood, 2005.106(2):p.584-92.
52. Lawson, C. and S. Wolf, ICAM-1 signaling in endothelial cells. Pharmacol Rep, 2009.61(1):p.22-32.
53. Lawson, C., et al., Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells:induction of VCAM-1 but no proliferation. Cardiovasc Res,2001.50(3):p.547-55.
54. Lawson, C., et al., Ligation of ICAM-1 on endothelial cells leads to expression of VCAM-1 via a nuclear factor-kappaB-independent mechanism. J Immunol, 1999.162(5):p.2990-6.
55. Liu, G., et al., Src phosphorylation of endothelial cell surface intercellular adhesion molecule-1 mediates neutrophil adhesion and contributes to the mechanism of lung inflammation. Arterioscler Thromb Vasc Biol,2011.31(6): p.1342-50.
56. Kumar, S., et al., Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma production. Cytokine,2002.18(2):p.61-71.
57. Bufler, P., et al., A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity. Proc Natl Acad Sci U S A,2002.99(21):p. 13723-8.
58. Bufler, P., et al., Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide. Biochem J,2004.381(Pt 2):p.503-10.