| BackgroundCalcific 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. ObjectiveTo investigate the effect of crosstalk between the TLR4and Notchl pathways on the inflammatory response in the interstitial cells of stenotic human aortic valves.MethodHuman 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. ResultsLPS 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.ConclusionLPS 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 ObjectiveTo investigate the mechanism by which Notchl signaling pathway modulates TLR4-mediated inflammatory response in human aortic valve interstitial cells. MethodsHuman 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.ResultsLPS 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.ConclusionLPS 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 cellsObjectiveTo investigate the effect of IL-37on the modulation of TLR4-mediated inflammatory response in human aortic valve interstitial cells and the underlying mechanisms.MethodsHuman 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.ResultsHuman 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.ConclusionsHuman 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.Summary1. 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. |
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