Role of the TLR4 signaling in smooth muscle cell phenotypic transition

TLR4信号在平滑肌细胞表型转变中的作用

• 批准号: 10448240
• 负责人: Olga Cherepanova
• 金额: $ 40.25万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10448240
• 关键词: ATP binding cassette transporter 1; ApoE knockout mouse; Apolipoprotein E; Area; Arterial Fatty Streak; Arteries; Atherosclerosis; Automobile Driving; Blood Vessels; Cell Lineage; Cells; Cholesterol; Coronary artery; Data; Differentiation Antigens; Disease; Endogenous Factors; Epigenetic Process; Feedback; Genetic; Genetic Transcription; Human; IRF3 gene; Immune; Impairment; In Vitro; Inflammatory; Inflammatory Response; Investments; Knock-out; Knockout Mice; LXRalpha protein; Lead; Lesion; Lipids; Lipopolysaccharides; Mediating; Molecular; Mus; Natural Immunity; Oxides; Pathogenesis; Pathway interactions; Phagocytosis; Pharmacology; Phenotype; Phospholipids; Play; Production; Reporting; Research Project Grants; Role; Signal Transduction; Smooth Muscle Myocytes; Somatic Cell; Specimen; Stem Cell Factor; Stem cell pluripotency; Stimulus; TLR4 gene; Testing; Time; Toll-like receptors; Up-Regulation; antagonist; atheroprotective; cell motility; chromatin immunoprecipitation; cytokine; embryonic stem cell; in vivo; indexing; induced pluripotent stem cell; innate immune mechanisms; insight; macrophage; member; mouse model; novel; overexpression; pluripotency factor; promoter; response; transdifferentiation; virtual

Abstract There is a growing body of evidence that smooth muscle cell (SMC) phenotypic transitions play an important role in the pathogenesis of atherosclerosis. However, there is still little understanding of molecular mechanisms responsible for these transitions in vivo. Recently, we found that the embryonic stem cell/induced pluripotency stem cell (iPSC) factor OCT4, which was believed to be silenced in somatic cells, plays an atheroprotective role in SMC, in that genetic inactivation of Oct4 in SMC led to marked increases in lesion size and multiple indices of plaque instability in Apoe‒/‒ mice. While we showed that OCT4 is required for SMC migration and investment into the protective fibrous cap, we also have evidence that its loss in SMC was associated with SMC transition toward a macrophage (MФ)-like state, including increased MФ marker expression, lipid accumulation, and phagocytosis. Given the difficulty in overexpressing targeting iPSC factors, it is crucial to identify endogenous molecular mechanisms responsible for OCT4 activation in SMC that can be potentially used to mediate beneficial SMC-OCT4-dependent effects. It has been reported that activation of the innate immune Toll-like receptor (TLR)/TRIF-dependent IRF3/NFkB signaling is required for efficient reprogramming to iPSC, as well as in cell trans-differentiation to other cell lineages by driving epigenetic plasticity of cells. While mechanisms and the critical role of the innate immunity in the progression of atherosclerosis are well characterized in the immune cells, virtually nothing is known about the role of TLRs in the phenotypic transition of SMC in atherosclerosis. Our data demonstrate that TLR4 signaling is responsible for hydroxymethylation of the Oct4 promoter and reactivation of Oct4 in SMC. Intriguingly, Oct4 deficiency in SMC, in turn, up-regulates Tlr4, and leads to dysregulation of both classical TLR4-downstream mechanisms, including MyD88-dependent (NFkB-mediated inflammatory response) and TRIF-dependent (IRF3/LXRa-mediated cholesterol efflux), in vitro and in vivo. In addition, Chromatin Immunoprecipitation (ChIP)-seq analysis on specimens isolated from mouse atherosclerotic arteries identified Tlr4 as a putative target of OCT4. Together, these results demonstrate the importance of TLR4- signaling in SMC both upstream and downstream of OCT4. Our central hypothesis is that TLR4 signaling mediates atheroprotective changes in SMC by regulating levels of the pluripotency factors OCT4 via a feedback mechanism. To test this hypothesis, we propose the following aims: Aim 1 will test the hypothesis that TLR4 signaling regulates levels of OCT4 in SMC via a feedback mechanism. Aim 2 will test the hypothesis that TLR4 signaling in SMC is atheroprotective. This Aim will utilize genetic and pharmacological approaches to define the role of TLR4 signaling in SMC at different stages of atherosclerosis. Aim 3 will test the hypothesis that the up- regulation of TLR4 in Oct4 deficient SMC promotes SMC transition toward МФ-like state. These studies will establish for the first time the mechanistic connection between innate immunity and pluripotency factors in SMC and provide insight into the role of TLR4 in SMC phenotypic transition.

摘要 越来越多的证据表明，平滑肌细胞（SMC）表型转换在血管生成中起重要作用。 在动脉粥样硬化发病机制中的作用。然而，对分子机制的了解仍然很少 在体内负责这些转变。最近，我们发现胚胎干细胞/诱导多能性 据信在体细胞中沉默的干细胞（iPSC）因子OCT 4发挥动脉粥样硬化保护作用 在SMC中，SMC中Oct 4基因失活导致病变大小和多个 ApoE/ApoE小鼠中的斑块不稳定性。虽然我们表明SMC迁移和投资需要OCT 4， 进入保护性纤维帽，我们也有证据表明，它的损失在SMC与SMC转型 向巨噬细胞（M β）样状态发展，包括M β标志物表达增加、脂质蓄积和 吞噬作用鉴于过度表达靶向iPSC因子的困难， 负责SMC中OCT 4激活的分子机制，可潜在地用于介导有益的 SMC-OCT 4依赖性效应。据报道，先天性免疫Toll样受体的激活 (TLR)/TRIF依赖性IRF 3/NFkB信号传导是有效重编程为iPSC以及在细胞内重编程所必需的。 通过驱动细胞的表观遗传可塑性转分化为其他细胞谱系。虽然机制和 先天免疫在动脉粥样硬化进展中的关键作用在免疫学中得到了很好的表征。 细胞，几乎没有什么是已知的TLRs的作用，在表型转变的SMC在动脉粥样硬化。 我们的数据表明，TLR 4信号转导负责Oct 4启动子的羟甲基化， SMC中Oct 4的再活化。有趣的是，SMC中的Oct 4缺陷反过来上调Tlr 4，并导致 两种经典的TLR 4下游机制的失调，包括MyD 88依赖性（NF κ B介导的 炎症反应）和TRIF依赖性（IRF 3/LXRa介导的胆固醇流出）。在 此外，对从小鼠动脉粥样硬化中分离的标本进行染色质免疫沉淀（ChIP）-seq分析 动脉将Tlr 4鉴定为OCT 4的推定靶点。总之，这些结果证明了TLR 4的重要性。 在OCT 4的上游和下游的SMC中的信令。我们的中心假设是TLR 4信号传导 通过反馈调节多能性因子OCT 4的水平介导SMC中的动脉粥样硬化保护性变化 机制为了检验这一假设，我们提出了以下目标：目标1将检验TLR 4 信号传导通过反馈机制调节SMC中OCT 4的水平。目标2将检验TLR 4 SMC中的信号传导具有动脉粥样硬化保护作用。本目标将利用遗传学和药理学方法来定义 TLR 4信号在动脉粥样硬化不同阶段SMC中的作用。目标3将检验这一假设： 在Oct 4缺陷的SMC中，TLR 4的调节促进SMC向平滑肌样状态转变。这些研究将 首次建立了SMC中先天免疫和多能性因子之间的机制联系 并提供TLR 4在SMC表型转变中的作用的见解。