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)的表型转变在 在动脉粥样硬化发病机制中的作用。然而，对其分子机制仍知之甚少。 负责体内的这些转变。最近，我们发现胚胎干细胞/诱导的多能性 干细胞(IPSC)因子OCT4被认为是在体细胞中沉默的，它发挥着动脉粥样硬化的保护作用 在SMC中，在SMC中Oct4基因失活导致SMC的病变大小和多项指数显著增加 APOE-/-小鼠的斑块不稳定性。虽然我们展示了SMC迁移和投资需要OCT4 进入保护性纤维帽，我们也有证据表明它在SMC中的丢失与SMC的转变有关 走向巨噬细胞(MФ)样状态，包括MФ标志物表达增加，脂质堆积，以及 吞噬作用。鉴于过度表达靶向IPSC因子的困难，确定内源性是至关重要的 SMC中负责OCT4激活的分子机制可能被用来介导有益的 SMC-OCT4依赖效应。据报道，先天免疫Toll样受体的激活 (TLR)/TRIF依赖的IRF3/NFkB信号对于高效地重新编程到IPSC以及在细胞中都是必需的 通过驱动细胞的表观遗传可塑性向其他细胞系的转分化。虽然机构和 天然免疫在动脉粥样硬化进展中的关键作用在免疫中有很好的特征 对于TLRs在动脉粥样硬化中SMC表型转变中的作用，我们几乎一无所知。 我们的数据表明TLR4信号负责Oct4启动子的羟甲基化和 SMC中Oct4的重新激活。有趣的是，SMC中Oct4缺乏反过来上调TLR4，并导致 两种经典的TLR4下游机制调节失调，包括MyD88依赖(NFkB介导 炎症反应)和TRIF依赖(IRF3/LXRa介导的胆固醇外流)。在……里面 染色质免疫沉淀(CHIP)-序列分析在小鼠动脉粥样硬化中的应用 动脉发现TLR4可能是OCT4的靶点。综上所述，这些结果证明了TLR4- OCT4上游和下游的SMC中的信令。我们的中心假设是TLR4信号 通过反馈调节多能因子OCT4水平介导SMC的动脉粥样硬化保护变化 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TLR4的假设 信号通过反馈机制调节SMC中OCT4的水平。目标2将检验TLR4的假设 SMC中的信号是抗动脉粥样硬化的。这一目标将利用遗传学和药理学方法来定义 TLR4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，即上行- 在Oct4缺陷的SMC中，TLR4的调节促进SMC向МФ样状态转变。这些研究将 首次建立SMC天然免疫与多能性因子之间的机制联系 并深入了解TLR4在SMC表型转变中的作用。