Defining SMC phenotypes critical in late stage atherosclerosis pathogenesis

动脉粥样硬化是一种进行性疾病，是西方世界死亡的主要原因。 值得注意的是，尽管经过了几十年的研究，关于平滑的作用仍然存在很大的模糊性。 肌细胞（SMC）在病变发病机制中的作用，以及控制斑块稳定性和 斑块破裂的可能性，可能伴有心肌梗死（MI）或卒中。一般的教条是， SMC主要参与晚期而非早期病变，其主要作用是动脉粥样硬化保护 通过促进纤维帽的形成。然而，我们实验室最近的自然医学研究， 同时SMC特异性谱系追踪和干细胞多能性基因敲除（KO），Oct 4 或Klf 4提供了令人信服的证据，表明SMC在病变发病机制中发挥的作用比 得到普遍赞赏。关键发现包括我们的研究结果：1）>80%的SMC来源的细胞在 ApoE-/-小鼠的晚期病变缺乏典型SMC标志物的可检测表达; 2）30-40%的SMC- 晚期小鼠和人类病变中的衍生细胞缺乏可检测的SMC标记物， 活化的M β s标志物; 3）SMC可对病变产生主要的有益或有害影响， 发病机制取决于其表型转变的性质。本提案中的研究将测试 假设SMC表型转变可以发挥显性动脉粥样硬化保护或动脉粥样硬化促进作用， 对晚期病变发病机制的影响，并代表了治疗晚期病变的新治疗靶点。 动脉粥样硬化目的1将检验SMC表型和功能中Klf 4依赖性转变的假设， 在动脉粥样硬化的早期和晚期加重病变的发病机制，并将包括定义不同的 SMC特异性Klf 4条件性敲除诱导SMC表型保护作用的研究 对源自我们的体内RNAseq的独特标志物组进行细胞计数和高分辨率共聚焦分析， 晚期头臂型（BCA）病变的Klf 4/Oct 4 ChIPseq基因组分析和交叉参考基因 与人类CAD风险增加有关。我们还将确定Klf 4的延迟SMC特异性条件性KO是否在 晚期病变的建立也引起病变发病机制的有利变化。目标2将测试 假设SMC表型和功能中Oct 4依赖性转变通过以下方式促进斑块稳定 增强SMC向保护性纤维帽的投资，并减少其向促炎性细胞的转化， 状态我们还将测试在我们的小鼠研究中鉴定的SMC表型是否也发生在人类病变中，并确定 这些表型与稳定病变和不稳定病变相关。目的3将确定SMC表型是否发生变化 在晚期病变中，如果治疗晚期病变的SMC谱系示踪小鼠， PCSK 9抑制剂促进SMC表型和/或整体病变发病机制的有益变化。研究可能 导致识别用于减少晚期临床并发症的新的治疗方法， 动脉粥样硬化通过诱导SMC表型和功能的有益（斑块稳定）变化。

Atherosclerosis is a progressive disease that is a leading cause of death in the Western world. Remarkably, despite decades of research, there remain major ambiguities regarding the role of smooth muscle cells (SMC) in lesion pathogenesis, as well as mechanisms that control plaque stability and the probability of plaque rupture with possible myocardial infarction (MI) or stroke. The general dogma is that SMC are primarily involved in late not early stage lesions, and that their primary role is atheroprotective by contributing to formation of a fibrous cap. However, recent Nature Medicine studies by our lab involving simultaneous SMC-specific lineage tracing and knockout (KO) of the stem cell pluripotency genes, Oct4 or Klf4 provided compelling evidence that SMC play a much greater role in lesion pathogenesis than has been generally appreciated. Key findings included our showing that: 1) >80% of SMC-derived cells within advanced lesions of ApoE-/- mice lack detectable expression of typical SMC markers; 2) 30-40% of SMC- derived cells within both advanced mouse and human lesions lack detectable SMC markers and have activated markers of MФs; and 3) SMC can have major beneficial or detrimental effects on lesion pathogenesis depending on the nature of their phenotypic transitions. Studies in this proposal will test the hypothesis that SMC phenotypic transitions can exert dominant atheroprotective or atheropromoting effects on late stage lesion pathogenesis and represent a novel therapeutic target for treating advanced atherosclerosis. Aim 1 will test the hypothesis that Klf4-dependent transitions in SMC phenotype and function exacerbate lesion pathogenesis in early and late stages of atherosclerosis, and will include defining distinct atheroprotective SMC phenotypes induced by SMC-specific conditional KO of Klf4 based on complementary flow cytometric and high resolution confocal analyses of unique marker panels derived from our in vivo RNAseq and Klf4/Oct4 ChIPseq genomic analyses of advanced brachiocephalic (BCA) lesions and cross referenced to genes linked to increased human CAD risk. We will also determine if delayed SMC specific conditional KO of Klf4 after establishment of advanced lesions also induces favorable changes in lesion pathogenesis. Aim 2 will test the hypothesis that Oct4-dependent transitions in SMC phenotype and function promote plaque stabilization by enhancing investment of SMC into a protective fibrous cap and reducing their transition to a pro-inflammatory state. We will also test if SMC phenotypes identified in our mouse studies also occur in human lesions and define which phenotypes correlate with stable versus unstable lesions. Aim 3 will determine if SMC phenotypic changes within advanced lesions are reversible, and if treatment SMC lineage tracing mice with advanced lesions with a PCSK9 inhibitor promotes beneficial changes in SMC phenotype and/or overall lesion pathogenesis. Studies may lead to identification of novel therapeutic approaches for reducing late stage clinical complications of atherosclerosis by inducing beneficial (plaque stabilizing) changes in SMC phenotype and function.