Defining SMC phenotypes critical in late stage atherosclerosis pathogenesis

动脉粥样硬化是一种进行性疾病，在西方世界是导致死亡的主要原因。 值得注意的是，尽管进行了数十年的研究，但关于Smooth的作用仍然存在重大模糊 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 斑块破裂与可能的心肌梗死(MI)或中风的可能性。一般的教条是 SMC主要参与晚期而不是早期病变，它们的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涉及 干细胞多能性基因的SMC特异性谱系追踪和敲除(KO)的同步进行，Oct4 或者KLF4提供了令人信服的证据，表明SMC在病变发病中所起的作用比 得到了普遍的赞赏。我们的主要发现包括：1)80%的SMC来源的细胞 ApoE-/-小鼠的晚期病变缺乏典型的SMC标志物的表达；2)30%-40%的SMC- 晚期小鼠和人类病变中的来源细胞缺乏可检测到的SMC标志物，并具有 激活的MФ标志物S；3)SMC对病变具有重大的有利或不利影响 发病机制取决于其表型转变的性质。这项提案中的研究将测试 SMC表型转变可发挥主要的动脉粥样硬化保护作用或促进动脉粥样硬化的假说 对晚期病变发病机制的影响及治疗晚期病变的新靶点 动脉硬化。目标1将检验以下假设：SMC表型和功能中依赖KLF4的转换 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 基于互补流的KLF4 SMC特异性条件性KO诱导的动脉粥样硬化保护表型 细胞学和高分辨率共聚焦分析来自我们体内RNAseq和 晚期头臂(BCA)病变的KLF4/Oct4 ChIPseq基因组分析及其与基因的交叉参照 与人类冠心病风险增加有关。我们还将确定KLF4的延迟SMC特定条件KO是否在 晚期病变的建立也会导致病变发病机制的有利变化。目标2将测试 SMC表型和功能依赖于Oct4的转变通过以下途径促进斑块稳定 增加SMC对保护性纤维帽的投资，减少其向促炎因子的转变 州政府。我们还将测试在我们的小鼠研究中发现的SMC表型是否也出现在人类皮损中，并确定 哪些表型与稳定和不稳定的皮损相关。目标3将确定SMC是否发生表型变化 晚期病变内是可逆的，如果治疗SMC谱系追踪患有晚期病变的小鼠 PCSK9抑制剂促进SMC表型和/或整体病变发病机制的有益改变。研究可能 导致确定减少晚期临床并发症的新治疗方法 通过诱导SMC表型和功能发生有益的(稳定斑块)改变而导致动脉粥样硬化。

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.