Role of Smooth Muscle-derived Vascular Progenitor Cells (AdvSca1-SM) in Vasa Vasorum Expansion and Atherosclerotic Plaque Progression

项目摘要/摘要 动脉粥样硬化是一种复杂的炎症性疾病，是全球发病率和死亡率的主要原因，但 目前的治疗方法不能充分满足临床需求。直到最近，大多数动脉粥样硬化的研究都集中在 关于血管内皮细胞激活、免疫细胞募集和内膜内脂质滞留，但正在出现的证据 提示血管外膜参与动脉粥样硬化的发病机制。具体地说，有人建议 外膜微血管的扩张，血管(VV)，通过促进动脉粥样硬化的进展 炎性细胞渗入血管壁，形成动脉粥样硬化。本项目关注的角色是 平滑肌来源的血管前体细胞，称为AdvSca1-SM细胞，在血管扩张中发挥作用 和斑块新生血管。我们的实验室之前发现AdvSca1-SM细胞是驻留血管的一个子集 在KLF4依赖的过程中，由成熟的平滑肌细胞在原位重新编程的前体细胞。这些细胞 在Hedgehog/Wnt/β-Catenin通路中富含基因，特别是Gli1，并具有多谱系 分化潜力。在急性血管损伤的背景下，AdvSca1-SM细胞强劲扩张做出贡献 与纤维化重塑有关，但目前尚不清楚它们在动脉粥样硬化等慢性损伤中起什么作用。工作地点： 几个小组已经提出外膜祖细胞可以促进VV的扩张，而我们的小组 已经证明AdvSca1-SM细胞能够通过内皮或血管内皮细胞形成新生血管 Matrigel Plug实验中的平滑肌细胞分化。此外，我们还鉴定了AdvSca1-SM样细胞 在人冠状动脉周围的微血管中，提示在室上性心动过速中可能起作用。这样做的目的是 项目是确定AdvSca1-SM细胞在VV扩张、斑块新生血管和 动脉粥样硬化进展。我们假设AdvSca1-SM细胞是动脉粥样硬化的主要贡献者， 而这些细胞的调节可能会改变疾病的进展。具体目标1将定义功能 利用高度特异的AdvSca1-SM细胞在VV扩张和/或斑块进展中的作用 SM遗传谱系追踪小鼠模型；AdvSca1-SM的贡献将使用免疫荧光进行评估 显微镜和流式细胞术。特定目标2将定义控制AdvSca1-SM细胞在 动脉粥样硬化的背景，重点是Gli/Wnt/β-Catenin/Klf4信号轴 急性血管损伤时AdvSca1-SM细胞的调节作用我们将使用以下工具来调制此信号轴 AdvSca1-SM特异性Gli1过表达或Klf4基因敲除；这些修饰对AdvSca1-SM的影响 维持或分化将使用免疫荧光显微镜、流式细胞术和单个 细胞RNA序列归根结底，这里提出的工作代表了一种新的研究途径，将有助于填补 目前关于AdvSca1-SM细胞在动脉粥样硬化疾病过程中所起作用的知识空白。

PROJECT SUMMARY/ABSTRACT Atherosclerosis is a complex inflammatory disease and a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Until recently, most atherosclerosis research has focused on endothelial activation, immune cell recruitment, and lipid retention within the intima, but emerging evidence implicates the vessel adventitia in the pathogenesis of atherosclerosis. Specifically, it has been suggested that expansion of adventitial microvessels, the vasa vasorum (VV), drives atherosclerosis progression by facilitating inflammatory cell infiltration to the vessel wall and developing atheroma. This project focuses on the role that smooth muscle-derived vascular progenitor cells, termed AdvSca1-SM cells, play in vasa vasorum expansion and plaque neovascularization. Our lab previously identified AdvSca1-SM cells as a subset of resident vascular progenitor cells reprogrammed from mature smooth muscle cells in situ in a Klf4 depended process. These cells are enriched for genes in the hedgehog/Wnt/β-catenin pathways, particularly Gli1, and have multilineage differentiation potential. In the setting of acute vascular injury, AdvSca1-SM cells expand robustly to contribute to fibrotic remodeling, but it is unclear what role they play in chronic injuries such as atherosclerosis. Work from several groups has suggested that adventitial progenitor cells can contribute to VV expansion, and our group has previously demonstrated that AdvSca1-SM cells are capable of de novo vessel formation via endothelial or smooth muscle cell differentiation in Matrigel plug assays. Additionally, we have identified AdvSca1-SM-like cells in the microvessels surrounding human coronary arteries, suggesting a potential role in the VV. The aim of this project is to determine the contribution of AdvSca1-SM cells to VV expansion, plaque neovascularization, and atherosclerotic progression. We hypothesize that AdvSca1-SM cells are major contributors to atherosclerosis, and that modulation of these cells could alter disease progression. Specific Aim 1 will define the functional contribution of AdvSca1-SM cells to VV expansion and/or plaque progression using a highly specific AdvSca1- SM genetic lineage tracing mouse model; AdvSca1-SM contribution will be assessed using immunofluorescent microscopy and flow cytometry. Specific Aim 2 will define the mechanisms controlling AdvSca1-SM cells in the setting of atherosclerosis, with an emphasis on the Gli/Wnt/β-catenin/Klf4 signaling axis previously identified as a regulator of AdvSca1-SM cells in the setting of acute vascular injury. We will modulate this signaling axis using AdvSca1-SM-specific Gli1 overexpression or Klf4 knockout; the effects of these modifications on AdvSca1-SM maintenance or differentiation will be assessed using immunofluorescent microscopy, flow cytometry, and single cell RNA-seq. Ultimately, the work proposed here represents a novel avenue of research that will help fill the current knowledge gap regarding the role AdvSca1-SM cells play in the atherosclerotic disease process.