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

项目总结/摘要 动脉粥样硬化是一种复杂的炎症性疾病，是世界范围内发病率和死亡率的主要原因， 目前的治疗不能充分满足临床需要。直到最近，大多数动脉粥样硬化研究都集中在 对内皮激活、免疫细胞募集和内膜内脂质滞留的影响，但新出现的证据表明， 提示血管外膜参与动脉粥样硬化的发病机制。具体而言，有人建议， 血管外膜微血管（vasavasorum，VV）的扩张通过促进动脉粥样硬化的进展而驱动动脉粥样硬化的进展。 炎性细胞浸润到血管壁并发展成动脉粥样化。该项目的重点是角色， 平滑肌来源的血管祖细胞，称为AdvSca 1-SM细胞，在血管扩张中发挥作用 和斑块新血管形成。我们的实验室先前将AdvSca 1-SM细胞鉴定为常驻血管内皮细胞的亚群。 在Klf 4依赖的过程中，祖细胞从成熟平滑肌细胞原位重编程。这些细胞 富含hedgehog/Wnt/β-连环蛋白途径中的基因，特别是Gli 1，并且具有多谱系 分化潜能在急性血管损伤的情况下，AdvSca 1-SM细胞强烈扩增， 纤维化重塑，但目前还不清楚它们在慢性损伤如动脉粥样硬化中发挥什么作用。工作从 几个研究小组认为外膜祖细胞可以促进VV扩张，我们的研究小组认为， 先前已经证明，AdvSca 1-SM细胞能够通过内皮细胞或血管内皮细胞重新形成血管。 基质胶塞测定中的平滑肌细胞分化。此外，我们还鉴定了AdvSca 1-SM样细胞， 在人冠状动脉周围的微血管中，表明在VV中的潜在作用。的目的 该项目旨在确定AdvSca 1-SM细胞对VV扩增、斑块新生血管形成和 动脉粥样硬化进展。我们假设AdvSca 1-SM细胞是动脉粥样硬化的主要贡献者， 并且这些细胞的调节可以改变疾病的进展。具体目标1将定义功能 使用高度特异性的AdvSca 1-SM细胞对VV扩增和/或斑块进展的贡献， SM遗传谱系追踪小鼠模型;将使用免疫荧光法评估AdvSca 1-SM贡献 显微镜和流式细胞术。具体目标2将定义控制AdvSca 1-SM细胞的机制， 动脉粥样硬化的背景下，重点是Gli/Wnt/β-catenin/Klf 4信号轴先前确定为 急性血管损伤背景下AdvSca 1-SM细胞的调节剂。我们将使用 AdvSca 1-SM特异性Gli 1过表达或Klf 4敲除;这些修饰对AdvSca 1-SM的影响 维持或分化将使用免疫荧光显微镜、流式细胞术和单克隆抗体进行评估。 细胞RNA测序最终，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AdvSca 1-SM细胞在动脉粥样硬化疾病过程中所起作用的知识缺口。

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.