Primed smooth muscle cells acting as first responder cells in disease. Primed smooth muscle cells acting as first responder cells in disease

Vascular smooth muscle cell (VSMC) dysregulation is a hallmark of vascular disease, including atherosclerosis. In particular, the majority of cells within atherosclerotic lesions are generated from pre-existing VSMCs and a clonal nature has been documented for VSMC-derived cells in multiple disease models. However, the mechanisms underlying the generation of oligoclonal lesions and the phenotype of proliferating VSMCs are unknown.Here we analyse clonal dynamics in multi-color lineage-traced animals over time after vessel injury to understand the cellular mechanisms underlying clonal VSMC expansion in disease.We demonstrate that VSMC proliferation is initiated in a small fraction of VSMCs that initially expand clonally in the medial layer and then migrate to form the oligoclonal neointima. Selective activation of VSMC proliferation also occurs in vitro, suggesting that this is a cell-autonomous feature. Mapping of VSMC trajectories using single-cell RNA-sequencing reveals a continuum of cellular states after injury and suggests that VSMC proliferation initiates in cells that have downregulated the contractile phenotype and show evidence of pronounced phenotypic switching. We show that proliferation is associated with induced expression of stem cell antigen 1 (SCA1) and the expression signature previously identified in SCA1+ VSMCs in healthy arteries. A remarkably increased proliferation of SCA1+ VSMCs, directly validated in functional assays, indicates that SCA1+ VSMCs act as "first responders" in vascular injury. Early atherosclerotic lesions also had clonal VSMC contribution and we show that the proliferation-associated injury response is conserved in plaque VSMCs, extending these findings to atherosclerosis. Finally, we identify VSMCs in healthy human arteries that correspond to the SCA1+ state in mouse VSMCs and show that genes identified as differentially expressed in this human VSMC subpopulation are enriched for genes showing genetic association with cardiovascular disease. We show that cell-intrinsic, selective VSMC activation drives clonal proliferation after injury and in atherosclerosis. Our study suggests that healthy mouse and human arteries contain VSMCs characterised by expression of disease-associated genes that are predisposed for proliferation. Targeting such "first responder" cells in patients undergoing vascular surgery could effectively prevent injury-associated VSMC activation and neoatherosclerosis. Overall design: This entry contains data from the following analyses: (1) 10X single-cell RNA-seq analysis of VSMC-lineage labeled cells isolated by flow-assisted cell sorting (FACS) from ligated left carotid arteries of Myh11-EYFP-Ki67/RFP animals 5 days after surgery. (2) 10X single-cell RNA-seq analysis of human medial cells isolated from a healthy aorta (65-year-old male). (3) 10X single-cell RNA-seq analysis of VSMC-lineage labeled cells isolated by flow-assisted cell sorting (FACS) from ligated left carotid arteries of Myh11-EYFP-Ki67/RFP animals 7 days after surgery. (4) Single-cell RNA-seq of index-sorted cells from Myh11-EYFP-Ki67/RFP animals 7 days after surgery and control unligated animals. (5) 10X single-cell RNA-seq analysis of VSMC-lineage labeled cells and other arterial cells generated 5 days after surgery.

血管平滑肌细胞（vascular smooth muscle cell, VSMC）功能异常是包括动脉粥样硬化在内的血管疾病的标志性特征。具体而言，动脉粥样硬化病灶内的绝大多数细胞均由预先存在的VSMC分化而来，且在多种疾病模型中，VSMC来源的细胞已被证实具有克隆特性。然而，寡克隆病灶的形成机制以及增殖性VSMC的表型特征仍未明确。

本研究通过对血管损伤后不同时间点的多色谱系示踪动物模型进行克隆动力学分析，旨在阐明疾病中VSMC克隆扩增的细胞机制。研究发现，VSMC增殖起始于少量VSMC亚群：这些细胞最初在血管中膜层发生克隆性扩增，随后迁移至内膜层形成寡克隆新内膜。VSMC增殖的选择性激活同样可在体外实验中观测到，提示这一过程是细胞自主特性的体现。

通过单细胞RNA测序（single-cell RNA-sequencing）对VSMC轨迹进行绘制，结果显示损伤后细胞状态呈连续过渡状态，且VSMC增殖起始于收缩表型已下调、表现出显著表型转换特征的细胞亚群。研究表明，VSMC增殖与干细胞抗原1（stem cell antigen 1, SCA1）的诱导表达以及健康动脉中SCA1阳性VSMC此前已被鉴定的表达特征密切相关。功能实验直接证实，SCA1阳性VSMC的增殖能力显著增强，提示该亚群在血管损伤过程中发挥“第一响应者”的作用。

早期动脉粥样硬化病灶同样存在VSMC的克隆性贡献，且研究证实增殖相关的损伤应答在斑块VSMC中保守存在，从而将本研究发现拓展至动脉粥样硬化疾病场景。最后，本研究在健康人类动脉中鉴定出与小鼠SCA1阳性VSMC状态相对应的VSMC亚群，并发现该人类VSMC亚群中差异表达的基因显著富集于与心血管疾病存在遗传关联的基因集。

研究证实，细胞内在的选择性VSMC激活可驱动损伤及动脉粥样硬化过程中的克隆性增殖。本研究提示，健康小鼠及人类动脉中存在一类以疾病相关基因表达为特征的VSMC亚群，该亚群易于发生增殖。针对血管手术患者体内的这类“第一响应者”细胞进行靶向干预，可有效预防损伤相关的VSMC激活及新动脉粥样硬化形成。

整体实验设计：本数据集包含以下分析产生的数据：
1. 对术后5天的Myh11-EYFP-Ki67/RFP小鼠结扎左侧颈总动脉后，通过流式细胞分选（flow-assisted cell sorting, FACS）分离得到的VSMC谱系标记细胞进行10X单细胞RNA测序分析；
2. 对从一名65岁男性健康主动脉中分离得到的人类中膜细胞进行10X单细胞RNA测序分析；
3. 对术后7天的Myh11-EYFP-Ki67/RFP小鼠结扎左侧颈总动脉后，通过流式细胞分选（FACS）分离得到的VSMC谱系标记细胞进行10X单细胞RNA测序分析；
4. 对术后7天的Myh11-EYFP-Ki67/RFP小鼠及未结扎对照小鼠的指数分选细胞进行单细胞RNA测序分析；
5. 对术后5天生成的VSMC谱系标记细胞及其他动脉细胞进行10X单细胞RNA测序分析。