Macrophage extracellular traps originated from the vascular smooth muscle cells play key roles in atherosclerosis progression [scRNA-seq]

Smooth muscle cells (SMC) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC transdifferentiation into macrophage-like SMCs. Furthermore, during transdifferentiation, the SMCs' expression of PADI4 upregulating and SMC generated extracellular trap, a web-like structure, which plays key role in the development of atherosclerosis plaque. To reveal the function of SMC's generating ETs during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of mouse atherosclerotic plaques. SMC-lineage tracing mice, B6-G/R; Myh11-CreERT2;Padi4(flox/flox) mice, were crossed onto B6-G/R Myh11cre group as control group and B6-G/R Myh11cre Padi4flox/flox group as case group to specific knock out the Padi4 within SMC. Mouse single cells digested from arterial tissues (including ascending aorta, brachiocephalic artery and thoracic aorta) for scRNA-seq were prepared from six mice of each group at the same timepoint of Western diet (WD) feeding (26 weeks). Alive tdTomato+ cells (SMC-lineage cells) and ZsGreen cells (none-SMC linage cells) were sorted separately and were immediately subjected to scRNA-seq using Chromium Single Cell Gene Expression system (10x Genomics) in parallel.

平滑肌细胞（Smooth muscle cells, SMC）通过表型转换在动脉粥样硬化进程中发挥关键作用。表型转换是一类病理过程，在此过程中SMC转分化为巨噬细胞样平滑肌细胞。此外，在转分化进程中，SMC的PADI4表达会上调，且SMC会生成细胞外陷阱（extracellular trap, ET）——一种网状结构，该结构在动脉粥样硬化斑块的发生发展中发挥核心作用。为阐明SMC生成ET在动脉粥样硬化中的功能，并筛选疾病治疗的潜在分子靶点，本研究结合了SMC命运图谱分析与小鼠动脉粥样硬化斑块的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。我们将SMC谱系示踪小鼠B6-G/R; Myh11-CreERT2;Padi4(flox/flox)分别与B6-G/R Myh11cre组（作为对照组）以及B6-G/R Myh11cre Padi4flox/flox组（作为实验组，用于特异性敲除SMC内的Padi4基因）进行杂交。实验样本采集自每组6只小鼠，所有小鼠均在西式饮食（Western diet, WD）喂养26周的同一时间点取材，从其动脉组织（包括升主动脉、头臂干动脉和胸主动脉）中消化分离得到单细胞用于scRNA-seq。我们分别分选存活的tdTomato阳性（tdTomato+）细胞（即SMC谱系细胞）与ZsGreen阳性（ZsGreen+）细胞（即非SMC谱系细胞），随后立即使用Chromium单细胞基因表达系统（10x Genomics）同步开展scRNA-seq实验。