Single-Cell Analysis of the Normal Mouse Aorta Reveals Functionally Distinct Endothelial Cell Populations. Single-Cell Analysis of the Normal Mouse Aorta Reveals Functionally Distinct Endothelial Cell Populations

The cells that form the arterial wall contribute to multiple vascular diseases. The extent of cellular heterogeneity within these populations has not been fully characterized. Recent advances in single-cell RNA-sequencing make it possible to identify and characterize cellular subpopulations. Clustering analysis of gene expression from aortic cells identified 10 populations of cells representing each of the main arterial cell types: fibroblasts, vascular smooth muscle cells, endothelial cells (ECs), and immune cells, including monocytes, macrophages, and lymphocytes. The most significant cellular heterogeneity was seen in the 3 distinct EC populations. Gene set enrichment analysis of these EC subpopulations identified a lymphatic EC cluster and 2 other populations more specialized in lipoprotein handling, angiogenesis, and extracellular matrix production. These subpopulations persist and exhibit similar changes in gene expression in response to a Western diet. Immunofluorescence for Vcam1 and Cd36 demonstrates regional heterogeneity in EC populations throughout the aorta. We present a comprehensive single-cell atlas of all cells in the aorta. By integrating expression from >1900 genes per cell, we are better able to characterize cellular heterogeneity compared with conventional approaches. Gene expression signatures identify cell subpopulations with vascular disease-relevant functions. Overall design: We validate a method for generating a droplet-based single-cell atlas of gene expression in a normal blood vessel. Enzymatic dissociation of 4 whole mouse aortas was followed by single-cell sequencing.

构成动脉管壁的细胞参与多种血管疾病的发生发展。这类细胞群体内部的细胞异质性程度尚未得到完全阐明。近年来，单细胞RNA测序（single-cell RNA-sequencing）技术的进步使得鉴定并表征细胞亚群成为可能。对主动脉细胞的基因表达开展聚类分析，共鉴定出10类细胞群体，涵盖动脉壁的主要细胞类型：成纤维细胞、血管平滑肌细胞、内皮细胞（endothelial cells，ECs），以及单核细胞、巨噬细胞、淋巴细胞等免疫细胞。其中细胞异质性最为显著的是3个不同的内皮细胞亚群。对这些内皮细胞亚群进行基因集富集分析（Gene Set Enrichment Analysis），鉴定出1个淋巴内皮细胞簇，以及另外2个分别专长于脂蛋白代谢、血管生成及细胞外基质生成的亚群。这些亚群在小鼠接受西式饮食后仍稳定存在，且基因表达模式发生了相似的改变。针对血管细胞黏附分子1（Vcam1）与CD36的免疫荧光实验证实，整个主动脉的内皮细胞亚群存在区域异质性。本研究构建了主动脉内所有细胞的全面单细胞图谱。通过整合每个细胞中超过1900个基因的表达数据，相较于传统实验方法，本研究能够更精准地表征细胞异质性。基因表达特征可用于鉴定具有血管疾病相关功能的细胞亚群。总体实验设计：本研究验证了一种可构建正常血管基因表达液滴型单细胞图谱的方法。研究首先通过酶解法解离4个完整的小鼠主动脉，随后开展单细胞测序。