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

Abstract

Background-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 makes it possible to identify and characterize cellular subpopulations.Methods-We validate a method for generating a droplet-based single cell atlas of gene expression in a normal blood vessel. Enzymatic dissociation of four whole mouse aortas was followed by single cell sequencing of over 10,000 cells.Results-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 (VSMCs), 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 two 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.

Endothelial reprogramming by disturbed flow revealed by single-cell RNA and chromatin accessibility study

“…Current single-cell analysis has now addressed this limitation. We also compared our carotid endothelial gene expression data to the previously published endothelial heterogeneity data from murine ECs from various tissue types and arteries (Kalluri et al, 2019;Kalucka et al, 2020). While we found that most of the highly enriched genes (Fbln5,Mecom,Gja4,Azin1,Sox17,Mgp,Nrp1,Kcnj8,Cytl1,Bmx,Eln,Fbln2,Gkn3) found in the arteries were also present in all of our EC clusters showing no flow dependency.…”

“…Our UMAP analysis identified 16 unique cell clusters distributed in a flow-and timedependent manner ( Figure 1A and B). Each cell cluster was identified using well-established marker genes ( Figure 1C) (Cochain et al, 2018;Kalluri et al, 2019;Roberts et al, 2020;Winkels et al, 2018). We found 8 endothelial cell clusters (E1-E8), vascular smooth muscle cells (SMCs), fibroblasts (Fibro), 4 monocytes/macrophages (M1-4), dendritic cells (DCs) and T-cells.…”

“…After euthanizing the mice using CO2, the LCAs and RCAs were dissected, cleaned, and perfused with normal saline solution before a dissociation buffer was injected. The dissociation buffer was slightly modified from a previously reported recipe (Kalluri et al, 2019) by including 600 U ml -1 Type II Collagenase (02100502-CF, MP biomedicals, Irvine, CA) and 60 U ml -1 DNase I (M0303S, New England Biolabs Inc, Ipswich, MA) in 0.5 % fetal bovine serum (FBS) of 1X phosphatebuffered saline (PBS). Immediately after the introduction of dissociation buffer into the carotid lumens, the ends of the RCAs and LCAs were clamped and dissected out.…”

“…Since gene expression is regulated both at the transcriptional and epigenomic levels, the unprecedented power of carrying out concomitant and integrated analyses of scRNAseq and scATACseq data are increasingly evident. These new powerful tools have revealed the heterogeneity of cell types in the liver (Zhao et al, 2020), lungs (Domingo-Gonzalez et al, 2020), heart (Bykov et al, 2020), immune cells (Villani et al, 2017), and ECs (Kalluri et al, 2019;Kalucka et al, 2020) under various physiological and pathological conditions.…”

Disrupted Endothelial Cell Heterogeneity and Network Organization Impairs Vascular Function in Prediabetic Obesity

“…This arrangement allows the endothelium to detect and process multiple stimuli in parallel and to generate stimulusspecific responses [30][31][32][33] . These findings, together with studies that describe molecular [34][35][36][37][38][39][40][41] and functional heterogeneity 40,42 in endothelial cells across vascular beds, and within single vessel segments, raise the possibility that dysfunctional vascular responses could arise from altered endothelial cell heterogeneity and disrupted network dynamics. Indeed, altered mulitcellular network behavior underlies disease development in a variety of physiological systems.…”