High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis

Abstract

Background: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, we have limited understanding of the comprehensive transcriptional and phenotypical landscape of the cells within these lesions. Methods: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. Results: We identified 25 distinct cell populations each having a unique multi-omic signature, including macrophages, T cells, NK cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Within the macrophage populations, we identified 2 proinflammatory subsets that were enriched in IL1B or C1Q expression, 2 distinct TREM2 positive foam cell subsets, one of which also expressed inflammatory genes, as well as subpopulations displaying a proliferative gene expression signature and one expressing SMC-specific genes and upregulation of fibrotic pathways. An in-depth characterization uncovered several subsets of SMCs and fibroblasts, including a SMC-derived foam cell. We localized this foamy SMC to the deep intima of coronary atherosclerotic lesions. Using CITE-seq data, we also developed the first flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Last, we found that the proportion of efferocytotic macrophages, classically activated endothelial cells, contractile and modulated SMC-derived cell types were reduced, and inflammatory SMCs were enriched in plaques of clinically symptomatic vs. asymptomatic patients. Conclusions: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. This facilitates both the mapping of cardiovascular disease susceptibility loci to specific cell types as well as the identification of novel molecular and cellular therapeutic targets for treatment of the disease.

Single-Cell Multimodal Profiling of Atherosclerosis Identifies CD200 as a Cell Surface Lineage Marker of Vascular Smooth Muscle Cells and Their Derived Cells

“…2 Therefore, these data support that CD45 − , CD31 − , CD200 + cells can identify VSMC and VSMC-derived cells extremely effectively and that the expression of CD200 is maintained on VSMC-derived cells throughout atherosclerosis. In support of clinical relevance and translational utility, we corroborated these findings in human atherosclerosis with CITE-seq data from a previously reported carotid endarterectomy data set and observed a similar pattern (Figure [I]), 3 and there was an increase in expression within symptomatic plaques (Figure [J]). We also performed immunohistochemistry on human coronary arteries to localize CD200-expressing cells.…”

Single-cell multimodal profiling of atherosclerosis identifies CD200 as a cell surface lineage marker of vascular smooth muscle cells and their derived cells

“…These data support that CD45-, CD31-, CD200+ cells can identify VSMC and VSMC-derived cells extremely effectively and that the expression of CD200 is maintained on VSMCs and VSMC-derived cells throughout atherosclerosis. In support of clinical relevance and translational utility, we corroborated these findings in human atherosclerosis with CITE-seq data from a previously reported carotid endarterectomy dataset and observed a similar pattern ( Fig [I] ) [3]. We also performed immunohistochemistry on human coronary arteries to localize CD200-expressing cells within atherosclerotic lesions.…”

ADAMTS7 Promotes Smooth Muscle Cell Foam Cell Expansion in Atherosclerosis

“…Despite the substantial body of literature connecting SMCs with ADAMTS7, our SMC conditional knockout mouse did not reveal a change in peripheral atherosclerosis 7 . Our analysis of the most extensive carotid single-cell data set to date highlights that SMCs and other cell types, including ECs and mast cells, all express ADAMTS7 18 . As ADAMTS7 is a secreted protein, the cells that respond to ADAMTS7 do not necessarily have to produce ADAMTS7 .…”

“…Given the clear role of Adamts7 in modulating SMC function during atherogenesis in mice, we next sought to determine if human atherosclerotic SMCs express ADAMTS7 . We interrogated the largest human atherosclerotic carotid artery scRNA-seq dataset generated to date 18 and found to our surprise that the strongest ADAMTS7 expression is in endothelial cells (ECs). There was also ADAMTS7 expression in SMCs, fibroblasts, and mast cells, but the expression in these cells was much lower than that of ECs (Figure 5A).…”