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.

BACKGROUND: Smooth muscle cells (SMCs) substantially contribute to the development of atherosclerosis through a process called phenotypic switching. Our previous work identified an SMC-derived intermediate cell type, termed SEM cells, which plays a crucial role in SMC transition to other cell types and in lesion development. Activation of retinoic acid (RA) signaling by all-trans retinoic acid (ATRA) attenuates atherosclerosis in mice coincident with suppression of SEM cell formation. However, the effect of RA signaling on advanced disease and the underlying molecular mechanisms by which RA modulates SMC transition to SEM cells are largely unknown. METHODS: We applied SMC lineage tracing atheroprone mice and biochemistry and cell and molecular biology techniques (e.g., RNA sequencing, quantitative reverse transcription PCR, co-immunoprecipitation, and chromatin immunoprecipitation-quantitative PCR) to reveal the regulatory mechanisms of RA signaling in SMC transition to SEM cells. RESULTS: Activation of RA signaling with ATRA in established atherosclerosis significantly reduced SEM cells and lesion size while increasing fibrous cap thickness. Mechanistically, retinoic acid receptor alpha (RARa) directly targets the promoters of Ly6a and Ly6c1 in mouse SMCs, and activation of RA signaling recruits EZH2 to the regulatory elements triggering local H3K27me3. Distinct from a molecular model that reported for RA recruitment of HDAC1 during embryogenesis, RARa/EZH2 complex recruits SIRT1 and SIRT6, rather than classical HDACs, to the regulatory regions of key SEM cell marker genes. This subsequently reduces multiple acetylated histone modifications (e.g., H3K27ac, H3K4ac, H3K9ac, H3K14ac, H3K56ac) with recruitment of the transcription corepressor, NCOR1, to repress downstream SEM cell marker genes. CONCLUSIONS: Our findings provide novel mechanistic insights into RA modulating SMC phenotypic switching in atherosclerosis, suggesting molecular targets for preventive and therapeutic interventions for atherosclerosis and its clinical complications.

Atherosclerosis, the leading cause of cardiovascular disease, is a chronic inflammatory disease involving pathological activation of multiple cell types, such as immunocytes (e.g., macrophage, T cells), smooth muscle cells (SMCs), and endothelial cells. Multiple lines of evidence have suggested that SMC "phenotypic switching" plays a central role in atherosclerosis development and complications. Yet, SMC roles and mechanisms underlying the disease pathogenesis are poorly understood. Here, employing SMC lineage tracing mice, comprehensive molecular, cellular, histological, and computational profiling, coupled to genetic and pharmacological studies, we reveal that atherosclerosis, in terms of SMC behaviors, share extensive commonalities with tumors. SMC-derived cells in the disease show multiple characteristics of tumor cell biology, including genomic instability, replicative immortality, malignant proliferation, resistance to cell death, invasiveness, and activation of comprehensive cancer-associated gene regulatory networks. SMC-specific expression of oncogenic KrasG12D accelerates SMC phenotypic switching and exacerbates atherosclerosis. Moreover, we present a proof of concept showing that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and induces regression of lesions in advanced disease in mouse models. Our work provides systematic evidence that atherosclerosis is a tumor-like disease, deepening the understanding of its pathogenesis and opening prospects for novel precision molecular strategies to prevent and treat atherosclerotic cardiovascular disease.

Background: Smooth muscle cells (SMCs) substantially contribute to atherosclerosis through “phenotypic switching.” Our previous work identified an intermediate SMC-derived cell type, termed “SEM” cells, which was multipotent and activated in inflammatory response. Activation of retinoic acid (RA) signaling by all-trans retinoic acid (ATRA) attenuated atherosclerosis in mice coincident with dramatic suppression of SEM cell formation from SMCs. However, the regulatory mechanisms by which RA signaling modulates SMC transition to SEM cells are largely unknown. Methods: We employed molecular and cell biology techniques, SMC-linage tracing and atheroprone mouse models, and next-generation sequencing (e.g., RNA-seq, ChIP-seq) to reveal how RA signaling modulates SMC transition to SEM cells. Results: Activation of RA signaling with ATRA significantly reduced SEM cells in established atherosclerosis, as well as downregulated the expression of SEM cell marker genes (e.g., Ly6a , Ly6c1 ) and repressed inflammatory response in ex vivo SEM cells, whereas inhibition of the signaling with antagonist, BMS49334, showed opposite results. RARα occupied the promoter regions of SEM cell marker genes, and ATRA treatment significantly increased the enrichment of NCOR1 at promoters of these genes. These findings suggest that RA signaling suppresses SMC transition to SEM cells via directly repressing the expression of SEM cell marker genes. Furthermore, we found EZH2, one of the subunits of PRC2, physically interacted with RARα in SMCs and occupied the promoters of SEM cell marker genes, and its methyltransferase activity at the promoter regions was responsible for the repression of SEM cell maker genes. Moreover, activation of RA signaling inhibited SEM cell inflammatory response through LXR-mediated suppression of a series of inflammatory genes. Finally, multiple epigenetic signatures (e.g., H3K27me3, H3K27ac, H3K4me3, etc.) at TSS of SEM cell marker genes and inflammatory genes were extensively altered in response to the activation of RA signaling. Conclusions: Our findings indicate that RA signaling modulates maintenance of SEM cell identity and inflammatory function in atherosclerosis by epigenetic regulation of gene expression.