Single-Cell Genomics Reveals a Novel Cell State During Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human

Abstract

Background: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive.

Methods: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro.

Results: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability.

Conclusions: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

Keywords: atherosclerosis; cardiovascular disease; retinoic acid signaling; single-cell RNA sequencing; smooth muscle cell.

Figure 1.. scRNA-seq identified multiple SMC-derived cell types/states, especially SEM cell, during atherosclerosis.

A, Schematic of scRNA-seq using 10x Chromium with both ZsGreen1⁺ and ZsGreen1⁻ cells from aortas of mice fed with Western diet (WD) for various timepoints (0, 8, 16, 26 weeks). ROSA26^ZsGreen1/+; Ldlr^−/−; Myh11-CreER^T2 mice at 7.5-week old are induced with tamoxifen (TAM) for 2 days, followed by chow diet. Afterwards, mice at 8-week old are fed WD for various timepoints as indicated. Arterial tissues (including ascending aorta, BCA and thoracic aorta) with atherosclerotic lesions are isolated and digested to single cells for fluorescence activated cell sorting (FACS) of ZsGreen1⁺ and ZsGreen1⁻ cells. Single cells are subsequently loaded to 10x Chromium for scRNA-seq. B-D, UMAP visualization of all scRNA-seq data from ROSA26^ZsGreen1/+; Ldlr^−/−; Myh11-CreER^T2 mice, including both ZsGreen1⁺ and ZsGreen1⁻ cells. For combined data of all timepoints (0, 8, 16, 26 weeks), representative cell type/state for each cluster (B) and ZsGreen1 status (ZsGreen1⁺ and ZsGreen1⁻) of cell clusters (C) are indicated. For each timepoint, representative cell types/states for cell clusters stratified by ZsGreen1 status are shown in D. SMC, smooth muscle cell; ICS, intermediate cell state, which is afterwards termed “SEM” cell; FC, fibrochondrocyte; EC, endothelial cell. E, Heatmap showing top 100 upregulated and top 100 downregulated DEGs in SEM cells versus SMC identified in ZsGreen1⁺ scRNA-seq data of Ldlr^−/− mice fed 16-week WD. SEM cell markers (Vcam1, Ly6a, Ly6c1) (green), FC-related genes (pink), complement and inflammation-related genes (orange) and SMC markers (Myh11, Cnn1, Acta2) (blue) are indicated. F-H, Expression levels of Vcam1 (F), Ly6a (G) and Ly6c1 (H) in each ZsGreen1⁺ cell type/state are indicated by color scales.

Figure 2.. SMC-derived intermediate SEM cells exhibit complex molecular and cellular features.

A and B, Gene expression tendencies of SMC marker, Myh11 (A) and FC-related gene, Fn1 (B) through SMC-SEM-FC axis shown as normalized expression versus UMAP_1. C, GO pathway analysis of DEGs (n = 149 upregulated and 159 downregulated genes in SEM cells, fold change ≥ 1.5, Bonferroni corrected P-value < 0.05) upregulated (pink) or downregulated (blue) in SEM cells versus SMC. D, IPA analysis of DEGs (the same with those in C) upregulated and downregulated in SEM cells versus SMC. Top 20 ranking “Diseases and Biological Functions” significantly altered in SEM cells from SMC are shown. E, RNAscope assay using probe set targeting the mRNA of SEM cell marker, Vcam1. BCA sections are from ROSA26^ZsGreen1/+; Ldlr^−/−; Myh11-CreER^T2 mice fed WD for 16 weeks. DAPI (blue), ZsGreen1 protein (green) and Vcam1 mRNA (red) are indicated. Yellow broken lines and arrows highlight ZsGreen1⁺Vcam1⁺ cells in media and intima regions, respectively. Scale bars, 50 μm. F, Induction of SEM cells to fibroblast-like cells. FACS sorted ZsGreen1⁺LY6A⁺LY6C1⁺ SEM cells are induced by CTGF (100 ng/mL; +CTGF) or control (PBS; -CTGF) for 4 weeks. The cells were from mice fed WD for 26 weeks. Relative mRNA levels of fibroblast markers (Col1a1, Col3a1, Fn1, Fsp1, Tnc, Vim) are measured by RT-qPCR and normalized against Actb. Values are shown as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, n=3. G, Induction of SEM cells towards SMC phenotype. FACS sorted ZsGreen1⁺LY6A⁺LY6C1⁺ SEM cells and ZsGreen1⁺LY6A⁻LY6C1⁻ non-SEM cells from mice fed WD for 26 weeks are incubated with TGFβ1 (10 ng/mL; +TGFβ1) or control (PBS; -TGFβ1) for 3 days. Immunofluorescence (IF) stained ACTA2⁺ cells are presented. Two-way ANOVA analysis indicates greater increase in the proportion of ZsGreen1⁺ACTA2⁺/ZsGreen1⁺ cells in SEM cells compared to that in non-SEM cells after 3-day TGFβ1 treatment. Scale bars, 100 μm. Values are shown as mean ± s.d. **P < 0.01, ****P < 0.0001, n=3. H, Schematic of SMC transdifferentiation into SEM cell state during atherosclerosis and the SEM cells differentiation into SMC-derived fibrochondrocyte and SMC-derived macrophage-like cell, and back towards SMC phenotype depending on conditions.

Figure 3.. Counterpart of mouse intermediate SEM cell state is identified in human atherosclerotic carotid arteries.

A and B, Reference-based integration analysis of combined scRNA-seq data of Ldlr^−/− mice fed 16-week WD (n=3 mice; 7029 cells) and human atherosclerotic carotid arteries scRNA-seq data (n=3 patients; 8867 cells). Mouse cell clusters are used as reference and human scRNA-seq data are then projected onto the mouse data. Combined mouse and human scRNA-seq data are visualized by UMAP (A). Representative mouse cell types/states are indicated, and a cell population (red circled) in human scRNA-seq data overlaps with the intermediate SEM cell state found in mouse atherosclerotic scRNA-seq data (B). C, Joint analysis of human atherosclerotic carotid arteries scRNA-seq data (n=3 patients) demonstrates an intermediate cell state (ICS) between SMC and fibrochondrocyte (FC). Representative cell type/state for each cluster is indicated. D and E, Gene expression tendencies of SMC marker, MYH11 (D) and FC-related gene, FN1 (E) through SMC-ICS-FC axis are shown as normalized expression versus UMAP_1.

Figure 4.. metaVIPER analysis identifies multiple master regulators of SMC to SEM cell transition during atherosclerosis, among which RA signaling is also found to be dysregulated in human atherosclerosis development and progression.

A, ARACNe network showing top 50 activated (red large dots) and repressed (blue large dots) MRs and their predicted target genes (light blue small dots) identified via metaVIPER using ZsGreen1⁺ scRNA-seq data of Ldlr^−/− mice fed 16-week WD. Some MRs-involved canonical cell signaling pathways are highlighted. Vcam1 and Ly6c1, in dark red text, are shown as predicted target genes of CRABP2, a transducer of retinoic acid (RA) signaling. B, Heatmap showing significantly up and downregulated (fold change ≥ 1.2, Bonferroni corrected P-value < 0.05) RA target genes (36 genes) in SEM cells relative to SMC. C, Heatmap showing 41 RA signaling target genes that were differentially expressed (fold change ≥ 1.2, FDR adjusted P-value < 0.05) in human unstable plaques (visible zone of plaque rupture) of atherosclerotic carotid arteries compared to stable plaques (macroscopically normal adjacent areas). Paired unstable and stable plaques were from 4 individual patients. D, Predicted protein activity of RA signaling transducer, RARB (retinoic acid receptor beta), in early (intimal thickening and xanthoma, n=13) and advanced (fibrous cap atheroma, n=16) human atherosclerotic lesions of carotid arteries. Protein activity was estimated using metaVIPER. Values are shown as box and whisker plot (min to max). ****P < 0.0001.

Figure 5.. Multiple RA signaling target gene loci are associated with risk of human CAD.

A, CAD GWAS enrichment summary based on a set of 226 RA signaling genes and its subsets. B-E, Regional association plots showing 1000-Genomes based GWAS of CAD/MI in four of RA signaling target gene loci, PRTG (B), ITGA1 (C), SKI (D) and TAGLN2 (E). −log₁₀(p-value) of SNP association for CAD/MI is shown for each plot. Linkage disequilibrium (LD) with the top SNP from each region is color coded by r². F-I, eQTL data from GTEx for SNPs, rs488986 (F and G) and rs4561398 (H and I), localized within the PRTG locus. Representative violin plots showing normalized expression of PRTG by genotype in CAD-relevant tissues (aorta (F and H) and tibial artery (G and I)). Numbers below the genotypes indicate sample size. Risk genotypes and eQTL and GWAS P-values are indicated. Reduced expression of PRTG is associated with risk alleles in both CAD-relevant tissues.

Figure 6.. Activation of RA signaling via all-trans retinoic acid (ATRA) inhibits SEM cell marker expression in vitro and suppresses SMC to SEM cell transition and atherosclerosis in vivo.

A, Relative mRNA levels of SEM cell markers, Ly6a and Vcam1, in cultured mouse SMC treated with TNFα (25 ng/mL), cholesterol (40 μg/mL) or vehicle control (PBS) in the absence or presence of ATRA (10 μM) for 72 hours, are measured by RT-qPCR and normalized against Actb. Values are shown as mean ± s.d. **P < 0.01, ***P < 0.001, n=3. B, ROSA26^ZsGreen1/+; Ldlr^−/−; Myh11-CreER^T2 mice induced by 2-day TAM are fed chow diet for 2 days, followed by WD. Vehicle (corn oil, control) or ATRA (2.5 mg/kg mice) administration is started after 4 weeks of WD, 3 times/week. Mice are sacrificed after 16-week WD. Arterial tissues (including ascending aorta, BCA and thoracic aorta) with atherosclerotic lesions are isolated and digested to single cells for flow cytometry analysis of the proportion of ZsGreen1⁺LY6A⁺LY6C1⁺ SEM cells among total ZsGreen1⁺ cells (control, n=6 mice; ATRA-treated, n=8 mice). Values are shown as mean ± s.d. P-value is indicated. C, RNAscope stained representative BCA sections from control (n=6) and ATRA-treated (n=7) mice indicate ZsGreen1⁺Vcam1⁺ SEM cells, regions of media, intima and fibrous cap (defined as the region within 30 μm of the luminal surface). Scale bars, 50 μm. D-G, ZsGreen1⁺Vcam1⁺ SEM cell number in BCA sections (D), ZsGreen1⁺ cell number within lesions (intima+fibrous cap) (E), percentage of ZsGreen1⁺ cells in fibrous cap/lesion (F) and percentage of ZsGreen1⁺ cells in intima/lesion (G) are calculated. Values are shown as mean ± s.d. P-values are indicated.