In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human arteries and found that these cells transform into unique fibroblast-like cells, termed ‘fibromyocytes’, rather than into a classical macrophage phenotype. SMC-specific knockout of TCF21—a causal CAD gene—markedly inhibited SMC phenotypic modulation in mice, leading to the presence of fewer fibromyocytes within lesions as well as within the protective fibrous cap of the lesions. Moreover, TCF21 expression was strongly associated with SMC phenotypic modulation in diseased human coronary arteries, and higher levels of TCF21 expression were associated with decreased CAD risk in human CAD-relevant tissues. These results establish a protective role for both TCF21 and SMC phenotypic modulation in this disease.
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High-throughput sequencing data (FASTQ) files for all scRNA-Seq, CITE-Seq and ChIP-Seq, as well as cell–gene count matrices for all scRNA-Seq and CITE-Seq experiments, have been deposited in the Gene Expression Omnibus database with the SuperSeries reference number GSE131780. These data were used to generate the images in Figs. 1–5 and Extended Data Figs. 2–5. FASTQ files and processed data are also available from the corresponding author upon request.
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The authors are grateful to G. Owens for providing TgMyh11-Cre, ApoE−/− animals and experimental protocols. The authors are also grateful to L. Román for assistance with developing the human coronary digestion protocol. This work was supported by NIH grant F32HL129670 and AHA grant 18CDA34110206 (both to R.C.W.), and NIH grants K08HL133375 (to J.B.K.), R00HL125912 (to C.L.M.), R01HL109512 (to T.Q.), R01HL134817 (to T.Q.), R33HL120757 (to T.Q.), R01DK107437 (to T.Q.), R01HL139478 (to T.Q.), 1R01HL141371 (to J.C.W.) and 17GRNT33660474 AHA (to M.D.T.). The sequencing data were generated on an Illumina HiSeq 4000 that was purchased with funds from NIH under award number S10OD018220. Cell sorting was performed on an instrument in the Shared FACS Facility obtained using NIH S10 Shared Instrument Grant S10RR025518-01.
The authors declare no competing interests.
Peer review information: Michael Basson was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Alleles present in SMClin and SMClin-KO mice. KO (knockout) refers to Tcf21, lin, lineage tracing; Tg, transgene; ΔSMC, SMC cell-specific KO. b, Mice were maintained on a chow diet from birth until 7 weeks of age, then underwent gavage and HFD treatment. For the scRNA-Seq, RNAscope, CITE-Aeq, histology involving BODIPY and FACS staining experiments (upper timeline), mice were gavaged only at 7 weeks of age, before the onset of HFD, as denoted by red arrows. For the scRNA-Seq experiments, mice were sacrificed at baseline (72 h after initial tamoxifen gavage), or after 8 or 16 weeks of HFD. For the RNAscope experiments, mice were sacrificed after either 8 or 16 weeks of HFD. For the CITE-Seq experiment, mice were sacrificed after 16 weeks of HFD. For the BODIPY studies, mice were sacrificed after 16 weeks of HFD. For the FACS staining experiment, two mice (one after 12 weeks HFD and another after 15 weeks HFD) were used. For quantitative histology experiments (lower timeline), mice were gavaged at 7 weeks of age, after 8 weeks of HFD and after 16 weeks of HFD (48 h before sacrifice), as denoted by red arrows. For these quantitative histology experiments, all mice were sacrificed after 16 weeks of HFD. c, FACS workflow for isolating single cells from the mouse aortic root.
a–d, t-SNE visualization of cell types present in the WT mouse aortic root from all time points overlaid with the expression of Col1a1, Acta2, Sca1 and Lgals3 (n = 9 mice). e–g, RNAscope staining for Lum (green) and tdT (red) in a plaque after 8 weeks of HFD (e), the non-diseased media of a mouse on 16 weeks HFD (f) and a baseline healthy aorta (g). h, RNAscope negative control. Images in e–h are representative of two experiments and scale bars indicate 25 μm. i, t-SNE visualization of cell types present in the WT mouse aortic root from all time points overlaid with osteopontin (Spp1) expression (n = 9 mice). j,k, RNAscope co-localization of Spp1 (green) and tdT (red) in a plaque after 16 weeks of HFD. Yellow arrows indicate co-localization of Spp1 and tdT. l, RNAscope negative control. Images from j–l are representative of four experiments, and scale bars indicate 50 μm. m, Heat map representation of the Euclidean distance between cell cluster centroids in 20-dimensional principal component space with the smallest distances in yellow and the largest distances in black. Data are after 16 weeks of HFD. n, Staining of a single-cell suspension from the atherosclerotic mouse aortic root and ascending aorta with antibodies against the macrophage markers Cd16 and Cd32, and analysis of co-expression with the tdT SMC lineage marker. Data are from one experiment and n = 2 mice (after 12 and 15 weeks of HFD). o–t, Single cells from the atherosclerotic mouse aortic root and ascending aorta at 16 weeks HFD were incubated with DNA-barcoded antibodies against the macrophage markers Cd16, Cd32, Cd11b, Cd64, Cd86 and F4/80 before undergoing scRNA-Seq (CITE-Seq), yielding simultaneous transcriptomic and antibody binding data within each individual cell. o, Cell type assignments were determined with scRNA-Seq as described previously. p–t, Quantitative antibody binding within each cell type. Results are from one experiment and n = 2 mice.
a,b, Tcf21 expression in SMC lineage-labeled cells from SMClin (WT) and SMClin-KO (KO) mice from all time points combined (n = 13 mice). a, Tcf21 expression for all WT cells (left; minimum = 0; maximum = 2.55; mean = 0.071) and all KO cells (right; minimum = 0; maximum = 1.97; mean = 0.004). b, Mean Tcf21 expression visualized for all SMC lineage-labeled WT and KO cells. c, Total Lgals3+ area in the lesion is reduced in SMClin-KO mice. d, Cd68 immunohistochemistry quantification (left) and representative images (right). Scale bars represent 100 μm. e, Lesion area, normalized to the total vessel area. Data from c–e are after 16 weeks HFD, and analyzed using a two-sided Student’s t-test. Error bars indicate standard error. Source data
a, Joint clustering of mouse and human datasets using canonical correlation analysis (CCA) as per the Seurat package. b, The shared mouse/human cluster containing bona fide SMC lineage-traced, phenotypically modulated SMCs (fibromyocytes) from the mouse is highlighted in red. c, Mouse cells in the shared mouse/human fibromyocyte cluster in b are highlighted in the independently clustered mouse dataset, confirming their location within the known fibromyocyte cell cluster. d, Human cells in the shared mouse/human fibromyocyte cluster in b are highlighted in the independently clustered human dataset, illustrating their location predominantly in the ‘fibromyocyte’ cluster (also shown in brown in Fig. 4d). e, All joint mouse/human clusters in a were mapped back to the human dataset. Agreement is identified in cell type assignment between the joint clustering approach and the independently clustered human dataset.
a, t-SNE visualization of cell types in the right coronary artery of four patients, overlaid with LUM expression. Expression levels are indicated by scales in the lower right. b, TNFRSF11B RNAscope staining in a human coronary artery section. Hybridization events are seen as red dots. c, A negative control RNAscope probe shows no staining. The images in b and c are representative of four experiments, and the scale bars represent 50 μm. d, Heat map representation of the Euclidean distance between cell cluster centroids in 20-dimensional principal component space, with the smallest distances in yellow and the largest distances in black. The relationship between ‘fibromyocyte’ and ‘fibroblast 2’ clusters is highlighted with white asterisks. The ‘fibromyocyte’, ‘SMC’ and the main ‘macrophage’ clusters are denoted by black asterisks. e,f, t-SNE visualization of cell types in the right coronary artery of four patients overlaid with CD68 expression (e) and TCF21 expression (f). g, UCSC Genome Browser shots of representative TCF21 ChIP-Seq peaks within the PRELP and MYH11 genes, which are highly correlated and anticorrelated, respectively, with TCF21 and the fibromyocyte phenotype. Images are from one ChIP-Seq experiment. Source data
Extended Data Fig. 6 Association of genome-wide significant CAD risk SNPs at the 6q23.2 locus with TCF21 expression.
Seven SNPs in the 6q23.2 locus were associated with CAD at genome-wide significance. The association between risk and protective genotypes and TCF21 expression for each of these SNPs was determined using the GTEx database in CAD-relevant tissues and a cohort of 52 HCASMC lines. The number of independent tissue samples included for each SNP is indicated in the GTEx data (‘N’), and n = 52 cell lines for the HCASMC data. In each box plot, the middle line represents the median, the box represents the first to third quartile range, and whiskers represent 1.5× the interquartile range.
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Wirka, R.C., Wagh, D., Paik, D.T. et al. Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis. Nat Med 25, 1280–1289 (2019). https://doi.org/10.1038/s41591-019-0512-5
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