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Smad3 regulates smooth muscle cell fate and mediates adverse remodeling and calcification of the atherosclerotic plaque

Nature Cardiovascular Research volume 1, pages 322–333 (2022)Cite this article

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Abstract

Atherosclerotic plaques consist mostly of smooth muscle cells (SMCs), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of SMAD3 in SMCs. However, the mechanism by which this gene modifies CAD risk remains poorly understood. Here we show that SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more outward remodeling and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: an SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells as well as a chondromyocyte fate. Together, the findings reveal that Smad3 expression in SMCs inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including outward remodeling, monocyte recruitment and vascular calcification.

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Fig. 1: Smad3ΔSMC mice have increased lesion burden in the ApoE null model.
Fig. 2: Loss of Smad3 alters cell fate decisions of transition SMCs in atherosclerotic lesions.
Fig. 3: A cluster of Mmp3-expressing R-SMC transition cells promote remodeling and inflammation.
Fig. 4: Smad3 regulates a transcriptional program associated with vascular outward expansion in conjunction with Sox9 and HoxB2.

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Data availability

Primary and processed data, along with all relevant metadata, have been deposited to the National Center of Biotechnology Information Gene Expression Omnibus under accession project number PRJNA794806.

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Acknowledgements

Special thanks to Y. Ryan, K. Hennig, P. McGuire and H. Chaib at the Stanford Genomic Sequencing and Service Center for performing 10x capture, library construction and sequencing. We also thank the Stanford shared FACS facility for required FACS analysis and experiments. Also, thanks to the Matzuk laboratory for providing us with conditional Smad3 knockout mice. Illustrations were made with BioRender software. D. Dichek (University of Washington) is acknowledged for advice regarding data interpretation.

This work was supported by National Institutes of Health grants F32HL143847 (P.C.), K08HL153798 (P.C.), K08HL152308 (R.W.), K08HL133375 (J.B.K.), F32HL154681 (A.P.), R01AR066629 (M.F.), R01HL109512 (T.Q.), R01HL134817 (T.Q.), R33HL120757 (T.Q.), R01HL139478 (T.Q.), R01HL156846 (T.Q.), R01HL151535 (T.Q.) and R01HL145708 (T.Q.) as well as a Human Cell Atlas grant from the Chan Zuckerberg Foundation. This work was also supported by American Heart Association grants 20CDA35310303 (P.C.) and 18CDA34110206 (R.W.).

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  1. Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Paul Cheng, Robert C. Wirka, Juyong Brian Kim, Hyun-Jung Kim, Trieu Nguyen, Ramendra Kundu, Quanyi Zhao, Disha Sharma, Manabu Nagao, Dharini Iyer & Thomas Quertermous

  2. Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA, USA

    Albert Pedroza & Michael P. Fischbein

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Contributions

P.C. and T.Q.: designing research studies, conducting experiments, acquiring data, analyzing data, providing reagents and writing the manuscript. R.W., J.K., T.N. and R.K.: conducting experiments and acquiring data. Q.Z., A.P., D.S., D.I. and M.F.: analyzing data and providing other critical scientific input.

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Correspondence to Thomas Quertermous.

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Extended data

Extended Data Fig. 1 Experimental model validation.

(A) Measured weight of experimental mice used in sections at time of sacrifice. (B) Percent of total experimental mice cohort that survived to final time point at 24 weeks. (C) Schematic indicating the location of aortic root section used in the study (dashed line). (D) Aligned captured mRNA sequence of tdTomato positive cells in control (top) and Smad3ΔSMC tdTomato cells, showing absence of reads mapping to exon 2-3 which are flanked by LoxP sites. Atherosclerotic plaque in control (E) and Smad3ΔSMC (F) mice stained for phospho-Smad3 (brown), demonstrating loss of Smad3 in SMC lineage cells in the plaque. Scale bar: 50um.

Extended Data Fig. 2 Vascular lesion analysis.

(A) Representative image of Oil Red O-stained aortic root in control and Smad3ΔSMC mice, (B) quantified total Oil Red O positive area, and (C) quantified fraction of plaque Oil Red O staining (total Oil Red-O positive area over plaque area). (D) Representative image of trichrome stained aortic root in control and Smad3ΔSMC, with (E) quantified total acellular area, and (F) quantified normalized plaque acellular fraction (total acellular area over plaque area). Scale bar: 50um.

Extended Data Fig. 3 SMC cell state changes.

(A) Mesenchymal proliferation score of de-differentiated SMC in control and SMC specific Smad3ΔSMC mice. (B) Number of TUNEL labeled apoptotic cells per high magnification field (HPF) in control and Smad3ΔSMC mice.

Extended Data Fig. 4 R-SMC gene expresson.

(A) Expression of Mmp3 in lineage labeled (Cre positive) and non-lineage labelled (Cre negative) cells based on scRNAseq data. (B) Measured Mmp3 activity detected in isolated aortic tissue from wild type (brown) vs Smad3ΔSMC mice (orange). (C) Featureplot of lineage-traced cells expressing Mmp3 in control and Smad3ΔSMC aortic root. (D) High magnification of a section of atherosclerotic plaque with region of broken elastic lamina stained for Mmp3 expression (orange-brown color). Scale bar: 75um. (E) Fraction of transition SMC in control and Smad3ΔSMC lesions with R-SMC fate as defined by unbiased clustering (left) and by concurrent high Mmp3 and CxCl12 expression (right). (F) Number of migrated THP-1 cells after 3 hours of transwell-incubation with no cells, control HCASMC, and SMAD3-deficient HCASMC.

Extended Data Fig. 5 (A - H) FeaturePlot of Smad3, Smad2, Col2a1, Mki67, Lox, Mfap5, HoxB2, and Sox9 expression in SMC in atherosclerotic lesions in the aortic root.

(I) Individual cell expression of HoxB2, HoxB3, HoxB4, and Sox9 in lineage labeled cells in control and Smad3ΔSMC aortic root. (J) Control reporter luciferase activity in response to SOX9 and HOXB2 overexpression.

Extended Data Fig. 6

Replicate of Flag-SOX9 immunoprecipitation with endogenous SMAD3 immunodetection, with additional controls for anti-flag antibody.

Source data

Supplementary information

Source data

Source Data Fig. 1

Numerical data for all graphs and all figures.

Source Data File 2

FACS gating strategy for single-cell studies.

Source Data Fig. 4

Western blot used for Fig. 4f with mol weight markers.

Source Data Extended Data Fig. 6

Raw western blot gel.

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Cheng, P., Wirka, R.C., Kim, J.B. et al. Smad3 regulates smooth muscle cell fate and mediates adverse remodeling and calcification of the atherosclerotic plaque. Nat Cardiovasc Res 1, 322–333 (2022). https://doi.org/10.1038/s44161-022-00042-8

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