Re-analysis of single-cell transcriptomics reveals a critical role of TNS1 gene in driving contractile VSMC transdifferentiation into macrophage-like SMC and atherosclerotic plaque instability

Abstract

Background: Vascular smooth muscle cell (VSMC) phenotype switching plays a significant role in the pathogenesis of atherosclerosis (AS). However, the subtypes of VSMC transdifferentiation and their impact on AS progression and atherosclerotic plaque instability remains unclear.

Methods: We reanalysed scRNA-seq datasets of GSE155513 and GSE253903 and performed single-sample gene set enrichment analysis (ssGSEA) in three transcriptome datasets from unstable plaques to determine the major subtypes contributing the most to plaque instability. Using high-dimensional weighted gene co-expression network analysis (hdWGCNA), we identified hub genes in macrophage (MP)-like smooth muscle cells (SMCs) of unstable plaques. We conducted cell communication analysis according to tensin1 (TNS1) gene levels in VSMCs. TNS1 expression was analysed in human AS plaques. Finally, an AS model was established in VSMC-specific Tns1 knockout ApoE^−/− mice to validate the causative role of TNS1 on atherosclerotic lesions.

Results: MP-like SMC was identified as the key subtype for plaque instability. hdWGCNA analysis for MP-like SMC identified blue module as the key gene module involved in unstable plaques. Decreased TNS1 expression in VSMCs was positively correlated with the down-regulation of contractile VSMC marker genes, SRF and MYCOD genes, negatively correlated with the up-regulation of CD68 and KLF4 genes, and activated VCAM, PDGF, THBS and CXCL signalling pathways. TNS1 mRNA expression levels were lower in human atherosclerotic arteries than in healthy arteries, and even lower in unstable plaques than in early and stable plaques. TNS1 protein levels in VSMCs were lower in human atherosclerotic plaques than in healthy arteries, and even lower in advanced plaques than in early plaques. VSMC-specific Tns1 gene deficiency aggravated AS progression and enhanced plaque instability with increased MP-like SMC transdifferentiation.

Conclusion: The reduction of TNS1 gene in VSMCs might drive contractile VSMC transdifferentiation into MP-like SMC, the major subtype contributing to plaque instability. In vivo experimental results confirmed the role of Tns1 gene in contractile VSMC transdifferentiation into MP-like SMC and plaque instability.

Keywords

atherosclerotic plaque instability / macrophage (MP)-like SMC / single-cell sequencing (scRNA-seq) dataset / tensin1 (TNS1) gene / VSMC transdifferentiation

References

[1] Tsao CW, Aday AW, Almarzooq ZI, et al. Heart disease and stroke statistics—2023 update: a report from the American Heart Association. Circulation. 2023; 147(8): e93-e621.

[2] Finn AV, Nakano M, Narula J, Kolodgie FD, Virmani R. Concept of vulnerable/unstable plaque. Arterioscler Thromb Vasc Biol. 2010; 30(7): 1282-1292.

[3] Virmani R, Burke AP, Farb A, Kolodgie FD. Pathology of the vulnerable plaque. J Am Coll Cardiol. 2006; 47(8) suppl: C13-C18.

[4] Cao G, Xuan X, Hu J, Zhang R, Jin H, Dong H. How vascular smooth muscle cell phenotype switching contributes to vascular disease. Cell Commun Signal. 2022; 20(1): 180.

[5] Shankman LS, Gomez D, Cherepanova OA, et al. KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat Med. 2015; 21(6): 628-637.

[6] Francis GA. The greatly under-represented role of smooth muscle cells in atherosclerosis. Curr Atheroscler Rep. 2023; 25(10): 741-749.

[7] Harman JL, Jørgensen HF. The role of smooth muscle cells in plaque stability: therapeutic targeting potential. Br J Pharmacol. 2019; 176(19): 3741-3753.

[8] Albarrán-Juárez J, Kaur H, Grimm M, Offermanns S, Wettschureck N. Lineage tracing of cells involved in atherosclerosis. Atherosclerosis. 2016; 251: 445-453.

[9] Feil S, Fehrenbacher B, Lukowski R, et al. Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circ Res. 2014; 115(7): 662-667.

[10] Allahverdian S, Chehroudi AC, McManus BM, Abraham T, Francis GA. Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis. Circulation. 2014; 129(15): 1551-1559.

[11] Wang Y, Dubland JA, Allahverdian S, et al. Smooth muscle cells contribute the majority of foam cells in apoe (apolipoprotein E)-deficient mouse atherosclerosis. Arterioscler Thromb Vasc Biol. 2019; 39(5): 876-887.

[12] Misra A, Feng Z, Chandran RR, et al. Integrin beta3 regulates clonality and fate of smooth muscle-derived atherosclerotic plaque cells. Nat Commun. 2018; 9: 2073.

[13] Dobnikar L, Taylor AL, Chappell J, et al. Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels. Nat Commun. 2018; 9: 4567.

[14] Chen R, McVey DG, Shen D, Huang X, Ye S. Phenotypic switching of vascular smooth muscle cells in atherosclerosis. J Am Heart Assoc. 2023; 12(20):e031121.

[15] Grootaert MOJ, Bennett MR. Vascular smooth muscle cells in atherosclerosis: time for a re-assessment. Cardiovasc Res. 2021; 117(11): 2326-2339.

[16] Gong X, Liu Y, Liu H, et al. Re-analysis of single-cell transcriptomics reveals a critical role of macrophage-like smooth muscle cells in advanced atherosclerotic plaque. Theranostics. 2024; 14(4): 1450-1463.

[17] Wang Z, Wang Y, Yuan R, et al. Sentrin-specific protease 3 (SENP3)-mediated Krüppel-like factor 4 (KLF4) deSUMOylation regulates vascular smooth muscle cell phenotypic switching in atherosclerosis. Mol Biomed. 2025; 6(1): 124.

[18] Li Y, Zhu H, Zhang Q, et al. Smooth muscle-derived macrophage-like cells contribute to multiple cell lineages in the atherosclerotic plaque. Cell Discov. 2021; 7(1): 111.

[19] Lee YRJ, Yamada S, Lo SH. Phase transition of tensin-1 during the focal adhesion disassembly and cell division. Proc Natl Acad Sci U S A. 2023; 120(15):e2303037120.

[20] Dina C, Bouatia-Naji N, Tucker N, et al. Genetic association analyses highlight biological pathways underlying mitral valve prolapse. Nat Genet. 2015; 47(10): 1206-1211.

[21] Liu Y, Zeng L, Cai Q, et al. Cytoskeletal-related genes function as checkpoints for the maintenance of VSMC contractile phenotype and prevent pathological remodeling in arterial diseases. J Adv Res. 2026; 81: 689-707. Published online June 20, 2025:S2090-1232(25)00386-8.

[22] Hou Q, Liu Y, Hou J, et al. miR-3154: novel pathogenic and therapeutic target in abdominal aortic aneurysm. Circ Res. 2025; 137(5): 587-604.

[23] Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1995; 92(5): 1355-1374.

[24] Elmarasi M, Elmakaty I, Elsayed B, et al. Phenotypic switching of vascular smooth muscle cells in atherosclerosis, hypertension, and aortic dissection. Journal Cellular Physiology. 2024; 239(4):e31200.

[25] Yu Y, Cai Y, Yang F, et al. Vascular smooth muscle cell phenotypic switching in atherosclerosis. Heliyon. 2024; 10(18):e37727.

[26] Libby P. The changing landscape of atherosclerosis. Nature. 2021; 592(7855): 524-533.

[27] Bashore AC, Yan H, Xue C, et al. High-dimensional single-cell multimodal landscape of human carotid atherosclerosis. Arterioscler Thromb Vasc Biol. 2024; 44(4): 930-945.

[28] Morabito S, Reese F, Rahimzadeh N, Miyoshi E, Swarup V. hdWGCNA identifies co-expression networks in high-dimensional transcriptomics data. Cell Rep Methods. 2023; 3(6):100498.

[29] Jin S, Guerrero-Juarez CF, Zhang L, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 2021; 12(1): 1088.

[30] Nofer JR. Estrogens and atherosclerosis: insights from animal models and cell systems. J Mol Endocrinol. 2012; 48(2): R13-29.

[31] Jiang S, Li H, Zhang L, et al. Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. Nucleic Acids Res. 2025; 53(D1): D1670-D1676.

[32] Cui Y, Chen Y, Li H, et al. PCSK9 promotes atherosclerotic plaque instability by inducing VSMC ferroptosis through the YAP1-NUPR1 axis. Research (Wash D C). 2025; 8: 0922.

[33] Zhang F, Guo X, Xia Y, Mao L. An update on the phenotypic switching of vascular smooth muscle cells in the pathogenesis of atherosclerosis. Cell Mol Life Sci. 2022; 79(1): 6.

[34] Pryma CS, Ortega C, Dubland JA, Francis GA. Pathways of smooth muscle foam cell formation in atherosclerosis. Curr Opin Lipidol. 2019; 30(2): 117-124.

[35] Alencar GF, Owsiany KM, Karnewar S, et al. Stem cell pluripotency genes Klf4 and Oct4 regulate complex SMC phenotypic changes critical in late-stage atherosclerotic lesion pathogenesis. Circulation. 2020; 142(21): 2045-2059.

[36] Gil-Yarom N, Radomir L, Sever L, et al. CD74 is a novel transcription regulator. Proc Natl Acad Sci U S A. 2017; 114(3): 562-567.

[37] Winter H, Winski G, Busch A, et al. Targeting long non-coding RNA NUDT6 enhances smooth muscle cell survival and limits vascular disease progression. Mol Ther. 2023; 31(6): 1775-1790.

[38] Menendez-Castro C, Cordasic N, Neureiter D, et al. Under-expression of α8 integrin aggravates experimental atherosclerosis. J Pathol. 2015; 236(1): 5-16.

[39] Daniel JM, Prock A, Dutzmann J, et al. Regulator of G-protein signaling 5 prevents smooth muscle cell proliferation and attenuates neointima formation. Arterioscler Thromb Vasc Biol. 2016; 36(2): 317-327.

[40] Xu X, Zhang DD, Kong P, et al. Sox10 escalates vascular inflammation by mediating vascular smooth muscle cell transdifferentiation and pyroptosis in neointimal hyperplasia. Cell Rep. 2023; 42(8):112869.

[41] Perisic Matic L, Rykaczewska U, Razuvaev A, et al. Phenotypic modulation of smooth muscle cells in atherosclerosis is associated with downregulation of LMOD1, SYNPO2, PDLIM7, PLN, and SYNM. Arterioscler Thromb Vasc Biol. 2016; 36(9): 1947-1961.

[42] Wang X, Hu G, Betts C, et al. Transforming growth factor-β1-induced transcript 1 protein, a novel marker for smooth muscle contractile phenotype, is regulated by serum response factor/myocardin protein. J Biol Chem. 2011; 286(48): 41589-41599.

[43] Chen J, Chu Y, Lan Y, et al. TRPV4 maintains the contractile phenotype of VSMCs by regulating CPI-17. Biochem Pharmacol. 2025; 240:117107.

[44] Miao G, Han Y, Chen J, et al. Vascular smooth muscle cell-derived KIF13B inhibits proinflammatory responses to protect against atherosclerosis. J Clin Invest. 2026; 136(6):e194175.

[45] Chatterjee P, Chakraborty R, Sizer AJ, et al. SUV39H1 regulates KLF4 and chromatin remodeling in smooth muscle cell phenotypic plasticity. Arterioscler Thromb Vasc Biol. 2025; 45(11): 2015-2033.

[46] Farina FM, Hall IF, Serio S, et al. miR-128-3p is a novel regulator of vascular smooth muscle cell phenotypic switch and vascular diseases. Circ Res. 2020; 126(12): e120-e135.

[47] Chen J, Kitchen CM, Streb JW, Miano JM. Myocardin: a component of a molecular switch for smooth muscle differentiation. J Mol Cell Cardiol. 2002; 34(10): 1345-1356.

[48] Wang Z, Wang DZ, Hockemeyer D, McAnally J, Nordheim A, Olson EN. Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression. Nature. 2004; 428(6979): 185-189.

[49] Miano JM. Role of serum response factor in the pathogenesis of disease. Lab Invest. 2010; 90(9): 1274-1284.

[50] Zheng JP, He X, Liu F, et al. YY1 directly interacts with myocardin to repress the triad myocardin/SRF/CArG box-mediated smooth muscle gene transcription during smooth muscle phenotypic modulation. Sci Rep. 2020; 10(1):21781.

[51] Gonen A, Miller YI. From inert storage to biological activity-in search of identity for oxidized cholesteryl esters. Front Endocrinol (Lausanne). 2020; 11:602252.

[52] Bogan BJ, Williams HC, Holden CM, et al. The role of fatty acid synthase in the vascular smooth muscle cell to foam cell transition. Cells. 2024; 13(8): 658.

[53] Castro R, Adair JH, Mastro AM, Neuberger T, Matters GL. VCAM-1-targeted nanoparticles to diagnose, monitor and treat atherosclerosis. Nanomedicine (Lond). 2024; 19(8): 723-735.

[54] Folestad E, Kunath A, Wågsäter D. PDGF-C and PDGF-D signaling in vascular diseases and animal models. Mol Aspects Med. 2018; 62: 1-11.

[55] Wågsäter D, Zhu C, Björck HM, Eriksson P. Effects of PDGF-C and PDGF-D on monocyte migration and MMP-2 and MMP-9 expression. Atherosclerosis. 2009; 202(2): 415-423.

[56] Gao JH, He LH, Yu XH, et al. CXCL12 promotes atherosclerosis by downregulating ABCA1 expression via the CXCR4/GSK3β/β-cateninT120/TCF21 pathway. J Lipid Res. 2019; 60(12): 2020-2033.

[57] Li L, Du Z, Rong B, et al. Foam cells promote atherosclerosis progression by releasing CXCL12. Biosci Rep. 2020; 40(1):BSR20193267.

[58] Chistiakov DA, Melnichenko AA, Myasoedova VA, Grechko AV, Orekhov AN. Thrombospondins: a role in cardiovascular disease. Int J Mol Sci. 2017; 18(7): 1540.

[59] Zhang T, Li Z, Ming Y, et al. A positive feedback loop between TNS1 and ZEB1 promotes TGFβ-induced epithelial-to-mesenchymal transition in lung cancer. Commun Biol. 2025; 8(1): 1686.

[60] Bennett MR, Sinha S, Owens GK. Vascular smooth muscle cells in atherosclerosis. Circ Res. 2016; 118(4): 692-702.

[61] Wirka RC, Wagh D, Paik DT, et al. Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis. Nat Med. 2019; 25(8): 1280-1289.

[62] Dong R, Li J, Jiang G, Tian Y, Bi W. Allograft inflammatory Factor-1 induces the dedifferentiation of vascular smooth muscle cells into a macrophage-like phenotype both in vivo and in vitro. Exp Cell Res. 2025; 446(1):114475.

[63] Lo SH. Tensins. Curr Biol. 2017; 27(9): R331-R332.

[64] Shih YP, Sun P, Wang A, Lo SH. Tensin1 positively regulates RhoA activity through its interaction with DLC1. Biochim Biophys Acta. 2015; 1853(12): 3258-3265.

[65] Chen X, Fang LT, Chen Z, et al. A benchmarking study of copy number variation inference methods using single-cell RNA-sequencing data. Precis Clin Med. 2025; 8(2):pbaf011.

RIGHTS & PERMISSIONS

2026 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.