Cardiovascular Research (2025) 121, 1448–1463 https://doi.org/10.1093/cvr/cvaf102 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Premature cell senescence promotes vascular smooth muscle cell phenotypic modulation and resistance to re-differentiation Anuradha Kaistha †, Sebnem Oc †, Abel Martin Garrido, James CK Taylor, Maria Imaz, Matthew D Worssam, Anna Uryga , Mandy Grootaert, Kirsty Foote, Alison Finigan, Nichola Figg, Helle F Jørgensen ‡, and Martin Bennett *‡ Section of Cardiorespiratory Medicine, University of Cambridge, Victor Phillip Dahdaleh Heart & Lung Research Institute, Papworth Road, Cambridge Biomedical Campus, Cambridge CB2 0BB, UK Received 5 August 2024; revised 23 December 2024; accepted 11 April 2025; online publish-ahead-of-print 10 June 2025 Time of primary review: 40 days Aims Human atherosclerotic plaque cells display DNA damage that if left unrepaired can promote premature cell senescence. Vascular smooth muscle cells (VSMCs) predisposed to senescence promote atherogenesis and features of unstable plaques and increase neointima formation after injury. However, how premature VSMC senescence promotes vascular disease and its effects on VSMC phenotype are unknown. Methods and results Bulk RNA-seq of primary human VSMCs identified 126 significantly up- or down-regulated genes after both DNA damage-induced (D + R) or replicative senescence (RS). Up-regulated genes included senescence markers CDKN2A (p16) and ICAM1 and genes ex pressed by phenotypically modulated de-differentiated/’fibromyocytic’ VSMCs [osteoprotegerin (TNFRSF11B), fibromodulin (FMOD)] as well as transmembrane protein 178B (TMEM178B) and secreted frizzle-related protein 4 (SFRP4). Mouse VSMCs also up-regulated genes associated with de-differentiated VSMC phenotype, Tmem178b and Sfrp4 after D + R. Single-cell RNA- sequencing of lineage-traced VSMCs in mouse plaques or human plaques showed that VSMCs expressing Cdkn2a had lower con tractile marker expression and higher expression of de-differentiated VSMC markers. Mice expressing a VSMC-restricted mutant telomere protein (TRF2T188A) that induces premature senescence showed increased atherosclerosis, expression of multiple de- differentiation genes in plaques and after injury, and differential regulation of pathways associated with extracellular matrix organ ization, inflammation and Transforming Growth Factor-β (Tgfb). Trf2T188A VSMCs were more resistant to re-differentiation and had dysregulated Tgfb signalling at multiple levels with down-regulated ligands, receptors, and coactivators and up-regulated co- repressor expression. Trf2T188A VSMCs also showed cytosolic DNA and activation of the STING–TBK1–IRF3 pathway that sup pressed Tgfb signalling. Silencing IRF3 restored expression of Tgfb pathway components and VSMC contractile markers after TGFb administration. Conclusion DNA damage and senescence induce genes associated with de-differentiated/fibromyocytic VSMCs, and persistence of these cells in vivo. Failure of senescent VSMCs to re-express contractile markers during re-differentiation suggests that VSMC senescence may promote atherosclerosis and neointima formation in part by inhibiting their re-differentiation. * Corresponding author. Tel: +441223331504; fax: +441223331505, E-mail: mrb24@cam.ac.uk † Equal first author ‡ Equal senior author contribution © The Author(s) 2025. Published by Oxford University Press on behalf of the European Society of Cardiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 https://orcid.org/0000-0002-2209-1535 https://orcid.org/0000-0003-3476-3359 https://orcid.org/0000-0002-0206-1435 https://orcid.org/0000-0002-7909-2977 https://orcid.org/0000-0002-2565-1825 mailto:mrb24@cam.ac.uk https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1093/cvr/cvaf102 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Graphical Abstract Senescence affects phenotype of neointimal VSMCs in vivo. Model of how persistent DNA damage/cell senescence affects VSMC phenotype. VSMCs under go de-differentiation in culture or in vivo (e.g. in atherosclerosis or injury) regulated by factors such as Myocd and Tcf21. These de-differentiated VSMCs can undergo further phenotypic modulation, for example to fibromyocytes, macrophage-like cells, or osteochondrogenic cells, which express some de-differ entiation markers but also some more cell state-specific markers, such as Fmod, Sfrp4 and Tnfrsf11b for fibromyocytes. Both de-differentiated and fibro myocyte VSMCs may re-differentiate to a contractile phenotype. However, senescence results in maintenance of a de-differentiated/fibromyocyte state, potentially due to cytosolic DNA-mediated STING/TBK1/IRF3 upregulation that suppresses multiple components of Tgfb signalling and contractile genes. Silencing IRF3 partly restores TGFb-mediated re-differentiation of VSMCs. Keywords Atherosclerosis • smooth muscle • senescence • DNA damage 1. Introduction Vascular smooth muscle cells (VSMCs) in human atherosclerotic plaques display a wide range of different types of DNA damage, including double- strand breaks,1 telomere damage,2,3 and oxidative DNA damage.4 Telomeres are particularly sensitive to DNA damage, in part because they are not targeted by most general DNA repair pathways, and VSMCs in human plaques show reduced telomere length and lower ex pression and binding of specific protective telomere proteins such as telo mere repeat binding factor 2 (TRF2).2,3 VSMC telomere damage occurs during replication or after stimuli such as oxidant stress,2 and progressive DNA damage results in premature senescence characterized by growth ar rest, activation of a persistent DNA damage response (DDR), expression of different senescence marker genes, and secretion of a panel of pro-inflammatory cytokines (the senescence-associated secretory pheno type—SASP). Cell senescence has been described in atherosclerosis and after vascular injury, including in endothelial cells and VSMCs (e.g.5–9). Furthermore, a number of studies have shown that VSMCs predisposed to senescence promote atherogenesis and features of plaque vulnerability, including reduced fibrous caps and increased neointima formation after VSMC senescence in atherosclerosis 1449 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 injury,3,4,10,11 but the underlying mechanisms are unclear and may be mul tiple. For example, senescent VSMCs show loss of proliferation and re duced migration and express genes with roles in inflammation, tissue remodelling and vascular calcification.12,13 DNA damage promotes expression of pro-inflammatory cytokines in VSMCs,4,10 similar to those seen after VSMC de-differentiation,14 but does not affect the number of VSMC-derived cells in the neointima or VSMC clonality after injury,10 suggesting that primary defects in initial de- differentiation, proliferation, or migration are not responsible. However, how DNA damage affects VSMC phenotypic modulation is unknown, par ticularly in those cells that retain or regain contractile markers after injury or in fibrous caps in atherosclerosis. VSMCs in culture, atherosclerosis and after injury exhibit a range of different phenotypes, described as ‘contract ile’, ‘synthetic’, ‘adipocyte-like’, ‘foam cell’, ‘macrophage-like’, ‘osteocyte- like’, and ‘chondrocyte-like’ cells, with relative proportions differing ac cording to context (reviewed in.15) VSMC lineage tracing studies combined with single-cell RNA-sequencing (scRNA-seq) have described the tran scriptomic heterogeneity of VSMCs underlying these phenotypes, and also revealed similarity of transcriptomic states between mouse and human atherosclerosis (e.g.16,17–19) These studies also identified a transitional de-differentiated multipotent population known as ‘stem cell, endothelial cell and monocyte’ (SEM) cells18 or ‘pioneer’ cells,17 that may represent an intermediate VSMC phenotypic switching state.18,20 Specific popula tions of these cells, for example those expressing the progenitor cell mark er Stem cell antigen 1 (SCA1), may then generate many other different phenotypes,21 including ‘fibromyocytes’ that may contribute to VSMCs that express contractile markers and overlie plaque or neointima.19 However, VSMC-derived plaque cells represent a continuum with relative rather than absolute changes in the expression of specific genes, and both regulation and markers of specific subsets may differ between human and mouse VSMCs. We examined the VSMC senescence and phenotype markers gene using two models of senescence of human VSMCs, scRNA-seq data from human and mouse atherosclerotic plaques, and mice with VSMC telomere damage with atherosclerosis and after injury. VSMC senescence up-regulated mul tiple de-differentiation/fibromyocytic markers in human and mouse VSMCs. VSMCs predisposed to undergo senescence expressed higher le vels of de-differentiation markers in vivo and were more resistant to re- differentiation in vitro. We suggest that VSMC senescence may promote atherosclerosis or neointima formation in part by retaining VSMCs in a de-differentiated phenotype. 2. Methods 2.1 Human tissue Human tissue was obtained under written informed consent using proto cols approved by Cambridge or Huntingdon Research Ethical Committees, conforming to the principles outlined in Declaration of Helsinki. Carotid endartectomy samples were obtained from Royal Papworth Hospital tis sue bank with ethical committee approval. Primary human aortic VSMCs were isolated from explants as described in Supplementary Material online. 2.2 Animal experiments Animal experiments were performed under Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 and approved by Cambridge Animal Welfare and Ethical Review Body (AWERB). Mice were anaesthetized with 2.5% inhalable isoflurane (maintained at 1.5%), monitoring respiratory and heart rates, muscle tone and reflexes, and eu thanized by CO2 overdose. Male and female C56BL/6J mice were used for all experiments, except Myh11-CreERt2/Confetti/ApoE−/− mice where only males expressed the tamoxifen-inducible CRE. Primary mouse aortic VSMCs were isolated, and EdU and SAβG activity assays were performed as described in Supplementary Material online. 2.3 Molecular analysis RNA isolation, cDNA preparation, qPCR primers and quantification con ditions are described in Supplementary Material online and Supplementary material online, Table S1. 2.4 Western blots Western blots and antibodies used are as described in Supplementary Material online and Supplementary material online, Table S2. 2.5 Confocal microscopy Immunofluorescence and imaging of formalin-fixed, paraffin-embedded human carotid endarterectomy sections, control and Trf2T188A VSMCs, li gated carotid artery sections and quantification of confetti-positive fibrous cap cells were performed as described in Supplementary Material online. 2.6 Immunohistochemistry Bright-field imaging was performed as described in Supplementary Material online. 2.7 RNA-seq Bulk RNA-seq was performed on human primary aortic VSMC cultures treated with Doxorubicin for 1d + 21d recovery (D + R), replicative senes cence (RS), or Dox 1d, or on control 1d replicating cells. Sample prepar ation, sequencing and data processing were performed as described previously10 and in Supplementary Material online. Atherosclerosis scRNA-seq profiles from human coronary lesions19 (GSE131778) were analysed as described in Supplementary Material online22 and murine VSMC-derived plaque and medial cells isolated from fat-fed Myh11-creERT2/Rosa 26-Confetti/ApoE−/− mice16 (GSE117963) were clustered and gene expression analysis performed as previously described.19,21 For scRNA-seq profiles from Trf2T188A/ApoE−/− vs. ApoE−/− mice, pla ques were micro-dissected from the aorta (root and arch) and carotid ar teries and underwent enzymatic digestion. Adventitia and non-remodelled arterial sections were removed before enzymatic digestion from ligated ca rotid arteries. Single-cell suspensions were analysed using a Chromium sys tem (10 × Genomics). Raw sequencing reads were aligned to the GRCm38 mouse genome using the 10 × Genomics Cell Ranger pipeline (v.7.0.0 and v.6.1.2, respectively, GSE210406). Data analysis was performed as described in Supplementary Material online. 2.8 Mouse carotid artery ligation Carotid artery ligation was performed as described previously;10 4- to 5-m-old male C57Bl6/J Sm22a-TRF2T188A or wild-type littermate control mice backcrossed >10 × received pre-operative buprenorphine (0.1 mg/ kg subcutaneously) and inhalable isoflurane. The left common carotid artery (LCCA) was ligated just below the bifurcation with a 6–0 silk suture. Following 28 days of recovery, the LCCA was collected and processed as described for scRNA-seq16 or confocal microscopy.10 2.9 Mouse atherosclerosis studies Transgenic mice generation, genotyping protocols, power calculation and animal randomization procedures were as described previously.16 C57Bl6/J Sm22a-Trf2T188A/ApoE−/− or wild-type littermate ApoE−/− mice were crossed with Myh11-CreERt2/Confetti/ApoE−/− mice to generate Myh11-CreERt2/Confetti/Sm22a-Trf2T188A/ApoE−/− (Trf2T188A/ApoE−/−) or Myh11-CreERt2/Confetti/ApoE−/− (ApoE−/−) mice, and 10 injections of 100 µL of 1 mg/mL tamoxifen (Sigma-Aldrich) administered over 2 weeks at 6 weeks of age. Following one-week rest, mice were administered high fat diet (HFD, 829-100, Special Diet Services, UK, 21% total fat, 0.2% chol esterol, 0% sodium cholate) for 14weeks. Blood pressure, serum lipids and cytokines were analysed as described in Supplementary Material online. 1450 A. Kaistha et al. D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data 2.10 Re-differentiation and exogenous DNA transfection protocol Control or Trf2T188A VSMCs were seeded in DMEM + 10% serum and 1% v/v L-glutamine/penicillin/streptomycin solution, attached for 24 h, washed with warm PBS and serum-free medium, and then serum-starved for 4–6 h. Cells were then treated with recombinant TGFb1 (10 ng/mL, R&D Systems 240-B-002) or vehicle for 1, 24, or 48 h in DMEM containing 0.5% serum or left in 2.5% serum medium overnight before administering 50 nM Rapamycin (Millipore Sigma- 553211) for 24 h in DMEM + 2.5% serum. For exogenous DNA transfection experiments, cells were transfected with 1 ug/mL Herring testis (HT) dsDNA (Millipore Sigma-D6898) for 24 h using Lipofectamine™ 3000 (Thermo Fisher Scientific) or Lipofectamine alone, and then administered TGFb1 for 48 h. 2.11 siRNA transfection For IRF3 silencing, control and Trf2T118A VSMCs were transfected with 50 nM of SMARTPool Irf3 (L-041095-00-0005, Horizon) or non-targeting control siRNAs (D-001810-10-05, Horizon) with Lipofectamine RNAiMAX (Thermo Fisher Scientific), cultured for 48 h, and administered recombinant TGFb1 or vehicle for 48 h. 2.12 Statistics RNA-seq data statistical analysis is described in Supplementary Material online. GraphPad Prism 9.00 (GraphPad Software Inc.) was used for other statistical analysis. After outlier identification (ROUT method Q = 1%) and Shapiro–Wilk test for normality distribution, statistical significance was de termined by unpaired Student’s t-test for normally distributed data with similar SDs, or Welch’s t-test without similar SDs. Mann–Whitney test was used for data that were not normally distributed. One-way ANOVA followed by Tukey’s or Bonnferroni’s multiple comparison was used for comparisons of more than two groups. Kruskal–Wallis H-test with Dunn’s multiple comparisons test was used if data were not normally dis tributed, as detailed in figure legends. Experimental reproducibility was achieved by the following actions: (i) the operator conducting carotid liga tion surgery was blinded to mouse genotype, and (ii) tissue sections were analysed blindly. Confounding factors were reduced by (i) wild-type litter mates from Sm22a-Trf2T188A mice were used as controls, (ii) both geno types were housed in the same cages during colony expansion and experiments, and (iii) underwent carotid ligation surgery and tissue collec tion on the same day. All data are shown in dot plots to demonstrate data distribution and represent individual biological not technical replicates. Data are presented as mean ± SD and statistically significant difference considered as P < 0.05. 3. Results 3.1 Models of human VSMC senescence We examined gene expression in two models of human VSMC senescence characterized by persistent telomere DNA damage. Human VSMCs trea ted with doxorubicin for 24-h manifest widespread DNA damage; cells normalize global DNA damage markers after 21-day recovery, but telo mere damage persists with reduced TRF2 expression.10 Similarly, cultured human VSMCs progressively shorten telomeres and ultimately enter rep licative senescence (RS). We have previously shown that Dox 1d + 21d treatment or RS results in <10% EdU incorporation over 48 h, no increase in cell number over 14d, and >70% senescence-associated beta- galactosidase’ (SAβG) expression.23 These protocols also reduced Lamin B1 and increased p16 expression, established markers of senescence, but not p53 or p21 DNA damage markers (see Supplementary Material online, Figure S1A and B). 3.2 VSMC senescence induces genes associated with a de-differentiated ‘fibromyocyte’ phenotype Human primary passage 4–5 ascending aortic VSMCs from four donors (two male, two female; average age, 65years) underwent Dox 1d, Dox 1d + 21d, or RS treatments, followed by bulk RNA-seq to compare gene expression against Control 1d (replicating) cells. 126 genes showed statis tically significant increased or decreased expression after both Dox 1d + 21d and RS compared with Control 1d replicating cells, but were not affected by acute DNA damage (Dox 1d) (Figure 1A, Supplementary Material online, Table S3). Gene Ontology (GO) analysis (P-adjusted < 0.05) showed enrichment of pathways associated with extracellular matrix (ECM) organization and stress responses after Dox 1d + 21d and RS. Up-regulated genes were also significantly associated with cell adhesion while down-regulated genes were significantly associated with cell cycle, DNA replication, response to DNA damage, and DNA repair, consistent with senescence (Figure 1B, Supplementary Material online, Table S4). Dox 1d + 21d and RS up-regulated some genes previously associated with VSMC or fibroblast senescence, including p16/CDKN2A (LogFC 1.5– 1.6), intercellular adhesion molecule 1 (ICAM1, LogFC 2.1–2.3), transmem brane protein 178B (TMEM178B) and secreted frizzle-related protein 4 (SFRP4)10,24,25 (Figure 1C, Supplementary Material online, Table S3), but im portantly several genes associated with a de-differentiated ‘fibromyocyte’ VSMC subset,19 including tumour necrosis factor receptor superfamily member 11b (TNFRSF11B)/osteoprotegerin (LogFC 2.0–2.5) and fibromo dulin (FMOD, LogFC 1.3–1.6) (Figure 1C, Supplementary Material online, Table S3), which have been implicated in fibrous cap formation,19 suggest ing that senescence may affect VSMC phenotypic switching. 3.3 Validation and expression of SAGs in human atherosclerotic plaques TNFRSF11B, FMOD, and SFRP4 were included for further study as they have been identified as genes expressed by fibromyocytic VSMCs (TNFRSF11B, FMOD) and/or have a role in atherosclerosis (TNFRSF11B,26,27,28 FMOD,29 SFRP4,30,31) or up-regulated in fibroblast senescence (TMEM178B, SFRP4).25 5–7 additional human VSMC isolates were used to validate changes in these genes in senescence. RS increased both TNFRSF11B and FMOD mRNA levels, and Dox 1d + 21d also increased TNFRSF11B. Although TMEM178B and SFRP4 expression were slightly increased in con trol (1d + 21d) cells, most likely reflecting continued culture, both tran scripts increased very significantly in Dox 1d + 21d and RS vs. Control 1d cells (Figure 1D). These data indicate that TNFRSF11B, FMOD, TMEM178B, and SFRP4 are ‘senescence-associated genes’ (SAGs) in human VSMCs, and that senescence may promote a de-differentiated/fibromyocy tic phenotype in VSMCs. VSMCs expressing senescence markers are located particularly in the fi brous cap in human plaques,2,3 and although TNFRSF11B, FMOD, and SFRP4 proteins are all expressed by VSMCs and fibroblasts in normal hu man vessels, human protein atlas data indicate that endothelial cells can also express TMEM178B (https://www.proteinatlas.org/). Confocal microscopy including Z-series reconstruction showed that all four genes co-localized with αSMA/ACTA2 in human plaques, particularly in fibrous caps, and were not found in luminal ECs (Figure 2A–C). No signals were observed with relevant isotype control antibodies or no antibodies to exclude auto fluorescence (see Supplementary Material online, Figure S2). 3.4 De-differentiation genes, Tmem178b, and Sfrp4 are expressed by mouse VSMCs undergoing senescence Senescence-associated genes (SAGs) vary with species, inducer and cell type and VSMC phenotype markers are often inconsistent across species; VSMC senescence in atherosclerosis 1451 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data https://www.proteinatlas.org/ http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data A C D B Figure 1 Replicative and stress-induced premature senescence of human vascular smooth muscle cells induce genes associated with a de-differentiated phenotype, TMEM178B and SFRP4 . (A) Venn diagram of RNA-seq data showing numbers of genes unique to RS, Dox 1d + 21d, or Dox 1d, or genes common to two or three groups. (B) Chord plots for selected Gene ontology (GO) terms enriched within the up-regulated or down-regulated genes (P-adjusted < 0.05) in cells following Dox 1 + 21d treatment or replicative senescence (RS). (C ) Volcano plots (left and middle) showing differential gene expression in human VSMCs after treatment with Dox 1d + 21d or RS compared to Control 1d, or scatter plot (right). TNFRSF11B, FMOD, TMEM178B, SFRP4, ICAM1, and CDKN2A (p16) are marked. False/True indicates False Discovery Rate (FDR)-adjusted P-value < 0.05, Fold change (FC) relative to control 1d. (D) Transcript levels of TNFRSF11B, FMOD, TMEM178B, and SFRP4 measured by RT-QPCR in human VSMC samples shown relative to Control [1d (n = 5–7 human VSMC isolates)]. Dot plots represent individual samples, mean and SD. 1-Way ANOVA. 1452 A. Kaistha et al. D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 we therefore examined SAG expression in mouse VSMCs undergoing sen escence. Primary mouse VSMCs treated with increasing Dox concentra tions for 1d + 7d recovery (Dox 1 + 7d) show <6% EdU+ and >85% % SAβG+ cells,23 and reduced Lamin B1 expression but increased p16, p53 and p21 (see Supplementary Material online, Figure S3A) consistent with senescence. Tnfrsf11b, Tmem178b, and Sfrp4 mRNA and protein expres sion showed a significant concentration-dependent increase after Dox 1d + 7d vs. vehicle control (Figure 3A–E, Supplementary Material online, A B C Figure 2 De-differentiated phenotype genes, TMEM178B, and SFRP4 are expressed by VSMCs in atherosclerosis. Confocal microscopy images of human plaques co-stained for TNFRSF11B, FMOD, TMEM178B, or SFRP4, together with aSMA/ACTA2 (A), CD31 (B), or CD68 (C ) with 4′,6-diamidino-2-pheny lindole (DAPI) and merged image with Z-series reconstruction. Scale bars = 10μm and 5μm (merged Z-stack). VSMC senescence in atherosclerosis 1453 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data Figure S3B), suggesting that like human VSMCs, DNA damage-induced sen escence promotes a de-differentiated/fibromyocytic phenotype in mouse VSMCs. Increased gene expression in senescent cells can be induced by senes cence itself or paracrine effects of multiple SASP cytokines.10 However, al though the pro-inflammatory stimulus (lipopolysaccharide) administered A E F G H I J B C D K L M N Figure 3 VSMC de-differentiation genes, Tmem178b, and Sfrp4 are induced in mouse VSMC senescence. (A–D) Transcript levels of Tnfrsf11b, Fmod, Tmem178b, and Sfrp4 measured by RT-QPCR in mouse VSMCs treated with increasing concentrations of Dox (1d + 7d) relative to vehicle control. n = 3–6, one-way ANOVA. (E) Western blot for TNFRSF11B, FMOD, TMEM178B, and SFRP4 for cells in (A–D). (F ) % SAβG+ mouse VSMCs after control or Dox 1d + 7d treatment, ± ABT-263 for 48 h; n = 5, unpaired t-test (G–J) Fold change in mRNA expression ± ABT-263 treatment against the housekeeping gene Hmbs for Tnfrsf11b (G), Fmod (H ), Tmem178b (I ), and Sfrp4 (J ); n = 4–5, unpaired t-test. (K–N) Transcript levels of Dcn, Lum, Mgp, and Tcf21 measured by RT-QPCR in mouse VSMCs treated with increasing concentrations of Dox (1d + 7d) relative to vehicle control. n = 6, one-Way ANOVA. 1454 A. Kaistha et al. D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data for 1d or 1d + 7d recovery significantly increased Il6 mRNA, Cdkn2a/p16, Tnfrsf11b, Tmem178b, Sfrp4, and Fmod mRNA expression were unchanged (see Supplementary Material online, Figure S4), suggesting that their induc tion is not caused by external pro-inflammatory signals. To examine whether these genes are preferentially expressed in senescent VSMCs, we removed these cells using the senolytic ABT-263.8,32,33 SAβG+ VSMCs were very infrequent in control cultures and ABT-263 did not af fect Tnfrsf11b, Fmod, Tmem178b, and Sfrp4 expression; however, ABT-263 significantly reduced %SAβG+ VSMCs (Figure 3F, Supplementary material online, Figure S5), and Tnfrsf11b, Tmem178b, and Sfrp4 mRNA expression in Dox 1d + 7d samples (Figure 3G–J). Finally, we examined a range of add itional ‘de-differentiation’ genes in mouse VSMCs after Dox 1d + 7d treat ment. Lum, Mgp, and Tcf21 were also significantly induced by Dox 1d + 7d (Figure 3K–N), together indicating that senescence of mouse VSMCs also upregulates de-differentiation/fibromyocytic genes. 3.5 Expression of senescence genes in VSMCs in human and mouse atherosclerosis To assess whether these SAGs are also expressed in senescent VSMCs in vivo, we examined a previously published human coronary plaque scRNA-seq dataset from 4 separate donors19 (see Supplementary Material online, Figure S6A–D). Typical contractile VSMC markers (MYH11, ACTA2, CNN1, and TAGLN) were highly expressed in the (con tractile) VSMC cluster and reduced in the Fibromyocyte cluster (see Supplementary Material online, Figure S6A–C). TNFRSF11B, FMOD, and SFRP4 were mostly expressed in the Fibromyocyte population, which also expressed CDKN2A/p16, MGP, FN1, COL1A1, and COL3A1, while LUM and DCN were expressed in both Fibroblasts and Fibromyocyte clus ters. Correlation analysis confirmed that p16/CDKN2A expression positive ly correlates with TNFRSF11B, FMOD and SFRP4 (see Supplementary Material online, Figure S6D) but not the VSMC contractile markers. TMEM178B was not detected. We also examined a scRNA-seq dataset of lineage-traced VSMC-derived plaque cells from ApoE−/− mice expres sing the Confetti reporter induced by a Myh11-driven recombinase (Myh11-CreERt2/Confetti/ApoE−/−)16 (see Supplementary Material online, Figure S7). Contractile VSMC markers (Myh11, Acta2, Cnn1, and Tagln) were expressed in most clusters, but reduced in Cluster 6, Cluster 9 (which also expressed the chondrocytic markers chondroadheren (Chad) and Sox9), and Cluster 11 (which also expressed Cd68) (see Supplementary Material online, Figure S7). Tnfrsf11b, Fmod, Sfrp4, Lum, Dcn, Fn1, Col1a1, and Col3a1 were also mostly expressed in Clusters 6 and 9, and Cdkn2a/p16 in Clusters 6, 8, 9, and 11 (see Supplementary Material online, Figure S7). Tmem178b was not detected. Expression of multiple de-differentiation marker genes with Cdkn2a in Clusters 6 and 9 may therefore represent VSMCs with a de-differentiated phenotype, and some of which are senescent VSMCs. This analysis suggests that several senescence and ‘fibromyocyte’ associated genes are expressed together in human and mouse atherosclerotic plaques. 3.6 Senescence promotes de-differentiated/ ’fibromyocyte’ VSMCs in atherosclerosis To directly examine the effects of VSMC senescence on VSMC phenotypes in vivo, we used a mouse model of accelerated VSMC senescence due to telomere damage. Sm22a-Trf2T188A mice express a TRF2 point mutant dri ven by the arterial VSMC-specific minimal Sm22a promoter whose activity is maintained during phenotypic switching,34 and, unlike the full-length Sm22a promoter, is not expressed in bone marrow, peripheral blood cells or spleen.4 Cultured Sm22a-Trf2T188A VSMCs show normal proliferation initially but undergo progressive telomere damage during replication and cells undergo premature senescence.3,10 Sm22a-Trf2T188A/ApoE−/− were crossed with Myh11-CreERt2/Confetti/ApoE−/− mice to generate Myh11-CreERt2/Confetti/Sm22a-Trf2T188A/ApoE−/− (Trf2T188A/ApoE−/−) or Myh11-CreERt2/Confetti/ApoE−/− (ApoE−/−) littermate control mice, admi nistered tamoxifen to label VSMCs, and fed high fat diet for 14w. Blood pressure and serum lipids were similar between Trf2T188A/ApoE−/− and ApoE−/− mice, but serum IL1B and CXCL1 were increased or borderline increased (P = 0.05), respectively, in Trf2T188A/ApoE−/− mice (see Supplementary Material online, Table S5). Absolute and relative aortic root atherosclerosis areas and necrotic core and cap areas were analysed on serial sections across the valve (Figure 4A-4D). Atherosclerosis areas were significantly increased in Trf2T188A/ApoE−/− vs. ApoE−/− mice, with no overall change in core or cap areas (Figure 4A–D). However, despite similar numbers of Confetti-positive cells in the fibrous cap (see Supplementary Material online, Figure S8), aSMA/ACTA2-positive cells were reduced and TNFRSF11B-positive cells increased in caps in Trf2T188A/ApoE−/− vs. ApoE−/− mice. This analysis suggests that although Trf2T188A/ApoE−/− mice show similar VSMC numbers in fibrous caps, these cells have reduced contractile and increased fibromyocyte marker protein expression. We generated scRNA-seq data from dissected plaques and identified 14 clusters of VSMC-derived cells after subsetting Confetti+ cells (929 cells from Trf2T188A/ApoE−/− and 1502 from control animals) (Figure 5A, Supplementary Material online, Table S6 and Supplementary material online, Figure S9). Clusters 12 and 13 had <25 cells and were not consid ered further. Typical contractile VSMC markers (Myh11, Cnn1, Acta2, and Tagln) were detected at similarly high levels in Clusters 0, 1, 6, 9, and 11 (Figure 5B and C, Supplementary Material online, Table S7). In contrast, Clusters 2 and 8 showed lower contractile marker expression and higher expression of de-differentiation/fibromyocyte markers (Tnfrsf11b, Fmod, Sfrp4, Lum, Dcn, Fn1, Col1a1, and Col3a1) and Cdkn2a, suggesting that these clusters contain VSMCs with a de-differentiated phenotype and senescent VSMCs (Figure 5B and C, Supplementary Material online, Table S7). We as sessed co-expression between fibromyocyte and senescence marker genes using Spearman’s rank correlation. There was significant positive correlation (FDR > 0.05) in expression of 52 of 60 possible gene-gene combinations, suggesting that senescence and fibromyocyte markers are also co-expressed by the same cells. Specifically, correlation analysis con firmed that p16/Cdkn2a expression correlates with Tnfrsf11b, Fmod, and Sfrp4 (see Supplementary Material online, Figure S10A). Differential gene expression analysis between Trf2T188A/ApoE−/− and ApoE−/− VSMCs showed reduced Acta2 and Tagln expression in Trf2T188A/ApoE−/− VSMCs in Cluster 2, and increased Lum and Dcn in Trf2T188A/ApoE−/− VSMCs in Cluster 8 (Figure 5D, Supplementary Material online, Table S7). GO analysis of genes with reduced expression in Trf2T188A/ApoE−/− vs. ApoE−/− VSMCs showed enrichment for actin-filament-based processes in Cluster 2 and muscle contraction or development in both Clusters 2 and 8, respectively, whereas tissue migration and regulation of inflammatory responses were enriched for genes with increased expression in Trf2T188A/ApoE−/− com pared to control mice in Cluster 8 (see Supplementary Material online, Figure S10B, Supplementary material online, Table S8). Overall, these data indicate that senescence reduces VSMC expression of contractile markers and increases expression of de-differentiation/fibromyocyte markers in vivo in atherosclerosis. 3.7 Senescence promotes a de-differentiated/’fibromyocyte’ VSMC state after artery ligation In addition to senescence, VSMCs in atherosclerosis are continually exposed to inflammation and mitogens, stimuli that promote de- differentiation; it is therefore unclear whether senescence enhances de- differentiation, reduces re-differentiation or both. We therefore used carotid artery ligation, an established and less complex model of transient VSMC phenotypic switching that exhibits re-expression of contractile genes 28 days after surgery,16,35 when Trf2T188A mouse lesions also show increased senescence markers.10 Trf2T188A mice showed increased neointi ma formation, but a smaller proportion of Confetti+ and aSMA/ACTA2 + Confetti+ cells compared to total neointimal cells, and higher proportion of aSMA/ACTA2−Confetti− cells (see Supplementary Material online, Supplementary material online, Figure S11A), consistent with previous studies showing increased inflammatory infiltrate.10 Control and VSMC senescence in atherosclerosis 1455 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data http://academic.oup.com/cardiovascres/article-lookup/doi/10.1093/cvr/cvaf102#supplementary-data A B D E C Figure 4 VSMC senescence promotes atherosclerosis and reduces ACTA2 + cells in the fibrous cap. (A) Aortic root atherosclerosis in Myh11-CreERt2/ Confetti/ApoE−/− (ApoE−/−) or Myh11-CreERt2/Confetti/Sm22a-Trf2T188A/ApoE−/− (Trf2T188A/ApoE−/−) mice fed a high fat diet from 8-22w stained with H + E or Masson’s, or immunohistochemistry for aSMA/ACTA2 or TNFRSF11B. Dashed lines in insets indicate fibrous caps, and arrows indicate positive cells. Scale bar = 200 µm in lower and 50 µm in high power views. (B–C) Quantification of plaque area under the curve (AUC) at increasing distance across the aortic valve (B) and aortic root plaque area/total aortic root area (C ). (D) Necrotic core or fibrous cap area in ApoE−/− or Trf2T188A/ApoE−/− mice. (E) % aSMA/ACTA2-positive or TNFRSF11B-positive cells in fibrous caps in ApoE−/− or Trf2T188A/ApoE−/− mice (n = 8–14, unpaired t-test). 1456 A. Kaistha et al. D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 A C D B Figure 5 Expression of lineage and de-differentiation markers in mouse atherosclerosis. (A) UMAP of VSMCs isolated from atherosclerotic plaques of high fat-fed control Myh11-CreERt2/Confetti/ApoE−/− (ApoE−/−) or Myh11-CreERt2/Confetti/Sm22a-Trf2T188A/ApoE−/− (Trf2T188A/ApoE−/−) mice showing clustering (top panel) or labelled by genotype (lower panel). (B) Dot plot of selected contractile (Myh11/Cnn), senescence (Cdkn1a/Cdkn2a) and fibromyocyte/de- differentiation markers (Tcf21/Tnfrsf11b/Fmod/Srfrp4/Lum/Mgp). Dot size represents the fraction of cells in each cluster that express the gene. Shade of colour represents scaled expression levels. (C ) UMAP showing expression levels of selected genes associated with VSMC subsets, senescence- or fibromyocyte/de- differentiation genes on a log-transformed scale. (D) Violin plot of selected contractile (Acta2, Tagln) or de-differentiation marker (Lum, Dcn) gene expression in Clusters 0, 2, and 8 for Trf2T188A/ApoE−/− (green) and control ApoE−/− mice (orange). *adjusted P < 0.05, Wilcoxon Rank Sum test, |log2 FC|>0.25. VSMC senescence in atherosclerosis 1457 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 Trf2T188A mice also showed similar absolute numbers of neointimal Confetti+ cells, but Trf2T188A mice showed fewer Confetti+ cells expressing aSMA/ACTA2 or MYH11, and more expressing TNFRSF11B (see Supplementary Material online, Supplementary material online, Figure S11B–D). This analysis suggests that although neointimal VSMC num bers are similar, Trf2T188A VSMCs show reduced contractile and increased fibromyocyte marker protein expression. ScRNA-seq analysis of cells from control (n = 4967) and Trf2T188A (n = 5392) mice formed six clusters with contributions from both genotypes, re presenting ECs (Cluster 4), immune cells (Cluster 5) and VSMC-derived cells (expressing Myh11, Cnn1, Acta2, and Tagln; Clusters 0–3) (Figure 6A– C, Supplementary Material Online, Supplementary material online, Table S9, Supplementary Material Online, Supplementary material online, Figure S12). Cdkn2a was detected predominantly in Clusters 1 and 2 (Figure 6B and C). Compared to Cluster 0, Cluster 1 cells had higher expres sion of some de-differentiation-associated genes [Fn1, Col1a1, Col3a1 (Figure 6B,C, Supplementary Material Online, Supplementary material online, Table S10)], while Cluster 2 cells had reduced contractile marker ex pression compared to other VSMC clusters, but increased levels of de- differentiation markers [Fmod, Sfrp4, Mgp, Tnfrsf11b, Lum, Dcn (Figure 6B, C, Supplementary Material Online, Supplementary material online, Table S10)]. Correlation analysis for senescence and fibromyocyte marker genes showed a significant positive correlation in the expression of 49 of 54 possible gene-gene combinations, suggesting their co-expression. Specifically, this analysis confirmed the positive correlation of p16/Cdkn2a with Fmod and Sfrp4 (see Supplementary Material Online, Supplementary material online, Figure S13). Differential gene expression analysis revealed increased expression of de-differentiation-associated genes Lum, Eln, Dcn, Col1a1, Col3a1, and Sfrp4 and lower expression of Myh11 in Trf2T188A vs. wild-type cells in both Clusters 1 and 2, and Eln expression was higher in Cluster 0 in Trf2T188A cells (Figure 6D, Supplementary Material Online, Supplementary material online, Table S10). GO enrichment analysis of genes showing differential expression in Trf2T188A mice suggested differ ences in ECM organization, cartilage development, ossification, and TGFb response/signalling in Cluster 1 and Cluster 2 cells and acute inflammatory response in Cluster 1 (Figure 6E, Supplementary Material Online, Supplementary material online, Table S11). Overall, these data indicate that senescence results in VSMCs with reduced contractile markers and in creased de-differentiation/fibromyocyte markers after injury, and that this might be related to perturbed TGFb signalling. 3.8 Trf2T188A VSMCs show reduced re-differentiation to a contractile phenotype To understand the relative contribution from enhanced de-differentiation vs. reduced re-differentiation, we examined contractile marker expression in uninjured aortas and cultured VSMCs of Sm22a-Trf2T188A and wild-type mice, and response of cultured VSMCs to TGFb or rapamycin, potent in ducers of re-differentiation in VSMCs in culture. Myh11, Smtn, Tagln, and Acta2 transcripts were detected at similar levels in uninjured aortas and cultured VSMCs of Trf2T188A vs. control mice (Figure 7A and B). However, upregulation of Myh11, Cnn1, Smtn and Acta2 after TGFb or rapamycin treatment was significantly blunted in Trf2T188A VSMCs vs. control cells (Figure 7C, Supplementary material online, Figure S14A), and upregulation of Tnfrsf11b, Tmem178b and Sfrp4 were significantly enhanced by TGFb (see Supplementary material online, Figure S14B). These data suggest that whereas DNA damage in VSMCs does not significantly affect VSMC phenotype in intact aortas (where cells are not proliferating) or initial de-differentiation of contractile VSMCs in culture, the ability of de-differentiated VSMCs to re-express contractile markers is impaired. We therefore examined receptor levels and expression of a range of intracellular regulators of Tgfb. Tgfb signalling occurs via both canonical and non-canonical pathways (see Supplementary Material Online, Supplementary material online, Figure S15A), the latter via activation/ phosphorylation of p38, ERK1/2 and AKT kinases, but expression/ phosphorylation of these kinases was similar in Trf2T188A vs. VSMCs (see Supplementary Material Online, Supplementary material online, Figure S15B). However, multiple components of the canonical (Smad) path way were different between genotypes. For example, compared to control VSMCs, Trf2T188A had reduced levels of ligand (Tgfb1), transmembrane re ceptors (Tgfbr2 and Tgfbr1/Alk5, that transduce Tgfb-mediated expression of contractile genes in human VSMCs36), and the transcriptional regulators myocardin (Myocd) and p300.37,38 In contrast, transcriptional co-repressors Ski and SnoN39 were significantly up-regulated in Trf2T188A vs. control VSMCs (Figure 7D). Western blots further showed that adminis tered TGFb significantly up-regulated TGFBR2 and total and phosphory lated forms of TGFBR1/ALK5 in control but not Trf2T188A VSMCs, and similarly for SMAD2 phosphorylation, an early event following TGFb bind ing, MYOCD, and p300. In contrast, the active histone modification mark H3K9ac and MYH11 protein levels were down-regulated in Trf2T188A VSMCs (Figure 7E, Supplementary Material Online, Supplementary material online, Figure S16A). These results suggest that dysregulation of multiple components of canonical Tgfb signalling in Trf2T188A VSMCs could underlie their inability to re-express contractile markers after TGFb treat ment (Figure 7F). 3.9 Trf2T188A VSMCs show cytoplasmic DNA and activation of STING/TBK1/IRF3 We have previously shown that human VSMCs expressing Trf2T188A show cytoplasmic DNA and activation of cGAS/STING/TBK1 intracellular DNA sensing pathways.10 cGAS/STING-induced activation of interferon-related factor (IRF3), the histone methyltransferase Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), and epigenetic modula tion of VSMC contractile protein expression are all implicated in aortic an eurysm formation, which also shows DNA damage and VSMC phenotypic modulation.40 Activated IRF3 can also suppress Tgfb/SMAD signalling by preventing the association of SMAD proteins with Tgfb receptors and forming functional Smad transcriptional complexes.41 Cytoplasmic dsDNA expressing γH2AX was evident in Trf2T188A but not control VSMCs (Figure 7G), associated with upregulation of Sting, Tbk1 and Irf3 transcripts (Figure 7H). Both total and phosphorylated STING, TBK1, and IRF3 proteins were also up-regulated in Trf2T188A VSMCs cells and ex ogenous TGFb treatment did not alter their expression levels, suggesting pathway activation irrespective of TGFb treatment. In addition, TGFb treat ment increased EZH2 and the repressive H3K27me3 epigenetic mark that it induces42 in Trf2T188A but not control VSMCs (Figure 7I, Supplementary Material Online, Supplementary material online, Figure S16B). To examine the role of IRF3 in the impaired re-differentiation response to TGFb in Trf2T188A VSMCs, we transiently transfected Trf2T188A VSMCs with Irf3 or control siRNA before TGFb administration. Silencing Irf3 sig nificantly increased TGFb-induced expression of contractile marker tran scripts (Figure 7J), Smad2 phosphorylation and protein expression of MYOCD, p300 and MYH11 (Figure 7K, Supplementary Material Online, Supplementary material online, Figure S17). These results suggest that re sistance of Trf2T188A VSMCs to TGFb-induced re-differentiation may be due to Irf3-induced repression of contractile proteins directly (Figure 7L) and/or TGFb receptors. Finally, we examined whether cytoplasmic DNA without senescence could directly regulate the expression of VSMC contractile genes in re sponse to Tgfb. Exogenous DNA transfection reduced TGFb-mediated upregulation of Myh11, Smtn, Tagln, Acta2 without inducing senescence (p16, Lamin b1) or global DNA damage (p21, p53) markers in the whole population (see Supplementary Material Online, Supplementary material online, Figure S18). This suggests that cytoplasmic DNA, for example from DNA damage, can induce phenotypic switching even without senescence. 4. Discussion We examined how DNA damage and senescence changes VSMC pheno type after replicative senescence (RS) of human VSMCs, doxorubicin 1458 A. Kaistha et al. 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(A) UMAP of carotid artery cells isolated 28 days post ligation of control or Trf2T188A mice showing clustering (top) or labelled by genotype (lower panel). (B) Dot plot of lineage and de-differentiation markers. Dot size re presents fraction of cells in each cluster that express the gene. Shade of blue represents scaled expression levels. (C ) UMAP showing expression levels of genes associated with VSMC subsets, including selected contractile (Myh11, Cnn1), senescence− (Cdkn2a) or fibromyocyte/de-differentiation (Tnfrsf11b, Sfrp4, Lum) genes on a log-transformed scale. (D) Violin plot of selected contractile (Myh11) or fibromyocyte/de-differentiation marker (Sfrp4/Lum/Dcn/Eln/Col1a1/Col3a1) gene expression in Clusters 0–3 for Trf2T188A (green) and control mice (orange). Adjusted *P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon Rank Sum test, |log2 FC|>0.25. (E) Chord plots for selected Gene Ontology terms that are enriched in up- or down-regulated genes after carotid artery ligation in TRF2T188A vs. control mouse VSMCs in Clusters 1 and 2. *adjusted P < 0.05, Wilcoxon Rank Sum test, |log2 FC|>0.25. VSMC senescence in atherosclerosis 1459 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 A B C D G H I J K L E F Figure 7 Senescence induces resistance to TGFb-mediated re-differentiation of VSMCs. (A–B) mRNA expression of VSMC contractile genes in aortas (A) or cultured VSMCs (B) derived from littermate control (wild-type) or Trf2T188A mice. (C ) mRNA expression of contractile genes in cultured VSMCs from control or Trf2T188A mice treated with 10 ng/mL TGFb for 48 h vs. control treatment. (D) RT-QPCR for Tgfb, Tgfbr1/Alk5, Tgfbr2, and selected downstream regulators of Tgfb signalling. (E) Representative Western blot of Tgfb receptors, phospho-SMAD2, Myocardin, p300 and MYH11 in TGFb-treated VSMCs from control and Trf2T188A mice. (F ) Schematic depicting changes in canonical Tgfb signalling in Trf2T188A VSMCs. (G) Immunocytochemistry of γH2AX+ cytosolic dsDNA (arrows) in control or Trf2T188A VSMCs. Scale bar = 5 µm. (H ) RT-QPCR of STING pathway genes in VSMCs from control or Trf2T188A mice. (I ) Representative Western blot of STING pathway components and H3K27me3 modification in control and Trf2T188A VSMCs. (J ) RT-QPCR for VSMCs contractile markers, Myocd and p300 in Trf2T188A VSMCs treated with Irf3-targeting or control siRNA followed by 10 ng/mL TGFb vs. control treatment. (K ) Representative Western blot showing SMAD2 phosphorylation, MYOCD, p300 and MYH11 expression after treatment in (J ). (L) Schematic illustrating components of cGAS/STING pathway downregulating VSMC contractile genes in Trf2T188A VSMCs. Data are means (SD) n = 5–7. Unpaired t-test. 1460 A. Kaistha et al. D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 treatment and recovery (D + R) in human and mouse VSMCs, and TRF2T118A in mouse VSMCs in vitro and in vivo. Our important findings are as follows: (i) D + R and RS of human VSMCs induce many common genes, including de-differentiation/’fibromyocyte’ markers (TNFRSF11B, FMOD) and genes expressed by human fibroblast senescence (TMEM178B, SFRP4): (ii) D + R induces Tmem178b, Sfrp4, and Tnfrsf11b in mouse VSMCs; (iii) SFRP4, TNFRSF11B and FMOD are expressed in hu man atherosclerotic plaques predominantly by VSMCs; (iv) SFRP4, TNFRSF11B, FMOD and CDKN2A are expressed in scRNA-seq clusters re presenting de-differentiated and senescent VSMCs in human and mouse plaques in vivo; (v) Sm22a-Trf2T188A mice have increased atherosclerotic plaque size and neointimal areas after arterial injury, but similar VSMC numbers in fibrous caps or injury-induced neointimas compared to con trols; however, VSMCs at these sites have reduced aSMA/ACTA2 and MYH11 and increased TNFRSF11B protein expression; (vi) Trf2T188A ex pression affects expression of multiple genes associated with Tgfb signal ling, and reduces canonical TGFb signalling and the resulting induction of VSMC contractile markers after TGFb treatment; (vii) Trf2T188A VSMCs have cytosolic dsDNA and activation of the STING/TBK1/IRF3 pathway, while silencing IRF3 upregulates VSMC contractile gene expression after TGFb treatment; (viii) cytosolic dsDNA can downregulate contractile pro teins even without senescence. Our data suggest that DNA damage and premature senescence cause VSMCs to maintain a de-differentiated phenotype, and that this may be partly due to impaired re-differentiation to a contractile phenotype. 4.1 Effect of cell senescence on VSMC phenotypic switching DNA damage and senescent VSMCs accumulate during atherogenesis, and particularly in advanced lesions, most likely due to repeated rounds of rep lication, inflammation and cellular stress. VSMC de-differentiation is also seen in culture and during atherogenesis and early arterial injury, while par tial re-differentiation can occur with the withdrawal of de-differentiation stimuli such as mitogens or oxidized lipids. It is therefore difficult to separ ate a direct effect of senescence on VSMC phenotypic switching from those due to common stimuli. However, our data shows that VSMC sen escence has a direct effect on expression of contractile and de-differentiated VSMC markers and impairs re-differentiation, associated with activation and repression of specific intrinsic cell signalling pathways. Senescence reduces VSMC proliferation and migration and induces in flammation, all of which may affect phenotypic switching.3,4,10,11 However, Trf2T188A did not reduce VSMC contractile marker expression in uninjured aortas or during de-differentiation in vitro, suggesting that Trf2T188A-induced DNA damage does not affect initial proliferation and de- differentiation of VSMCs. In contrast, Trf2T188A VSMCs showed reduced upregulation of many contractile markers in vitro after TGFb or rapamycin-induced re-differentiation. While confirmation of this finding in vivo will require detailed re-differentiation protocols and suppression of DNA damage signalling in senescent VSMCs, our data suggest that pro gressive DNA damage during replication may affect VSMC re- differentiation, causing persistence of de-differentiated VSMCs. 4.2 Fibromyocyte genes as senescence markers and potential targets in atherogenesis We identified TNFRSF11B, FMOD, TMEM178B and SFRP4 as ‘senescence-associated genes’, and significant correlation in the expression of many possible fibromyocyte and senescence gene-gene combinations in our scRNA-seq analysis; however, these 4 genes do not represent a specif ic VSMC ‘senescence signature’. Several studies have attempted to identify senescent cells based on gene expression data, including gene panels (e.g. SenMayo42 and Sencid43). However, panel performance has profound vari ation,43 indicating that senescent cell transcriptomes (e.g. in scRNA-seq da tasets) are highly context-dependent, with considerable differences in expression signatures between cell types, disease state and severity. Furthermore, these 4 genes are up-regulated during VSMC phenotypic switching from multiple other stimuli and may have roles in atherogenesis and plaque stability that are not related to senescence. For example, in mice Tnfrsf11b can inhibit atherogenesis and promote fibrous cap forma tion,26,27,28 fibromodulin can promote LDL accumulation in plaques29 and Sfrp4 reduces inflammation, oxidative stress, and plaque forma tion.30,31 In humans, both SFRP4 and FMOD are related to ruptured pla ques,44 but FMOD was reduced in ruptured vs. stable plaques while SFRP4 was unchanged, and TNFRSF11B serum protein levels are associated with increased extent and progression of atherosclerosis and events (re viewed in45). TMEM178B is up-regulated in senescence of cultured human fibroblasts,25 but its function is unknown, although it may be a useful sen escence marker in cultured human VSMCs. 4.3 DNA damage, cytoplasmic dsDNA and phenotypic modulation Loss of lamin B1 in senescence results in cytoplasmic leakage of chromatin fragments, which is sensed by cGAS, resulting in activation of cGAS/ STING/TBK1/IRF3 pathways.46 We find increased expression of STING, TBK1 and IRF3 transcripts and upregulation of their total and phosphory lated protein forms in Trf2T188A VSMCs, suggesting that DNA damage ac tivates this pathway, and show that IRF3 suppresses Tgfb signalling and contractile gene expression in Trf2T188A VSMCs. IRF3 shows structural similarity with SMAD proteins and interacts with inactive SMADs, inhibiting their activation/phosphorylation induced by Tgfb receptors.41 IRF3 also uses p300 as a transcriptional co-activator and competes for its use,41 su pressing p300 and MYOCD availability and interacting with EZH2 to in duce repressive epigenetic changes on contractile gene promoters. Although silencing IRF3 increases contractile gene expression in Trf2T188A VSMCs, manipulating IRF3 may not be useful therapeutically. IRF3 regulates the Type I interferon response that prevents/limits virus infections,47 and STING can also promote neointima formation via proliferation, migration, and phenotypic switching in VSMCs through NF-kB signalling.48 4.4 Limitations of our study Dox1d + 21d and RS induced many similar genes. However, our studies do not identify whether VSMC DNA damage and senescence in atheroscler osis is due to RS or stressors such as oxidative stress or lipids, or both. Our findings also differ from other studies suggesting that senescent cells re duce VSMC migration through soluble growth factor inhibitors such as IGFBP3,9 while we find similar numbers of VSMCs in fibrous caps, and IGFBP3 was not induced by senescent human VSMCs. Our atherosclerosis, ligation, and re-differentiation studies utilized Sm22aTRF2T188A mice and VSMCs, which may not completely recapitulate the phenotype of senes cent VSMCs in vivo, and TRF2T188A might also be expressed in cells from other lineages that adopt a SMC-like phenotype in vivo, including endothe lial cells (EndoMT) and bone marrow-derived cells.49 However, similar to TRF2T188A VSMCs, human plaque VSMCs show extensive telomere damage and premature senescence,2 and lineage tracing bone marrow-derived cells expressing specific SMC promoters showed only very rare cells in ad vanced lesions, and not in the fibrous cap.49 In addition, although we cannot exclude the possibility that Sm22a-Trf2T188A subtly affects SMC progenitor differentiation and arterial development, we find no difference in the ex pression of contractile or de-differentiation markers in adult vessels before fat feeding or injury. Finally, we find that CDKN2A expression (and by im plication senescence) is low in human and mouse atherosclerosis and mouse carotid injury artery datasets compared with VSMC contractile and de-differentiated genes. However, scRNA-seq may underestimate cel lular senescence, in part because senescent cells are under-represented due to their larger size, increased sensitivity to extraction conditions, are cleared by both killing and phagocytosis, and CDKN2A upregulation in sen escence is mostly post-translational. VSMC senescence in atherosclerosis 1461 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 Translational perspective Risk factors for atherosclerosis such as smoking, diabetes and hyperlipidaemia induce DNA damage and senescence in vascular smooth muscle cells (VSMCs), both of which promote atherogenesis and features of unstable plaques. Senescence causes VSMCs to maintain a de-differentiated/fibromyo cyte phenotype in atherosclerosis and after injury, associated with cytosolic DNA, dysregulation of multiple Tgfb signalling proteins, and resistance to re-differentiation. Preventing senescence may delay atherogenesis and promote plaque stability, in part by promoting re-differentiation to contractile VSMCs. 5. Conclusions We find that senescence downregulates contractile genes and upregulates genes associated with a de-differentiated fibromyocyte phenotype in hu man and mouse VSMCs and maintains VSMCs expressing de-differentiated markers in vivo. Resistance to TGFb-mediated re-differentiation to a con tractile phenotype due to cytosolic DNA-mediated activation of the cGAS/STING/TBK1/IRF3 pathway may partly explain how VSMC DNA damage reduces expression of contractile proteins in fibrous caps in ath erosclerotic plaques, potentially promoting plaque instability and neointi mal formation. Supplementary material Supplementary material is available at Cardiovascular Research online. Author contribution A.K. and A.M.G. undertook the cell/molecular biology experiments and im munohistochemistry; S.O., J.C.K.T., M.I. and M.D.W. undertook scRNA-seq experiments and analysis; A.U., M.G., K.F., A.F., and N.F. under took in vivo experiments; H.F.J. and M.R.B. conceived and directed experi ments. All authors were involved in data collection, analysis, and manuscript preparation. Conflict of interest: None declared Funding This work was supported by the British Heart Foundation (BHF, grants RG71070, RG8455, BHF Centres for Research Excellence (RE/18/1/ 34212, RE/24/130011) and Regenerative Medicine (RM/13/3/30159) and the National Institute of Health Research Cambridge Biomedical Research Centre. Data availability The data underlying this article will be shared on reasonable request to the corresponding author. 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Bone marrow-derived smooth muscle-like cells are infrequent in advanced primary atherosclerot ic plaques but promote atherosclerosis. Arterioscler Thromb Vasc Biol 2011;31:1291–1299. VSMC senescence in atherosclerosis 1463 D ow nloaded from https://academ ic.oup.com /cardiovascres/article/121/9/1448/8159934 by U niversity of C am bridge user on 29 August 2025 Premature cell senescence promotes vascular smooth muscle cell phenotypic modulation and resistance to re-differentiation 1. Introduction 2. Methods 2.1 Human tissue 2.2 Animal experiments 2.3 Molecular analysis 2.4 Western blots 2.5 Confocal microscopy 2.6 Immunohistochemistry 2.7 RNA-seq 2.8 Mouse carotid artery ligation 2.9 Mouse atherosclerosis studies 2.10 Re-differentiation and exogenous DNA transfection protocol 2.11 siRNA transfection 2.12 Statistics 3. Results 3.1 Models of human VSMC senescence 3.2 VSMC senescence induces genes associated with a de-differentiated ‘fibromyocyte’ phenotype 3.3 Validation and expression of SAGs in human atherosclerotic plaques 3.4 De-differentiation genes, Tmem178b, and Sfrp4 are expressed by mouse VSMCs undergoing senescence 3.5 Expression of senescence genes in VSMCs in human and mouse atherosclerosis 3.6 Senescence promotes de-differentiated/’fibromyocyte’ VSMCs in atherosclerosis 3.7 Senescence promotes a de-differentiated/’fibromyocyte’ VSMC state after artery ligation 3.8 Trf2T188A VSMCs show reduced re-differentiation to a contractile phenotype 3.9 Trf2T188A VSMCs show cytoplasmic DNA and activation of STING/TBK1/IRF3 4. Discussion 4.1 Effect of cell senescence on VSMC phenotypic switching 4.2 Fibromyocyte genes as senescence markers and potential targets in atherogenesis 4.3 DNA damage, cytoplasmic dsDNA and phenotypic modulation 4.4 Limitations of our study 5. Conclusions Supplementary material Author contribution Conflict of interest Funding Data availability References