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Single-cell and spatial transcriptomics map senescent vascular cells in arterial remodeling during atherosclerosis in mice

Nature Aging volume 5, pages 1528–1547 (2025)Cite this article

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Abstract

Growing evidence suggests that the induction of cellular senescence in vascular cells is causally linked to the etiology of cardiovascular diseases. To investigate systematically the heterogeneity of senescent vascular cells in atherosclerosis, we used a high-fat diet and PCSK9 overexpression to induce atherosclerosis in a senescence reporter mouse model (p16-tdTomato+/−) and performed single-cell RNA sequencing on whole aortas. Using the SenMayo and CellAge gene sets, we identified four clusters of vascular smooth muscle cells (VSMCs), fibroblasts and T cells enriched in features of senescence, which were reduced upon treatment with the senolytic agent ABT-737. We then derived a global senescence signature of atherosclerosis including Spp1, Ctsb and Tnfrsf11b mRNAs. We validated the enrichment of these mRNAs in senescence by using spatial transcriptomics in a second mouse model of atherosclerosis and senolysis (Ldlr−/−; p16-3MR), as well as by analyzing in vitro models of human VSMC senescence. Our results uncover a vascular-specific transcriptomic signature of senescence that may be exploited for tracking and treating age-related vascular diseases.

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Fig. 1: Senolytics improve ECM remodeling during atherosclerosis.
Fig. 2: A subset of aortic cell clusters is enriched for senescence-associated genes.
Fig. 3: Identifying senescent subpopulations in VSMC clusters.
Fig. 4: Identifying senescent subpopulations in fibroblast and T cell clusters.
Fig. 5: Validation of mouse vascular senescence markers in diverse models of vascular senescence.
Fig. 6: Spatial transcriptomic profiling confirms the vascular senescence signature in the cap and core of atherosclerotic plaques.
Fig. 7: smFISH visualization of vascular senescence-associated mRNAs in the atherosclerotic core.
Fig. 8: smFISH visualization of vascular senescence-associated mRNAs in the atherosclerotic cap.

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Data availability

RNA-sequencing data are deposited in the Gene Expression Omnibus under accession nos. GSE239591 and GSE239602. Commercially available reagents and their catalog numbers are listed in Supplementary Table 4. Source data are provided with this paper. All other data will be provided upon request.

Code availability

No unique code was used in this study. All code used in this article is open source and listed, and additional information can be provided upon request.

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Acknowledgements

This work was supported in part by the NIA IRP, NIH and National Science Foundation grant number 2244127 (to M.B.), and the Paul F. Glenn Center for Biology of Aging Research at the Mayo Clinic (B.G.C. and D.J.B.). We would like to thank the sequencing and data processing support from the CCR Genomics Core at the National Cancer Institute/NIH.

Author information

Author notes

  1. These authors contributed equally: Krystyna Mazan-Mamczarz, Dimitrios Tsitsipatis.

Authors and Affiliations

  1. Laboratory of Genetics and Genomics, National Institute on Aging (NIA) Intramural Research Program (IRP), National Institutes of Health (NIH), Baltimore, MD, USA

    Krystyna Mazan-Mamczarz, Angelica E. Carr, Carlos Anerillas, Jordan M. Gregg, Charnae’ Henry-Smith, Ada N. Okereke, Marc Michel, Rachel Munk, Jennifer L. Martindale, Yulan Piao, Jinshui Fan, Supriyo De & Myriam Gorospe

  2. Laboratory of Cardiovascular Science, NIA IRP, NIH, Baltimore, MD, USA

    Dimitrios Tsitsipatis, Carla Rocha Dos Santos, Olga V. Fedorova & Allison B. Herman

  3. Department of Biochemistry and Molecular Biology, Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA

    Bennett G. Childs & Darren J. Baker

  4. Department of Medical and Molecular Sciences, University of Delaware, Newark, DE, USA

    Brigette Romero & Mona Batish

  5. Center for Cancer Research, National Cancer Institute IRP, NIH, Bethesda, MD, USA

    Maria O. Hernandez, Noemi Kedei & Madeline M. F. Wong

  6. Seqmatic LLC, Fremont, CA, USA

    Michael L. Viacheslavov

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Contributions

A.B.H., D.T. and K.M.-M. conceptualized the study; A.B.H., D.T., K.M.-M., B.G.C., N.K., O.V.F., D.J.B., S.D., M.B. and M.G. designed experiments; A.B.H., K.M.-M., D.T., A.E.C., B.R., C.R.D.S., C.A., J.M.G., C.H.-S., A.N.O., M.M., R.M., J.L.M., Y.P., J.F., M.O.H., N.K., M.L.V. and M.M.F.W. performed and analyzed experiments and analyzed data; A.B.H. and M.G. wrote the manuscript.

Corresponding authors

Correspondence to Myriam Gorospe or Allison B. Herman.

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Competing interests

B.G.C. and D.J.B. have potential financial interests related to this research. They are co-inventors on patents held by the Mayo Clinic and patent applications licensed to or filed by Unity Biotechnology. They are also Unity Biotechnology shareholders. Research in the laboratory of D.J.B. has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with the Mayo Clinic conflict of interest policies. The other authors declare no competing interests.

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Nature Aging thanks Minna Kaikkonen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Atherosclerotic plaque pathology analysis and quality control analysis.

a, Quantification of plaque size presented by sex and condition (ND, HFD, and HFD + ABT-737). For female mice, ND (n = 4), HFD (n = 3), and HFD + ABT-737 (n = 2) and HFD vs. ND, p = 0.0006 and HFD + ABT-737 vs. HFD, p = 0.576, whereas for male mice, ND (n = 4), HFD (n = 2), and HFD + ABT-737 (n = 3) and HFD vs. ND, p = 0.0004 and HFD + ABT-737 vs. HFD, p = 0.8876. b, Quantification of necrotic core area presented by sex and condition (ND, HFD, and HFD + ABT-737). For female mice, HFD (n = 3) and HFD + ABT-737 (n = 2) HFD + ABT-737 vs. HFD, p = 0.1929, whereas for male mice, HFD (n = 2) and HFD + ABT-737 (n = 2) and HFD + ABT-737 vs. HFD, p = 0.4985. c, Quantification by plaque fibrous cap thickness presented by sex and condition (ND, HFD, and HFD + ABT-737). For female mice, HFD (n = 3) and HFD + ABT-737 (n = 2) and HFD + ABT-737 vs. HFD, p = 0.2033, whereas for male mice, HFD (n = 2) and HFD + ABT-737 (n = 2) and HFD + ABT-737 vs. HFD, p = 0.4552. d, Representative H&E staining of aortic roots from ND, HFD, and HFD + ABT-737. The scale bar is 200 µm. e, Quantification of collagen fibers (Aniline blue) area / area of aortic root presented by sex and condition (ND, HFD, and HFD + ABT-737). For female mice, HFD (n = 2) and HFD + ABT-737 (n = 2) HFD + ABT-737 vs. HFD, p = 0.2467, whereas for male mice, HFD (n = 2) and HFD + ABT-737 (n = 2) and HFD + ABT-737 vs. HFD, p = 0.7036. f, Representative senescence-associated beta galactosidase (SA-β-gal) activity staining of aortic roots from ND, HFD, and HFD + ABT-737. The scale bar is 200 µm. g, Quantification of SA-β-gal (ND, HFD, and HFD + ABT-737). ND (n = 5), HFD (n = 5) and HFD + ABT-737 (n = 5) and for significance, HFD vs. ND, p ≤ 0.0001 and HFD + ABT-737 vs. HFD, p = 0.069. h, Pulse wave velocity (PWV) measurements presented by sex and condition (ND, HFD, and HFD + ABT-737). For female mice, ND (n = 3), HFD (n = 2), and HFD + ABT-737 (n = 4) and HFD vs. ND, p = 0.748 and HFD + ABT-737 vs. HFD, p = 0.9597, whereas for male mice, ND (n = 3), HFD (n = 2), and HFD + ABT-737 (n = 2) and HFD vs. ND, p = 0.0294 and HFD + ABT-737 vs. HFD, p = 0.1001. i, Quality control table of the scRNA-seq samples including estimated number of cells, fraction reads in cells, mean reads per cell, median UMI counts per cell, median genes per cell, and total genes detected. j, Heatmap of normalized expression levels of classical senescence associated mRNAs either enriched in HFD and reduced by ABT-737 treatment, or conversely, reduced by HFD and increased by ABT-737 treatment in mice (Lmnb1 and Lbr mRNAs). Significance was established using Two-Way ANOVA (for data in graphs a-c, e, and h) and One-Way ANOVA (for data in graph g) with multiple comparisons. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Source data

Extended Data Fig. 2 scRNA-seq analysis of senescence gene expression.

a, UMAPs of p16-tdTomato mRNA expression in ND, HFD, and HFD + ABT-737 treated mice. b, UMAP plot of Cdkn2a mRNA expression across all clusters and conditions (left). Violin plot of Cdkn2a mRNA expression across all clusters (right). c, UMAP plot of Cdkn1a mRNA expression across all clusters and conditions (left). Violin plot of Cdkn1a mRNA expression across all clusters (right). d, GSEA SenMayo plots comparing HFD vs ND and HFD + ABT-737 vs HFD for Clusters 15, 20, 21, 3, 16, and 27. e, GSEA CellAge plots comparing HFD vs ND and HFD + ABT-737 vs HFD for Clusters 6, 17, 7, and 11.

Extended Data Fig. 3 VSMC phenotype analysis.

a, Dot plot representation of the gene expression profile for VSMC phenotype mRNA markers for cluster 0 subclustered VSMCs. b, Dot plot representation of the gene expression profile for VSMC phenotype mRNA markers for cluster 12 subclustered VSMCs.

Extended Data Fig. 4 Analysis of UMI counts, mitochondrial and ribosomal gene expression in VSMC, fibroblast, and T cell clusters with senescence features.

a-c, UMAPs of scRNA-seq UMI counts (a), mitochondrial gene expression (b) and ribosomal gene expression (c) across cluster 0 subclustered VSMCs in ND, HFD, and HFD + ABT-737 treated mice. d-f, UMAPs of scRNA-seq UMI counts (d), mitochondrial gene expression (e) and ribosomal gene expression (f) across cluster 12 subclustered VSMCs in ND, HFD, and HFD + ABT-737 treated mice. g-i, UMAPs of scRNA-seq UMI counts (g), mitochondrial gene expression (h) and ribosomal gene expression (i) across cluster 2 subclustered fibroblasts in ND, HFD, and HFD + ABT-737 treated mice. j-l, UMAPs of scRNA-seq UMI counts (j), mitochondrial gene expression (k) and ribosomal gene expression (l) across Cluster 9 subclustered T cells in ND, HFD, and HFD + ABT-737 treated mice.

Extended Data Fig. 5 Vascular senescence score on individual cell clusters.

a, UMAPs of vascular-senescence scoring across cluster 0 VSMCs in ND, HFD, and HFD + ABT-737. b, UMAPs of vascular-senescence scoring across cluster 12 VSMCs in ND, HFD, and HFD + ABT-737. c, UMAPs of vascular-senescence scoring across cluster 2 fibroblasts in ND, HFD, and HFD + ABT-737. d, UMAPs of vascular-senescence scoring across cluster 9 T cells in ND, HFD, and HFD + ABT-737.

Extended Data Fig. 6 Gene score of the most prominent transcripts expressed in senescent vascular cells.

UMAPs visualization of the expression of the 17 vascular senescent transcripts individually across all clusters. The arrows indicate the senescent cell clusters for VSMCs (V), fibroblasts (F), and T cells (T) as well as non-senescent macrophages (M) in which the transcripts were expressed.

Extended Data Fig. 7 Doxorubicin-induced cellular senescence in wild type mice.

a, RT-qPCR analysis of the levels of Cdkn2a and Lmnb1 mRNAs in aortic roots of mice treated with vehicle, doxorubicin, and doxorubicin followed by ABT-737. b, RT-qPCR analysis of the levels of Ltbp2, Sparc, Mgp, Lum, and Cd9 mRNAs in aortic root tissue as described (a). c, RT-qPCR analysis of the levels of Apoe, Lgals3, Cst3, Tnfrsf11b, and Prg4 mRNAs in aortic root tissue as described (a). d, RT-qPCR analysis of the levels of Mmp3, Ctss, Lcp1, and Comp mRNAs in aortic root tissue as described (a). In a-d, data represent the means ± SD for DMSO, n = 4; Doxo, n = 4; Doxo + ABT-737, n = 3 (females), and DMSO, n = 4; Doxo, n = 4; Doxo + ABT-737, n = 4 (males). n represents biological replicates. In panel (a), Cdkn2a, Doxo vs. DMSO, p = 0.0162 (females) and p = 0.002 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0226 (females) and p = 0.0034 (males); Lmnb1, Doxo vs. DMSO, p = 0.5863 (females) and p = 0.1807 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.1013 (females) and p = 0. 3449 (males). In panel (b), Ltbp2, Doxo vs. DMSO, p = 0.0004 (females) and p ≤ 0.0001 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0007 (females) and p ≤ 0.0001 (males); Sparc, Doxo vs. DMSO, p ≤ 0.0001 (females) and p ≤ 0.0001 (males), whereas Doxo + ABT-737 vs. Doxo, p ≤ 0.0001 (females) and p = 0.0002 (males); Mgp, Doxo vs. DMSO, p = 0.9928 (females) and p = 0.9928 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.7181 (females) and p > 0.9999 (males); Lum, Doxo vs. DMSO, p = 0.0018 (females) and p = 0. 0002 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0018 (females) and p = 0.001 (in males); Cd9, Doxo vs. DMSO, p = 0.0014 (females) and p = 0. 0011 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0031 (females) and p = 0.0019 (males). In panel (c), Apoe, Doxo vs. DMSO, p = 0.0194 (females) and p = 0. 3607 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.5746 (females) and p = 0.4692 (males); Lgals3, Doxo vs. DMSO, p ≤ 0.0001 (females) and p ≤ 0.0001 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0002 (females) and p ≤ 0.0001 (males); Cst3, Doxo vs. DMSO, p = 0.0008 (females) and p ≤ 0.0001 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0021 (females) and p ≤ 0.0001 (males); Tnfrsf11b, Doxo vs. DMSO, p = 0.3051 (females) and p = 0.9339 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.1425 (females) and p = 0.9833 (males); Prg4, Doxo vs. DMSO, p ≤ 0.0001 (females) and p = 0.0177 (males), whereas Doxo + ABT-737 vs. Doxo, p ≤ 0.0001 (females) and p = 0.0283 (males). In panel (d), Mmp3, Doxo vs. DMSO, p ≤ 0.0001 (females) and p ≤ 0.0001 (males), whereas Doxo + ABT-737 vs. Doxo, p ≤ 0.0001 (females) and p ≤ 0.0001 (males); Ctss, Doxo vs. DMSO, p = 0.0007 (females) and p = 0.0025 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0017 (females) and p = 0.005 (males); Lcp1, Doxo vs. DMSO, p ≤ 0.0001 (females) and p ≤ 0.0001 (males), whereas Doxo + ABT-737 vs. Doxo, p ≤ 0.0001 (females) and p ≤ 0.0001 (males); Comp, Doxo vs. DMSO, p = 0.0005 (females) and p = 0.0029 (males), whereas Doxo + ABT-737 vs. Doxo, p = 0.0027 (females) and p = 0.004 (males). Significance was established using One-Way Anova for each sex, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

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Extended Data Fig. 8 Human VSMC senescence validation.

a, Phase contrast micrographs of SA-βgal activity (blue) staining in proliferating (P), replicative senescence (RS), doxo-treated, IR-treated, oxLDL-treated, and CoCl2-treated human VSMCs for 7–10 days. b, RT-qPCR analysis of the levels of CDKN2A and IL8 mRNAs in human VSMCs treated as described in (a). c, RT-qPCR analysis of the levels of LTBP2, SPARC, and MGP mRNAs in human VSMCs treated as described in (a). d, RT-qPCR analysis of the levels of LUM, CD9, APOE, LGALS3, CST3 mRNAs in human VSMCs treated as described in (a). e, RT-qPCR analysis of the levels of TNFRSF11b, PRG4, MMP3, CTSS, and LCP1 mRNAs in human VSMCs treated as described in (a). f, Table of human scRNA-seq metadata analysis from atherosclerotic tissue. Each column represents a VSMC phenotype identified in the metadata analysis, and each column includes the vascular senescent scoring genes that were expressed in the indicated VSMC phenotype. In b-e, data represent the means ± SD for P, n = 3; IR, n = 3; oxLDL, n = 3; CoCl2, n = 3; Doxo, n = 3; RS, n = 3; n represents biological replicates. In panel (b), CDKN2A, IR (p = 0.1334), oxLDL (p = 0.0191), CoCl2 (p = 0.001), Doxo (p = 0.4446), RS (p ≤ 0.0001) vs. P; IL8, IR (p = 0.0007), oxLDL (p = 0.0014), CoCl2 (p = 0.0003), Doxo (p = 0.0008), RS (p = 0.0027) vs. P. In panel (c), LTBP2, IR (p = 0.002), oxLDL (p = 0.0021), CoCl2 (p ≤ 0.0001), Doxo (p ≤ 0.0001), RS (p = 0.4192) vs. P; SPARC, IR (p = 0.2171), oxLDL (p ≤ 0.0001), CoCl2 (p = 0.0002), Doxo (p = 0.0106), RS (p = 0.0019) vs. P; MGP, IR (p = 0.0201), oxLDL (p = 0.001), CoCl2 (p = 0.0134), Doxo (p = 0.0015), RS (p = 0.0043) vs. P. In panel (d), LUM, IR (p ≤ 0.0001), oxLDL (p = 0.0002), CoCl2 (p ≤ 0.0001), Doxo (p = 0.0003), RS (p ≤ 0.0001) vs. P; CD9, IR (p = 0.0002), oxLDL (p = 0.0296), CoCl2 (p = 0.0151), Doxo (p = 0.0291), RS (p = 0.0002) vs. P; APOE, IR (p = 0.0171), oxLDL (p = 0.0004), CoCl2 (p = 0.016), Doxo (p = 0.0486), RS (p = 0.1703) vs. P; LGALS3, IR (p = 0.2702), oxLDL (p = 0.0014), CoCl2 (p = 0.007), Doxo (p = 0.0284), RS (p = 0.0014) vs. P; CST3, IR (p = 0.9809), oxLDL (p ≤ 0.0001), CoCl2 (p = 0.1202), Doxo (p = 0.087), RS (p = 0.0209) vs. P. In panel (e), TNFRSF11b, IR (p = 0.0019), oxLDL (p ≤ 0.0001), CoCl2 (p = 0.001), Doxo (p = 0.0003), RS (p ≤ 0.0001) vs. P; PRG4, IR (p = 0.0595), oxLDL (p = 0.0005), CoCl2 (p = 0.0008), Doxo (p = 0.0011), RS (p = 0.0021) vs. P; MMP3, IR (p = 0.0054), oxLDL (p = 0.0007), CoCl2 (p = 0.0003), Doxo (p = 0.0024), RS (p = 0.0098) vs. P; CTSS, IR (p = 0.0085), oxLDL (p ≤ 0.0001), CoCl2 (p = 0.0922), Doxo (p = 0.0019), RS (p = 0.0003) vs. P; LCP1, IR (p = 0.0029), oxLDL (p = 0.0033), CoCl2 (p = 0.0003), Doxo (p = 0. 0008), RS (p = 0.0006) vs. P. Significance was established using Shapiro-Wilk test first, followed by Welch’s t-test. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Source data

Extended Data Fig. 9 Human WI-38 fibroblast senescence validation.

a, Phase contrast micrographs of SA-βgal activity (blue) staining in human WI-38 fibroblasts that were either proliferating (P), rendered senescent by replicative senescence (RS), or by treatment with Doxo, IR and additional culture for 7–10 days. b, RT-qPCR analysis of the levels of CDKN2A, CDKN1A, GDF15, LMNB1 and IL8 mRNAs in WI-38 fibroblasts treated as described in (a). c-f, RT-qPCR analysis of the levels of SPP1, SERPINE2, CTSB, TNFRSF11B, PRG4, LTPBP2, FTH1, MGP, LUM, CD9, APOE, LGALS3, CST3, SPARC, THBS1, MMP3, CTSS, and LCP1 mRNAs in human WI-38 fibroblasts treated as described in (a). In b-f, data represent the means ± SD for P, n = 3; IR, n = 3; n = 3; Doxo, n = 3; RS, n = 3; n represents biological replicates. In panel (b), CDKN2A, IR (p = 0.0099), Doxo (p ≤ 0.0001), RS (p = 0.0005) vs. P; CDKN1A, IR (p ≤ 0.0001), Doxo (p ≤ 0.0001), RS (p ≤ 0.0001) vs. P; GDF15, IR (p = 0.001), Doxo (p ≤ 0.0001), RS (p ≤ 0.0021) vs. P; LMNB1, IR (p = 0.001), Doxo (p = 0.0002), RS (p = 0.0009) vs. P; IL8, IR (p ≤ 0.0001), Doxo (p = 0.0005), RS (p = 0.0003) vs. P. In panel (c), SPP1, IR (p = 0.0085), Doxo (p = 0.0035), RS (p = 0.0071) vs. P; SERPINE2, IR (p = 0.0014), Doxo (p = 0.0025), RS (p = 0.0026) vs. P; CTSB, IR (p = 0.0003), Doxo (p = 0.0002), RS (p = 0.0007) vs. P; TNFRSF11B, IR (p = 0.0013), Doxo (p = 0.0003), RS (p ≤ 0.0001) vs. P; PRG4, IR (p = 0.0135), Doxo (p = 0.0043), RS (p = 0.0039) vs. P. In panel (d), LTPBP2, IR (p = 0.0003), Doxo (p = 0.0012), RS (p = 0.0006) vs. P; FTH1, IR (p = 0.0025), Doxo (p = 0.0038), RS (p = 0.0016) vs. P; MGP, IR (p = 0.0007), Doxo (p ≤ 0.0001), RS (p ≤ 0.0001) vs. P; LUM, IR (p = 0.0005), Doxo (p = 0.0059), RS (p = 0.0014) vs. P; CD9, IR (p = 0.0051), Doxo (p = 0.7688), RS (p = 0.0008) vs. P. In panel (e), APOE, IR (p = 0.0008), Doxo (p = 0.007), RS (p ≤ 0.0001) vs. P; LGALS3, IR (p = 0.0108), Doxo (p = 0.0229), RS (p ≤ 0.0001) vs. P; CST3, IR (p = 0.0026), Doxo (p ≤ 0.0001), RS (p ≤ 0.0001) vs. P; SPARC, IR (p = 0.0021), Doxo (p = 0.002), RS (p = 0.0004) vs. P; THBS1, IR (p = 0.0003), Doxo (p = 0.0114), RS (p ≤ 0.0001) vs. P. In panel (f), MMP3, IR (p = 0.0015), Doxo (p = 0.0007), RS (p = 0.0003) vs. P; CTSS, IR (p = 0.0002), Doxo (p = 0.0023), RS (p = 0.0019) vs. P; LCP1, IR (p = 0.0003), Doxo (p = 0.9169), RS (p ≤ 0.0001) vs. P.Significance was established using Shapiro-Wilk test first, followed by Welch’s t-test. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

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Extended Data Fig. 10 Spatial transcriptomic profiling of arterial segments.

a, H&E staining (top) and Masson’s trichrome staining (bottom) of brachiocephalic arterial sections from Ldlr−/− + GCV, Ldlr−/−;3MR + Vehicle, and Ldlr−/−;3MR + GCV treated mice. The scale bar is 100 µm. b, Plaque cross sectional area, c, plaque fibrous cap thickness measurements and d, Masson’s trichrome analysis of Aniline blue-positive area over total plaque area in brachiocephalic arterial sections from Ldlr−/− + GCV (n = 4), Ldlr−/−;3MR + Vehicle (n = 4), and Ldlr−/−;3MR + GCV (n = 4) treated mice. In panel (b), Ldlr−/−;3MR + Vehicle vs. Ldlr−/− + GCV (p = 0.8299) and Ldlr−/−;3MR + GCV vs. Ldlr−/−;3MR + Vehicle (p = 0.7933). In panel (c), Ldlr−/−;3MR + Vehicle vs. Ldlr−/− + GCV (p = 0.5444) and Ldlr−/−;3MR + GCV vs. Ldlr−/−;3MR + Vehicle (p = 0.3969). In panel (b), Ldlr−/−;3MR + Vehicle vs. Ldlr−/− + GCV (p = 0.7628) and Ldlr−/−;3MR + GCV vs. Ldlr−/−;3MR + Vehicle (p = 0.9994). e, H&E staining (top) and Masson’s trichrome staining (bottom) of the descending aortic sections from Ldlr−/− + GCV, Ldlr−/−;3MR + Vehicle, and Ldlr−/−;3MR + GCV treated mice. The scale bar is 50 µm. f-h, Plaque cross sectional area (f), plaque fibrous cap thickness measurements (cap is underlined with yellow dashed line) (g), and Masson’s trichrome analysis of Aniline blue-positive area over total plaque area in sections of the descending aorta (h) from Ldlr−/− + GCV, Ldlr−/−;3MR + Vehicle, and Ldlr−/−;3MR + GCV treated mice. In panel (f) and (g), Ldlr−/− + GCV (n = 5), Ldlr−/−;3MR + Vehicle (n = 4), and Ldlr−/−;3MR + GCV (n = 6); in panel (f), Ldlr−/−;3MR + Vehicle vs. Ldlr−/− + GCV (p = 0.9706) and Ldlr−/−;3MR + GCV vs. Ldlr−/−;3MR + Vehicle (p = 0.08), whereas in (g), Ldlr−/−;3MR + Vehicle vs. Ldlr−/− + GCV (p = 0.379) and Ldlr−/−;3MR + GCV vs. Ldlr−/−;3MR + Vehicle (p = 0.0021). In panel (h), Ldlr−/− + GCV (n = 4), Ldlr−/−;3MR + Vehicle (n = 4), and Ldlr−/−;3MR + GCV (n = 5), and significance Ldlr−/−;3MR + Vehicle vs. Ldlr−/− + GCV (p = 0.9867) and Ldlr−/−;3MR + GCV vs. Ldlr−/−;3MR + Vehicle (p = 0.0263) i, Reactome pathway analysis of genes upregulated in cap compared to normal wall across all conditions (left) and of genes upregulated in the normal wall compared to the cap (right). j, Reactome pathway analysis of genes upregulated in the core compared to the normal wall across all conditions. In b-d and f-h, data represent the means ± SD and n represents biological replicates. Significance was established using One-Way Anova with multiple comparisons (b-d and f-h) or Fisher-Irwin test (i and j). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

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Supplementary tables containing information on GSEA results (Supplementary Table 1), set of primers (Supplementary Table 2), smFISH probes (Supplementary Table 3) and reagents used in the study (Supplementary Table 4).

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Raw measurements for the assays indicated in the figure legend.

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RT–qPCR results.

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Raw measurements for smFISH.

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Raw measurements for smFISH.

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Raw measurements for the assays indicated in the figure legend.

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RT–qPCR results.

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RT–qPCR results.

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RT–qPCR results.

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Raw measurements for the assays indicated in the figure legend.

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Mazan-Mamczarz, K., Tsitsipatis, D., Childs, B.G. et al. Single-cell and spatial transcriptomics map senescent vascular cells in arterial remodeling during atherosclerosis in mice. Nat Aging 5, 1528–1547 (2025). https://doi.org/10.1038/s43587-025-00889-z

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