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The age of bone marrow dictates the clonality of smooth muscle-derived cells in atherosclerotic plaques

Nature Aging volume 3, pages 64–81 (2023)Cite this article

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Abstract

Aging is the predominant risk factor for atherosclerosis—the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand, giving rise to up to ~70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells noncell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are decreased, which epigenetically silences integrin β3, resulting in increased tumor necrosis factor-α (TNFα) signaling. TNFα signals through TNF receptor 1 on SMCs to promote proliferation, and induces the recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin β3 overexpression in aged BM preserves the dominance of the lineage of a single SMC progenitor and attenuates the plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

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Fig. 1: The age of mice dictates the clonality of SMC- and macrophage-derived atherosclerotic plaque cells.
Fig. 2: Aged BM promotes the expansion of multiple smooth muscle myosin heavy chain-positive progenitors in the plaque.
Fig. 3: Tet2-null BM induces the expansion of multiple SMC progenitors in the plaque.
Fig. 4: Decreased TET2 decreases integrin β3 in aged myeloid cells, and Itgb3-deficient myeloid clones expand in the plaque.
Fig. 5: Itgb3−/− monocytes and macrophages induce inflammatory pathways.
Fig. 6: In the context of Itgb3−/− BM, TNFα antagonism preserves the predominance of a single SMC clone in plaques.
Fig. 7: Aged BM induces SMC polyclonal expansion, proliferation and atherosclerosis via TNFα–TNFR1.
Fig. 8: Overexpression of Itgb3 in aged BM preserves the dominance of a single SMC lineage in the plaque.

Data availability

Data associated with this study are presented within the paper and its Supplementary Information files. The bulk and scRNA-seq data generated in this study are available from the Gene Expression Omnibus (accession code GSE206431). Trimmed bulk RNA-seq reads were aligned to the reference mouse genome mm10 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/). Reagents and materials associated with this study are available from the corresponding author D.M.G. Source data are provided with this paper.

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Acknowledgements

We thank members of the D.M.G. laboratory and J. Testani for input. Schematics created with BioRender.com (Figs. 1, 2a, 3a, 6a,f, 7c, 8a and Extended Data Figs. 2a, 10) I.K. was supported by a Postdoctoral Fellowship (18POST34030015) and Career Development Award (938472) from the American Heart Association. Funding was also provided by the National Institutes of Health (R35HL150766, R01HL125815, R01HL142674, R21AG062202 and 1R21NS123469 to D.M.G.) and American Heart Association (Established Investigator Award 19EIA34660321 to D.M.G.).

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Authors and Affiliations

  1. Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University, New Haven, CT, USA

    Inamul Kabir, Jui M. Dave, Raja Chakraborty, Rachana R. Chandran, Aglaia Ntokou, Eunate Gallardo-Vara, John Hwa, Kathleen A. Martin & Daniel M. Greif

  2. Department of Genetics, Yale University, New Haven, CT, USA

    Inamul Kabir, Jui M. Dave, Rachana R. Chandran, Aglaia Ntokou, Eunate Gallardo-Vara & Daniel M. Greif

  3. Department of Comparative Medicine, Yale University, New Haven, CT, USA

    Xinbo Zhang, Binod Aryal, Noemi Rotllan & Carlos Fernández-Hernando

  4. Department of Pathology, Yale University, New Haven, CT, USA

    Rihao Qu, Yuval Kluger & Carlos Fernández-Hernando

  5. Bioinformatics Support Program, Yale University, New Haven, CT, USA

    Rolando Garcia-Milian

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Contributions

I.K., J.M.D., R.C., R.Q., N.R., K.A.M., C.F.-H. and D.M.G. conceived of and designed the experiments. I.K., X.Z., J.M.D., R.C., R.R.C., A.N., E.G.-V., B.A. and N.R. performed them. R.Q. and Y.K. conducted the scRNA-seq analysis. R.G.-M. helped with the bulk RNA-seq analysis. J.H. provided infrastructure for access to human blood. I.K. and D.M.G. analyzed the results, prepared the figures and wrote the manuscript. All authors reviewed and provided input on the manuscript.

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Correspondence to Inamul Kabir or Daniel M. Greif.

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Extended data

Extended Data Fig. 1 Aged BMT promotes polyclonal SMC progenitor expansion.

a–d, Young and aged Myh11-CreERT2, ROSA26R(Rb/+) mice were injected with AAV-Pcsk9 and fed a WD for 16 weeks. Brachiocephalic artery sections were imaged for Rb colors (a). Percent of DAPI+ plaque cells marked by any Rb color was quantified (b). For marked plaque (c) or underlying media (d) cells, the percent of cells of each color was quantified for each age group. In a given plaque, color 1 has greatest number of cells, color 2 is second most common color and color 3 is least frequent. In c, n = 5 (young) and 7 (aged) mice and 10 plaques per age group, 5 sections with a total of ~1100–1350 cells and spanning 200 μm per plaque. In d, n = 5 (young) and 7 (aged) mice and media underlying 14 plaques analyzed per group. e-i, Young Ldlr(−/−), Myh11-CreERT2, ROSA26R(Rb/+) mice underwent young or aged BMT. In e, after 4 weeks CD feeding, peripheral leukocyte counts were analyzed. n = 6 mice. In f,g, following 16 weeks WD feeding, transverse aortic root sections were stained with SMA or Sirius red, with dashed lines demarcating cap. Percent of cap cells that are SMA+ and percent of plaque area stained with Sirius red (h,i). n = 6 mice. j–q, Young Apoe(−/−), Myh11-CreERT2, ROSA26R(Rb/+) mice underwent young or aged BMT, AAV-Pcsk9 injection and 16 weeks WD feeding. Aortic sections were stained with H&E and Oil Red O (j,k), with quantification of lesion area (l; demarcated by dashed lines in j), lipid content (m), necrotic core area (n). Sections were imaged for Rb colors (o), and of the marked plaque (p) or medial cells (q), the percent of cells of each color was quantified. n = 5 mice, 12 plaques per group, 5–6 sections with a total of ~1000–1150 cells and spanning 200 μm per plaque. r,s, Murine aortic SMCs were cultured for 8 h with EdU in medium pre-conditioned by BM-derived macrophages of young vs. aged mice. In r, SMCs were stained for EdU and DAPI, and in s, percent of DAPI+ nuclei expressing EdU was quantified. n = 6 mice. Lu, lumen; Med, tunica media; Pl; plaque. All data are means ± SD. Two-tailed Student’s t-test. Scale bars, 100 µm (j,k), 50 µm (a,f,g,o), 25 µm (r).

Extended Data Fig. 2 Aged BM induces polyclonal expansion of SMMHC+ progenitors in the plaque.

Aged Myh11-CreERT2, ROSA26R(Rb/+) mice were induced with tamoxifen, irradiated, transplanted with BM from young or aged mice, injected with AAV-Pcsk9 and fed a WD for 16 weeks. Transverse aortic root sections were analyzed. a, Schematic of experiments. b–e, Sections were stained with H&E (dashed lines demarcate lesion; b) and Oil Red O (c) to determine lesion area (d) and lipid content (e). n = 5 and 7 mice per young and aged BMT group, respectively. Triplicate measurements from each mouse. f–i, Sections were stained with DAPI and directly imaged for Rb colors (mCh, mOr, Cer). In f, representative sections are shown with close-ups of boxed regions displayed on the right. In g, percent of DAPI+ plaque cells that were marked by any of the Rb colors was quantified. Of the marked plaque (h) or underlying medial cells (i), percent of cells of each color was quantified for each BMT group. In a given plaque (or media), color 1 is color with the greatest number of cells in plaque (or media), color 2 is second most common color and color 3 is least frequent color. n = 6 and 8 mice per young and aged BMT group, respectively, 14 plaques per BMT group, 5 sections with a total of ~1000–1200 cells and spanning 200 μm per plaque. Lu, lumen; Med, media; Pl, plaque. All data are means ± SD, and two-tailed Student’s t-test was used. Scale bars, 100 μm (b,c), 50 μm (f).

Extended Data Fig. 3 Tet2(−/−) BMT promotes recruitment and expansion of multiple SMC progenitors in the plaque.

Apoe(−/−), Myh11-CreERT2, ROSA26R(Rb/+) mice were induced with tamoxifen, irradiated and transplanted with Tet2(+/+) or Tet2(−/−) BM, injected with AAV-Pcsk9 to reduce hepatic LDLR receptor level and fed a WD for 16 weeks. a, Transverse aortic root sections were stained with DAPI and directly imaged for Rb colors (mCh, mOr, Cer). b, Percent of DAPI+ plaque cells that were marked by one of the Rb colors was quantified. n = 5 mice per group. c,d, Of the marked plaque (c) or medial cells (d), the percent of cells of each color was quantified for each BMT group. In a given plaque, color 1 is the most dominant color of cells in a plaque, color 2 is the second most common color and color 3 is least frequent. n = 5 mice per group, 9 plaques per genotype, 5 sections with a total of ~950–1100 cells and spanning 200 μm per plaque were analyzed. e,f, Murine aortic SMCs were cultured for 8 h in the presence of EdU in a medium conditioned by BM-derived macrophages of Tet2(+/+) and Tet2(−/−) mice. In e, SMCs were stained for EdU and nuclei (DAPI), and in f, the percent of DAPI+ nuclei that also express EdU was quantified. n = 6 mice. Lu, lumen; Med, tunica media; Pl; plaque. All data are means ± SD. Two-tailed Student’s t-test. Scale bar, 50 µm (a), 25 µm (e).

Extended Data Fig. 4 Isolation of mouse BM-derived and human peripheral blood monocytes and pathway analysis of young vs old monocytes.

a,c,d, Dot plots showing gating schemes of monocyte isolation by FACS. In a, BM from the femurs of young and aged mice was harvested and subjected to FACS to isolate CD3−CD19−Cd11b+ Ly6C+ monocytes (n = 9 mice per group). In c, human peripheral blood monocytes were harvested from young and aged subjects, and CD14+ monocytes were sorted (n = 12 humans per age group). In d, BM from Tet2(+/+) and Tet2(−/−) mice was harvested and subjected to FACS to isolate CD3−CD19−Cd11b+Ly6C+ monocytes (n = 9 mice per group). b, Lysates of monocytes isolated from young or aged mice underwent bulk RNA-seq (n = 3 mice per age group), and the transcriptome was subjected to gene set enrichment analysis (GSEA). Activated pathways are shown with gene ratio indicating the number of activated genes relative to the total number of genes in the corresponding pathways and size of the dot representing number of activated genes. The GSEA algorithm as implemented in clusterProfiler R package was used to perform a one-sided test to determine the significance level of pathway enrichment.

Extended Data Fig. 5 Deletion of Itgb3 in CSF1R+ cells exacerbates atherosclerosis and induces proliferation and accumulation of this cell lineage in the plaque.

a–j, Apoe(−/−), Csf1r-Mer-iCre-Mer mice also carrying Itgb3(fl°x/flox) or wild type for Itgb3 were induced with tamoxifen. In a–f, mice were then fed a WD for 16 weeks, and transverse aortic root sections were stained with H&E (dashed lines demarcate lesion and necrotic core in a,e, respectively) and Oil Red O (b). Quantification of lesion area (c), lipid content (d) and necrotic core area (f). n = 5 mice and 10–12 plaques per genotype (triplicate measurements per plaque). BM-derived GFP+ myeloid cells (Ly6C+) were isolated by FACS after 5 days of tamoxifen induction and subjected to qRT-PCR for Itgb3 (g). Itgb3 mRNA is relative to Gapdh and normalized to control. n = 6 mice per group, and qRT-PCR was done in triplicate. Four weeks following tamoxifen induction, peripheral blood total cholesterol and triglycerides (after a 16 h fast; n = 5–7 mice per group; h,i) and leukocyte counts (j) were determined. n = 5 and 7 mice in Itgb3(+/+) and Itgb3(flox/flox) groups, respectively. k–o, Apoe(−/−), Csf1r-Mer-iCre-Mer, ROSA26R(mTmG/+) mice also carrying Itgb3(+/+) or Itgb3(flox/flox) were induced with tamoxifen and fed a WD for 16 weeks. In k,l, after 16 weeks of WD feeding, EdU was injected intraperitoneally 12 h prior to euthanasia. Transverse aortic root sections were stained for markers of fate (GFP), SMCs (SMA), nuclei (DAPI) and either proliferation (EdU) in k or CD68 in m. Boxed regions in k are shown as close-ups below with arrowheads indicating GFP+EdU+ cells. In l, the percentage of GFP+ plaque cells that are EdU+ is shown. In, n,o, percentage of plaque cells that are GFP+ or CD68+ was quantified. n = 5 and 7 mice in Itgb3(+/+) and Itgb3(flox/flox) groups, respectively, 10 plaques per genotype, 4 sections with a total of ~1000–1250 cells and spanning 150 μm per plaque (l,n,o). All data are means ± SD, and two-tailed Student’s t-test was used. Lu, lumen; Med, media; Pl, plaque. Scale bars, 100 μm (a,b,e), 50 μm (k,m).

Extended Data Fig. 6 SMC-specific Itgb3 deletion attenuates atherosclerosis and SMC contribution to the plaque without altering plasma lipids.

Apoe(−/−), Myh11-CreERT2 mice also carrying Itgb3(flox/flox) or Itgb3(+/+) and either ROSA26R(mTmG/+) (a–k) or ROSA26R(Rb/+) (l–n) were induced with tamoxifen. a–e and i–n, Mice were rested, subjected to WD for 16 weeks, and aortic root transverse sections were analyzed. In a–e, sections were stained with H&E [dashed lines demarcating lesion; (a)] and Oil Red O (b), and lesion area (c), lipid content (d) and necrotic core area (e) were quantified. n = 7 mice (c–e) in Itgb3(+/+) and n = 8 (c,d), n = 7 (e) mice in Itgb3(flox/flox) groups. In i, EdU was injected 12 h prior to euthanasia. Sections were stained for SMA, GFP (fate marker), EdU and nuclei (DAPI). Close-ups of boxed regions are shown. Arrowheads indicate GFP+EdU+ cells. Percent of total plaque cells that are GFP+ and percent of total plaque GFP+ cells that are EdU+ are shown in j,k, respectively. n = 5 mice and 10 plaques per genotypes, 5–6 sections with a total of ~1200–1500 cells and spanning 200 μm per plaque. In l, sections were imaged for Rb colors with close-ups of boxed regions. Of the marked plaque (m) or underlying medial cells (n), the percent of cells of each color was quantified. Colors #1–3 are defined as in previous figures. n = 5 mice and 12 plaques per genotype, 6 sections with a total of ~1000–1300 cells and spanning 250 μm per plaque. Lu, lumen; Med, media; Pl, plaque. f, After 5 days of tamoxifen induction, GFP+ SMCs were isolated by FACS and subjected to qRT-PCR for Itgb3. Itgb3 mRNA is represented relative to Gapdh and normalized to Itgb3(+/+) group. n = 6 mice per group, and qRT-PCR was done in triplicate. g,h, Four weeks following tamoxifen induction, plasma total cholesterol and triglycerides were measured after fasting 16 h; n = 5 and 7 mice in Itgb3(+/+) and Itgb3(flox/flox) groups, respectively. All data are means ± SD, and two-tailed Student’s t-test was used. Scale bars, 100 μm (a,b), 50 μm (i,l).

Extended Data Fig. 7 Integrin β3 reduction in aortic SMCs attenuates phosphorylation of AKT and ERK and PDGF-B-induced dorsal ruffles.

a,b, Aortic lysates of wild type (WT) or Itgb3(−/−) mice were subjected to Western blot for integrin β3, GAPDH and phosphorylated (p-) and total AKT (a) with densitometry of each protein relative to GAPDH and normalized to WT (b). n = 6 mice per genotype. c–f, Mouse aortic SMCs were isolated and treated with Itgb3 siRNA or scrambled (Scr) RNA. Cell lysates were subjected to Western blot for integrin β3, GAPDH as well as phosphorylated and total AKT, PAK1 and ERK (c) with densitometry of each protein relative to GAPDH and normalized to Scr (d). In e, following siRNA treatment, SMCs were serum starved overnight, treated with PDGF-B (10 ng/mL) for the indicated times and stained with FITC-phalloidin. Arrowheads indicate dorsal ruffles with boxed regions shown as close-ups below. Scale bar, 25 μm. Quantification of dorsal ruffles is shown in (f). n = 6 experiments. All data are means ± SD, and two-tailed Student’s t-test was used.

Source data

Extended Data Fig. 8 scRNA-seq analysis of Apoe(−/−), Itgb3(−/−) and Apoe(−/−), Itgb3(+/+) BM cells, focusing on monocyte/macrophage cluster.

BM was isolated from Apoe(−/−) mice carrying Itgb3(−/−) or Itgb3(+/+) and subjected to scRNA-seq. a, Proportion of leukocyte sub-types in BM cells from scRNA-seq data. b-h, t-SNE plots of pooled scRNA-seq data from BM of both genotypes showing the expression of monocyte/macrophage markers. i, IPA predicts the effects of differentially expressed genes on the inflammatory responses and monocyte/macrophage migration or proliferation. n = 3 mice pooled per genotype. j, Differential abundance analysis of sub-clustered monocytes/macrophages show abundance of Apoe(−/−), Itgb3(−/−) cells in circled regions. k, l, Dot plot analysis of monocytes/macrophage sub-cluster showing differentially expressed genes in TNFα and NF-kB signaling pathways, respectively. Dot size represents the fraction of cells expressing the gene, and shade of blue color represents average gene expression.

Extended Data Fig. 9 Aged BM, TNFα and SMC biology.

a–c, Young Ldlr(−/−), Myh11-CreERT2, ROSA26R(Rb/+) mice underwent young or aged BMT. In a, DNA isolated from peripheral blood of recipients before (lanes 1–3) or after (lanes 4–6) aged BMT or of WT (lane 7) or ROSA26R(Rb/+) (lane 8) mice was amplified using primers for ROSA26R Rainbow (Rb) and WT alleles. In b,c, BMT recipients were fed a WD feeding for 12 weeks with concomitant twice weekly anti-TNFα antibody or control IgG2a injections. Transverse aortic root sections were stained with SMA and DAPI, and the percent of SMA+ cells in the cap area was assessed (cap area demarcated by dashed lines in b). n = 6 mice per group. Lu, lumen; Med, media; Pl, plaque. d,e, BM of young or aged wild type mice was harvested and differentiated into macrophages and isolated mouse aortic SMCs were cultured with macrophage conditioned medium. Anti-TNFα blocking antibody or control IgG was added to conditioned medium, 1 h prior to incubation with SMCs. SMCs were incubated with conditioned medium with EdU for 8 h. SMCs were stained for EdU and DAPI, and percent of EdU+ cells was quantified. n = 3 mice per group, each experiment done in triplicate. f,g, Mouse aortic SMCs were isolated and treated with Tnfr1 siRNA or scrambled (Scr) RNA. Cell lysates were subjected to Western blot for TNFR1 and GAPDH with densitometry relative to GAPDH and normalized to Scr. n = 3 mice per group, each experiment done in triplicate. h–k, Tnfr1-silenced SMCs were exposed to methyl-β-cyclodextrin complexes and TNFα for 3 days, and then mRNA levels were assessed by qRT-PCR. n = 3 experiments, each experiment done in triplicate. l,m, BM harvested from aged wild type mice was infected with Itgb3-expressing or control lentivirus and transplanted into aged Myh11-CreERT2, ROSA26R(Rb/+) mice. Four weeks after BMT, fasting total cholesterol and triglycerides levels were assessed in blood plasma. n = 4 mice in control and n = 6 in Itgb3 o/e groups. All data are means ± SD. Two-tailed Student’s t-test. Scale bars, 50 μm (b), 25 μm (d).

Source data

Extended Data Fig. 10 Age of BM-derived monocytes/macrophages determines clonality of SMC lineage in the atherosclerotic plaque.

Atherogenesis is depicted in a young (a) or aged (b) host. Aged monocytes/macrophages have decreased levels of the epigenetic regulator TET2, leading to reduction of the 5-hydroxymethylcytosine (5hmC) mark on the Itgb3 promoter. The resulting low integrin β3 levels in aged monocytes/macrophages induces high TNFα levels, facilitating recruitment and expansion of multiple SMC progenitors (polyclonality) in the atherosclerotic plaque and worse disease burden. In contrast, the young control is characterized by mono/oligoclonal SMC expansion in a smaller plaque.

Supplementary information

Supplementary Information

Supplementary Tables 1–5 and Supplementary Methods.

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Supplementary Data

Bulk RNA-seq of BM-derived monocytes isolated from aged mice relative to young mice.

Source data

Source Data Fig. 4

Unprocessed Western blots.

Source Data Extended Data Fig. 7

Unprocessed Western blots.

Source Data Extended Data Fig. 9

Unprocessed Western blots.

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Kabir, I., Zhang, X., Dave, J.M. et al. The age of bone marrow dictates the clonality of smooth muscle-derived cells in atherosclerotic plaques. Nat Aging 3, 64–81 (2023). https://doi.org/10.1038/s43587-022-00342-5

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