Senescent Vascular Cells Mapped in Atherosclerosis Using Single-Cell and Spatial Transcriptomics
Researchers identify four senescence-enriched vascular cell clusters in atherosclerotic mouse aortas and derive a conserved transcriptomic signature including Spp1, Ctsb, and Tnfrsf11b.
Summary
Using single-cell RNA sequencing of whole aortas from atherosclerotic mice, researchers identified four distinct clusters of vascular smooth muscle cells, fibroblasts, and T cells enriched for senescence features. These clusters expanded under high-fat diet conditions and were reduced by treatment with the senolytic drug ABT-737. A core senescence signature—including Spp1, Ctsb, and Tnfrsf11b mRNAs—was derived and validated using spatial transcriptomics in a second mouse model and in human vascular smooth muscle cell cultures. ABT-737 treatment improved plaque stability, reduced necrotic core area, increased fibrous cap thickness, and lowered vascular stiffness, suggesting that clearing senescent cells promotes beneficial arterial remodeling without necessarily reducing plaque size.
Detailed Summary
Atherosclerosis, a hallmark age-related disease, is increasingly linked to the accumulation of senescent vascular cells that drive inflammation and tissue dysfunction through their senescence-associated secretory phenotype (SASP). Despite this recognized connection, a detailed transcriptomic map of which vascular cell types become senescent during atherosclerosis—and what genes define that state—has been lacking.
Researchers engineered an 'athero-senescent' reporter mouse model by crossing p16-tdTomato reporter mice with a PCSK9 gain-of-function AAV8 vector system to induce atherosclerosis via high-fat diet (HFD). Three groups were compared: normal diet (ND), HFD, and HFD treated with the BCL-2/BCL-XL inhibitor senolytic ABT-737. Whole aortas were dissected, enzymatically digested, and subjected to single-cell RNA sequencing (scRNA-seq). Gene set enrichment analysis (GSEA) using the SenMayo (123 conserved senescence genes) and CellAge curated panels was applied across 28 identified cell clusters spanning nine major cell types.
Four clusters—two VSMC clusters (0 and 12), one fibroblast cluster, and one T cell cluster—showed robust enrichment of both SenMayo and CellAge gene sets under HFD conditions, which was significantly reduced by ABT-737 treatment. Notably, p16 mRNA itself was below the detection sensitivity of the 10x Genomics platform, underscoring the necessity of broader gene panels. Within VSMC cluster 0, subcluster 5 was entirely absent in ND mice (0%), rose to 13.07% under HFD, and nearly disappeared after ABT-737 treatment (0.42%). Pathway analyses of senescence-enriched clusters highlighted extracellular matrix remodeling, TGFβ signaling, epithelial-mesenchymal transition, complement and coagulation signaling as dominant features. A core vascular senescence signature was distilled, prominently featuring Spp1 (osteopontin), Ctsb (cathepsin B), and Tnfrsf11b (OPG/osteoprotegerin) mRNAs.
Histological analyses confirmed that ABT-737 did not reduce overall plaque size but significantly increased fibrous cap thickness, reduced necrotic core area, reorganized collagen deposition, decreased SA-β-galactosidase activity, and lowered pulse wave velocity (a marker of arterial stiffness). Pseudobulk RNA-seq revealed that HFD-elevated transcripts in pathways such as ECM-receptor interaction and TGFβ signaling were reduced by ABT-737. Validation in a second mouse model (Ldlr−/−; p16-3MR) using spatial transcriptomics, and in vitro human VSMC senescence models, confirmed the enrichment of the Spp1/Ctsb/Tnfrsf11b signature in senescent vascular cells.
These findings establish a vascular-specific transcriptomic senescence atlas that goes beyond canonical p16/p21 markers and identifies actionable molecular targets. The demonstrated benefit of senolysis on plaque stability rather than size has important implications for cardiovascular disease treatment strategies focused on plaque vulnerability rather than simple plaque burden reduction.
Key Findings
- Four cell clusters (two VSMC, one fibroblast, one T cell) showed HFD-induced senescence enrichment reversed by ABT-737.
- VSMC subcluster 5 rose from 0% in normal diet to 13.07% under HFD and dropped to 0.42% with ABT-737 treatment.
- A three-gene vascular senescence signature—Spp1, Ctsb, Tnfrsf11b—was validated across two mouse models and human VSMCs.
- ABT-737 improved plaque stability (thicker fibrous cap, smaller necrotic core, lower vascular stiffness) without reducing plaque size.
- p16 mRNA was undetectable by scRNA-seq, highlighting the need for multi-gene senescence panels over single-marker approaches.
Methodology
Atherosclerosis was induced in p16-tdTomato reporter mice via PCSK9 AAV8 injection and high-fat diet feeding; a subset received senolytic ABT-737. Whole aortas underwent enzymatic digestion and live-cell FACS sorting before 10x Genomics scRNA-seq; senescence was assessed by GSEA with SenMayo and CellAge panels across 28 clusters. Findings were validated in a second model (Ldlr−/−; p16-3MR) using spatial transcriptomics and in human VSMC in vitro senescence assays.
Study Limitations
The study was conducted entirely in mouse models; translation of the senescence signature to human atherosclerotic tissue requires further validation. ABT-737 is a research tool compound not approved for clinical use, and the senolytic effects observed may not fully replicate those of clinically available agents like dasatinib plus quercetin. p16 mRNA was undetectable by current scRNA-seq sensitivity, limiting direct reporter-based validation of cell-level senescence assignment.
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