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

May combine two claims.

Pseudobulk RNA-seq showed that classical senescence markers (Il6, Bcl2l2, Ccnd1, Ccnd2, Cdkn1a, Lmnb1, Lbr mRNAs) were increased by HFD and reduced by ABT-737.

“The pseudobulk RNA-seq assessment also revealed that the levels of classical senescence markers, including Il6, Bcl2l2, Ccnd1, Ccnd2, Cdkn1a, Lmnb1 and Lbr mRNAs, were increased by HFD and reduced by ABT-737 (Extended Data Fig. 1j)22,23.”

• cites [22] Herman, A. B. et al. Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin, promotes cell senescence and enhances senolysis. Nucleic Acids Res. 49, 7389–7405 (2021). sourceCould not verify

  The cited paper’s senolytic experiments (p. 13) test ABT-737 in pre-senescent WI-38 fibroblasts and measure viability and caspase activity, not the claimed pseudobulk mRNA changes. Our copy does not report an HFD comparison or show that ABT-737 reduced the listed mRNAs; searches for HFD, pseudobulk RNA-seq, and the marker list found no such analysis. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [23] Wechter, N. et al. Single-cell transcriptomic analysis uncovers diverse and dynamic senescent cell populations. Aging (Albany NY) 15, 2824–2851 (2023). sourcePartly supported

  The Results section on senescent-cell clusters (page not provided) identifies several listed mRNAs as senescence-associated markers in WI-38 fibroblasts. It reports changes after etoposide treatment, not HFD or ABT-737, and reports decreases—not increases—in LMNB1 and LBR mRNAs. The cited paper therefore does not support the claimed pseudobulk treatment effects. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

SASP-associated complement and coagulation signaling pathways have been linked to senescence, particularly in vascular cells, where they are critical for cell survival.

“In addition, SASP-associated complement and coagulation signaling pathways were recently linked to senescence, particularly in vascular cells, for which they are critical for cell survival20,21.”

• cites [20] Herman, A. B. et al. DPP4 inhibition impairs senohemostasis to improve plaque stability in atherosclerotic mice. J. Clin. Invest. https://doi.org/10.1172/JCI165933 (2023). sourceSupported
• cites [21] Wiley, C. D. et al. SILAC analysis reveals increased secretion of hemostasis-related factors by senescent cells. Cell Rep. 28, 3329–3337 (2019). sourcePartly supported

  The Results section links senescence-associated secreted factors to hemostasis and reports increased expression of several such factors in senescent vascular endothelial cells. It does not establish a complement pathway or show that these pathways are critical for vascular-cell survival; the Results instead report no significant effect of senescent cells on the coagulation cascade. No page numbers were provided in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

The serum biomarker results further validate atherosclerosis development in the model, and senolytics do not strongly alter known secreted proteins.

“These results further validate the development of atherosclerosis in our model; however, as others have demonstrated16, senolytics do not appear to strongly alter the profile of known secreted proteins.”

• cites [16] Garrido, A. M. et al. Efficacy and limitations of senolysis in atherosclerosis. Cardiovasc Res. https://doi.org/10.1093/cvr/cvab208 (2022). sourcePartly supported

  The Results (§3.8; page unavailable) report that ABT-263 left most measured serum cytokines unchanged, supporting the limited-profile-change point. But it markedly reduced serum IL-6, and the paper also reports inflammation following genetic senolysis; its findings therefore do not establish the claim for senolytics generally. The citing authors’ statement about validating their own model is not attributed to this source. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

GPNMB-expressing cells have been found to be highly senescent, and eliminating them reduced senescence burden and vascular disease.

“Gpnmb mRNA was recently found to be highly expressed in senescent cells, and eliminating glycoprotein NMB (GPNMB)-expressing cells reduced the burden of senescence and vascular disease37”

• cites [37] Lear, T. B. & Finkel, T. Senolytic vaccination: a new mandate for cardiovascular health? J. Cardiovasc. Aging 2, 17 (2022). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

A recent study attributed vascular stiffening to accumulation of senescent vascular cells in naturally aged mice.

“This finding is further supported by a recent study that attributed vascular stiffening to the accumulation of senescent vascular cells in naturally aged mice47.”

• cites [47] Clayton, Z. S. et al. Cellular senescence contributes to large elastic artery stiffening and endothelial dysfunction with aging: amelioration with senolytic treatment. Hypertension 80, 2072–2087 (2023). sourceSupported

May combine two claims.

In primary human VSMCs, senescence induced by ionizing radiation, doxorubicin, oxidized LDL, cobalt chloride, or replicative exhaustion produced a phenotype with increased SA-β-gal activity, increased p21/CDKN1A, GDF15, and IL8 mRNAs, and reduced LMNB1 mRNA.

“we observed an elevation of SA-β-gal activity, an increase in classical markers of cellular senescence (including p21/CDKN1A, GDF15 and IL8 mRNAs) and a reduction in LMNB1 mRNA, all consistent with a senescent phenotype”

• cites [29] Freund, A., Laberge, R.-M., Demaria, M. & Campisi, J. Lamin B1 loss is a senescence-associated biomarker. Mol. Biol. Cell 23, 2066–2075 (2012). sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [30] Park, H., Kim, C.-H., Jeong, J.-H., Park, M. & Kim, K. S. GDF15 contributes to radiation-induced senescence through the ROS-mediated p16 pathway in human endothelial cells. Oncotarget 7, 9634–9644 (2016). sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [31] Basisty, N. et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol. 18, e3000599 (2020). sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [32] Coppé, J.-P., Desprez, P.-Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118 (2010). sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

May combine two claims.

The findings are consistent with previous studies suggesting senescent cells contribute to fibrous cap thinning, although differences in other aspects could be due to different animal models, detection methods, traits examined, and time points.

“our findings are consistent with previous studies suggesting that senescent cells contribute to fibrous cap thinning, although differences in other aspects of these studies, such as gene expression changes, could be attributed to the use of different animal models, methods of detection, traits examined and time points studied48.”

• cites [48] Childs, B. G. et al. Senescent cells suppress innate smooth muscle cell repair functions in atherosclerosis. Nat. Aging 1, 698–714 (2021). sourcePartly supported

  Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

May combine two claims.

H&E staining showed that HFD mice treated with ABT-737 had increased fibrous cap thickness and reduced necrotic core area compared to HFD alone.

“Importantly, hematoxylin and eosin (H&E) staining revealed an increase in fibrous cap thickness in HFD mice treated with ABT-737, as well as a reduction in the necrotic core area, compared to mice that received HFD alone (Fig. 1d,e and Extended Data Fig. 1b–d).”

May combine two claims.

Serum levels of SERPINE1, CD93, BAFF, MMP3, and CXCL1 were significantly higher in HFD mice than controls; ABT-737 modestly reduced them but not significantly.

“The levels of serpin family E member 1 (SERPINE1), cluster of differentiation 93 (CD93), B cell activating factor (BAFF), MMP3 and C–X–C motif chemokine ligand 1 (CXCL1) were significantly higher in the serum of HFD mice; treatment with ABT-737 modestly reduced the levels of these cytokines, but the differences were not statistically significant (Fig. 1h).”

May combine two claims.

Masson's trichrome staining showed disorganized collagen accumulation in HFD mice, with significant necrotic areas.

“Masson’s trichrome staining revealed that collagen accumulated in a disorganized manner in HFD mice, maintaining significant areas of necrosis that promote plaque vulnerability.”

May combine two claims.

In senescence-enriched subclusters, VSMCs and fibroblasts showed mRNAs in ECM remodeling/organization pathways, and senescent T cells showed enrichment in inflammation-related pathways.

“Within subclusters exhibiting features of senescence, we then identified mRNAs encoding proteins in pathways related to extracellular matrix (ECM) remodeling and organization among vascular smooth muscle cells (VSMCs) and fibroblasts, as well as inflammation-related pathways enriched in senescent T cells.”

May combine two claims.

Pulse wave velocity measurements showed increased vascular stiffness in HFD mice, and ABT-737 reduced stiffness to ND levels.

“Pulse wave velocity (PWV) measurements revealed that HFD mice had increased vascular stiffness, whereas ABT-737 treatment reduced stiffness to the levels observed in ND mice (Fig. 1g and Extended Data Fig. 1h).”

May combine two claims.

Approximately 30 RNAs increased with HFD and decreased with ABT-737, belonging to five pathways: complement signaling, coagulation signaling, EMT, TGFβ signaling, and ECM-receptor interactions.

“We identified approximately 30 RNAs that reflected this pattern (increasing with an HFD and decreasing with drug treatment), which belonged to five different pathways: complement signaling, coagulation signaling, epithelial–mesenchymal transition (EMT), transforming growth factor-β (TGFβ) signaling and ECM–receptor interactions (Fig. 1k,l).”

May combine two claims.

In whole-aorta scRNA-seq of atherosclerotic p16-tdTomato+/− mice, four clusters of VSMCs, fibroblasts, and T cells were enriched for senescence features using SenMayo and CellAge gene sets, and these clusters were reduced by ABT-737 treatment.

“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.”

• AssumptionRests on an assumption: Senescent vascular cells are heterogeneous and dysfunctional, exhibiting growth arrest and inflammation.
• AssumptionRests on an assumption: Senescence in tissues is poorly characterized in both humans and other species.
• AssumptionRests on an assumption: Existing senescence-modulating mouse strains do not inform about the temporal expression of senescence markers or the heterogeneity of senescent cells, including those that do not express p16 or p21.
• AssumptionRests on an assumption: The p16-tdTomato+/− reporter model can distinguish senescent cells from all other cells in whole-aorta scRNA-seq.

May combine two claims.

In HFD mice treated with ABT-737, collagen deposition was highly compacted and collagen fibers increased, preventing necrotic core formation.

“However, in HFD mice treated with ABT-737, collagen deposition was highly compacted, preventing necrotic core formation as evidenced by the increase in collagen fibers (Fig. 1f and Extended Data Fig. 1e).”

May combine two claims.

Oil Red O staining showed increased plaque accumulation in aortic valves of HFD-fed p16-tdTomato+/− mice compared to ND-fed mice, and ABT-737 treatment did not affect plaque deposition.

“Oil Red O staining of aortic root sections demonstrated enhanced plaque accumulation in the aortic valves of p16-tdTomato+/− mice fed an HFD compared to mice fed an ND; treatment with ABT-737 did not affect plaque deposition (Fig. 1b,c and Extended Data Fig. 1a).”

May combine two claims.

Senescence-associated gene expression was validated in human VSMCs and fibroblasts and by spatial transcriptomic profiling of another atherosclerotic mouse and senolytic treatment model.

“We validated senescence-associated gene expression in human VSMCs (hVSMCs) and fibroblasts and by spatial transcriptomic profiling of another atherosclerotic mouse and senolytic treatment model system.”

May combine two claims.

The enrichment of the senescence signature mRNAs was validated by spatial transcriptomics in a second mouse model (Ldlr−/−; p16-3MR) and in in vitro models of human VSMC senescence.

“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.”

May combine two claims.

ABT-737 revealed prominent senescent cell populations but did not completely eliminate them, leaving areas with remaining senescent cells.

“ABT-737 revealed prominent senescent cell populations but did not completely eliminate them, leaving areas with remaining senescent cells.”

May combine two claims.

Reactome pathway analysis of core versus normal wall revealed increases in inflammatory pathways including Toll-like receptor activity and interleukin-1 regulation, and senescence-related pathways such as lysosome activity, ECM organization, and complement activation.

“Reactome pathway analysis revealed increases in inflammatory pathways, including Toll-like receptor activity and interleukin-1 regulation, and senescence-related pathways, such as lysosome activity, ECM organization and complement activation”

May combine two claims.

VSMC clusters 0 and 12 were enriched for SenMayo and CellAge RNAs and reduced by ABT-737, indicating heterogeneity of the senescence phenotype within a cell type.

“Multiple VSMC clusters (0 and 12) were enriched for SenMayo and CellAge RNAs and reduced by ABT-737 treatment, indicating that the senescence phenotype may be heterogeneous even among a given cell type.”

• AssumptionRests on an assumption: Senescent vascular cells are heterogeneous and dysfunctional, exhibiting growth arrest and inflammation.

May combine two claims.

Unique pathways in this fibroblast subcluster included FGFR signaling and RUNX2 regulation, which may be specific to senescent vascular fibroblasts.

“However, there were also unique pathways identified, such as fibroblast growth factor receptor (FGFR) signaling and Runt-related transcription factor 2 (RUNX2) regulation, which may be specific to senescent vascular fibroblasts (Fig. 4d).”

May combine two claims.

The vascular senescence scoring system developed in the atherosclerosis mouse model was applicable to a mouse model of doxorubicin-induced cellular senescence and to human senescent VSMCs and fibroblasts.

“While this score was developed in an atherosclerosis mouse model, we found that the scoring system was applicable to a mouse model of doxorubicin-induced cellular senescence and to human senescent VSMCs and fibroblasts.”

May combine two claims.

A second heatmap visualized significantly reduced RNAs in media and core across conditions, identifying increased senescence-associated RNAs with development of atherosclerotic core and their reduction by GCV treatment.

“A second heatmap was used to visualize the significantly reduced RNAs in the media and core across conditions, to identify the increased senescence-associated RNAs with the development of the atherosclerotic core and to visualize their reduction by GCV treatment”

May combine two claims.

Overlaying the vascular senescence score on UMAP plots showed senescent cells developing with HFD in VSMC, fibroblast, and T cell clusters, and these were strongly reduced by ABT-737.

“An overlay of our vascular senescence score on the UMAP (Uniform Manifold Approximation and Projection) plots of all cells from each condition aided in the visualization of senescent cells developing with HFD in VSMC, fibroblast and T cell clusters, which were strongly reduced in all clusters by treatment with ABT-737 (Fig. 5b).”

May combine two claims.

Treatment of HFD mice with ABT-737 did not significantly affect plaque accumulation but contributed to physiological ECM remodeling, as evidenced by reduced arterial stiffness, increased collagen, and altered gene expression patterns.

“treatment of HFD mice with the senolytic drug ABT-737 did not significantly affect plaque accumulation but contributed to physiological remodeling of the ECM, as evidenced by reduced arterial stiffness, increased collagen and altered gene expression patterns”

May combine two claims.

Reactome pathway analysis suggested a role for these cells in ECM organization, collagen degradation, and integrin interactions.

“Reactome pathway analysis suggested a potential role of these cells in ECM organization, collagen degradation and integrin interactions (Fig. 3d).”

May combine two claims.

In VSMC cluster 12, subcluster 4 increased from 0.37% in ND to 19.86% under HFD and decreased to 9.62% after ABT-737.

“Subclustering analysis recognized subcluster 4 as potentially senescent. Its proportion increased from 0.37% in the ND condition to 19.86% under the HFD condition and decreased to 9.62% following ABT-737 treatment (Fig. 3e).”

May combine two claims.

Transcripts in a small subpopulation were strong enough to drive expression in the larger cluster under HFD, and this was reduced by ABT-737.

“It also revealed that the expression of these transcripts in a small population of cells was strong enough to drive their expression in the HFD condition in the larger cluster, and this expression was subsequently reduced by ABT-737 (Fig. 3b,c).”

May combine two claims.

GCV treatment of Ldlr−/−; 3MR animals increased fibrous cap thickness and collagen accumulation in the descending aorta.

“GCV treatment of the Ldlr−/−; 3MR animals increased fibrous cap thickness and collagen accumulation, consistent with our earlier results”

May combine two claims.

In doxorubicin-treated mice, RT-qPCR showed significantly increased aortic root levels of Cdkn1a, Il6, Gdf15, and Cdkn2a mRNAs, and ABT-737 significantly reduced their expression in both sexes.

“We then performed reverse transcription followed by qPCR (RT–qPCR) analysis on the aortic roots and found that doxorubicin significantly increased the levels of the traditional senescence markers Cdkn1a, Il6, Gdf15 (Fig. 5d) and Cdkn2a mRNAs (Extended Data Fig. 7a), whereas treatment with ABT-737 significantly reduced their expression in both male and female mice.”

May combine two claims.

In the atherosclerotic core (CD45-positive cells), Spp1 and Ctsb mRNAs were highly expressed in the HFD group and significantly reduced upon senolytic treatment.

“The expression levels of the vascular senescence markers Spp1 and Ctsb mRNAs were high in the core region of the aortic roots in the HFD group, and they were significantly reduced upon senolytic treatment in the atherosclerotic core”

May combine two claims.

In fibroblast cluster 2, subcluster 3 increased from 0% in ND to 7.13% under HFD and decreased to 0.28% after ABT-737; its enriched transcripts overlapped with VSMC senescent subclusters, including Spp1, Comp, Ltbp2, and Lum mRNAs.

“However, through unbiased subclustering, we identified subcluster 3 as being enriched under the HFD condition (increasing from 0% with ND to 7.13% with HFD and decreasing to 0.28% after ABT-737 treatment; Fig. 4a); many enriched transcripts overlapped with those identified in VSMC senescent subclusters, including Spp1, Comp, Ltbp2 and Lum mRNAs.”

May combine two claims.

In brachiocephalic arterial cross-sections from Ldlr−/− + GCV, Ldlr−/−; 3MR + vehicle, and Ldlr−/−; 3MR + GCV mice, there was no difference in plaque accumulation, plaque fibrous cap thickness, or ECM organization among conditions.

“we found no difference in plaque accumulation, plaque fibrous cap thickness or ECM organization among the conditions”

May combine two claims.

Of the 17 vascular senescence RNAs, 16 encoded predicted secreted proteins and 5 were previously identified by the SenMayo panel.

“Additionally, of the top 17 RNAs strongly linked to vascular senescence, 16 encoded proteins that were predicted to be secreted and 5 were previously identified by the SenMayo panel.”

May combine two claims.

Senescent T cells are strongly inflammatory, with top pathways featuring immune system activation and interleukin signaling.

“Senescent T cells are strongly inflammatory, with the top pathways featuring immune system activation and interleukin signaling.”

May combine two claims.

ECM-related RNAs were highly abundant specifically in this senescent population, contributing to increased expression under HFD, and this was mitigated by ABT-737.

“Notably, RNAs expressed from these genes were highly abundant specifically within this potentially senescent population, contributing to their increased expression in the HFD condition; however, this effect was mitigated by the administration of ABT-737 (Fig. 3f,g).”

May combine two claims.

These mRNAs were highly abundant in HFD and diminished after ABT-737.

“As observed, these mRNAs were highly abundant in the HFD group and diminished after ABT-737 treatment (Fig. 4b,c).”

May combine two claims.

Atherosclerosis induced by PCSK9 overexpression and HFD exposure led to increased plaque deposition and pathological ECM remodeling, including large necrotic core areas, disorganized and reduced collagen, and smooth muscle-rich caps.

“We found that atherosclerosis induced by PCSK9 overexpression and HFD exposure led to increased plaque deposition and pathological ECM remodeling, including large necrotic core areas, disorganized and reduced collagen, and smooth muscle-rich caps.”

May combine two claims.

In WI-38 fibroblasts, the vascular senescence markers showed lower consistency than in hVSMCs, although many mRNAs appeared to be reliable markers of senescence.

“We observed lower consistency than in hVSMCs, although many mRNAs appeared to be reliable markers of senescence.”

May combine two claims.

Unsupervised clustering identified 28 unique clusters representing nine main cell types across all aortas, with multiple clusters for VSMCs, monocytes, T cells, endothelial cells, and B cells.

“We identified 28 unique clusters representing nine main cell types among all aortas; a detailed dot plot of the top 4 mRNAs driving each cluster’s cell type demonstrated the heterogeneity of vascular cells, including multiple clusters for VSMCs, monocytes, T cells, endothelial cells and B cells (Fig. 2a,b).”

May combine two claims.

HFD increased VSMC and monocyte populations, and ABT-737 reduced these populations.

“Such changes included HFD-mediated increases in VSMCs and monocytes and subsequent reductions of these populations with ABT-737 (Fig. 2d).”

May combine two claims.

WI-38 fibroblasts rendered senescent by IR, doxorubicin, or replicative exhaustion showed general senescence gene expression and increased SA-β-gal activity.

“We confirmed general senescence gene expression and increased SA-β-gal activity in WI-38 fibroblasts rendered senescent 10 days after treatment with IR (15 Gy) or doxorubicin (250 nM) or after reaching replicative exhaustion”

May combine two claims.

In T cell cluster 9, which had the highest SenMayo enrichment, subcluster 5 increased from 2% in ND to 23.29% under HFD and decreased to 8.49% after ABT-737.

“In cluster 9 (T cells), which exhibited the highest enrichment of SenMayo genes according to GSEA, unbiased subclustering uncovered a responsive subcluster (subcluster 5), which increased from 2% in the ND condition to 23.29% under the HFD condition and subsequently decreased to 8.49% following treatment with ABT-737 (Fig. 4e).”

May combine two claims.

Four clusters showed increased SenMayo and CellAge enrichment with HFD and were reduced by ABT-737.

“Using this approach, we narrowed our focus to the four clusters with increased SenMayo and CellAge enrichment by HFD, which were also reduced by ABT-737.”

May combine two claims.

Mitochondrial and ribosomal RNAs were equally distributed among clusters or not abundant enough to affect cell classification.

“The results indicated that mitochondrial and ribosomal RNAs were equally distributed among the clusters, or their abundance was not pronounced enough to have an impact on the classification of cells.”

May combine two claims.

In doxorubicin-treated mice, 14 of 17 vascular senescence-associated transcripts plus Mmp3 and Lcp1 mRNAs were significantly increased by doxorubicin and reduced by ABT-737 in both male and female mice.

“As shown, 14 of 17 transcripts and 2 additional transcripts that showed specificity for VSMCs (Mmp3 and Lcp1 mRNAs) were significantly increased by doxorubicin and reduced by ABT-737, including Spp1, Serpine2, Ctsb, Fth1 and Thbs1 mRNAs (Fig. 5e), as well as Ltbp2, Sparc, Lum, Cd9, Lgals3, Cst3, Prg4, Mmp3, Ctss, Lcp1 and Comp mRNAs (Extended Data Fig. 7b–d) in male and female mice.”

May combine two claims.

In the atherosclerotic cap (αSMA-positive cells) of the aortic root, Spp1, Ctsb, and Fth1 mRNAs were highly abundant in the HFD condition and significantly reduced following ABT-737 treatment.

“in the atherosclerotic cap (αSMA-positive cells) of the aortic root, all three markers (Spp1, Ctsb and Fth1 mRNAs) were highly abundant in the HFD condition and were significantly reduced following treatment with ABT-737”

May combine two claims.

Reactome pathway analysis showed normal wall gene expression was consistent with VSMC contraction and healthy function, whereas cap VSMCs were enriched for senescence-associated pathways such as ECM degradation and collagen assembly and degradation.

“Reactome pathway analysis reinforced the notion that normal wall gene expression was consistent with VSMC contraction and healthy function, whereas cap VSMCs were enriched for pathways associated with senescence, such as ECM degradation and collagen assembly and degradation”

May combine two claims.

Cluster 12 senescent cells (subcluster 4) were similar to cluster 0's senescent subpopulation but additionally exhibited foam-like cell gene expression, underscoring heterogeneity.

“While similar to cluster 0’s senescent subpopulation, cluster 12 senescent cells (subcluster 4) additionally exhibited foam-like cell gene expression, underscoring the heterogeneous properties of senescent cells.”

• AssumptionRests on an assumption: Senescent vascular cells are heterogeneous and dysfunctional, exhibiting growth arrest and inflammation.

May combine two claims.

Gpnmb mRNA was significantly increased in the cap, although it was not identified in the scRNA-seq data.

“While not identified in our scRNA-seq data, but significantly increased in the cap, Gpnmb mRNA was recently found to be highly expressed in senescent cells”

May combine two claims.

Doxorubicin caused loss of Lmnb1 mRNA expression, with a slight non-significant increase after ABT-737.

“We also observed a loss of Lmnb1 mRNA expression with doxorubicin and a slight (but not significant) increase with ABT-737 (Extended Data Fig. 7a).”

May combine two claims.

In VSMC cluster 0, subcluster 5 was 0% in ND, increased to 13.07% under HFD, and decreased to 0.42% after ABT-737.

“In particular, subcluster 5 increased in the HFD condition; the proportion of this subcluster was 0% in the ND condition, increased to 13.07% of the entire population during HFD feeding and decreased to 0.42% after ABT-737 treatment (Fig. 3a).”

May combine two claims.

Fth1 mRNA was highly abundant in the atherosclerotic core but its expression remained unchanged after ABT-737 treatment.

“Fth1 mRNA exhibited high abundance in the atherosclerotic core, but its expression remained unchanged after ABT-737 treatment”

May combine two claims.

Heatmap visualization showed significant increases in many senescence-associated transcripts (Spp1, Apoe, Ctsb, Thbs1, Sparc, Ctss) with development of atherosclerotic cap, which were subsequently reduced by senolytic treatment.

“Heatmap visualization of these results points to significant increases in many senescence-associated transcripts (Spp1, Apoe, Ctsb, Thbs1, Sparc and Ctss mRNAs) with the development of the atherosclerotic cap, which were subsequently reduced by senolytic treatment”

May combine two claims.

Among highly represented RNAs were those encoding SPP1, CTSB and the cathepsin family, CD9, LUM, TNFRSF11B, and THBS1, all showing high conservation in human senescent cells and human atherosclerosis scRNA-seq metadata in disease-associated VSMC populations.

“Among the highly represented RNAs were those that encode SPP1, CTSB and the cathepsin family, CD9, LUM, TNFRSF11B and THBS1, all showing high conservation in human senescent cells and human atherosclerosis scRNA-seq metadata in disease-associated VSMC populations.”

May combine two claims.

The changed pathways are heavily implicated in ECM remodeling, consistent with ABT-737 fostering physiological arterial remodeling.

“Further, these pathways are heavily implicated in ECM remodeling, consistent with the finding that ABT-737 treatment in HFD mice fostered physiological arterial remodeling (Fig. 1b,f,g).”

May combine two claims.

The identified transcriptomic signature is vascular-specific and may be useful for tracking and treating age-related vascular diseases.

“Our results uncover a vascular-specific transcriptomic signature of senescence that may be exploited for tracking and treating age-related vascular diseases.”

Classified as probably not a substantial claim.

Prior research has established an association between cellular senescence and the development and progression of atherosclerosis.

“Previous research has established an association between cellular senescence and the development and progression of atherosclerosis1–3.”

• cites [1] Matthews, C. et al. Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress. Circ. Res. 99, 156–164 (2006). sourceNot checked — no full text of the source
• cites [2] Minamino, T. et al. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation 105, 1541–1544 (2002). sourceNot checked — no full text of the source
• cites [3] Kunieda, T. et al. Angiotensin II induces premature senescence of vascular smooth muscle cells and accelerates the development of atherosclerosis via a p21-dependent pathway. Circulation 114, 953–960 (2006). sourceNot checked — no full text of the source

May combine two claims.

Two distinct mouse models of atherosclerosis and senolysis validated the same vascular senescence RNA markers, suggesting the senescence signature is vascular and not specific to the model used.

“An important finding of this study is that two distinct mouse models of atherosclerosis and senolysis validated the same vascular senescence RNA markers, suggesting that our senescence signature is vascular and not specific to the model used.”

May combine two claims.

The potentially senescent cells are connected to HFD-associated ECM remodeling, which was ameliorated by ABT-737.

“This link suggests a connection between these potentially senescent cells and the observed ECM remodeling associated with HFD, which was ameliorated by ABT-737.”

May combine two claims.

The data indicate that cellular senescence emerges in the aortas of HFD mice and is diminished by ABT-737 treatment.

“Together, these data point to the emergence of cellular senescence in the aortas of HFD mice, which was subsequently diminished by ABT-737 treatment.”

May combine two claims.

T cell cluster 9 had reduced total cell number under HFD, suggesting most cells may have died while a subset potentially underwent senescence.

“Notably, cluster 9 had an overall reduced total number of cells under the HFD condition, suggesting that most of these cells may have undergone cell death, whereas a subset of the remaining cells potentially underwent senescence.”

Classified as probably not a substantial claim.

The finding that oxLDL was the strongest trigger points to the importance of using physiologically relevant senescence inducers in culture.

“which points to the importance of using physiologically relevant senescence inducers in culture.”

May combine two claims.

Spatial transcriptomic data validated scRNA-seq findings that a subset of mRNAs indicates senescent cells, that these mRNAs are present in atherosclerotic cap and core where senescent cells accumulate, and that their abundance decreases with senolytic treatment.

“The spatial transcriptomic data thus validated the scRNA-seq findings that a subset of mRNAs is indicative of senescent cells, that these mRNAs are present in regions of the atherosclerotic artery (cap and core) where senescent cells accumulate, and that their abundance decreases with senolytic treatment.”

May combine two claims.

Validation of scRNA-seq results in the Ldlr−/−; 3MR model, which relies on p16-positive senescent cells, increased confidence that senescent vascular cells are likely p16-positive, as evidenced by shared reduction of senescence-associated RNAs after ABT-737 or GCV.

“By validating our scRNA-seq results in a model that relies on p16-positive senescent cells (Ldlr−/−; 3MR), we were more confident in concluding that senescent vascular cells are likely p16-positive, as evidenced by the shared reduction of senescence-associated RNAs in response to senolytic treatment (ABT-737) or p16-positive cell removal (GCV).”

May combine two claims.

The effect of ABT-737 on arterial stiffness may be independent of plaque remodeling because PWV measurements are taken from the abdominal aorta, suggesting reduced necrotic core area may be important for arterial elasticity but likely more indicative of improved plaque stability.

“The effect on arterial stiffness may be independent of plaque remodeling, as PWV measurements are taken from the abdominal aorta, suggesting that the reduced necrotic core area may be important for arterial elasticity but is likely more indicative of improved plaque stability.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Cst3, Ctsb, and Ctss mRNAs are commonly expressed in macrophages and have functions overlapping senescent cells, including increased lysosomal activity.

“Interestingly, Cst3, Ctsb and Ctss mRNAs are commonly expressed in macrophages, with functions that overlap those observed in senescent cells, including increased lysosomal activity27.”

• cites [27] Frendéus, K. H., Wallin, H., Janciauskiene, S. & Abrahamson, M. Macrophage responses to interferon-γ are dependent on cystatin C levels. Int. J. Biochem. Cell Biol. 41, 2262–2269 (2009). sourceNot checked — no full text of the source

May combine two claims.

The finding supports a functional role of senescent VSMCs and emphasizes the need for more specific markers.

“This finding not only supports the functional role of senescent VSMCs but also emphasizes the need for increasingly specific markers to identify such cells.”

May combine two claims.

HFD feeding and ABT-737 treatment may substantially influence the composition and transcriptomic profiles of aortic cells.

“These results suggest that HFD feeding and ABT-737 treatment may substantially influence the composition and transcriptomic profiles of aortic cells.”

May combine two claims.

Some senescence markers may be unique to vascular cells and their environments, while others may be broader markers of cellular senescence in the body.

“These results indicate that, while some senescence markers may be unique to vascular cells and the environments they are exposed to, others may constitute broader markers of cellular senescence in the body.”

May combine two claims.

p16 mRNA levels were too low to confidently identify senescent cells, and ABT-737 did not directly reduce any subset of p16-positive cells.

“Therefore, the levels of p16 mRNA were too low to confidently identify senescent cells, and treatment with ABT-737 did not directly reduce any subset of p16-positive cells (Extended Data Fig. 2b).”

• AssumptionRests on an assumption: The 10x Genomics scRNA-seq platform detects approximately 20–30% of all transcripts.

May combine two claims.

Transgene-mediated senolysis by removing p16-positive cells significantly modified senescence-associated transcriptomes in atherosclerotic conditions in both cap and core, restoring patterns to those seen in healthy cells.

“transgene-mediated senolysis by removing p16-positive cells significantly modified the senescence-associated transcriptomes seen in atherosclerotic conditions in both the cap and the core, restoring the patterns to those seen in areas of healthy cells.”

May combine two claims.

Fth1 mRNA appears to be a strong marker of senescence, preferentially expressed in nonimmune-like cells of the vasculature.

“Further, Fth1 mRNA appears to be a strong marker of senescence, preferentially expressed in nonimmune-like cells of the vasculature.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Sample sizes were selected based on previous experiments.

“Sample sizes were chosen based on previous experiments”

Probably not a substantial claim: may not pass S3 (load-bearing).

No statistical method was used to predetermine sample sizes for all experiments.

“no statistical method was used to predetermine sample sizes for all experiments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Each aPWV measurement in each animal was the average of five independent recordings.

“Each measurement of aPWV in each animal represents the average of five independent recordings.”

Probably not a substantial claim: may not pass S3 (load-bearing).

No data points were excluded from the analyses.

“no data points were excluded from the analyses.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All plaque areas in a given section above the innermost elastic fiber were scored blinded to the treatment group.

“In all cases, all plaque areas in a given section above the innermost elastic fiber were scored blinded to the treatment group.”

Classified as probably not a substantial claim.

Ldlr−/−; 3MR mice on a high-fat diet were randomly assigned to receive GCV or vehicle control by intraperitoneal injection.

“At 90 days of HFD feeding, Ldlr−/−; 3MR mice were randomized to receive GCV (5 mg kg−1, APP Pharmaceuticals) or vehicle control (Dulbecco’s PBS (DPBS), Corning) by IP injection on a repeating cycle of 5 days on and 14 days off until animal killing.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Animals were assigned to experimental groups using simple randomization.

“Simple randomization was used to assign animals to experimental groups.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All plaques in a given section were scored blinded to the treatment group.

“In all cases, all plaques in a given section were scored blinded to the treatment group.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Data collection and analysis were not performed blind to the experimental conditions.

“Data collection and analysis were not performed blind to the conditions of the experiments”