Efficacy and limitations of senolysis in atherosclerosis

Abstract

Aims: Traditional markers of cell senescence including p16, Lamin B1, and senescence-associated beta galactosidase (SAβG) suggest very high frequencies of senescent cells in atherosclerosis, while their removal via 'senolysis' has been reported to reduce atherogenesis. However, selective killing of a variety of different cell types can exacerbate atherosclerosis. We therefore examined the specificity of senescence markers in vascular smooth muscle cells (VSMCs) and the effects of genetic or pharmacological senolysis in atherosclerosis.

Methods and results: We examined traditional senescence markers in human and mouse VSMCs in vitro, and in mouse atherosclerosis. p16 and SAβG increased and Lamin B1 decreased in replicative senescence and stress-induced premature senescence (SIPS) of cultured human VSMCs. In contrast, mouse VSMCs undergoing SIPS showed only modest p16 up-regulation, and proliferating mouse monocyte/macrophages also expressed p16 and SAβG. Single cell RNA-sequencing (scRNA-seq) of lineage-traced mice showed increased p16 expression in VSMC-derived cells in plaques vs. normal arteries, but p16 localized to Stem cell antigen-1 (Sca1)+ or macrophage-like populations. Activation of a p16-driven suicide gene to remove p16+ vessel wall- and/or bone marrow-derived cells increased apoptotic cells, but also induced inflammation and did not change plaque size or composition. In contrast, the senolytic ABT-263 selectively reduced senescent VSMCs in culture, and markedly reduced atherogenesis. However, ABT-263 did not reduce senescence markers in vivo, and significantly reduced monocyte and platelet counts and interleukin 6 as a marker of systemic inflammation.

Conclusions: We show that genetic and pharmacological senolysis have variable effects on atherosclerosis, and may promote inflammation and non-specific effects respectively. In addition, traditional markers of cell senescence such as p16 have significant limitations to identify and remove senescent cells in atherosclerosis, suggesting that senescence studies in atherosclerosis and new senolytic drugs require more specific and lineage-restricted markers before ascribing their effects entirely to senolysis.

Keywords: Ageing; Atherosclerosis; Cell senescence.

Graphical Abstract

Effects of p16-induced genetic senolysis or ABT-263 drug-induced senolysis on atherosclerosis.

Figure 1

Senescence markers in primary human and mouse VSMCs undergoing senescence. (A) % EdU⁺ in cultured human VSMCs (Control), after 24h treatment with 500 nM doxorubicin (Dox 1d), after an additional 21 days recovery in control conditions (control 21d) or after doxorubicin (Dox 1d+ 21d), or at replicative senescence (RS). (B–D) mRNA levels of Lamin B1, p16, and p21 in cell populations described in (A) relative to control (1d) cells. (E) Western blot for Lamin B1, p16, p21, and p53 for cells treated in (A). n = 6–8 human VSMC isolates. (F and G) EdU⁺ % (F) or SAβG⁺ % (G) of mouse p16-3MR VSMCs treated increasing concentrations of Doxorubicin for 1 day followed by 7 days recovery vs. vehicle control. (I–K) qPCR for Lamin B1, IL6, p16, or p21 mRNA expression for cells treated in (F). (L) Western blot of mouse cells as treated in (F) for Lamin B1, p16, or p21. n = 3–8 mouse VSMC isolates. Data are means (SD), one-way ANOVA with correction for multiple comparisons (A) or unpaired Student’s t-test vs. Control 1d (B–D) or vs. Vehicle (Dox 0 nM) (F–K).

Figure 2

p16/Cdkn2a is detected in VSMCs in mouse atherosclerotic plaques. (A and B) UMAP plots showing scRNA-seq profiles of unsorted aortic cells from Myh11Cre^ERt2+/Confetti⁺ mice (A), or sorted Confetti⁺ VSMCs from atherosclerotic plaque and media of fat-fed Myh11Cre^ERt2/Confetti⁺/ApoE^−/− mice (B). Log-transformed expression levels of Myh11 and p16/Cdkn2a are shown alongside e-Cadherin/Cdh5 and Pdgfrα (A) or Cd68 and Ly6a/Sca1 (B) using a scale from white to dark red. Insets show high power regions of clusters 6, 8, and 9 and expression of p16 in (B). Feature plots show log-normalized expression levels. (C) % cells in each cluster with detectable expression of p16/cdkn2a after 14 weeks or 18 weeks or high-fat feeding, or combined.

Figure 3

GCV treatment of p16-3MR mice does not affect atherosclerosis, but induces inflammation. (A) Aortic root plaques in ApoE→ApoE, p16→ApoE, ApoE→p16, or p16→p16 mice + GCV, or p16→p16 mice + saline, stained with Masson’s trichrome, TUNEL, or Mac3. Scale bar = 300 µm. High power inset shows apoptotic cell and nuclear debris from outlined area. (B) Plaque area for mice in (A). (C and D) Number of TUNEL⁺ cells/aortic root plaque (C) or %Mac3⁺ cells (D) for mice in (A). (E–G) Relative mRNA expression for p16, IL18, or TNFα in experimental mice. Data are means (SD) n = 5–10 mice. One-way ANOVA with correction for multiple comparisons (B–D) or Kruskal–Wallis H test followed by Dunn’s multiple comparisons test (E–G).

Figure 4

ABT-263 (Navitoclax) selectively reduces senescent VSMCs. (A and B) Photomicrographs (A) or quantification (B) of mouse VSMCs stained for SAβG, as replicating control cells or after dox1 + 7 days treatment, or each group ±1 µM ABT-263 treatment for 48 h. (C) Western blot and quantification for p16 in cells treated in (A and B). (D) Fold change in mRNA expression compared with control replicating cells for p16 and a range of SASP cytokines against the housekeeping gene HMBS. Data are means (SD), n = 4–5. Unpaired Student’s t-test. (E) Mouse macrophages cultured for 28 days, then treated with 1 µM ABT-263 for 48 h and stained for SAβG. Data are means (SD), n = 3. Unpaired Student’s t-test.

Figure 5

ABT-263 reduces atherosclerosis, but not local SASP cytokine expression. (A) ORO staining of mouse descending aorta treated with control (vehicle) or ABT-263, and quantification of %ORO area (n = 11–14). Scale bar = 3 mm. (B) Masson’s trichrome histochemistry of aortic root atherosclerotic plaque from mice treated in (A). Panels below show high power view of outlined area. Arrow shows necrotic core. Scale bar = 200 µm. (C–E) Aortic root plaque area/total area (C) Cap area (D), or Core area (E) for mice in (A). n = 11–13. (F) qPCR for relative expression of p16 or SASP cytokines in aortic arches of experimental mice against the housekeeping gene HMBS (n = 7). Data are means (SD), n = 10. Unpaired Student’s t-test (A, C–E) or Mann–Whitney U test (F).

Figure 6

Effects of ABT-263 on peripheral blood counts and overview of effects of senolysis on atherosclerosis. (A–F) Total red blood cell (RBC) and white blood cell (WBC) counts and differential WBC and platelet counts in experimental mice at baseline and end of study after control treatment or with ABT-263. Data are means (SD), n = 12–15, Student’s t-test between baseline and end for each group. (G and H) Schematic of predicted effects of GCV treatment on experimental mice and observed or predicted consequences (G), or of ABT-263 on atherosclerosis and peripheral blood counts (H).