Endothelial senescent-cell-specific clearance alleviates metabolic dysfunction in obese mice

Abstract

Accumulation of senescent cells is a key contributor to multiple diseases across the lifespan, including metabolic dysfunction. We previously demonstrated that elimination of senescent cells using senolytic drugs alleviates obesity-induced metabolic dysfunction. However, the contribution of senescent endothelial cells to metabolic disorders remains elusive. Hence, we crossed mice that allow selective elimination of senescent cells (p16^Ink4a-LOX-ATTAC mice) with Tie2-Cre mice (Tie2-Cre;p16^Ink4a-LOX-ATTAC) to enable identification and inducible, selective elimination of p16^Ink4a+ senescent endothelial cells. Targeted removal of senescent endothelial cells from obese Tie2-Cre;p16^Ink4a-LOX-ATTAC mice attenuated the pro-inflammatory senescence-associated secretory phenotype and alleviated metabolic dysfunction. Conversely, transplanting senescent endothelial cells into lean mice caused adipose tissue inflammation and metabolic dysfunction. Consistent with these findings, the senolytic, fisetin, which targets senescent endothelial cells among other senescent cell types, reduced adipose tissue senescent endothelial cell abundance and improved glucose metabolism in obese mice or mice transplanted with senescent mouse endothelial cells. Our results indicate that specifically eliminating p16^Ink4a+ senescent endothelial cells is a potential therapeutic strategy for metabolic disease.

Keywords: SASP factors; TNFα; cellular senescence; diabetes; endothelial cells; fisetin; glucose intolerance; obesity; p16(Ink4a); senolytics.

Figure 1.. AP20187 treatment reduced senescent ECs in HFD-fed Tie2-Cre+/–;p16-LOX-ATTAC mice

(A) t-distributed stochastic neighbor embedding (t-SNE) plots of CyTOF-defined clusters showing total SVF, CD45^- CD31⁺ ECs, and Tie2⁺ (CD202⁺) ECs in the SVF of gWAT from HFD-fed Tie2-Cre^+/- ;p16-LOX-ATTAC mice. (B) t-SNE plots of p16⁺ Ki67^-, gH2A-X⁺, Tnfα⁺, Il-6⁺, and p21⁺ Ki67^- cells in Tie2⁺ EC populations. (C) The population of p16⁺ p21⁺ Ki67^-, p16⁺ p21^- Ki67^-, and p16^- p21⁺ Ki67^- subsets in Tie2⁺ ECs from HFD-fed Tie2-Cre^+/- ;p16-LOX-ATTAC mice (n = 6). (D) Heatmap of SASP-high populations in Tie2⁺, CD45⁺, or Pdgfra⁺ Ki67^- cells. (E and F) t-SNE plots and (F) immunohistochemistry of p16⁺ Ki67^- ECs from chow- and HFD-fed Tie2-Cre^+/- ;p16-LOX-ATTAC mice after treatment with vehicle or AP20187. p16 (red), CD31 (green), Ki67 (yellow), and DAPI (blue). (G–J) Quantification p16⁺ Ki67^- (n = 6,6), (H) p21⁺ Ki67^- (n = 5,6), (I) p16⁺ p21⁺ Ki67^- ECs, and (J) SA-β-gal⁺ cells (arrows) in gWAT (n = 4,5). Scale bars: 50 μm and 25 μm from vehicle- or AP20187-treated HFD-fed Tie2-Cre^+/- ;p16-LOX-ATTAC mice, respectively. Data are means ± SEM in plots of all individual data. *p < 0.05, ***p < 0.001 by two-tailed, unpaired Student’s t tests (G, H, and J). See also Data S1.

Figure 2.. Targeting senescent ECs by AP20187 treatment alleviated metabolic dysfunction in HFD-fed Tie2-Cre+/–p16-LOX-ATTAC mice

(A and B) Changes in lean and fat mass from baseline (n = 12,13). (C) Representative images and quantification of lipid droplet size in gWAT (HE stain, scale bars: 200 μm) (n = 4,4). (D) GTT curves (mean ± SEM) and area under the GTT curves (AUC) (n = 12,13). (E) Representative images of liver fibrosis (Masson trichrome stain, scale bars: 200 μm). (F) Quantification of hepatic lipid accumulation (n = 4,5). (G and H) (G) Activity (n = 12,13) and (H) representative images and quantification of EC density in gWAT (n = 5,4) of vehicle- or AP20187-treated obese Tie2-Cre^+/- ;p16-LOX-ATTAC mice. CD31 (red), WGA lectin (green), and DAPI (blue). Scale bars: 200 mm. Data are means ± SEM in plots of all individual data. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed, unpaired Student’s t tests (A–C, E, and H) and one-way ANOVA with Tukey’s post hoc comparison (G). See also Data S1.

Figure 3.. Senescent ECs promoted adipose tissue inflammation through their SASP

(A) Heatmap of SASP factor expression in total SVF, p16⁺ Ki67^-, and p16⁺ Ki67^- Tie2⁺ cells in SVF from vehicle-treated obese Tie2-Cre^+/- ;p16-LOX-ATTAC mice analyzed by CyTOF (n = 6). (B and C) Percent of p16⁺ Ki67^- Tnfα⁺ cells in ECs (n = 6,5), and (C) representative images and quantification of CLS in gWAT (n = 4,4) from vehicle- or AP20187- treated obese Tie2-Cre^+/- ;p16-LOX-ATTAC mice. F4/80 (red), WGA lectin (green), and DAPI (blue). Scale bars: 200 μm. (D and E) (D) Heatmap of relative SASP factors and (E) senescence markers in human preadipocytes exposed to CM from non-irradiated (non-IR) or irradiated (IR) HUVECs (n = 4,4,4,4). (F) SASP factors in preadipocytes exposed to CM from non-IR or si-Cont-, si-TNFα-, or si-RELA-treated IR HUVECs (n = 3,3,3,3). (G–K) GTT curve (mean ± SEM) and AUC (n = 8,9), (H) SA-β-gal (n = 5,7), (I) senescence markers, (J) SASP factors (n = 7,9), and (K) representative images and quantification of CLS in gWAT from non-IR or IR EC-transplanted mice (n = 8,9). Data are means ± SEM in plots of all individual data. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed, unpaired Student’s t tests (B, E, and G–K) and one-way ANOVA with Tukey’s post hoc comparison (F). See also Data S1.

Figure 4.. Reducing senescent ECs by fisetin alleviated adipose tissue inflammation and glucose intolerance

(A–C) Representative images and quantification of p16⁺ cells (n = 6.6), p16 (red), WGA lectin (green), and DAPI (blue), scale bars: 50 μm; (B) SA-β-gal⁺ cells around blood vessels, scale bars: 25 μm (n = 3,3); and (C) heatmap of SASP factors (n = 5,5) in vehicle- or fisetin-treated human fat explants. (D–H) Representative images and quantification of luciferase activity (n = 8,6); (E) GTT curve (means ± SEM) and AUC (n = 4, 4); (F) senescence markers (n = 4,4); (G) SASP factors; and (H) representative images and quantification of CLS (n = 4,4) in vehicle- or fisetin-treated IR EC-transplanted mice. F4/80 (red), WGA lectin (green), and DAPI (blue). Scale bars: 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed, unpaired Student’s t tests (A, B, and D–H). See also Data S1.