Senescent intimal foam cells are deleterious at all stages of atherosclerosis

Abstract

Advanced atherosclerotic lesions contain senescent cells, but the role of these cells in atherogenesis remains unclear. Using transgenic and pharmacological approaches to eliminate senescent cells in atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr^-/-) mice, we show that these cells are detrimental throughout disease pathogenesis. We find that foamy macrophages with senescence markers accumulate in the subendothelial space at the onset of atherosclerosis, where they drive pathology by increasing expression of key atherogenic and inflammatory cytokines and chemokines. In advanced lesions, senescent cells promote features of plaque instability, including elastic fiber degradation and fibrous cap thinning, by heightening metalloprotease production. Together, these results demonstrate that senescent cells are key drivers of atheroma formation and maturation and suggest that selective clearance of these cells by senolytic agents holds promise for the treatment of atherosclerosis.

Fig. 1. p16^Ink4a+ senescent cells drive formation of atherosclerotic plaques

(A) (Left) X-Gal electron microscopy images showing three types of senescent cells in plaques of Ldlr^−/− mice on a HFD for 88 days. Cell outlines are traced by dashed yellow lines. Endothelial-like cells are elongated and adjacent to the lumen. VSMC-like cells are elongated spindle-shaped cells or irregularly shaped ramified cells. Macrophage-like cells are highly vacuolated circular cells. (Right) Senescent cell quantification in plaques. Mac, macrophage; EC, endothelial cell. (B) (Left) Experimental design for testing the effect of senescent cell clearance on atherogenesis. Veh, Vehicle. (Right) Sudan IV–stained descending aortas (not including the aortic arch). (C) Quantification of total descending aorta plaque burden, number, and lesion size. Scale bars: 2 μm (A); 500 nm [(A), insets]. Data represent mean ± SEM (error bars) [biological n is indicated on graphs and refers to individual plaques in (A) (one per mouse) and aortas in (C)]. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t tests with Welch’s correction).

Fig. 2. Intimal senescent foamy macrophages form during early atherogenesis and foster production of proatherogenic factors

(A) (Left) Schematic of the inner curvature of the aortic arch. LV, left ventricle. (Middle) Examples of SA β-Gal–stained 9-day fatty streaks with and without senescent cell clearance and quantification. (Right) Measurements of streak size. Treatment involved the administration of 25 mg/kg of GCV once daily. BCA, brachiocephalic artery. (B) TEM images of Ldlr^−/− mice after 9-day HFD feeding, showing fatty-streak foci with X-Gal–positive foam cell macrophages (artificial coloring articulates cell boundaries in the multilayer). (C) Quantification of multilayer foci in day-9 fatty streaks with and without senescent cells. (D) Quantification of foam cell macrophages with X-Gal crystal–containing vesicles without and with clearance. (E) (Left) Representative SA β-Gal–stained 12-day fatty streaks without and with GCV treatment (25 mg/kg of GCV three times daily) for the last 3 days. (Right) Quantification of lesion burden. (F) RT-qPCR analysis of senescence marker expression in fatty streaks collected from Ldlr^−/− and Ldlr^−/−;3MR mice on a 12-day HFD and treated with GCV for the last 3 days. Scale bars: 1 mm (A) and (E); 2 μm (B); and 500 nm [(B), insets]. Bar graphs represent mean ± SEM (error bars) [biological n is indicated directly on all graphs and refers to individual aortic arches in (A) and (C) to (E) or dissected aortic arch inner curvatures in (F)]. *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed t tests with Welch’s correction).

Fig. 3. Removal of p16^Ink4a+ cells in established plaques perturbs the proatherogenic microenvironment

(A) (Left) Experimental design. (Middle) Sudan IV–stained descending aortas (not including the arch). (Right) Quantification of Sudan IV⁺ areas and plaque number. (B) RT-qPCR for senescence markers in aortic arches from indicated cohorts. Aortic arches from Ldlr^−/−;3MR females fed a LFD until 258 days of age and treated with Veh for the last 100 days were used to assess baseline expression levels. Treatments in (A) and (B) involved daily injections of 5 mg/kg of GCV (or Veh) for 5 days, followed by 14 days off; this cycle was repeated for 100 days. Bar graphs represent mean ± SEM (error bars) [biological n is indicated directly on all graphs and refers to individual aortas in (A) or aortic arches in (B)]. *P < 0.05; **P < 0.01; ***P < 0.001 [analysis of variance (ANOVA) with Sidak’s post-hoc correction for family-wise error in (A) and unpaired two-tailed t test with Welch’s correction in (B)].

Fig. 4. Senescent cells promote plaque instability by elevating metalloprotease production

(A) Representative sections from descending aorta plaques of mice with the indicated genotypes, treatments, diets, and histological stainings. Red dashed lines trace the fibrous cap; red arrowheads indicate ruptured aortic elastic fibers. H–E, hematoxylin and eosin. (B) Quantification of fibrous cap thickness in plaques from (A). (C) (Top) Experimental overview. (Bottom) RT-qPCR analysis of senescence markers in GFP⁺ and GFP⁻ cells. EGFP, enhanced green fluorescent protein. Bar graphs represent mean ± SEM (error bars) [biological n is indicated directly on all graphs and refers to individual descending aorta mouse plaques in (B) and flow-sorted cell fractions isolated from individual mice in (C)]. *P < 0.05; **P < 0.01; ns, not significant [ANOVA with Sidak’s post-hoc correction for familywise error in (B) and ratio paired t test in (C)].