Senescent Vascular Smooth Muscle Cells Drive Inflammation Through an Interleukin-1α-Dependent Senescence-Associated Secretory Phenotype

Abstract

Objective: Vascular smooth muscle cells (VSMCs) that become senescent are both present within atherosclerotic plaques and thought to be important to the disease process. However, senescent VSMCs are generally considered to only contribute through inaction, with failure to proliferate resulting in VSMC- and collagen-poor unstable fibrous caps. Whether senescent VSMCs can actively contribute to atherogenic processes, such as inflammation, is unknown.

Approach and results: We find that senescent human VSMCs develop a proinflammatory state known as a senescence-associated secretory phenotype. Senescent human VSMCs release high levels of multiple cytokines and chemokines driven by secreted interleukin-1α acting in an autocrine manner. Consequently, the VSMC senescence-associated secretory phenotype promotes chemotaxis of mononuclear cells in vitro and in vivo. In addition, senescent VSMCs release active matrix metalloproteinase-9, secrete less collagen, upregulate multiple inflammasome components, and prime adjacent endothelial cells and VSMCs to a proadhesive and proinflammatory state. Importantly, maintaining the senescence-associated secretory phenotype places a large metabolic burden on senescent VSMCs, such that they can be selectively killed by inhibiting glucose utilization.

Conclusions: Senescent VSMCs may actively contribute toward the chronic inflammation associated with atherosclerosis through the interleukin-1α-driven senescence-associated secretory phenotype and the priming of adjacent cells to a proatherosclerotic state. These data also suggest that inhibition of this potentially important source of chronic inflammation in atherosclerosis requires blockade of interleukin-1α and not interleukin-1β.

Keywords: aging; atherosclerosis; inflammation; interleukin-1; muscle, smooth, vascular.

Figure 1.

Vascular smooth muscle cell (VSMC) senescence, which occurs in mature plaques, can be modeled in vitro. A, Human carotid plaque stained for senescence with senescence-associated β-galactosidase (SABG; blue) and VSMCs with α-smooth muscle actin (αSMA; brown), as indicated. Most SABG +ve cells are in the fibrous cap region, whereas dual staining shows the majority of senescent cells to be VSMCs (arrows), although some senescent cells with a VSMC-like morphology do not stain for αSMA (arrow heads). B–F, Replicative and induced senescent VSMCs were stained and enumerated for SABG (B and C) and BrdU (5-bromo-2’-deoxyuridine; D and E) +ve cells, or stained for persistent DNA damage with γH2AX (F). Data represent mean±SD, representative of n=≥3. Scale bars represent 200 mm (low power) and 50 mm (high power).

Figure 2.

Senescent vascular smooth muscle cells (VSMCs) secrete cytokines and chemokines in an interleukin-1α (IL-1α)–dependent manner. Cytokine and chemokine content of conditioned media from control, replicative (A) and induced (B) senescent VSMC cultures measured by ELISA. IL-6 and IL-8 content of conditioned media from control and senescent VSMCs incubated ±neutralizing IL-1α (C and D) or IL-1β antibodies (E and F). Data represent mean±SEM of n=5 (A–C, E, and F), 3 (D); *P≤0.05, **P≤0.02, and ***P≤0.005. NS indicates not significant.

Figure 3.

Secretion of interleukin-1α (IL-1α) by senescent vascular smooth muscle cells (VSMCs) drives the senescence-associated secretory phenotype in an autocrine manner. A, Lactate dehydrogenase (LDH) activity in conditioned media (CM) from control and senescent VSMCs, lysed cells (+ve) or buffer only (−ve). B, IL-6 content of conditioned media from control and senescent VSMCs incubated with the indicated % of necrotic control and senescent VSMCs. C, Flow cytometry analysis of senescent VSMCs and lipopolysaccharide (LPS)-treated THP-1 cells stained with isotype control-FITC (fluorescein-5-isothiocyanate; red) or anti-IL-1α−FITC (blue). D, IL-1–dependent IL-2 production by EL4 cells incubated with senescent VSMC conditioned media (CM), ±neutralizing IL-1α pAb. E, IL-6 content of media conditioned by senescent VSMCs incubated ±IL-1α pAb as a pretreatment or during supernatant collection. Data represent mean±SEM of n=3 (B and E), 4 (D); *P≤0.05, **P≤0.02, and ***P≤0.005.

Figure 4.

Senescent vascular smooth muscle cells (VSMCs) induce chemotaxis, secrete active matrix metalloproteinase-9 (MMP-9), and upregulate inflammasome components. A, Number of THP-1 cells migrating toward control or senescent VSMCs, ±neutralizing interleukin-1α (IL-1α), MCP-1, or IL-8 antibodies. B, Enumeration of immune cells recruited to the peritoneum of mice in response to control or senescent VSMCs. C, Gelatin gel zymography showing level and activity of MMPs in the conditioned media of control, IL-1α–treated or senescent VSMCs. D, Amount of collagen produced by equal numbers of control or senescent VSMCs. E, Relative expression of transcripts for inflammasome-associated components in control or senescent VSMCs. Data represent mean±SEM of n=4 (A), 3 (B and D), 2 (E); *P≤0.05, **P≤0.02, ***P≤0.005. ND indicates not detected.

Figure 5.

Vascular smooth muscle cell (VSMC) senescence-associated secretory phenotypes (SASPs) can prime adjacent control cells to a proinflammatory state. A and B, Interleukin-6 (IL-6) and IL-8 content of cell lysates from control VSMCs (A) or human umbilical vein endothelial cells (HUVECs; B) incubated with IL-1α or conditioned media (CM) from control or 2 senescent VSMC cultures, ±neutralizing IL-1α pAb. IL-8 values are scaled 2.5-fold lower. C, Flow cytometry plots showing IL-1α–dependent increase in surface E-selectin expression after treatment as indicated. D, Mean fluorescent intensities (MFIs) of adhesion molecules on HUVEC populations analyzed by flow cytometry after treatment as indicated, ±neutralizing IL-1α pAb (Ab). Data represent mean±SD of n=3 (A), 2 (B–D); **P≤0.02 and *** P≤0.005.

Figure 6.

Senescent vascular smooth muscle cells (VSMCs) adopt a hypermetabolic state that enables their selective elimination. A and B, Measurement of glycolysis by the extracellular acidification rate (ECAR; A) and oxidative phosphorylation by the oxygen consumption rate (OCR; B) in control (gray) and senescent (black) VSMCs treated at times indicated with: I=oligomycin; II=FCCP; III=2-deoxyglucose (2-DG); and IV=rotenone/antimycin. C, Level of XBP-1 splicing in control and senescent VSMCs measured by qPCR (quantitative polymerase chain reaction) and RT-PCR (reverse transcription-polymerase chain reaction). D, Cell number measured by crystal violet following treatment of control (gray) and senescent (black) VSMCs with 2-DG for 4 days. E, Propidium iodide staining reveals increased levels of cell death in senescent VSMCs after 2-DG treatment. F, Senescent VSMC treated with 2-DG displaying cellular blebbing, pyknotic nuclear condensation, and propidium iodide negativity indicative of apoptosis. Data represent mean±SD of n=2; *P≤0.05, **P≤0.02. Scale bars represent 50 mm.

Figure 7.

Senescent cells in human carotid plaques express interleukin-1α (IL-1α) and colocalize with IL-6 and CD68. Human carotid endarterectomy samples stained for senescence-associated β-galactosidase (SABG): showing senescent vascular smooth muscle cell (VSMC)-like cells in the fibrous cap that coexpress IL-1α (A); a discrete region containing many senescent cells showing localized IL-6 expression, in contrast to an adjacent region with no senescent cells or IL-6 (B); area within a fibrous cap (FC) showing high levels of local IL-6 expression adjacent to senescent cells (C); a large composite image showing localization between senescent and CD68 +ve cells (outlined and enlarged below; D); a region showing focal accumulation of senescent and CD68 +ve cells (E). Only eosin counterstain was used. Scale bars represent 100 and 25 mm (B; high power). L indicates lumen; and NC, necrotic core.