Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis

Abstract

Autophagy is triggered in vascular smooth muscle cells (VSMCs) of diseased arterial vessels. However, the role of VSMC autophagy in cardiovascular disease is poorly understood. Therefore, we investigated the effect of defective autophagy on VSMC survival and phenotype and its significance in the development of postinjury neointima formation and atherosclerosis. Tissue-specific deletion of the essential autophagy gene Atg7 in murine VSMCs (atg7^-/- VSMCs) caused accumulation of SQSTM1/p62 and accelerated the development of stress-induced premature senescence as shown by cellular and nuclear hypertrophy, CDKN2A-RB-mediated G₁ proliferative arrest and senescence-associated GLB1 activity. Transfection of SQSTM1-encoding plasmid DNA in Atg7^+/+ VSMCs induced similar features, suggesting that accumulation of SQSTM1 promotes VSMC senescence. Interestingly, atg7^-/- VSMCs were resistant to oxidative stress-induced cell death as compared to controls. This effect was attributed to nuclear translocation of the transcription factor NFE2L2 resulting in upregulation of several antioxidative enzymes. In vivo, defective VSMC autophagy led to upregulation of MMP9, TGFB and CXCL12 and promoted postinjury neointima formation and diet-induced atherogenesis. Lesions of VSMC-specific atg7 knockout mice were characterized by increased total collagen deposition, nuclear hypertrophy, CDKN2A upregulation, RB hypophosphorylation, and GLB1 activity, all features typical of cellular senescence. To conclude, autophagy is crucial for VSMC function, phenotype, and survival. Defective autophagy in VSMCs accelerates senescence and promotes ligation-induced neointima formation and diet-induced atherogenesis, implying that autophagy inhibition as therapeutic strategy in the treatment of neointimal stenosis and atherosclerosis would be unfavorable. Conversely, stimulation of autophagy could be a valuable new strategy in the treatment of arterial disease.

Keywords: atherosclerosis; autophagy; neointima formation; senescence; sequestosome 1/p62; vascular smooth muscle cells.

Figure 1.

Autophagy is defective in Atg7^F/FTagln-Cre⁺ VSMCs. (A) VSMCs were isolated from the aorta of Atg7^+/+Tagln-Cre⁺ (+/+) and Atg7^F/FTagln-Cre⁺ (−/−) mice. Western blot analysis of ATG7, SQSTM1, ATG12–ATG5 and MAP1LC3B in untreated Atg7^+/+ and atg7^−/− VSMCs. ACTB was used as loading control. Bands are shown in duplicate. (B,C) Atg7^+/+ and atg7^−/− VSMCs were treated with EBSS for 48 h, followed by western blot analysis of MAP1LC3B (B) or transmission electron microscopy (C). Autophagic vesicles, characterized by incorporated dense degraded material and indicated by arrows, were quantified per cell (***, P < 0.001 vs. atg7^−/−; ^###, P < 0.001 vs. control; two-way ANOVA with genotype and treatment as category factors). Scale bar: 1 µm. The right panel shows a high-power image of autophagosomes in EBSS-treated Atg7^+/+ VSMCs. Scale bar: 500 nm.

Figure 2.

Defective autophagy in VSMCs results in increased protection against oxidative stress-induced cell death. (A) Atg7^+/+Tagln-Cre⁺ (+/+) and Atg7^F/FTagln-Cre⁺ (−/−) VSMCs were treated with 25 µmol/l tBHP or 50 µg/ml oxLDL for 24 h (***, P < 0.001 vs. Atg7^+/+; ^###, P < 0.001 vs. control; n = 3 experiments in triplicate; two-way ANOVA with genotype and treatment as category factors and Dunnett Post Hoc test). To measure ROS production, Atg7^+/+ and atg7^−/− VSMCs were left untreated or treated with 100 µmol/l tBHP for 6 h, followed by a DCFDA staining (***, P < 0.001 vs. Atg7^+/+; n = 200 cells/condition in duplicate; two-way ANOVA with genotype and treatment as category factors). (B) Atg7^+/+ and atg7^−/− VSMCs were treated with 10 µmol/l puromycin (PM) for 12 h or exposed to UV-irradiation for 10 min (NS, not significant vs. Atg7^+/+; ^###, P < 0.001 vs. control; n = 2 experiments in triplicate; two-way ANOVA with genotype and treatment as category factors). (C) Analysis of Gsta and Nqo1 expression in Atg7^+/+ and atg7^−/− VSMCs by real time RT-PCR (***, P < 0.001; n = 2 experiments in duplicate; Student t test) and western blotting. (D) Western blot analysis of NFE2L2 in cytoplasmic and nuclear fractions of Atg7^+/+ and atg7^−/− VSMCs. (E) atg7^−/− VSMCs were transfected with 100 nmol/l siRNA against Nfe2l2 (siNfe2l2) or nontargeting siRNA (siCtrl). After 72 h, silencing efficiency was confirmed by western blotting by assessment of NFE2L2, GSTA and NQO1 expression and VSMCs were treated with tBHP or oxLDL for 24 h (***, P < 0.001 vs. siCtrl; ^###, P < 0.001 vs. control; n = 3 experiments in duplicate; two-way ANOVA with genotype and treatment as category factors and Dunnett Post Hoc test).

Figure 3.

Defective autophagy in VSMCs elicits cellular hypertrophy, and increases migration capacity and total collagen amount. (A) VSMCs isolated from Atg7^+/+Tagln-Cre⁺ (+/+) and Atg7^F/FTagln-Cre⁺ (−/−) aorta were labeled with calcein AM and visualized by confocal fluorescence microscopy. Scale bar: 10 µm. Cell size was measured using z-stack images (*, P < 0.05; n = 2 experiments; Student t test). (B) Thoracic aorta of Atg7^+/+ and atg7^−/− mice were stained with H&E to measure the width of the media (white arrows) (**, P < 0.01; n = 6 regions/aorta; Univariate). Scale bar: 25 µm. Note that the number of VSMC layers between Atg7^+/+ and atg7^−/− aorta was not different. (C) Migratory capacity of Atg7^+/+ and atg7^−/− VSMCs was analyzed using an Oris Migration Assay (***, P < 0.001; n = 2 experiments in triplicate; Student t test). Western blot analysis of TGFB and CXCL12 in Atg7^+/+ and atg7^−/− VSMCs. (D) Atg7^+/+ and atg7^−/− VSMCs were left untreated or treated with 10 ng/ml TGFB for 48 h and stained with Sirius red to examine total collagen amount (***, P < 0.001 vs. Atg7^+/+; ^###, P < 0.001 vs. control; n = 4 experiments in triplicate; two-way ANOVA with genotype and treatment as category factors).

Figure 4.

Defective autophagy in VSMCs accelerates senescence. (A) Atg7^+/+Tagln-Cre⁺ (+/+) and Atg7^F/FTagln-Cre⁺ (−/−) VSMCs were treated with BrdU to examine proliferation (***P < 0.001; n = 9 counting regions of 300 cells/region/condition; Student t test). The size of BrdU-positive nuclei was measured to detect nuclear hypertrophy (**, P < 0.01; n = 50 nuclei; Student t test). (B) DNA cell cycle analysis of Atg7^+/+ (blue) and atg7^−/− (red) VSMCs. The bar graph shows the percentage of Atg7^+/+ and atg7^−/− nuclei in the G₁ and G₂/M phase of the cell cycle (**, P < 0.01; ***, P < 0.001; Student t test). (C) Atg7^+/+ and atg7^−/− VSMCs were stained with X-gal mixture for 24 h, followed by Nuclear Fast Red staining. Scale bar: 125 µm. The number of senescence-associated GLB1-positive VSMCs was quantified. (***, P < 0.001; n = 2 counting regions of 200 cells/region/condition; Student t test) (D) Western blot analysis of CDKN2A, phospho RB, total RB, acetylated TP53 and CDKN1A in Atg7^+/+ and atg7^−/− VSMCs. (E) Detection of DNA damage in Atg7^+/+ and atg7^−/− VSMCs by comet assay. Atg7^+/+ VSMCs treated with .5 mmol/l H₂O₂ for 10 min were used as positive control. Scale bar: 25 µm.

Figure 5.

SQSTM1 accumulation links defective VSMC autophagy to senescence. (A) Atg7^+/+ VSMCs were transfected with 5 µg plasmid DNA encoding SQSTM1 (SQSTM1+). Four d after transfection, VSMCs were analyzed for SQSTM1, CDKN2A, phospho RB and total RB expression by western blotting. (B,C) SQSTM1 overexpressing VSMCs were incubated with BrdU (B) to examine proliferation capacity (***, P < 0.001; n = 2 counting regions of 1000 cells/region/condition in duplicate; Student t test) or (C) stained with X-gal mixture for 24 h to quantify the number of senescence-associated GLB1-positive VSMCs (***, P < 0.001; n = 3 counting regions of 150 cells/region/condition in duplicate; Student t test). Scale bar: 50 µm.

Figure 6.

Defective VSMC autophagy promotes upregulation of MMP9, TGFB and CXCL12, 5 d after ligation-induced injury. (A) The left common carotid artery (LCCA) of Atg7^+/+Tagln-Cre⁺ (+/+) and Atg7^F/FTagln-Cre⁺ (−/−) mice (n = 3) was ligated for 5 d. Gelatin zymographic analysis of the LCCA to detect MMP9 and MMP2 activity followed by densitometric analysis (***, P < 0.001; NS, not significant; Student t test). (B) Western blot analysis of the LCCA for TGFB, CXCL12 and GAPDH. Relative expression of TGFB/GAPDH and CXCL12/GAPDH was determined by densitometric analysis (*, P < 0.05; ***, P < 0.001; Student t test).

Figure 7 (See previous page).

Defective VSMC autophagy promotes neointima formation 5 wk after ligation-induced injury. (A,B) The left common carotid artery (LCCA) of Atg7^+/+Tagln-Cre⁺ (+/+) (n = 10) and Atg7^F/FTagln-Cre⁺ (−/−) mice (n = 12) was ligated for 5 wk. Sections of the LCCA were stained with anti-ACTA2 antibody (A) or Sirius red (B) to quantify the degree of stenosis and total collagen deposition, respectively. Scale bar: 100 µm. (**P < 0.01; *P < 0.05; Student t test). (C) Sections of the LCCA were immunostained for CDKN2A to quantify CDKN2A-positive nuclei (black arrowheads). Scale bar: 25 µm. (**, P < 0.01; Mann Whitney test). (D) The LCCA was stained for senescence-associated GLB1 activity ex vivo (right panel). Scale bar: 250 µm. Sections of the LCCA were then counterstained with periodic acid-Schiff (PAS) to identify senescent neointimal VSMCs (left panel). Note that the neointimal VSMCs are surrounded by a cage of PAS-positive basal lamina. Scale bar: 10 µm.

Figure 8.

Defective VSMC autophagy accelerates atherogenesis after 10 wk of western-type diet. (A) Atg7^+/+Tagln-Cre⁺ apoe^−/− (+/+) and Atg7^F/FTagln-Cre⁺,apoe^−/− (−/−) mice (n = 16) were fed a western-type diet for 10 wk. Sections of the brachiocephalic artery were stained with H&E to quantify plaque size and percentage of necrosis. (*, P<0.05; Student t test). (B to D) Consecutive sections were immunostained for cleaved CASP3 (B), LAMP2 (C) and ACTA2 (D) to measure the percentage of apoptosis (indicated by a black arrowhead in the high-power photograph of the boxed area in the left corner of each image), percentage of macrophages and fibrous cap thickness (indicated by black arrows), respectively (*, P < 0.05; Student t test (B); ***, P < 0.001; Student t test (C); *P < 0.05; n = 10 measurements/mouse, Repeated Measure (D)). (E) Consecutive sections were stained with Sirius red to quantify total collagen. (*, P < 0.05; Student t test). *, necrotic core. Scale bar: 100 µm.

Figure 9.

Defective VSMC autophagy promotes formation of a thick fibrous cap and enhances total collagen deposition after 14 wk of western-type diet. (A) Atg7^+/+Tagln-Cre⁺,apoe^−/− (+/+) and Atg7^F/FTagln-Cre⁺,apoe^−/− (−/−) mice (n = 16) were fed a western-type diet for 14 wk. Sections of the brachiocephalic artery were stained with H&E to quantify plaque size and percentage of necrosis. (NS, not significant; Student t test). (B to D) Consecutive sections were immunostained for cleaved CASP3 (B), LAMP2 (C) and ACTA2 (D) to measure the percentage of apoptosis, percentage of macrophages and fibrous cap thickness (indicated by black arrows), respectively. (NS, not significant; Student t test (B,C); **, P < 0.01; n = 10 measurements/mouse, Repeated Measure (D)). (E) Consecutive sections were stained with Sirius red to quantify total collagen. (*, P < 0.05; Student t test). Scale bar: 100 µm. *, necrotic core.

Figure 10.

Atherosclerotic plaques of Atg7^F/FTagln-Cre⁺,apoe^−/− mice show several features of VSMC senescence. (A) Sections of the aortic root were stained for senescence-associated (black arrowheads) and compared with serial TAGLN staining (not shown) to locate the fibrous caps. Scale bar: 10 µm. (B) Consecutive sections of the aortic root were double stained for phospho RB (red; white arrowheads) and ACTA2 (green). Scale bar: 25 µm (C) Sections of the brachiocephalic artery were double stained with phospho RB and periodic acid-Schiff (PAS) to quantify phospho RB-positive VSMC nuclei in the media (**, P < 0.01; Mann Whitney test). Scale bar: 50 µm.

Figure 11.

Defective autophagy in macrophages triggers neither an antioxidative backup mechanism nor senescence. Bone marrow-derived macrophages were isolated from Atg7^+/+Lysm-Cre⁺ (+/+) and Atg7^F/FLysm-Cre⁺ (−/−) mice. (A) Western blot analysis of ATG7 and SQSTM1 in untreated Atg7^+/+ and atg7^−/− macrophages. (B) Real-time RT-PCR and western blot analysis of GSTA and NQO1 in Atg7^+/+ and atg7^−/− macrophages (NS, not significant; n = 2 experiments in duplicate; Student t test). (C) Atg7^+/+ and atg7^−/− macrophages were treated with 50 µmol/l tBHP for 24 h (***, P < 0.001 vs. Atg7^+/+; ^###, P < 0.001 vs. control; n = 2 experiments in duplicate; Two-way ANOVA with genotype and treatment as category factors). (D) Western blot analysis of NFE2L2 in cytoplasmic and nuclear fractions of Atg7^+/+ and atg7^−/− macrophages. (E) Proliferation of Atg7^+/+ and atg7^−/− macrophages were examined by BrdU incorporation assay (NS, not significant; n = 6 counting regions of 500 cells/region/condition; Student t test) (F) DNA cell cycle analysis of Atg7^+/+ (blue) and atg7^−/− (red) macrophages. The bar graph shows the percentage of Atg7^+/+ and atg7^−/− nuclei in G₁ and G₂/M phase of the cell cycle (NS, not significant; Student t test). (G) Western blot analysis of CDKN2A in Atg7^+/+ and atg7^−/− macrophages.

Figure 12.

Overview of the mechanisms by which defective VSMC autophagy accelerates senescence and promotes postinjury neointima formation and diet-induced atherogenesis. SQSTM1 accumulation in autophagy defective VSMCs triggers NFE2L2 activation and transcription of multiple antioxidative enzymes including GSTA and NQO1. Upregulation of GSTA and NQO1 promotes VSMC survival against oxidative stress under defective autophagy conditions. SQSTM1 accumulation in autophagy defective VSMCs triggers the development of stress-induced premature senescence. Autophagy defective VSMCs are characterized by CDKN2A-RB-mediated G₁ proliferation arrest, increased migration and changes in VSMC phenotype. Enhanced migration is associated with increased secretion of MMP9, TGFB and CXCL12. The phenotype of autophagy defective VSMCs is defined by nuclear and cellular hypertrophy, and by increased collagen content. Defective autophagy in VSMCs accelerates postinjury neointima formation and diet-induced atherogenesis.