. 2019 Feb 11;10(2):119.

doi: 10.1038/s41419-019-1400-0.

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

Hripsimé Nahapetyan^{1

2}, Manon Moulis^{1

2}, Elisa Grousset^{1

2}, Julien Faccini¹, Marie-Hélène Grazide^{1

2}, Elodie Mucher^{1

2}, Meyer Elbaz^{1

2

3}, Wim Martinet⁴, Cécile Vindis^{5

6}

Affiliations

¹ Inserm, UMR 1048, Institut des Maladies Métaboliques et Cardiovasculaires, F-31342, Toulouse, France.
² Université de Toulouse III, F-31342, Toulouse, France.
³ Fédération des Services de Cardiologie, Hôpital Rangueil, F-31342, Toulouse, France.
⁴ Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium.
⁵ Inserm, UMR 1048, Institut des Maladies Métaboliques et Cardiovasculaires, F-31342, Toulouse, France. cecile.vindis@inserm.fr.
⁶ Université de Toulouse III, F-31342, Toulouse, France. cecile.vindis@inserm.fr.

PMID: 30741928
PMCID: PMC6370858
DOI: 10.1038/s41419-019-1400-0

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

Hripsimé Nahapetyan et al. Cell Death Dis. 2019.

. 2019 Feb 11;10(2):119.

doi: 10.1038/s41419-019-1400-0.

Authors

Affiliations

¹ Inserm, UMR 1048, Institut des Maladies Métaboliques et Cardiovasculaires, F-31342, Toulouse, France.
² Université de Toulouse III, F-31342, Toulouse, France.
³ Fédération des Services de Cardiologie, Hôpital Rangueil, F-31342, Toulouse, France.
⁴ Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium.
⁵ Inserm, UMR 1048, Institut des Maladies Métaboliques et Cardiovasculaires, F-31342, Toulouse, France. cecile.vindis@inserm.fr.
⁶ Université de Toulouse III, F-31342, Toulouse, France. cecile.vindis@inserm.fr.

PMID: 30741928
PMCID: PMC6370858
DOI: 10.1038/s41419-019-1400-0

Abstract

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE^-/-) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Characterization of Atg7 deletion in *Atg7*^F/FTagln-Cre⁺, ApoE^-/- tissues and vascular smooth muscle cells (VSMCs), plaque size and type 3 collagen content are increased in ApoE^−/− mice deleted for Atg7 in VSMCs.
a Western blot analyses of ATG7 in different tissue homogenates from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice. b Western blot analyses of ATG7, P62, and LC3-I/II protein expression in VSMCs isolated from the aorta of *Atg7* ^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice. α-SMA was used as a loading control and is a classical marker for VSMCs. Bands are shown in duplicate for two different primary VSMC cultures. c Representative images of consecutive aortic sinus sections stained with oil red after 10 weeks of high-fat diet (HFD) in *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice. The graph represents the % of plaque area and the data are the mean ± SEM. **P < 0.01; Student’s t-test, n = 10 mice/group. Scale bar, 100 µm. d Representative images of en face staining of the whole aorta from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 18 weeks of HFD. The graph represents the % of plaque area and the data are the mean ± SEM. *P < 0.05; Student’s t-test, n = 3 mice/group. e Representative images of consecutive aortic sinus sections stained with Masson’s trichrome to assess the total collagen content in *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. The graph represents the % of total collagen content and the data are the mean ± SEM. ns, nonsignificant; Student’s t-test, n = 10 mice/group. Scale bar, 100 µm. f Representative images of consecutive aortic sinus sections stained with Picrosirius red and analyzed using polarized light microscopy to distinguish between type 1 (red) and 3 (green) collagen after 10 weeks of HFD. The graph represents the % of type 3 collagen content and the data are the mean ± SEM. ***P < 0.001; Student’s t-test, n = 8 mice/group. Scale bar, 100 µm

**Fig. 2. Enhanced inflammation and apoptosis in atherosclerotic lesions of ApoE^−/− mice deleted for Atg7 in vascular smooth muscle cells (VSMCs).**
a Representative images of consecutive aortic sinus sections immunostained with MOMA 2 antibody to quantify the content of macrophages/monocytes in the plaque area of *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of high-fat diet (HFD). DAPI (blue, nucleus). The graph represents the % of MOMA 2 staining and the data are the mean ± SEM, **P < 0.01; Student’s t-test, n = 8 mice/group. Scale bar, 200 µm. b Representative images of apoptotic cell detection by TUNEL staining in aortic sinus sections of *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7* ^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. TUNEL (green), DAPI (blue, nucleus). The graph represents the number of TUNEL-positive cells measured in the plaque area and the data are the mean ± SEM. **P < 0.01; Student’s t-test, n = 8 mice/group. Scale bar, 50 µm. c Representative images of consecutive aortic sinus sections immunostained with cleaved-caspase 3 (green) and α-SMA (red) antibodies to detect apoptotic VSMCs of *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. DAPI (blue, nucleus). The graph represents the % of cleaved-caspase 3-positive VSMCs measured in the plaque area and the data are the mean ± SEM. **P < 0.01; Student’s t-test, n = 8 mice/group. Scale bar, 50 µm

Fig. 3. Increased mitochondrial reactive oxygen species (ROS) production and reduced mitochondrial membrane potential in plaques from ApoE^−/− mice deleted for Atg7 in vascular smooth muscle cells (VSMCs).
a Representative images of consecutive aortic sinus sections stained with MitoSOX (red) and DAPI (blue, nucleus) of *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of high-fat diet (HFD). Scale bar, 20 µm. b The graph represents the % of MitoSox staining in the plaque area per section and the data are the mean ± SEM. ***P < 0.001; Student’s t-test, n = 5 mice/group. c Measurement of mitochondrial ROS production in VSMCs isolated from the aorta of *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. The graph represents the quantification of MitoSOX Red fluorescence at baseline or after antimycin A (AA, 10 µM) stimulation. Data are the median with interquartile range of four independent experiments from different primary VSMC cultures per group. **P < 0.01; ^##P < 0.01; ^#P < 0.05; ns, nonsignificant; one-way ANOVA, Kruskal–Wallis non-parametric test. d Measurement of the mitochondrial membrane potential (ΔΨm) with the JC-1 dye in VSMCs isolated from the aorta of *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. The graph represents the quantification of the potential-dependent accumulation of the JC-1 dye in mitochondria at baseline and after CCCP (20 µM) stimulation. Data are the median with interquartile range of four independent experiments from different primary VSMC cultures per group. ***P < 0.001; ^##P < 0.01; ns, nonsignificant; one-way ANOVA, Kruskal–Wallis non-parametric test

**Fig. 4. Altered mitochondrial network and bioenergetic functions in ApoE^−/− mice deleted for Atg7 in vascular smooth muscle cells (VSMCs).**
a Representative images of aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of high-fat diet (HFD) and stained with MitoTtracker Deep Red (MitoTR, red), DAPI (blue, nucleus). Scale bar, 5 µm. b The graph represents the calculated average size of mitochondria (µm²) in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. Data are the mean ± SEM of 10 cells analyzed from three independent experiments from different primary VSMC cultures per group.***P < 0.001; Student’s t-test. c Representative images of structured illuminated confocal microscopy (SIM) followed by an image processing algorithm deconvolution and 3D reconstruction. The graph represents the calculated average volume of mitochondria (µm³) stained with MitoTR, in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. Data are the mean ± SEM of six cells analyzed from two independent experiments from different primary VSMC cultures per group. ***P < 0.001; Student’s t-test. d Representative images of Ser616 phosphorylated Drp-1 (P-Drp-1, green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. The graph represents the intensity of P-Drp-1 fluorescence. Data are the median with interquartile of six cells analyzed from four independent experiments from different primary VSMC cultures per group. **P < 0.01; Mann–Whitney non-parametric test. e Seahorse profile for oxygen consumption rate (OCR) in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD with treatment with oligomycin, FCCP, and antimycin A/rotenone. Data are the mean ± SEM of five replicates from three independent experiments from different primary VSMC cultures per group. The graphs represent the basal respiration (last rate measurement before first injection - non mitochondrial respiration rate) and ATP production (last rate measurement before first injection – minimum rate measurement after oligomycin injection) in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD, the data are the mean ± SEM of five replicates from three independent experiments from different primary VSMC cultures per group. *P < 0.05; Student’s t-test

Fig. 5. Increased expression of the mitophagy proteins PTEN-induced putative kinase 1 (PINK1) and Parkin functions in VSMCs from atherosclerotic lesions of ApoE^−/− mice deleted for Atg7 in vascular smooth muscle cells (VSMCs).
a Representative images of consecutive aortic sinus sections immunostained with PINK1, Parkin, P62 (red), α-SMA (green) antibodies, and DAPI (blue, nucleus) from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of high-fat diet (HFD). The graph represents the % of PINK1, Parkin, or P62 staining in VSMCs within the plaque area and the data are the mean ± SEM from n = 8 mice/group. ***P < 0.001; Student’s t-test. Scale bar, 20 µm. b Western blot analyses of the expression of PINK1 and Parkin proteins in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD, β-actin was used as the loading control. Bands are shown in duplicate. The graph represents the densitometric analysis of the expression level of PINK1 and Parkin proteins. The data are the mean ± SEM of three independent experiments from different primary VSMC cultures per group. **P < 0.01; *P < 0.05; Student’s t-test

**Fig. 6. Atg7 deficiency in vascular smooth muscle cells (VSMCs) alters mitochondrial quality control and enhances apoptosis.**
a Flow cytometry analyses of mitophagy flux in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/−mice after 10 weeks of high-fat diet (HFD). Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL, left graph) or CCCP (20 µM, right graph) for 8 h and treated either with or without the lysosomal inhibitor Bafilomycin A1 (BafA1) (100 nM). VSMCs were then stained with MitoTR for flow cytometry analysis. The data are expressed as mean ± SEM of five independent experiments (left graph); *P < 0.05; Wilcoxon signed-rank test, ns nonsignificant; and as mean ± SEM of three independent experiments (right graph) *P < 0.05; Mann–Whitney non-parametric test. b Western blot analyses of the expression of TOMM 40 and VDAC1 proteins in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 16 h and treated either with or without the lysosomal inhibitor Bafilomycin A1 (BafA1) (100 nM). β-Actin was used as the loading control. The graph represents the densitometric analysis of the expression level of TOMM 40 and VDAC1 proteins. The data are expressed as mean ± SEM of four independent experiments from different primary VSMC cultures. *P < 0.05; ***P < 0.01; one-way ANOVA, Bonferroni’s multiple comparison test. c Western blot analyses of the expression of TFEB and PGC-1-α proteins in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 8 h. β-Actin was used as the loading control. The graph represents the densitometric analysis of the expression level of TFEB and PGC-1-α proteins. The data are expressed as mean ± SEM of four independent experiments from different primary VSMC cultures. *P < 0.05; ***P < 0.001; Student’s t-test. (d) Representative images of TFEB (green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. Cells were incubated either with or without oxidized LDL (200 μg ApoB/mL) for 8 h. The graph represents the intensity of nuclear TFEB fluorescence and the data are the mean ± SEM of three independent experiments from different primary VSMC cultures. *P < 0.05; ^###P < 0.001; one-way ANOVA, Kruskal–Wallis non-parametric test. e Representative images of cleaved-caspase 3 (green) and DAPI (blue, nucleus) immunostaining in aortic VSMCs isolated from *Atg7*^+/+ *Tagln-Cre*⁺, ApoE^−/− and *Atg7*^F/F *Tagln-Cre*⁺, ApoE^−/− mice after 10 weeks of HFD. Cells were incubated with or without oxidized LDL (200 μg ApoB/mL) for 16 h. Scale bar, 20 µm. The graph represents the % of cleaved-caspase 3-positive cells and the data are the mean ± SEM of three independent experiments from different primary VSMC cultures. *P < 0.05; two-way ANOVA, Bonferroni’s multiple comparison test. Scale bar, 20 µm

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Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

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Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

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