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. 2016 May 17;7(20):28821-35.
doi: 10.18632/oncotarget.8936.

Mitophagy acts as a safeguard mechanism against human vascular smooth muscle cell apoptosis induced by atherogenic lipids

Audrey Swiader  1   2 , Hripsime Nahapetyan  1   2 , Julien Faccini  1   2 , Romina D'Angelo  1   2 , Elodie Mucher  1   2 , Meyer Elbaz  1   2 , Patricia Boya  3 , Cécile Vindis  1   2
Affiliations

Mitophagy acts as a safeguard mechanism against human vascular smooth muscle cell apoptosis induced by atherogenic lipids

Audrey Swiader et al. Oncotarget. .

Abstract

Mitophagy is a critical cellular process that selectively targets damaged mitochondria for autophagosomal degradation both under baseline conditions and in response to stress preventing oxidative damage and cell death. Recent studies have linked alterations in mitochondria function and reduced autophagy with the development of age-related pathologies. However, the significance of mitochondrial autophagy in vessel wall in response to atherogenic lipid stressors is not known. In the present study, we investigated the role of mitophagy on human vascular smooth muscle cells (VSMC) apoptosis induced by oxidized low-density lipoproteins (LDL). We reported for the first time that the engulfment of defective mitochondria by autophagosomes occurred in human VSMC in response to oxidized LDL. The molecular mechanism mediating mitophagy in human VSMC involved dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, accumulation of PTEN-induced putative kinase 1 (PINK1) and the recruitment of the E3 ubiquitin ligase Parkin to mitochondria. Likewise, we found increased voltage-dependent anion channel 1 (VDAC1) and mitofusin 2 (Mnf2) mitochondrial proteins ubiquitination and LC3 association to mitochondria. Using flow cytometry in the presence of lysosomal inhibitors, we showed that PINK1 and Parkin silencing impaired mitophagy flux and enhanced oxidized LDL-induced VSMC apoptosis. In addition, overexpression of PINK1 and Parkin were protective by limiting cell death. Moreover, reduced Bax levels found in VSMC-overexpressing Parkin indicated cross talk among mitophagy and mitochondrial apoptotic signalling pathways. Altogether these data demonstrate that mitophagy is a safeguard mechanism against human VSMC apoptosis induced by atherogenic stressors and highlight mitophagy as a potential target to stabilize atherosclerotic plaque.

Keywords: apoptosis; atherosclerosis; human vascular smooth muscle cell; mitophagy; oxidized lipoproteins.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. Oxidized LDL induced mitochondrial depolarization, dysfunction and Drp-1 dependent fission in human VSMC
A. Time course analysis of the mitochondrial membrane potential (ΔΨm). Human VSMC were labelled with the JC-1 dye and stimulated with oxidized or native LDL (respectively oxLDL and nLDL) (200 μg ApoB/mL), at the indicated times or with CCCP (20 μM). JC-1 exhibits potential-dependent accumulation in mitochondria. At low membrane potentials, JC-1 exists as a monomer and produces a green fluorescence (emission at 520 nm). At high membrane potentials or concentrations, JC-1 forms aggregates and produces a red fluorescence (emission at 590 nm). The ratio 590/520 is indicative of mitochondrial membrane depolarization. The data are expressed as mean ± SEM of 5 separate experiments, ** P < 0.01 and ## P < 0.01 indicate significance, n.s indicates no significance. B. Mitochondrial superoxide formation was detected using MitoSOX Red dye (excitation/emission at λ = 510 nm/580 nm) in human VSMC treated with oxidized or native LDL (respectively oxLDL and nLDL) (200 μg ApoB/mL) or Antimycin A (AA) (10 μM) at the indicated times and the relative fluorescence intensity is quantified. The data are expressed as mean ± SEM of 5 separate experiments, * P < 0.05, ** P < 0.01 and ## P < 0.01 indicate significance, n.s indicates no significance. C. Immunoblot analysis of the fission protein Drp-1 following oxidized or native LDL treatment. Human VSMC were stimulated with oxidized (oxLDL) or native LDL (nLDL) (200 μg ApoB/mL) at the indicated times and Western blot experiments were performed on total protein extracts using anti-phosphorylated Drp-1(Ser616) antibody and total Drp-1 expression was used as loading control. Blots are representative of 4 independent experiments. The graph represents values of phosphorylated Drp-1(Ser616) band intensity after normalization for total Drp-1 band intensity by densitometry, * P < 0.05 and ** P < 0.01 indicate significance. D. Representative images of mitochondrial fragmentation/fission. Reversal of the mitochondrial fragmentation in human VSMC was achieved using siRNA mediated knockdown of Drp-1 expression. Images are representative of human VSMC treated with oxidized LDL (200 μg ApoB/mL) for 5 h, undergoing siRNA Drp-1 or siRNA scramble transfection. Mitochondria were stained using an antibody against the outer mitochondrial membrane-localized protein TOMM20 (red). Nuclei (blue) were stained with DAPI (4ʹ,6-diamidino-2-phenylindole). The graph represents the quantification of the MFC and shows a significant reduction in oxidized LDL stimulated cells transfected with siRNA Drp-1. Data are expressed as mean ± SEM of 3 separate experiments, ** P < 0.01 and ## P < 0.01 indicate significance, n.s indicates no significance.
Figure 2
Figure 2. Oxidized LDL induced the recruitment of PINK1 and Parkin to the damaged mitochondria and increased mitochondrial ubiquitinylated proteins in human VSMC
A. Immunoblot analysis of the 60-kDa full-length and the 50-kDa cleaved endogenous PINK1 expression in cytosolic and mitochondrial fractions of human VSMC treated with oxidized LDL (200 μg ApoB/mL) at the indicated times. The cellular fractions were probed for PINK1 and the cytosol and mitochondrial markers β-actin and TOMM20, respectively. Blots are representative of 4 independent experiments. The graph represents values (means ± SEM) of PINK1 band intensity after normalization for β-actin and TOMM20 by densitometry, * P < 0.05, # P < 0.05, ## P < 0.01 and ### P < 0.001 indicate significance. B. Representative images of Parkin translocation to the mitochondria, human VSMC were stimulated with oxidized LDL (200 μg ApoB/mL) for 5 h. Mitochondria were immunostained with Mito Tracker Deep Red (MTDR, red) and Parkin antibody. C. The Pearson coefficient indexes between Parkin and MTDR fluorescence intensity were determined in regions of interest (ROI) for 10 or more cells in 3 independent experiments. The values are the means ± SEM; ** P < 0.01 indicates significance. D. Immunoblot analysis of total ubiquitinated proteins in mitochondrial fractions of human VSMC treated with oxidized LDL (200 μg ApoB/mL) at the indicated times. The cellular fractions were probed for ubiquitin and immunoblots are representative of 3 independent experiments. The graph represents the densitometric analysis of mitochondrial ubiquitinated protein intensity relative to untreated control, # P < 0.05 and ## P < 0.01 and indicate significance. E. Immunoprecipitations of VDAC1 and Mnf2 from human VSMC confirm ubiquitylation of both proteins. Oxidized LDL (200 μg ApoB/mL, 5 h) treatment increases high molecular weight (HMW) ubiquitylated species of VDAC1 and Mnf2. Using specific antibodies VDAC1 and Mnf2 were immunoprecipitated from lysates, western blotted and probed with anti-ubiquitin, anti-VDAC1 and anti-Mnf2 antibodies.
Figure 3
Figure 3. Mitophagy monitoring in oxidized LDL-stimulated human VSMC
A. Analysis of mitochondria engulfment by autophagosomes using dual fluorescence. Human VSMC were transfected with GFP-LC3 (green) and the colocalization of GFP-LC3 with the mitochondria marker TOMM20 (red) was determined following oxidized LDL (200 μg ApoB/mL, 8h) treatment with or without 3-MA. B. The Pearson coefficient indexes between GFP-LC3 intensity and TOMM20 intensity were determined in regions of interest (ROI) for 5 or more cells in 3 independent experiments. The values are the means ± SEM; *** P < 0.001 and ## P < 0.01 indicate significance. C. Three-dimensional reconstruction of confocal fluorescence microscopy z-stack slices showed that mitochondria are present within autophagosomes following oxidized LDL treatment in human VSMC. D. Immunoblot analysis of the mitochondrial proteins TOMM40, COX IV-1 and TOMM20 in human VSMC treated with oxidized LDL (200 μg ApoB/mL) for the indicated times. The blots are representative of 3 independent experiments. The graph represents values (means ± SEM) of TOMM40, COX IV and TOMM20 band intensities after normalization for β-actin by densitometry. ** P < 0.01 and * P < 0.05 indicate significance. E. Flow cytometry analysis of mitophagy in human VSMC after oxidized LDL treatment. Cells were incubated with oxidized LDL (200 μg ApoB/mL) for 8 h and treated with or without the lysosomal inhibitors Baf1 (10 nM) and chloroquine CQ (10μM) 3 h before analysis to block lysosomal degradation. Human VSMC were then stained with MTDR for flow cytometry analysis. The data are expressed as mean ± SEM of 4 separate experiments, *** P < 0.001, ## P < 0.01 and §§ P < 0.01 indicate significance. F. Additional aspects of mitophagy analysis. The preloading of MTDR shows a mitophagy dependent reduction in fluorescence levels. Human VSMC were preincubated with MTDR for 20 min to load mitochondria, washed and then treated with oxidized LDL (200 μg ApoB/mL) for 8 h and treated with or without Baf1 (10 nM) 3 h before flow cytometry analysis to block lysosomal degradation. The data are expressed as mean ± SEM of 4 separate experiments, *** P < 0.01 and ## P < 0.01 indicate significance.
Figure 4
Figure 4. PINK1 and Parkin knockdown enhances human VSMC apoptosis mediated by oxidized LDL
Analysis of oxidized LDL-induced apoptosis in human VSMC transfected with scrambled (si-Scr, 100 nM), (si-Parkin, 100 nM) or (si-PINK1 100 nM), siRNA for 24 h and then incubated with oxidized LDL (200 μg ApoB/mL) for 16 h. A. shows the western-blot analysis of PINK1 and Parkin expressions in human VSMC after siRNA transfection. B. Assessment of caspases activity in PINK1 and Parkin-deficient human VSMC. Whole cell detection of caspase activity in apoptotic or caspase-positive cells was performed using the membrane-permeant, fluorescent inhibitor-based FLICA caspase probes as described under “Materials and Methods”. The data are expressed as mean ± SEM of 3 separate experiments, ** P < 0.01 and # P < 0.05 indicate significance. C. Apoptosis of control, PINK1 and Parkin-deficient human VSMC stimulated with oxidized LDL (200 μg ApoB/mL) for 16 h was determined by Annexin V/PI staining followed by flow cytometry analysis. The graph represents the quantitative analysis of the percentage of Annexin V-FITC positive cells. The data are expressed as mean ± SEM of 5 separate experiments, * P < 0.05 and ** P < 0.01 indicate significance. Mitophagy assessment in D. human VSMC transfected with scrambled siRNA (si-Scr); E. human VSMC transfected with PINK1 siRNA (si-PINK1) and F. human VSMC transfected with Parkin siRNA (si-Parkin), and incubated with oxidized LDL (200 μg ApoB/mL) for 8 h. Cells were treated with or without Baf1 (10 nM) 3 h before analysis to block lysosomal degradation. Human VSMC were then stained with MTDR for flow cytometry analysis. The data are expressed as mean ± SEM of 6 separate experiments, ** P < 0.01 indicates significance.
Figure 5
Figure 5. PINK1 and Parkin overexpression protects human VSMC against oxidized LDL-induced apoptosis
A., B. Analysis of oxidized LDL-induced apoptosis in cells transfected with EGFP vector, YFP-Parkin vector or GFP-PINK1 vector. A. Immunoblot analysis of PINK1 and Parkin overexpression in human VSMC after 24 h of transfection. B. Apoptosis of control, PINK1 and Parkin-overexpressing VSMC stimulated with oxidized LDL (200 μg ApoB/mL) for 16 h was determined by Annexin V-APC/PI staining followed by flow cytometry analysis. The graph represents the quantitative analysis of the percentage of Annexin V-APC positive cells. The data are expressed as mean ± SEM of 5 separate experiments, * P < 0.05 and ** P < 0.01 indicate significance. Mitophagy assessment in C. human VSMC transfected with EGFP vector; D. human VSMC transfected with GFP-PINK1 vector and E. human VSMC transfected with YFP-Parkin vector, and incubated with oxidized LDL (200 μg ApoB/mL) for 8 h. Cells were treated with or without Baf1 (10 nM) 3 h before analysis to block lysosomal degradation. Cells were then stained with MTDR for flow cytometry analysis and the MTDR fluorescence was determined in the GFP-positive population. The data are expressed as mean ± SEM of 6 separate experiments, * P < 0.05 and ** P < 0.01 indicate significance, n.s (non significant). F. Mitophagy flux determination in human VSMC transfected with EGFP vector or GFP-PINK1 or YFP-Parkin, and incubated with oxidized LDL (200 μg ApoB/mL) for 8 h in the presence of Baf1 (10 nM). The data are expressed as mean ± SEM of 6 separate experiments. *P < 0.05 indicates significance, n.s indicates no significance.
Figure 6
Figure 6. Parkin overexpression decreases Bax levels
A. Immunoblot analysis of Bax and Parkin expression in human VSMC transfected with YFP-Parkin or control vector, and incubated with oxidized LDL (200 μg ApoB/mL) for 8 h. Blots are representative of 3 independent experiments. The graph represents values (means ± SEM) of Bax band intensity after normalization for β-actin by densitometry. *** P < 0.001 and # P < 0.05 indicate significance. B. Immunoblot analysis of Bax and Parkin expression in human VSMC transfected with YFP-Parkin or control vector, pretreated with a proteasome inhibitor MG132 (10 μM) and incubated with oxidized LDL (200 μg ApoB/mL) for 8 h. Blots are representative of 3 independent experiments. The graph represents values (means ± SEM) of Bax band intensity after normalization for β-actin by densitometry. *** P < 0.001 and ## P < 0.01 indicate significance.
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