7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling

Cheikh Seye, Michiel W M Knaapen, Danièle Daret, Claude Desgranges, Arnold G. Herman, Mark M. Kockx, Hidde Bult

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Objective: 7-Ketocholesterol, a major oxysterol in oxidized low-density lipoproteins in advanced atherosclerotic plaques, induces vascular smooth muscle cell (SMC) death. We investigated whether cytochrome c release participated in SMC death induced by 7-ketocholesterol and whether the processes were reversible. Methods: SMC cultures derived from the rabbit aorta were exposed to 25 μM 7-ketocholesterol. Cytochrome c and Bax were studied by means of immunofluorescence and immunoblotting, apoptosis by the TUNEL technique and mitochondrial structure by transmission electron microscopy. Results: 7-Ketocholesterol induced rapid upregulation of the proapoptotic protein Bax and its translocation from cytosol into the mitochondria (4 h). This was followed by mitochondrial cytochrome c release (65% at 8 h) into the cytosol, which was almost complete at 16 h. The mitochondria became spherical and ultracondensed, without showing signs of lysis. They clustered around the nucleus and were wrapped by wide cisternae of the rough endoplasmic reticulum. Cytochrome c release was not blocked by the pan-caspase inhibitor zVAD-fmk, in contrast to DNA fragmentation and SMC loss. Interestingly, upon removal of 7-ketocholesterol after 16 h and re-exposure to serum for 24 h, the mitochondrial cytochrome c content, their transmembrane potential and TUNEL labelling normalised and SMC loss decreased. However, none of these cell death markers was rescued when the SMCs had been exposed to the oxysterol for 24 h. Conclusion: The results indicate that cytochrome c release during oxysterol-induced SMC apoptosis is not caspase-dependent and occurs as a result of a reversible mitochondrial conformational change rather than swelling and rupture of the outer membrane. The reversibility of these events suggests that the apoptotic cascade could be arrested before a point of no return.

Original languageEnglish (US)
Pages (from-to)144-153
Number of pages10
JournalCardiovascular Research
Volume64
Issue number1
DOIs
StatePublished - Oct 1 2004
Externally publishedYes

Fingerprint

Mitochondrial Swelling
Cytochromes c
Smooth Muscle Myocytes
Cell Death
In Situ Nick-End Labeling
Cytosol
Mitochondria
Apoptosis
bcl-2-Associated X Protein
Caspase Inhibitors
Rough Endoplasmic Reticulum
DNA Fragmentation
Atherosclerotic Plaques
Caspases
Transmission Electron Microscopy
Vascular Smooth Muscle
Immunoblotting
Membrane Potentials
Fluorescent Antibody Technique
Aorta

Keywords

  • Apoptosis
  • Atherosclerosis
  • Cholesterol
  • Mitochondria
  • Rabbit smooth muscle

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling. / Seye, Cheikh; Knaapen, Michiel W M; Daret, Danièle; Desgranges, Claude; Herman, Arnold G.; Kockx, Mark M.; Bult, Hidde.

In: Cardiovascular Research, Vol. 64, No. 1, 01.10.2004, p. 144-153.

Research output: Contribution to journalArticle

Seye, Cheikh ; Knaapen, Michiel W M ; Daret, Danièle ; Desgranges, Claude ; Herman, Arnold G. ; Kockx, Mark M. ; Bult, Hidde. / 7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling. In: Cardiovascular Research. 2004 ; Vol. 64, No. 1. pp. 144-153.
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abstract = "Objective: 7-Ketocholesterol, a major oxysterol in oxidized low-density lipoproteins in advanced atherosclerotic plaques, induces vascular smooth muscle cell (SMC) death. We investigated whether cytochrome c release participated in SMC death induced by 7-ketocholesterol and whether the processes were reversible. Methods: SMC cultures derived from the rabbit aorta were exposed to 25 μM 7-ketocholesterol. Cytochrome c and Bax were studied by means of immunofluorescence and immunoblotting, apoptosis by the TUNEL technique and mitochondrial structure by transmission electron microscopy. Results: 7-Ketocholesterol induced rapid upregulation of the proapoptotic protein Bax and its translocation from cytosol into the mitochondria (4 h). This was followed by mitochondrial cytochrome c release (65{\%} at 8 h) into the cytosol, which was almost complete at 16 h. The mitochondria became spherical and ultracondensed, without showing signs of lysis. They clustered around the nucleus and were wrapped by wide cisternae of the rough endoplasmic reticulum. Cytochrome c release was not blocked by the pan-caspase inhibitor zVAD-fmk, in contrast to DNA fragmentation and SMC loss. Interestingly, upon removal of 7-ketocholesterol after 16 h and re-exposure to serum for 24 h, the mitochondrial cytochrome c content, their transmembrane potential and TUNEL labelling normalised and SMC loss decreased. However, none of these cell death markers was rescued when the SMCs had been exposed to the oxysterol for 24 h. Conclusion: The results indicate that cytochrome c release during oxysterol-induced SMC apoptosis is not caspase-dependent and occurs as a result of a reversible mitochondrial conformational change rather than swelling and rupture of the outer membrane. The reversibility of these events suggests that the apoptotic cascade could be arrested before a point of no return.",
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AU - Herman, Arnold G.

AU - Kockx, Mark M.

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AB - Objective: 7-Ketocholesterol, a major oxysterol in oxidized low-density lipoproteins in advanced atherosclerotic plaques, induces vascular smooth muscle cell (SMC) death. We investigated whether cytochrome c release participated in SMC death induced by 7-ketocholesterol and whether the processes were reversible. Methods: SMC cultures derived from the rabbit aorta were exposed to 25 μM 7-ketocholesterol. Cytochrome c and Bax were studied by means of immunofluorescence and immunoblotting, apoptosis by the TUNEL technique and mitochondrial structure by transmission electron microscopy. Results: 7-Ketocholesterol induced rapid upregulation of the proapoptotic protein Bax and its translocation from cytosol into the mitochondria (4 h). This was followed by mitochondrial cytochrome c release (65% at 8 h) into the cytosol, which was almost complete at 16 h. The mitochondria became spherical and ultracondensed, without showing signs of lysis. They clustered around the nucleus and were wrapped by wide cisternae of the rough endoplasmic reticulum. Cytochrome c release was not blocked by the pan-caspase inhibitor zVAD-fmk, in contrast to DNA fragmentation and SMC loss. Interestingly, upon removal of 7-ketocholesterol after 16 h and re-exposure to serum for 24 h, the mitochondrial cytochrome c content, their transmembrane potential and TUNEL labelling normalised and SMC loss decreased. However, none of these cell death markers was rescued when the SMCs had been exposed to the oxysterol for 24 h. Conclusion: The results indicate that cytochrome c release during oxysterol-induced SMC apoptosis is not caspase-dependent and occurs as a result of a reversible mitochondrial conformational change rather than swelling and rupture of the outer membrane. The reversibility of these events suggests that the apoptotic cascade could be arrested before a point of no return.

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