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

doi:10.1016/j.cardiores.2004.05.016

Cardiovascular Research 2004 64(1):144-153; doi:10.1016/j.cardiores.2004.05.016
© 2004 by European Society of Cardiology

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

Cheikh I. Seye^a^,1, Michiel W.M. Knaapen^b, Danièle Daret^c, Claude Desgranges^c, Arnold G. Herman^a, Mark M. Kockx^b and Hidde Bult^*^,a

^aLaboratory of Pharmacology, University of Antwerp, Campus Drie Eiken (CDE), Wilrijk, B-2610 Antwerp, Belgium
^bDepartment of Pathology, AZ Middelheim, Lindendreef 1, B-2020 Antwerp, Belgium
^cINSERM Unité 441 d'Athérosclérose, Avenue du Haut Lévêque, 33600 Pessac, France

^* Corresponding author. Tel.: +32-3-820-27-38; fax: +32-3-820-25-67. E-mail address: seyec{at}missouri.edu, hidde.bult{at}ua.ac.be

Objective: 7-Ketocholesterol, a major oxysterol in oxidizedlow-density lipoproteins in advanced atherosclerotic plaques,induces vascular smooth muscle cell (SMC) death. We investigatedwhether cytochrome c release participated in SMC death inducedby 7-ketocholesterol and whether the processes were reversible.Methods: SMC cultures derived from the rabbit aorta were exposedto 25 µM 7-ketocholesterol. Cytochrome c and Bax werestudied by means of immunofluorescence and immunoblotting, apoptosisby the TUNEL technique and mitochondrial structure by transmissionelectron microscopy. Results: 7-Ketocholesterol induced rapidupregulation of the proapoptotic protein Bax and its translocationfrom cytosol into the mitochondria (4 h). This was followedby mitochondrial cytochrome c release (65% at 8 h) into thecytosol, which was almost complete at 16 h. The mitochondriabecame spherical and ultracondensed, without showing signs oflysis. They clustered around the nucleus and were wrapped bywide cisternae of the rough endoplasmic reticulum. Cytochromec 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-exposureto serum for 24 h, the mitochondrial cytochrome c content, theirtransmembrane potential and TUNEL labelling normalised and SMCloss decreased. However, none of these cell death markers wasrescued when the SMCs had been exposed to the oxysterol for24 h. Conclusion: The results indicate that cytochrome c releaseduring oxysterol-induced SMC apoptosis is not caspase-dependentand occurs as a result of a reversible mitochondrial conformationalchange rather than swelling and rupture of the outer membrane.The reversibility of these events suggests that the apoptoticcascade could be arrested before a point of no return.

KEYWORDS Rabbit smooth muscle; Mitochondria; Cholesterol; Apoptosis; Atherosclerosis

¹ Current address: Department of Biochemistry, University of Missouri,Columbia, MO 65212, USA.

Time for primary review 21 days

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