on September 30, 2004
Published online before print September 30, 2004, doi: 10.1161/01.ATV.0000146266.65820.a1
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Submitted on January 17, 2004
Accepted on August 26, 2004
7-Ketocholesterol Induces Protein Ubiquitination, Myelin Figure Formation, and Light Chain 3 Processing in Vascular Smooth Muscle Cells
Wim Martinet *;
From the Division of Pharmacology (W.M., M.D., D.M.S., G.R.Y.D., A.G.H., M.M.K.), University of Antwerp, Wilrijk, Belgium; and the Department of Pathology (M.M.K.), General Hospital Middelheim, Antwerp, Belgium.
* To whom correspondence should be addressed. E-mail: wim.martinet{at}ua.ac.be.
Objective--Oxysterols such as 7-ketocholesterol (7-KC) are important mediators of cell death in atherosclerosis. Therefore, in vitro studies of human smooth muscle cell (SMC) death in response to 7-KC were undertaken to investigate the potential mechanisms.
Methods and Results--Human aortic SMCs treated with 7-KC showed enhanced immunoreactivity for the oxidative stress marker 4-hydroxy-2-nonenal and upregulated several stress genes (70-kDa heat shock protein 1, heme oxygenase 1, and growth arrest and DNA damage-inducible protein 153) at the mRNA but not at the protein level. 7-KC-treated SMCs rapidly underwent cell death as determined by neutral red, counting of adherent cells, and depolarization of the mitochondrial inner membrane. Cell death was associated with upregulation of ubiquitin mRNA and ubiquitination of cellular proteins. Inhibition of the proteasome by lactacystin potentiated considerably the toxicity of 7-KC. Transmission electron microscopy revealed formation of myelin figures, extensive vacuolization, and depletion of organelles. Formation of autophagosomes was suggested by labeling cells with LysoTracker and monitoring processing of microtubule-associated protein 1 light chain 3 (LC3). Analogous to our in vitro studies, human atherosclerotic plaques showed signs of ubiquitination in SMCs.
Conclusions--7-KC activates the ubiquitin-proteasome system and induces a complex mode of cell death associated with myelin figure formation and processing of LC3-evocating autophagic processes.
Key words: 7-ketocholesterol • ubiquitination • smooth muscle cells • myelin figures • LC3 • autophagy • atherosclerosis
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