Interactions between cell death induced by statins and 7-ketocholesterol in rabbit aorta smooth muscle cells.
OBJECTIVE
7-Ketocholesterol, an oxysterol present in atherosclerotic lesions, induces smooth muscle cell (SMC) death, thereby destabilizing plaques. Statins protect patients from myocardial infarction, though they induce SMC apoptosis. We investigated whether statins and 7-ketocholesterol exerted additive cell death effects.


METHODS
Cultured rabbit aorta SMCs (passage 2-6) were exposed to 7-ketocholesterol with or without fluvastatin, simvastatin or pravastatin. Uptake of neutral red (NR), monolayer protein, cleavage of the pan-caspase substrate Asp-Glu-Val-Asp-rhodamine110, cell morphology (light and electron microscopy) and processing of microtubule-associated protein 1 light chain 3 (LC3, immunoblot) were determined.


RESULTS
NR uptake declined upon 18 h exposure to 25 microM 7-ketocholesterol (-41+/-3%, n=13), 100 microM fluvastatin (-59%) or 30-100 microM simvastatin (-28 to -74%). Oxysterol and high statin concentrations exerted additive effects, but lower concentrations (fluvastatin 10-30 microM, simvastatin 1-10 microM) partly reversed viability loss. 7-Ketocholesterol caused intense cytoplasmic vacuolization, processing of LC3-I to LC3-II, but little caspase activation (increase 29.5%). Fluvastatin (10-100 microM, 70-545% increase) and simvastatin (3-100 microM 43-322% increase) induced caspase activation without LC3 processing, but failed to activate caspases in 7-ketocholesterol-treated SMCs. Pravastatin up to 100 microM was always inactive.


CONCLUSIONS
7-Ketocholesterol caused SMC death, mainly via autophagic vesicle formation with LC3 processing, whereas lipophilic statins evoked SMC apoptosis. Cell death following 7-ketocholesterol and low statin concentrations were not additive, presumably because the autophagic process interfered with statin-induced caspase activation. This further illustrates that drug effects in normal SMCs are not necessarily predictive for activities in atherosclerotic settings.
Martinet
W
W
Division of Pharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Wilrijk, Belgium.
Schrijvers
D M
DM
Timmermans
J-P
JP
Bult
H
H
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D005434:Flow Cytometry; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000633:toxicity*; D066298:In Vitro Techniques; D007653:Ketocholesterols / Q000633:toxicity*; D046529:Microscopy, Electron, Transmission; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000648:ultrastructure; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009499:Neutral Red; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D013778:Tetrazolium Salts; D013844:Thiazoles
D017304:Annexin A5; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007653:Ketocholesterols; D013778:Tetrazolium Salts; D013844:Thiazoles; D009243:NAD; D009499:Neutral Red; D009249:NADP; D020169:Caspases; C022616:thiazolyl blue; C003001:7-ketocholesterol