Martinet W, Schrijvers D M, Timmermans J-P, Bult H
Division of Pharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Wilrijk, Belgium.
Br J Pharmacol. 2008 Jul;154(6):1236-46. doi: 10.1038/bjp.2008.181. Epub 2008 May 12.
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.
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.
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.
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.
7-酮胆固醇是一种存在于动脉粥样硬化病变中的氧化甾醇，可诱导平滑肌细胞（SMC）死亡，从而使斑块不稳定。他汀类药物可保护患者免受心肌梗死的影响，尽管它们会诱导SMC凋亡。我们研究了他汀类药物和7-酮胆固醇是否具有相加的细胞死亡效应。
将培养的兔主动脉SMC（传代2-6代）暴露于含或不含氟伐他汀、辛伐他汀或普伐他汀的7-酮胆固醇中。测定中性红（NR）摄取、单层蛋白、泛半胱天冬酶底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-罗丹明110的裂解、细胞形态（光学和电子显微镜）以及微管相关蛋白1轻链3（LC3，免疫印迹）的处理情况。
暴露于25μM 7-酮胆固醇18小时后，NR摄取下降（-41±3%，n = 13），100μM氟伐他汀（-59%）或30 - 100μM辛伐他汀（-28%至-74%）也导致NR摄取下降。氧化甾醇和高浓度他汀类药物具有相加效应，但较低浓度（氟伐他汀10 - 30μM，辛伐他汀1 - 10μM）部分逆转了活力丧失。7-酮胆固醇导致强烈的细胞质空泡化，LC3-I加工为LC3-II，但半胱天冬酶激活很少（增加29.5%）。氟伐他汀（10 - 100μM，增加70 - 545%）和辛伐他汀（3 - 100μM，增加43 - 322%）诱导半胱天冬酶激活而无LC3加工，但在7-酮胆固醇处理的SMC中未能激活半胱天冬酶。高达100μM的普伐他汀始终无活性。
7-酮胆固醇主要通过自噬小泡形成及LC3加工导致SMC死亡，而亲脂性他汀类药物诱发SMC凋亡。7-酮胆固醇和低浓度他汀类药物引起的细胞死亡并非相加效应，可能是因为自噬过程干扰了他汀类药物诱导的半胱天冬酶激活。这进一步说明正常SMC中的药物效应不一定能预测动脉粥样硬化环境中的活性。
