Seye Cheikh I, Knaapen Michiel W M, Daret Danièle, Desgranges Claude, Herman Arnold G, Kockx Mark M, Bult Hidde
Laboratory of Pharmacology, University of Antwerp, Campus Drie Eiken, Wilrijk, B-2610 Antwerp, Belgium.
Cardiovasc Res. 2004 Oct 1;64(1):144-53. doi: 10.1016/j.cardiores.2004.05.016.
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.
SMC cultures derived from the rabbit aorta were exposed to 25 microM 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.
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.
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.
7-酮胆固醇是晚期动脉粥样硬化斑块中氧化型低密度脂蛋白的主要氧化甾醇，可诱导血管平滑肌细胞（SMC）死亡。我们研究了细胞色素c释放是否参与7-酮胆固醇诱导的SMC死亡以及这些过程是否可逆。
将源自兔主动脉的SMC培养物暴露于25μM的7-酮胆固醇。通过免疫荧光和免疫印迹研究细胞色素c和Bax，通过TUNEL技术研究细胞凋亡，通过透射电子显微镜研究线粒体结构。
7-酮胆固醇诱导促凋亡蛋白Bax快速上调并从胞质溶胶转运至线粒体（4小时）。随后线粒体细胞色素c释放至胞质溶胶（8小时时为65%），16小时时几乎完全释放。线粒体变为球形且高度浓缩，未显示出裂解迹象。它们聚集在细胞核周围并被粗面内质网的宽池包裹。与DNA片段化和SMC损失相反，泛半胱天冬酶抑制剂zVAD-fmk未阻断细胞色素c释放。有趣的是，在16小时后去除7-酮胆固醇并重新暴露于血清24小时后，线粒体细胞色素c含量、其跨膜电位和TUNEL标记恢复正常，SMC损失减少。然而，当SMC暴露于氧化甾醇24小时时，这些细胞死亡标志物均未恢复。
结果表明，氧化甾醇诱导的SMC凋亡过程中细胞色素c释放不依赖于半胱天冬酶，是线粒体构象可逆性变化的结果，而非外膜肿胀和破裂所致。这些事件的可逆性表明凋亡级联反应可能在不可逆转点之前被阻断。
