Jian Wenying, Arora Jasbir S, Oe Tomoyuki, Shuvaev Vladimir V, Blair Ian A
Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA.
Free Radic Biol Med. 2005 Nov 1;39(9):1162-76. doi: 10.1016/j.freeradbiomed.2005.06.008.
Endothelial dysfunction is considered to be the earliest event in atherogenesis. Oxidative stress, inflammation, and apoptosis play critical roles in its progression and onset. Lipid peroxidation, which occurs during oxidative stress, results in the formation of lipid hydroperoxide-derived bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal that induce apoptosis. In this study, recently identified lipid hydroperoxide-derived bifunctional electrophiles 4-oxo-2(E)-nonenal (ONE; 5-30 microm) and 4,5-epoxy-2(E)-decenal (EDE; 10-20 microM) were shown to cause a dose- and time-dependent apoptosis in EA.hy 926 endothelial cells. This was manifest by morphological changes, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Bifunctional electrophiles caused cytochrome c release from mitochondria into the cytosol, implicating a mitochondrial pathway of apoptosis in the endothelial cells. The novel carboxylate-containing lipid hydroperoxide-derived bifunctional electrophile 9,12-dioxo-10(E)-dodecenoic acid was inactive because it could not translocate across the plasma membrane. However, its less polar methyl ester derivative (2-10 microM) was the most potent inducer of apoptosis of any bifunctional electrophile that has been tested. An acute decrease in intracellular glutathione (GSH) preceded the onset of apoptosis in bifunctional electrophile-treated cells. The ability of ONE and EDE to deplete GSH was directly correlated with their predicted reactivity toward nucleophilic amino acids. Liquid chromatography/mass spectrometry methodology was developed in order to examine the intracellular and extracellular concentrations of bifunctional electrophile-derived GSH adducts. Relative intracellular/extracellular ratios of the GSH adducts were identical with the rank order of potency for inducing caspase 3 activation. This suggests that there may be a role for the bifunctional electrophile-derived GSH adducts in the apoptotic response. N-Acetylcysteine rescued bifunctional electrophile-treated cells from apoptosis, whereas the GSH biosynthesis inhibitor d,l-buthionine-(R,S)-sulfoximine sensitized the cells to apoptosis. These data suggest that lipid hydroperoxide-derived bifunctional electrophiles may play an important role in cardiovascular pathology through their ability to induce endothelial cell apoptosis.
内皮功能障碍被认为是动脉粥样硬化发生过程中最早出现的事件。氧化应激、炎症和细胞凋亡在其进展和发病过程中起着关键作用。氧化应激期间发生的脂质过氧化作用会导致脂质氢过氧化物衍生的双功能亲电试剂形成，如4-羟基-2（E）-壬烯醛，可诱导细胞凋亡。在本研究中，最近鉴定出的脂质氢过氧化物衍生的双功能亲电试剂4-氧代-2（E）-壬烯醛（ONE；5 - 30微摩尔）和4,5-环氧-2（E）-癸烯醛（EDE；10 - 20微摩尔）在EA.hy 926内皮细胞中可引起剂量和时间依赖性的细胞凋亡。这表现为形态学改变、半胱天冬酶-3激活和聚（ADP-核糖）聚合酶裂解。双功能亲电试剂导致细胞色素c从线粒体释放到细胞质中，提示内皮细胞凋亡存在线粒体途径。新型含羧酸盐的脂质氢过氧化物衍生的双功能亲电试剂9,12-二氧代-10（E）-十二碳烯酸无活性，因为它不能穿过质膜。然而，其极性较小的甲酯衍生物（2 - 10微摩尔）是所有已测试的双功能亲电试剂中最有效的细胞凋亡诱导剂。在双功能亲电试剂处理的细胞中，细胞内谷胱甘肽（GSH）的急性减少先于细胞凋亡的发生。ONE和EDE消耗GSH的能力与其对亲核氨基酸的预测反应性直接相关。开发了液相色谱/质谱方法以检测双功能亲电试剂衍生的GSH加合物的细胞内和细胞外浓度。GSH加合物的相对细胞内/细胞外比率与诱导半胱天冬酶3激活的效力等级顺序相同。这表明双功能亲电试剂衍生的GSH加合物在凋亡反应中可能起作用。N-乙酰半胱氨酸可使双功能亲电试剂处理的细胞免于凋亡，而GSH生物合成抑制剂d，l-丁硫氨酸-（R，S）-亚砜亚胺使细胞对凋亡敏感。这些数据表明，脂质氢过氧化物衍生的双功能亲电试剂可能通过诱导内皮细胞凋亡在心血管病理过程中起重要作用。
