Martinet W, Croons V, Timmermans J-P, Herman A G, De Meyer G R Y
Division of Pharmacology, University of Antwerp, Wilrijk, Belgium.
Br J Pharmacol. 2007 Oct;152(4):493-500. doi: 10.1038/sj.bjp.0707426. Epub 2007 Aug 13.
Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism.
Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis.
Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin.
Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.
动脉粥样硬化斑块中的巨噬细胞对动脉粥样硬化的发生发展以及斑块不稳定具有重大影响。我们之前证明，用一氧化氮（NO）供体莫西赛利处理喂食胆固醇的兔子的斑块，可优先清除巨噬细胞，从而有利于斑块稳定。在本研究中，我们探究了其潜在机制。
巨噬细胞和平滑肌细胞（SMC）在体外分别用NO供体亚精胺NONOate或S-亚硝基-N-乙酰青霉胺（SNAP）以及著名的内质网（ER）应激诱导剂毒胡萝卜素、衣霉素、二硫苏糖醇或布雷菲德菌素A进行处理。通过中性红活力测定法分析细胞活力。检测半胱天冬酶-3的切割、DNA片段化和超微结构变化，以表征巨噬细胞死亡的类型。通过测量C/EBP同源蛋白（CHOP）表达、真核起始因子2α（eIF2a）的磷酸化、X盒结合蛋白1（XBP1）的剪接以及蛋白质合成的抑制来评估ER应激的诱导情况。
用亚精胺NONOate或SNAP处理的巨噬细胞和SMC表现出多种ER应激迹象，包括CHOP表达上调、eIF2α过度磷酸化、从头蛋白质合成抑制以及XBP1 mRNA的剪接。这些效应在巨噬细胞和SMC中相似，但只有巨噬细胞发生凋亡。与安慰剂相比，用莫西赛利处理的动脉粥样硬化兔子的斑块中CHOP表达增加了2.7倍。除了NO之外，毒胡萝卜素和衣霉素也可引发巨噬细胞死亡的选择性诱导。
ER应激的诱导解释了NO供体对动脉粥样硬化斑块中巨噬细胞的选择性消耗，可能是通过抑制蛋白质合成实现的。
