Oxidative lipidomics of pulmonary endothelial apoptosis in hyperoxia

描述（由申请人提供）：急性肺损伤（ALI），包括高氧肺损伤，是一种具有重大公共卫生意义的综合征。虽然ALI的发病机制尚不清楚，但活性氧和氮的产生和消除之间的不平衡被公认为是一个促成因素。肺内皮细胞是高氧肺损伤早期结构和功能改变的发生部位，肺内皮细胞凋亡是高氧肺损伤发生和维持的重要因素。尽管如此，高氧诱导肺内皮细胞凋亡的早期分子途径尚不清楚，ALI的治疗仍然完全是姑息性的。我们最近表明，在与阴离子磷脂，特别是与特定的心磷脂（CL），细胞色素c（cyt c）失去其三级结构和过氧化物酶活性显着增加。CL诱导的细胞色素c的过氧化物酶活性已被发现是重要的选择性CL氧化在细胞进行程序性死亡。在凋亡过程中，过氧化物酶活性和CL结合的cyt c的分数显着增加，表明CL作为一个开关，调节cyt c的线粒体功能。在初步的数据中，使用离子阱电喷雾电离质谱法为基础的脂质组学首次在肺，我们表明，CL的选择性氧化是一个早期事件在高氧肺，也发生在简单的模型（如LPS）培养的肺内皮细胞凋亡。此外，我们还设计了一种抗氧化剂/电子清除剂（XJB-5-131;一种半短杆菌肽-4-氨基-2，2，6，6-四甲基哌啶-N-氧基）和一种过氧化物酶可激活的一氧化氮供体（HVTP，2-羟基氨基-乙烯基-三苯基-磷），它们特异性靶向线粒体，并显示它们可以有效抑制多种细胞类型的凋亡，改善体内急性器官功能障碍。因此，本研究的具体目的是确定：1）CL诱导的细胞色素c过氧化物酶活性在高氧小鼠肺内皮细胞凋亡中的分子作用; 2）氧化阴离子磷脂在肺内皮细胞凋亡的信号转导途径中的分子作用，包括巨噬细胞清除凋亡的肺内皮细胞;（3）XJB-5-131和HVTP这两种靶向抗氧化剂/抑制剂在防止肺内皮细胞CL氧化、凋亡和有利地影响高氧肺损伤过程中的治疗潜力。 公共卫生相关性：虽然抗氧化剂长期以来一直被认为是潜在的有效的治疗干预措施，阐明特定的机制，通过细胞色素c和CL氧化产物连接自由基的生产与早期凋亡事件的线粒体将有助于路线图，以新的有效的药物发现目标。

DESCRIPTION (provided by applicant): Acute lung injury (ALI), including hyperoxic lung injury, is a syndrome of great public health importance. Although the pathogenesis of ALI remains unclear, an imbalance between production and elimination of reactive oxygen and nitrogen species is well accepted as a contributing factor. Pulmonary endothelium is the locus of early structural and functional changes in hyperoxic lung injury and apoptosis of pulmonary endothelium is a contributing factor to genesis and maintenance of the injury. Nonetheless, the early molecular pathways that account for hyperoxic induced pulmonary endothelial cell apoptosis are unknown and therapies for ALI remain entirely palliative. We have recently shown that upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL acts as a switch to regulate cyt c's mitochondrial functions. In preliminary data, using ion trap electrospray ionization mass spectrometry based lipidomics for the first time in lung, we show that selective oxidation of CL is an early event in hyperoxic lung and also occurs in simple models (e.g. LPS) of apoptosis in cultured pulmonary endothelial cells. Furthermore we have designed an antioxidant/electron scavenger (XJB-5-131; an hemigramicidin-4-amino-2,2,6,6- tetramethylpiperidine-N-oxyl) and a peroxidase-activatable nitric oxide donor (HVTP, 2- hydroxylamino-vinyl-triphenyl-phosphonium) that are specifically targeted to mitochondria and show that they can effectively inhibit apoptosis in a variety of cell types and ameliorate acute organ dysfunction in vivo. Accordingly, the SPECIFIC AIMS of this proposal are to determine: 1) the molecular role of CL-induced peroxidase activity of cytochrome c in pulmonary endothelial cell apoptosis in hyperoxic mouse lung; 2) the molecular role of oxidized anionic phospholipids in the signal transduction pathway of pulmonary endothelial cell apoptosis including scavenging of apoptotic pulmonary endothelium by macrophages; 3) the therapeutic potential of XJB-5-131 and HVTP, two mitochondria-targeted antioxidants/inhibitors of cyt c/CL peroxidase, in preventing pulmonary endothelial cell CL oxidation, apoptosis and favorably affecting the course of hyperoxic lung injury. PUBLIC HEALTH RELEVANCE: While antioxidants have been long viewed as potentially effective for therapeutic interventions, elucidation of specific mechanisms through which cyt c and CL oxidation products link free radical production with early apoptotic events in mitochondria will contribute to a roadmap to new effective drug discovery targets.