Platelet-derived exosomes of septic individuals possess proapoptotic NAD(P)H oxidase activity: A novel vascular redox pathway
Platelet-derived exosomes of septic individuals possess proapoptotic NAD(P)H oxidase activity: A novel vascular redox pathway
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DOI：
10.1097/01.ccm.0000114829.17746.19
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发表时间：
2004-03-01
影响因子：
8.8
通讯作者：
Laurindo, FRM
中科院分区：
文献类型：
--
作者：
Janiszewski, M;do Carmo, AO;Laurindo, FRM
客观，脓毒症的血管功能障碍可能涉及通过氧化还原信号机制的血管细胞凋亡，这仍然是研究不足。血小板已被证明产生活性氧并释放微粒，与血栓形成和炎症过程有关。本研究旨在研究在严重脓毒症中，血小板源性微粒是否可以通过吞噬细胞型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶产生活性氧，以及此类颗粒是否可以通过活性氧诱导血管细胞凋亡依赖性机制。设计。实验研究。设置。与三级医院相关的分子和细胞生物学实验室。主题：从脓毒症患者和健康个体中获得的微粒进行了调查，了解其生化特性及其对培养的血管内皮细胞和平滑肌细胞的影响。干预措施：通过流式细胞术研究微粒表面抗原，并通过Western印迹分析研究NADPH氧化酶亚基的存在。通过超氧化物歧化酶可降解的细胞色素c还原和5 μ M光泽精化学发光研究微粒活性氧的产生。微粒对血管细胞凋亡率的影响进行了分析，免疫荧光显微镜的基础上膜联蛋白V-荧光素5（6）-异硫氰酸酯assay.Measurements和主要结果：流式细胞术分析的微粒从脓毒症患者和健康人的表面抗原模式相似的外来体和强烈提示血小板起源。这些微粒还显示吞噬细胞-NADPH氧化酶的p22（phox）和gp91（phox）亚基，并表现出内在的活性氧产生。内皮细胞和血管平滑肌细胞与微粒的孵育提高了凋亡率。与来自健康受试者的外泌体相比，来自脓毒症个体的外泌体的活性氧产生和前列腺增生诱导活性显著更大。这些影响被超氧化物歧化酶或NADPH氧化酶抑制剂二苯碘鎓和氧化苯胂减弱。血小板源性外泌体NADPH氧化酶活性似乎有助于血管细胞凋亡，并可能代表一种新的血管氧化还原信号通路参与脓毒症的病理生理。
Objective., Vascular dysfunction in sepsis may involve apoptosis of vascular cells through redox signaling mechanisms, which are still poorly investigated. Platelets have been shown to produce reactive oxygen species and to release microparticles, related to thrombotic and inflammatory processes. The present study was undertaken to investigate whether, in severe sepsis, platelet-derived microparticles could produce reactive oxygen species through a phagocyte-type nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and if such particles may induce vascular cell apoptosis through a reactive oxygen species-dependent mechanism.Design. Experimental study.Setting. Molecular and cell biology laboratories related to tertiary hospitals.Subjects: Microparticles obtained from septic patients and from healthy individuals were investigated concerning their biochemical properties and their effects on vascular endothelial and smooth muscle cells in culture.Interventions: Microparticle surface antigens were studied by flow cytometry and the presence of NADPH oxidase subunits by Western blot analysis. Microparticle reactive oxygen species generation was investigated through superoxide dismutase-inhibitable cytochrome c reduction and 5 muM lucigenin chemiluminescence. The effects of microparticles on vascular cell apoptosis rates were analyzed by immunofluorescence microscopy based on annexin V-fluorescein 5(6)-isothiocyanate assay.Measurements and Main Results: Flow cytometry analysis of microparticles obtained from septic patients and healthy individuals showed a surface antigenic pattern similar to exosomes and strongly suggestive of platelet origin. Those microparticles also displayed the p22(phox) and gp91(phox) subunits of phagocyte-simile NADPH oxidase and exhibited intrinsic reactive oxygen species production. Incubation of endothelial and vascular smooth muscle cells with microparticles enhanced apoptosis rates. Reactive oxygen species generation and apoptosis-inducing activity were markedly greater with exosomes from septic individuals than with exosomes from healthy subjects. These effects were diminished by the addition of superoxide dismutase or the NADPH oxidase inhibitors diphenylene iodonium and phenilarsine oxide.Conclusions. Platelet-derived exosome NADPH oxidase activity seems to contribute to vascular cell apoplosis and may represent a new vascular redox-signaling pathway involved in the pathophysiology of sepsis.