Teoh Narci C, Ajamieh Hussam, Wong Heng Jian, Croft Kevin, Mori Trevor, Allison Anthony C, Farrell Geoffrey C
Australian National University Medical School at The Canberra Hospital, Canberra, ACT, Australia.
School of Medicine and Pharmacology, University of Western Australia, Perth, WA, Australia.
PLoS One. 2014 Sep 15;9(9):e104376. doi: 10.1371/journal.pone.0104376. eCollection 2014.
BACKGROUND & AIMS: Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis.
C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min-24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function.
Microparticles were detected in the circulation 15-30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8+ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-α increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory properties of microparticles involved activation of NF-κB and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin.
Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.
缺血再灌注损伤（IRI）可导致肝手术后或肝移植后的肝衰竭。IRI引发氧化应激，损伤肝血窦内皮细胞（SECs），导致库普弗细胞、血小板募集和活化以及微循环障碍。我们研究了缺血后再灌注期间受损的SECs和其他细胞类型是否释放微粒，以及这些微粒是否具有促炎、血小板活化和促损伤作用，从而可能导致IRI发病机制。
C57BL6小鼠经历60分钟的部分肝缺血，随后进行15分钟至24小时的再灌注。我们收集血液和肝脏样本，分离循环微粒，并测定蛋白质和脂质含量。为了确定微粒产生的机制，我们将小鼠原代肝细胞进行缺氧复氧处理。由于微粒表达外翻的磷脂酰丝氨酸残基，这是膜联蛋白V的作用靶点，我们分析了膜联蛋白V-同二聚体（Diannexin或ASP8597）对缺血后微粒产生和功能的影响。
缺血后再灌注15 - 30分钟后在循环中检测到微粒，其含有SECs、血小板、自然杀伤T细胞和CD8 +细胞的标志物；4小时后，它们含有巨噬细胞的标志物。微粒含有F2 -异前列腺素，表明膜脂质受到氧化损伤。给小鼠注射TNF-α可增加微粒形成，而Diannexin可显著减少微粒释放并预防IRI。缺氧复氧通过涉及氧化应激的过程从原代肝细胞产生微粒。将培养的肝细胞暴露于IRI期间从循环中分离的微粒制剂会导致涉及线粒体膜通透性转换的损伤。微粒还在体外激活血小板并诱导中性粒细胞迁移。微粒的炎症特性涉及NF-κB和JNK的激活，E-选择素、P-选择素、ICAM-1和VCAM-1表达增加。所有这些过程都可通过用Diannexin包被微粒来阻断。
肝IRI后，微粒在循环中存在并可被肝细胞摄取，在肝细胞中它们激活介导炎症和肝细胞损伤的信号通路。Diannexin可预防微粒形成及随后的炎症。
