Microparticles Mediate Hepatic Ischemia-Reperfusion Injury and Are the Targets of Diannexin (ASP8597)

Abstract

Background & AimsIschemia–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.MethodsC57BL6 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.ResultsMicroparticles 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.ConclusionsFollowing 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.

Lipotoxic lethal and sublethal stress signaling in hepatocytes: relevance to NASH pathogenesis

“…EVs are constitutively released under physiologic conditions into various body fluids (125). Pathogenic stimuli may further increase the number of released vesicles and modify their cargo, as demonstrated by an elevated number of circulating EVs in mouse models of liver ischemia reperfusion injury (126) and NASH (33,127).…”

Extracellular Vesicles from Red Blood Cell Products Induce a Strong Pro-Inflammatory Host Response<b>, </b> Dependent on Both Numbers and Storage Duration

“…In addition to EVs derived from blood bags, endogenous circulating EVs also appear to have pro-inflammatory properties. In an animal model of ischemia reperfusion injury, EVs from endothelium and inflammatory cells are shed into the circulation, which can subsequently promote neutrophil migration [19]. Moreover, we showed in an earlier investigation that in trauma patients endogenously circulating EVs can induce an inflammatory host response [20].…”

Ischemia/Reperfusion

“…Although originally dismissed as cellular debris, it is now clear that microparticles display strong procoagulant activity and encapsulate a number of molecular entities that contribute to the pathogenesis of I/R by shuttling signaling agents amongst cell types (27, 353, 487, 545, 652, 772). These include bioactive lipids, chemokines, cytokines, tissue factor, arachidonic acid, integrins, receptors, RNA, proteases, growth factors, and caspases.…”

“…Moreover, I/R-induced microparticle release from endothelial cells, platelets, NK cells, and CD8 + T cells activate JNK and NFκB and increase the expression of endothelial cell adhesion molecules (772). Exciting recent work indicates that adherent platelets form extremely long (up to 250 µm), negatively charged protrusions (FLIPRs) under conditions of flow.…”