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.
Highlights
Hepatic ischemia-reperfusion injury (IRI) remains an important complication of liver surgery and transplantation [1,2]
The late phase (6–24 hr) is mediated by hepatic recruitment of leukocytes and macrophages. These adhere to sinusoidal endothelial cells (SECs) that express adhesion molecules (E-selectin, P-selectin, ICAM-1, VCAM-1) and secrete chemokines [5,6,7,8] in response to hypoxia, oxidative stress, and tumor necrosis factor-a (TNF) [8]
We have described extensive blebbing of SEC plasma membranes during the first 20 min of post-IR [10], leading us to conceive the hypothesis that MPs originating from perturbed plasma membranes of SECs contribute to the microvascular inflammatory and platelet activating responses in hepatic IRI
Summary
Hepatic ischemia-reperfusion injury (IRI) remains an important complication of liver surgery and transplantation [1,2]. In the early phase (first 2 hr), hepatic damage is caused by oxidative stress, generated by Kupffer cells (KCs) with damage to sinusoidal endothelial cells (SECs) [1,2,3,4,5,6]. The late phase (6–24 hr) is mediated by hepatic recruitment of leukocytes and macrophages These adhere to SECs that express adhesion molecules (E-selectin, P-selectin, ICAM-1, VCAM-1) and secrete chemokines [5,6,7,8] in response to hypoxia, oxidative stress, and tumor necrosis factor-a (TNF) [8]. 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, plateletactivating and pro-injurious effects that could contribute to IRI pathogenesis
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