Rho-kinase as a novel gene therapeutic target in treatment of cold ischemia/reperfusion-induced acute lethal liver injury: effect on hepatocellular NADPH oxidase system

Abstract

In the transplant surgery, reactive oxygen species (ROS) from the reperfused tissue cause ischemia-reperfusion injury, resulting in the primary graft failure. We have recently reported that Rho-kinase, an effecter of the small GTPase Rho, plays an important role in the ROS production in the hyperacute phase of reperfusion; however, the sources and mechanisms of the ROS production remain to be elucidated. The aim of this study was to investigate the source of ROS production with a special reference to Rho-kinase to develop a new strategy against ischemia-reperfusion injury. In an in vivo rat model of liver transplantation, Kupffer cells in the graft were depleted using liposome-encapsulated dichloromethylene diphosphonate to examine the source of ROS production. The effect of adenoviral-mediated overexpression of a dominant-negative Rho-kinase (AdDNRhoK) in hepatocytes in the graft was also examined. Kupffer cells were not involved in the ROS production, whereas the AdDNRhoK transfection to hepatocytes significantly suppressed the ROS production. Furthermore, the ROS production was dose-dependently inhibited by apocynin, an NADPH oxidase inhibitor. Expression of DNRhoK also suppressed the release of pro-inflammatory cytokines, and ameliorated the lethal liver injury with a significant prolongation of the survival. These results suggest that the Rho-kinase-mediated pathway plays a crucial role in the ROS production through NADPH oxidase in hepatocytes during the hyperacute phase of reperfusion in vivo. Thus, Rhokinase in hepatocytes may be a new therapeutic target for the prevention of primary graft failure in liver transplantation.

Which NADPH Oxidase Isoform Is Relevant for Ischemic Stroke? The Case for Nox 2

“…Pharmacological Nox inhibition with the unspecific apocynin attenuated necrotic and apoptotic cell death, O 2 -production, and lipid peroxidation in the murine and rat models of hepatic I/R (104,150). Moreover, apocynin and the Nox2-inhibitory peptide gp91ds-tat (41) prevented remote liver damage in a murine model of bilateral hind limb I/R (52).…”

Toll-like receptors 2 and 4: initiators of non-septic inflammation in critical care medicine?

“…However, in the setting of ischemia-reperfusion and other processes related to critical illness, there is increased production of ROS through mitochondrial electron transport mechanisms, activation of the purine/xanthine oxidase system, and NADPH oxidase activation [9–11]. …”

“…We and others have demonstrated that inhibition of superoxide production by xanthine oxidase, through allopurinol treatment, or from NADPH oxidase, as a result of blocking translocation to the membrane of the NADPH phox 47 subunit, protected mice from proinflammatory processes initiated by hemorrhage or ischemia-reperfusion injury [9–11, 27]. We have also shown that extracellular superoxide derived from xanthine oxidase is able to activate neutrophils and induce neutrophil-mediated proinflammatory responses through a TLR4-dependent mechanism [28].…”

Hepatic dysfunction after shock: Clinical parameters and biological pathways for therapeutic intervention

“…In non-phagocytic cells, ROS can be produced by the NADPH oxidase homolog Nox. Rho-kinase is associated with Nox, and Rho-kinase-dependent ROS production was found in I/R (65,66). Rho-kinase can also attenuate NO from endogenous nitric oxide synthase (eNOS), as well as regulate NF-kB through phosphorylation of IkB, and cause production of IL-6, monocyte chemoattractant protein (MCP-1), and migration inhibitory factor.…”

“…ROS are produced in the hyperacute phase by hepatocytes, and later by KC (65). Sources of ROS include the mitochondrial electron transport chain, cyclooxygenase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and xanthine oxidase (66).…”