Absence of toll-like receptor 4 (TLR4) signaling in the donor organ reduces ischemia and reperfusion injury in a murine liver transplantation model.

This study analyzes how toll-like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild-type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4 degrees C for 24 hours. Unlike WT OLTs, TLR4-deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzuki's grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)-gamma-inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM-1), as well as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, and IFN-gamma, yet increased IL-4 and IL-10 expression. The well-functioning OLTs from TLR4 KO donors revealed attenuated activity of capase-3, and enhanced heme oygenase-1 (HO-1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI-dependent responses.

Heme oxygenase-1 mediated cytoprotection against liver ischemia and reperfusion injury: inhibition of type-1 interferon signaling.

BACKGROUND: Toll-like receptor (TLR)-4 signaling plays a key role in initiating exogenous antigen-independent innate immunity-dominated liver ischemia/reperfusion injury (IRI). Heme oxygenase (HO)-1, a heat-shock protein 32, exerts potent adaptive anti-oxidant and anti-inflammatory functions. Signal transducers and activator of transcription (STAT)-1 activation triggers interferon (IFN)-inducible protein 10 (CXCL-10), one of major products of type-1 IFN pathway downstream of TLR4. This study focuses on the role of type-1 IFN pathway in the mechanism of HO-1 cytoprotection during liver IRI. METHODS AND RESULTS: Cobalt protoporphyrin (CoPP)-induced HO-1 overexpression ameliorated liver damage in a well-defined mouse model of liver warm IRI, as evidenced by improved hepatic function (serum alanine aminotransferase levels) and liver histology (Suzuki's scores). HO-1 downregulated phospho-STAT-1 and its key product, CXCL-10. In contrast, TLR4 expression remained elevated regardless of the IRI status. To dissect the mechanism of HO-1 upon CXCL-10, we cultured RW 264.7 (macrophage) cells with exogenous rIFN-beta to stimulate CXCL-10 production via TLR4 pathway in vitro. Indeed, CoPP-induced HO-1 suppressed otherwise highly upregulated rIFN-beta-triggered CXCL-10. Moreover, consistent with our in vitro data, CoPP pretreatment diminished rIFN-beta-induced CXCL-10 production in normal mouse livers. CONCLUSION: Hepatic IRI activates TLR4 signaling in vivo to elaborate CXCL-10. HO-1 overexpression downregulates activation of STAT1 via type-1 IFN pathway downstream of TLR4, which in turn decreases CXCL-10 production. This study provides evidence for a novel mechanism by which HO-1 exerts adaptive cytoprotective and anti-inflammatory functions in the context of innate TLR4 activation.

Viral interleukin-10 gene transfer prevents liver ischemia-reperfusion injury: Toll-like receptor-4 and heme oxygenase-1 signaling in innate and adaptive immunity.

Ischemia-reperfusion injury (IRI) contributes to early and late dysfunction of liver transplants. We have shown that sentinel Toll-like receptor-4 (TLR4) plays a key role in the activation of T cell immune responses during hepatic IRI. We have also documented that overexpression of heme oxygenase-1 (HO-1) exerts potent cytoprotective effects. This study analyzes how adenovirus (Ad)-based viral interleukin-10 (vIL-10) gene transfer affects TLR4 and HO-1 signaling in host innate and adaptive immunity during liver IRI. Using a partial lobar warm IRI model, groups of wild-type and HO-1(+/-) knockout (KO) mice were assessed for severity of hepatocellular damage after 90 min of warm ischemia followed by 6 hr of reperfusion. Both wild-type and HO-1 (+/-) KO mice treated with Ad-vIL-10 have shown improved hepatic function (serum glutamic-oxaloacetic transaminase levels), ameliorated histological signs of IRI (Suzuki's score), decreased neutrophil accumulation (myeloperoxidase activity), and depressed tumor necrosis factor-alpha/IL-1beta, IL-2/interferon-gamma, E-selectin, and macrophage inflammatory protein-2 expression. These effects were IL-10 dependent as treatment with neutralizing antibody re-created liver IRI. In contrast, untreated wild-type and HO-1 (+/-) KO mice, as well as wild-type and HO-1 (+/-) KO mice treated with Ad-beta-Gal, showed severe hepatocellular damage due to IRI. Unlike in controls, wild-type and HO-1 (+/-) KO mice treated with Ad-vIL-10 revealed markedly depressed TLR4 and NF-kappaB expression, along with increased HO-1 and Bcl-2/Bcl-x(L) expression, as compared with respective controls. Thus, vIL-10 gene transfer prevents hepatic IRI in association with depressed expression of innate TLR4, and adaptive Th1 cytokine/chemokine programs. The induction of antioxidant HO-1 and anti-apoptotic Bcl-2/Bcl-x(L) by vIL-10 exerts synergistic cytoprotective function against antigen-independent hepatic inflammatory response triggered by IRI.

Basal rather than induced heme oxygenase-1 levels are crucial in the antioxidant cytoprotection.

Heme oxygenase-1 (HO-1) overexpression protects against tissue injury in many inflammatory processes, including ischemia/reperfusion injury (IRI). This study evaluated whether genetically decreased HO-1 levels affected susceptibility to liver IRI. Partial warm ischemia was produced in hepatic lobes for 90 min followed by 6 h of reperfusion in heterozygous HO-1 knockout (HO-1(+/-)) and HO-1(+/+) wild-type (WT) mice. HO-1(+/-) mice demonstrated reduced HO-1 mRNA/protein levels at baseline and postreperfusion. This corresponded with increased hepatocellular damage in HO-1(+/-) mice, compared with WT. HO-1(+/-) mice revealed enhanced neutrophil infiltration and proinflammatory cytokine (TNF-alpha, IL-6, and IFN-gamma) induction, as well as an increase of intrahepatic apoptotic TUNEL(+) cells with enhanced expression of proapoptotic genes (Bax/cleaved caspase-3). We used cobalt protoporphyrin (CoPP) treatment to evaluate the effect of increased baseline HO-1 levels in both WT and HO-1(+/-) mice. CoPP treatment increased HO-1 expression in both animal groups, which correlated with a lower degree of hepatic damage. However, HO-1 mRNA/protein levels were still lower in HO-1(+/-) mice, which failed to achieve the degree of antioxidant hepatoprotection seen in CoPP-treated WT. Although the baseline and postreperfusion HO-1 levels correlated with the degree of protection, the HO-1 fold induction correlated instead with the degree of damage. Thus, basal HO-1 levels are more critical than the ability to up-regulate HO-1 in response to the IRI and may also predict the success of pharmacologically induced cytoprotection. This model provides an opportunity to further our understanding of HO-1 in stress defense mechanisms and design new regimens to prevent IRI.

Vascular endothelial growth factor antagonist modulates leukocyte trafficking and protects mouse livers against ischemia/reperfusion injury.

Although hypoxia stimulates the expression of vascular endothelial growth factor (VEGF), little is known of the role or mechanism by which VEGF functions after ischemia and reperfusion (I/R) injury. In this report, we first evaluated the expression of VEGF in a mouse model of liver warm ischemia. We found that the expression of VEGF increased after ischemia but peaked between 2 and 6 hours after reperfusion. Mice were treated with a neutralizing anti-mouse VEGF antiserum (anti-VEGF) or control serum daily from day -1 (1 day before the initiation of ischemia). Treatment with anti-VEGF significantly reduced serum glutaminic pyruvic transaminase levels and reduced histological evidence of hepatocellular damage compared with controls. Anti-VEGF also markedly decreased T-cell, macrophage, and neutrophil accumulation within livers and reduced the frequency of intrahepatic apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells. Moreover, there was a reduction in the expression of pro-inflammatory cytokines (tumor necrosis factor-alpha and interferon-gamma), chemokines (interferon-inducible protein-10 and monocyte chemoattractant protein-1) and adhesion molecules (E-selectin) in parallel with enhanced expression of anti-apoptotic genes (Bcl-2/Bcl-xl and heme oxygenase-1) in anti-VEGF-treated animals. In conclusion, hypoxia-inducible VEGF expression by hepatocytes modulates leukocyte trafficking and leukocyte-induced injury in a mouse liver model of warm I/R injury, demonstrating the importance of endogenous VEGF production in the pathophysiology of hepatic I/R injury.

Molecular characterization of rat leukocyte P-selectin glycoprotein ligand-1 and effect of its blockade: protection from ischemia-reperfusion injury in liver transplantation.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-alpha, IL-1beta, IL-6, IFN-gamma, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.

Inflammatory responses in a new mouse model of prolonged hepatic cold ischemia followed by arterialized orthotopic liver transplantation.

The current models of liver ischemia/reperfusion injury (IRI) in mice are largely limited to a warm ischemic component. To investigate the mechanism of hepatic "cold" IRI, we developed and validated a new mouse model of prolonged cold preservation followed by syngeneic orthotopic liver transplantation (OLT). Two hundred and forty-three OLTs with or without rearterialization and preservation in University of Wisconsin solution at 4 degrees C were performed in Balb/c mice. The 14-day survivals in the nonarterialized OLT groups were 92% (11/12), 82% (9/11), and 8% (1/12) after 1-hour, 6-hour and 24-hour preservation, respectively. In contrast, hepatic artery reconstruction after 1-hour, 6-hour, and 24-hour preservation improved the outcome as evidenced by 2-week survival of 100% (12/12), 100% (10/10), and 33% (4/12), respectively, and diminished hepatocellular damage (serum alanine aminotransferase /histology). Moreover, 24-hour (but not 1-h) cold preservation of rearterialized OLTs increased hepatic CD4+ T-cell infiltration and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin 2, interferon-gamma) production, as well as enhanced local apoptosis, and Toll-like receptor 4/caspase 3 expression. These cardinal features of hepatic IRI validate the model. In conclusion, we have developed and validated a new mouse model of IRI in which hepatic artery reconstruction was mandatory for long-term animal survival after prolonged (24-h) OLT preservation. With the availability of genetically manipulated mouse strains, this model should provide important insights into the mechanism of antigen-independent hepatic IRI and help design much needed refined therapeutic means to combat hepatic IRI in the clinics.

Heme oxygenase system in ischemia and reperfusion injury.

Ischemia/reperfusion injury (IRI) is a multifactorial antigen-independent process that affects both early and late graft function after transplantation. The complex mechanism of IRI can be attributed to neutrophil accumulation at the site of tissue injury, release of pro-inflammatory mediators such as oxygen free radicals (OFRs), and cytokines, which lead to cellular injury that culminates in the ultimate graft failure. The heme oxygenase-1 (HO-1) system is among the most critical of cytoprotective mechanisms activated during the cellular stress. The cytoprotection often seen in the transplanted organ following local HO-1 overexpression may include several factors, such as: a) antioxidant function, b) maintenance of microcirculation, c) anti-apoptotic function, and d) anti-inflammatory function. The role of enhanced endogenous HO-1 overexpression, and HO-1 downstream mediators (bilirubin, ferritin, CO), which protect against the IRI sequel, remain currently one of the most active areas of investigation. Indeed, HO-1, which functions to amplify multiple intracellular cytoprotective pathways, may serve as a novel therapeutic concept in transplantation to maximize organ donor pool through their safer use despite prolonged periods of cold ischemia.

Induction of specific stress response increases resistance of rat liver allografts to cold ischemia and reperfusion injury.

Heme oxygenase-1 (HO-1) has been shown to increase cellular resistance against oxidative injury, but the functional significance of this is currently obscure. We investigated the protective role of HO-1, induced by tin-protoporphyrin IX (SnPP), in attenuating liver transplantation injury. Lewis rats were intraperitoneally treated with saline as control, 50 micro mol/kg of SnPP, or 2 mg/kg of cycloheximide (CHX) before SnPP injection. Gene expression of HO-1 was induced after either treatment with SnPP- or CHX + SnPP instead of saline, whereas HO-1 protein synthesis was enhanced in Kupffer-like dendritic cells of the SnPP-treated group. Following reperfusion of liver grafts preserved for 30 h, there were fewer intercellular adhesion molecule-1-positive cells in SnPP-treated livers, significantly reduced numbers of dead cells, and enhanced graft viability. The present data suggest that increased synthesis of HO-1 protein by SnPP pre-conditioning is linked to the improved liver graft viability through inhibition of inflammatory adhesion molecules.

Pyrrolidine dithiocarbamate provides protection against hypothermic preservation and transplantation injury in the rat liver: the role of heme oxygenase-1.

BACKGROUND: Pyrrolidine dithiocarbamate (PDTC) represents a class of antioxidants and is a potent inducer of the heme oxygenase-1 (HO-1) gene and an inhibitor of nuclear factor-kappa B (NF-kappa B). We examined the impact of PDTC preconditioning against cold ischemia and reperfusion injury in the rat liver. METHODS: Lewis rats were treated subcutaneously with saline or PDTC solution 24 hours before harvesting. Some animals pretreated with PDTC were also given zinc protoporphyrin IX intravenously immediately after reperfusion. HO-1 expression and enzyme activity in liver tissues were analyzed at different time points after each treatment. After transplantation of 24-hour preserved livers, serum levels of transaminases and gene expression of tumor necrosis factor-alpha, interleukin-1 beta, and NF-kappa B were measured. Animal survival and cellular viability were monitored. RESULTS: HO-1 gene expression and protein synthesis were enhanced in PDTC-treated livers, leading to increased enzyme activity (P <.05). The PDTC treatment group showed lower transaminase levels (P <.05), lower cytokine and NF-kappa B messenger RNA expression (P <.05), and fewer nonviable cells (P <.05) than did the control group, whereas these PDTC effects were abolished with zinc protoporphyrin injection after reperfusion (P <.05). The best animal survival rate was observed in the PDTC group (P <.05). CONCLUSION: PDTC preconditioning reduces inflammatory responses during reperfusion. PDTC appears to exert this protective effect by induction of an antioxidative stress protein and inhibition of proinflammatory cytokines.

Preconditioning with tin-protoporphyrin IX attenuates ischemia/reperfusion injury in the rat kidney.

BACKGROUND: Heme oxygenase (HO)-1 is induced as a unique stress response and leads to a transient resistance against oxidative damage, including ischemia and reperfusion (I/R) injury. In the present study, we examined whether HO-1 induction may confer a protection against I/R injury in the rat kidney. METHODS: Lewis rats were divided into four groups as follows: (1) vehicle group; (2) group treated with ferri-protoporphyrin IX (hemin), an inducer of HO; (3) group treated with low-dose tin-protoporphyrin IX (SnPP), an inhibitor of HO; and (4) group treated with high-dose SnPP. Renal warm ischemia for 60 minutes was performed 24 hours after each treatment. RESULTS: At 24 hours after treatment, hemin induced a significant increase in renal HO activity, but failed to induce HO-1 protein synthesis. Although both low- and high-dose SnPP reduced HO activity, a marked HO-1 expression was observed only in the high-dose SnPP-treated kidney. Hemin exacerbated the renal function after reperfusion, while high-dose SnPP significantly suppressed the intercellular adhesion molecule (ICAM)-1 expression, the infiltration of ED-1-positive macrophages and the expression of activated caspase-3, which resulted in attenuation of apoptotic cell death and ameliorated I/R injury. CONCLUSION: These results suggest that prior induction of HO-1 protein by high-dose SnPP may lead to anti-inflammatory and antiapoptotic effects on warm renal I/R injury independently of its enzyme activity, and that HO enzyme activation may not always act as an antioxidant, especially under I/R-induced oxidative stress.