Sei-ichiro Tsuchihashi

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°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 Suzukis grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)‐γ‐inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM‐1), as well as tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐2, and IFN‐γ, 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. Liver Transpl 13:1435–1443, 2007.

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-&bgr; to stimulate CXCL-10 production via TLR4 pathway in vitro. Indeed, CoPP-induced HO-1 suppressed otherwise highly upregulated rIFN-&bgr;-triggered CXCL-10. Moreover, consistent with our in vitro data, CoPP pretreatment diminished rIFN-&bgr;-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.

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-α, IL-1β, IL-6, IFN-γ, 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.

Cyclooxygenase-2 Deficiency Enhances Th2 Immune Responses and Impairs Neutrophil Recruitment in Hepatic Ischemia/Reperfusion Injury

Cyclooxygenase-2 (COX-2) is a prostanoid-synthesizing enzyme that is critically implicated in a variety of pathophysiological processes. Using a COX-2-deficient mouse model, we present data that suggest that COX-2 has an active role in liver ischemia/reperfusion (I/R) injury. We demonstrate that COX-2-deficient mice had a significant reduction in liver damage after I/R insult. The inability of COX-2−/− to elaborate COX-2 products favored a Th2-type response in these mice. COX-2−/− livers after I/R injury showed significantly decreased levels of IL-2, as well as IL-12, a cytokine known to have a central role in Th1 effector cell differentiation. Moreover, such livers expressed enhanced levels of the anti-inflammatory cytokine IL-10, shifting the balance in favor of a Th2 response in COX-2-deficient mice. The lack of COX-2 expression resulted in decreased levels of CXCL2, a neutrophil-activating chemokine, reduced infiltration of MMP-9-positive neutrophils, and impaired late macrophage activation in livers after I/R injury. Additionally, Bcl-2 and Bcl-xL were normally expressed in COX-2−/− livers after injury, whereas respective wild-type controls were almost depleted of these two inhibitors of cell death. In contrast, caspase-3 activation and TUNEL-positive cells were depressed in COX-2−/− livers. Therefore, our data support the concept that COX-2 is involved in the pathogenic events occurring in liver I/R injury. The data also suggest that potential valuable therapeutic approaches in liver I/R injury may result from further studies aimed at identifying specific COX-2-derived prostanoid pathways.

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-α, IL-6, and IFN-γ) 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.

Inducible nitric oxide synthase deficiency impairs matrix metalloproteinase-9 activity and disrupts leukocyte migration in hepatic ischemia/reperfusion injury.

Matrix metalloproteinase 9 (MMP-9) is a critical mediator of leukocyte migration in hepatic ischemia/reperfusion (I/R) injury. To test the relevance of inducible nitric oxide synthase (iNOS) expression on the regulation of MMP-9 activity in liver I/R injury, our experiments included both iNOS-deficient mice and mice treated with ONO-1714, a specific iNOS inhibitor. The inability of iNOS-deficient mice to generate iNOS-derived nitric oxide (NO) profoundly inhibited MMP-9 activity and depressed leukocyte migration in livers after I/R injury. While macrophages expressed both iNOS and MMP-9 in damaged wild-type livers, neutrophils expressed MMP-9 and were virtually negative for iNOS; however, exposure of isolated murine neutrophils and macrophages to exogenous NO increased MMP-9 activity in both cell types, suggesting that NO may activate MMP-9 in leukocytes by either autocrine or paracrine mechanisms. Furthermore, macrophage NO production through the induction of iNOS was capable of promoting neutrophil transmigration across fibronectin in a MMP-9-dependent manner. iNOS expression in liver I/R injury was also linked to liver apoptosis, which was reduced in the absence of MMP-9. These results suggest that MMP-9 activity induced by iNOS-derived NO may also lead to detachment of hepatocytes from the extracellular matrix and cell death, in addition to regulating leukocyte migration across extracellular matrix barriers. These data provide evidence for a novel mechanism by which MMP-9 can mediate iNOS-induced liver I/R injury.

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°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‐α, interleukin 2, interferon‐γ) 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. (Liver Transpl 2005;11:1273–1281.)

FK330, a Novel Inducible Nitric Oxide Synthase Inhibitor, Prevents Ischemia and Reperfusion Injury in Rat Liver Transplantation

Nitric oxide (NO), produced via inducible NO synthase (iNOS), is implicated in the pathophysiology of liver ischemia/reperfusion injury (IRI). We examined the effects of a novel iNOS inhibitor, FK330 (FR260330), in well‐defined rat liver IRI models. In a model of liver cold ischemia followed by ex vivo reperfusion, treatment with FK330 improved portal venous flow, increased bile production and decreased hepatocellular damage. FK330 prevented IRI in rat model of 40‐h cold ischemia followed by syngeneic orthotopic liver transplantation (OLT), as evidenced by: (1) increased OLT survival (from 20% to 80%); (2) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic pyruvic transaminase levels); (3) improved histological features of IRI; (4) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P‐selectin/intracellular adhesion molecule 1, ED‐1/CD3 cells and neutrophils; (5) depressed lymphocyte activation, as evidenced by expression of pro‐inflammatory cytokine (TNF‐α, IL‐1β, IL‐6) and chemokine (IP‐10, MCP‐1, MIP‐2) programs; (6) prevented hepatic apoptosis and down‐regulated Bax/Bcl‐2 ratio. Thus, by modulating leukocyte trafficking and cell activation patterns, treatment of rats with FK330, a specific iNOS inhibitor, prevented liver IRI. These results provide the rationale for novel therapeutic approaches to maximize organ donor pool through the safer use of liver grafts despite prolonged periods of cold ischemia.

Diannexin, a novel annexin V homodimer, protects rat liver transplants against cold ischemia-reperfusion injury.

Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4°C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 μg/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF‐α, IL‐1β, IP‐10 expression, diminished expression of P‐selectin, endothelial ICAM‐1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO‐1/Bcl‐2/Bcl‐xl, and reduced Caspase‐3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool.

The membrane attack complex (C5b‐9) in liver cold ischemia and reperfusion injury

Activation of the complement cascade represents an important event during ischemia/reperfusion injury (IRI). This work was designed to investigate the role of the membrane attack complex (MAC; C5b‐9) in the pathogenesis of hepatic IRI. Livers from B&W/Stahl/rC6(+) and C6(−) rats were harvested, stored for 24 hours at 4°C, and then transplanted [orthotopic liver transplantation (OLT)] to syngeneic recipients. There were 4 experimental groups: (1) C6(+)→C6(+), (2) C6(+)→C6(−), (3) C6(−)→C6(+), and (4) C6(−)→C6(−). At day +1, C6(−) OLTs showed decreased vascular congestion/necrosis, contrasting with extensive necrosis in C6(+) livers, that was independent of the recipient C6 status (Suzuki score: 7.2 ± 0.9, 7.3 ± 1.3, 4.5 ± 0.6, and 4.8 ± 0.4 for groups 1‐4, respectively, P < 0.05). The liver function improved in recipients of C6(−) grafts (serum glutamic oxaloacetic transaminase: 2573 ± 488, 1808 ± 302, 1170 ± 111, and 1188 ± 184 in groups 1‐4, respectively, P < 0.05). Intragraft macrophage infiltration (ED‐1 immunostaining) and neutrophil infiltration (myeloperoxidase activity) were reduced in C6(−) grafts versus C6(+) grafts (P = 0.001); these data were confirmed by esterase staining (naphthol). The expression of proinflammatory interferon‐γ, interleukin‐1β, and tumor necrosis factor messenger RNA/protein was also reduced in C6(−) OLTs in comparison with C6(+) OLTs. Western blot–assisted expression of proapoptotic caspase‐3 was decreased in C6(−) OLTs versus C6(+) OLTs (P = 0.006), whereas antiapoptotic Bcl‐2/Bag‐1 was enhanced in C6(−) OLTs compared with C6(+) OLTs (P = 0.001). Terminal deoxynucleotidyl transferase–mediated dUTP nick end‐labeling staining of apoptotic cells was enhanced (P < 0.05) in C6(+) OLTs compared with C6(−) OLTs. Thus, the terminal products of the complement system are essential in the mechanism of hepatic IRI. This is the first report using a clinically relevant liver cold ischemia model to show that local MAC inhibition attenuates IRI cascade in OLT recipients. Liver Transpl 14:1133–1141, 2008.