Kengo Kawai

Lipopolysaccharide triggered TNF-α-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model ☆

BACKGROUND/AIMS Endogenous gut-derived bacterial endotoxins have been implicated as an important cofactor in the pathogenesis of liver injury, although their contribution to the progression of non-alcoholic steatohepatitis (NASH) remains unclear. METHODS Male C57BL/6 mice were fed a methionine-choline-deficient (MCD) diet or a standard diet for 17 days, following which they were injected with lipopolysaccharide (LPS) intraperitoneally and sacrificed after 6h. In an in vitro experiment, RAW264.7 cells, a mouse macrophage cell line, and primary mouse hepatocytes were co-treated with hydrogen peroxide (H(2)O(2)) and LPS or tumour necrosis factor (TNF)-alpha. RESULTS Compared to the control mice, LPS treatment significantly increased hepatic TNF-alpha production in MCD mice. LPS also significantly increased TUNEL-positive cells, which were especially observed in the perivenular area. The apoptotic change was inhibited by co-treatment with a neutralizing anti-mouse TNF receptor antibody or pentoxifylline. In an in vitro experiment, treatment with H(2)O(2) synergistically enhanced LPS-induced TNF-alpha production in RAW264.7 cells, accompanied by an up-regulation of CD14 mRNA. Moreover, co-treatment with TNF-alpha- and H(2)O(2)-induced apoptosis in primary hepatocytes, although neither TNF-alpha nor H(2)O(2) could do so independently. CONCLUSIONS LPS up-regulated TNF-alpha production, which induced hepatocyte apoptosis in a murine NASH model. LPS may play a key role in the pathogenesis of NASH.

Telmisartan attenuates progression of steatohepatitis in mice: role of hepatic macrophage infiltration and effects on adipose tissue.

Background/Aims: Non‐alcoholic fatty liver disease and non‐alcoholic steatohepatitis (NASH) are the hepatic manifestation of metabolic syndrome. However, its therapeutic strategy has not been established. Recently, an angiotensin II type 1 receptor blocker, telmisartan (Tel), has received a great deal of attention as a therapeutic tool for metabolic syndrome. The aim of this study was to investigate the efficacy and mechanisms of Tel on a murine NASH model.

Tumor necrosis factor-α accelerates apoptosis of steatotic hepatocytes from a murine model of non-alcoholic fatty liver disease

Non-alcoholic steatohepatitis (NASH) develops in a subset of patients with non-alcoholic fatty liver disease (NAFLD), but the exact mechanisms involved in the progression of NAFLD to NASH remain poorly understood. We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the apoptosis of hepatocytes that is related to the severity of NASH. We separated primary hepatocytes from the NAFLD liver caused by a high-fat diet. The production of intracellular reactive oxygen species was increased in steatotic hepatocytes, which were also sensitive to TNF-alpha. This factor induced significant apoptosis through the signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) pathway. We describe here a novel culture model of steatotic hepatocytes separated from the NAFLD liver, and demonstrate that TNF-alpha induces their apoptosis in vitro.

IFN-γ deficiency attenuates hepatic inflammation and fibrosis in a steatohepatitis model induced by a methionine- and choline-deficient high-fat diet

Cytokines play important roles in all stages of steatohepatitis, including hepatocyte injury, the inflammatory response, and the altered function of sinusoidal cells. This study examined the involvement of a major inflammatory cytokine, interferon-γ (IFN-γ), in the progression of steatohepatitis. In a steatohepatitis model by feeding a methionine- and choline-deficient high-fat (MCDHF) diet to both wild-type and IFN-γ-deficient mice, the liver histology, expression of genes encoding inflammatory cytokines, and fibrosis-related markers were examined. To analyze the effects of IFN-γ on Kupffer cells in vitro, we examined the tumor necrosis factor-α (TNF-α) production by a mouse macrophage cell line. Forty two days of MCDHF diet resulted in weight loss, elevated aminotransferases, liver steatosis, and inflammation in wild-type mice. However, the IFN-γ-deficient mice exhibited less extensive changes. RT-PCR revealed that the expression of tumor necrosis factor-α (TNF-α), transforming growth factor-β, inducible nitric oxide synthase, interleukin-4 and osteopontin were increased in wild-type mice, although they were suppressed in IFN-γ-deficient mice. Seventy days of MCDHF diet induced much more liver fibrosis in wild-type mice than in IFN-γ-deficient mice. The expression levels of fibrosis-related genes, α-smooth muscle actin, type I collagen, tissue inhibitor of matrix metalloproteinase-1, and matrix metalloproteinase-2, were dramatically increased in wild-type mice, whereas they were significantly suppressed in IFN-γ-deficient mice. Moreover, in vitro experiments showed that, when RAW 264.7 macrophages were treated with IFN-γ, they produced TNF-α in a dose-dependent manner. The present study showed that IFN-γ deficiency might inhibit the inflammatory response of macrophages cells and subsequently suppress stellate cell activation and liver fibrosis. These findings highlight the critical role of IFN-γ in the progression of steatohepatitis.

Development of osteomalacia in a post-liver transplant patient receiving adefovir dipivoxil

We report the case of a patient treated with living donor-related liver transplantation who suffered from osteomalacia during adefovir dipivoxil (ADV)-containing antiviral therapy for lamivudine-resistant hepatitis B virus infection. The patient had generalized bone pain, with severe hypophosphatemia after 20 mo of ADV therapy. Radiographic studies demonstrated the presence of osteomalacia. The peak plasma ADV level was 38 ng/mL after administration of ADV at 10 mg/d. It was also found that ADV affected the metabolism of tacrolimus, a calcineurin-inhibitor, and caused an increase in the plasma levels of tacrolimus. The disability was reversed with the withdrawal of ADV and with mineral supplementation. ADV can cause an elevation of plasma tacrolimus levels, which may be associated with renal dysfunction. High levels of ADV and tacrolimus can cause nephrotoxicity and osteomalacia. This case highlights the importance of considering a diagnosis of osteomalacia in liver transplantation recipients treated with both ADV and tacrolimus.

Theaflavin attenuates ischemia–reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.

Metron factor‐1 prevents liver injury without promoting tumor growth and metastasis

Hepatocyte growth factor (HGF) is the most powerful hepatotrophic factor identified so far. However, the ability of HGF to promote tumor cell “scattering” and invasion raises some concern about its therapeutic safety. We compared the therapeutic efficacy of HGF with that of Metron Factor‐1 (MF‐1), an engineered cytokine derived from HGF and the HGF‐like factor macrophage stimulating protein (MSP), in mouse models of acute and chronic liver injury. At the same time, we tested the ability of HGF and MF‐1 to promote tumor growth, angiogenesis, and invasion in several mouse models of cancer. We show that (1) MF‐1 and HGF stimulate hepatocyte proliferation in vitro; (2) MF‐1 and HGF protect primary hepatocytes against Fas‐induced and drug‐induced apoptosis; (3) HGF but not MF‐1 induces scattering and matrigel invasion of carcinoma cell lines in vitro; (4) HGF but not MF‐1 promotes migration and extracellular matrix invasion of endothelial cells in vitro; (5) MF‐1 and HGF prevent CCl4‐induced acute liver injury as measured by alanine aminotransferase (ALT) levels, histology, terminal deoxynucleotidyl transferase‐mediated nick‐end labeling (TUNEL) analysis, and phospho‐histone‐3 immunostaining; (6) MF‐1 and HGF attenuate liver fibrosis caused by chronic CCl4 intoxication and promote regeneration as measured by Sirius red staining, alpha‐smooth muscle actin immunostaining, and Ki‐67 analysis; (7) HGF but not MF‐1 promotes tumor growth, angiogenesis, and metastasis in a variety of xenograft models; (8) HGF but not MF‐1 promotes intrahepatic dissemination of hepatocarcinoma cells injected orthotopically. Conclusion: These data suggest that MF‐1 is as effective as HGF at preventing liver injury and at promoting hepatocyte regeneration, but therapeutically safer than HGF because it lacks proangiogenic and prometastatic activity. (HEPATOLOGY 2008;47:2010–2025.)

Neutrophil/lymphocyte ratio as a prognostic indicator of hepatic arterial infusion chemotherapy with arterial cisplatin plus continuous 5-fluorouracil.

Hepatic arterial infusion (HAIC) therapy may be a therapeutic option for advanced hepatocellular carcinoma (HCC) in addition to administration of sorafenib, which is the only currently established standard regimen for this disease. Survival benefit of HAIC has been reported in patients positive for antitumor response. Therefore, the prediction of antitumor response is important in decision‐making for HAIC treatment.

Matrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in the injured liver.

Effective mobilization of hematopoietic stem cells (HSCs) in injured organs has not been established. Matrix metalloproteinase-9 (MMP-9) is known to release HSCs from bone marrow (BM) into the peripheral blood, but its role in the recruitment of HSCs to injured organs is unclear. In this study we tried to clarify the role of the host MMP-9 in trafficking of HSCs toward the injured liver, especially the relation of MMP-9 with the chemokine receptor 4 (CXCR4)–chemokine ligand 12 (CXCL12) axis, and to examine whether MMP-9 deficiency affects BM cell trafficking to the injured liver in mice. In vitro, we investigated the effect of MMP-9 on migration activity and CXCR4 expression on lineage-negative (Lin-) BM cells. In vivo, we induced acute and chronic liver injury in MMP-9 knockout (KO) and control mice by inoculation of carbon tetrachloride, followed by transplantation of Lin- BM cells obtained from enhanced green fluorescent protein (EGFP)-transgenic mice, and counted the BM cells mobilized in the injured liver. In a migration assay, active MMP-9, but not proMMP-9, increased the number of migrated Lin- BM cells, which was inhibited by tissue inhibitor of metalloproteinase-1 or a MMP inhibitor. This chemoattractant function by MMP-9 was synergistic when cotreated with CXCL12. CXCR4 expression on Lin- BM cells was dose- and time-dependently increased by active MMP-9. At the same time, treatment with MMP-9 enhanced CXCL12 expression, and CXCL12 reciprocally increased MMP-9 expression in BM cells. In in vivo studies, many EGFP+ cells were seen in control recipient mice. In contrast, few EGFP+ cells were observed in MMP-9 KO mice. BM cells tended to differentiate into desmin+ cells. In conclusion, MMP-9 contributes to the mobilization of BM cells in the injured liver by upregulating the expression of CXCR4 on Lin- BM cells and attracting BM cells along its gradient of CXCL12. Therefore, host MMP-9 plays an important role in BM cell migration in the injured liver.

A case of primary biliary cirrhosis that progressed rapidly after treatment involving rituximab.

Primary biliary cirrhosis (PBC) is a progressive liver disease for which limited therapies are recommended. Rituximab, an anti-CD20 monoclonal antibody, is expected to be a useful therapeutic regimen for PBC. Previous studies indicated biochemical and immunological improvement in PBC after rituximab treatment. Although rituximab shows therapeutic potential for PBC, few cases have been reported and histological improvement and long-term outcome remain uncertain. Here, we report a case of PBC in a 66-year-old Japanese female patient who presented with a gastric lymphoma and who had been treated with a regimen containing rituximab for incidental malignant lymphoma. She showed biochemical and immunological improvements, and liver histology before and after rituximab treatment confirmed a decrease in liver inflammation. However, she developed liver cirrhosis a short time after rituximab treatment without biochemical or immunological worsening. Rituximab treatment for PBC might be considered and careful observation is required after treatment.