Yuji Iimuro

NFkappaB prevents apoptosis and liver dysfunction during liver regeneration.

Although NFkappaB binding activity is induced during liver regeneration after partial hepatectomy, the physiological consequence of this induction is unknown. We have assessed the role of NFkappaB during liver regeneration by delivering to the liver a superrepressor of NFkappaB activity using an adenoviral vector expressing a mutated form of IkappaBalpha. This adenovirus (Ad5IkappaB) was almost exclusively expressed in the liver and inhibited NFkappaB DNA binding activity and transcriptional activity in cultured cells as well as in the liver in vivo. After partial hepatectomy, infection with Ad5IkappaB, but not a control adenovirus (Ad5LacZ), resulted in the induction of massive apoptosis and hepatocytes as demonstrated by histological staining and TUNEL analysis. In addition, infection with Ad5IkappaB but not Ad5LacZ decreased the mitotic index after partial hepatectomy. These two phenomena, increased apoptosis and failure to progress through the cell cycle, were associated with liver dysfunction in animals infected with the Ad5IkappaB but not Ad5LacZ, as demonstrated by elevated serum bilirubin and ammonia levels. Thus, the induction of NFkappaB during liver regeneration after partial hepatectomy appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

NF-κB inactivation converts a hepatocyte cell line TNF-α response from proliferation to apoptosis

Toxins convert the hepatocellular response to tumor necrosis factor-α (TNF-α) stimulation from proliferation to cell death, suggesting that hepatotoxins somehow sensitize hepatocytes to TNF-α toxicity. Because nuclear factor-κB (NF-κB) activation confers resistance to TNF-α cytotoxicity in nonhepatic cells, the possibility that toxin-induced sensitization to TNF-α killing results from inhibition of NF-κB-dependent gene expression was examined in the RALA rat hepatocyte cell line sensitized to TNF-α cytotoxicity by actinomycin D (ActD). ActD did not affect TNF-α-induced hepatocyte NF-κB activation but decreased NF-κB-dependent gene expression. Expression of an IκB superrepressor rendered RALA hepatocytes sensitive to TNF-α-induced apoptosis in the absence of ActD. Apoptosis was blocked by caspase inhibitors, and TNF-α treatment led to activation of caspase-2, caspase-3, and caspase-8 only when NF-κB activation was blocked. Although apoptosis was blocked by the NF-κB-dependent factor nitric oxide (NO), inhibition of endogenous NO production did not sensitize cells to TNF-α-induced cytotoxicity. Thus NF-κB activation is the critical intracellular signal that determines whether TNF-α stimulates hepatocyte proliferation or apoptosis. Although exogenous NO blocks RALA hepatocyte TNF-α cytotoxicity, endogenous production of NO is not the mechanism by which NF-κB activation inhibits this death pathway.

Management of Adrenal Metastasis from Hepatocellular Carcinoma

Abstract.Purpose: Although the adrenal gland is a common site of extrahepatic metastasis from hepatocellular carcinoma (HCC), there are no definitive guidelines for the treatment of adrenal metastasis. This study examines the effectiveness of various treatments for this disease. Methods: We retrospectively analyzed 20 patients treated for adrenal metastasis of HCC by adrenalectomy (n = 13), transarterial chemoembolization (TACE), or percutaneous ethanol injection therapy (PEIT) (n = 7). Results: There were no significant differences in cumulative survival rates between patients given adrenalectomy and those given TACE or PEIT, either after completing treatment for primary HCC or after the first treatment for adrenal metastasis. Six of seven patients with tumor thrombi in the inferior vena cava (IVC) from adrenal metastasis underwent adrenalectomy combined with intracaval thrombectomy, five of whom survived for more than 1 year after surgery, and two of whom are still alive without any recurrence more than 3 years after surgery. PEIT showed good results for small adrenal metastasis. Conclusion: These findings suggest that therapeutic modalities should be chosen according to the clinical features of each individual, including the size of the metastatic tumor, whether there is invasion into the IVC, the function of the remaining liver, and the existence of intra- and/or nonadrenal extrahepatic lesions. Furthermore, intracaval tumor thrombectomy could be indicated for patients with IVC thrombus if they are suitable candidates for surgery.

Regulation of cultured rat hepatocyte proliferation by stellate cells

BACKGROUND/AIMS This study using primary culture models was aimed to reveal the stellate cell-derived factors that regulate hepatocyte proliferation. METHODS Rat hepatocytes and stellate cells were cultured in serum-free Williams-E medium. We prepared hepatocyte mono-culture and two different co-cultures of hepatocytes and stellate cells; (1) co-culture on the same surface (Co-mix.) and (2) co-culture without contact between hepatocytes and stellate cells using a culture insert (Co-sep.). The change in the number and the DNA synthesis of hepatocytes was evaluated. RESULTS The number of hepatocytes decreased to 76% of the original number after 48 h of starting mono-culture, while it remained at 106% in mixed co-culture (Co-mix.) and increased to 135% in separated co-culture (Co-sep.). The hepatocyte DNA synthesis was enhanced by carbenoxolone in Co-mix. and reduced by NK1 in each co-culture. PD153035 had no effect. Heparitinase-I (20 mU/ml) and sodium chrolate (25 mM) reduced the hepatocyte DNA synthesis in Co-sep. to 71.8 and 61.6%, respectively. Activation of mitogen-activated protein kinase was induced in hepatocytes stimulated by conditioned mediums. CONCLUSIONS Hepatocyte proliferation was stimulated in the presence of stellate cells through hepatocyte growth factor, extracellular heparan sulfate (HS), and HS proteoglycan, and might be negatively regulated by gap junction-dependent mechanism.

Inactivation of the small GTPase Rac1 protects the liver from ischemia/reperfusion injury in the rat.

BACKGROUND In ischemia/reperfusion (I/R) injury, a massive generation of reactive oxygen species (ROS) after reperfusion is a critical factor. Rac, a member of the Rho GTPase superfamily, plays important roles in the production of ROS and activation of nuclear factor-kappaB (NF-kappaB) in vitro. However, the exact role of Rac in the ROS production and NF-kappaB activation in vivo after I/R is still obscure. METHODS We blocked Rac1 activity in the rat liver using adenovirus encoding a dominant negative rac1 mutant (Ad5N17Rac1) and examined whether inactivation of Rac1 could prevent ROS generation in the hepatic I/R injury. Seventy-two hours after the adenoviral infection, hepatic I/R was induced by Pringles maneuver for 20 minutes, followed by reperfusion in the rats. RESULTS Ad5N17Rac1 infection significantly attenuated ROS production after reperfusion and suppressed the hepatic injury. Furthermore, N17Rac1 suppressed NF-kappaB activation and messenger RNA expression of tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthetase (iNOS). Ad5LacZ, a control adenovirus, had no effect on the induced hepatic I/R injury, nor did it affect NF-kappaB activation. Immunohistochemical analysis of NF-kappaB (p65) revealed that translocation of p65 to the nucleus after reperfusion was blocked in many of non-parenchymal cells (NPCs) and in hepatocytes in the Ad5N17Rac1-infected liver. CONCLUSION We conclude that Rac1 is required in ROS generation and NF-kappaB activation after hepatic I/R in vivo, and that inactivation of NF-kappaB in NPCs and suppression of ROS generation in NPCs and hepatocytes possibly account for the protective effect of N17Rac1 in this study.

Percutaneous Radiofrequency Ablation Therapy after Intrathoracic Saline Solution Infusion for Liver Tumor in the Hepatic Dome

Two liver tumors undetected by ultrasonography (US) because they were located in the hepatic dome were treated with radiofrequency (RF) ablation therapy after intrathoracic saline solution infusion. After administration of local anesthesia, artificial pneumothorax was produced by needle thoracentesis and a drainage catheter was inserted into the right thoracic cavity. After saline solution (450-500 mL) was injected into the thoracic cavity via the catheter, US-guided RF ablation was performed. No severe complications occurred and complete therapeutic effects were obtained. Percutaneous RF ablation therapy with intrathoracic saline solution injection seems to be a feasible alternative to other ablation therapies.

Effect of Kupffer cell inactivation on ethanol-induced protein adducts in the liver.

Tissue deposition of protein adducts derived from ethanol metabolism and lipid peroxidation, has been suggested to play a role in the initiation of alcoholic liver disease. The mechanisms modulating adduct formation have, however, remained unclear. We used immunohistochemical methods to examine acetaldehyde (AA) and malondialdehyde (MDA) adducts and cytochrome P4502E1 and P4503A2 expression in rats after administration of (i) an ethanol-diet (n = 6), (ii) ethanol-diet plus gadolinium chloride (GdCl(3)), a selective Kupffer cell toxicant (n = 7), or (iii) control diet (n = 6). A 4 week ethanol treatment resulted in liver steatosis, necrosis, and inflammation and deposition of protein adducts with both AA and MDA, which colocalized with areas of fatty change. The intensities (mean +/- SD) of the immunohistochemical reactions for both AA (2.50 +/- 1.23) and MDA (3.00 +/- 1.10) adducts were significantly higher in the ethanol-fed animals than in the controls (0.083 +/- 0.20) (0.16 +/- 0.25) (p <.001). GdCl(3) prevented adduct accumulation, the mean immunohistochemistry scores being 0.86 +/- 1.07 for AA and 1.64 +/- 0.63 for MDA, the former showing a more striking reduction (p <.01). The hepatic cytochrome enzymes were not different in the ethanol-fed groups with or without GdCl(3). The data indicates that Kupffer cells are involved in the generation of protein adducts with both acetaldehyde and ethanol-induced lipid peroxidation products in alcoholic liver disease.

Continuous intravenous infusion of deleted form of hepatocyte growth factor attenuates hepatic ischemia–reperfusion injury in rats

BACKGROUND/AIMS Although beneficial roles of hepatocyte growth factor (HGF) and its variants on several hepatic disorders have been reported, their effects on hepatic ischemia-reperfusion (IR) injury remain undetermined. We investigated the action of a deleted form of HGF (dHGF) on hepatic IR injury in rats. METHODS dHGF or phosphate-buffered saline was continuously infused intravenously for 20 h prior to a 20-min occlusion of hepatic vessels. Samples were taken before and after IR, for measurement of serum dHGF and released enzymes, liver gamma-glutamylcysteinyl glycine (GSH) level, as well as histological and immunohistochemical examinations. RESULTS After reperfusion, histological injury, as well as increase in the serum activities of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatine kinase-BB were significantly attenuated in the dHGF-treated rats. dHGF maintained a high GSH level and suppressed oxidative stress and intercellular adhesion molecule-1 (ICAM-1) expression on sinusoidal endothelial cells (SECs), on which c-Met was not detected. IR caused activation of c-Met expression, which was milder in the dHGF-treated group, in hepatocytes at the pericentral region. CONCLUSIONS dHGF attenuated liver injury after IR. It also maintained a higher GSH level, depressed oxidative stress and inhibited ICAM-1 expression on c-Met negative SECs, suggesting a paracrine effect of dHGF.

Increased expression of collagenase in the liver induces hepatocyte proliferation with cytoplasmic accumulation of β-catenin in the rat

BACKGROUND/AIMS Since the hepatic extracellular matrix is remodeled in liver regeneration, we investigated whether increased collagenase activity in the liver can induce hepatocyte proliferation in vivo. METHODS To increase hepatic collagenase activity, human matrix metalloproteinase-1 was delivered to the rat liver by the recombinant adenoviral vector Ad5MMP-1. RESULTS Hepatic delivery of Ad5MMP-1 increased the 5-bromo-2-deoxyuridine labeling index and mitotic index in hepatocytes, causing an increase in the dry liver weight; control adenovirus, Ad5LacZ, had minimal effect. Hepatocyte proliferation started approximately 48 h after infection with Ad5MMP-1 and ended after about 2 weeks. The increase in the dry liver weight also returned to baseline after 2 weeks. Transient liver injury by Ad5MMP-1, reflected by increased aspartate and alanine aminotransferase levels, peaked around 1 week, and was associated with hepatocyte apoptosis. Collagenase-induced hepatocyte proliferation was accompanied by cytoplasmic accumulation of beta-catenin and a transient decrease in E-cadherin expression. CONCLUSIONS Modification of the hepatic extracellular matrix by collagenase induces transient hepatocyte proliferation in vivo, suggesting that the condition of the hepatic extracellular matrix per se plays a pivotal role in regulating hepatocyte proliferation.