Innate immune reactivity of the ileum-liver axis in nonalcoholic steatohepatitis.

BACKGROUND: Over-proliferation and bacterial translocation of Gram-negative bacilli within the intestinal flora, and increased portal venous levels of endotoxins, are involved in nonalcoholic steatohepatitis (NASH). AIM: To evaluate the innate immune response in the small intestine and liver using the rat NASH model. METHODS: We produced the NASH model by administering a choline-deficient amino acid-defined diet to F344 rats. We analyzed the serum and liver tissue to assess the effects of innate immune reactivity in this NASH model. RESULTS: Significant increases were detected in serum ALT levels and in the portal venous serum and whole-liver levels of TNF-α and IFN-γ in the NASH group. Strong Sirius red staining and TNF-α immune staining were seen in the NASH group, and real-time PCR revealed significantly increased expression of TNF-α and TLR4 mRNA in the NASH group. Higher TNF-α levels were detected in the Kupffer cells isolated culture supernatant in the NASH group than in the control group. Immune staining of the ileal tissue specimens resulted in greater staining of TNF-α, TLR4, and macrophage/dendritic cells, mainly in the submucosa, in the NASH group than in the control group. CONCLUSIONS: In the small intestine and liver of the rat NASH model, the possibility that enhancement of the innate immune response, mediated by the TLR4 signal, led to increased production of TNF-α was suggested. This interaction between the small intestine and liver may be involved in the onset and progression of NASH.

Therapeutic effects of cytokine modulator Y-40138 in the rat alcoholic liver disease model.

BACKGROUND AND AIM: Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ), induce liver injury in the rat alcoholic liver disease (ALD) model. Y-40138 is known to suppress the pro-inflammatory cytokines and augment the anti-inflammatory cytokines. We investigated whether or not Y-40138 may be effective as a novel immunotherapy in the rat ALD model. METHODS: Male Wistar rats were fed Lieber-DeCarli ethanol liquid diet. The effects of Y-40138 treatment in the ALD models were assessed by analyzing the serum and the liver tissues. RESULTS: The serum levels of alanine aminotransferase (ALT), TNF-α, and IFN-γ, and the liver levels of TNF-α and IFN-γ were significantly higher in the ethanol-fed group than in the pair-fed group. The immunohistochemistry of the liver TNF-α and 4-hydroxynonenal (4HNE), and the expressions of TNF-α and IFN-γ mRNA were increased, too. The gene expressions of interleukin-10 (IL-10) in the ethanol-fed group were suppressed as compared with the pair-fed group. The serum triglyceride (TG) and liver TG were increased, and Oil Red O and α-smooth muscle actin (α-SMA) staining showed greater expression by ethanol-fed feeding. After administration of Y-40138, enzyme linked immunosorbent assay and real-time polymerase chain reaction of the liver showed that the increased TNF-α and IFN-γ were suppressed, and that IL-10 was augmented. Moreover, ethanol-induced lipid accumulation in the liver was suppressed by administering Y-40138. CONCLUSIONS: Y-40138 decreased the inflammation, fibrosis, oxidative stress, and lipid synthesis, and augmented the anti-inflammatory cytokines of the liver. These results indicate that the multiple cytokine production modulator, Y-40138, is a promising novel therapy for ALD.

Rapid detection of spontaneous bacterial peritonitis by granulocyte elastase latex immunoassay and reagent strip.

Spontaneous bacterial peritonitis (SBP) is a serious complication in patients with liver cirrhosis that requires rapid recognition for effective antibiotic therapy. Elevated levels of granulocyte elastase (GE), an enzyme that is released from degranulated polymorphonuclear neutrophils(PMN), have been reported in ascitic fluid of SBP patients. The aim of this study was to assess the utility of GE measurement by a latex immunoassay (LIA) and by reagent strips for rapid diagnosis of SBP. In 26 ascitic samples which had differing GE concentrations, the results of this LIA method closely correlated with those of a GE/alpha1-PI complex ELISA and an EIA using monoclonal antibodies against GE. The evaluation parameters of linearity (r > 0.99), analytical recovery (96-107%) and within-assay variation[coefficient of variation(CV): 0.97-2.35%] were found to be satisfactory. In 58 ascitic samples from patients with liver cirrhosis, GE levels confirmed by LIA in SBP ascites (n=14) at the time of diagnosis were higher (1436.9 +/- 715.1 ng/ml) than those in non SBP ascites (n=44)(13.1 +/- 3.9 ng/ml). The receiver operating characteristic (ROC) curve showed that ascitic GE by LIA enabled discrimination between SBP and non-SBP, and a cut-off value of 49.5 ng/ml had a sensitivity of 85.7% and specificity of 97.7%. In addition, the usefulness of reagent strips designed for testing cervical mucus for rapid bedside detection of SBP was assessed for GE. The sensitivity, specificity, and positive and negative predictive values of the reagent strips for diagnosis of SBP were 92.9%, 90.9%, 76.5%, and 97.6%, respectively. These results indicate that GE-LIA and GE reagent strips are rapid and sensitive and can aid diagnosis of SBP.

Potential role of enhanced cytokinemia and plasma inhibitor on the decreased activity of plasma ADAMTS13 in patients with alcoholic hepatitis: relationship to endotoxemia.

BACKGROUND: Deficiency of ADAMTS13 (adisintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13) results in an increase in unusually large von Willebrand factor multimer (UL-VWFM) of the plasma and finally causes microcirculatory disturbance. Our previous study demonstrated that the imbalance of increased UL-VWFM over decreased ADAMTS13 activity may contribute to the development of multiorgan failure in patients with alcoholic hepatitis (AH). The aim of this study was to explore the potential mechanism to reduce the activity of plasma ADAMTS13. METHODS: Plasma cytokine levels including interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha), plasma endotoxin concentration, and the plasma inhibitor against ADAMTS13 were determined together with ADAMTS13 activity, VWF antigen (VWF:Ag), and UL-VWFM in 24 patients with AH and 5 patients with severe alcoholic hepatitis (SAH). RESULTS: The concentrations of IL-6, IL-8, and TNF-alpha on admission were significantly higher in patients with SAH than in those with AH and controls. The ADAMTS13 activity concomitantly decreased, and the VWF:Ag progressively elevated with increasing concentrations of these cytokines from normal range to over 100 pg/ml. Plasma endotoxin concentration was markedly higher in patients with SAH (mean 52.3 pg/ml) and AH (21.7 pg/ml) than in controls (7.9 pg/ml). The endotoxin concentration inversely correlated with ADAMTS13 activity and was higher in patients with UL-VWFM than those without. The inhibitor was detected in 4 patients with SAH (0.9 to 2.1 BU/ml) and 6 patients with AH (0.5 to 1.6 BU/ml). Patients with the inhibitor showed lower functional liver capacity, higher endotoxin concentration, and marked inflammatory signs than those without. At the recovery stage, the ADAMTS13 activity increased to normal range, the VWF:Ag decreased, and the UL-VWFM disappeared with the decrease in the concentrations of cytokines and endotoxin, and the disappearance of the inhibitor. CONCLUSION: Decreased ADAMTS13 activity and increased VWF:Ag could be induced not only by pro-inflammatory cytokinemia, but also by its inhibitor, both of which may be closely related to enhanced endotoxemia in patients with AH and SAH.

Combination of vitamin K2 and angiotensin-converting enzyme inhibitor ameliorates cumulative recurrence of hepatocellular carcinoma.

BACKGROUND/AIMS: No chemopreventive agent has been approved against hepatocellular carcinoma (HCC) yet. Since neovascularization plays a pivotal role in HCC, an angiostatic agent is considered as one of the promising approaches. The aim of this study was to elucidate the combined effect of the clinically used vitamin K(2) (VK) and angiotensin-converting enzyme inhibitor (ACE-I) on cumulative recurrence after curative treatment on a total of 87 patients, especially in consideration of neovascularization. METHODS: VK (menatetrenone; 45 mg/day) and/or ACE-I (perindopril; 4 mg/day) were administered for 36-48 months after curative therapy for HCC. The cumulative recurrence and several indices were analyzed. RESULTS: A 48-month follow-up revealed that the combination treatment with VK and ACE-I markedly inhibited the cumulative recurrence of HCC in association with suppression of the serum level of the vascular endothelial growth factor (VEGF); a central angiogenic factor. The serum level of lectin-reactive alpha-fetoprotein was also suppressed almost in parallel with VEGF. These beneficial effects were not observed with single treatment using VK or ACE-I. CONCLUSIONS: The combination treatment of VK and ACE-I may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the VEGF-mediated neovascularization.

Branched-chain amino acids suppress insulin-resistance-based hepatocarcinogenesis in obese diabetic rats.

BACKGROUND: Branched-chain amino acids (BCAAs) reportedly inhibit the incidence of hepatocellular carcinoma (HCC) in patients with liver cirrhosis and obesity that is frequently associated with insulin resistance (IR). However, the possible mechanism is still obscure. The aim of the present study was to examine the effect of BCAAs, especially in conjunction with angiogenesis, on hepatocarcinogenesis under the condition of IR. METHODS: The effect of BCAAs on the development of liver enzyme-altered preneoplastic lesions and angiogenesis was examined in obese diabetic Otsuka Long-Evans Tokushima Fatty rats. We also performed an in vitro study to elucidate the possible mechanisms involved. RESULTS: Treatment with BCAAs markedly inhibited glutathione-S-transferase placental form (GST-P)-positive preneoplastic lesions along with suppression of neovascularization in the liver. The hepatic expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, was also attenuated. BCAA treatment significantly suppressed glucose- and insulin-induced in vitro angiogenesis in the presence of VEGF. CONCLUSIONS: In obese diabetic rats BCAAs exerted a chemopreventive effect against HCC, associated with the suppression of VEGF expression and hepatic neovascularization. Since BCAA preparations are widely used in clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against HCC in the future.

Comprehensive analysis of ADAMTS13 in patients with liver cirrhosis.

Decreased plasma ADAMTS13 activity (ADAMTS13:AC) results in the accumulation of unusually large von Willebrand factor multimer (UL-VWFM) and the formation of platelet thrombi. It remains controversial whether or not plasma ADAMTS13:AC decreases in patients with liver cirrhosis (LC), and its relationship to clinical features has not been fully investigated. We measured ADAMTS13:AC and its related parameters in plasma in 33 patients with chronic hepatitis (CH) and in 109 patients with LC. ADAMTS13:AC decreased with increasing severity of liver disease (controls means 100%, CH 87%, Child A-LC 79%, Child B-LC 63%, and Child C-LC 31%), and showed severe deficiency (<3% of controls) in five end-stage LC. Activities measured by act-ELISA strongly correlated with those determined by the VWFM assay and ADAMTS13 antigen. Multivariate analysis showed Child-Pugh score and spleen volume independent factors contributing to ADAMTS13:AC. VWFM patterns were normal in 53% of cases, degraded in 31%, and unusually large in 16%. Patients with unusually large VWFM had the lowest ADAMTS13:AC as well as the highest Child-Pugh score, serum creatinine and blood ammonia levels. Plasma inhibitor against ADAMTS13 detected in 83% of patients with severe to moderate ADAMTS13:AC deficiency mostly showed marginal zone between 0.5 and 1.0 BU/ml. The IgG-type autoantibodies specific to plasma derived-ADAMTS13 was detected by Western blot in only five end-stage LC with severe ADAMTS13:AC deficiency. In conclusion, both plasma ADAMTS13 activity and antigen levels decreased with increasing severity of cirrhosis. An imbalance between the decreased ADAMTS13:AC and its increased substrate may reflect the predisposing state for platelet thrombi formation in patients with advanced LC.

Different tumoricidal effects of interferon subclasses and p53 status on hepatocellular carcinoma development and neovascularization.

Interferon (IFN) is known as a multifunctional cytokine. The aim of this study was to examine the different effects of IFN subclass; namely, IFN-alpha and IFN-beta, on hepatocellular carcinoma (HCC) growth especially in conjunction with angiogenesis that is known to play a pivotal role in the tumor growth. Furthermore, we also examined whether the p53 status in the tumor would alter the anti-tumoral effect of IFN against HCC growth since the p53 status reportedly affected the therapeutic effect of anti-angiogenic agents against cancer. When compared with IFN-alpha, IFN-beta exerted a more potent inhibitory effect on HCC growth, even after the tumor was established, along with suppression of neovascularization in the tumor. A single treatment with clinically comparable low doses of IFN-beta significantly inhibited HCC growth whereas the same dose of IFN-alpha did not. IFN-beta also significantly suppressed the tumor growth both in the p53-wild and p53-mutant HCC cells. Our in vitro study revealed that IFN-beta showed a more potent inhibitory effect on the endothelial cell proliferation than IFN-alpha as in the in vivo study. Collectively, IFN may be an alternative anti-angiogenic agent against HCC since it exerted a significant tumoricidal effect regardless of the host p53 status even at a low dose. A cautious approach may be also required in the clinical practice since even in a same IFN subclass (class-I), IFN-alpha and IFN-beta exert tumoricidal effects of different magnitudes on HCC.

Plasma ADAMTS13 activity parallels the APACHE II score, reflecting an early prognostic indicator for patients with severe acute pancreatitis.

OBJECTIVE: Severe acute pancreatitis (SAP) frequently progresses to pancreatitis-associated multiorgan failure (MOF) with high mortality. Decreased plasma ADAMTS13 activity (ADAMTS13:AC) results in the accumulation of unusually large von Willebrand factor multimers (UL-VWFM) and the formation of platelet thrombi, ultimately leading to MOF. The purpose of the study was to investigate the potential role of ADAMTS13:AC in the severity of SAP. MATERIAL AND METHODS: Plasma ADAMTS13:AC and its related parameters were sequentially determined in 13 SAP patients. ADAMTS13:AC was determined by the chromogenic act-ELISA. RESULTS: Within 1 or 2 days after admission, ADAMTS13:AC was lower in SAP patients (mean 28%) than in healthy controls (99%), and gradually recovered in the 11 survivors but further decreased in the 2 non-survivors. Patients with higher sepsis-related organ failure assessment (SOFA) scores showed lower ADAMTS13:AC than those without these scores. The inhibitor against ADAMTS13 was undetectable. On day 1, von Willebrand factor antigen (VWF:Ag) was higher (402%, p<0.001) in SAP patients than in controls (100%). VWF:Ag gradually decreased in the survivors, except in the 3 patients needing a necrosectomy, but remained high in the non-survivors. ADAMTS13:AC was inversely correlated with the APACHE II score (r=-0.750, p<0.005), and increased plasma concentrations of interleukin 6 (IL-6) and IL-8 at admission. UL-VWFM-positive patients had lower ADAMTS13:AC and decreased serum calcium concentrations, but higher VWF:Ag and IL-8 concentrations than UL-VWFM-negative patients. CONCLUSIONS: Plasma ADAMTS13:AC was closely related to the APACHE II score. This intimate relationship may serve as an early prognostic indicator for SAP patients. The imbalance between decreased ADAMTS13:AC and increased UL-VWFM could contribute to SAP pathogenesis through enhanced thrombogenesis.

Impact of insulin resistance on the progression of chronic liver diseases.

Recent studies have revealed a close relationship between insulin resistance (IR) and the progression of chronic liver diseases, although relatively little is known regarding the possible mechanisms involved. The aim of this study was to elucidate the impact of IR on the development of liver fibrosis and hepatocarcinogenesis using obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Liver fibrosis development and glutathione-S-transferase placental form (GST-P)-positive pre-neoplastic lesions were both markedly accelerated in OLETF rats, being induced by pig serum and diethylnitrosamine (DEN), respectively. In the fibrosis experiment, alpha-smooth muscle actin-positive activated hepatic stellate cells (HSCs) also significantly increased in OLETF rats along with augmentation of the hepatic collagen content and transforming growth factor-beta1. Our in vitro study showed that both glucose and insulin stimulated the proliferation of activated HSCs, and the combination treatment exerted an additive effect. In the DEN model, neovascularization, which plays a pivotal role in hepatocarcinogenesis, was up-regulated in OLETF rats almost in parallel with pre-neoplastic lesion development and a potent angiogenic factor, vascular endothelial growth factor. High glucose and insulin also significantly augmented the in vitro neovascularization via extracellular signal-regulated kinase 1/2 phosphorylation. Similar to the effect on the activated HSCs, co-existence of both factors exerted a more potent effect than either single factor. In conclusion, these results indicated that the IR status directly accelerated liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated HSC proliferation and hepatic neovascularization, respectively, in the rat.

The role of radixin in altered localization of canalicular conjugate export pump Mrp2 in cholestatic rat liver.

AIM: Cholestasis has been associated with the endocytic retrieval of multidrug resistance protein 2 (Mrp2), but its mechanism is still unclear. Recent studies have indicated that radixin, a cross-linker between the actin filaments and membrane proteins, may be activated by phosphorylation and may be required for the canalicular localization of Mrp2. METHODS: We investigated the role of radixin in the altered localization of Mrp2 in rat models of intrahepatic (ethinyl estradiol treatment) and extrahepatic (bile duct ligation) cholestasis using immunofluorescence microscopy. The changes in localization and expression were analyzed using Scion Image for Windows. RESULTS: In both models, Mrp2 was localized outside as well as inside the ZO-1 staining, indicating partial dislocation from the canalicular membrane. In contrast to the steep elevation of the immunostaining intensity curves for Mrp2 in the controls, the corresponding curves in both models were broadened and flattened, confirming endocytic retrieval into the hepatocytes. Mrp2 and radixin were colocalized at the canalicular domain in the controls, whereas in both cholestatic rats they were dissociated at some canaliculi, indicating the disturbed colocalization of Mrp2 and radixin in cholestasis. The fluorescence of phosphorylated radixin, an active form of radixin, markedly decreased in both cholestatic models, which was supported by the reduced peak fluorescence intensities. CONCLUSION: The disturbed colocalization of Mrp2 and radixin may contribute to the endocytic retrieval of Mrp2 in cholestasis due to the failure to anchor Mrp2 in the canalicular membrane, in which the phosphorylated radixin may play a major role.

Disturbed colocalization of multidrug resistance protein 2 and radixin in human cholestatic liver diseases.

BACKGROUND AND AIM: Endocytic retrieval of multidrug resistance protein 2 (MRP2) is closely associated with cholestasis and may be attributed to the disturbed linking of MRP2 and radixin, a cross-linker between actin filaments and membrane proteins. This study aimed to investigate the role of radixin in the altered localization of MRP2 in various human cholestatic liver diseases. METHODS: Using immunofluorescence microscopy, we investigated the localization and expression of MRP2 and radixin in various cholestatic liver diseases, such as drug-induced liver injury, obstructive jaundice, primary sclerosing cholangitis and autoimmune hepatitis. Changes in localization and expression were analyzed using Scion Image (software). RESULTS: In the icteric drug-induced liver injury, MRP2 was localized outside as well as inside of ZO-1 staining, indicating endocytic retrieval from the canalicular membrane into the pericanalicular compartments of the hepatocytes. The colocalization of MRP2 and radixin observed in the controls was disturbed, and MRP2 fluorescence disappeared in the canaliculi with disrupted radixin staining. Disturbed colocalization of MRP2 and radixin as well as endocytic retrieval of MRP2 was found in the poorly drained obstructive jaundice. When drainage was good, MRP2 was exclusively colocalized with radixin. Similar findings were observed in autoimmune hepatitis and primary sclerosing cholangitis. In the controls, the immunostaining intensity curves for MRP2 and radixin were steeply elevated in the canaliculi. The intensity curves for MRP2 and radixin were broadened in the icteric drug-induced liver injury and poorly drained obstructive jaundice, indicating endocytic retrieval into the hepatocytes. The peak fluorescence intensities for MRP2 and radixin decreased in the icteric liver. CONCLUSION: Disturbed colocalization of MRP2 and radixin was common in various cholestatic liver diseases, which may be associated with endocytic retrieval of MRP2 due to failure in anchoring MRP2 in the canalicular membrane.

Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model.

AIM: To investigate Kupffer cell dynamics and phagocytic activity, using a rat nonalcoholic steatohepatitis (NASH) model. METHODS: Male F344 rats were fed either a control diet or a choline-deficient L-amino acid-defined (CDAA) diet, followed by contrast enhanced ultrasonography (CEUS) using Levovist. The uptake of latex beads by the Kupffer cells was determined by fluorescent microscopy. The status of the Kupffer cells was compared between the two groups, using the immunohistochemical staining technique. RESULTS: After 4 or more wk of the CDAA diet, CEUS examination revealed a decrease in the signal intensity, 20 min after intravenous Levovist. Fluorescent microscopic examination showed that the uptake of latex beads by the Kupffer cells was reduced at week 1 and 2 in the study group, compared with the controls, with no further reduction after 3 wk. Immunohistochemical staining revealed no significant difference in the Kupffer cell counts between the control group and the CDAA group. CONCLUSION: CEUS examination using Levovist demonstrated reduced contrast effect and phagocytic activity in the liver parenchymal phase, although the Kupffer cell numbers were unchanged, indicating reduced phagocytic function of the Kupffer cells in the rat NASH model. We believe that CEUS examination using Levovist is a useful screening modality, which can detect NASH in fatty liver patients.

Innate immune reactivity of the liver in rats fed a choline-deficient L-amino-acid-defined diet.

AIM: To investigate the innate immune reactivity of tumor necrosis factor-alpha (TNF-alpha), Toll-like receptor 4 (TLR4), and CD14 in the liver of non-alcoholic steatohepatitis (NASH) model rats. METHODS: Male F344 rats were fed a choline-deficient L-amino-acid-defined (CDAA) diet. The rats were killed after 4 or 8 wk of the diet, and their livers were removed for immunohistochemical investigation and RNA extraction. The liver specimens were immunostained for TNF-alpha, TLR4, and CD14. The gene expressions of TNF-alpha, TLR4, and CD14 were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Kupffer cells were isolated from the liver by Percoll gradient centrifugation, and were then cultured to measure TNF-alpha production. RESULTS: The serum and liver levels of TNF-alpha in the CDAA-fed rats increased significantly as compared with the control group, as did the immunohistochemical values and gene expressions of TNF-alpha, TLR4, and CD14 with the progression of steatohepatitis. TNF-alpha production from the isolated Kupffer cells of the CDAA-fed rats was elevated by lipopolysaccharide stimulation. CONCLUSION: The expressions of TNF-alpha, TLR4, and CD14 increased in the NASH model, suggesting that TLR4 and CD14-mediated endotoxin liver damage may also occur in NASH.

Renin-angiotensin system inhibitors as therapeutic alternatives in the treatment of chronic liver diseases.

The renin-angiotensin system (RAS) is frequently activated in the patients with chronic liver diseases, and recent studies have shown that RAS plays a pivotal role in the progression of chronic liver diseases, i.e., liver fibrosis and hepatocellular carcinoma (HCC). Angiotensin-II (AT-II) reportedly stimulates contractility and proliferation of the activated hepatic stellate cells, and increases the transforming growth factor-beta (TGF-betabeta expression through angiotensin type-I receptors (AT1-R). Many studies have demonstrated that the clinically used angiotensin-converting enzyme inhibitors (ACE-I) and AT1-R blockers (ARB) significantly attenuated the liver fibrosis development in the experimental studies and clinical practice. AT-II also strongly promotes neovascularization, which plays a pivotal role in tumor development. AT-II induces a potent angiogenic factor; namely, the vascular endothelial growth factor (VEGF). It has been reported that ACE-I significantly attenuated the experimental HCC growth and hepatocarcinogenesis along with suppression of neovascularization. The VEGF expression in the tumor was suppressed by ACE-I, too. The combined treatment of ACE-I with other clinically used agents, such as interferon, imatinib mesylate, and vitamin K, shows more potent inhibitory effects on the development of liver fibrosis and HCC. Since RAS inhibitors are widely used in the clinical practice without serious side effects, they may represent a potential new therapeutic strategy against the progression of chronic liver diseases.

Mitochondrial abnormality and oxidative stress in nonalcoholic steatohepatitis.

BACKGROUND: Oxidative stress plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Mitochondrial abnormality may be associated with the onset and progression of NASH via excessive formation of mitochondrial reactive oxygen species. This study aimed to investigate the role of mitochondrial abnormality in NASH in relation to oxidative stress. METHODS: Twenty-six patients with NASH, 11 with simple steatosis, and 10 healthy volunteers underwent clinico-pathological analysis. The liver/spleen ratio, an index of the hepatic fat content, was evaluated with computed tomography. Plasma glutathione levels were measured as an antioxidative marker, and the urinary 8-isoprostane levels and 3-nitrotyrosine staining in the liver as an oxidative stress marker. Mitochondrial abnormality was estimated by serum levels of mitochondria aspartate transaminase (mAST) and the mitochondrial staining in the liver. RESULTS: Urinary 8-isoprostane levels were higher in NASH than in the healthy volunteers, whereas plasma glutathione levels were similar in the 2 groups. In NASH, urinary 8-isoprostane levels positively correlated with alanine transaminase levels and negatively with the liver/spleen ratio. The 3-nitrotyrosine staining was more advanced in simple steatosis and NASH than in the normal liver, but was similar in simple steatosis and NASH. In contrast to the normal mAST levels in the healthy volunteers and simple steatosis, serum mAST levels were elevated in one-fourth of the NASH patients and positively correlated with urinary 8-isoprostane levels in NASH. Most cases of NASH showed diffuse or focal but intense mitochondrial staining in the liver in contrast to scattered staining in simple steatosis. CONCLUSIONS: Our present study demonstrated that in NASH, the enhanced oxidative stress may be associated with hepatic inflammation and the degree of fat infiltration in the liver. However, simple steatosis and NASH were both exposed to oxidative stress, while NASH alone was associated with mitochondrial abnormality. These findings indicate that mitochondrial abnormality may play a role in the onset and progression of NASH in correlation with oxidative stress.

Increased von Willebrand factor over decreased ADAMTS13 activity may contribute to the development of liver disturbance and multiorgan failure in patients with alcoholic hepatitis.

BACKGROUND: Severe alcoholic hepatitis (SAH) in addition to alcoholic hepatitis (AH) is a life-threatening complication of alcohol abuse, and its pathogenesis remains unclear. The deficiency of ADAMTS13 results in an increase of the plasma unusually large von Willebrand factor multimer (UL-VWFM) and finally causes microcirculatory disturbance and multiorgan failure. We investigated the relationship of ADAMTS13 and von Willebrand factor antigen (VWF:Ag) with the clinical features of AH and SAH. METHODS: The plasma levels of ADAMTS13 activity, VWF:Ag, and UL-VWFM were determined in 24 patients with AH, 5 with SAH, and 10 with alcoholic liver cirrhosis (LC). RESULTS: The ADAMTS13 activity was significantly lower in SAH (mean 24%), AH (62%), and LC (76%) than in the healthy subjects (102%, n=62). The VWF:Ag levels were higher in SAH (806%), AH (405%), and LC (514%) than in the healthy subjects (100%), resulting in a higher ratio of VWF:Ag to ADAMTS13 activity in SAH (102.2), AH (8.9), and LC (8.6) compared with the healthy subjects (1.0). In 3 nonsurvivors with SAH and multiorgan failure, the protease activity markedly decreased (from 4.5 to 16%), and VWF:Ag remarkably increased (from 560 to 1,202%), resulting in an extremely high ratio of VWF:Ag to the activity (from 35.0 to 240.4). At the recovery stage in the survivors with SAH and AH, the protease activity increased and the VWF:Ag decreased, whereas in a nonsurvivor with SAH, the activity remained extremely low and the VWF:Ag was still high. Unusually large von Willebrand factor multimer was detected in 80.0% of SAH and 55.6% of AH. Multivariate analysis showed that the serum albumin and platelet count independently correlated with VWF:Ag. CONCLUSION: The enhanced production of UL-VWFM over deficient activity of ADAMTS13 may, in part, contribute to not only the progression of liver injury but also the development of multiorgan failure through microcirculatory disturbance in SAH in addition to AH. The imbalance between the plasma ADAMTS13 activity and VWF:Ag could be a useful prognostic marker in AH.

Synergistic inhibition of hepatocellular carcinoma growth and hepatocarcinogenesis by combination of 5-fluorouracil and angiotensin-converting enzyme inhibitor via anti-angiogenic activities.

Therapies aimed at destruction of the tumor vasculature are now recognized as a promising approach against cancer, and it has been reported that the combination treatment with an angiogenic inhibitor and conventional chemotherapeutic drug exerted synergistic anti-cancerous effects. We previously reported that the clinically used angiotensin-converting enzyme inhibitor (ACE-I) exerted potent-anti-angiogenic activities. The aim of our current study was to examine the combined effect of ACE-I and 5-fluorouracil (5-FU), which is widely used for hepatogastrointestinal tumors, on hepatocellular carcinoma (HCC) growth and hepatocarcinogenesis. When used individually at low doses, neither 5-FU nor ACE-I exerted significant inhibitory effects on the HCC growth. However, the combination treatment of 5-FU and ACE-I showed a potent inhibitory effect on HCC growth along with suppression of neovascularization in the tumor. The expression level of the vascular endothelial growth factor, a potent angiogenic factor, was also suppressed, almost in conjunction with tumor growth inhibition. Furthermore, 5-FU and ACE-I treatment resulted in a marked increase of apoptosis in the tumor. In the hepatocarcinogenesis model, the combination treatment with 5-FU and ACE-I also showed a marked inhibitory effect on the development of preneoplastic lesions. The in vitro study demonstrated that this combination treatment inhibited endothelial cell tubular formation. Collectively, the combination treatment of 5-FU and ACE-I exerted a marked synergistic inhibitory effect on HCC growth via suppression of angiogenesis. This regimen also showed a chemopreventive effect against hepatocarcinogenesis. Since both 5-FU and ACE-I are widely used in clinical practice, this combination therapy may be an effective new therapeutic strategy against HCC in the future.

Crosstalk between high-molecular-weight adiponectin and T-cadherin during liver fibrosis development in rats.

Adiponectin, a circulating adipocyte-derived secretory protein, reportedly plays an important role in liver fibrosis development, although the biological role of adiponectin in liver fibrogenesis is still controversial. Adiponectin is present in the serum as three oligometric complexes; namely, high-, middle-, and low-molecular weight (HMW, MMW, and LMW, respectively). Adiponectin exerts different biological activities in an oligomerization-dependent manner. The aim of our current study was to examine the alteration of each isoform of adiponectin and its receptors (AdipoR1, AdipoR2, and T-cadherin) during the choline-deficient L-amino acid-defined (CDAA) diet-induced rat liver fibrosis development. We also elucidated the methylation status of all receptors. The serum level of total adiponectin significantly increased during the liver fibrosis development. Among the three isoforms, only HMW adiponectin was significantly up-regulated whereas MMW and LMW were not. The expression of T-cadherin, which exclusively binds with HMW adiponectin, was significantly augmented as well. The AdipoR2 expression was markedly decreased and showed no marked difference from that of AdipoR1. No obvious methylation change was observed in all three receptors, suggesting that another mechanism is involved in the alteration of receptor gene expression. Collectively, since the specific ligand and receptor were augmented together, crosstalk between HMW adiponectin and T-cadherin may play an important role during liver fibrosis development in rats.

Combined treatment of vitamin K2 and angiotensin-converting enzyme inhibitor ameliorates hepatic dysplastic nodule in a patient with liver cirrhosis.

Although it is well known that the hepatocellular carcinoma (HCC) is an ominous complication in patients with liver cirrhosis, there has been no approved drug to prevent the development of HCC to date. We previously reported that the combined treatment of vitamin K2 (VK) and angiotensin-converting enzyme inhibitor (ACE-I) significantly suppressed the experimental hepatocarcinogenesis. A 66-year-old Japanese woman with hepatitis C virus (HCV)-related liver cirrhosis developed a dysplastic nodule in the liver detected by enhanced computed tomography along with elevation of the tumor markers, namely, alpha-fetoprotein (AFP) and lectin-reactive demarcation (AFP-L3), suggesting the presence of latent HCC. After oral administration of VK and ACE-I, the serum levels of both AFP and AFP-L3 gradually decreased without any marked alteration of the serum aminotransferase activity. After one-year treatment, not only the serum levels of AFP and AFP-L3 returned to the normal ranges, but also the dysplastic nodule disappeared. Since both VK and ACE-I are widely used without serious side effects, this combined regimen may become a new strategy for chemoprevention against HCC.