Tatsuhiro Tsujimoto

Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats†

Nonalcoholic steatohepatitis (NASH) may cause fibrosis, cirrhosis, and hepatocellular carcinoma (HCC); however, the exact mechanism of disease progression is not fully understood. Angiogenesis has been shown to play an important role in the progression of chronic liver disease. The aim of this study was to elucidate the role of angiogenesis in the development of liver fibrosis and hepatocarcinogenesis in NASH. Zucker rats, which naturally develop leptin receptor mutations, and their lean littermate rats were fed a choline‐deficient, amino acid–defined diet. Both Zucker and littermate rats showed marked steatohepatitis and elevation of oxidative stress markers (e.g., thiobarbital acid reactive substances and 8‐hydroxydeoxyguanosine). In sharp contrast, liver fibrosis, glutathione‐S‐transferase placental form (GST‐P)‐positive preneoplastic lesions, and HCC developed in littermate rats but not in Zucker rats. Hepatic neovascularization and the expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, only increased in littermate rats, almost in parallel with fibrogenesis and carcinogenesis. The CD31‐immunopositive neovessels were mainly localized either along the fibrotic septa or in the GST‐P–positive lesions. Our in vitro study revealed that leptin exerted a proangiogenic activity in the presence of VEGF. In conclusion, these results suggest that leptin‐mediated neovascularization coordinated with VEGF plays an important role in the development of liver fibrosis and hepatocarcinogenesis in NASH. (HEPATOLOGY 2006;44:983–991.)

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.

The Effect of Inflammatory Cytokines in Alcoholic Liver Disease

Alcohol is the most common cause of liver disease in the world. Chronic alcohol consumption leads to hepatocellular injury and liver inflammation. Inflammatory cytokines, such as TNF-α and IFN-γ, induce liver injury in the rat model of alcoholic liver disease (ALD). Hepatoprotective cytokines, such as IL-6, and anti-inflammatory cytokines, such as IL-10, are also associated with ALD. IL-6 improves ALD via activation of the signal transducer and activator of transcription 3 (STAT3) and the subsequent induction of a variety of hepatoprotective genes in hepatocytes. IL-10 inhibits alcoholic liver inflammation via activation of STAT3 in Kupffer cells and the subsequent inhibition of liver inflammation. Alcohol consumption promotes liver inflammation by increasing translocation of gut-derived endotoxins to the portal circulation and activating Kupffer cells through the LPS/Toll-like receptor (TLR) 4 pathways. Oxidative stress and microflora products are also associated with ALD. Interactions between pro- and anti-inflammatory cytokines and other cytokines and chemokines are likely to play important roles in the development of ALD. The present study aims to conduct a systemic review of ALD from the aspect of inflammation.

Particle-mediated gene transfer into murine livers using a newly developed gene gun

Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases.

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

BackgroundBranched-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.MethodsThe 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.ResultsTreatment 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.ConclusionsIn 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.

Losartan, an angiotensin-II type 1 receptor blocker, attenuates the liver fibrosis development of non-alcoholic steatohepatitis in the rat

BackgroundApart from simple steatosis, the non-alcoholic steatohepatitis (NASH) can progress into liver fibrosis and cirrhosis. To date, however, no widely accepted therapeutic modalities have been established against NASH in the clinical practice. To find out promising new therapeutic agents, it is important to employ an appropriate experimental model of NASH, such as association with insulin resistance.FindingsIn the current study, we found that losartan, a clinically used angiotensin-II type 1 receptor blocker, significantly attenuated a choline-deficient L-amino acid-defined (CDAA) diet-induced steatohepatitis in obese diabetic- and insulin resistance-associated Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The transforming growth factor-beta, a well-known major fibrogenic cytokine, was also suppressed in a similar magnitude to that of the fibrosis area. Noteworthy was the finding that these inhibitory effects were achieved even at a clinically comparable low dose.ConclusionSince losartan is widely used without serious side effects in the clinical practice, this agent may be an effective new therapeutic strategy against NASH.

Significance of increased plasma adrenomedullin concentration in patients with cirrhosis

BACKGROUND/AIMS Adrenomedullin recently discovered in human pheochromocytoma is a potent vasodilatory peptide mainly derived from vascular endothelial and smooth muscle cells. Hyperdynamic circulation, ultimately leading to ascites formation, has been attributed to peripheral vasodilatation in liver cirrhosis. However, little is known about the role of adrenomedullin in this condition. METHODS Plasma adrenomedullin concentrations were measured by radioimmunoassay after extraction and purification in 28 cirrhotic patients without ascites, 12 cirrhotic patients with ascites and 10 healthy subjects. RESULTS Plasma adrenomedullin concentrations in cirrhotic patients with ascites (12.7+/-4.5 fmol/ml) were significantly higher than those in cirrhotic patients without ascites (8.2+/-2.3 fmol/ml, p<0.005) and healthy subjects (5.8+/-0.8 fmol/ml, p<0.005). Interestingly, plasma adrenomedullin concentrations were highest in patients with refractory ascites (n=5, 15.8+/-3.0 fmol/ml) and were positively correlated with the Child-Pugh score (r=0.44, p<0.01). Moreover, plasma adrenomedullin concentrations were positively correlated with plasma renin activity (r=0.63, p<0.0001), plasma aldosterone (r=0.60, p<0.0001) and plasma norepinephrine concentrations (r=0.60, p<0.0001), and negatively correlated with creatinine clearance (r=-0.61, p<0.0005) and urinary sodium excretion (r=-0.44, p<0.02). Stepwise multiple regression analysis using certain independent variables, including Pughs score, vasoactive substances, renal function and hemodynamic parameters, showed that the adjusted R square was highest when plasma renin activity and creatinine clearance (standard coefficient=0.53, -0.49, respectively) were considered (adjusted R square=0.61, p<0.0001). CONCLUSIONS Plasma adrenomedullin concentrations increased with the progression of liver cirrhosis and were highest in cirrhotic patients with refractory ascites. In addition, elevated adrenomedullin was associated with activation of the renin-angiotensin-aldosterone and sympathetic nervous systems, and with functional renal impairment in cirrhosis. Considering the potent vasodilatory action of adrenomedullin, increased adrenomedullin may participate in the hyperdynamic circulation, ultimately leading to ascites formation, in patients with liver cirrhosis.

Mixed endothelin receptor antagonist, SB209670, decreases portal pressure in biliary cirrhotic rats in vivo by reducing portal venous system resistance

BACKGROUND/AIMS This study aimed to evaluate the hemodynamic effects of endothelin-1 or mixed endothelin receptor antagonist, SB209670 in cirrhotic rats, and to elucidate the role of endothelin in cirrhotic portal hypertension. METHODS Secondary biliary cirrhosis was induced by bile duct ligation. Hemodynamics were studied using the radioactive microsphere technique. RESULTS Plasma and hepatic endothelin levels in cirrhotic rats were significantly higher than those in normal rats (plasma, 9.0+/-1.3 vs. 2.6+/-0.5 pg/ml, p<0.001; liver, 74.8+/-13.3 vs. 12.6+/-2.5 pg/g wet tissue, p<0.001). Intraportal administration of endothelin-1 (3 nmol/kg) progressively raised portal pressure without an initial transient reduction, which was observed in systemic arterial pressure, in both cirrhotic and normal rats. SB209670 (5.4 micromol/kg) reduced portal pressure in cirrhotic rats (-19+/-5%, p<0.01) without modifying systemic arterial pressure and renal blood flow, but not in normal rats. This reduction was associated with reduced portal venous system resistance (vehicle, 2.5+/-0.2 vs. SB209670, 1.7+/-0.1 mmHg x min x 100 g bw/ml, p<0.01), but not with change in portal venous inflow and collateral blood flow. CONCLUSIONS Mixed endothelin antagonist, SB209670, decreased portal pressure by reducing portal venous system resistance without modifying systemic arterial pressure and renal blood flow in cirrhotic rats. This result, together with the findings that plasma and hepatic endothelin levels were elevated in cirrhotic rats and that exogenous endothelin-1 increased portal pressure, provides further support for a role of endothelin in portal hypertension and suggests a potential use of mixed endothelin antagonist in the pharmacological treatment of portal hypertension.

Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers

BACKGROUND When recombinant adenoviruses are infused directly into the circulation, transgene expression is almost completely restricted to the liver. AIMS Efficiency and safety of adenovirus mediated gene transfer into damaged livers were examined in mice with liver cirrhosis or fulminant hepatitis. METHODS Liver cirrhosis and fulminant hepatitis were induced by intraperitoneal administration of thioacetamide and d-galactosamine followed by lipopolysaccharide, respectively. Mice were infused with adenoviruses carrying the Escherichia coliβ-galactosidase gene, lacZ gene, into the tail vein. Transduction efficiency of thelacZ gene was estimated histochemically by X-gal staining and quantitatively using a chemiluminescent assay. Activation of adenovirus specific T cells and development of neutralising antibodies against adenovirus were also examined. RESULTS Histochemical evaluation revealed that approximately 40%, 80%, and 40% of cells in normal, cirrhotic, and fulminant hepatitis livers, respectively, were stained blue using X-gal staining. Quantitative analyses revealed that levels of lacZ expression in cirrhotic livers were approximately 2.5-fold and sixfold greater than those in normal and fulminant hepatitis livers, respectively. Although transgene expression in fulminant hepatitis livers was significantly lower than that in normal livers, marked levels of transgene expression were achieved even in fulminant hepatitis livers. Significant adverse effects of adenoviruses were not observed in damaged livers. There were no significant differences in cellular or humoral immune responses to adenoviruses among animals with normal, cirrhotic, and fulminant hepatitis livers. CONCLUSIONS Our results suggest that gene therapy with adenoviruses may be used efficiently and safely, even in patients with severe liver disease.