Masahito Uemura

Changes in the expression and localization of hepatocellular transporters and radixin in primary biliary cirrhosis

BACKGROUND/AIMS Expression and localization of human hepatocellular transporters and of radixin, cross-linking actin with some membrane transporters, may change in cholestatic liver diseases. METHODS We investigated the uptake transporters OATP2 (SLC21A6), OATP8 (SLC21A8), and NTCP (SLC10A1), the export pumps MRP2 (ABCC2), MRP3 (ABCC3), MRP6 (ABCC6), and P-glycoproteins (ABCB1, ABCB4, ABCB11), and radixin, in non-icteric primary biliary cirrhosis (PBC stages I-III) and control human liver needle-biopsies using immunofluorescence microscopy and semi-quantitative RT-PCR. RESULTS Expression and localization of all transporters were unchanged in PBC I-II. Immunostaining intensities of uptake transporters decreased in PBC III with a concomitant decrease in mRNA levels. Immunostaining intensities and mRNA levels of export pumps were similar in controls and PBC I-III, however, irregular MRP2 immunostaining suggested redistribution of MRP2 into intracellular structures in PBC III. Areas of irregular MRP2 immunostaining showed largely reduced radixin immunostaining, whereas normal hepatocytes had MRP2 and radixin confined to the canalicular membrane. Disrupted localization of radixin and MRP2 supports the concept that radixin contributes to the canalicular localization of MRP2. CONCLUSIONS Down-regulation of uptake transporters may contribute to the impaired hepatobiliary elimination in advanced PBC, and partially altered localization of MRP2 may reflect the onset of changes leading to icteric PBC.

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.

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.

Endothelin-1 plays a major role in portal hypertension of biliary cirrhotic rats through endothelin receptor subtype B together with subtype A in vivo

BACKGROUND/AIMS Endothelin-1 has been suggested to play a key role in cirrhotic portal hypertension, but a role of its receptors in vivo is not fully elucidated. METHODS Biliary cirrhosis was induced by bile duct ligation. Expressions of endothelin-1 and its receptors were evaluated by radioimmunoassay and/or reverse-transcription polymerase chain reaction. Hemodynamics were studied using endothelin receptor agonist or antagonist. RESULTS Portal pressure and hepatic endothelin-1 concentrations progressively increased in parallel after bile duct ligation. Gene expression of hepatic prepro-endothelin-1 and endothelin B receptor enhanced after bile duct ligation, while that of endothelin A receptor was unchanged. Intraportal administration of endothelin-1 or endothelin B receptor agonist sarafotoxin 6c (0.5 nmol/kg, respectively) progressively raised portal pressure in both sham and cirrhotic rats. Portal hypertensive effect of sarafotoxin 6c was more intense in cirrhotic rats than sham animals. Neither endothelin A receptor antagonist FR139317 (1 mg/kg) nor endothelin B receptor antagonist BQ788 (1 mg/kg) alone ameliorated cirrhotic portal hypertension. Only the combined endothelin A and B blockade was associated with a decrease in portal pressure in cirrhotic rats. CONCLUSIONS These results indicate that endothelin-1 plays a major role in cirrhotic portal hypertension through endothelin receptor subtype B together with subtype A in vivo.

Pivotal role of ADAMTS13 function in liver diseases

The liver is a major source of clotting and fibrinolytic proteins, and plays a central role in thrombo-regulation. Patients with advanced liver diseases tend to bleed because of reduced plasma levels of several clotting factors and thrombocytopenia, but they do also exhibit thrombotic complications. ADAMTS13 is a metalloproteinase, produced exclusively in hepatic stellate cells, and specifically cleaves highly multimeric von Willebrand factor (VWF). VWF plays a pivotal role in hemostasis and thrombosis, and its function is dependent on its multimeric state. Deficiency of ADAMTS13 results in accumulation of unusually large VWF multimers (UL-VWFM) in plasma, in turn induces platelet clumping or thrombi under high shear stress, followed by microcirculatory disturbances. Considering that UL-VWFM, the substrate of ADAMTS13, is produced in transformed vascular endothelial cells at sites of liver injury, decreased ADAMTS13 activity may be involved in not only sinusoidal microcirculatory disturbances, but also subsequent progression of liver injuries, eventually leading to multiorgan failure. This concept can be applied to the development or aggravation of liver diseases, including liver cirrhosis, alcoholic hepatitis, veno-occlusive disease, and adverse events after liver transplantation. These results promise to bring further understanding of the pathophysiology of liver diseases, and offer new insight for development of therapeutic strategies.

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.

Plasma ADAMTS13 activity may predict early adverse events in living donor liver transplantation: Observations in 3 cases

A disintegrin‐like and metalloproteinase with thrombospondin type‐1 motifs 13 (ADAMTS13) is a metalloproteinase that specifically cleaves the multimeric von Willebrand factor (VWF). Deficiency of ADAMTS13 increases the unusually large VWF multimers (UL‐VWFM), which leads to platelet clumping and/or thrombus formation, resulting in microcirculatory disturbance. We serially determined the activity of plasma ADAMTS13, together with VWF antigen (VWF:Ag) and UL‐VWFM, in association with the development of early graft dysfunction in 3 liver transplant recipients and 4 patients with major hepatectomy as controls. In case 1, ADAMTS13 activity decreased markedly from 108% to less than 3% with concomitant thrombocytopenia on posttransplantation day 7, when acute rejection occurred. Simultaneously, UL‐VWFM were detected. During the second episode of rejection, VWF:Ag increased to 368% with the appearance of UL‐VWFM, while ADAMTS13 activity was as low as 18%, indicating an imbalance between a large amount of UL‐VWFM and low activity of ADAMTS13. Administration of fresh frozen plasma (FFP) together with treatment for acute rejection resulted in an improvement of ADAMTS13 activity and disappearance of the UL‐VWFM. In case 2, ADAMTS13 activity promptly decreased to 9% with thrombocytopenia on day 1, when ischemia‐reperfusion injury occurred. Subsequently, the ADAMTS13 activity increased steadily without appearance of UL‐VWFM, and the patient recovered uneventfully. ADAMTS13 activity decreased to 15% immediately after transplantation in case 3 as well. In contrast, ADAMTS13 activity never decreased below 20% in 4 patients with major hepatectomy as controls. In conclusion, these results indicate that the kinetics of ADAMTS13 and UL‐VWFM could be good indicators of adverse events after liver transplantation. Our findings not only suggest a novel mechanism for thrombocytopenia, but also provide a useful tool for diagnosis of graft dysfunction in the early stage after transplantation. Liver Transpl 12:859–869, 2006.

Clinical features of liver disturbance in rheumatoid diseases: clinicopathological study with special reference to the cause of liver disturbance.

Background:Background: Liver disturbance in rheumatoid diseases results not only from liver disease associated with the rheumatoid diseases themselves but also from various other causes. This study aimed to elucidate the clinical features of liver disturbance in rheumatoid diseases, focusing on the cause of this disturbance. Methods: A clinicopathological study was performed in 306 patients (106 with systemic lupus erythematosus, 71 with Sjögrens syndrome, 59 with rheumatoid arthritis, 27 with scleroderma, 30 with polymyositis, and 13 with polyarteritis nodosa). Results: Liver disturbance occurred in 43% of these patients and resulted from various causes. Its degree and duration varied from one cause to another. Liver disease associated with rheumatoid diseases was the leading cause of the liver disturbance in these patients and was characterized by mild and transient liver disturbance (maximum alanine aminotransferase [ALT] level during the study period, 68 ± 8 IU/ml; maximum alkaline phosphatase [ALP] level, 410 ± 31 IU/ml; duration of liver disturbance, 6 ± 2 months). Most patients with this type of liver disease showed minimal change in liver histology, although two-thirds of those evaluated by the international scoring system for autoimmune hepatitis (AIH) were classified as “probable” or “definite”. Eight of 14 patients with histologically proven chronic hepatitis or cirrhosis were infected with hepatotropic virus (7 with hepatitis C virus [HCV] and 1 with hepatitis B virus [HBV]). Five of 9 patients in whom the hepatic lesion progressed had hepatotropic virus infection (4 with HCV and 1 with HBV), and the other 4 patients suffered from autoimmune liver diseases. Conclusions: Liver disease associated with rheumatoid diseases was the leading cause of liver disturbance in these patients and was characterized by mild and transient liver disturbance, whereas progressive liver diseases were often associated with hepatotropic virus, mainly HCV, or autoimmune liver diseases. Liver histology is indispensable for differentiating AIH from liver disease associated with rheumatoid diseases.