Sadataka Inuzuka

Therapeutic effects of restricted diet and exercise in obese patients with fatty liver

BACKGROUND/AIMS The incidence of obese patients with fatty liver has recently increased in Japan as well as in the United States and Europe. Fatty liver may occasionally progress to liver cirrhosis. In this study, we have compared the effects of restricted diet and exercise versus no treatment in obese patients with fatty liver. METHODS Twenty-five obese patients with fatty liver were divided into treated and control groups. Fifteen obese patients followed a program of restricted diet (ideal weight x 25 Cal x kg(-1)) and exercise (walking or jogging) for a trial period of 3 months. No changes in diet or lifestyle were made by the other 10 patients during the same trial period. Blood biochemical tests and liver histology were compared in all patients before and after the trial. RESULTS In the treated group, weight, blood biochemical data such as aminotransferase, albumin, cholinesterase, total cholesterol and fasting blood glucose values, and steatosis were significantly decreased after the trial. In the control group, there were no significant differences in the clinical and histological findings before and after the trial. CONCLUSIONS These results indicate that restricted diet and exercise therapy, such as walking and jogging, are useful means of improving blood biochemical data and histological findings in liver tissues related to fatty liver.

Serum hyaluronate reflects hepatic sinusoidal capillarization

BACKGROUND Most of circulating hyaluronate has been commonly degraded by hepatic sinusoidal endothelial cells (SECs). In hepatic sinusoidal capillarization, SECs morphologically change and also seem to decrease hyaluronate degradation. This work expands on the relationship between serum hyaluronate levels and changes in hepatic SECs accompanying hepatic sinusoidal capillarization. METHODS Serum hyaluronate levels were determined using an enzyme binding assay system. Liver biopsy specimens were collected to examine basement-membrane formation, the localization of Weibel-Palade bodies, and the localization of factor VIII-related antigen (FVIIIRAg) in SECs. RESULTS Serum hyaluronate levels increased with the progression of liver disorder, being high in all patients with liver cirrhosis. Patients showing markedly high serum hyaluronate levels, 200 ng/mL or more, had liver cirrhosis involving the SECs, which showed basement-membrane formation, Weibel-Palade bodies, and FVIIIRAg and closely resembled vascular endothelial cells. CONCLUSIONS Measurement of the serum hyaluronate concentration allows the evaluation of morphological and functional changes that occur in SEC accompanying hepatic sinusoidal capillarization in various liver disorders. The findings also suggest that patients with high serum hyaluronate levels, 200 ng/mL or more, have liver cirrhosis with typical hepatic sinusoidal capillarization formed by SECs containing FVIIIRAg.

Sinusoidal capillarization in small hepatocellular carcinoma.

The morphological and phenotypic changes of sinusoidal endothelial cells in hepatocellular carcinoma were investigated along with the hemodynamic changes. Hepatocellular carcinomas, which were 10–20 mm in diameter, were obtained by liver aspiration biopsy. Twenty specimens of well differentiated and 20 specimens of moderately differentiated hepatocellular carcinoma were used. These were classified into two groups of hepatocellular carcinoma: with and without hypervascularity. Electron microscopy, immunohistochemistry using antibodies against factor VIII‐related antigen, type IV collagen and laminin and lectin histochemistry using Ulex europaeus agglutinin I (UEA‐I) were performed. Factor VIII‐related antigen and UEA‐1 binding sites were present in the sinusoidal endothelial cells in two groups. In hepatocelMar carcinoma with hypervascularity, type IV collagen and laminin were present corresponding to basement membranes along the endothelial cells, which had few fenestrae. In another group, although type IV collagen was present along the endothelial cells that had a few fenestrae, laminin and basement membranes were rarely observed. These results suggest that the sinusoidal endothelial cells tend to show phenotypic changes in the early stage of hepatocarcinogenesis. As the arterial blood supply for hepatocellular carcinoma increases, the sinusoidal endothelial cells may form basement membranes and take on the morphological appearance of capillaries.

Basic fibroblast growth factor regulates proliferation and motility of human hepatoma cells by an autocrine mechanism

BACKGROUND/AIMS Basic fibroblast growth factor has mitogenic and angiogenic properties. In this study, we aimed to evaluate the role of fibroblast growth factor in the development and progression of human hepatocellular carcinoma. METHODS The expression of basic fibroblast growth factor, fibroblast growth factor receptor-1, and a receptor isoform was investigated by in situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction, Western blot analysis and confocal laser-scanning microscopy. The influence of exogenous basic fibroblast growth factor on DNA synthesis and motility of human hepatoma cells were also evaluated. RESULTS Basic fibroblast growth factor and fibroblast growth factor receptor-1 messenger RNAs were present mainly in tumor cells and less so in hepatocytes from noncancerous liver tissue. Immunoreactive products of basic fibroblast growth factor and fibroblast growth factor receptor-1 were observed in tumor cells. The isoform IIIc was expressed in hepatocellular carcinoma tissue and hepatoma cell lines. Exogenous basic fibroblast growth factor stimulated DNA synthesis and motility of hepatoma cells. The effect was more marked in poorly-differentiated hepatoma cells than in well-differentiated hepatoma cells. Fibroblast growth factor-1 expression on hepatoma cells was also more marked in poorly-differentiated hepatoma cells than in well-differentiated hepatoma cells. The stimulated motility on basic fibroblast growth factor was suppressed by an anti-fibroblast growth factor receptor-1 antibody. CONCLUSIONS Basic fibroblast growth factor may play an important role in the development and progression of hepatocellular carcinoma via an autocrine mechanism involving fibroblast growth factor and its receptor.

Significance of serum type-IV collagen levels in various liver diseases : measurement with a one-step sandwich enzyme immunoassay using monoclonal antibodies with specificity for pepsin-solubilized type-IV collagen

Serum type-IV collagen levels determined with a one-step sandwich enzyme immunoassay (EIA) using monoclonal antibodies with specificity for pepsin-solubilized type-IV collagen were compared with histologic changes in liver biopsy specimens from 107 patients with various liver diseases. Serum type-IV collagen levels were increased in the groups with liver diseases compared with controls. The serum type-IV collagen levels in the group with alcoholic cirrhosis showed significantly higher values than the other groups (P less than 0.05). A significant positive correlation was found between the serum type-IV collagen level and the degree of fibrosis or cell infiltration in 107 patients. Immunolocalization of type-IV collagen was observed around blood vessels and bile ducts increased in number in the portal tracts, with cell infiltration and fibrosis, increased around vessels in fibrous septa, and sinusoidal walls of areas with cell infiltration or necrosis in hepatic lobules, and along the boundary between fibrous septa and hepatocytes. The present data indicate that serum type-IV collagen may be a sensitive marker for active fibrosis and that the elevation of serum type-IV collagen level primarily reflects the enhancement of type-IV collagen synthesis and deposition in the liver tissue at the stage of active fibrosis in liver disease.

The extracellular matrix in hepatocellular carcinoma shows different localization patterns depending on the differentiation and the histological pattern of tumors: immunohistochemical analysis

This study investigated cells producing type I, III, and IV collagens, laminin, and fibronectin, the major components of the extracellular matrix, and compared their localization patterns in relation to the grade of tumor differentiation and the histological pattern of hepatocellular carcinoma. Type I, III, and IV collagens, laminin, and fibronectin were produced by tumor, endothelial, and Ito cells. Regarding their localization pattern in relation to the histological pattern of tumors, although the extracellular matrix was present in the subendothelial spaces of sinusoids in every histological pattern, the localization of these components in the intercellular spaces of tumor cells was most marked in hepatocellular carcinoma with a compact pattern. These results suggest that the extracellular matrix produced by tumor, endothelial, and Ito cells is deposited in appropriate positions in hepatocellular carcinoma to sustain the tissue structure showing different histological patterns. In relation to the grade of tumor differentiation, in most cases, Type I, III, and IV collagens and fibronectin were present in the subendothelial spaces of sinusoids and the intercellular spaces of some tumor cells, while little laminin was observed in well-differentiated small hepatocellular carcinoma (less than 10 mm diameter). In undifferentiated hepatocellular carcinoma, little extracellular matrix was observed, except around vessels. These results suggest that sinusoidal capillarization may not yet have occurred in the early stage of hepatocarcinogenesis, although it develops as the tumors increase in size and the tumor cells dedifferentiate. In undifferentiated hepatocellular carcinoma, tumor cells are too atypical to produce each extracellular matrix component.

Coordinated expression of integrin α6β1 and laminin in hepatocellular carcinoma

Abstract The interaction between tumor cells and laminin mediated by laminin-binding integrins is critical for tumor invasion and metastasis. The aim of this study was to clarify the altered expression of lamininbinding integrins with the change of laniinin deposition in hepatocellular carcinoma (HCC) in comparison with cirrhotic or normal liver by immunohistochemistry. In HCC, hepatoma cells and sinusoidal endothelial cells expressed integrins α1β1, α2β1, α3β1, and α6β1. Integrins α1β1 and α6β1 were detected in a continuous pattern along the sinusoids in accordance with laminin assembly. Integrins α2β1 and α3β1 were detected in a discontinuous pattern at these sites. Integrin α6β4 was not detected. In cirrhotic liver, although integrins α1β1 and α6β1 as well as laminin were detected in a continuous pattern along the sinusoids, integrins α2β1, α3β1, and α6β4 were not detected. In normal liver, although integrin α1β1 was detected in a continuous pattern along the sinusoids, neither integrins α2β1, α3β1, α6β1, α6β4, nor laminin were detected. We have clarified that, of laminin-binding integrins, the localization of integrin α6β1 shows the best correspondence with the localization of laminin. These results suggest that of laminin-binding integrins, integrin α6β1 is very important for cell-laminin interactions in HCC.

Cultured rat hepatic sinusoidal endothelial cells express intercellular adhesion molecule-1 (ICAM-1) by tumor necrosis factor-α or interleukin-1α stimulation

This study investigated the expression of intercellular adhesion molecule-1, a leukocyte adhesion molecule, on cultured rat hepatic sinusoidal endothelial cells during stimulation with tumor necrosis factor- α or interleukin-1 α . Using immunoelectron microscopy and the immunogold technique against intercellular adhesion molecule-1, gold particles were shown to increase significantly on the surface of sinusoidal epithelial cells treated with tumor necrosis factor- α (100 U/ml) or interleukin-1 α (10 U/ml) for 8 h compared with unstimulated cells. In addition, semi-quantitative analysis of intercellular adhesion molecule-1 on the sinusoidal endothelial cells was performed by cytofluorometer. Even without stimulation, intercellular adhesion molecule-1 was weakly expressed. However, 8 h after tumor necrosis factor- α or interleukin-1 α treatment, the expression of intercellular adhesion molecule-1 on cells was increased in a dose-dependent manner. Kinetic analysis showed that the expression of intercellular adhesion molecule-1 on sinusoidal endothelial cells treated with these cytokines increased gradually from the beginning of stimulation to 24 h. These findings suggest that hepatic sinusoidal endothelial cells may mediate the direct interaction between leukocytes and sinusoidal endothelial cells by expressing leukocyte adhesion molecules such as intercellular adhesion molecule-1.

Integrin α6β1 plays a significant role in the attachment of hepatoma cells to laminin

Abstract Background/Aims: Tumor invasion and metastasis consist of a series of complex events. During this process, the ability of tumor cells to adhere to laminin, a major component of basement membranes, is required at various steps. The expression of laminin-binding integrins and the extent of tumor metastasis and progression appear to be related. In hepatocellular carcinoma, increased expression of laminin-binding integrins is observed. However, little is known concerning the possible functional interactions between laminin-binding integrins and laminin. Therefore, we investigated the participation of laminin-binding integrins in the attachment of hepatoma cells to laminin. Methods: Human hepatoma cell lines (KIM-1, KYN-1,2) were used. We investigated the expression of integrin α 1 , α 2 , α 3 , α 6 , β 1 and β 4 subunits on hepatoma cells by immunocytochemical and flow cytometric analysis. Participation of these integrin subunits in the attachment of hepatoma cells to laminin was evaluated by an inhibition of cell adhesion assay. Results: Integrin α 1 , α 2 , α 3 , α 6 and β 1 subunits were expressed at the marginal areas of hepatoma cells, while the integrin β 4 subunit was scarcely detected. Laminin promoted the attachment of hepatoma cells in a dose-dependent manner. Although anti-integrin α 1 , α 2 , α 3 and β 4 subunit antibodies did not inhibit cell attachment to laminin, anti-integrin α 6 and β 1 subunit antibodies inhibited the attachment by 50% or more. Conclusions: These findings indicate that integrin α 6 β 1 is very important in the attachment of hepatoma cells to laminin, suggesting the participation of this integrin in metastasis and invasion of hepatoma cells.

Significance of serum tissue inhibitor of metalloproteinases-1 in various liver diseases

BACKGROUND/AIMS This study was performed to assess the significance of elevated serum tissue inhibitor of metalloproteinases-1 concentration in various liver diseases. METHODS Tissue inhibitor of metalloproteinases-1 levels were measured in patients with various liver diseases, and were compared with serum type III procollagen-N-peptide (P III P), type IV collagen and laminin P1 levels, as well as with the histology of liver biopsy specimens. RESULTS Mean tissue inhibitor of metalloproteinases-1 levels were significantly higher in subjects with acute viral hepatitis, cirrhosis, alcoholic hepatitis, and alcoholic cirrhosis than in the control group (p < 0.05). Serum tissue inhibitor of metalloproteinases-1 levels in the various liver diseases showed positive correlation with serum type IV collagen, P III P, and laminin P1 levels. Regarding the relationship between tissue inhibitor of metalloproteinases-1 and liver histology, serum tissue inhibitor of metalloproteinases-1 levels correlated with the degree of hepatic fibrosis and inflammation, such as focal necrosis and cell infiltration. Furthermore, elevated serum tissue inhibitor of metalloproteinases-1 levels were especially related to the cell infiltration, focal necrosis, portal fibrosis, and serum type IV collagen level. CONCLUSIONS These findings suggest that the measurement of the serum tissue inhibitor of metalloproteinases-1 level in various liver diseases may be useful to estimate the active hepatic fibrogenesis associated with the active inflammatory stage of the liver injury.