Motoaki Kin

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.

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.

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.

Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPases, inhibits the receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

BACKGROUND/METHODS The role of vacuolar type H(+)-ATPases (v-ATPases) and pH gradient between the endocytic compartments and cytoplasm in the endocytosis of asialoglycoproteins was morphologically investigated in isolated rat hepatocytes using bafilomycin A1, a specific inhibitor of v-ATPases. RESULTS Fluorescent staining by acridine orange showed that bafilomycin A2 inhibited the acidification of the endocytic compartments. Uptake of gold-conjugated asialofetuin was significantly inhibited by bafilomycin A1. However, bafilomycin A1 did not significantly inhibit uptake of a fluid phase marker, horseradish peroxidase. The number of autophagic vacuoles increased after the bafilomycin A1 treatment. However, materials in the autophagic vacuoles were rapidly degraded after the removal of bafilomycin A1. CONCLUSIONS Results suggest that: (a) v-ATPases are necessary for acidification of the endocytic compartments; (b) the pH gradient between the endocytic compartments and the cytoplasm which is generated by v-ATPases is necessary for the receptor-mediated endocytosis of asialoglycoproteins, and (c) v-ATPases may contribute to the degradation of the materials in autophagic vacuoles.

Estrogen upregulates nitric oxide synthase expression in cultured rat hepatic sinusoidal endothelial cells

BACKGROUND/AIMS Estrogen receptor (ER) is present in vascular endothelial cells and estrogen promotes nitric oxide (NO) synthesis, which relaxes smooth muscle cells. It is also speculated that NO is synthesized by estrogen in hepatic sinusoidal endothelial cells (SECs). Here we investigated the localization of ER and endothelial cell nitric oxide synthase (ecNOS), and determined 17beta-estradiol (E2)-induced ecNOS expression in normal rat SECs. METHODS Cultured SECs were used. Fluorescence intensities of ecNOS were measured by immunofluorescence using a confocal laser-scanning microscope. E2 was added (100 pg/ml) to the culture medium, and the expressions of ecNOS mRNA and protein were analyzed by reverse-transcription polymerase chain reaction and Western blotting. NO production in cultured SECs was examined using diaminofluorescein-2 diacetate as a fluorescent indicator for NO. RESULTS Immunolocalization of ER and ecNOS in normal liver was demonstrated in endothelial cells lining the hepatic sinusoids. ER and ecNOS were localized in the nuclei and cytoplasm of cultured SECs, respectively. The mRNA expression of ecNOS in cultured SECs was increased after 6 h, and the protein expression of ecNOS was increased 24 h after E2 stimulation. The fluorescence intensity of NO in cultured SECs was increased by E2 stimulation compared with untreated control cells. CONCLUSIONS These results suggested that ER is present in SECs, and estrogen upregulates NO production in SECs. E2 may be involved in the regulation of the hepatic sinusoidal microcirculation.

Laminin deposition to type IV collagen enhances haptotaxis, chemokinesis, and adhesion of hepatoma cells through β1-integrins

BACKGROUND/AIMS In hepatocellular carcinoma, laminin deposition to type IV collagen along the sinusoids is observed with the development of arterial network, coinciding with intrahepatic metastasis. We investigated the influence of laminin deposition to type IV collagen on hepatoma cell adhesion, motility and secretion of matrix metalloproteinases (MMPs), which are indispensable behaviors for tumor metastasis. METHODS Hepatoma cell lines (KYN-1, -2 and -3) were used. The expression of integrin subunit mRNAs in hepatoma cells was confirmed by RT-PCR. The influence of laminin addition to type IV collagen on the adhesion, chemokinesis, and migration of KYN-1, -2 and -3 was evaluated by the haptotactic migration, phagokinetic track motility, and cell adhesion assays. The effects of integrin subunits on these activities were evaluated using the function-blocking antibodies for integrins. Phosphorylation of MEK1/2 and secretion of MMPs were investigated by Western blotting and gelatin zymography. RESULTS Integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunit mRNAs were detected. The combination of type IV collagen and laminin enhanced the migration, chemokinesis, and adhesion of hepatoma cells compared to that of type IV collagen when used alone. The enhanced activity was significantly suppressed by function-blocking antibodies for integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunits. Hepatoma cells cultured on the combination of type IV collagen and laminin showed phosphorylation of MEK1/2 and increased secretion of MMPs. CONCLUSIONS The addition of laminin to type IV collagen enhances hepatoma cell adhesion and motility through beta1-integrins.

Ultrastructural observation on hepatocellular carcinoma

In 31 cases of hepatocellular carcinoma, electron microscopic observation and morphometry on the cell organelles were carried out to evaluate the usefulness of electron microscopy for the diagnosis of well differentiated hepatocellular carcinoma. The cell organelles in well differentiated tumor cells were very similar to those in normal hepatocytes or hepatocytes with liver cirrhosis (LC). We found that in poorly differentiated tumor cells, the nuclear area, N/C ratio, nucleolar area, the amount of dispersed chromatin, and the number of free ribosomes had increased, but the cellular area, degree of nuclear roundness, and mitochondrial area had decreased. These results seem to indicate that electron microscopy is not as useful as light microscopy in the diagnosis of well differentiated hepatocellular carcinoma, but is useful in the study of poorly differentiated tumor cells, which indicated that the cell proliferation through mitoses was activated.