Ryuichiro Sakata

Mechanical stretch induces TGF‐β synthesis in hepatic stellate cells

Background  It is known that mechanical stress induces extracellular matrix via transforming growth factor‐β (TGF‐β) synthesis in vascular smooth muscle cells. Activated hepatic stellate cells (HSCs) are an important source of TGF‐β in the liver. However, it remains unclear whether mechanical stress induces TGF‐β in HSCs. The Rho small GTP‐binding protein (Rho) has recently emerged as an important regulator of actin and cytoskeleton. We examined whether TGF‐β is expressed in stretched HSCs and whether Rho is involved in stretch‐induced TGF‐β synthesis.

Hepatic stellate cells and intralobular innervation in human liver cirrhosis

In normal and cirrhotic human liver tissues, we examined immunolocalization of alpha-smooth muscle actin (alpha-SMA), endothelin-1 receptor (ET-1R), and S-100 protein, with special emphasis on the intralobular spaces, using immunohistochemical methods. The ratio of the number of hepatic stellate cells (HSCs) with closely apposing nerve endings to the total number of HSCs in normal livers was compared with that in cirrhotic livers by electron microscopy. Immunolocalization of alpha-SMA and ET-1R was obviously recognized along the sinusoidal walls in cirrhotic liver and was significantly increased in cirrhotic liver compared with that in normal liver. Immunoreactive products for these substances were mainly localized in HSCs. However, immunolocalization of S-100 protein in intralobular spaces was markedly decreased in cirrhotic liver compared with that in normal liver. Nerve fibers were ultrastructurally hardly visible in intralobular spaces of cirrhotic livers. The ratio of the number of HSCs with closely apposing nerve endings to the total number of HSCs was significantly reduced in cirrhotic liver compared with that in normal liver. These results indicate that in liver cirrhosis, alpha-SMA-positive HSCs may play an important role in hepatic sinusoidal microcirculation through vasoactive agents such as ET-1 rather than through intralobular innervation.

Green tea with high-density catechins improves liver function and fat infiltration in non-alcoholic fatty liver disease (NAFLD) patients: A double-blind placebo-controlled study

Catechins, a major component of green tea extract, have anti-hyperlipidemic effects. The present study investigated the effects of consumption of green tea with high-density catechins in non-alcoholic fatty liver disease (NAFLD) patients. Seventeen patients with NAFLD consumed green tea with high-density catechins, low-density catechins or a placebo for 12 weeks in a randomized double-blind study. Ultrasonography and computed tomography (CT) were performed at baseline and after 12 weeks. Serum alanine aminotransferase (ALT) levels and urine 8-isoprostane were monitored and compared to baseline at 4, 8 and 12 weeks. Body fat was significantly decreased in the high-density catechin group compared with the placebo and low-density catechin groups after 12 weeks of consumption. All the patients in the high-density catechin group showed a significantly improved liver-to-spleen CT attenuation ratio compared with the placebo and low-density catechin groups after 12 weeks of consumption. The high-density catechin group significantly decreased serum ALT levels and reduced urinary 8-isoprostane excretion compared with the placebo and low-density catechin group after 12 weeks of consumption. Based on a reduced proportion of body fat as estimated by bioimpedance measurement, increased liver-to-spleen CT attenuation ratio, decreased serum ALT levels and reduced urinary 8-isoprostane excretion, we concluded that 12 weeks of 700 ml per day of green tea containing >1 g catechin improved liver fat content and inflammation by reducing oxidative stress in patients with NAFLD.

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.

The significance of colocalization of plasminogen activator inhibitor-1 and vitronectin in hepatic fibrosis.

BACKGROUND We examined the relationships among vitronectin (VN), plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor beta 1 (TGF-beta 1) in liver diseases to evaluate the presence of plasmin cascade in human hepatic fibrosis. METHODS Blood and liver tissues were obtained from 57 patients with liver disease. Plasma VN, PAI-1 antigen, and PAI-1 activity levels were evaluated. Biopsied liver specimens were observed by light and electron microscopy after immunohistochemical staining. Morphometric analysis was performed on these specimens. RESULTS Plasma VN and PAI-1 activity levels decreased significantly with the progression of hepatic fibrosis and were particularly marked in the liver cirrhosis group. Plasma PAI-1 antigen level increased significantly. The immunolocalization of the active form of TGF-beta became more intense with the progression of hepatic fibrosis, whereas that of the dual-stained positive areas of PAI-1 and VN (PAI-1.VN) decreased. There was a positive correlation between TGF-beta and PAI-1, whereas there was a negative correlation between TGF-beta and PAI-1.VN. Immunoelectron microscopy showed the localization of PAI-1-VN in the extracellular space around the sinusoidal cells or surface of aggregating platelets, TGF-beta mainly in Ito cells, and VN in hepatocytes near the focal necrotic area or fibrous septa. CONCLUSIONS These findings suggest that VN and PAI-1 are related to the active form of TGF-beta and that it is possible that the plasmin cascade is present in the human liver.

Improvement of portal hypertension and hepatic blood flow in cirrhotic rats by oestrogen

Background  In this study, we investigated the effects of oestrogen on nitric oxide synthase activity and nitric oxide production using the cirrhotic rat liver.

Radiographic Evidence of Cholecystokinin Octapeptide Receptors in the Hamster Gallbladder

The distribution of Cholecystokinin receptors in the hamster gallbladder was investigated by 125I-labeled Bolton-Hunter-cholecystokinin octapeptide autoradiography. Light microscopic examination showed a marked accumulation of radiolabeled Cholecystokinin within the domain of the muscle layer of the gallbladder. The electron microscopic study further disclosed the presence of radiolabeling mostly in those areas corresponding to cell-to-cell junctions of smooth-muscle cells. Our results suggest that contraction of the gallbladder may primarily be induced by cholecystokinin interacting with its specific receptor in smooth-muscle cells. That Cholecystokinin receptors were more abundant in the junctional complexes of smooth-muscle cells suggests that Cholecystokinin may have a major role in muscle contraction of the gallbladder, which eventually produces an effective bile emptying.

Formation of black pigment gallstone in a hamster model of experimental cirrhosis

The relationship between liver cirrhosis and the pathogenesis of black pigment stones has not been clarified. We attempted to induce black pigment stone formation in the gallbladders of hamsters. Male golden hamsters were divided into a cirrhosis group and a control group. Liver cirrhosis was induced by administering drinking water containing thioacetamide. The control group was given tap water. Gallstones at 48 weeks after treatment were examined by stereoscopic microscopy and scanning electron microscopy. The copper content of the black pigment stones was analysed by atomic absorption spectrophotometry. Black pigment stones in the gallbladder were detected in 25% of the cirrhosis group. Their surface and cross‐section appeared amorphous. Black pigment stones contained copper. We confirmed the formation of gallstones in an animal model of cirrhosis by thioacetamide. Our findings may contribute to the clarification of the relationship between the pathogenesis of black pigment stones and the pathophysiology of liver cirrhosis.

Morphological observation on extrahepatic bile duct of golden hamsters fed a lithogenic diet : histochemical, ultrastructural and cell kinetic studies

Cholelithiasis is often accompanied with disorders of the extrahepatic bile duct and pancreas. However, studies on changes of the extrahepatic bile duct in cholecystolithiasis have not shown this clearly. We therefore investigated sequential histologic changes, mucous secretion and DNA synthetic activity of the extrahepatic bile duct epithelium in cholecystolithiasis. Serial changes in the mucosal epithelial cells of the extrahepatic bile duct in golden hamsters treated with a lithogenic diet were examined by light and electron microscopy and an ultrastructural quantitative technique. In addition, epithelial cell kinetics were studied using bromodeoxyuridine (BrdU). After the 2nd week of diet, the extrahepatic bile duct showed an increase in goblet cells of the mucosal epithelium, a large number of secretory granules in the upper nuclear area of the epithelial cells and an increase in the BrdU-labeling index compared with the controls. These findings indicate that mucous secretion and cell turnover were enhanced in the mucosal epithelial cells of the extrahepatic bile duct in cholelithiasis, suggesting that the epithelial cells of the bile duct were protected and regenerating.

Reduced Contractility and Histological Changes in the Gallbladder due to Portal Hypertension in a Hamster Cirrhosis Model

BACKGROUND/AIMS Little is known about the relationship of portal venous pressure to contractility and histological changes in the gallbladder. In this study, we investigated the relationship between portal hypertension and contractility and histological changes in the gallbladder in a hamster cirrhosis model. METHODS Liver cirrhosis was induced in the hamsters (n = 20) by thioacetamide (TAA). Portal venous pressure was directly measured using a pressure-measuring instrument. The contractility of the gallbladder was appraised by the diameter before and after caerulein treatment. Gallbladder wall thickness and vessel areas in tissues were evaluated in relation to the portal venous pressure. RESULTS The portal venous pressure, gallbladder wall thickness with submucosal edema and area of vessels in the gallbladder wall in the cirrhosis group were significantly increased compared with those in the control group (n = 20, receiving saline instead of TAA). The gallbladder contraction rate in the cirrhosis group was significantly decreased compared with that in the control group. In the cirrhosis group, there were positive correlations between the portal venous pressure and the gallbladder contraction rate, gallbladder wall thickness, and area of vessels. CONCLUSIONS In the gallbladders of experimental cirrhotic hamsters, portal hypertension caused dilatation of the vessels as well as submucosal edema, and was an important factor in the increased thickness and reduced contractility of the gallbladder wall.