Hideaki Enzan

Altered expression of fatty acid–metabolizing enzymes in aromatase-deficient mice

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Immunohistochemical identification of Ito cells and their myofibroblastic transformation in adult human liver

To identify Ito cells in normal and pathological adult human livers, immunohistochemical studies were performed by the avidin-biotin-peroxidase complex method using monoclonal antibodies for α-smooth muscle actin (ASMA), desmin, and vimentin. Fifty one needle biopsies, 7 surgically resected specimens, and 5 autopsy specimens were studied. In the normal adult liver vascular smooth muscle cells and pericytes, together with perisinusoidal cells with thin cytoplasmic processes were positive for ASMA. These latter cells formed a loose and discontinuous layer along the sinusoidal walls. Immunoelectron microscopy showed that the ASMA-positive perisinusoidal cells were Ito cells containing fat droplets. The other sinusoidal lining cells were negative for ASMA. In chronic liver disease, ASMA-positive Ito cells showed an increase in number, size, and the intensity of immunostaining in areas of piecemeal necrosis), and formed a continuous cellular network. These cells were dendritic in shape with irregularly elongated cytoplasmic processes and contained an increased amount of microfilaments, in association with loss of the characteristic fat droplets. Thus, their ultrastructural features corresponded to those of myofibroblastic cells. Ito cells showed no staining for desmin in both normal and pathological livers. These results indicate that immunohistochemistry using an anti-ASMA antibody is a sensitive and reliable method for the identification of both normal and transformed Ito cells in adult human livers.

Development of hepatic sinusoidal structure with special reference to the Ito cells

To elucidate sinusoidal cell structure and function under normal conditions and their behavior in diseased settings, an understanding of their developmental aspects is needed.

Differential expression of CD34 in normal colorectal tissue, peritumoral inflammatory tissue, and tumour stroma

Aims—To investigate the role of CD34 positive stromal cells, namely dendritic interstitial cells, in the desmoplastic stroma formation of malignant epithelial neoplasms the distribution of CD34 positive stromal cells was examined in human colorectal adenocarcinomas, peritumoral inflammatory tissue, and normal tissue. Methods—Forty one surgically resected human colorectal adenocarcinomas and their corresponding peritumoral inflammatory and normal tissues were examined. To distinguish CD34 positive stromal cells from vascular endothelial cells, immunostaining for both CD34 and CD31 was performed. The distribution of myofibroblasts was also analysed immunohistochemically, and double staining with CD34 and α smooth muscle actin (ASMA) was performed. Results—Most of the stromal cells in the normal colorectal submucosa, muscularis propria, subserosa, and perirectal tissue were positive for CD34. In contrast, the peritumoral inflammatory tissue and the tumour stroma had no CD34 positive stromal cells. The distribution of myofibroblasts was almost the same as in the aforementioned series. No stromal cells double positive for CD34 and ASMA were detected in the peritumoral inflammatory tissues. Conclusions—Most stromal fibroblasts are CD34 positive stromal cells (dendritic interstitial cells). In colorectal adenocarcinomas, a lack of CD34 expression in stromal cells is associated with desmoplastic reaction.

PROLIFERATION OF ITO CELLS (FAT‐STORING CELLS) IN ACUTE CARBON TETRACHLORIDE LIVER INJURY: A Light and Electron Microscopic Autoradiographic Study

The proliferative activity of Ito cells in acute liver injury induced by carbon tetrachloride (CCl4) was studied by light and electron microscopic autoradio‐graphy. At 48 hours after a single intraperitoneal injection of CCl4, the livers of the mice given vitamin A per os for preceding 9 days and those of the mice without vitamin A‐pretreatment were removed. Small tissue blocks of each group were respectively incubated at 37°CC for 1 hour in culture medium containing 3H‐thymidine. After CC14, injection, perisinusoidal and sinusoidal cells adjacent to centrilobular necrotic liver cells increased in number and size. Some of them were labelled by 3H‐thymidine. On the other hand, the perisinusoidal and sinusoidal cells in the peripheral zone in which liver cells are not markedly degenerated nor necrotic showed no noticeable increase in number. They contained very few or no silver grains after 3H‐thymidine. In control mice the labelling of perisinusoidal cells was hardly observed. Electron microscopic autoradiography revealed that most of the labelled perisinusoidal cells in the centrilobular zone possess characteristics of Ito cells in their location and in the fine structures such as the presence of small fat droplets, well‐developed rough endoplasmic reticulum, and Golgi complex in the cytoplasm. These findings indicate that Ito cells incorporate 3H‐thymidine in DNA synthesis after hepatocellular necrosis resulting in cell proliferation. ACTA PATHOL. JPN. 35: 1301–1308, 1985.

FINE STRUCTURE OF HEPATIC SINUSOIDS and THEIR DEVELOPMENT IN HUMAN EMBRYOS and FETUSES

The fine structure of the hepatic sinusoids of 81 human embryos and fetuses and their development from 5 to 12 weeks gestation were studied. At 5 weeks gestation, sinusoid‐like structures and Kupffer‐like cells were observed between liver cell cords. Between 6 and 8 weeks gestation the sinuosids were completely developed. Definite Kupffer cells appear at this developmental stage, when the bone marrow has not yet formed. Floating macrophages form cell aggregates in the sinusoids which contact endothelial cells and settle as Kupffer cells. Erythroblastophagia is observed in Kupffer cells and macrophages. The endothelial linings are closed, with the attenuated cell processes and intercellular junctions between the adjoining endothelial cells. No transition was observed between Kupffer cells and endothelial cells. The findings suggest that Kupffer cells in the human embryo are extrahepatic in origin and that they reach the sinusoids via the circulatory system. Ito cells, which store fat, originate from mesenchymal cells in the septum transversum.

Super paramagnetic iron oxide MRI shows defective Kupffer cell uptake function in non-alcoholic fatty liver disease

Background The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is incompletely understood. Kupffer cells (KCs), phagocytic liver-resident macrophages, provide a protective barrier against egress of endotoxin from the portal to the systemic circulation. It is not known if KC phagocytic function is impaired in NAFLD. Super-paramagnetic iron oxide (SPIO) magnetic resonance imaging is a comparative technology dependent on KC phagocytic function. Objective To evaluate KC uptake function, in patients and experimental animals with NAFLD, using SPIO. Methods Abdominal CT and histological examination of liver biopsy specimens were used to estimate the degree of steatosis in patients with NAFLD and controls with chronic hepatitis C. SPIO-MRI was then performed in all patients. Normal rats fed a methionine-choline-deficient diet to induce non-alcoholic steatohepatitis (NASH), the more severe stage of NAFLD, and obese, insulin resistant, Zucker fa/fa rats with steatohepatitis, were also studied with SPIO-MRI and analysed for hepatic uptake of fluorescent microbeads. Immunohistochemical analysis evaluated the numbers of KCs in patients and rat livers. Results Relative signal enhancement (RSE), inversely proportional to KC function, was higher in patients with NAFLD than in controls and with the degree of steatosis on CT. RSE also positively correlated with the degree of steatosis on histology and was similarly higher in rats with induced severe NAFLD (NASH). On immunohistochemistry, defective phagocytic function was the result of reduced phagocytic uptake and not due to reduced KC numbers in rats or patients with NAFLD. Conclusions KC uptake function is significantly impaired in patients with NAFLD and experimental animals with NASH, worsens with the degree of steatosis and is not due to a reduction of KC numbers.

Dielectric properties of rat liver in vivo: analysis by modeling hepatocytes in the tissue architecture

Abstract The audio/radiofrequency dielectric dispersion of rat liver is analyzed by applying several competent mixture theories combined with a realistic model of hepatocytes. A naive application of the Maxwell–Wagner (interfacial-polarization) theory could not fully explain the observed dielectric behavior especially at frequencies below 1 MHz. The Bruggeman–Hanai-type effective-medium theory (EMT) proved competent to some extent in simulating the observation on liver at low frequencies, although some sizeable discrepancies between theory and observation still remained unexplained. These remaining discrepancies, however, were minimized when we applied a novel theory developed in this study. This theory introduces second-order corrections for possible dipole–dipole interaction (DDI) effects to the classical EMT for a concentrated suspension of particles. Analysis by simulations based on the EMT-DDI revealed that this theory can predict: (i) the effective size and shape of hepatic cell plates within the liver tissue, (ii) values of the specific capacitance for plasma, nuclear and mitochondrial membranes associated with the hepatocyte, and (iii) cytosolic as well as nucleoplasmic conductivities of physiological interest.

Vinculin: Its Possible Use as a Marker of Normal Collecting Ducts and Renal Neoplasms with Collecting Duct System Phenotype

Vinculin is a cytoskeletal protein associated with membrane actin-filament-attachment sites of cell-cell and cell-matrix adherens-type junctions. In this article, we examine the expression of vinculin to elucidate its role in human renal neoplasms. We reviewed surgically resected specimens and selected available tissue from 79 renal tumors in 78 patients. There were 55 men and 23 women. Their mean age was 61 years and the mean size of the renal tumors was 6.1 cm. All renal tumors were examined by immunohistochemistry using a monoclonal antibody against vinculin. Overall, 17 (21.5%) renal tumor samples reacted with vinculin. The positive ratio in various types of renal tumors was as follows: conventional-type (clear cell), 0/54; papillary-type, 5/12; chromophobe-type, 5/5; sarcomatoid-type, 3/4; collecting duct carcinoma, 3/3; and oncocytoma, 1/1. The positive rate of conventional-type renal cell carcinomas (RCCs) is significantly different from that of other renal tumors (P < .01). Normal kidney, conventional, and papillary-type RCCs exhibited positive signals in Western blot analysis. These results suggest that vinculin may serve as a useful marker of renal neoplasms with collecting duct system phenotype such as chromophobe-type RCC.

ELECTRON MICROSCOPIC STUDIES OF MACROPHAGES IN EARLY HUMAN YOLK SACS

Distribution and fine structure of macrophages were studied in 10 human embryos in the 6th and 7th week of gestation, 5.5 to 12 mm in crown‐rump length. The yolk sac macrophages were found in the extravascular mesenchymal tissues and intravascular spaces long before the first appearance of bone marrow and lymphatic tissues in the embryos. In addition to the macrophages, the fibroblastic cells and the cells of erythropoietic series were also present in the extravascular space. The macrophages showed a variety of cellular structures suggesting transition from immature cell type with no heterophagolysosomes to mature cell type in phagocytosis. The mature macrophages avidly phagocytized the primitive erythroblasts and occasionally platelets. They were positively stained for lysosomal enzymes and were characterized by numerous pleomorphic heterophagolysosomes which exhibited various stages of digestion of phagocytized blood cells. The origin of intravascular macrophages may be in either migrated extravascular macrophages or phagocytic endothelial cells. The phagocytosis and degradation of erythroblasts appear to be one of the main functions of yolk sac macrophages. The presence of the macrophages in mitosis indicates their proliferation in situ.