Mitsutaka Miura

Hepatic stellate cell (vitamin A-storing cell) and its relative - past, present and future.

HSCs (hepatic stellate cells) (also called vitamin A‐storing cells, lipocytes, interstitial cells, fat‐storing cells or Ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store 50–80% of vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of vitamin A homoeostasis. In pathological conditions, such as hepatic fibrosis or liver cirrhosis, HSCs lose vitamin A and synthesize a large amount of extracellular matrix components including collagen, proteoglycan, glycosaminoglycan and adhesive glycoproteins. Morphology of these cells also changes from the star‐shaped SCs (stellate cells) to that of fibroblasts or myofibroblasts. The hepatic SCs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. HSCs are activated by adhering to the parenchymal cells and lose stored vitamin A during hepatic regeneration. Vitamin A‐storing cells exist in extrahepatic organs such as the pancreas, lungs, kidneys and intestines. Vitamin A‐storing cells in the liver and extrahepatic organs form a cellular system. The research of the vitamin A‐storing cells has developed and expanded vigorously. The past, present and future of the research of the vitamin A‐storing cells (SCs) will be summarized and discussed in this review.

Three-dimensional structure of extracellular matrix reversibly regulates morphology, proliferation and collagen metabolism of perisinusoidal stellate cells (vitamin A-storing cells)

The three‐dimensional structure of the extracellular substratum was found to regulate reversibly the morphology, proliferation and collagen synthesis of perisinusoidal stellate cells (lipocytes, i.e. fat‐storing ‘Ito’ cells). On non‐coated polystyrene and type I collagen‐coated culture dishes, the cells spread well and extended their cellular processes. On the surface of type I collagen gels, the cells gathered and formed a mesh‐like structure. However, in type I collagen gel where the cells were surrounded by type I collagen three‐dimensionally, the cells extended their fine cellular processes and resembled the star‐shaped stellate cells seenin vivo. The cell proliferation was more prominent in culture dishes coated with type I collagen or in polystyrene culture dishes than on or in type I collagen gels. The collagen synthesis was affected in the same manner. These data indicate that the nature and the three‐dimensional structure of the extracellular matrix (ECM) can regulate morphology, proliferation and functions of the perisinusoidal stellate cells. In order to examine the reversibility of these regulations, we liberated cultured cells with trypsin or with purified bacterial collagenase and re‐seeded them onto or into each substratum. The cells changed their shape, rate of proliferation and collagen synthesis according to each new substratum. These results indicate that the three‐dimensional structure of ECM reversibly regulates the morphology, proliferation rate and functions of the perisinusoidal stellate cells.

Pharmacological preconditioning protects lung injury induced by intestinal ischemia/reperfusion in rat

Intestinal ischemia/reperfusion (I/R) is a critical and triggering event in the development of distal organ dysfunction, frequently involving the lungs. Respiratory failure is a common cause of death and complications after intestinal I/R. Stress protein heme oxygenase-1 (HO-1) confers the protection against a variety of oxidant-induced cell and tissue injuries. The aim of this study was to investigate the hypothesis that the induced HO-1 expression by pharmacological preconditioning with anticancer drug doxorubicin (Dox) could protect the lung injury induced by intestinal I/R. Intravenous administration of Dox induced HO-1 expression in the lungs and high levels of the expression were sustained at least to 48 h after the injection. Therefore, as pharmacological preconditioning, a low dose of Dox was injected intravenously into rats at 48 h before the start of intestinal ischemia. Rats underwent intestinal I/R by superior mesenteric artery occlusion for 120 min followed by 120 min of reperfusion. Preconditioning with Dox significantly ameliorated the lung injury induced by the intestinal I/R. Administration of a specific inhibitor of HO activity reduced the efficacy of the preconditioning. Our results suggest that this improvement may be mediated at least in part by the HO-1 induction. These findings may offer interesting perspectives for patient management in intestinal surgical operation and intestine transplantation.

Induction of cellular processes containing collagenase and retinoid by integrin-binding to interstitial collagen in hepatic stellate cell culture

Cultered hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively. The process induction was inhibited by several reagents as follows: (1) anti‐integrin α2 antibody; (2) an oligopeptide, DGEA, an integrin‐binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3‐kinase inhibitor. Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel. Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin. Thus, the process extension was found to depend on integrin‐binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly. The cellular processes included interstitial collagenase and vitamin A‐containing lipid droplets. The lipid droplets and vitamin A‐autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function.

Storage of Lipid Droplets in and Production of Extracellular Matrix by Hepatic Stellate Cells (Vitamin A- Storing Cells) in Long-Evans Cinnamon-Like Colored (LEC) Rats

LEC rats spontaneously develop hepatocellular carcinoma with cholangiofibrosis after chronic hepatitis, but the mechanism of development of the hepatic injury is not clear. To investigate the role of hepatic stellate cells in induction or suppression of hepatic fibrosis, we morphologically examined the liver of LEC rats. Accumulation of copper was analyzed by the Danscher‐Timms sulfide‐silver method. Histopathological changes were evaluated by hematoxylin and eosin staining, and by Massons trichrome method. Activated stellate cells were identified by immunostaining method for α‐smooth muscle actin. Cytological alterations of the stellate cells were investigated by transmission electron microscopy. To evaluate the lipid content in the stellate cells, we analyzed the area of lipid droplets of the cells by morphometric analysis. Also for evaluation of the changes in the number of stellate cells, the numbers of nucleated stellate cells and parenchymal cells were counted and statistically analyzed. Hepatic parenchymal cells showed excessive accumulation of copper at 5 weeks of age. Submassive necrosis was observed at 19 weeks of age. The liver of LEC rats 1.5 years of age showed cholangiofibrosis and subcellular injury of hepatic parenchymal cells. However, no diffuse hepatic fibrosis was observed in the liver, and hepatic stellate cells around the regions of cholangiofibrosis were negative for α‐smooth muscle actin. The area of lipid droplets of a stellate cell in the liver of LEC rats was 1.6 to 1.8 times as large as that of normal Wistar rats. The hepatic stellate cells did not participate in the accumulation of collagen fibers around themselves when the cells contained a large amount of vitamin A‐lipid droplets, even though the development of hepatic lesions was in progress. Our present data are consistent with our previous hypothesis that there is an antagonistic relationship between the storage of vitamin A and the production of collagen in stellate cells. Anat Rec 258:338–348, 2000.

Hepatic stellate cells (vitamin A-storing cells) change their cytoskeleton structure by extracellular matrix components through a signal transduction system

Abstract When cultured on a polystyrene surface or aminoalkylsilane-coated cover glasses, rat and human hepatic stellate cells exhibit a flattened, fibroblast-like shape with well-developed stress fibers. However, culturing the cells on type I collagen gel results in the elongation of long, multipolar cellular processes, whereas cells cultured on Matrigel maintain their round shapes. Dual fluorescence staining of microtubules and fibrillar actin indicated that the processes extend together with collagen fibers and contained microtubules as the core, whereas the periphery contained fibrillar actin. Immunofluorescence staining of vinculin showed that the focal adhesions were distributed mainly in lamellipodia when cultured on aminoalkylsilane-coated cover glasses, whereas in the cells cultured on type I collagen gel they were localized to the tips of the processes and along their bottom surface contacting collagen fibers. Wortmannin, as well as staurosporin and herbimycin A, inhibited the elongation process and induced the retraction of elongated processes. The wortmannin treatment also resulted in an alteration in focal adhesion distribution from the processes to cell bodies. These results indicate that the cell surface integrin binding to interstitial collagen fibers induces the elongation of processes through signaling events and the subsequent cytoskeleton assembly in hepatic stellate cells.

Cell–cell junctions between mammalian (human and rat) hepatic stellate cells

To investigate intercellular junctions between mammalian hepatic stellate cells, we examined cultured human and rat hepatic stellate cells at the ultrastructural and molecular levels. Intercellular junctions between cultured human stellate cells, which developed irrespective of the type of culture substratum, were detected by transmission electron microscopy. On the basis of their characteristic ultrastructure, these junctions were identified in cultured human hepatic stellate cells as adherens junctions but not as tight junctions, desmosomes, or gap junctions. N-cadherin, α-catenin and β-catenin, and p120ctn were detected by Western blotting in rat stellate cells as molecular components of the intercellular adhesive structures. Immunofluorescence for pan-cadherin, α-catenin, and β-catenin were also detected in cultured human stellate cells. Moreover, pan-cadherin and β-catenin were co-localized at the contact regions between the cultured human stellate cells. These data suggest that the junctional adhesion between the stellate cells can be formed both in vivo and in vitro. Thus, hepatic stellate cells may participate in the structural organization of the cells in liver lobules through the formation of intercellular adherens junctions. This is the first description of the presence of cell–cell junctions between hepatic stellate cells in mammals at the fine structural and molecular levels.

Insoluble, speckled cytosolic distribution of retinoic acid receptor alpha protein as a marker of hepatic stellate cell activation in vitro.

Hepatic stellate cells (HSCs) are the major site of retinoid storage, and their activation is a key process in liver fibrogenesis. We have previously shown that expression of the retinoic acid receptor alpha (RARα) is upregulated in activated rat HSCs at a post-transcriptional level and that these RARα proteins showed a speckled distribution in the cytosol, despite their possession of a nuclear localization signal (NLS). In this report, we further characterize these cytosolic RARα proteins by using exogenously expressed RARα protein fragments or mutants tagged with a green fluorescent protein. Substitution of four amino acids, 161–164 from lysine to alanine, abolished the NLS. Exogenously expressed RARα protein fragments containing an NLS were localized exclusively in the nuclei of activated rat HSCs and never colocalized with the endogenous RARα proteins in the cytosol, suggesting that the NLS of endogenous RARα proteins is masked. Biochemical analysis showed that 65% of RARα proteins in activated HSCs were insoluble in a mixture of detergents. The insolubility of RARα proteins makes it difficult to identify RARα proteins in activated HSCs. Therefore, we propose that insoluble, speckled cytosolic distribution of RARα proteins represents a new marker of HSC activation. (J Histochem Cytochem 57:687–699, 2009)

Correlation between the expression of methionine adenosyltransferase and the stages of human colorectal carcinoma

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet) from ATP and l-methionine. AdoMet is the major methyl donor in most transmethylation reactions in vivo, and it is also the propylamino donor in the biosynthesis of polyamines. In the present study, we assessed MAT activity in human colons with colorectal carcinoma and the values were compared with those of morphologically normal adjacent mucosa. Higher levels of MAT activity were observed in the colorectal carcinoma than in the normal colon. The ratio of MAT activity in tumor tissue versus normal tissue seemed to be correlated well will the stage of the colorectal tumor. Furthermore, immunoblot analysis showed that the high levels of MAT activity observed in colorectal carcinoma were due to the increased amounts of MAT protein. Immunohistochemical analysis revealed that MAT was most abundant in goblet cells, particularly in granules in the supranuclear area of these cells. In the colorectal carcinoma tissues, MAT was strongly stained in the cancerous cells and localized in granules in the supranuclear region. The results of this preliminary study suggest that determination of the relative ratio of MAT activity in both normal and tumor regions in human colorectal carcinoma could be a clinically useful tool for determining the stage of malignancy of colorectal carcinomas.

Onset of Apoptosis in the Cystic Duct During Metamorphosis of a Japanese Lamprey, Lethenteron reissneri

A nonparasitic lamprey in Japan, Lethenteron reissneri, stops feeding prior to the commencement of metamorphosis. Resumption of feeding cannot take place due to major alterations in the digestive system, including loss of the gall bladder (GB) and biliary tree in the liver. This degeneration of bile ducts is considered to depend on programmed cell death or apoptosis, but molecular evidence of apoptosis remains lacking. Using terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining and immunohistochemistry with an antibody against active caspase‐3, we showed that epithelial cells of the cystic duct (CD) and GB became TUNEL‐positive by the early metamorphosing stage. Immunohistochemical staining of active caspase‐3, a key mediator in the apoptotic cascade, showed that the apoptotic signal was initiated in the region around the CD in the late larval phase. In later stages, active caspase‐3‐positive epithelial cells were also observed in the large intrahepatic bile duct (IHBD) and peripheral small IHBDs. At the early metamorphosing stage, bile canaliculi between hepatocytes were dilated and displayed features resembling canaliculi in cholestasis. Onset of apoptosis around the CD, which is the pathway for the storage of bile juice, and progression of apoptosis towards the large IHBD, which is the pathway for the secretion of bile juice, may lead to temporary intrahepatic cholestasis. The present study represents the first precise spatial and temporal analysis of apoptosis in epithelial cells of the biliary tract system during metamorphosis of any lamprey species. Anat Rec 293:1155–1166, 2010.