Kiwamu Yoshikawa

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.

Acute effects on the lung and the liver of oral administration of cerium chloride on adult, neonatal and fetal mice

We evaluated tissue changes associated with cerium chloride administration via gavage to adult mice, via milk to neonatal mice and transplacentally to fetal mice. Change in adults consisted of extensive pulmonary hemorrhage, pulmonary venous congestion, thickened alveolar septae, hepatic necrosis and neutrophil infiltrations. Those in fetal mice consisted of pulmonary and hepatic congestion. These results indicate that gavage cerium administration elicited subtle tissue changes, though oral toxicity is rather low. These changes were less severe in neonatal and fetal mice. When cerium was injected into adult mice through the tail vein, cerium was distributed mainly to the liver, spleen and lung dose-dependently with the cerium concentration gradually decreasing after 3 days. A study of cerium anticoagulation in mouse plasma showed that clotting time was significantly prolonged when cerium was added to plasma. These results suggest that cerium may disturb blood coagulation and cause pulmonary and hepatic vascular congestion.

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)

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.

Characterization of a cellular retinol-binding protein from lamprey, Lethenteron japonicum.

Lampreys are ancestral representatives of vertebrates known as jawless fish. The Japanese lamprey, Lethenteron japonicum, is a parasitic member of the lampreys known to store large amounts of vitamin A within its body. How this storage is achieved, however, is wholly unknown. Within the body, the absorption, transfer and metabolism of vitamin A are regulated by a family of proteins called retinoid-binding proteins. Here we have cloned a cDNA for cellular retinol-binding protein (CRBP) from the Japanese lamprey, and phylogenetic analysis suggests that lamprey CRBP is an ancestor of both CRBP I and II. The lamprey CRBP protein was expressed in bacteria and purified. Binding of the lamprey CRBP to retinol (Kd of 13.2 nM) was identified by fluorimetric titration. However, results obtained with the protein fluorescence quenching technique indicated that lamprey CRBP does not bind to retinal. Northern blot analysis showed that lamprey CRBP mRNA was ubiquitously expressed, although expression was most abundant in the intestine. Together, these results suggest that lamprey CRBP has an important role in absorbing vitamin A from the blood of host animals.

Elevated expression of transforming growth factor β3 in carbon tetrachloride-treated rat liver and involvement of retinoid signaling

Transforming growth factor (TGF) β is a pro-fibrotic cytokine. While three isoforms (TGF-β1, 2 and 3) are known, the functional differences between them are obscure. To investigate the roles of TGF-β isoforms during liver fibrogenesis, male Wistar rats were administrated carbon tetrachloride (CCl4) subcutaneously twice a week for two months. Livers were excised and sectioned for histochemical examinations. These livers were also used to quantitate the expression of genes associated with fibrogenesis, including TGF-β isoforms, as well as those associated with retinoid metabolism. Expression levels of Tgfb1 and Tgfb3 were up-regulated in CCl4-treated rat livers while that of Tgfb2 was not changed. The mRNAs for lecithin-retinol acyltransferase (Lrat) and retinoic acid hydroxylase, Cyp26a1, were also elevated. By immunohistochemical staining, TGF-β3 protein was found to be localized mainly in liver parenchymal cells (hepatocytes). These results indicate that retinoid mobilization likely takes place within the rats liver following CCl4 treatment, and suggest the possibility that the expression of Tgfb mRNA is regulated by retinoic acid receptors. Reporter analyses of a region of the Tgfb3 gene were performed using the rat liver parenchymal cell line, RLC-16, and a positively responsive region was identified within its intron.

Differential Increases in the Expression of Intermediate Filament Proteins and Concomitant Morphological Changes of Transdifferentiating Rat Hepatic Stellate Cells Observed In Vitro

The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.

Massive bowel resection upregulates the intestinal mRNA expression levels of cellular retinol-binding protein II and apolipoprotein A-IV and alters the intestinal vitamin A status in rats

Short bowel (SB) syndrome causes the malabsorption of various nutrients. Among these, vitamin A is important for a number of physiological activities. Vitamin A is absorbed by epithelial cells of the small intestine and is discharged into the lymphatic vessels as a component of chylomicrons and is delivered to the liver. In the present study, we used a rat model of SB syndrome in order to assess its effects on the expression of genes associated with the absorption, transport and metabolism of vitamin A. In the rats with SB, the intestinal mRNA expression levels of cellular retinol-binding protein II (CRBP II, gene symbol Rbp2) and apolipoprotein A-IV (gene symbol Apoa4) were higher than those in the sham-operated rats, as shown by RT-qPCR. Immunohistochemical analysis revealed that absorptive epithelial cells stained positive for both CRBP II and lecithin retinol acyltransferase, which are both required for the effective esterification of vitamin A. In the rats with SB, the retinol content in the ileum and the retinyl ester content in the jejunum were lower than those in the sham-operated rats, as shown by quantitative analysis of retinol and retinyl esters by high performance liquid chromatography. These results suggest that the elevated mRNA expression levels of Rbp2 and Apoa4 in the rats with SB contribute to the effective esterification and transport of vitamin A.

The role of retinoic acid receptors in activated hepatic stellate cells

Hepatic stellate cells (HSCs), also known as Ito cells, fat-storing cells, vitamin A-storing cells or lipocytes, reside in the spaces between hepatocytes and liver sinusoids. Vitamin A storage within the HSCs is achieved through the cooperative action of two proteins, cellular retinol-binding protein (CRBP) I and lecithin:retinol acyltransferase (LRAT). After the discovery that HSCs are responsible not only for the storage of vitamin A, but also for the development of liver fibrosis and subsequent liver cirrhosis, HSCs have been considered a therapeutic target for prevention or reversal of liver fibrogenesis. We have reported that HSCs acquire retinoid responsiveness after in vitro activation by post-transcriptional upregulation of retinoic acid receptor α gene expression. Here we extend this observation in relation to the functions of CRBP I and LRAT, and propose a hypothesis that increased retinoid signaling in activated HSCs forms a feedback loop toward vitamin A restoration in the liver.

How Do Lampreys Avoid Cholestasis After Bile Duct Degeneration

Biliary atresia (BA) is the most common cause of cholestasis during infancy for which an etiology remains undetermined. Patients require hepatic portoenterostomy within the first 2-3 months of life in order to restore bile flow from the liver into the intestinal tract. Even with successful surgery, in most patients the disease advances to end-stage cirrhosis due to the progressive destruction of bile ducts, and requires liver transplantation in order for long term survival to be viable (Mack & Sokol, 2005). Without a better understanding of the etiology and pathogenesis of the progressive cholangitic mechanisms in BA, improvement in the prognosis of non-transplantation patients should not be expected. Despite the importance of understanding these underlying mechanisms, research of BA has been hindered by the lack of a suitable animal model.