Preservation of collagen in the soft tissues of frozen mammoths.

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.

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.

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.

Uptake and storage of vitamin A as lipid droplets in the cytoplasm of cells in the lamina propria mucosae of the rat intestine.

Vitamin A (retinyl palmitate) was injected subcutaneously or administered to rats by tube feeding. After subcutaneous injection, vitamin A was taken up and stored in cells of the lamina propria mucosae of the rat intestine. After oral administration, vitamin A was absorbed by the intestinal absorptive epithelial cells and transferred to cells of the lamina propria mucosae, where cells took up and stored the transferred vitamin A. The morphology of these cells was similar to that of hepatic stellate cells (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells). Thus, these cells in the intestine could take up vitamin A from the systemic circulation and as well as by intestinal absorption, and store the vitamin in the lipid droplets in their cytoplasm. The data suggest that these cells are extrahepatic stellate cells of the digestive tract that may play roles in both the absorption and homeostasis of vitamin A.

Accumulation of vitamin A in the hepatic stellate cell of arctic top predators.

We performed a systematic characterization of the hepatic vitamin A storage in mammals and birds of the Svalbard Archipelago and Greenland. The liver of top predators, including polar bear, Arctic fox, bearded seal, and glaucous gull, contained about 10-20 times more vitamin A than the liver of all other arctic animals studied, as well as their genetically related continental top predators. The values are also high compared to normal human and experimental animals like mouse and rat. This massive amount of hepatic vitamin A was located in large autofluorescent lipid droplets in hepatic stellate cells (HSCs; also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, or Ito cells). The droplets made up most of the cells' cytoplasm. The development of such an efficient vitamin A-storing mechanism in HSCs may have contributed to the survival of top predators in the extreme environment of the arctic. These animals demonstrated no signs of hypervitaminosis A. We suggest that HSCs have capacity to take-up and store large amounts of vitamin A, which may play a pivotal role in maintenance of the food web, food chain, biodiversity, and eventually ecology of the arctic.

Elevated expression of transforming growth factor ß3 in carbon tetrachloride-treated rat liver and involvement of retinoid signaling.

Transforming growth factor (TGF) ß is a pro-fibrotic cytokine. While three isoforms (TGF-ß1, 2 and 3) are known, the functional differences between them are obscure. To investigate the roles of TGF-ß 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-ß 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-ß3 protein was found to be localized mainly in liver parenchymal cells (hepatocytes). These results indicate that retinoid mobilization likely takes place within the rat's 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.

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.

Induction of an incomplete autophagic response by cancer-preventive geranylgeranoic acid (GGA) in a human hepatoma-derived cell line.

GGA (geranylgeranoic acid) is a natural polyprenoic acid, derivatives of which has been shown to prevent second primary hepatoma. GGA induces mitochondria-mediated PCD (programmed cell death), which may be relevant to cancer prevention. To gain further insights into GGA-induced PCD, autophagy processes were examined in human hepatoma-derived HuH-7 cells. Treatment of HuH-7/GFP (green fluorescent protein)-LC3 cells with GGA induced green fluorescent puncta in the cytoplasm within 30 min and their massive accumulation at 24 h. After 15 min of GGA treatment, a burst of mitochondrial superoxide production occurred and LC3ß-I was appreciably converted into LC3ß-II. GGA-induced early stages of autophagy were unequivocally confirmed by electron-microscopic observation of early/initial autophagic vacuoles. On the other hand, LC3ß-II as well as p62/SQSTM1 (sequestosome 1) continuously accumulated and co-localized in the cytoplasmic puncta after GGA treatment. Furthermore, GGA treatment of HuH-7/mRFP (monomeric red fluorescent protein)-GFP-LC3 cells showed yellow fluorescent puncta, whereas glucose deprivation of the cells gave red fluorescent puncta. These results strongly suggest that GGA induces the initial phase of autophagy, but blocks the maturation process of autolysosomes or late stages of autophagy, insomuch that GGA provides substantial accumulation of autophagosomes under serum-starvation conditions in human hepatoma cells.

Antiproliferative and proapoptotic effects of tocopherol and tocol on activated hepatic stellate cells.

Activated hepatic stellate cells (HSCs) play crucial roles in liver fibrosis. In the course of liver injury, HSCs, which reside in perisinusoidal spaces and lose lipid droplets, morphologically change into a myofibroblastic phenotype and acquire an increased proliferation activity in what is known as the activated state. We have investigated therapeutic strategies for liver fibrosis by promoting spontaneous reversion or inducing apoptosis in activated HSCs. Vitamin E consists of four tocopherols and four tocotrienols, all of which are well-known antioxidants. In this study, the antiproliferative and proapoptotic effects of a tocol, which lacks methyl groups attached to the chromanol ring, and four tocopherols were investigated using activated HSCs. δ-Tocopherol and tocol exhibited relatively high proliferation inhibitory and proapoptotic abilities. However, they did not show proliferation inhibition ability on primary hepatocytes or HepG2 cells. Significant cell detachment was also observed in δ-tocopherol- and tocol-treated HSCs. Decreased protein expressions of α-smooth muscle actin and ß1 integrin were observed in a dose-dependent manner. These results indicate that δ-tocopherol and tocol induce anoikis in activated HSCs.

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.

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.

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 (RARalpha) is upregulated in activated rat HSCs at a posttranscriptional level and that these RARalpha 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 RARalpha proteins by using exogenously expressed RARalpha 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 RARalpha protein fragments containing an NLS were localized exclusively in the nuclei of activated rat HSCs and never colocalized with the endogenous RARalpha proteins in the cytosol, suggesting that the NLS of endogenous RARalpha proteins is masked. Biochemical analysis showed that 65% of RARalpha proteins in activated HSCs were insoluble in a mixture of detergents. The insolubility of RARalpha proteins makes it difficult to identify RARalpha proteins in activated HSCs. Therefore, we propose that insoluble, speckled cytosolic distribution of RARalpha proteins represents a new marker of HSC activation.

Rat hepatic stellate cells acquire retinoid responsiveness after activation in vitro by post-transcriptional regulation of retinoic acid receptor alpha gene expression.

Activation of hepatic stellate cells (HSCs) is a key process in liver fibrogenesis and retinoid loss is a remarkable feature of activated HSCs. However, roles of retinoids in liver fibrogenesis are obscure. We show that mRNA levels of RARalpha, beta and gamma were decreased during rat HSC activation in vitro. However, protein levels of RARalpha and beta were increased during HSC activation. A retinoic acid response element-containing luciferase assay indicated that HSCs became responsive to retinoids only after activation in vitro and that this response was mediated by, at least in part, RARalpha subtype. Immunocytochemical analysis showed that RARalpha proteins were mainly distributed in cytosol as many spots. All-trans retinoic acid treatment strongly lowered the cytosolic RARalpha protein levels. These results indicate that rat HSCs become retinoid responsive after activation in vitro, through post-transcriptional up-regulation of RARalpha gene expression.

Distribution of retinylester-storing stellate cells in the arrowtooth halibut, Atheresthes evermanni.

Hepatic stellate cells play a major role in retinylester storage in mammals, but the retinoid-storing state in nonmammalian vertebrates remains to be elucidated. In this study, we examined retinoids and retinoid-storing cells in the arrowtooth halibut, Atheresthes evermanni. High-performance liquid chromatography analyses revealed the highest concentrations of stored retinoids (retinol and retinylester, 6199 nmol/g) in the pyloric cecum, a teleost-specific organ protruding from the intestine adjacent to the pylorus. Considerable amounts of retinoids were also stored in the intestine (3355 nmol/g) and liver (1891 nmol/g), and small amounts in the kidney (102 nmol/g). Very small amounts or no retinoids were detected in the heart, gill, skeletal muscle, and gonads (less than 2 nmol/g). Use of gold chloride staining and fluorescence microscopy to detect retinoid autofluorescence showed that, in the pyloric cecum and intestine, retinoid-storing cells were localized in the lamina propria mucosae. Under electron microscopy, cells containing well-developed lipid droplets, which are common morphological characteristics of the hepatic stellate cells of mammals, were observed in the lamina propria mucosae of the pyloric cecum. Thus, the distribution of stellate cells with retinoid-storing capacity differs between this halibut and mammals, suggesting that the retinoid-storing site has shifted during vertebrate evolution.

Further application of a two-step heparin affinity chromatography method using divalent cations as eluents: purification and identification of membrane-bound heparin binding proteins from the mitochondrial fraction of HL-60 cells.

Membrane proteins were obtained from the mitochondrial fraction of HL-60 cells by solubilization with octyl glucoside and bound to heparin-gels. Bound proteins were successively eluted with solutions containing increasing concentrations of Mg(2+) in the first and increasing concentrations of Ca(2+) in the second chromatography. After SDS-PAGE and subsequent N-terminal amino acid analysis of proteins on each band, 13 proteins were identified. Fifteen out of the 37 proteins analysed were modified at their N-termini. These results show that this two-step affinity chromatography method using divalent cations as eluents can be applied to a variety of membranes for the isolation of specific proteins.

Degradation of extracellular matrix by extrahepatic stellate cells in the intestine of the lamprey, Lampetra japonica.

To investigate the mechanisms involved in the atrophy of the intestines in lampreys (Lampetra japonica) during the spawning migration stage, we examined by morphological methods their intestines with special reference to degradation of extracellular matrix (ECM) components. Stellate cells are known to be distributed not only in the liver (hepatic stellate cells) but also in other organs, such as the pancreas, intestine, lung, and kidney (extrahepatic stellate cells). Hepatic stellate cells are well known to be able to biosynthesize, secrete, and degrade ECM. Therefore, we investigated the cellular and molecular mechanisms involved in the atrophy of the intestines by focusing on these intestinal extrahepatic stellate cells. The cells were found to contain phagocytosed and degraded collagen fibrils, which are one of the ECM components. A positive reaction for trimetaphosphatase (TMPase, a cytochemical marker of lysosomes) was preferentially detected in round or elongated vesicles in the intestinal extrahepatic stellate cells and the deposits of the reaction products coexisted with the degraded collagen fibrils. However, the basement membrane of the intestine, which membrane is also an ECM component, was preserved throughout the spawning migration stage of the lamprey and accumulated as a mass of thick membrane, suggesting the existence of a special mechanism for selective digestion of ECM components. These results indicate that the intestinal extrahepatic stellate cells in Lampetra japonica during its spawning migration stage might play an important mechanistic role in the atrophy of lamprey intestines by phagocytizing collagen fibrils and digesting the phagocytized collagen fibrils in their lysosomes.

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, alpha-catenin and beta-catenin, and p120ctn were detected by Western blotting in rat stellate cells as molecular components of the intercellular adhesive structures. Immunofluorescence for pan-cadherin, alpha-catenin, and beta-catenin were also detected in cultured human stellate cells. Moreover, pan-cadherin and beta-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.

Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells.

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.

3-D structure of extracellular matrix regulates gene expression in cultured hepatic stellate cells to induce process elongation.

HSCs showed myofibroblast-like shapes when cultured on polystyrene surface or on type I collagen-coated surface, whereas HSCs cultured on type I collagen gel were induced to elongate cellular processes, suggesting that HSCs recognize 3-D structure of extracellular type I collagen fibrils and change their morphology and function. In this study we examined the differentially regulated gene expression by extracellular matrix (ECM) components by PCR-differential display (PCR-DD) analysis followed by cloning and FASTA homology search, and identified the mRNA species as a transcription factor SP1, breast cancer resistant protein (BCRP), dystonin, and KAP3B. Regulation of dystonin and KAP3B expression was confirmed by RT-PCR analysis. Thus, cell surface-binding to extracellular interstitial collagen may trigger intracellular signaling and alteration in gene expression, and HSCs not only produce various ECM components but also change their morphology and gene expression in response to ECM components adhering to the cells.

Intercellular Adhesive Structures Between Stellate Cells - An Analysis in Cultured Human Hepatic Stellate Cells.

To investigate whether or not hepatic stellate cells can form intercellular junctions with each other, we cultured human stellate cells (LI90) on different kinds of substrata. Intercellular junctions were detected between these cultured stellate cells by transmission electron microscopy (TEM). The molecular components of the intercellular adhesive structures were identified by immunofluorescence microscopy. Immunofluorescence for cadherin and catenins was detected at the adhesion sites between the cultured stellate cells. Thus, the intercellular junctions were indicated to be adherens junctions at the molecular level. The junctions developed in the cultured stellate cells irrespective of the type of substratum. These data suggest that the junctional formation between the stellate cells occurs in vivo as well as in vitro.