Dynamically Programmed Switchable DNA Hydrogels Based on a DNA Circuit Mechanism.

Biological stimuli-responsive DNA hydrogels have attracted much attention in the field of medical engineering owing to their unique phase transitions from gel to sol through cleavage of DNA cross-linking points in response to specific biomolecular inputs. In this paper, a new class of biological stimuli-responsive DNA hydrogels with a dynamically programmed DNA system that relies on a DNA circuit system through cascading toehold-mediated DNA displacement reactions is constructed, allowing the catalytic cleavage of cross-linking points and main chains in response to an appropriate DNA input. The dynamically programmed DNA hydrogels exhibit a significant sharp phase transition from gel to sol in comparison to another DNA hydrogel showing noncatalytic cleavage of cross-linking points due to synchronization of the catalytic cleavage of cross-linking points and the main chains. Further, the sol-gel phase transitions of the DNA hydrogels in response to the DNA input are easily tunable by changing the cross-linking density. Additionally, with a structure-switching aptamer, DNA hydrogels encapsulating PEGylated gold nanoparticles can be used as enzyme-free signal amplifiers for the colorimetric detection of adenosine 5'-triphosphate (ATP); this detection system provides simplicity and higher sensitivity (limit of detection: 5.6 × 10-6 m at 30 min) compared to other DNA hydrogel-based ATP detection systems.

Attenuation of acute and chronic liver injury in rats by iron-deficient diet.

Oxidative stress due to iron deposition in hepatocytes or Kupffer cells contributes to the initiation and perpetuation of liver injury. The aim of this study was to clarify the association between dietary iron and liver injuries in rats. Liver injury was initiated by the administration of thioacetamide or ligation of the common bile duct in rats fed a control diet (CD) or iron-deficient diet (ID). In the acute liver injury model induced by thioacetamide, serum levels of aspartate aminotransferase and alanine aminotransferase, as well as hepatic levels of lipid peroxide and 4-hydroxynonenal, were significantly decreased in the ID group. The expression of 8-hydroxydeoxyguanosine and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling positivity showed a similar tendency. The expression of interleukin-1beta and monocyte chemotactic protein-1 mRNA was suppressed in the ID group. In liver fibrosis induced by an 8-wk thioacetamide administration, ID suppressed collagen deposition and smooth muscle alpha-actin expression. The expressions of collagen 1A2, transforming growth factor beta, and platelet-derived growth factor receptor beta mRNA were all significantly decreased in the ID group. Liver fibrosis was additionally suppressed in the bile-duct ligation model by ID. In culture experiments, deferoxamine attenuated the activation process of rat hepatic stellate cells, a dominant producer of collagen in the liver. In conclusion, reduced dietary iron is considered to be beneficial in improving acute and chronic liver injuries by reducing oxidative stress. The results obtained in this study support the clinical usefulness of an iron-reduced diet for the improvement of liver disorders induced by chronic hepatitis C and alcoholic/nonalcoholic steatohepatitis.

Erythrophagocytosis by liver macrophages (Kupffer cells) promotes oxidative stress, inflammation, and fibrosis in a rabbit model of steatohepatitis: implications for the pathogenesis of human nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. Here, we report a molecular aspect of this disease elucidated using rabbits fed a cholesterol-rich high-fat diet and exhibiting insulin resistance. The liver in this model showed steatohepatitis with fibrosis and high mRNA expression for some cytokines, heme oxygenase-1, transforming growth factor-beta1, and collagen alpha1(I). Erythrocytes isolated from the model showed marked fragility and the externalization of phosphatidylserine (PS) on the outer leaflet of the membrane and were frequently engulfed by Kupffer cells/macrophages in the hepatic sinusoids. Expression of milk fat globule-epidermal growth factor (EGF)-factor 8, a PS-binding protein, was augmented in the liver. In culture, RAW 264.7 cells engulfed erythrocytes oxidized by tert-butyl hydroperoxide, a process that was inhibited by anti-milk fat globule-EGF-factor 8 antibody. In addition, PS-positive erythrocytes appeared entrapped in the model liver in ex vivo perfusion experiments. Finally, in specimens from NASH patients, the aggregation of erythrocytes in inflammatory hepatic sinusoids was notable. These results indicate that the engulfment of PS-externalized, apoptotic signal-positive, erythrocytes by hepatic macrophages may lead to the deposition of iron derived from hemoglobin in the liver and be involved in the pathogenesis of steatohepatitis.

Rho kinase inhibitor Y27632 affects initial heart myofibrillogenesis in cultured chick blastoderm.

During early vertebrate development, Rho-associated kinases (ROCKs) are involved in various developmental processes. Here, we investigated spatiotemporal expression patterns of ROCK1 protein and examined the role of ROCK during initial heart myofibrillogenesis in cultured chick blastoderm. Immunohistochemistry showed that ROCK1 protein was distributed in migrating mesendoderm cells, visceral mesoderm of the pericardial coelom (from which cardiomyocytes will later develop), and cardiomyocytes of the primitive heart tube. Pharmacological inhibition of ROCK by Y27632 did not alter the myocardial specification process in cultured posterior blastoderm. However, Y27632 disturbed the formation of striated heart myofibrils in cultured posterior blastoderm. Furthermore, Y27632 affected the formation of costamere, a vinculin/integrin-based rib-like cell adhesion site. In such cardiomyocytes, cell-cell adhesion was disrupted and N-cadherin was distributed in the perinuclear region. Pharmacological inactivation of myosin light chain kinase, a downstream of ROCK, by ML-9 perturbed the formation of striated myofibrils as well as costameres, but not cell-cell adhesion. These results suggest that ROCK plays a role in the formation of initial heart myofibrillogenesis by means of actin-myosin assembly, and focal adhesion/costamere and cell-cell adhesion.

[Dissection of the heart: a useful guide to understanding the three-dimensional gross anatomy].

In order to understand the three-dimensional gross anatomy of the heart, it is important to observe the inner structures of the chambers and the spatial relation between the valves and the ventricles. In our dissecting laboratory, we designed a guide to dissection of the heart according to the following procedures. First, we observe the surface anatomy of the heart in the pericardial cavity, remove the heart and then identify the coronary vessels, open the four chambers and observe the intra-cardiac structures. Next, we remove the atria from the ventricles and examine the relation between the valves and the chambers. Our guide is useful for learning the three-dimensional gross anatomy which is fundamental to understanding the normal function and disease of the heart.

Derivation and characterization of hepatocytes from embryonic stem cells in vitro.

From a therapeutic perspective on liver, the use of embryonic stem (ES) technology in the generation of a large number of high-functional hepatocytes developed from ES cells for cell transplantation is anticipated. We have explored a three-dimensional culture system in which hepatocytes differentiated from mouse ES cells by transfection with the hepatocyte nuclear factor-3beta possess high metabolic functions that can be maintained long term.

Expression of NCAM in activated portal fibroblasts during regeneration of the rat liver after partial hepatectomy.

In the portal tract of the regenerating liver after partial hepatectomy, vascular and bile ductular remodeling takes place in response to the portal hyperdynamic state and parenchymal hyperplasia. In order to reveal phenotypical changes in the portal fibroblasts, we immunohistochemically investigated neural cell adhesion molecules (NCAM) and alpha smooth muscle actin (alphaSMA) expression and the ultrastructural changes in them during liver regeneration. In the control rat liver, portal fibroblasts were negative for both NCAM and alphaSMA. They became positive for both markers two days after partial hepatectomy, increased in staining intensity, reached a maximum at three to four days, then decreased, being still clearly positive at 14 days. Under an electron microscope, portal fibroblasts from the regenerating liver had larger amounts of cytoplasm and rough endoplasmic reticulum than those from the control liver; thus they might be activated. Additionally, periportal hepatic stellate cells in the regenerating liver were activated with alphaSMA, but without NCAM. The present study has demonstrated that portal fibroblasts express NCAM and alphaSMA in the regenerating liver after partial hepatectomy via transformation into myofibroblasts following reconstruction of the portal tracts.

Rho kinases regulate endothelial invasion and migration during valvuloseptal endocardial cushion tissue formation.

Rho-associated kinase (ROCK) is a downstream effector of small Rho-GTPases, and phosphorylates several substrates to regulate cell functions, including actin cytoskeletal reorganization and cellular motility. Endothelial-mesenchymal transformation (EMT) is a critical event in the formation of valves and septa during cardiogenesis. It has been reported that ROCK plays an important role in the regulation of endocardial cell differentiation and migration during mouse cardiogenesis (Zhao and Rivkees [2004] Dev. Biol. 275:183-191). Immunohistochemistry showed that, during chick cardiogenesis, ROCK1 and -2 were expressed in the transforming and migrating endothelial/mesenchymal cells in the outflow tract (OT) and atrioventricular (AV) canal regions from which valvuloseptal endocardial cushion tissue would later develop. Treatment with Y27632, a specific ROCK inhibitor, of cultured AV explants or AV endothelial monolayers of stage 14-minus heart (preactivated stage for EMT) on three-dimensional collagen gel perturbed the seeding of mesenchymal cells into the gel lattice. In these experiments, Y27632 did not suppress the expression of an early transformation marker, smooth muscle alpha-actin. Moreover, Y27632 inhibited the mesenchymal invasion in stage 14-18 AV explants, in which endothelial cells had committed to undergo EMT. ML-9, a myosin light chain kinase inhibitor, also inhibited the mesenchymal invasion in cultured AV explants. These results suggest that ROCKs have a critical role in the mesenchymal cell invasion/migration that occurs at the late onset of EMT.

Heart myofibrillogenesis occurs in isolated chick posterior blastoderm: a culture model.

Early cardiogenesis including myofibrillogenesis is a critical event during development. -Recently we showed that prospective cardiomyocytes reside in the posterior lateral blastoderm in the chick embryo. Here we cultured the posterior region of the chick blastoderm in serum-free medium and observed the process of myofibrillogenesis by immunohistochemistry. After 48 hours, explants expressed sarcomeric proteins (sarcomeric alpha-actinin, 61%; smooth muscle alpha-actin, 95%; Z-line titin, 56%; sarcomeric myosin, 48%); however, they did not yet show a mature striation. After 72 hours, more than 92% of explants expressed I-Z-I proteins, which were incorporated into the striation in 75% of explants or more (sarcomeric alpha-actinin, 75%; smooth muscle alpha-actin, 81%; Z-line titin, 83%). Sarcomeric myosin was -expressed in 63% of explants and incorporated into A-bands in 37%. The percentage incidence of expression or striation of I-Z-I proteins was significantly higher than that of sarcomeric myosin. Results suggested that the nascent I-Z-I components appeared to be generated independently of A-bands in the cultured posterior blastoderm, and that the process of myofibrillogenesis observed in our culture model faithfully reflected that in vivo. Our blastoderm culture model appeared to be useful to investigate the mechanisms regulating the early cardiogenesis.

Induction of initial cardiomyocyte alpha-actin--smooth muscle alpha-actin--in cultured avian pregastrula epiblast: a role for nodal and BMP antagonist.

During early cardiogenesis, endoderm-derived bone morphogenetic protein (BMP) induces the expression of both heart-specific transcription factors and sarcomeric proteins. However, BMP antagonists do not inhibit the expression of the "initial heart alpha-actin"--smooth muscle alpha-actin (SMA)--which is first expressed in the anterior lateral mesoderm and then recruited into the initial myofibrils (Nakajima et al. [2002] Dev. Biol. 245:291-303). Therefore, mechanisms that regulate the expression of SMA in the heart-forming mesoderm are not well-understood. Regional explantation experiments using chick blastoderm showed that the posterolateral region of the epiblast differentiated into cardiomyocytes. Posterior epiblast cultured with or without the associated hypoblast showed that interaction between the tissues of these two germ layers at the early pregastrula stage (stages X-XI) was a prerequisite for the expression of SMA. Posterior epiblast that is cultured without hypoblast could also be induced to express SMA if TGF-beta or activin was added to the culture medium. However, neither neutralizing antibodies against TGF-betas nor follistatin perturbed the expression of SMA in cultured blastoderm. Adding BMP to the cultured blastoderm inhibited the expression of SMA, whereas BMP antagonists, such as chordin, were able to induce the expression of SMA in cultured posterior epiblast. Furthermore, adding lefty-1, a nodal antagonist, to the blastoderm inhibited the expression of SMA, and nodal plus BMP antagonist up-regulated the expression of SMA in cultured posterior epiblast. Results indicate that the interaction between the tissues of the posterior epiblast and hypoblast is necessary to initiate the expression of SMA during early cardiogenesis and that nodal and BMP antagonist may play an important role in the regulation of SMA expression.

Pit cells as liver-associated natural killer cells: morphology and function.

Pit cells are one type of hepatic sinusoidal cells, defined morphologically as large granular lymphocytes (LGLs) and functionally as liver-associated natural killer (NK) cells. They are situated inside the sinusoidal lumen, adhering to the endothelial cells and Kupffer cells. They contain multivesicular body-related dense granules and rod-cored vesicles. The number and size of granules and vesicles differ between hepatic NK cells and peripheral blood cells, suggesting possible differentiation of the latter into the former in the microenvironment of the liver. In addition to NK cells, natural killer T (NKT) cells are also abundant in the liver. They share several morphological properties with NK cells, and at least some are probably observed as pit cells under an electron microscope. NK cells recognize target cells with their surface receptors such as inhibitory and activating receptors. They exert antitumor functions by exocytosis of perforin/granzyme-containing granules, induction of death receptor-mediated apoptosis in target cells, and production of various cytokines that augment the activities of other immune cells. NKT cells play important roles in initiating and assisting the function of NK cells by producing interferon-gamma.

Cytoglobin/STAP, its unique localization in splanchnic fibroblast-like cells and function in organ fibrogenesis.

Cytoglobin/stellate cell activation-associated protein (Cygb/STAP) consists of a new class of hexacoordinate globin superfamily, which was recently discovered by a proteome analysis on the rat hepatic stellate cells. Unlike haemoglobin, myoglobin, and neuroglobin, Cygb/STAP is ubiquitously expressed in several organs, although its detailed localization has not been clarified. Immunohistochemistry and immunoelectron microscopy revealed that Cygb/STAP is uniquely localized in fibroblast-like cells in splanchnic organs, namely the vitamin A-storing cell lineage, but neither in epithelial cells, endothelial cells, muscle cells, blood cells, macrophages, nor dermal fibroblasts. The expression of Cygb/STAP was upregulated in fibrotic lesions of the pancreas and kidney in which activated fibroblast-like cells or myofibroblasts are known to increase in number. In cultured hepatic stellate cells, Cygb/STAP expression was augmented by the stimulation with sera, platelet-derived growth factor-BB, and transforming growth factor-beta 1. Overexpression of Cygb/STAP in NIH 3T3 cells induced the cells to lessen migratory activities and increase the expression of collagen alpha1(I) mRNA. These results indicate that Cygb/STAP is a tissue globin uniquely localized in splanchnic fibroblastic cell lineage and may play a role in fibrotic organ disorder.

Apoptosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells.

Apoptosis of T cells contributes to the immune homeostasis in inflamed organs. A prominent T-cell infiltration is usually seen in human chronic active hepatitis, being associated with liver fibrosis. In order to demonstrate T-cell apoptosis in the hepatic fibrotic tissue, we induced T-cell infiltration in the fibrotic liver of the rat by injecting concanavalin A (Con A), a T-cell mitogen. Lymphocytes increased in number with a peak at 1 day, preferentially distributing in the fibrotic tissue rather than the parenchyma. They consisted of CD4-positive and CD8-positive cells, and gave the feature of lymphoblasts. Double staining for CD3 and TUNEL demonstrated that T cells underwent apoptosis. Apoptotic cells were more frequent in the fibrotic livers than the normal livers, and were spatially associated with alpha-smooth muscle actin-positive myofibroblast-like cells that possibly derived from hepatic stellate cells (HSCs) and portal fibroblasts through activation. In vitro experiments demonstrated that lymphocyte apoptosis was more frequently induced in the co-culture of Con A-activated splenic T cells/activated HSCs compared to that induced in activated T cells/quiescent HSCs or resting T cells/activated HSCs. The present results indicate that T cells which have extravasated and infiltrated the hepatic fibrotic tissue undergo apoptosis probably through an interaction with myofibroblast-like cells, suggesting the regulatory role of the latter cells in T-cell accumulation in the fibrotic liver.

Localization of lymphocyte apoptosis in murine lymphoid tissues after stimulation of natural killer T cells with alpha-galactosylceramide.

Alpha-galactosylceramide (alpha-GalCer) has been reported to induce activation-induced cell death (AICD) in natural killer T (NKT) cells. We undertook this study to demonstrate the distribution of AICD of NKT cells in the lymphoid tissues and differences in frequency between the central and peripheral lymphoid tissues, histologically and quantitatively analyzing the apoptotic figure in the murine spleen, lymph node, and thymus after alpha-GalCer treatment. Lymphocyte apoptosis was identified as a cluster of nuclei with chromatin condensation in hematoxylin-eosin-stained sections, and was confirmed by TUNEL staining, double staining for TUNEL and CD4, and electron microscopy. In the spleen, it appeared at 12 h after alpha-GalCer administration, increased in number, reaching a peak at 24 h, and then falling to a normal level at 72 h. It was preferentially found in the white pulp, especially in the periarterial lymphoid sheath, but was sparse in the red pulp. Alpha-GalCer-induced lymphocyte apoptosis was seen in tumor-necrosis factor (TNF)-deficient mice as well, but was not in lpr/lpr (Fas-deficient) or gld/gld (Fas ligand-deficient) mice. As for the tissue distribution, lymphocyte apoptosis was frequently seen in the paracortex of the lymph node, whereas it was rare in the thymus. These data indicate that alpha-GalCer-induced AICD of NKT cells takes place in the T-cell area of peripheral lymphoid tissues (i.e., the spleen and lymph node) through the Fas/Fas ligand, but not the TNF pathway.

Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.

Secreted protein, acidic and rich in cysteine (SPARC), which functions in tissue remodeling, has been reported to be expressed by myofibroblasts in liver cirrhosis and hepatocellular carcinoma. This study aimed to reveal its expression in chronic hepatitis. Immuno-light and electron microscopy demonstrated that SPARC was expressed by nerve fibers and hepatic stellate cells (HSCs) in the liver parenchyma and myofibroblasts in the fibrous septa. Reaction products were localized in the rough endoplasmic reticulum and nuclear envelope. Serial section analysis demonstrated that SPARC, platelet-derived growth factor receptor-beta, and alpha-smooth muscle actin were co-expressed by HSCs. Quantitative analysis demonstrated that, while SPARC-positive HSCs were sparse in control livers, they significantly increased in number in the livers with chronic hepatitis. There were, however, no significant differences in number among the grades of activity, the stages of fibrosis, or etiology (virus-infected or autoimmune, hepatitis B virus or hepatitis C virus). In liver cirrhosis, however, they significantly decreased in number. The present results indicate that SPARC is expressed by activated HSCs in chronic hepatitis, suggesting the involvement of SPARC in hepatic fibrogenesis after chronic injuries.

Characterization of human stellate cell activation-associated protein and its expression in human liver.

This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.

Development of hepatocytes from ES cells after transfection with the HNF-3beta gene.

We have attempted to generate embryonic stem (ES) cell-derived hepatocytes expressing liver-specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)-3beta-transfected ES cells are cultured in alpha-MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 in the three-dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF-3beta-transfected ES cells also had hepatocyte-like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases.

BACKGROUND/AIMS: Although oxidative stress is an important candidate in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), the localization and pathological significance of oxidative stress-induced cellular damage in NAFLD remains unclear. METHODS: Hepatic expression of 4-hydroxy-2'-nonenal (HNE) and 8-hydroxydeoxyguanosine (8-OHdG), as reliable markers of lipid peroxidation and oxidative DNA damage, respectively, was immunohistochemically investigated in NAFLD and the results were compared with histological findings. RESULTS: While no HNE adducts were observed in control livers, they were frequently detected in NAFLD. In NASH, the localization of the adducts was in the cytoplasm of sinusoidal cells and hepatocytes with a predominance in zone 3. The grade of necro-inflammation as well as the stage of fibrosis significantly correlated with the HNE index. Regarding 8-OHdG, although no 8-OHdG expression was observed in normal liver and only a few in fatty liver, 11 of 17 cases (64.7%) with NASH exhibited nuclear expression of 8-OHdG in hepatocytes and sinusoidal cells in areas of active inflammation. The 8-OHdG index significantly correlated with the grade of necro-inflammation. CONCLUSIONS: Oxidative cellular damage occurs frequently in livers with NAFLD and may be associated with some clinico-pathological features of NAFLD including liver fibrosis and possibly, hepatocarcinogenesis.

Fatty change in human hepatocellular carcinoma cell lines derived from patients with antibodies to hepatitis C virus.

BACKGROUND/AIMS: Evidence suggesting a relationship between fatty change in normal or malignant hepatocytes and hepatitis C virus has gradually accumulated, but less is known about the relationship between cell proliferation and fatty change in human hepatocellular carcinoma. METHODOLOGY: We studied the latter issue in two human hepatocellular carcinoma cell lines (OCUH-16 and Nuk-1) derived from hepatitis C virus-associated tumors. We examined the relationship between degree of fatty change assessed by oil-red-O staining and electron microscopy, actively proliferating cells counted using a monoclonal antibody to MIB-1 protein, and apoptotic cells counted using DNA nick-end labeling in the above two hepatocellular carcinoma cell lines with time lapse. RESULTS: On day 1 in culture, fatty change was present randomly in cytoplasm of some Nuk-1 cells, but was not found in OCUH-16 cells. Over time, fat droplets were found more frequently in large hepatocellular carcinoma cells in both Nuk-1 and OCUH-16 lines. Most of these cells were located in the periphery of hepatocellular carcinoma cell nests or islands as opposed to the small hepatocellular carcinoma cells located in the centers of nests in both lines. According to MIB-1 staining, these small cells proliferate more actively than the large, peripherally located hepatocellular carcinoma cells. Only a few apoptotic hepatocellular carcinoma cells were detected during culture. CONCLUSIONS: Fatty change in large hepatocellular carcinoma cells seems to be associated with less proliferative activity than was seen in small hepatocellular carcinoma cells without fatty change, located in more centrally cell nests, in these hepatocellular carcinoma cell lines.