Kinji Asahina

Near Completely Humanized Liver in Mice Shows Human-Type Metabolic Responses to Drugs

Human hepatocytes were transplanted into urokinase-type plasminogen activator-transgenic SCID mice (uPA/SCID mice), which are immunodeficient and undergo liver failure. The transplanted cells were characterized in terms of their in vivo growth potential and functions. The human hepatocytes progressively repopulated the murine host liver. However, the recipients died when the replacement index (RI) of the human hepatocytes exceeded 50%. The hosts (chimeric mice) survived at RI >50% when treated with a drug that has anti-human complement factor activity, and these mice developed livers with RI values as high as 96%. In total, 36 chimeric mice were generated, and the rate of successful engraftment was as high as 92%. The yield of chimeric mice with RI >70% was 32%. The human hepatocytes in the murine host liver expressed mRNAs for a variety of human cytochrome P450 (hCYP) subtypes, in a manner that was similar to the donor liver. The mRNAs for hCYP3A4 and hCYP1A1/2 were induced in the liver in a CYP type-specific manner when the mice were treated with rifampicin and 3-methylcholanthrene, respectively. These results indicate that human hepatocytes that propagate in mice retain their normal pharmacological responses. We conclude that the chimeric mouse developed in the present study is a useful model for assessing the functions and pharmacological responses of human hepatocytes.

Characterization of a Stellate Cell Activation-associated Protein (STAP) with Peroxidase Activity Found in Rat Hepatic Stellate Cells

A proteome approach for the molecular analysis of the activation of rat stellate cell, a liver-specific pericyte, led to the discovery of a novel protein named STAP (stellate cellactivation-associated protein). We cloned STAP cDNA. STAP is a cytoplasmic protein with molecular weight of 21,496 and shows about 40% amino acid sequence homology with myoglobin. STAP was dramatically induced in in vivo activated stellate cells isolated from fibrotic liver and in stellate cells undergoingin vitro activation during primary culture. This induction was seen together with that of other activation-associated molecules, such as smooth muscle α-actin, PDGF receptor-β, and neural cell adhesion molecule. The expression of STAP protein and mRNA was augmented time dependently in thioacetamide-induced fibrotic liver. Immunoelectron microscopy and proteome analysis detected STAP in stellate cells but not in other hepatic constituent cells. Biochemical characterization of recombinant rat STAP revealed that STAP is a heme protein exhibiting peroxidase activity toward hydrogen peroxide and linoleic acid hydroperoxide. These results indicate that STAP is a novel endogenous peroxidase catabolizing hydrogen peroxide and lipid hydroperoxides, both of which have been reported to trigger stellate cell activation and consequently promote progression of liver fibrosis. STAP could thus play a role as an antifibrotic scavenger of peroxides in the liver.

Hepatic stellate cells in liver development, regeneration, and cancer

Hepatic stellate cells are liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells. It is becoming increasingly clear that hepatic stellate cells have a profound impact on the differentiation, proliferation, and morphogenesis of other hepatic cell types during liver development and regeneration. In this Review, we summarize and evaluate the recent advances in our understanding of the formation and characteristics of hepatic stellate cells, as well as their function in liver development, regeneration, and cancer. We also discuss how improved knowledge of these processes offers new perspectives for the treatment of patients with liver diseases.

Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol.

Septum transversum‐derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver

The septum transversum mesenchyme (STM) signals to induce hepatogenesis from the foregut endoderm. Hepatic stellate cells (HSCs) are sinusoidal pericytes assumed to originate from the STM and participate in mesenchymal‐epithelial interaction in embryonic and adult livers. However, the developmental origin of HSCs remains elusive due to the lack of markers for STM and HSCs. We previously identified submesothelial cells (SubMCs) beneath mesothelial cells (MCs) as a potential precursor for HSCs in developing livers. In the present study, we reveal that both STM in embryonic day (E) 9.5 and MC/SubMCs in E12.5 share the expression of activated leukocyte cell adhesion molecule (Alcam), desmin, and Wilms tumor 1 homolog (Wt1). A cell lineage analysis using MesP1Cre/Rosa26lacZflox mice identifies the mesodermal origin of the STM, HSCs, and perivascular mesenchymal cells (PMCs). A conditional cell lineage analysis using the Wt1CreERT2 mice demonstrates that Wt1+ STM gives rise to MCs, SubMCs, HSCs, and PMCs during liver development. Furthermore, we find that Wt1+ MC/SubMCs migrate inward from the liver surface to generate HSCs and PMCs including portal fibroblasts, smooth muscle cells, and fibroblasts around the central veins. On the other hand, the Wt1+ STM and MC/SubMCs do not contribute to sinusoidal endothelial cells, Kupffer cells, and hepatoblasts. Conclusion: our results demonstrate that HSCs and PMCs are derived from MC/SubMCs, which are traced back to mesodermal STM during liver development. (HEPATOLOGY 2011;.)

Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development†

The knowledge concerning fetal hepatic stellate cells (HSCs) is scarce, and their cell lineage and functions are largely unknown. The current study isolated fetal liver mesenchymal cells from a mouse expressing β‐galactosidase under the control of Msx2 promoter by fluorescence‐activated cell sorting (FACS) and surveyed marker genes by microarray analysis. Based on the location and immunostaining with conventional and newly disclosed markers, we have identified three distinct populations of fetal liver mesenchymal cells expressing both desmin and p75 neurotrophin receptor (p75NTR): HSCs in the liver parenchyma; perivascular mesenchymal cells expressing α‐smooth muscle actin (α‐SMA); and submesothelial cells associated with the basal lamina beneath mesothelial cells and expressing activated leukocyte cell adhesion molecule (ALCAM) and platelet‐derived growth factor receptor α. A transitional cell type from the submesothelial cell phenotype to fetal HSCs was also identified near the liver surface. Mesothelial cells expressed podoplanin and ALCAM. Ki‐67 staining showed that proliferative activity of the submesothelial cells is higher than that of mesothelial cells and transitional cells. Using anti‐ALCAM antibodies, submesothelial and mesothelial cells were isolated by FACS. The ALCAM+ cells expressed hepatocyte growth factor and pleiotrophin. In culture, the ALCAM+ cells rapidly acquired myofibroblastic morphology and α‐SMA expression. The ALCAM+ cells formed intracellular lipid droplets when embedded in collagen gel and treated with retinol, suggesting the potential for ALCAM+ cells to differentiate to HSCs. Finally, we demonstrated that fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are all derived from mesoderm by using MesP1‐Cre and ROSA26 reporter mice. Conclusion: Fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are mesodermal in origin, and ALCAM+ submesothelial cells may be a precursor for HSCs in developing liver. (HEPATOLOGY 2009.)

Expression of the liver-specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells

Hepatic differentiation from mouse embryonic stem (ES) cells via the formation of embryoid bodies (EBs) has been revealed by the expression of hepatocyte‐related genes such as α‐fetoprotein and albumin. It is known, however, that the visceral endoderm differentiates in early EBs and expresses these hepatocyte‐related genes. Thus, it remains unclear whether ES cells are capable of differentiating into hepatocytes derived from definitive endoderm in vitro. In the present study, yolk sac tissues isolated from the foetal mouse were found to express many hepatocyte‐related genes. Among the hepatocyte‐related genes examined, cytochrome P450 7A1 (Cyp7a1) was identified as a liver‐specific gene that was not expressed in the yolk sac. Cyp7a1 was induced in developing EBs, and hepatic differentiation was preferentially observed in the developing EBs in attached culture as compared to those in suspension culture. Leukaemia inhibitory factor permitted the differentiation of visceral endoderm, but inhibited the expression of gastrulation‐related genes and the hepatic differentiation in cultured EBs. ES cells expressing green fluorescent protein (GFP) under the control of the Cyp7a1 enhancer/promoter showed that cultured EBs contained GFP‐positive epithelial‐like cells. These results demonstrate that ES cells can differentiate in vitro into hepatocytes derived from definitive endoderm.

Mesothelial cells give rise to hepatic stellate cells and myofibroblasts via mesothelial–mesenchymal transition in liver injury

In many organs, myofibroblasts play a major role in the scarring process in response to injury. In liver fibrogenesis, hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts, but the origins of both HSCs and myofibroblasts remain elusive. In the developing liver, lung, and intestine, mesothelial cells (MCs) differentiate into specific mesenchymal cell types; however, the contribution of this differentiation to organ injury is unknown. In the present study, using mouse models, conditional cell lineage analysis has demonstrated that MCs expressing Wilms tumor 1 give rise to HSCs and myofibroblasts during liver fibrogenesis. Primary MCs, isolated from adult mouse liver using antibodies against glycoprotein M6a, undergo myofibroblastic transdifferentiation. Antagonism of TGF-β signaling suppresses transition of MCs to mesenchymal cells both in vitro and in vivo. These results indicate that MCs undergo mesothelial–mesenchymal transition and participate in liver injury via differentiation to HSCs and myofibroblasts.

Effect of Poly(N-vinyl-pyrrolidone)-block-poly(d,l-lactide) as Coating Agent on the Opsonization, Phagocytosis, and Pharmacokinetics of Biodegradable Nanoparticles

The effect of the coating polymer poly(N-vinyl-pyrrolidone) (PVP) on the protein adsorption, phagocytosis, and pharmacokinetics of poly(D,L-lactide)-based nanoparticles was evaluated in vitro and in vivo. Control poly(ethylene glycol) (PEG)-coated nanoparticles were included for comparison. While no difference between PEG- and PVP-decorated nanoparticles in terms of amount of adsorbed protein was evident upon incubation in single protein solutions (BSA, IgG), incubation in serum revealed a protein adsorption pattern both quantitatively and qualitatively distinct. Larger amounts of complement components and immunoglobulins were found to adhere to PVP-coated particles, whereas PEG particles showed preferential adsorption of apolipoproteins. Furthermore, preopsonization in fresh rather than heat-inactivated serum enhanced uptake of both types of particles by murine RAW 264.7 macrophages. However, when isolated rat Kupffer cells were employed, activation of the complement system significantly enhanced the uptake of PVP-coated nanoparticles compared to PEG particles. Ultimately, PVP-coated nanoparticles exhibited considerably shorter circulation times compared to their PEG counterparts when administered intravenously to rats.

Rosmarinic acid and baicalin epigenetically derepress peroxisomal proliferator‐activated receptor γ in hepatic stellate cells for their antifibrotic effect

Hepatic stellate cells (HSCs) undergo myofibroblastic transdifferentiation (activation) to participate in liver fibrosis and identification of molecular targets for this cell fate regulation is essential for development of efficacious therapeutic modalities for the disease. Peroxisomal proliferator‐activated receptor γ (PPARγ) is required for differentiation of HSCs and its epigenetic repression underlies HSC activation. The herbal prescription Yang‐Gan‐Wan (YGW) prevents liver fibrosis, but its active ingredients and molecular mechanisms are unknown. Here we demonstrate YGW prevents and reverses HSC activation by way of epigenetic derepression of Pparγ involving reductions in MeCP2 expression and its recruitment to Pparγ promoter, suppressed expression of PRC2 methyltransferase EZH2, and consequent reduction of H2K27di‐methylation at the 3′ exon. High‐performance liquid chromatography / mass spectrometry (HPLC/MS) and nuclear magnetic resonance (NMR) analyses identify polyphenolic rosmarinic acid (RA) and baicalin (BC) as active phytocompounds. RA and BC suppress the expression and signaling by canonical Wnts, which are implicated in the aforementioned Pparγ epigenetic repression. RA treatment in mice with existing cholestatic liver fibrosis inhibits HSC activation and progression of liver fibrosis. Conclusion: These results demonstrate a therapeutic potential of YGW and its active component RA and BC for liver fibrosis by way of Pparγ derepression mediated by suppression of canonical Wnt signaling in HSCs. (Hepatology 2012)