Hypo-osmolarity induces apoptosis resistance via TRPV2-mediated AKT-Bcl-2 pathway.

In cirrhosis, several molecular alterations such as resistance to apoptosis could accelerate carcinogenesis. Recently, mechanotransduction has been attracting attention as one of the causes of these disturbances. In patients with cirrhosis, the serum sodium levels progressively decrease in the later stage of cirrhosis, and hyponatremia leads to serum hypo-osmolality. Since serum sodium levels in patients with cirrhosis with liver cancer are inversely related to cancer's number, size, stage, and cumulative survival, we hypothesized that hypo-osmolality-induced mechanotransduction under cirrhotic conditions might contribute to oncogenesis and/or progression of hepatocellular carcinoma (HCC). In this study, we adjusted osmosis of culture medium by changing the sodium chloride concentration and investigated the influence of hypotonic conditions on the apoptosis resistance of an HCC cell line, HepG2, using a serum-deprivation-induced apoptosis model. By culturing the cells in a serum-free medium, the levels of an antiapoptotic protein Bcl-2 were downregulated. In contrast, the hypotonic conditions caused apoptosis resistance by upregulation of Bcl-2. Next, we examined which pathway was involved in the apoptosis resistance. Hypotonic conditions enhanced AKT signaling, and constitutive activation of AKT in HepG2 cells led to upregulation of Bcl-2. Moreover, we revealed that the enhancement of AKT signaling was caused by intracellular calcium influx via a mechanosensor, TRPV2. Our findings suggested that hyponatremia-induced serum hypotonic in patients with cirrhosis promoted the progression of hepatocellular carcinoma.NEW & NOTEWORTHY Our study first revealed that hypo-osmolarity-induced mechanotransduction enhanced calcium-mediated AKT signaling via TRPV2 activation, resulting in contributing to apoptosis resistance. The finding indicates a possible view that liver cirrhosis-induced hyponatremia promotes hepatocellular carcinogenesis.

Hexa Histidine-Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species.

BACKGROUND AND AIMS: Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH AND RESULTS: Cygb-deficient mice that had bile duct ligation-induced liver cholestasis or choline-deficient amino acid-defined diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb-overexpressing mice. We produced hexa histidine-tagged recombinant human CYGB (His-CYGB), traced its biodistribution, and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes through a clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type 1 alpha 1 production and α-smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-ß secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA-sequencing analysis revealed the down-regulation of inflammation- and fibrosis-related genes and the up-regulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in chimeric mice with humanized livers. CONCLUSIONS: His-CYGB could have antifibrotic clinical applications for human chronic liver diseases.

TGF-ß1-driven reduction of cytoglobin leads to oxidative DNA damage in stellate cells during non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species. The molecular role of CYGB in human hepatic stellate cell (HSC) activation and human liver disease remains uncharacterised. The aim of this study was to reveal the mechanism by which the TGF-ß1/SMAD2 pathway regulates the human CYGB promoter and the pathophysiological function of CYGB in human non-alcoholic steatohepatitis (NASH). METHODS: Immunohistochemical staining was performed using human NASH biopsy specimens. Molecular and biochemical analyses were performed by western blotting, quantitative PCR, and luciferase and immunoprecipitation assays. Hydroxyl radicals (•OH) and oxidative DNA damage were measured using an •OH-detectable probe and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA. RESULTS: In culture, TGF-ß1-pretreated human HSCs exhibited lower CYGB levels - together with increased NADPH oxidase 4 (NOX4) expression - and were primed for H2O2-triggered •OH production and 8-OHdG generation; overexpression of human CYGB in human HSCs reversed these effects. Electron spin resonance demonstrated the direct •OH scavenging activity of recombinant human CYGB. Mechanistically, pSMAD2 reduced CYGB transcription by recruiting the M1 repressor isoform of SP3 to the human CYGB promoter at nucleotide positions +2-+13 from the transcription start site. The same repression did not occur on the mouse Cygb promoter. TGF-ß1/SMAD3 mediated αSMA and collagen expression. Consistent with observations in cultured human HSCs, CYGB expression was negligible, but 8-OHdG was abundant, in activated αSMA+pSMAD2+- and αSMA+NOX4+-positive hepatic stellate cells from patients with NASH and advanced fibrosis. CONCLUSIONS: Downregulation of CYGB by the TGF-ß1/pSMAD2/SP3-M1 pathway brings about •OH-dependent oxidative DNA damage in activated hepatic stellate cells from patients with NASH. LAY SUMMARY: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species and protects cells from oxidative DNA damage. Herein, we show that the cytokine TGF-ß1 downregulates human CYGB expression. This leads to oxidative DNA damage in activated hepatic stellate cells. Our findings provide new insights into the relationship between CYGB expression and the pathophysiology of fibrosis in patients with non-alcoholic steatohepatitis.

Involvement of ERK1/2 activation in the gene expression of senescence-associated secretory factors in human hepatic stellate cells.

Senescent hepatic stellate cells (senescent HSCs) are found in patients with liver cirrhosis and have been thought to be involved in the development of hepatocellular carcinoma (HCC) in mice via the senescence-associated secretory proteins. However, in humans, which secretory proteins are involved and what regulate their expression remain unclear. In the current study, we characterized senescence-associated ß-galactosidase-positive senescent human HSCs (hHSCs) induced by repetitive passaging. They exhibited enhanced expression of 14 genes for secretory protein and persistent phosphorylation of ERK1/2 protein but not JNK or p38 MAPK proteins. Enhanced nuclear ERK1/2 phosphorylation was observed in senescent hHSCs. Treatment of the senescent hHSCs with ERK1/2 inhibitor, SCH772984, significantly decreased the levels of angiopoietin like 4 (ANGPTL4), C-C motif chemokine ligand 7 (CCL7), Interleukin-8 (IL-8), platelet factor 4 variant 1 (PF4V1), and TNF superfamily member 15 (TNFSF15) mRNA levels in a dose-dependent manner. The enhanced phosphorylation of ERK1/2 and expression of ANGPTL4, IL-8 and PF4V1 genes were observed in both of senescent human dermal fibroblasts and X-ray-induced senescent hHSCs. However, transient ERK1/2 activation induced by epidermal growth factor could not mimic the gene profile of the senescent hHSCs. These results revealed involvement of ERK1/2 signaling in the regulation of senescence-associated secretory factors, suggesting that simultaneous induction of ANGPTL4, IL-8, and PF4V1 genes is a marker of hHSC senescence. This study will contribute to understanding roles of senescent hHSCs in liver diseases.

Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling.

Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of CYGB gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN-targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5'-TGA(C/G)TCA), located -218 to -222 bases from the transcription initiation site in the CYGB promoter. Of note, in bile duct-ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers CYGB gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.

Interstitial fluid flow-induced growth potential and hyaluronan synthesis of fibroblasts in a fibroblast-populated stretched collagen gel culture.

BACKGROUND: Tensioned collagen gels with dermal fibroblasts (DFs) as a dermis model are usually utilized in a static culture (SC) that lacks medium flowing. To make the model closer to its in vivo state, we created a device to perfuse the model with media flowing at a physiological velocity and examined the effects of medium flow (MF) on the cultures. METHODS: We constructed a medium perfusion device for human DF-embedded stretched collagen gels (human dermis model), exposed the model to media that flows upwardly at ~1mL/day, and examined water retention of the gels, cells' growth ability, metabolic activity, expression profiles of nine extracellular matrix (ECM)-related genes. The obtained data were compared with those from the model in SC. RESULTS: MF increases the gels' water retention and cells' growth potential but had little effect on their metabolic activities. MF robustly enhanced hyaluronan synthase 2 (HAS2) and matrix metalloprotease 1 (MMP1) gene expressions but not of the other genes (MMP2, HYAL1, HYAL2, HYAL3, COL1A1, COL3A1, and CD44). MF significantly increased the amounts of cellular hyaluronan and adenosine triphosphate. CONCLUSIONS: The MF at a physiological speed significantly influences the nature of ECMs and their resident fibroblasts and remodels ECMs by regulating hyaluronan metabolism. GENERAL SIGNIFICANCE: Fibroblasts in tensioned collagen gels altered their phenotypes in a MF rate-dependent manner. Collagen gel culture with tension and MF could be utilized as an appropriate in vitro model of interstitial connective tissues to evaluate the pathophysiological significance of mechanosignals generated by fluid flow and cellular/extracellular tension.

MiR-29a Assists in Preventing the Activation of Human Stellate Cells and Promotes Recovery From Liver Fibrosis in Mice.

The microRNA-29 (miR-29) family is known to suppress the activation of hepatic stellate cells (HSCs) and reversibly control liver fibrosis; however, the mechanism of how miR-29a controls liver fibrosis remains largely unknown. This study was conducted to clarify the mechanism of anti-fibrotic effect of miR-29a and to explore if miR-29a is a promising candidate for nucleic acid medicine against liver fibrosis. Two liver fibrosis murine models (carbon tetrachloride or thioacetamide) were used. MiR-29a mixed with atelocollagen was systemically administered. Hepatic fibrosis was evaluated by histological analysis and the expression levels of fibrosis-related genes. We observed that miR-29a treatment dramatically accelerated the reversion of liver fibrosis in vivo. Additionally, miR-29a regulated the mRNA expression of collagen type I alpha 1 (COL1A1) and platelet-derived growth factor C (PDGFC). We also noted that miR-29a significantly suppressed COL1A1 mRNA expression and cell viability and significantly increased caspase-9 activity (P < 0.05) in LX-2 cells. Pretreatment of miR-29a inhibited activation of LX-2 cell by transforming growth factor beta treatment. MiR-29a exhibited anti-fibrotic effect without cell toxicity in vivo and directly suppressed the expression of PDGF-related genes as well as COL1A1 and induced apoptosis of LX-2 cells. MiR-29a is a promising nucleic acid inhibitor to target liver fibrosis.

Human Cytotoxic T Lymphocyte-Mediated Acute Liver Failure and Rescue by Immunoglobulin in Human Hepatocyte Transplant TK-NOG Mice.

UNLABELLED: Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are critical in eliminating infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by HBV-specific CTLs. After HBV inoculation in human hepatocyte-transplanted herpes simplex virus type-1 thymidine kinase-NOG mice, human peripheral blood mononuclear cells (PBMCs) were administered, and albumin, HBV DNA, alanine aminotransferase (ALT), and cytokine levels were analyzed. Histopathological and flow-cytometric analysis of infiltrating human immune cells were performed, and the efficacy of CTL-associated antigen-4 immunoglobulin (CTLA4Ig) against liver damage was evaluated. PBMC treatment resulted in massive hepatocyte damage with elevation of ALT, granzyme A, and gamma interferon and decrease in albumin and HBV DNA. The number of liver-infiltrating human lymphocytes and CD8-positive cells was significantly higher in HBV-infected mice. HBV-specific CTLs were detected by core and polymerase peptide-major histocompatibility complex-tetramer, and the population of regulatory T cells was significantly decreased in HBV-infected mice. Serum hepatitis B surface (HBs) antigen became negative, and HBs antibody appeared. CTLA4Ig treatment strongly inhibited infiltration of mononuclear cells. CTLA4Ig treatment will be used to treat patients who develop severe acute hepatitis B to prevent liver transplantation or lethality. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B. IMPORTANCE: Without liver transplantation, some HBV-infected patients will die from severe liver damage due to acute overreaction of the immune system. No effective treatment exists, due in part to the lack of a suitable animal model. An animal model is necessary to investigate the mechanism of hepatitis and to develop therapeutic strategies to prevent acute liver failure in HBV infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by human HBV-specific CTLs. In this model, HBV-infected human hepatocytes were transplanted into severely immunodeficient NOG mice in order to reconstruct elements of the human immune system. Using this model, we found that CTL-associated antigen-4 immunoglobulin was able to suppress damage to HBV-infected hepatocytes, suggesting an approach to treatment. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B.

Cytoglobin deficiency promotes liver cancer development from hepatosteatosis through activation of the oxidative stress pathway.

This study was conducted to clarify the role of cytoglobin (Cygb), a globin expressed in hepatic stellate cells (HSCs), in the development of liver fibrosis and cancer in nonalcoholic steatohepatitis (NASH). Cygb expression was assessed in patients with NASH and hepatocellular carcinoma. Mouse NASH model was generated in Cygb-deficient (Cygb(-/-)) or wild-type (WT) mice by giving a choline-deficient amino acid-defined diet and, in some of them, macrophage deletion and N-acetyl cysteine treatment were used. Primary-cultured mouse HSCs isolated from WT (HSCs(Cygb-wild)) or Cygb(-/-) (HSCs(Cygb-null)) mice were characterized. As results, the expression of CYGB was reduced in patients with NASH and hepatocellular carcinoma. Choline-deficient amino acid treatment for 8 weeks induced prominent inflammation and fibrosis in Cygb(-/-) mice, which was inhibited by macrophage deletion. Surprisingly, at 32 weeks, despite no tumor formation in the WT mice, all Cygb(-/-) mice developed liver cancer, which was ameliorated by N-acetyl cysteine treatment. Altered expression of 31 genes involved in the metabolism of reactive oxygen species was notable in Cygb(-/-) mice. Both HSCs(Cygb-null) and Cygb siRNA-transfected-HSCs(Cygb-wild) exhibited the preactivation condition. Our findings provide important insights into the role that Cygb, expressed in HSCs during liver fibrosis, plays in cancer development with NASH.

Discovery of cytoglobin and its roles in physiology and pathology of hepatic stellate cells.

Cytoglobin (CYGB), a new member of the globin family, was discovered in 2001 as a protein associated with stellate cell activation (stellate cell activation-associated protein [STAP]). Knowledge of CYGB, including its crystal, gene, and protein structures as well as its physiological and pathological importance, has increased progressively. We investigated the roles of oxygen (O2)-binding CYGB as STAP in hepatic stellate cells (HSCs) to understand the part played by this protein in their pathophysiological activities. Studies involving CYGB-gene-deleted mice have led us to suppose that CYGB functions as a regulator of O2 homeostasis; when O2 homeostasis is disrupted, HSCs are activated and play a key role(s) in hepatic fibrogenesis. In this review, we discuss the rationale for this hypothesis.

Involvement of hepatic stellate cell cytoglobin in acute hepatocyte damage through the regulation of CYP2E1-mediated xenobiotic metabolism.

Oxygen (O2) is required for cytochrome P450 (CYP)-dependent drug metabolism. Cytoglobin (CYGB) is a unique globin expressed exclusively in hepatic stellate cells (HSCs). However, its role in O2-dependent metabolism in neighboring hepatocytes remains unknown. This study provides evidence that CYGB in HSCs is involved in acetaminophen (N-acetyl-p-aminophenol; APAP)-induced hepatotoxicity. Serum alanine aminotransferase levels were higher in wild-type mice than in Cygb-null mice. Wild-type mice exhibited more severe hepatocyte necrosis around the central vein area compared with Cygb-null mice, thus indicating that CYGB deficiency protects against APAP-induced liver damage. Although no difference in the hepatic expression of CYP2E1, a key enzyme involved in APAP toxicity, was observed between wild-type and Cygb-null mice, the serum levels of the APAP metabolites cysteinyl-APAP and N-acetyl-cysteinyl-APAP were decreased in Cygb-null mice, suggesting reduced APAP metabolism in the livers of Cygb-null mice. In primary cultures, APAP-induced hepatocyte damage was increased by co-culturing with wild-type HSCs but not with Cygb-null HSCs. In addition, cell damage was markedly alleviated under low O2 condition (5% O2), suggesting the requirement of O2 for APAP toxicity. Carbon tetrachloride-induced liver injury (CYP2E1-dependent), but not lipopolysaccharide/D-galactosamine-induced injury (CYP2E1-independent), was similarly alleviated in Cygb-null mice. Considering the function of CYGB as O2 carrier, these results strongly support the hypothesis that HSCs are involved in the CYP2E1-mediated xenobiotic activation by augmenting O2 supply to hepatocytes. In conclusion, CYGB in HSCs contributes to the CYP-mediated metabolism of xenobiotics in hepatocytes by supplying O2 for enzymatic oxidation.

Restorative effect of hair follicular dermal cells on injured human hair follicles in a mouse model.

No model is available for examining whether in vivo-damaged human hair follicles (hu-HFs) are rescued by transplanting cultured hu-HF dermal cells (dermal papilla and dermal sheath cells). Such a model might be valuable for examining whether in vivo-damaged hu-HFs such as miniaturized hu-HFs in androgenic alopecia are improvable by auto-transplanting hu-HF dermal cells. In this study, we first developed mice with humanized skin composed of hu-keratinocytes and hu-dermal fibroblasts. Then, a 'humanized scalp model mouse' was generated by transplanting hu-scalp HFs into the humanized skin. To demonstrate the usability of the model, the lower halves of the hu-HFs in the model were amputated in situ, and cultured hu-HF dermal cells were injected around the amputated area. The results demonstrated that the transplanted cells contributed to the restoration of the damaged HFs. This model could be used to explore clinically effective technologies for hair restoration therapy by autologous cell transplantation.

Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptor-α.

Peroxisome proliferator (PP)-activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%. Fenofibrate induced hepatocellular hypertrophy, cell proliferation, and peroxisome proliferation in livers of severe combined immunodeficiency (SCID) mice, but not in the h-hep of chimeric mouse livers. Fenofibrate increased the expression of the enzymes of ß- and ω-hydroxylation and deoxygenation of lipids at both gene and protein levels in SCID mouse livers, but not in the h-heps of chimeric mouse livers, supporting the studies with h-PPARα-transgenic mice, a hitherto reliable model for studying the regulation of h-PPARα in the h-liver in most respects, except the induction of the peroxisome proliferation. This study indicates the importance of not only h-PPARα gene but also h-heps themselves to correctly predict effects of fibrates on h-livers, and, therefore, suggests that the chimeric mouse is a currently available, consistent, and reliable model to obtain pharmaceutical data concerning the effects of fibrates on h-livers.

Cytoglobin is expressed in hepatic stellate cells, but not in myofibroblasts, in normal and fibrotic human liver.

Cytoglobin (CYGB) is ubiquitously expressed in the cytoplasm of fibroblastic cells in many organs, including hepatic stellate cells. As yet, there is no specific marker with which to distinguish stellate cells from myofibroblasts in the human liver. To investigate whether CYGB can be utilized to distinguish hepatic stellate cells from myofibroblasts in normal and fibrotic human liver, human liver tissues damaged by infection with hepatitis C virus (HCV) and at different stages of fibrosis were obtained by liver biopsy. Immunohistochemistry was performed on histological sections of liver tissues using antibodies against CYGB, cellular retinol-binding protein-1 (CRBP-1), α-smooth muscle actin (α-SMA), thymocyte differentiation antigen 1 (Thy-1), and fibulin-2 (FBLN2). CYGB- and CRBP-1-positive cells were counted around fibrotic portal tracts in histological sections of the samples. The expression of several of the proteins listed above was examined in cultured mouse stellate cells. Quiescent stellate cells, but not portal myofibroblasts, expressed both CYGB and CRBP-1 in normal livers. In fibrotic and cirrhotic livers, stellate cells expressed both CYGB and α-SMA, whereas myofibroblasts around the portal vein expressed α-SMA, Thy-1, and FBLN2, but not CYGB. Development of the fibrotic stage was positively correlated with increases in Sirius red-stained, α-SMA-positive, and Thy-1-positive areas, whereas the number of CYGB- and CRBP-1-positive cells decreased with fibrosis development. Primary cultured mouse stellate cells expressed cytoplasmic CYGB at day 1, whereas they began to express α-SMA at the cellular margins at day 4. Thy-1 was undetectable throughout the culture period. In human liver tissues, quiescent stellate cells are CYGB positive. When activated, they also become α-SMA positive; however, they are negative for Thy-1 and FBLN2. Thus, CYGB is a useful marker with which to distinguish stellate cells from portal myofibroblasts in the damaged human liver.

Combination therapies with NS5A, NS3 and NS5B inhibitors on different genotypes of hepatitis C virus in human hepatocyte chimeric mice.

OBJECTIVE: We recently demonstrated that combination treatment with NS3 protease and NS5B polymerase inhibitors succeeded in eradicating the virus in genotype 1b hepatitis C virus (HCV)-infected mice. In this study, we investigated the effect of combining an NS5A replication complex inhibitor (RCI) with either NS3 protease or NS5B inhibitors on elimination of HCV genotypes 1b, 2a and 2b. DESIGN: The effects of Bristol-Myers Squibb (BMS)-605339 (NS3 protease inhibitor; PI), BMS-788329 (NS5A RCI) and BMS-821095 (NS5B non-nucleoside analogue inhibitor) on HCV genotypes 1b and 2a were examined using subgenomic HCV replicon cells. HCV genotype 1b, 2a or 2b-infected human hepatocyte chimeric mice were also treated with BMS-605339, BMS-788329 or BMS-821095 alone or in combination with two of the drugs for 4 weeks. Genotypic analysis of viral sequences was achieved by direct and ultra-deep sequencing. RESULTS: Anti-HCV effects of BMS-605339 and BMS-821095 were more potent against genotype 1b than against genotype 2a. In in-vivo experiments, viral breakthrough due to the development of a high prevalence of drug-resistant variants was observed in mice treated with BMS-605339, BMS-788329 and BMS-821095 in monotherapy. In contrast to monotherapy, 4 weeks of combination therapy with the NS5A RCI and either NS3 PI or NS5B inhibitor succeeded in completely eradicating the virus in genotype 1b HCV-infected mice. Conversely, these combination therapies failed to eradicate the virus in mice infected with HCV genotypes 2a or 2b. CONCLUSIONS: These oral combination therapies may serve as a Peg-alfa-free treatment for patients chronically infected with HCV genotype 1b.

Cytoglobin functions as a redox regulator of melanogenesis in normal epidermal melanocytes.

Epidermal melanocytes are continuously exposed to sunlight-induced reactive oxygen species (ROS) and oxidative stress generated during the synthesis of melanin. Therefore, they have developed mechanisms that maintain normal redox homeostasis. Cytoglobin (CYGB), a ubiquitously expressed intracellular iron hexacoordinated globin, exhibits antioxidant activity and regulates the redox state of mammalian cells through its activities as peroxidase and nitric oxide (NO) dioxygenase. We postulated that CYGB functions in the melanogenic process as a regulator that maintains oxidative stress within a physiological level. This was examined by characterizing normal human melanocytes with the knockdown (KD) of CYGB using morphological and molecular biological criteria. CYGB-KD cells were larger, had more dendrites, and generated more melanin granules in the advanced stages of melanogenesis than control cells. The expression levels of major melanogenesis-associated genes and proteins were higher in CYGB-KD melanocytes than in wild type (WT) cells. As expected, CYGB-KD melanocytes generated more ROS and NO than WT cells. In conclusion, CYGB physiologically contributes to maintaining redox homeostasis in the melanogenic activity of normal melanocytes by controlling the intracellular levels of ROS and NO.

Morphological and microarray analyses of human hepatocytes from xenogeneic host livers.

We previously produced mice with human hepatocyte (h-hep) chimeric livers by transplanting h-heps into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic severe combined immunodeficient (SCID) mice with liver disease. The chimeric livers were constructed with h-heps, mouse hepatocytes, and mouse hepatic sinusoidal cells (m-HSCs). Here, we investigated the morphological features of the chimeric livers and the h-hep gene expression profiles in the xenogeneic animal body. To do so, we performed immunohistochemistry, morphometric analyses, and electron microscopic observations on chimeric mouse livers, and used microarray analyses to compare gene expression patterns in hepatocytes derived from chimeric mouse hepatocytes (c-heps) and h-heps. Morphometric analysis revealed that the ratio of hepatocytes to m-HSCs in the chimeric mouse livers were twofold higher than those in the SCID mouse livers, corresponding to twin-cell plates in the chimeric mouse liver. The h-heps in the chimeric mouse did not show hypoxia even in the twin-cell plate structure, probably because of low oxygen consumption by the h-heps relative to the mouse hepatocytes (m-heps). Immunohistochemical and electron microscopic examinations revealed that the sinusoids in the chimeric mouse livers were normally constructed with h-heps and m-HSCs. However, a number of microvilli projected into the intercellular clefts on the lateral aspects of the hepatocytes, features typical of a growth phase. Microarray profiles indicated that ∼82% of 16 605 probes were within a twofold range difference between h-heps and c-heps. Cluster and principal component analyses showed that the gene expression patterns of c-heps were extremely similar to those of h-heps. In conclusion, the chimeric mouse livers were normally reconstructed with h-heps and m-HSCs, and expressed most human genes at levels similar to those in human livers, although the chimeric livers showed morphological characteristics typical of growth.

Transplantation of engineered chimeric liver with autologous hepatocytes and xenobiotic scaffold.

OBJECTIVE: Generation of human livers in pigs might improve the serious shortage of grafts for human liver transplantation, and enable liver transplantation without the need for deceased or living donors. We developed a chimeric liver (CL) by repopulation of rat hepatocytes in a mouse and successfully transplanted it into a rat recipient with vessel reconstruction. This study was designed to investigate the feasibility of CL for supporting the recipient after auxiliary liver grafting. METHODS: Hepatocytes from luciferase transgenic or luciferase/LacZ double-transgenic rats were transplanted into 20- to 30-day-old urokinase-type plasminogen activator/severe-combined immunodeficiency (uPA/SCID) mice (n = 40) to create CLs with rat-origin hepatocytes. After replacement of mouse hepatocytes with those from rats, the CLs were transplanted into wild-type Lewis (n = 30) and analbuminemia (n = 10) rats, followed by immunosuppression using tacrolimus (TAC) with/without cyclophosphamide (CPA) or no immunosuppression. Organ viability was traced by in vivo bioimaging and Doppler ultrasonography in the recipient rats for 4 to 6 months. Rat albumin production was also evaluated in the analbuminemia rats for 4 months. In addition, histological analyses including Ki67 proliferation staining were performed in some recipients. RESULTS: Both immunosuppressive protocols significantly improved graft survival and histological rejection of CLs as compared to the nonimmunosuppressed group. Although rat albumin production was maintained in the recipients for 4 months after transplantation, ultrasonography revealed patent circulation in the grafts for 6 months. Ki67 staining analysis also revealed the regenerative potential of CLs after a hepatectomy of the host native liver, whereas immune reactions still remained in the mouse-origin structures. CONCLUSIONS: This is the first report showing that engineered CLs have potential as alternative grafts to replace the use of grafts from human donors.

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections.

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA-SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA-SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA-SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8weeks after injection) was similar in both TK-NOG mice and uPA-SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.

Severe necroinflammatory reaction caused by natural killer cell-mediated Fas/Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse.

UNLABELLED: The necroinflammatory reaction plays a central role in hepatitis B virus (HBV) elimination. Cluster of differentiation (CD)8-positive cytotoxic T lymphocytes (CTLs) are thought to be a main player in the elimination of infected cells, and a recent report suggests that natural killer (NK) cells also play an important role. Here, we demonstrate the elimination of HBV-infected hepatocytes by NK cells and dendritic cells (DCs) using urokinase-type plasminogen activator/severe combined immunodeficiency mice, in which the livers were highly repopulated with human hepatocytes. After establishing HBV infection, we injected human peripheral blood mononuclear cells (PBMCs) into the mice and analyzed liver pathology and infiltrating human immune cells with flow cytometry. Severe hepatocyte degeneration was observed only in HBV-infected mice transplanted with human PBMCs. We provide the first direct evidence that massive liver cell death can be caused by Fas/Fas ligand (FasL) interaction provided by NK cells activated by DCs. Treatment of mice with anti-Fas antibody completely prevented severe hepatocyte degeneration. Furthermore, severe hepatocyte death can be prevented by depletion of DCs, whereas depletion of CD8-positive CTLs did not disturb the development of massive liver cell apoptosis. CONCLUSION: Our findings provide the first direct evidence that DC-activated NK cells induce massive HBV-infected hepatocyte degeneration through the Fas/FasL system and may indicate new therapeutic implications for acute severe/fulminant hepatitis B.