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Masahito Nagaki

Publications and source records attributed to Masahito Nagaki.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells.

The CD133 antigen, identified as a hematopoietic stem cell marker, appears in various human embryonic epithelia including the neural tube, gut, and kidney. We herein investigated whether CD133(+) cells isolated from human hepatocellular carcinoma cell lines possess cancer stem/progenitor cell-like properties. Among the three cell lines studied, the CD133 antigen was found to be expressed only on the surface of Huh-7 cells. CD133(+) cells from Huh-7 performed a higher in vitro proliferative potential and lower mRNA expressions of mature hepatocyte markers, glutamine synthetase and cytochrome P450 3A4, than CD133(-) population of Huh-7 cells. When either CD133(+) or CD133(-) cells were subcutaneously injected into SCID mice, CD133(+) cells formed tumors, whereas CD133(-) cells induced either a very small number of tumors or none at all. Taken together, the identification of CD133(+) cells could thus be a potentially powerful tool to investigate the tumorigenic process in the hepatoma system and to also develop effective therapies targeted against hepatocellular carcinoma.

AC133 Antigen↗
PubMed2006

Dual effects of heat stress on tumor necrosis factor-alpha-induced hepatocyte apoptosis in mice.

The major heat shock protein, HSP70, plays a critical role in cell survival in response to stress, possibly by inhibiting a number of antisurvival pathways. However, heat stress (HS) and HSPs also sensitize cells to certain apoptotic stimuli, such as TNF-alpha. To clarify the relations between HS and apoptosis, we examined the differential effects of the intensity of HS on liver injury and apoptosis induced by TNF-alpha in mice. TNF-alpha was injected into D-galactosamine (GalN)-sensitized mice that were pretreated with or without HS. Liver injury was assessed biochemically and histologically. In GalN-sensitized mice, application of HS for 7 days led to significant enhancement of TNF-alpha-induced hepatotoxicity, despite upregulation of HSP70 in the liver. In contrast, application of HS for 1 day led to attenuation of TNF-alpha-induced liver injury. Repeated HS decreased the levels of the FLICE inhibitory protein short (FLIP(S)) and activated caspase-8 in the liver. The caspase-8 inhibitor Z-IETD-FMK effectively protected both the nontreated and HS-pretreated mice from the hepatotoxicity induced by GalN/TNF-alpha. HS shows dual effects on TNF-alpha-induced hepatocyte apoptosis. Exposure to repeated HS, but not to single HS, leads to enhancement of TNF-alpha-induced hepatocyte apoptosis via the interaction of FLIP and caspase-8.

Animals↗
PubMed2006

Hepatocyte nuclear factor-4alpha and -1 small interfering RNA inhibits hepatocyte differentiation induced by extracellular matrix.

Liver-specific genes and hepatocyte nuclear factor (HNF)-4alpha and -1 are coordinately regulated by extracellular matrix (ECM). However, still are unclear interactions between liver-specific genes and these liver-enriched transcription factors in the mechanism of hepatocyte differentiation regulated by ECM. To elucidate the relationship, we used small interfering RNA (siRNA), which obtains strong and specific knockdown of gene expression in cell culture. Treatment with siHNF-4alpha and siHNF-1 declined the expression levels for HNF-4alpha mRNA and HNF-1 mRNA in primary rat hepatocytes, respectively. The mRNA expressions of albumin, transthyretin, and apolipoproteins that were up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, were decreased in the presence of siHNF-4alpha or siHNF-1. Moreover, siHNF-4alpha and siHNF-1 did not affect the morphology and actin assembly of hepatocytes. These findings demonstrated that HNF-4alpha and HNF-1 directly regulate liver-specific gene expression and might be downstream of cytoskeletal organization in the mechanism by which the differentiated phenotype of hepatocytes is regulated by EHS gel.

Journal Article↗
PubMed2006

Actin organization and hepatocyte differentiation are regulated by extracellular matrix via PI-4,5-bisphosphate in the rat.

Cell adhesion to the extracellular matrix (ECM) plays vital roles in both morphogenesis and regulation of gene expression in cells of adult organisms. How intracellular, cytoskeletal, and signaling factors connect and communicate with the ECM is a fundamental question. Using a cDNA microarray analysis, we identified phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) phosphatase mRNA as being up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, which led to the finding that the PI(4,5)P2 levels of hepatocytes decreased on EHS gel. These changes in hepatocytes on EHS gel were accompanied by promotion of actin depolymerization and differentiated phenotypes of the hepatocytes. Treatment with PI(4,5)P2 or a phospholipase C inhibitor, U73122, resulted in decreased mRNA expressions of albumin and hepatocyte nuclear factor 4 (HNF-4) in hepatocytes. In contrast, actin-disrupting agent gelsolin increased mRNA expressions of albumin and HNF-4. In conclusion, organization of the actin cytoskeleton via PI(4,5)P2 is involved in the regulation of hepatocyte differentiation by the ECM.

Actins↗
PubMed2006

Differential caspase-9-dependent signaling pathway between tumor necrosis factor receptor- and Fas-mediated hepatocyte apoptosis in mice.

BACKGROUND/AIMS: Two apoptosis signaling pathways, which are used by different cell types, are identified. The activation of caspases is critical for the apoptosis process. The aim of this study was to investigate the effects of the caspase-9 inhibitor Ac-LEHD-CHO on tumor necrosis factor receptor (TNFR)- and Fas-mediated hepatocyte apoptosis in vivo, in order to evaluate the similarities and distinctions between TNFR- and Fas-mediated signaling pathways. METHODS: BALB/c mice were intravenously injected with d-galactosamine (GalN, 20 mg/mouse)/tumor necrosis factor-alpha (TNF-alpha, 0.5 microg/mouse), or alphaFas (10 microg/mouse) 30 min after treatment with the caspase-9 inhibitor Ac-LEHD-CHO or pan-caspase inhibitor Z-VAD-fmk. Liver injury was assessed biochemically and histologically. Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using a fluorogenic peptide substrate. RESULTS: Pretreatment with Z-VAD-fmk prevented liver injury and hepatocyte apoptosis induced by either GalN/TNF-alpha or alphaFas. On the other hand, pretreatment with Ac-LEHD-CHO prevented GalN/TNF-alpha-induced hepatotoxicity and hepatocyte apoptosis but not alphaFas-induced liver injury and apoptosis. Both inhibitors reduced the activities of caspase-9 and -3 in the livers of mice administered by GalN/TNF-alpha. However, unlike Z-VAD-fmk, Ac-LEHD-CHO did not inhibit caspase-3 activation in alphaFas-treated mice, although this inhibitor attenuated caspase-9. CONCLUSION: Fas may rely on both caspase-8 activation (extrinsic pathway) and mitochondria (intrinsic pathway) to activate caspase-3. If the mitochondria-dependent pathway is blocked, the other pathway can compensate. In contrast, TNFR may mediate hepatocellular apoptosis mainly through the mitochondria-mediated caspase-9 activation pathway alone.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Prevention of hepatic fibrosis in nonobese diabetic mice: a critical role for interferon-gamma.

BACKGROUND/AIMS: Nonobese diabetic (NOD) mice, a model of type I diabetes mellitus, harbor certain unique defects in their immune system. The aim of this study was to investigate how NOD mice show hepatic injury and subsequent fibrogenic responses. METHODS: Hepatic fibrosis was induced by intraperitoneal injections of dimethylnitrosamine (DMN), and assessed biochemically and histologically. Expressions of cytokine messenger RNA (mRNA) in the liver were determined. RESULTS: In a model of liver cirrhosis induced by dimethylnitrosamine (DMN), we found that NOD mice had lower levels of hepatic fibrosis and better survival than control ICR mice. The resistance to DMN-induced lethality in NOD mice was independent of apoptosis and necrosis of hepatocytes, but apparently due to the prevention of hepatic fibrosis. We also found increased inductions of interferon-gamma (IFN-gamma) mRNA in the liver of NOD mice and of intracellular IFN-gamma from intrahepatic T cells following DMN administration. Treatment with neutralizing anti-IFN-gamma-antibody cancelled the inhibition of hepatic fibrosis in NOD mice. CONCLUSIONS: These results suggest that IFN-gamma is effective for inhibiting hepatic fibrosis and that genetic host factors may be important in determining differential responses to injury.

Animals↗
PubMed2006

Pathogenic role of B cells in anti-CD40-induced necroinflammatory liver disease.

Activated B cells function in antibody production and antigen presentation, but whether they perform any pathophysiological functions at sites of inflammation is not fully understood. Here, we report that intravenous injection of an agonistic anti-CD40 monoclonal antibody (alphaCD40) causes a biphasic inflammatory liver disease in inbred mice. The late phase of disease was suppressed in B-cell-deficient mice and by the depletion of macrophages, but not T cells or natural killer cells. We also report that SCID mice were not susceptible to alphaCD40-induced liver disease unless they were reconstituted with normal B cells and that B cells as well as macrophages played key roles in alphaCD40-induced late phase of liver inflammation. Finally, liver disease and the recruitment of inflammatory cells into the liver were mediated by interferon-gamma and tumor necrosis factor-alpha, but not by Fas. In conclusion, these results indicate that CD40 ligation can trigger a B-cell-mediated inflammatory response that can have pathogenic consequences for the liver.

Animals↗
PubMed2005

Prospective study on early virologic response to treatment with interferon alpha-2b plus ribavirin in patients with chronic hepatitis C genotype 1b.

To evaluate the predictive value of early virologic response (EVR) of achieving a sustained virologic response (SVR), an open, prospective trial including 42 patients with chronic hepatitis C genotype 1b was performed with directly observed 24-week treatment with interferon alpha-2b plus ribavirin. We assessed the predictive values of EVR at days 3, 7, 14, and weeks 4, 8, and 12 of the SVR. The SVR in an intention-to-treat analysis was 19.0%. Patients who reached SVR presented a significantly faster reduction in plasma viral load. Stepwise multiple logistic regression analysis of the factors (gender, age, IFN dosage, ribavirin dosage, HCV RNA, ISDR, and loss of HCV RNA at week 4) revealed that loss of HCV RNA at week 4 was the only independent variable of treatment outcome (P=0.0039). A viral load at treatment day 3 above 100kIU/ml, at day 7 above 50kIU/ml, and at day 14 above 10kIU/ml was 100% predictive for virologic non-response in all except 1 patient. The cutoff levels for HCV RNA at days 3 and 14 of treatment were associated with an algorithm of the failure to detect HCV RNA after 12 weeks of treatment. In conclusions, a very early virologic response assessment could be useful for prediction of later outcome of combination therapy in chronic hepatitis C genotype 1b.

Journal Article↗
PubMed2005

Adenovirus-mediated hepatocyte nuclear factor-4alpha overexpression maintains liver phenotype in cultured rat hepatocytes.

Hepatocyte nuclear factor 4alpha (HNF-4alpha) is a transcription factor that controls embryonal liver development and that maintains and regulates gene expression in adult liver cells. We have previously demonstrated that transient overexpression of HNF-4alpha up-regulates a number of liver-specific genes in hepatoma cell lines. In this study, we extend these studies by assessing the functional role of HNF-4alpha in regulating cellular viability and liver-specific functions of primary rat hepatocytes. In cells transfected with an adenovirus vector carrying rat HNF-4alpha cDNA, induction and maintenance of liver-specific genes and functions were observed over a long-term culture, which might be associated with the prevention of a rapid loss of the mitochondrial membrane potential. In addition, we demonstrated that transthyretin mRNA was up-regulated by HNF-4alpha in primary hepatocytes, but not in hepatoma cells. These results indicate that HNF-4alpha plays a role in the maintenance of morphologically and biochemically functional hepatocytes and that the difference in expression of liver-specific genes induced by HNF-4alpha may depend on a differentiation state of cells.

Adenoviridae↗
PubMed2005

Normal liver regeneration and liver cell apoptosis after partial hepatectomy in tumor necrosis factor-alpha-deficient mice.

AIMS/BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF-alpha also influences liver repair following hepatotoxic damage, and regeneration following partial hepatectomy (PH). The aim of this study was to assess the mechanism by which TNF-alpha influences liver cell apoptosis and regeneration following PH in TNF-alpha-deficient (TNF-alpha(-/-)) mice. METHODS: PH was performed in wild mice and TNF-alpha(-/-) mice. RESULTS: In both groups, serum alanine aminotransferase and serum total bilirubin levels comparably peaked at 6 and 48 h after PH, respectively. No differences were observed in hepatocyte proliferation, as determined by mitotic and the proliferating cell nuclear antigen labeling indices, between TNF-alpha(+/+) and TNF-alpha(-/-) mice. Few terminal deoxynucleotidyl transferase nick end-labeling-positive hepatocytes were seen in either type of mice. Nuclear factor-kappa B DNA binding activity in the remaining liver of TNF-alpha(-/-) mice after PH was similar to that of control mice. Ribonuclease protection assay showed that transforming growth factor beta1 mRNA was up-regulated comparably in the livers of the two groups, and that other cytokines were hardly seen in either. Interleukin-6/ signal transducer and activator of transcription-3-dependent pathway was not affected in TNF-alpha(-/-) mice. CONCLUSIONS: These findings suggest that TNF-alpha has little influence on liver regeneration and liver cell apoptosis after PH in mice.

Alanine Transaminase↗
PubMed2005

Effects of plasma from patients with fulminant hepatic failure on function of primary rat hepatocytes in three-dimensional culture.

BACKGROUND/AIM: As biotechnology continues to advance, a bioartificial liver is expected to be developed for the treatment of patients with fulminant hepatic failure (FHF) whose liver dysfunction is potentially reversible or for providing liver support as a bridge to liver transplantation. While monolayer-cultured hepatocytes rapidly lose their capacity to express many liver-specific functions over time when cultured, spherical-shaped hepatocytes in three-dimensional culture with the use of extracellular matrix components sustain long-term survival by maintaining differentiated hepatocyte functions. The aim of this study was to investigate whether sufficient functions of viable spherical-shaped hepatocytes could be maintained in plasma of patients with FHF in order to use these cells in an extracorporeal system. METHODS: Hepatocyte functions were evaluated under monolayer or three-dimensional culture in FHF plasma. RESULTS: Primary rat hepatocytes on poly-N-p-vinylbenzyl-D-lactonamide (PVLA) formed spheroids even in FHF plasma and maintained their spherical shapes in FHF plasma as long as in medium. Spherical-shaped hepatocytes on PVLA cultured in FHF plasma showed higher activity in albumin secretion, urea formation, and gluconeogenesis than those in normal human plasma or medium. As being cultured in medium, hepatocytes on PVLA cultured in plasma were also superior to cells on collagen in regard to albumin secretion, amino acid metabolism, and gluconeogenesis. CONCLUSIONS: These findings demonstrated that FHF plasma is not toxic to rat hepatocyte spheroids and that hepatocyte spheroids have potential use in the development of a bioartificial liver.

Albumins↗
PubMed2005

Blockade of neutrophil elastase attenuates severe liver injury in hepatitis B transgenic mice.

Serine proteinases produced by polymorphonuclear neutrophils play important roles in neutrophil-mediated tissue injury at inflammatory sites. Although neutrophil recruitment to the liver has been shown to be involved in the exacerbation of liver inflammation, the function of neutrophil elastase (NE) in liver injury remains unclear. Here, we found that administration of an NE inhibitor (NEI) reduced serum alanine aminotransferase (sALT) activity and inflammatory cell infiltration into the liver from 8 to 24 h after injection of antigen-specific cytotoxic T lymphocytes (CTLs) into hepatitis B virus transgenic mice. Furthermore, the NEI treatment reduced the expressions of inflammatory cytokines and chemokines in the liver and tumor necrosis factor alpha production by macrophages. In addition, the NEI treatment suppressed the mRNA expressions of CC chemokine ligand 3 (CCL-3), CCL-4, and macrophage inflammatory protein 2 (MIP-2) in neutrophils in the liver at 8 h after the CTL injection. In support of these results, we confirmed that administration of anti-CCL-3, anti-CCL-4, and anti-MIP-2 monoclonal antibodies suppressed sALT activity and leukocyte migration into the liver. In conclusion, the present results suggest that NE contributes to the early step of the inflammatory cascade in acute viral hepatitis and that NEIs may have potential as therapeutic drugs against acute severe viral hepatitis.

Animals↗
PubMed2004

Pivotal role of nuclear factor kappaB signaling in anti-CD40-induced liver injury in mice.

Nuclear factor kappaB (NF-kappaB) has a central role in coordinating the expression of a wide variety of genes that control immune responses and is also recognized as an antiapoptotic transcription factor. Here, we focused on the role of the NF-kappaB signaling pathway in the interaction between inflammatory cells and hepatocytes in liver inflammation. We found that pretreatment of mice with adenoviruses expressing a mutant form of the inhibitor kappaB superrepressor (Ad5IkappaB), a NF-kappaB inhibitor, reduced the migration of inflammatory cells and cytokine and chemokine expression in the liver 12 hours after a single intravenous injection of an anti-CD40 antibody (alphaCD40) compared with mice infected with control adenoviruses (Ad5LacZ). We also confirmed reductions in cytokine production by macrophages, T cells, and natural killer (NK) cells in the liver of Ad5IkappaB-treated mice by FACS analysis. However, alphaCD40 treatment in Ad5IkappaB-infected mice induced elevation of serum alanine aminotransferase at 24 hours, and the liver injury was associated with massive hepatocyte apoptosis. Furthermore, interferon gamma (IFN-gamma) production by NK cells and T cells was increased and stimulated tumor necrosis factor alpha (TNF-alpha) production by macrophages in the Ad5IkappaB-infected liver. Moreover, the liver injury was completely suppressed by the administration of anti-IFN-gamma and anti-TNF-alpha. These results suggest that inhibition of NF-kappaB activity suppressed alphaCD40-induced liver inflammation at an early phase, resulting in a reduction in cytokine and chemokine production, whereas it sensitized hepatocytes to TNF-alpha-induced apoptosis and exacerbated liver injury at the late phase. In conclusion, NF-kappaB exerts pivotal activities at inflammatory sites, and caution should be exercised in NF-kappaB-targeted therapy of liver disease.

Animals↗
PubMed2004

Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB.

Leflunomide is a novel immunosuppressive and anti-inflammatory agent for the treatment of autoimmune disease. The aim of this study was to investigate whether leflunomide protects from liver injury induced by concanavalin A (Con A), a T-cell-dependent model of liver damage. BALB/c mice were injected with 25 mg/kg Con A in the presence or absence of 30 mg/kg leflunomide. Liver injury was assessed biochemically and histologically. Levels of circulating cytokines and expressions of cytokine messenger RNA (mRNA) in the liver and the spleen were determined. Treatment with leflunomide markedly reduced serum transaminase activities and the numbers of dead liver cells. Leflunomide significantly inhibited increases in plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 2 concentrations, and also reduced TNF-alpha mRNA expression in the liver after administration of Con A. These findings were supported by the results in which leflunomide administration decreased the number of T lymphocytes infiltrating the liver as well as inhibiting their production of TNF-alpha. Activation of nuclear factor kappaB (NF-kappaB), which regulates TNF-alpha production, was inhibited in the liver of mice treated with leflunomide, resulting in a reduction of TNF-alpha production from lymphocytes infiltrating the liver. In conclusion, leflunomide is capable of regulating T-cell-mediated liver injury in vivo and that this event may depend on the decrease of TNF-alpha production in the liver through inhibition of NF-kappaB activation caused by leflunomide.

Animals↗
PubMed2004

Psychoneurological symptoms during interferon therapy in patients with chronic hepatitis: prospective study on predictive use of Cornell Medical Index and electroencephalogram.

BACKGROUND/AIMS: We assessed the usefulness of the Cornell Medical Index (CMI) and electroencephalogram (EEG) in the prediction and early detection of psychoneurological symptoms associated with interferon (IFN) therapy for chronic viral hepatitis. METHODS: Forty-eight consecutive patients received IFN for chronic viral hepatitis for 8-24 weeks. CMI was measured before IFN therapy. Serial EEGs were recorded before IFN therapy, 2, 4 weeks, and thereafter every 4 weeks in the therapy. RESULTS: Psychoneurological symptoms including insomnia, depression, and restlessness were seen in 11 (23%) of 48 patients. Five (13%) of 40 patients with CMI I and II and six (75%) of eight with CMI III developed psychoneurological symptoms (P<0.001). Sensitivity, specificity, and predictive accuracy of CMI III were 55%, 95%, and 75%, respectively. Abnormal EEG such as slow basic rhythm, appeared in 13 patients (27%) during IFN therapy. Psychoneurological symptoms were seen in six (46%) of the 13 patients with abnormal EEG, and in five (14%) of 35 in whom EEG remained normal (P<0.05). CONCLUSIONS: CMI is useful for the prediction of IFN-induced psychoneurological symptoms in patients with chronic viral hepatitis. Serial EEGs contribute to the screening and auxiliarily assessing the adverse effects of IFN on the central nervous system.

Adult↗