Language：English   Publishing type：Research paper (scientific journal)  

The two principal histological types of primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma, can coexist within a tumor, comprising combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Although the possible involvement of liver stem/progenitor cells has been proposed for the pathogenesis of cHCC-CCA, the cells might originate from transformed hepatocytes that undergo ductular transdifferentiation or dedifferentiation. We previously demonstrated that concomitant introduction of mutant HRASV12 (HRAS) and Myc into mouse hepatocytes induced dedifferentiated tumors that expressed fetal/neonatal liver genes and proteins. Here, we examine whether the phenotype of HRAS- or HRAS/Myc-induced tumors might be affected by the disruption of the Trp53 gene, which has been shown to induce biliary differentiation in mouse liver tumors. Hepatocyte-derived liver tumors were induced in heterozygous and homozygous p53-knockout (KO) mice by hydrodynamic tail vein injection of HRAS- or Myc-containing transposon cassette plasmids, which were modified by deleting loxP sites, with a transposase-expressing plasmid. The HRAS-induced and HRAS/Myc-induced tumors in the wild-type mice demonstrated histological features of HCC, whereas the phenotype of the tumors generated in the p53-KO mice was consistent with cHCC-CCA. The expression of fetal/neonatal liver proteins, including delta-like 1, was detected in the HRAS/Myc-induced but not in the HRAS-induced cHCC-CCA tissues. The dedifferentiation in the HRAS/Myc-induced tumors was more marked in the homozygous p53-KO mice than in the heterozygous p53-KO mice and was associated with activation of Myc and YAP and suppression of ERK phosphorylation. Our results suggest that the loss of p53 promotes ductular differentiation of hepatocyte-derived tumor cells through either transdifferentiation or Myc-mediated dedifferentiation.

DOI： 10.1111/cas.14996

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Differentiated epithelial cells show substantial lineage plasticity upon severe tissue injuries. In chronically injured mouse livers, part of hepatocytes become Sry-HMG box containing 9 (Sox9) (+) epithelial cell adhesion molecule (-) hepatocyte nuclear factor 4 alpha(+)biphenotypic hepatocytes. However, it is not clear whether all Sox9(+) hepatocytes uniformly possess cellular properties as hepatocyte progenitors. Here, we examined the microarray data comparing Sox9(+) hepatocytes with mature hepatocytes and identified CD24 as a novel marker for biphenotypic hepatocytes. Immunohistochemical analyses showed that part of Sox9(+) hepatocytes near expanded ductular structures expressed CD24 in the liver injured by 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet and by bile duct ligation. Indeed, Sox9(+) hepatocytes could be separated into CD24(-) and CD24(+) cells by fluorescence activated cell sorting. The ratio of CD24(+) cells against CD24(-) ones in Sox9+ hepatocytes gradually increased while DDC-injury progressed and colony-forming capability mostly attributed to CD24(+) cells. Although hepatocyte markers were remarkably downregulated in of Sox9(+) CD24(+) hepatocytes, they re-differentiated into mature hepatocytes in vitro and in vivo. Our current results demonstrate that the emergence of biphenotypic hepatocytes is a sequential event including the transition from CD24(-) and CD24(+) status, which may be a crucial step for hepatocytes to acquire progenitor properties.

DOI： 10.1038/srep39990

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Hepatocellular carcinoma develops in either chronically injured or seemingly intact livers. To explore the tumorigenic mechanisms underlying these different conditions, we compared the mRNA expression profiles of mouse hepatocellular tumors induced by the repeated injection of CCl4 or a single diethylnitrosamine (DEN) injection using a cDNA microarray. We identified tumor-associated genes that were expressed differentially in the cirrhotic CCl4 model (H19, Igf2, Cbr3, and Krt20) and the non-cirrhotic DEN model (Tff3, Akr1c18, Gpc3, Afp, and Abcd2) as well as genes that were expressed comparably in both models (Ly6d, Slpi, Spink3, Scd2, and Cpe). The levels and patterns of mRNA expression of these genes were validated by quantitative RT-PCR analyses. Most of these genes were highly expressed in mouse livers during the fetal/neonatal periods. We also examined the mRNA expression of these genes in mouse tumors induced by thioacetamide, another cirrhotic inducer, and those that developed spontaneously in non-cirrhotic livers and found that they shared a similar expression profile as that observed in CCl4-induced and DEN-induced tumors, respectively. There was a close relationship between the expression levels of Igf2 and H19 mRNA, which were activated in the cirrhotic models. Our results show that mouse liver tumors reactivate fetal/neonatal genes, some of which are specific to cirrhotic or non-cirrhotic modes of pathogenesis.

DOI： 10.1111/cas.12700

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Mature hepatocytes are suggested to possess a capacity for bile ductular transdifferentiation, but whether and how hepatocytes contribute to ductular reaction in chronic liver diseases has not been elucidated. We examined whether mouse hepatocytes can transdifferentiate into bile ductular cells in vitro, using a three-dimensional collagen gel culture method, and in vivo, using a liver repopulation model in which beta-galactosidase-positive hepatocytes from Alb-Cre x ROSA26R mice were transplanted into the liver of wild-type mice. We further examined the relative contribution of intrinsic hepatocytes in ductular reaction in a hepatocyte lineage-tracing model using Mx1-Cre x ROSA26R mice treated with polyinosinic-polycytidylic acid. Within collagen gels, hepatocytes exhibited branching morphogenesis associated with the emergence of bile duct-like phenotype. In the Liver repopulation model, many beta-galactosidase- positive, hepatocyte-derived bile ductular structures were identified; these markedly increased after liver injury. In Mx1-Cre x ROSA26R mice, relatively minor but significant contributions of hepatocyte-derived bile ductules were observed in both periportal and centrilobular ductular reaction. As the centrilobular ductular reaction progressed, the portal ducts or ductules migrated toward the injured area and joined with hepatocyte-derived ductules, Leaving the portal tract without biliary structures. We conclude that hepatocytes and bite ducts or ductules are important sources of ductular reaction and that the intrahepatic biliary system undergoes remarkable remodeling in response to chronic liver injury.

DOI： 10.1016/j.ajpath.2014.07.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Background: Biphenotypic hepatocytes expressing Sox9 emerge upon liver injuries associated with ductular reaction. Results: Sox9(+) biphenotypic hepatocytes are derived from mature hepatocytes (MHs). Some of them are incorporated into ductular structures, whereas they efficiently differentiate to functional hepatocytes. Conclusion: Biphenotypic hepatocytes not only terminally convert to cholangiocytes but also differentiate back to MHs. Significance: Mature epithelial cells can show plasticity upon severe injuries and contribute to regeneration.
It has been shown that mature hepatocytes compensate tissue damages not only by proliferation and/or hypertrophy but also by conversion into cholangiocyte-like cells. We found that Sry HMG box protein 9-positive (Sox9(+)) epithelial cell adhesion molecule-negative (EpCAM(-)) hepatocyte nuclear factor 4-positive (HNF4(+)) biphenotypic cells showing hepatocytic morphology appeared near EpCAM(+) ductular structures in the livers of mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. When Mx1-Cre:ROSA mice, which were injected with poly(I:C) to label mature hepatocytes, were fed with the DDC diet, we found LacZ(+)Sox9(+) cells near ductular structures. Although Sox9(+)EpCAM(-) cells adjacent to expanding ducts likely further converted into ductular cells, the incidence was rare. To know the cellular characteristics of Sox9(+)EpCAM(-) cells, we isolated them as GFP(+)EpCAM(-) cells from DDC-injured livers of Sox9-EGFP mice. Sox9(+)EpCAM(-) cells proliferated and could differentiate to functional hepatocytes in vitro. In addition, Sox9(+)EpCAM(-) cells formed cysts with a small central lumen in collagen gels containing Matrigel (R) without expressing EpCAM. These results suggest that Sox9(+)EpCAM(-) cells maintaining biphenotypic status can establish cholangiocyte-type polarity. Interestingly, we found that some of the Sox9(+) cells surrounded luminal spaces in DDC-injured liver while they expressed HNF4. Taken together, we consider that in addition to converting to cholangiocyte-like cells, Sox9(+)EpCAM(-) cells provide luminal space near expanded ductular structures to prevent deterioration of the injuries and potentially supply new hepatocytes to repair damaged tissues.

DOI： 10.1074/jbc.M113.517243

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：4  

We previously showed that mature hepatocytes could transdifferentiate into bile ductular cells when placed in a collagen-rich microenvironment. To explore the mechanism of transdifferentiation, we examined whether inflammatory cytokines affected the phenotype of hepatocytes in a three-dimensional culture system. Spheroidal aggregates of rat hepatocytes were embedded within a type I collagen gel matrix and cultured in the presence of various cytokines. In the control, hepatocytes gradually lost expression of albumin, tyrosine aminotransferase, and hepatocyte nuclear factor (HNF)-4α, while aberrantly expressed bile ductular markers, including cytokeratin 19 (CK 19) and spermatogenic immunoglobulin superfamily (SgIGSF). Among the cytokines examined, tumor necrosis factor (TNF)-α inhibited expression of albumin and HNF-4α, both at mRNA and protein levels. After culturing for 2 weeks with TNF-α, hepatocytic spheroids were transformed into extensively branching tubular structures composed of CK 19- and SgIGSF-positive small cuboidal cells. These cells responded to secretin with an increase in secretion and expressed functional bile duct markers. TNF-α also induced the phosphorylation of Jun N-terminal kinase (JNK) and c-Jun, and the morphogenesis was inhibited by SP600125, a specific JNK inhibitor. Furthermore, in chronic rat liver injury induced by CCl(4) , ductular reaction in the centrilobular area demonstrated strong nuclear staining of phosphorylated c-Jun. Our results demonstrate that TNF-α promotes the ductular transdifferentiation of hepatocytes and suggest a role of TNF-α in the pathogenesis of ductular reaction.

DOI： 10.1002/jcb.24424

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DOI： 10.1016/j.ajpath.2012.08.034

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC  

Orthovanadate (OV), an inhibitor of protein tyrosine phosphatases, affects various biological processes in a cell-type-specific manner. In this study, we investigated the effect of OV on hepatic stellate cells (HSCs). When primary rat HSCs were cultured in the presence of 10% serum, they spontaneously lost characteristic stellate morphology, proliferated, and were transformed into an activated state with the formation of abundant stress fibers and increased expression of both a-smooth muscle actin and collagen type I mRNA. OV treatment inhibited proliferation and activation of HSCs and partially reversed the phenotype of activated HSCs. Among the signaling molecules investigated, phosphorylation of the Src protein at tyrosine 416 was the most striking in OV-treated HSCs. Treatment of cells with Src family inhibitors partially abrogated the effects of OV. Furthermore, transfection of v-Src into activated HSCs induced a stellate morphology similar to that in the quiescent state. We then examined whether OV could effectively suppress HSC activation in vivo after liver injury induced by either carbon tetrachloride or dimethylnitrosamine. OV significantly reduced the appearance of a-smooth muscle actin-positive cells and decreased collagen deposition, concomitant with an improvement in liver function. Our study showed for the first time that OV was able to suppress die activation of HSCs, possibly through the modulation of Src activity, and attenuated fibrosis after chronic liver injury. (Am J Pathol 2009, 174:881-890; DOI. 10.2353/ajpath.2009.080261)

DOI： 10.2353/ajpath.2009.080261

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Spermatogenic immunoglobulin superfamily (SgIGSF) is an intercellular adhesion molecule of the nectin-like family. While screening its tissue distribution, we found that it was expressed in fetal liver but not adult liver. In the present study, we examined which cells in developing and regenerating liver express SgIGSF via immunohistochemistry and Western blot analysis. In developing mouse liver, SgIGSF expression was transiently upregulated at perinatal ages and was restricted to the lateral membrane of biliary epithelial cells (BECs). In regenerating rat livers from the 2-acetylaminofluorene/partial hepatectomy model, SgIGSF was detected exclusively in oval cells that aligned in ductal and trabecular patterns by the second week posthepatectomy. In human livers, fetal and newborn bile ducts and cirrhotic bile ductules were clearly positive for SgIGSF, whereas disease-free adult bile ducts were negative. To investigate the role of SgIGSF in bile duct/ductule formation, we used an in vitro model in which rat hepatocyte aggregates embedded in collagen gels containing insulin and epidermal growth factor extend epithelial sheets and processes in the first week and form ductules within a month. The process and ductular cells were continuously positive for SgIGSF and cytokeratin 19, a BEC marker. When the aggregate culture was started in the presence of a function-blocking anti-SgIGSF antibody, the number of epithelial processes per aggregate was reduced by 80%. Conclusion: We propose that SgIGSF is a novel and functional BEC adhesion molecule that is expressed for a limited time during active bile duct/ductule formation.

DOI： 10.1002/hep.21501

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC  

We investigated the mechanism of phenotypic plasticity of hepatocytes in a three-dimensional organoid culture system, in which hepatocytic spheroids were embedded within a collagen gel matrix. Hepatocytes; expressed several bile duct markers including cytokeratin (CK) 19 soon after culture and underwent branching morphogenesis within the matrix in the presence of insulin and epidermal growth factor. Cultured hepatocytes did not express Delta-like, a specific marker for oval cells and hepatoblasts. Furthermore, hepatocytes isolated from c-kit mutant rats (Ws/Ws), which are defective in proliferation of oval cells, showed essentially the same phenotypic changes as those isolated from control rats. The bile duct-like differentiation of hepatocytes was associated with increased expression of Jagged1, Jagged2, Notch1, and several Notch target genes. CK19 expression and branching morphogenesis were inhibited by dexamethasone, a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059), and a phosphatidyl inositol 3-kinase inhibitor (LY294002). After being cultured for more than 3 weeks within the gels, hepatocytes transformed into ductular structures surrounded by basement membranes. Our results suggest that hepatocytes might have the potential to transdifferentiate into bile duct-like cells without acquiring a stem-like phenotype and that this is mediated through specific protein tyrosine phosphorylationpathways.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background/Aims: H19 is a paternally imprinted gene that is believed to function as non-coding mRNA. While H19 is only faintly expressed in the normal adult liver, it is abundantly expressed during the fetal period. We explored the possibility that H19 might participate in the regulation of hepatocyte proliferation.
Methods: Adult male rats and mice were subjected to a two-thirds partial hepatectomy, and after various time periods, hepatocytes were isolated and analyzed for H19 gene expression. The expression was also examined in cultured rat hepatocytes.
Results: The expression of H19 was dramatically increased after 2 days (rat) and 4 days (mouse), peaked at 3 days (rat) and 6 days (mouse), and then gradually declined. In both species, the increase in U19 gene expression was preceded by the induction of proliferating cell nuclear antigen and DNA synthesis. An allele-specific RT-PCR analysis in the mouse showed that the paternally imprinted status of the gene was maintained after a partial hepatectomy. H19 was strongly induced in spheroid cultures after transient hepatocyte proliferation, but not in conventional monolayer cultures, in which persistent proliferation occurred.
Conclusions: Our results demonstrated that H19 gene expression was dynamically regulated in adult hepatocytes in close association with their proliferation. (C) 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jhep.2004.01.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background/Aims: To develop a specific isolation method of hepatic sinusoidal endothelial cells (SEC), we applied the immunomagnetic method using a monoclonal antibody (SE-1) that recognizes a membranous antigen expressed only in rat SEC.
Methods: Cells were isolated by incubating mixed non-parenchymal cells, which were obtained by collagenase digestion of the liver, with SE-1-conjugated superparamagnetic polystyrene beads. The conventional Percoll method was also performed in parallel to compare with the immunomagnetic method. The isolated cells were cultured on glass coverslips coated with type 1 collagen in the presence of various growth factors for 6 days.
Results: Approximately 98% of the isolated cells were positive for SE-1 and the contamination of Kupffer cells or stellate cells was less than 1%. The purity was significantly better than that obtained by the Percoll method. The cultured cells showed typical SEC features, such as sieve plates and uptake of acetylated low-density lipoprotein. Although the cells continuously underwent apoptotic cell death after 2 days, they started robust cell growth after 3 days and were well maintained during the culture period.
Conclusions: Our simple and specific isolation method enables us to culture SEC with high purity and should be useful for the biological analysis of SEC.
(C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0168-8278(02)00048-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We recently found that a thioether analog of K vitamin (Cpd 5) inhibited the activity of protein-tyrosine phosphatases (PTPases) and induced protein-tyrosine phosphorylation in a human hepatoma cell line (Hep3B), We have now examined the structural requirements for induction of protein-tyrosine phosphorylation and PTPase inhibition by several K vitamin analogs. Thioether analogs with sulfhydryl arylation capacity, especially those with a hydroxy (Cpd 5) or a methoxy group at the end of the side chain, induced protein-tyrosine phosphorylation, but non-arylating analogs, such as those with an all-carbon or O-ether side chain, did not. Among the receptor-tyrosine kinases, epidermal growth factor receptors were tyrosine-phosphorylated by treatment with thioether analogs, whereas insulin and hepatocyte growth factor receptors were not. An increase in tyrosine-phosphorylated ERK2 mitogen-activated protein kinase was also observed. The activity of purified T cell PTPase was inhibited only by the thioether analogs, but not by non-arylating analogs. Furthermore, the epidermal growth factor receptor dephosphorylation activity of Hep3B cell lysates was inhibited by Cpd 5 treatment. A similar induction of protein-tyrosine phosphorylation by Cpd 5 was seen in other human hepatoma cell lines together with growth inhibition. However, one cell line (HepG2), which was relatively resistant to growth inhibition by Cpd 5, did not increase its phosphorylation levels upon Cpd 5 treatment. These results suggest that cell growth inhibition by thioether analogs is closely associated with inhibition of PTPases by sulfhydryl arylation and with tyrosine phosphorylation of selected proteins.

DOI： 10.1074/jbc.274.49.34803

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

To clarify the role of transforming growth factor-beta (TGF-beta) and its receptors in hepatocyte growth, we studied the expression of TGF-beta 1 and its receptors and the sensitivity to growth inhibition by TCF-beta 1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-beta type II receptor (T beta R-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-beta 1 increased with time in primary culture. The cell surface TGF-beta receptor proteins (T beta R-I, II, and III), examined by the receptor affinity-labeling assay using I-125-TGF-beta 1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TCF-beta 1 protein was added at later times in culture, corresponding to the presence of increased TGF-beta receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of T beta R-I, T beta R-II, T beta R-III, IGF-II/M-6-PR, and TGF-beta 1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-beta receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in T beta R-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-beta 1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-beta receptors and sensitivity to growth inhibition by TGF-beta 1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176.612-623, 1998. (C) 1998 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-4652(199809)176:3<612::AID-JCP18>3.0.CO;2-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.

DOI： 10.1074/jbc.273.16.9906

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS  

We screened genes responsive to transforming growth factor-beta (TGF-beta(1)) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta(1) to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta(1) treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta(1) protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N-1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta(1). Spermine levels in Hep3B cells were decreased by TGF-beta(1) treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N-1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitory effects of TGF-beta(1) on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Congeners of vitamin K are known to inhibit cell growth, although the precise mechanisms of growth inhibition are not well understood. To investigate the mechanisms involved, we synthesized several vitamin K analogs and examined their growth inhibitory activities for a human hepatoma cell line (Hep3B). The analogs included 2-methyl-1,4-naphthoquinone and trimethylbenzoquinone, with and without aliphatic side chains at position 3. The side chains were all-carbon, thioethers, or O-ethers. Growth inhibition was potent in the compounds with short chains. The presence of a sulfur (thioether) or oxygen atom (O-ether) at the site of attachment of the side chain to the ring potentiated the activity. Apoptotic cell death was induced by the potent growth inhibitory compounds at low concentrations (20-60 mu M), whereas necrotic cell death followed treatment with the same compounds at high concentrations. Expression of c-myc, which is thought to be associated with apoptosis, was increased by most of the compounds tested. Both reduced glutathione and cysteine almost completely abrogated the growth inhibitory effects of the thioether analogs as well as of vitamin K-3. The effect of glutathione was less prominent for the all-carbon and O-ether analogs, and cysteine had no effect on these analogs. Catalase and deferoxamine mesylate had no significant effect on the thioether analogs, although they showed partial antagonistic effects on the growth inhibition of vitamin K-3 and the all-carbon and O-ether analogs. Other non-thiol antioxidants tested had no effect on any of the analogs. Our results indicated that vitamin K-related quinoid compounds cause growth inhibition and both apoptotic and necrotic cell death and that the effects may be mediated by interaction at position 3 of their quinoid nuclei with cellular thiols.

DOI： 10.1074/jbc.270.47.28304

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Language：English   Publisher：LIPPINCOTT-RAVEN PUBL  

We report an apocrine adenocarcinoma of the left axilla associated with hamartomatous apocrine gland hyperplasia of both axillae. The patient, a 69-year-old man, presented with no symptoms or complaints other than an oval mass felt in the left axilla. The mass was resected and histopathological examination revealed a papillary apocrine adenocarcinoma located within hyperplastic apocrine glands. Because gallium scintigraphy performed after the operation still showed bilateral abnormal uptakes, skin and subcutaneous tissues of the bilateral axillary areas were resected. Histological examination demonstrated marked multilobular hyperplasia of the apocrine glands. These hyperplastic glands did not show distinct atypia, and there was no evidence of tumor remnants in the left axilla. The patient has shown no signs of local recurrence or metastasis at 20 months' follow-up. To our knowledge, this is the first case of malignant transformation of hamartomatous apocrine gland hyperplasia (apocrine gland organic hamartoma or apocrine nevus).

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILLIAMS & WILKINS  

BACKGROUND: The phenotype of isolated rat hepatocytes changes during in vitro cultivation, but it is not clear whether this process turns back when the cells are returned to an in vivo milieu. Glutathione S-transferase placental form (GST-P), which is not expressed in hepatocytes in vivo, is induced within a few days of cultivation, whereas cytochrome P-450 constitutionally expressed in hepatocytes in vivo rapidly diminishes. We explored the dynamic nature of hepatocytic phenotypes using these two markers and intrahepatic and intrasplenic transplantation of cultured hepatocytes.
EXPERIMENTAL DESIGN: Hepatocytes of F344 rats were isolated and cultured for 5 days to form spheroidal aggregates and then implanted into the livers and spleens of congenic analbuminemic rats. Expression of GST-P and P-450 was then examined immunocytochemically. In the intrahepatic case, the implanted hepatocytes could be distinguished from surrounding host hepatocytes by albumin immunostaining.
RESULTS: The intrahepatically implanted hepatocytes turned GST-P-negative within 5 to 10 days, and re-expressed P-450 to a comparable level to that in surrounding host hepatocytes after 2 days. On the other hand, the hepatocytes implanted within spleens continued to express GST-P for 10 days, and started to express P-450 at extraordinarily high levels after 5 to 10 days.
CONCLUSIONS: The phenotype of hepatocytes under in vivo and in vitro conditions has proven to be changeable from one to another.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：HUMANA PRESS INC  

The redistribution of glutamate and GABA in postischemic brains was examined immunocytochemically using the gerbil model of unilateral 1 h cerebral ischemia. In the cerebral neocortex, the majority of neurons underwent recovery processes after 5 h of recirculation, while neurons in the hippocampus were irreversibly damaged. Glutamate-like immunoreactivity (LI) was highly increased in the degenerating hippocampal CA3 pyramidal tells after recirculation, while in the neocortex and the hippocampal CAI sector, the pyramidal cells showed only slightly increased glutamate-LI. GABA-LI-positive punctae in the neuropil, corresponding to neuronal processes of GABAergic neurons, were accentuated after recirculation both in the cerebral neocortex and the hippocampus. Although the astrocytes on the nonischemic side showed neither glutamate-LI nor GABA-LI, the swollen astrocytes and their foot processes, which were observed after recirculation, often showed strong glutamate-LI and GABA-LI. These data suggest (1) the accumulation of glutamate or glutamate-like substances, especially in the CA3 pyramidal cells, (2) the excitation of the GABAergic neurons and their subsequent uptake of GABA, and (3) the sequestration of the extracellular neurotransmitters by astrocytes in the postischemic period.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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Invasive fungal infections including invasive pulmonary aspergillosis (IPA) generally have a poor prognosis, because the fungi spread throughout various organs. Therefore, it is important to accurately identify the fungal species for treatment. In this article, we present the results of pathological and molecular morphological analyses that were performed to elucidate the cause of respiratory failure in a patient who died despite suspicion of IPA and treatment with micafungin (MCFG). Pathological analysis revealed the existence of cystic and linear fungi in lung tissue. The fungi were identified as Aspergillus fumigatus (A. fumigatus) by partial sequencing of genomic DNA. Correlative light microscopy and electron microscopy (CLEM) analysis confirmed that fungi observed with light microscopy can also be observed with scanning electron microscopy (SEM) using formalin-fixed paraffin-embedded tissue sections. SEM revealed an atypical ultrastructure of the fungi including inhomogeneous widths, rough surfaces, and numerous cyst-like structures of various sizes. The fungi showed several morphological changes of cultured A. fumigatus treated with MCFG that were previously reported. Our results indicate that integrated analysis of ultrastructural observation by SEM and DNA sequencing may be an effective tool for analyzing fungi that are difficult to identify by conventional pathological analysis.

DOI： 10.1007/s00795-024-00402-2

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BACKGROUND: The University of Wisconsin (UW) solution is the gold standard for preserving the liver, kidneys, and pancreas. For renal preservation, the addition of the flavonoid, quercetin (QE), to the preservation solution reduces damage to renal tubular cells, and the addition of sucrose (Suc) is also beneficial for preservation. The aim of this study was to investigate the protective effects of QE and Suc on porcine livers in terms of warm and cold injury and to evaluate whether their use improves ischemia-reperfusion (I/R) injury after simple cold storage (CS). METHODS: We tested porcine livers procured after 30 minutes of warm ischemia followed by preservation for 6 hours under the following 2 conditions: group 1, preserved with the CS/UW solution (n = 4); group 2, preserved with the CS/UW solution containing Que 33.1 μM and Suc 0.1 M (n = 6). All livers were evaluated using an ex vivo isolated liver reperfusion model with saline-diluted autologous blood. RESULTS: Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase levels in group 2 were significantly lower at 30 minutes of reperfusion than in group 1. Furthermore, histologic evaluation by hematoxylin and eosin staining showed significantly fewer morphologic changes in group 2 than in group 1, as indicated by the total Suzuki score. Group 2 also had significantly better scores for sinusoidal congestion and hepatocyte cytoplasmic vacuolization. CONCLUSION: Adding Que and Suc to the UW solution can effectively prevent cold injury in livers donated after circulatory death.

DOI： 10.1016/j.transproceed.2023.07.031

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l-Ornithine (Orn) is a core amino acid responsible for ammonia detoxification in the body via the hepatic urea cycle. Clinical studies in Orn therapy have focused on interventions for hyperammonemia-associated diseases, such as hepatic encephalopathy (HE), a life-threatening neurological symptom affecting more than 80% of patients with liver cirrhosis. However, its low molecular weight (LMW) causes Orn to diffuse nonspecifically and be rapidly eliminated from the body after oral administration, resulting in unfavorable therapeutic efficacy. Hence, Orn is constantly supplied by intravenous infusion in many clinical settings; however, this treatment inevitably decreases patient compliance and limits its application in long-term management. To improve the performance of Orn, we designed self-assembling polyOrn-based nanoparticles for oral administration through ring-opening polymerization of Orn-N-carboxy anhydride initiated with amino-ended poly(ethylene glycol), followed by acylation of free amino groups in the main chain of the polyOrn segment. The obtained amphiphilic block copolymers, poly(ethylene glycol)-block-polyOrn(acyl) (PEG-block-POrn(acyl)), enabled the formation of stable nanoparticles (NanoOrn(acyl)) in aqueous media. We employed the isobutyryl (iBu) group for acyl derivatization in this study (NanoOrn(iBu)). In the healthy mice, daily oral administration of NanoOrn(iBu) for one week did not induce any abnormalities. In the mice exhibiting acetaminophen (APAP)-induced acute liver injury, oral pretreatment with NanoOrn(iBu) effectively reduced systemic ammonia and transaminases levels compared to the LMW Orn and untreated groups. The results suggest that the application of NanoOrn(iBu) is of significant clinical value with the feasibility of oral delivery and improvement in APAP-induced hepatic pathogenesis. STATEMENT OF SIGNIFICANCE: Liver injury is often accompanied by hyperammonemia, a life-threatening condition characterized by elevated blood ammonia levels. Current clinical treatments for reducing ammonia typically entail the invasive approach of intravenous infusion, involving the administration of l-ornithine (Orn) or a combination of Orn and L-aspartate. This method is employed due to the poor pharmacokinetics associated with these compounds. In our pursuit of enhancing therapy, we have developed an orally administrable nanomedicine based on Orn-based self-assembling nanoparticle (NanoOrn(iBu)), which provides sustained Orn supply to the injured liver. Oral administration of NanoOrn(iBu) to healthy mice did not cause any toxic effects. In a mouse model of acetaminophen-induced acute liver injury, oral administration of NanoOrn(iBu) surpassed Orn in reducing systemic ammonia levels and liver damage, thereby establishing NanoOrn(iBu) as a safe and effective therapeutic option.

DOI： 10.1016/j.actbio.2023.07.005

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BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and has a poor prognosis. However, the molecular mechanisms underlying hepatocarcinogenesis and progression remain unknown. In vitro gain- and loss-of-function analyses in cell lines and xenografts revealed that dual-specificity tyrosine-regulated kinase 2 (DYRK2) influences tumour growth in HCC. METHODS: To investigate the role of Dyrk2 during hepatocarcinogenesis, we developed liver-specific Dyrk2 conditional knockout mice and an in vivo gene delivery system with a hydrodynamic tail vein injection and the Sleeping Beauty transposon. The antitumour effects of Dyrk2 gene transfer were investigated in a murine autologous carcinogenesis model. RESULTS: Dyrk2 expression was reduced in tumours, and that its downregulation was induced before hepatocarcinogenesis. Dyrk2 gene transfer significantly suppressed carcinogenesis. It also suppresses Myc-induced de-differentiation and metabolic reprogramming, which favours proliferative, and malignant potential by altering gene profiles. Dyrk2 overexpression caused Myc and Hras degradation at the protein level rather than at the mRNA level, and this degradation mechanism was regulated by the proteasome. Immunohistochemical analyses revealed a negative correlation between DYRK2 expression and MYC and longer survival in patients with HCC with high-DYRK2 and low-MYC expressions. CONCLUSIONS: Dyrk2 protects the liver from carcinogenesis by promoting Myc and Hras degradation. Our findings would pave the way for a novel therapeutic approach using DYRK2 gene transfer. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is one of the most common cancers, with a poor prognosis. Hence, identifying molecules that can become promising targets for therapies is essential to improve mortality. No studies have clarified the association between DYRK2 and carcinogenesis, although DYRK2 is involved in tumour growth in various cancer cells. This is the first study to show that Dyrk2 expression decreases during hepatocarcinogenesis and that Dyrk2 gene transfer is an attractive approach with tumour suppressive activity against HCC by suppressing Myc-mediated de-differentiation and metabolic reprogramming that favours proliferative and malignant potential via Myc and Hras degradation.

DOI： 10.1016/j.jhepr.2023.100759

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With the preponderance of a high-calorie diet and sedentary lifestyle, the prevalence of non-alcoholic steatohepatitis (NASH), a state of abnormally elevated lipid accumulation in the liver with chronic inflammation, is increasing at an alarming rate worldwide. Hence, cost-effective therapeutic interventions are required to manage this disease at an early stage. Numerous reports have suggested a link between gut microbial dysbiosis, particularly a decrease in the abundance of short-chain fatty acids (SCFA)-producing microbiota and NASH pathogenesis. Considering these low molecular weight (LMW) SCFAs such as acetic, propionic, and butyric acids have been used to inhibit hepatic steatosis in mouse models. However, the poor pharmacokinetic (PK) profile of SCFAs, caused due to their LMW, renders them therapeutically ineffective. Thus, to improve the PK characteristic-based therapeutic efficacy of LMW SCFAs, we designed SCFA-based prodrugs that possess self-assembling characteristics in aqueous media. The designed SCFA prodrugs consist of enzyme-metabolizable amphiphilic block copolymers, [poly(ethylene glycol)-b-poly(vinyl ester)s] conjugated to propionic acid (PA) or butyric acid (BA) by an ester linkage, which self-assemble into stable nanosized micelles several tens of nanometers in diameter (NanoPA and NanoBA). Via pharmacological analysis, we confirmed that, after oral administration, LMW BA decreased to a physiological level within 24 h in the liver, whereas BA liberated from NanoBA was observed until 72 h post-administration, implying a sustained release profile. Here, we evaluated the therapeutic efficacy of NanoSCFA in a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD)-induced NASH and liver fibrosis mouse model by ad libitum drinking. NanoSCFA, particularly NanoBA, exhibited the remarkable potential to ameliorate the phenotypic features of fatty liver disease by reducing hepatic lipogenesis and fibrosis, with negligible adverse effects. In contrast, conventional LMW SCFAs failed to prevent the pathogenesis of fatty liver disease, which plausibly can be explained by their rapid clearance and discernible adverse effects. Mechanistic studies revealed that NanoBA restored the nuclear expression of PPARα, a transcriptional factor regulating mitochondrial fatty acid oxidation, in the periportal hepatocytes and decreased the CPT1A expression level in the hepatic tissues, reflecting the therapeutic effects of NanoBA. Taken together, we confirmed that our NanoSCFA potentially improved the PK properties of SCFAs, and it consequently alleviated NASH symptoms and fibrotic liver compared to LMW SCFAs. Our study establishes NanoSCFA as a suitable nano-assembled prodrug for NASH treatment.

DOI： 10.1016/j.biomaterials.2023.122047

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BACKGROUND AND AIM: There are very few reports comparing the use of the University of Wisconsin solution and histidine-tryptophan-ketoglutarate solution as machine perfusion solutions for marginal liver grafts. We aimed to clarify whether the use of the histidine-tryptophan-ketoglutarate solution in hypothermic machine perfusion improves the split-liver graft function in a large animal model. METHODS: Porcine split-liver grafts were created by 75% liver resection. Hypothermic machine perfusion experimental groups were divided as follows: Group 1, perfusate, University of Wisconsin gluconate solution (UW group; n = 5), and Group 2, perfusate, histidine-tryptophan-ketoglutarate solution (HTK group; n = 4). After 4 h of preservation, the liver function was evaluated using an isolated liver reperfusion model for 2 h. RESULTS: In the HTK group, the portal vein and hepatic artery resistance during hypothermic machine perfusion and the portal vein resistance during isolated liver reperfusion were lower than those in the UW group. In addition, the total Suzuki score for hepatic ischemia-reperfusion injury in the HTK group was significantly better than that in the UW group. The number of anti-ETS-related genes staining-positive sinusoid epithelial cell nuclei in the HTK group was higher than that in the UW group (not significant). CONCLUSIONS: The histidine-tryptophan-ketoglutarate solution can be perfused with lower vascular resistance than the University of Wisconsin solution, reducing shear stress and preventing sinusoid epithelial cell injury in marginal grafts used as split-liver grafts.

DOI： 10.1111/jgh.16140

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Maternal overnutrition affects offspring susceptibility to nonalcoholic steatohepatitis (NASH). Male offspring from high-fat diet (HFD)-fed dams developed a severe form of NASH, leading to highly vascular tumor formation. The cancer/testis antigen HORMA domain containing protein 1 (HORMAD1), one of 146 upregulated differentially expressed genes in fetal livers from HFD-fed dams, was overexpressed with hypoxia-inducible factor 1 alpha (HIF-1alpha) in hepatoblasts and in NASH-based hepatocellular carcinoma (HCC) in offspring from HFD-fed dams at 15 weeks old. Hypoxia substantially increased Hormad1 expression in primary mouse hepatocytes. Despite the presence of three putative hypoxia response elements within the mouse Hormad1 gene, the Hif-1alpha siRNA only slightly decreased hypoxia-induced Hormad1 mRNA expression. In contrast, N-acetylcysteine, but not rotenone, inhibited hypoxia-induced Hormad1 expression, indicating its dependency on nonmitochondrial reactive oxygen species production. Synchrotron-based phase-contrast micro-CT of the fetuses from HFD-fed dams showed significant enlargement of the liver accompanied by a consistent size of the umbilical vein, which may cause hypoxia in the fetal liver. Based on these findings, a maternal HFD induces fetal origins of NASH/HCC via hypoxia, and HORMAD1 is a potential therapeutic target for NASH/HCC.

DOI： 10.1038/s41598-022-17501-8

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Hepatocellular carcinoma (HCC) activates platelets through the action of adjacent sinusoidal cells. Activated platelets bind to tumor-associated endothelial cells and release growth factors that promote tumor progression. We hypothesized that platelets encapsulated with tumor inhibitors would function as drug carriers for tumor therapy. We propose a therapeutic strategy for HCC using autologous platelets encapsulating multiple tyrosine kinase inhibitors in a rat chemically induced HCC model. Sorafenib or lenvatinib was encapsulated in platelets isolated from tumor-bearing rats in vitro. The rats were divided into groups that received repeated intravenous injections (twice a week for 10 weeks) of the following materials: placebo, sorafenib (SOR), lenvatinib (LEN), autologous platelets, autologous platelets encapsulating sorafenib (SOR-PLT) and autologous platelets encapsulating lenvatinib (LEN-PLT). The therapeutic effect was then analyzed by ultrasonography (US) and histopathological analysis. Histopathological and US analysis demonstrated extensive tumor necrosis in the tumor tissue of SOR-PLT or LEN-PLT, but not in other experimental groups. By liquid chromatography-mass spectrometry, more abundant sorafenib was detected in tumor tissues after SOR-PLT administration than in surrounding normal tissues, but no such difference in sorafenib level was observed with SOR administration. Therefore, the use of autologous platelets encapsulating drugs might be a novel therapeutic strategy for HCC.

DOI： 10.1002/ijc.33915

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Liver fibrosis is a chronic disease resulting from repetitive or prolonged liver injury with limited treatment options. Sorafenib has been reported to be a potential antifibrotic agent; however, its therapeutic effect is restricted because of its low bioavailability and severe adverse effects in the gastrointestinal (GI) tract. In this study, we developed sorafenib-loaded silica-containing redox nanoparticles (sora@siRNP) as an oral nanomedicine to treat liver fibrosis. The designed siRNP were prepared by self-assembly of amphiphilic block copolymers, which possess antioxidant nitroxide radicals as a side chain of the hydrophobic segment and porous silica particles in the nanoparticle core. The silica moieties in the core formed a crosslink between the self-assembling block copolymers to afford stable drug absorption, which could be useful in harsh GI conditions after oral drug administration. Based on in vitro evaluation, sora@siRNP exerted antiproliferative and antifibrotic effects against hepatic stellate cells (HSCs) and low toxicity against normal endothelial cells. A pharmacokinetic study showed that siRNP significantly improved the bioavailability and distribution of sorafenib in the liver. In an in vivo study using a mouse model of CCl4-induced liver fibrosis, oral administration of sora@siRNP significantly suppressed the fibrotic area in comparison to free sorafenib administration. In mice with CCl4-induced fibrosis, free sorafenib administration did not suppress the expression of α-smooth muscle actin; however, mice treated with sora@siRNP showed significantly suppressed expression of α-smooth muscle actin, indicating the inhibition of HSC activation, which was confirmed by in vitro experiments. Moreover, oral administration of free sorafenib induced severe intestinal damage and increased leakage into the gut, which can be attributed to the generation of reactive oxygen species (ROS). Our antioxidant nanocarriers, siRNP, reduced the adverse effects of local ROS scavenging in the GI tract. Our results suggest that sora@siRNP could serve as a promising oral nanomedicine for liver fibrosis.

DOI： 10.1016/j.jconrel.2022.04.002

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BACKGROUND: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. METHODS: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. RESULTS: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. CONCLUSIONS: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

DOI： 10.1007/s00535-021-01846-4

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This study examined the efficacy of end-ischemic hypothermic oxygenated machine perfusion preservation (HOPE) using an originally developed machine perfusion system for split-liver transplantation. Porcine split-liver grafts were created via 75% liver resection after 10 min of warm ischemia. In Group 1, grafts were preserved by simple cold storage (CS) for 8 h (CS group; n = 4). In Group 2, grafts were preserved by simple CS for 6 h and end-ischemic HOPE for 2 h (HOPE group; n = 5). All grafts were evaluated using an isolated ex vivo reperfusion model with autologous blood for 2 h. Biochemical markers (aspartate aminotransferase and lactate dehydrogenase levels) were significantly better immediately after reperfusion in the HOPE group than in the CS group. Furthermore, the HOPE group had a better histological score. The levels of inflammatory cytokines (tumor necrosis factor-α, interferon-γ, interleukin-1β, and interleukin-10) were significantly lower after reperfusion in the HOPE group. Therefore, we concluded that end-ischemic HOPE for split-liver transplantation can aid in recovering the graft function and reducing ischemia-reperfusion injury. HOPE, using our originally developed machine perfusion system, is safe and can improve graft function while attenuating liver injury due to preservation.

DOI： 10.1038/s41598-021-01467-0

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Language：Japanese   Publisher：(一社)日本移植学会  

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2021&ichushi_jid=J00083&link_issn=&doc_id=20210716280001&doc_link_id=10.11386%2Fjst.56.1_1&url=https%3A%2F%2Fdoi.org%2F10.11386%2Fjst.56.1_1&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

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BACKGROUND AND AIMS: Mitogen-activated protein kinase kinase (MKK) 7 and MKK4 are upstream activators of c-Jun NH2 -terminal kinases (JNKs) and have been shown to be required for the early development of the liver. Although it has been suggested that MKK7 might be involved in the regulation of hepatocyte proliferation, the functional role of MKK7 in the liver has remained unclear. APPROACH AND RESULTS: Here, we examined phenotypic alterations in liver-specific or hepatocyte/hematopoietic cell-specific MKK7 knockout (KO) mice, which were generated by crossing MKK7LoxP/LoxP with albumin-cyclization recombination (Alb-Cre) or myxovirus resistance protein 1-Cre mice, respectively. The livers of Alb-Cre-/+ MKK7LoxP/LoxP mice developed without discernible tissue disorganization. MKK7 KO mice responded normally to liver injuries incurred by partial hepatectomy or injection of CCl4 . However, tissue repair following CCl4 -induced injury was delayed in MKK7 KO mice compared with that of control mice. Furthermore, after repeated injections of CCl4 for 8 weeks, the liver in MKK7 KO mice showed intense fibrosis with increased protractive hepatocyte proliferation, suggesting that MKK7 deficiency might affect regenerative responses of hepatocytes in the altered tissue microenvironment. MKK7 KO hepatocytes demonstrated normal proliferative activity when cultured in monolayers. However, MKK7 KO significantly suppressed branching morphogenesis of hepatocyte aggregates within a collagen gel matrix. Microarray analyses revealed that suppression of branching morphogenesis in MKK7 KO hepatocytes was associated with a reduction in mRNA expression of transgelin, glioma pathogenesis related 2, and plasminogen activator urokinase-type (Plau); and forced expression of these genes in MKK7 KO hepatocytes partially recovered the attenuated morphogenesis. Furthermore, hepatocyte-specific overexpression of Plau rescued the impaired tissue repair of MKK7 KO mice following CCl4 -induced injury. CONCLUSIONS: MKK7 is dispensable for the regenerative proliferation of hepatocytes but plays important roles in repair processes following parenchymal destruction, possibly through modulation of hepatocyte-extracellular matrix interactions.

DOI： 10.1002/hep.31565

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In cancer, angiogenesis is a critical phenomenon of nascent blood vessel development to facilitate the oxygen and nutrient supply prerequisite for tumor progression. Therefore, targeting tumors at the angiogenesis step may be significant to prevent their advanced progression and metastasis. Although angiogenesis inhibitors can limit the further growth of tumors, complete eradication of tumors may not be possible by monotherapy alone. Therefore, a therapeutic regimen targeting both tumor growth and its vasculature is essential. Because reactive oxygen species (ROS) are fundamental to both angiogenesis and tumor growth, the use of antioxidants may be an effective dual approach to inhibit tumors. We previously confirmed that our original antioxidant nitroxide radical-containing nanoparticles (RNPs) such as pH-sensitive RNPN, and pH-insensitive RNPO, effectively attenuates the tumorigenic and metastasis potentials of triple-negative breast cancer. In this study, we further investigated the efficacy of RNPs to limit the tumor progression by inhibiting the ROS-regulated cancer angiogenesis in a triple-negative breast cancer model. Here, we confirmed that RNPs significantly inhibited in vitro angiogenesis, attributed to the downregulation of the ROS-regulated angiogenesis inducer, vascular endothelial growth factor (VEGF) in the breast cancer cell line (MDA-MB231) and human umbilical vein endothelial cells (HUVEC), which was consistent with decreased cellular ROS. TEMPOL, a low-molecular-weight (LMW) control antioxidant, exhibited anti-angiogenic effects accompanied by cytotoxicity to the endothelial cells. In an in vivo xenograft model for breast cancer, RNPs exerted significant anti-tumor effect due to the decreased expression of tumor VEGF, which prevented accumulation of the endothelial cells. It should be noted that such efficacy of RNPs was obtained with negligible off-target effects. On the other hand, TEMPOL, because of its size, exerted anti-angiogenesis effect accompanied with injuries to the kidneys, which corroborated with previous reports. Our findings imply that RNPs are more potential antioxidants than their LMW counterparts, such as TEMPOL, for the management of breast cancers.

DOI： 10.1016/j.biomaterials.2020.120645

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In organ transplantation, the University of Wisconsin (UW) solution has been the gold standard for organ preservation. Quercetin (Que) has numerous antioxidant and anti-inflammatory activities, and sucrose (Suc) may be effective for cold storage (CS). This study aimed to investigate the in vitro protective effect of Que and Suc on cold injury to the kidney and to determine whether Que + Suc could improve ischemia-reperfusion injury during CS and hypothermic oxygenated perfusion (HOPE) in autologous transplantation models. METHODS: BHK-21 cells were stored at 4°C for 3 days in UW solution for CS/machine perfusion (CS/MP-UW) with Que (33.1 μM, 3.3 μM, 0.33 μM) and Suc (0.1 M). In a porcine model of renal autologous transplantation, left kidney grafts were preserved under 3 conditions: group 1, CS preservation for 24 hours; group 2, CS preservation for 22 hours and HOPE with CS/MP-UW solution for 2 hours; and group 3, identical preservation as group 2, with Que and Suc added to the solution. Animals were euthanized on day 7 after autologous transplantation. RESULTS: After 3 days of CS preservation, the CS/MP-UW solution with Que (33.1 μM, 3.3 μM) and Suc showed significant cell protection against cold injury. In the porcine model of renal autologous transplantation, the last blood Cre level and the blood lipid hydroperoxide on posttransplantation day 2 were significantly different between group 1 and group 3. Moreover, the total endothelial, glomerular, tubular, interstitial (EGTI) histology score in the kidney tissue was also significantly different. Regarding the change in renal resistance in HOPE, the decrease observed in group 3 was significantly larger than that in group 2. CONCLUSIONS: Our results suggest that the addition of Que and Suc to a UW solution can improve kidney preservation and could potentially enhance the outcome of kidney transplantation.

DOI： 10.1097/TXD.0000000000001077

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The prevalence of non-alcoholic steatohepatitis (NASH) rapidly increases with metabolic disorders such as dyslipidaemia, high blood pressure, and hyperglycaemia. B cell lymphoma 6 (Bcl6), a transcriptional repressor, is essential for the formation of germinal centre B cells. In this study, we analysed the role of Bcl6 in NASH progression-associated pathological changes, such as hepatic lipid accumulation, liver fibrosis, and hepatocarcinogenesis. The roles of Bcl6 in NASH were analysed using liver-specific Bcl6 knockout (Bcl6-LKO) and control wild-type (WT) mice. The murine NASH model was established by feeding the mice with choline-deficient, L-amino-acid-defined, high-fat diet (CDAHFD). Feeding the WT mice with CDAHFD for 7 weeks induced the formation of histopathological features resembling human NASH, such as hepatic lipid accumulation, hepatocellular injury, and fibrosis. These histopathological changes were significantly attenuated in Bcl6-LKO mice. Additionally, feeding the male WT mice with CDAHFD for 38 weeks induced the formation of liver tumours, which was suppressed in Bcl6-LKO mice. These findings indicate that Bcl6 is involved in the progression of NASH and NASH-derived tumours.

DOI： 10.1038/s41598-020-66539-z

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Low bone mineral density (BMD)-diagnosed as osteoporosis or osteopenia-has been reported as a new characteristic feature of Fabry disease; however, the mechanism underlying the development of low BMD is unknown. We previously revealed that a mouse model of Fabry disease [GlatmTg(CAG-A4GALT)] exhibits impaired functioning of medullary thick ascending limb (mTAL), leading to insufficient Ca2+ reabsorption and hypercalciuria. Here, we investigated bone metabolism in GlatmTg(CAG-A4GALT) mice without marked glomerular or proximal tubular damage. Low BMD was detected by 20 weeks of age via micro-X-ray-computed tomography. Bone histomorphometry revealed that low BMD results by accelerated bone resorption and osteomalacia. Plasma parathyroid hormone levels increased in response to low blood Ca2+-not plasma fibroblast growth factor 23 (FGF-23) elevation-by 5 weeks of age and showed progressively increased phosphaturic action. Secondary hyperparathyroidism developed by 20 weeks of age and caused hyperphosphatemia, which increased plasma FGF-23 levels with phosphaturic action. The expression of 1α-hydroxylase [synthesis of 1α,25(OH)2D3] in the kidney did not decrease, but that of 24-hydroxylase [degradation of 1α,25(OH)2D3] decreased. Vitamin D deficiency was ruled out as the cause of osteomalacia, as plasma 1α,25(OH)2D3 and 25(OH)D3 levels were maintained. Results demonstrate that secondary hyperparathyroidism due to mTAL impairment causes accelerated bone resorption and osteomalacia due to hyperphosphaturia and hypercalciuria, leading to low BMD in Fabry model mice.

DOI： 10.1096/fba.2019-00080

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DOI： 10.1111/pin.12890

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BACKGROUND Split-liver transplantation can be useful in situations of limited donor resources. However, novel preservation methods are required to help the recipient recover from severe ischemic reperfusion injury incurred due to receiving a relatively small liver graft. MATERIAL AND METHODS Our experiment was performed using porcine livers without warm ischemia time, assuming a brain-dead organ. We made porcine split-liver grafts by 75% liver resection at the back table and divided the specimens into 4 groups. Group 1 was preserved with simple cold storage after splitting (CS; n=3), Group 2 was preserved with hypothermic perfusion preservation (HMP) after splitting (SBP; n=3), Group 3 was preserved with HMP after splitting under perfusion preservation (SDP; n=4), and Group 4 had the whole liver perfused as control grafts (Whole Liver; n=3). To assess potential methods of preservation and their effects, all grafts were evaluated by an ex vivo isolated liver reperfusion model using diluted autologous blood. RESULTS Portal vein pressure resistances during reperfusion were low in Group3 (SDP). Hepatic artery pressure resistances during reperfusion were markedly higher in Group 1(CS) than in the other groups. The levels of AST and LDH were high and increased at 2 h after reperfusion in Group 1 (CS). The histological findings show that the liver cell structure was irregular in Group 1 (CS) but remained regular in Groups 2 (SBP) and 3 (SDP). Histological Suzuki scores were also significantly better in Groups 2 (SBP) and 3 (SDP) compared with Group 1 (CS). CONCLUSIONS Splitting the liver under machine perfusion preservation may help restore the function and reduce ischemia-reperfusion injury.

DOI： 10.12659/AOT.919920

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BACKGROUND: The present study examined the impact of oxygenated machine perfusion on preservation of liver grafts donated after cardiac death by measuring sinusoidal endothelial injury and microcirculatory disturbances. MATERIALS AND METHODS: Fifteen porcine livers were retrieved 60 min after warm ischemia and allocated into three groups as follows: (1) CS group: static cold storage, (2) HMP group: oxygenated hypothermic perfusion preservation, (3) SNMP group: oxygenated subnormothermic perfusion preservation. The liver grafts donated after cardiac death were preserved for 4 h in different treatment conditions mentioned previously, then subject to ex vivo reperfusion for 2 h using diluted allogeneic blood. The hemodynamic parameters, liver function tests, tissue adenosine triphosphate (ATP) levels, and immunohistochemical findings were investigated. RESULTS: The number of sinusoidal epithelial cells and trabecular structures were maintained after 4 h of preservation in the CS, HMP, and SNMP group. Liver tissue ATP levels after 4 h of preservation in the HMP and SNMP groups were significantly higher compared with that in the CS group. The sinusoidal epithelial cells were significantly exfoliated to a more severe extent in the CS group than in the HMP and SNMP groups. Intrasinusoidal platelet aggregation occurred more frequently in the CS group than in the HMP and SNMP groups. CONCLUSIONS: The results indicated that oxygenated machine perfusion preservation was important to prevent the depletion of tissue ATP and maintain sinusoidal homeostasis regardless of the perfusate temperature. Our findings suggest oxygenated machine perfusion preservation as an effective alternative to static cold storage.

DOI： 10.1016/j.jss.2019.07.058

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Oligo-peptides, including monomeric amino acids, have received much attention as bioactive molecules and drugs. One of the biggest problems of these compounds, however, is their very short bioavailability due to instant metabolism and rapid excretion. To solve this problem, we newly designed a poly(ethylene glycol) (PEG)-block-polypeptide self-assembling based drug for the treatment of acute liver injury. Here, PEG-block-poly(L-Ornithine) (PEG-b-POrn) was synthesized via a ring opening polymerization, and a nano-sized polyion self-assembling complex (NanoOrn) was prepared by simply mixing polycationic PEG-b-POrn with polyanionic chondroitin sulfate. The obtained NanoOrn was quite stable under high ionic strength and different pH conditions and NanoOrn exhibited extremely low toxicity in vitro and in vivo as compared to the original PEG-b-POrn. As compared to monomeric L-ornithine, administration of NanoOrn to mice significantly improved bioavailability of liberated ornithine, especially in the liver. Interestingly, NanoOrn treatment in acetaminophen (APAP)-induced acute liver injury mice remarkably suppressed blood ammonia levels and liver injury markers, resulting in more effective improvement of liver damage compared to monomeric ornithine via activation of ornithine transcarbomylase. These results show that the self-assembling polypeptide NanoOrn may provide a new concept and promising therapeutics as nanomedicines.

DOI： 10.1016/j.jconrel.2019.08.011

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The patient was a 76-year-old man who was treated with nivolumab due to recurrent gastric cancer. A blood examination revealed grade 3 alkaline phosphatase (ALP) elevation. A histopathological examination revealed marked portal infiltration, including eosinophils and CD4+ and CD8+ T lymphocytes, suggesting nivolumab-related cholangitis accompanied by the features of both an immune-related adverse event (irAE) and drug-induced liver injury (DILI) with allergic reaction. The patient's ALP level immediately decreased after the administration of prednisolone. Although nivolumab-related cholangitis, a rare irAE, has been reported to be refractory to steroid therapy, patients with features of irAE and allergic DILI might immediately respond to prednisolone.

DOI： 10.2169/internalmedicine.2330-18

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Hepatocellular carcinoma often reactivates the genes that are transiently expressed in fetal or neonatal livers. However, the mechanism of their activation has not been elucidated. To explore how oncogenic signaling pathways could be involved in the process, we examined the expression of fetal/neonatal genes in liver tumors induced by the introduction of myristoylated v-akt murine thymoma viral oncogene (AKT), HRas proto-oncogene, guanosine triphosphatase (HRASV12), and MYC proto-oncogene, bHLH transcription factor (Myc), in various combinations, into mouse hepatocytes in vivo. Distinct sets of fetal/neonatal genes were activated in HRAS- and HRAS/Myc-induced tumors: aldo-keto reductase family 1, member C18 (Akr1c18), glypican 3 (Gpc3), carboxypeptidase E (Cpe), adenosine triphosphate-binding cassette, subfamily D, member 2 (Abcd2), and trefoil factor 3 (Tff3) in the former; insulin-like growth factor 2 messenger RNA binding protein 3 (Igf2bp3), alpha fetoprotein (Afp), Igf2, and H19, imprinted maternally expressed transcript (H19) in the latter. Interestingly, HRAS/Myc-induced tumors comprised small cells with a high nuclear/cytoplasmic ratio and messenger RNA (mRNA) expression of delta-like noncanonical Notch ligand 1 (Dlk1), Nanog homeobox (Nanog), and sex determining region Y-box 2 (Sox2). Both HRAS- and HRAS/Myc-induced tumors showed decreased DNA methylation levels of Line1 and Igf2 differentially methylated region 1 and increased nuclear accumulation of 5-hydroxymethylcytosine, suggesting a state of global DNA hypomethylation. HRAS/Myc-induced tumors were characterized by an increase in the mRNA expression of enzymes involved in DNA methylation (DNA methyltransferase [Dnmt1, Dnmt3]) and demethylation (ten-eleven-translocation methylcytosine dioxygenase 1 [Tet1]), sharing similarities with the fetal liver. Although mouse hepatocytes could be transformed by the introduction of HRAS/Myc in vitro, they did not express fetal/neonatal genes and sustained global DNA methylation, suggesting that the epigenetic alterations were influenced by the in vivo microenvironment. Immunohistochemical analyses demonstrated that human hepatocellular carcinoma cases with nuclear MYC expression were more frequently positive for AFP, IGF2, and DLK1 compared with MYC-negative tumors. Conclusion: The HRAS signaling pathway and its interactions with the Myc pathway appear to reactivate fetal/neonatal gene expression in hepatocytic tumors partly through epigenetic alterations, which are dependent on the tumor microenvironment.

DOI： 10.1002/hep4.1327

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Liver cancer consists of two main histological subtypes, hepatocellular carcinoma and cholangiocarcinoma, both of which have poor prognosis. Therefore, in searching for new therapeutic targets, adequate mouse models to develop and validate therapeutic strategies are urgently needed. Although there are mouse models of liver cancer, each model has shortcomings. To overcome these shortcomings, a mouse model using a hydrodynamic tail vein injection and the Sleeping Beauty transposon was developed. By inducing stable expression of oncogenes in mouse hepatocytes in vivo, the model can easily induce liver cancer with specific characteristics that depend on the oncogenes used to induce carcinogenesis. Here, we describe the details of the methods to induce hepatocellular carcinoma or cholangiocarcinoma from mouse hepatocytes.

DOI： 10.1007/978-1-4939-8961-4_20

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The recent clinical application of perfusion technology for the machine preservation of donation after cardiac death (DCD) grafts has some advantages. Oxygenation has been proposed for the preservation of DCD liver grafts. The aim of this study is to clarify whether the use of HbV-containing preservation solution during the subnormothermic machine perfusion (SNMP) of the liver graft improves the graft function of DCD porcine livers in an ex vivo reperfusion model. Pig livers were excised after 60 minutes of warm ischemic time and were preserved under one of three preservation conditions for 4 hours. The preservation conditions were as follows: 4°C cold storage (CS group; N = 5), Hypothermic machine preservation (HMP) with UW gluconate solution (HMP group; N = 5), SNMP (21°C) with UW gluconate solution (SNMP group; N = 5), SNMP (21°C) with HbVs (Hb; 1.8 mg/dl) perfusate (SNMP+HbV group; N = 5). Autologous blood perfusion was performed for 2 hours in an isolated liver reperfusion model (IRM). The oxygen consumption of the SNMP and SNMP+HbV group was higher than the HMP groups (p < 0.05). During the reperfusion, the AST level in the SNMP+HbV group was lower than that in the CS, HMP and SNMP groups. The changes in pH after reperfusion was significantly lower in SNMP+HbV group than CS and HMP groups. The ultrastructural findings indicated that the mitochondria of the SNMP+HbV group was well maintained in comparison to the CS, HMP and SNMP groups. The SNMP+HbVs preservation solution protected against metabolic acidosis and preserved the liver function after reperfusion injury in the DCD liver.

DOI： 10.1371/journal.pone.0226183

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BACKGROUND Machine perfusion techniques offer a solution to the serious organ shortage. However, to assess the effects of machine perfusion, many detailed studies are required. In this study, an ex vivo reperfusion model using diluted autologous blood was confirmed to evaluate the utility of machine preservation for livers donated after cardiac death (DCD). In particular, beneficial effects of the oxygenated hypothermic machine perfusion (HMP) for DCD porcine livers are evaluated. MATERIAL AND METHODS Porcine livers were procured under warm ischemia time (WIT) of 60 min. The livers were preserved by hypothermic machine perfusion (HMP) or static cold storage (CS) for 4 h. After the preservation, the livers were perfused for 2 h using the ex vivo reperfusion model with diluted blood oxygenated by a membrane oxygenator at 35-38°C. RESULTS At 2 h of ex vivo reperfusion with 60 min of warm ischemic time (WIT), the portal vein pressure for CS was higher than HMP (18.8±15.9 vs. 7.5±3.9 [mmHg] in 60 min). Furthermore, LDH in CS was higher than HMP (528.5±149.8 vs. 194.1±32.2 [IU/L/100 g liver] in 60 min. P<0.05). Lactate after CS (60) was significantly higher than HMP (60) (8.67±0.39 vs. 5.68±0.60 [mmol/L] at 60 min. p<0.01). CONCLUSIONS The ex vivo reperfusion model can be used to evaluate the utility of machine perfusion. Advantages of HMP for DCD livers are evaluated with this model.

DOI： 10.12659/AOT.910008

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Oxygenation is necessary for aerobic metabolism, which maintains adenosine triphosphate within the graft organ. In recent years, some studies have demonstrated that subnormothermic machine perfusion (SNMP) with hemoglobin-based oxygen carriers has the potential to improve oxygen metabolism. OBJECTIVE: The aim of this study was to evaluate the effectiveness of perfusate with human-derived hemoglobin vesicles (HbV) under SNMP in a pig model of donation after cardiac death. MATERIALS AND METHODS: In this study, pig livers were procured with a warm ischemic time of 60 minutes and were preserved in 3 groups for 240 minutes. The preservation conditions were as follows: 4°C cold storage (Group 1); SNMP with University of Wisconsin perfusate alone (Group 2); and SNMP (21°C) with University of Wisconsin solution and HbV (hemoglobin, 0.6 mg/dL) perfusate (Group 3). All livers were perfused for 120 minutes using pig autologous blood machine perfusion (reperfusion phase). We investigated the aspartate transaminase level and hemodynamics (portal vein resistance and oxygen consumption) in the preservation and reperfusion phases. A histologic study (hematoxylin-eosin staining) was performed after 240 minutes of preservation. RESULTS: The portal vein resistance of Group 3 was not increased in comparison with Group 2. During preservation, the oxygen consumption of Group 3 was higher than that of Group 2. However, the level of aspartate transaminase did not differ between Groups 2 and 3. CONCLUSION: The present study revealed that perfusate with HbV increased the oxygen consumption of the donor liver during SNMP.

DOI： 10.1016/j.transproceed.2018.02.184

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Recent molecular re-evaluation of Echinococcus granulosus, which causes cystic echinococcosis (CE), has revealed that it is not a single species, but instead consists of 5 cryptic species. Among them, E. granulosus (dog-sheep strain) is predominant (75%) followed by Echinococcus canadensis (22%). The major affected organs, in humans, are the liver (88%) and lungs (11%). Primary cerebral CE comprises less than 1% of all cases. As cerebral CE cases are rare, there are few reports with molecular confirmation of the causative species. This study reports mitochondrial gene analysis from 4 Mongolian pediatric cerebral CE cases. Molecular confirmation was obtained for 3 of the 4 cases, with all 3 cases determined to be due to E. canadensis (G6/G7) infection. None of the cases had other organ involvement. This is only the third report on the molecular identification of the Echinococcus species responsible for cerebral CE, and only the second report of E. canadensis (G6/G7) being the causative agent of cerebral CE.

DOI： 10.1016/j.parint.2018.05.006

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A main feature of Fabry disease is nephropathy, with polyuria an early manifestation; however, the mechanism that underlies polyuria and affected tubules is unknown. To increase globotriaosylceramide (Gb3) levels, we previously crossbred asymptomatic Glatm mice with transgenic mice that expressed human Gb3 synthase (A4GALT) and generated the GlatmTg(CAG-A4GALT) symptomatic Fabry model mice. Additional analyses revealed that these mice exhibit polyuria and renal dysfunction without remarkable glomerular damage. In the present study, we investigated the mechanism of polyuria and renal dysfunction in these mice. Gb3 accumulation was mostly detected in the medulla; medullary thick ascending limbs (mTALs) were the most vacuolated tubules. mTAL cells contained lamellar bodies and had lost their characteristic structure ( i.e., extensive infolding and numerous elongated mitochondria). Decreased expression of the major molecules-Na+-K+-ATPase, uromodulin, and Na+-K+-2Cl- cotransporter-that are involved in Na+ reabsorption in mTALs and the associated loss of urine-concentrating ability resulted in progressive water- and salt-loss phenotypes. GlatmTg(CAG-A4GALT) mice exhibited fibrosis around mTALs and renal dysfunction. These and other features were consistent with pathologic findings in patients with Fabry disease. Results demonstrate that mTAL dysfunction causes polyuria and renal impairment and contributes to the pathophysiology of Fabry nephropathy.-Maruyama, H., Taguchi, A., Nishikawa, Y., Guili, C., Mikame, M., Nameta, M., Yamaguchi, Y., Ueno, M., Imai, N., Ito, Y., Nakagawa, T., Narita, I., Ishii, S. Medullary thick ascending limb impairment in the GlatmTg(CAG-A4GALT) Fabry model mice.

DOI： 10.1096/fj.201701374R

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Authorship：Lead author   Language：Chinese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Activation of the phosphoinositide 3-kinase- AKT, Yes-associated protein (YAP), and MYC pathways is involved in human liver cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). However, the nature of the interactions among these pathways has remained poorly understood. Herein, we demonstrate the coordination of these pathways during the formation of mouse liver tumors induced by hepatocyte-specific somatic integration of myristoylated AKT, mutant YAP, Myc, or their combinations. Although the introduction of YAP or Myc alone was inefficient in inducing tumors, these proteins accelerated tumorigenesis induced by AKT. The generated tumors demonstrated various histological features: low-grade HCC by AKT/Myc, CC by AKT/YAP, and high-grade HCC by AKT/Myc/YAP. CC induced by AKT/YAP was associated with activation of the Notch pathway. Interestingly, the combination of Myc and YAP generated tumors composed of hepatoblast/stem-like cells expressing mRNA for Afp, D1k1, Nanog, and Sox2 and occasionally forming immature ducts. Finally, immunohistochemical analysis revealed that human HCC and CC were predominantly associated with phosphorylation of S6 and glycogen synthase kinase-3 beta, respectively, and &gt; 60% of CC cases were positive for both phosphorylated glycogen synthase kinase-3 beta and YAP. Our study suggests that hepatocyte-derived tumors demonstrate a wide spectrum of tumor phenotypes, including HCC, CC, and hepatoblastoma-Like, through the combinatory effects of the oncogenic pathways and that the state of the phosphoinositide 3-kinase-AKT pathway is a key determinant of differentiation.

DOI： 10.1016/j.ajpath.2017.07.022

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The effects of warm machine perfusion preservation of liver grafts donated after cardiac death on the intracellular three-dimensional ultrastructure of the organelles in hepatocytes remain unclear. Here we analyzed comparatively the ultrastructure of the endomembrane systems in porcine hepatocytes under warm ischemia and successive hypothermic and midthermic machine perfusion preservation, a type of the warm machine perfusion. Porcine liver grafts which had a warm ischemia time of 60 minutes were perfused for 4 hours with modified University of Wisconsin gluconate solution. Group A grafts were preserved with hypothermic machine perfusion preservation at 8 degrees C constantly for 4 hours. Group B grafts were preserved with rewarming up to 22 degrees C by warm machine perfusion preservation for 4 hours. An analysis of hepatocytes after 60 minutes of warm ischemia by scanning electron microscope revealed the appearance of abnormal vacuoles and invagination of mitochondria. In the hepatocytes preserved by subsequent hypothermic machine perfusion preservation, strongly swollen mitochondria were observed. In contrast, the warm machine perfusion preservation could preserve the functional appearance of mitochondria in hepatocytes. Furthermore, abundant vacuoles and membranous structures sequestrating cellular organelles like autophagic vacuoles were frequently observed in hepatocytes after warm machine perfusion preservation. In conclusion, the ultrastructure of the endomembrane systems in the hepatocytes of liver grafts changed in accordance with the temperature conditions of machine perfusion preservation. In addition, temperature condition of the machine perfusion preservation may also affect the condition of the hepatic graft attributed to autophagy systems, and consequently alleviate the damage of the hepatocytes.

DOI： 10.1371/journal.pone.0186352

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MYC activation at modest levels has been frequently found in hepatocellular carcinoma. However, its significance in hepatocarcinogenesis has remained obscure. Here we examined the role of Myc activation in mouse liver tumours induced by hepatocytic expression of myristoylated AKT (AKT) and/or mutant HRASV12 (HRAS) via transposon-mediated gene integration. AKT or HRAS alone required 5 months to induce liver tumours, whereas their combination generated hepatocellular carcinoma within 8 weeks. Co-introduction of AKT and HRAS induced lipid-laden preneoplastic cells that grew into nodules composed of tumour cells with or without intracytoplasmic lipid, with the latter being more proliferative and associated with spontaneous Myc expression. AKT/HRAS-induced tumorigenesis was almost completely abolished when MadMyc, a competitive Myc inhibitor, was expressed simultaneously. The Tet-On induction of MadMyc in preneoplastic cells significantly inhibited the progression of AKT/HRAS-induced tumours; its induction in transformed cells suppressed their proliferative activity with alterations in lipid metabolism and protein translation. Transposon-mediated Myc overexpression facilitated tumorigenesis by AKT or HRAS, and when it was co-introduced with AKT and HRAS, diffusely infiltrating tumours without lipid accumulation developed as early as 2 weeks. Examination of the dose-responses of Myc in the enhancement of AKT/HRAS-induced tumorigenesis revealed that a reduction to one-third retained enhancing effect but three-times greater introduction damped the process with increased apoptosis. Myc overexpression suppressed the mRNA expression of proteins involved in the synthesis of fatty acids, and when combined with HRAS introduction, it also suppressed the mRNA expression of proteins involved in their degradation. Finally, the MYC-positive human hepatocellular carcinoma was characterized by the cytoplasm devoid of lipid accumulation, prominent nucleoli and a higher proliferative activity. Our results demonstrate that in hepatocarcinogenesis induced by both activated AKT and HRAS, activation of endogenous Myc is an enhancing factor and adequate levels of Myc deregulation further facilitate the process with alterations in cellular metabolism.

DOI： 10.1038/onc.2017.114

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Singapore  

The bile duct system is a pathway of bile transportation from the liver to the intestine and plays a role of exocrine function of the liver. It consists of two different types of epithelial cells, hepatocytes and cholangiocytes. Anatomically as well as developmentally, the bile duct could be divided into intrahepatic bile duct (IHBD) and extrahepatic bile duct (EHBD
extrahepatic hepatic duct, gallbladder, cystic duct, and common bile duct) system. Initially, the secreted bile is transported through the apical side of hepatocytes called as bile canaliculus and then transferred to the duct system (IHBD and EHBD). EHBD characteristically develops the peribiliary glands (PBGs) which are suggested to be a niche of progenitor cell for the hepatobiliary system. Recent studies have revealed that development of IHBD and EHBD is differently regulated during developmental stage of the liver. EHBD arises from a part of the pancreatobiliary domain of foregut endodermal epithelium. In contrast, IHBD develops from the hepatoblasts inhabiting in the fetal liver as a common progenitor cell for hepatocytes and cholangiocytes. In this chapter, we first show histology of the bile duct system and review recent advances in the regulatory mechanisms of both IHBD and EHBD development.

DOI： 10.1007/978-981-10-3500-5_1

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The carcinogenic mechanism of extrahepatic cholangiocarcinoma (ECC) is unclear, due at least in part to the lack of an appropriate mouse model. Because human studies have reported frequent genetic alterations in the Ras- and TGFβ/SMAD-signaling pathways in ECC, mice with tamoxifen-inducible, duct-cell-specific Kras activation and a TGFβ receptor type 2 (TGFβR2) deletion were first generated by crossing LSL-KrasG12D , Tgfbr2flox/flox , and K19CreERT mice (KT-K19CreERT ). However, KT-K19CreERT mice showed only mild hyperplasia of biliary epithelial cells (BECs) in the extrahepatic bile duct (EHBD) and died within 7 wk, probably a result of lung adenocarcinomas. Next, to analyze the additional effect of E-cadherin loss, KT-K19CreERT mice were crossed with CDH1flox/flox mice (KTC-K19CreERT ). Surprisingly, KTC-K19CreERT mice exhibited a markedly thickened EHBD wall accompanied by a swollen gallbladder within 4 wk after tamoxifen administration. Histologically, invasive periductal infiltrating-type ECC with lymphatic metastasis was observed. Time-course analysis of EHBD revealed that recombined BECs lining the bile duct lumen detached due to E-cadherin loss, whereas recombined cells could survive in the peribiliary glands (PBGs), which are considered a BEC stem-cell niche. Detached dying BECs released high levels of IL-33, as determined by microarray analysis using biliary organoids, and stimulated inflammation and a regenerative response by PBGs, leading eventually to ECC development. Cell lineage tracing suggested PBGs as the cellular origin of ECC. IL-33 cooperated with Kras and TGFβR2 mutations in the development of ECC, and anti-IL-33 treatment suppressed ECC development significantly. Thus, this mouse model provided insight into the carcinogenic mechanisms, cellular origin, and potential therapeutic targets of ECC.

DOI： 10.1073/pnas.1619416114

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The BrafV637E mutation is frequently reported in mouse hepatic tumors, depending on the mouse strain, and corresponds to the human BrafV600E mutation. In this study, we detected the BrafV637E mutation by whole-exome analysis in 4/4 hepatic tumors induced by neonatal treatment with diethylnitrosamine (DEN) in male B6C3F1 mice. We also detected the BrafV637E mutation in 54/63 (85.7%) hepatic lesions, including microscopic foci and grossly visible tumors, by PCR-direct sequencing. Although the mutation was detected in 5/7 (71.4%) hepatic tumors induced by neonatal DEN treatment followed by repeated CCl4 administration, it was not detected in 24 tumors induced by CCl4 treatment without DEN or in eight spontaneous lesions in B6C3F1 mice, suggesting that the mutation is induced by the genotoxic action of DEN. The DEN-induced tumors exhibited hyperphosphorylation of ERK1 and Akt, suggesting that the BrafV637E mutation might activate the MAPK and Akt pathways. Moreover, the DEN-induced tumors overexpressed mRNAs for the oncogene-induced senescence (OIS) markers such as p15Ink4b and p19Arf as well as pro-survival/pro-proliferative cytokines/chemokines such as complement C5/C5a, ICAM-1, IL-1 receptor antagonist and CXCL9, suggesting that the BrafV637E mutation influences the expression of genes involved in either OIS or cellular growth/survival. Liver-specific expression of mutated Braf under control of the albumin enhancer/promoter resulted in an enlarged liver that consisted entirely of small basophilic hepatocytes resembling DEN-induced preneoplastic hepatocytes with ERK1/Akt hyperphosphorylation and C5/C5a overexpression. These results indicate that the BrafV637E mutation induces hepatocytic changes in DEN-induced hepatic tumors. © 2016 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

DOI： 10.1002/mc.22510

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Tumor hypoxia, which is associated with poor prognosis in cancer, is known to lead to resistance to radiotherapy and anticancer chemotherapy. Impaired drug penetration in hypoxic regions has been recognized as an essential barrier to drug development in solid tumors. Here, we propose novel hypoxia-activated prodrugs, which drastically improved the penetration property of commonly used anticancer drugs in the hypoxic region. In this design, conventional anticancer drugs were modified with 2-nitroimidazole derivatives. The most important point of this study was that the prodrug designed formed a 6-membered cyclic structure to allow liberation of the active drug in the hypoxic region. This design markedly increased the selectivity of the hypoxia-targeted prodrug, resulting in significant reduction of adverse effects in the normoxic region. In vitro studies confirmed the selective activation under hypoxic conditions. In vivo studies showed drastic reduction of adverse effects associated with conventional anticancer drugs and improvement of the survival rate of mice. Immunofluorescence analyses confirmed that the designed prodrug had a tendency to localize at the hypoxic region, in contrast to conventional anticancer drugs, which localize only at the normoxic region.

DOI： 10.1021/acs.molpharmaceut.6b00011

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.transproceed.2015.12.076

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DOI： 10.1016/j.transproceed.2015.12.087

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Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial-mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-β)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-β pathway may be effective treatment for cHC-CCs and ICCs.

DOI： 10.1073/pnas.1517188113

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DOI： 10.11378/organbio.22.121

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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E-cadherin is an important adhesion molecule whose loss is associated with progression and poor prognosis of liver cancer. However, it is unclear whether the loss of E-cadherin is a real culprit or a bystander in liver cancer progression. In addition, the precise role of E-cadherin in maintaining liver homeostasis is also still unknown, especially in vivo. Here we demonstrate that liver-specific E-cadherin knockout mice develop spontaneous periportal inflammation via an impaired intrahepatic biliary network, as well as periductal fibrosis, which resembles primary sclerosing cholangitis. Inducible gene knockout studies identified E-cadherin loss in biliary epithelial cells as a causal factor of cholangitis induction. Furthermore, a few of the E-cadherin knockout mice developed spontaneous liver cancer. When knockout of E-cadherin is combined with Ras activation or chemical carcinogen administration, E-cadherin knockout mice display markedly accelerated carcinogenesis and an invasive phenotype associated with epithelial-mesenchymal transition, up-regulation of stem cell markers, and elevated ERK activation. Also in human hepatocellular carcinoma, E-cadherin loss correlates with increased expression of mesenchymal and stem cell markers, and silencing of E-cadherin in hepatocellular carcinoma cell lines causes epithelial-mesenchymal transition and increased invasiveness, suggesting that E-cadherin loss can be a causal factor of these phenotypes. Thus, E-cadherin plays critical roles in maintaining homeostasis and suppressing carcinogenesis in the liver.

DOI： 10.1073/pnas.1322731111

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The phenotype of hepatocytes has been thought to be fixed once they are terminally differentiated. However, we and other investigators have demonstrated that mature hepatocytes can transform into bile duct/ductule cells in various experimental conditions in vitro. Since the normal bile duct system is almost invariably surrounded by dense periportal collagenous matrices, we placed isolated hepatocytes in a collagen-rich environment to address whether mature hepatocytes can transform into ductular cells. Here, we describe in detail our three-dimensional collagen culture method for the induction of transdifferentiation of mature rat hepatocytes into bile ductular cells. Our in vitro system might be useful for the elucidation of the mechanisms of the aberrant differentiation of hepatocytes in the diseased liver.

DOI： 10.1007/978-1-61779-468-1_13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Although the connexin32 (Cx32)-mediated gap junction is abolished in hepatocellular carcinoma (HCC), the expression of cytoplasmic Cx32 tends to increase in correspondence with the grade of malignancy. Establishing a Tet-off expression system in human nonmetastatic HuH7 HCC cells where cytoplasmic Cx32 was overexpressed by doxycycline (Dox) withdrawal, we previously demonstrated that overexpression of cytoplasmic Cx32 made HuH7 cells metastatic in mice. In our study, hypothesizing that the cytoplasmic Cx32-induced metastasis may involve expansion of the cancer stem cell (CSC) population, we examined whether cytoplasmic Cx32 controlled the size of the side population (SP) in HuH7 Tet-off Cx32 cells. Fluorescence-activated cell sorting revealed that SP was expanded in a Dox-free medium compared with a Dox-supplemented one. Although cytoplasmic Cx32 did not block maturation from SP to non-SP, purified SP reconstituted a larger SP fraction in the Dox-free medium than in the Dox-supplemented one. Furthermore, although SP from HuH7 Tet-off mock cells formed a similar number of CSC spheres of a similar size whether with or without Dox, SP from HuH7 Tet-off Cx32 cells developed a greater number of larger CSC spheres in the Dox-free medium than in the Dox-supplemented one. Taken together, these results suggest that accumulation of cytoplasmic Cx32 should enhance self-renewal of CSC to expand the CSC population in HCC.

DOI： 10.1002/ijc.25308

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Although hepatocyte transplantation (HCTx) is expected to become a useful therapy for human liver diseases, allogenic hepatocytes still tend to be rejected within a short period due to host immunosurveillance. In the present study, we investigated the effect of prior bone marrow transplantation (BMTx) for the engraftment of allogenic hepatocytes using the analbuminemic rat transplantation model. The hepatocytes of Lewis (LEW) rats were not accepted in the liver of retrorsine (RS)/partial hepatectomy (PH)-treated analbuminemic F344 (F344-alb) rats, which express the disparate major histocompatibility complex (MHC) against that of LEW rats. Prior BMTx with the LEW bone marrow cells (BMCs) after sublethal irradiation achieved acceptance and repopulation of LEW hepatocytes in the liver of the RS/PH-treated F344-alb rats, associated with elevation of serum albumin. The replacement of hepatic parenchyma with albumin positive (Alb(+)) donor hepatocytes and elevation of serum albumin levels were dependent on the bone marrow reconstitution by donor BMCs, which was more efficiently achieved by intrabone marrow (IBM)-BMTx than by intravenous (IV)-BMTx. Our results demonstrate that efficient bone marrow reconstitution by IBM-BMTx enables the replacement of the hepatic parenchyma with allogenic hepatocytes in RS/PH-treated analbuminemic rats without immunosuppressants.

DOI： 10.3727/096368910X547453

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DOI： 10.1111/j.1349-7006.2010.01640.x

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Language：English   Publisher：11  

DOI： 10.1111/j.1440-1827.2009.02451.x

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Language：English   Publisher：AMER ASSOC CANCER RESEARCH  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Down syndrome with transient myeloproliferative disorder (TMD) is often associated with perinatal liver fibrosis. The authors recently encountered an autopsy case of this disease with a characteristic severe perisinusoidal liver fibrosis. Osteopontin (OPN) is a molecule that plays an important role in diverse fibro-inflammatory diseases. The purpose of the present report was to examine the involvement of OPN in development of the Down syndrome-associated liver fibrosis. Histology indicated severe perisinusoidal fibrosis and ductular arrangements of hepatocytes in the liver. Appearance of atypical megakaryocytes in the liver, a feature of TMD associated with Down syndrome, was not evident. On immunohistochemistry expression of OPN was observed in hepatocytes often having ductular arrangements and infiltrating macrophages. In contrast, a small number of transforming growth factor-beta 1 (TGF-beta 1)-positive mononuclear cells were present in the liver. Numerous activated hepatic stellate cells (HSC) expressing alpha-smooth muscle actin (alpha-SMA) were seen in the perisinusoidal area. A recent report indicated that OPN could directly activate the HSC. Thus, it is suggested that OPN produced by hepatocytes and macrophages induces activation of the HSC, and leads to the development of perisinusoidal liver fibrosis.

DOI： 10.1111/j.1440-1827.2007.02191.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A considerable amount of evidence has established that gap junctional intercellular communication (GJIC) suppresses tumor development by halting the stage of tumor promotion. Consistently, GJIC is downregulated in tumors. The downregulation of GJIC is caused by not only the reduced expression level of connexin proteins but also their aberrant cytoplasmic localization. Although it has long been thought that cytoplasmic localization of connexin proteins is merely one of the mechanisms of the downregulation of GJIC, careful studies with human tumor samples have indicated that the expression level of intracytoplasmic connexin proteins correlates well with the grade of malignancy and the progression stage of tumors. Hypothesizing that intracytoplasmic connexin proteins should have their proper functions and that their increase should facilitate tumor progression such as cell migration, invasion and metastasis, we examined the effects of overexpressed connexin32 (Cx32) protein on the phenotype of human HuH7 hepatoma cells, which express a basal level of endogenous Cx32 only in cytoplasm. The cells were retrovirally transduced with the Tet-off Cx32 construct so that withdrawal of doxycycline from the culture medium could induce overexpression of Cx32 protein in cytoplasm. Even when overexpressed, Cx32 protein was retained in cytoplasm, i.e., Golgi apparatuses, and did not induce GJIC. However, overexpression of Cx32 protein in cytoplasm enhanced both the motility and the invasiveness of HuH7 cells and induced metastasis when the cells were xenografted into SCID mice. Taken together, cytoplasmic accumulation of connexin proteins may exert effects favorable for tumor progression.

DOI： 10.1007/s00232-007-9048-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Connexins have long been believed to suppress tumour developmerit during carcinogenesis by exerting gap junctional intercellular communication (GJIC). Although GJIC is abrogated in hepatocellular carcinoma (HCC), connexin32 (Cx32) protein tends to remain expressed in cytoplasm, but not in cell-cell contact areas; thus, it is incapable of forming gap junctions. Hypothesising that cytoplasmic Cx32 protein that has accumulated in HCC should have its proper functions, which ate independent of GJIC, we established an inducible expression system of Cx32 in human HuH7 HCC cells, which were unable to support the formation of Cx32-mediated gap junctions, so that Cx32 protein could be overexpressed by doxycycline (Dox) withdrawal. Although the established clone HuH7 Tet-off Cx32 cells exhibited a 4-fold increase in Cx32 expression after Dox withdrawal, none of them Were dye-coupled, and Cx32 protein was retained in the Golgi apparatus. However, the proliferation rate of the HuH7 Tet-off Cx32 cells was significantly higher in the Dox-free medium than in the Dox-supplemented one. Transwell assays also revealed that Dox withdrawal enhanced serum-stimulated motility and invasiveness into Matrigel of the HuH7 Tet-off Cx32 cells. Furthermore, when HuH7 Tet-off Cx32 cells were xenografted into the liver of SCID mice, only the mice to which no Dox was administered developed metastatic lesions, indicating that overexpression of cytoplasmic Cx32 protein induced meiastasis of HuH7 cells. Our results suggest that, while Cx32-mediated GJIC suppresses the development of HCCs, cytoplasmic Cx32 protein exerts effects favourable for HCC progression, such as invasion and metastasis, once the cells have acquired a malignant phenotype. (c) 2007 Wiley-Liss, Inc.

DOI： 10.1002/ijc.22696

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-beta 1) and VEGF plus SB-431542 (an inhibitor of the TGF-beta 1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.

DOI： 10.1007/s00441-007-0387-5

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NEUROL SOC INDIA  

The authors describe an extremely rare case with malignant peripheral nerve sheath tumor (MPNST) with focal epithelioid differentiation presenting as an intraosseous lesion of the spine. A 75-year-old woman presented with progressive paraplegia caused by epidural mass arising from the posterior element of the T7 vertebra. At surgery, the lesion was noted to originate from the T7 vertebra and separate from the dura and spinal nerve roots. The patient died of tumor metastases to the lungs six months after the initial presentation. Histological diagnosis was MPNST. However, the tumor also contained cystic structures lined by epithelioid cells, requiring differentiation from synovial sarcoma. From the histological and immunohistochemical features, as well as the absence of SYT-SSX fusion gene expression, the diagnosis of MPNST with focal epithelioid differentiation was made. This is the first case report of intraosseous MPNST of the spine with a peculiar biphasic appearance.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC  

To elucidate the mechanism of apoptosis of liver sinusoidal endothelial. cells (SECs), we examined the phosphorylation status of Bad and its upstream signaling molecules during apoptosis in culture and after ischemia-reperfusion injury. Rat SECs were isolated by the immunomagnetic method, and 2 days after culture, most SECs underwent apoptosis, which was associated with decreased tyrosine phosphorylation of cellular proteins. Addition of orthovanadate (OV), a protein tyrosine phosphatase inhibitor, sustained cellular protein phosphorylation and strongly inhibited apoptosis. Bad was dephosphorylated at Ser-112 and Ser-136 during apoptosis, but the phosphorylation status of Bad was maintained in the presence of OV. OV activated the Akt, extracellular signal-regulated protein kinase, and p38 mitogen-activated protein kinase pathways, which are involved in Bad phosphorylation. in the absence of OV, depletion of Bad by RNA interference conferred resistance to apoptosis. Hepatic injury after ischemia-reperfusion was alleviated by OV treatment, with significant inhibition of SEC apoptosis. SEC apoptosis in vivo was associated with dephosphorylation of Bad, Akt, and extracellular signal-regulated protein kinase, which was blocked by OV treatment. Our data suggest that maintenance of Bad phosphorylation is important in the prevention of SEC apoptosis and that the anti-apoptotic property of OV might have therapeutic utility.

DOI： 10.2353/ajpath.2006.050462

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Publisher：4  

DOI： 10.1007/s00795-004-0261-4

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Language：English   Publisher：JAPAN SOC CELL BIOLOGY  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

Background: p120catenin (p120ctn), a member of the Armadillo protein family, is ubiquitously expressed and binds to classical cadherins together with other catenins to participate in the adherens junction. p120ctn has numerous isoforms generated through extensive splicing and alternative usage of translation initiation codons. Although the involvement of p120ctn in cell growth control has been postulated, little has so far been known about its expression patterns in the skin and epidermal tumors. Objective: To identify the isoforms expressed in benign and malignant keratinocytes and to analyze the expression patterns of p120ctn in epidermal tumor specimens. Methods: HaCaT, DJM-1, BSCC-93, HSC-1, HSC-5 and A431 cells along with normal skin tissue were subjected to RT-PCR to identify the expressed isoforms. For immunofluorescence study, surgical specimens including 29 squamous cell carcinomas (SSCs), 4 basal cell carcinomas (BCCs) and 4 seborrheic keratoses as well as 3 normal skin samples were stained with specific antibodies to detect p120ctn and E-cadherin. Results: RT-PCR revealed that the isoform 3A was predominantly expressed with faint expression of the isoform 4 and that there was no difference in expressed isoforms between the examined cell tines. Immunofluorescent staining indicated that the expression of p120ctn was significantly reduced in all of the examined squamous cell carcinomas and that p120ctn was frequently localized in cytoplasm. In benign tumors and normal samples, p120ctn was property expressed in cell-cell boundaries. Furthermore, there were several SCCs where E-cadherin continued to be expressed with no expression of p120ctn. Conclusion: Reduced expression and aberrant localization of p120ctn are among the most common events during the development and/or progression of SCCs. (C) 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.jdermsci.2003.12.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

A high level of plasmid DNA expression in rat liver can be achieved by the rapid injection of a large volume of a naked DNA solution into the tail vein, called the 'hydrodynamics-based procedure.' The preparation of PCR-amplified DNA fragments is easier than that of naked DNA. In this paper we evaluated the effects of expressing the erythropoietin (Epo) gene in the rat liver by injecting fCAGGS-Epo, an Epo-expressing PCR-amplified DNA fragment, via the tail vein. After injection of 5 pmol fCAGGS-Epo (10 mug) or pCAGGS-Epo (18.4 mug), plasmid DNA, the serum Epo levels peaked at week 1, then persisted for at least 12 weeks. Transgene-derived Epo secretion resulted in significant erythropoiesis. These results demonstrated that transfer of PCR-amplified DNA fragments into the rat liver via rapid tail vein injection can be achieved. This method may provide a useful means for studying the physiologic function of a putative gene. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2003.08.087

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Gene therapy is expected to revolutionize the treatment of kidney diseases. Viral interleukin (vlL)-10 has a variety of immunomodulatory properties. We examined the applicability of vIL-10 gene transfer to the treatment of rats with crescentic glomerulonephritis, a T helper 1 (Th 1) predominant disease. To produce the disease, Wistar-Kyoto, rats were injected with a rabbit polyclonal anti-rat glomerular basement membrane antibody. After 3 h, a large volume of plasmid DNA expressing viL-10 (pCAGGS-vlL-10) solution was rapidly injected into the tail vein. pCAGGS solution was similarly injected into control rats (pCAGGS rats). We confirmed the presence of vector-derived vlL-10 mainly in the liver and observed high serum vIL-10 levels in pCAGGSvIL-10-injected rats. Compared with the pCAGGS rats, the pCAGGS-vlL-10 rats showed significant therapeutic effects: reduced frequency of crescent formation, decrease in the number of total cells, macrophages, and CD4(+) T cells in the glomeruli, decrease in urine protein, and attenuation of kidney dysfunction. Using quantitative real-time polymerase chain reaction, we also observed that this model was Th1-predominant in the glomeruli and that the ratio of the transcripts of CD4, interferon-gamma, tumor necrosis factor-a, and monocyte chemotactic protein-1 to the transcripts of glucose-6-phosphate dehydrogenase in the glomeruli were all significantly lower in the pCAGGS-vlL-10 rats than in the pCAGGS rats. These results demonstrate that pCAGGS-vlL-10 gene transfer by hydrodynamics-based transfection suppresses crescentic glomerulonephritis.

DOI： 10.1038/sj.gt.3301988

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE CANCER ASSOC  

A growing body of evidence indicates that the gap junction (GJ) plays a pivotal role in tumor suppression by exerting cell-cell communication. It has, however, been reported that expression of connexin26 (Cx26) protein is induced in human ductal carcinomas of the breast and that its amount increases in proportion to the grade of malignancy. We thus examined the effects of overexpressed Cx26 on growth characteristics in GJ-deficient human MCF-7 breast cancer cells that maintain the phenotype of early-stage cancers. MCF-7 cells were transfected with Cx26 cDNA, and several clones of stable transformants exhibiting a high level of cell-cell communication were established. When they were examined in terms of various growth characteristics in vitro, the proliferation rate and the saturation density were drastically reduced in Cx26-transfected clones compared with the mock-transfectant. The anchorage-independent growth capacity was also decreased by 50-75% after transfection of Cx26. Furthermore, the cell migration toward growth factors and cell invasion into Matrigel in a Boyden chamber were suppressed to 5-10% and 20-60%, respectively, of the control in Cx26-transfected clones. When implanted into the mammary fat pads of nude mice in the presence of an excess of 17beta-estradiol, Cx26-transfected clones tended to show slower tumor growth than the mock-transfectant, although the difference was not statistically significant. Our results strongly suggest that the induction of Cx26 protein observed in human breast cancers, reported previously, may not be very relevant to the development of breast cancers, and that Cx26 can function as a tumor suppressor in breast cancer cells.

DOI： 10.1111/j.1349-7006.2003.tb01473.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

Background High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the 'hydrodynamics-based procedure'. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.
Methods We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo.
Results We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 800 mug. Using quantitative real-time PCR, the vector-derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, beta-galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.
Conclusions These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright (C) 2002 John Wiley Sons, Ltd.

DOI： 10.1002/jgm.281

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Previous gene transfer methods using nonviral vectors administered via renal arterial, pelvic, or ureteric routes into the glomerulus, tubules, or interstitial fibroblasts have resulted in low-level expression for &lt;1 month. The peritubular capillaries (PTC) network is one of the main targets of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. To access the PTC, we retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats. We detected lacZ expression exclusively in the interstitial fibroblasts near the PTC of the injected kidney by immunoelectron microscopic analysis. Nephrotoxicity attributable to gene transfer was not apparent. We then used a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo, in a reporter assay. We obtained maximal Epo expression when the DNA solution was injected within 5 sec, and with a volume of 1.0 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 &mu;g. We detected the transgene-derived Epo mRNA by reverse transcription polymerase chain reaction only in the kidneys injected with pCAGGS-Epo. After an injection of 100 mg of pCAGGS-Epo, the serum Epo levels peaked at 208.3 +/- 71.8 mU/ml at week 5, and gradually decreased to 116.2 +/- 38.7 mU/ml at week 24. A similar pattern was obtained using smaller doses of plasmid, 2 &mu;g or 30 &mu;g of pCAGGS-Epo. Transgene-derived Epo secretion resulted in significant erythropoiesis. This novel technique is simple and safe, allowing high-level and long-term stable gene expression specific to the fibroblasts near the PTC, and should have therapeutic value for future applications in humans.

DOI： 10.1089/10430340252792585

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Language：English   Publisher：2  

DOI： 10.1046/j.1440-1827.2002.01325.x

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CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background/Aims: Compound 5 (Cpd 5), a vitamin K analog, inhibits rat hepatocyte DNA synthesis and hepatoma cell growth. The aim of this study was to determine if the inhibitory effect of Cpd 5 on cell growth was related to apoptosis.
Methods: Isolated rat hepatocytes were cultured with Cpd 5, and mitogen-activated signaling pathway and apoptosis pathway were investigated using Western blot analysis.
Results: When rat hepatocytes were cultured with Cpd 5 for 48 h, apoptosis was evident, which included characteristic morphological changes, DNA fragmentation, and the activation of caspase 3 (CPP 32)-like protease. Examination of upstream events of apoptosis pathway showed that the expression of Bax was induced and bcl-2 was inhibited by Cpd 5 treatment. Concomitant with the induction of apoptosis, Cpd 5 activated the extracellular signal-regulated kinase (ERK) signaling pathway. PD 98059, a mitogen-activated protein kinase kinase inhibitor, and glutathione, an anti-thiolo oxidant, not only blocked Cpd 5-induced ERK phosphorylation, but also antagonized the activation of CPP-32, the altered Bcl-2/Bax expression, and DNA fragmentation.
Conclusions: The data suggest that the ERK signaling pathway may be involved in the regulation of rat hepatocyte apoptosis induced by Cpd 5. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD  

Changes in expression of the two tight junction-associated proteins, barmotin/7H6 and ZO-1, as well as the adherence junction-associated protein, E-cadherin, were followed during hepatocyte growth factor/scatter factor (HGF/SC)-induced migration process of MDCK cells. Modulation of the HGF/SF-induced migration process by staurosporine, an inhibitor of protein kinase C (PKC), was also examined. Cell migration induced by HGF/SF consisted of two distinct phases, initial cell spreading between 2 and 9 h after the start of treatment, and the scattering phase which started similar to 12 h after treatment. Both ZO-1 and E-cadherin were expressed at the cell-cell border of adherent cells in the scattering phase, whereas barmotin/7H6, a barrier function-related tight junction protein, was not seen during the early spreading phase. Confluent cultures of MDCK cells, which did not spread after HGF/SF treatment, were positive for barmotin/7H6 expression at cell-cell borders. Blocking PKC activation during HGF/SF treatment with staurosporine inhibited cell spreading, and the cells retained barmotin/7H6 expression until at 6 h after HGF/SF treatment. The results indicate that disappearance of the tight junction protein, barmotin/7H6, is closely associated with cell spreading, with both barmotin/7H6 expression and cell spreading seemingly being regulated by PKC-mediated signaling. (C) 2000 Academic Press.

DOI： 10.1006/cbir.2000.0524

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

It has been suggested that hepatocytes have the ability to form bile ductal structures during normal development and in various pathological conditions of the liver. In the present study, we attempted to establish an in vitro model of ductal morphogenesis of hepatocytic cells by combining an aggregate culture and a type I collagen gel culture. When spheroidal aggregates of rat or mouse primary hepatocytes were embedded within the collagen gel matrix and then cultured with a medium containing a fibroblast-conditioned medium, the aggregates extended many dendritic processes composed of a trabecular arrangement of cells. Dendritic morphogenesis was also seen in embedded aggregates of immortal liver epithelial cell. lines, which spontaneously emerged during long-term cultures of mouse primary hepatocytes. A similar morphogenesis was induced by the presence of insulin in the medium. Although epidermal growth factor (EGF) and hepatocyte growth factor (HGF) showed only a small effect on the morphogenesis of most of the hepatocytic cells when used alone, these factors, especially EGF, enhanced the morphogenetic effect of insulin. Electron microscopical observations revealed luminal structures lined by microvilli within these dendritic processes, indicating ductal differentiation. Immunocytochemically, the dendritic processes were positive for cytokeratin 19, a marker for bile duct cells. On the other hand, an H-ras-transformed mouse liver epithelial cell line and rat hepatocellular carcinoma cell lines did not demonstrate the organized morphogenesis. Our results indicate that hepatocytic cells can produce bile duct-like structures in the presence of the type I collagenous matrix and soluble morphogenetic factors. (C) 1996 Academic Press, Inc.

DOI： 10.1006/excr.1996.0091

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF-alpha, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF-beta family proteins. Affinity crosslinking gels using I-125-labeled TGF-beta showed a significant decrease in the TGF-beta cell-surface type II receptor in Hep40 cells, compared to the TGF-beta-sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c-myc, c-jun, and prothrombin genes, in contrast to the actions of TGF-beta 1 protein, which caused a decrease in the level of c-myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF-beta with a unique TGF-beta receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF-beta as compared to vitamin K suggest that these two growth inhibitors act through differing pathways. (C) 1995 Wiley-Liss, Inc.

DOI： 10.1002/jcp.1041650303

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

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Authorship：Lead author   Language：English   Publisher：JAPANESE CANCER ASSOCIATION  

DOI： 10.1111/j.1349-7006.1990.tb02632.x

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DOI： 10.1111/j.1440-1827.1987.tb00436.x

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Language：Japanese   Book type：Scholarly book

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Language：English   Publisher：(一社)日本癌学会  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：文光堂  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：羊土社  

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Language：Japanese   Publisher：(一社)日本病理学会  

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Language：Japanese   Publisher：(一社)日本病理学会  

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Language：Japanese   Publisher：(一社)日本病理学会  

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Language：English   Publisher：(一社)日本癌学会  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：株式会社アークメディア  

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Language：Japanese   Publisher：(一社)日本移植学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(一社)日本外科学会  

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Language：Japanese   Publisher：(一社)日本内視鏡外科学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO  

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Event date： 2022.9 - 2022.10

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2022.9

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Venue：北海道大学医学部学友会館フラテ大研修室  

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Event date： 2022.8

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Venue：KFC Hall & Rooms（東京・両国）  

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Event date： 2022.8

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Venue：KFC Hall & Rooms（東京・両国）  

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Event date： 2022.4

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Venue：神戸コンベンションセンター  

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Venue：神戸コンベンションセンター  

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Venue：神戸コンベンションセンター  

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Venue：神戸コンベンションセンター  

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Venue：神戸コンベンションセンター  

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Event date： 2021.10

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Venue：札幌医科大学 教育研究棟D101  

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Event date： 2021.10

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Venue：札幌医科大学 教育研究棟D101  

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Event date： 2021.9 - 2021.10

Language：English   Presentation type：Poster presentation  

Venue：パシフィコ横浜+オンライン開催  

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Event date： 2021.9 - 2021.10

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Venue：パシフィコ横浜+オンライン開催  

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Event date： 2021.9

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Venue：札幌医科大学 教育研究棟D101  

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Event date： 2021.9

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Venue：淡路夢舞台・ハイブリッド開催(zoom)  

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Event date： 2021.9

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Event date： 2021.9

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Event date： 2021.9

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Event date： 2021.9

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Venue：淡路夢舞台・ハイブリッド開催(zoom)  

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Event date： 2021.7

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Venue：城山ホテル鹿児島+web開催  

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Event date： 2021.4

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Venue：京王プラザホテル・オンライン  

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Event date： 2021.4

Language：Japanese   Presentation type：Poster presentation  

Venue：京王プラザホテル・オンライン  

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Event date： 2021.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル・オンライン  

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Event date： 2021.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル・オンライン  

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Event date： 2021.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル・オンライン  

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Event date： 2020.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン開催  

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Event date： 2020.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン開催  

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Event date： 2020.11

Language：Japanese   Presentation type：Poster presentation  

Venue：アクトシティ浜松  

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Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フラテ」  

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Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フラテ」  

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Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：WEB開催  

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Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：WEB開催  

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Event date： 2020.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：WEB開催  

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Event date： 2020.7

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：WEB開催  

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Event date： 2020.7

Language：Japanese   Presentation type：Poster presentation  

Venue：WEB開催  

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Event date： 2020.7

Language：Japanese   Presentation type：Poster presentation  

Venue：WEB開催  

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Event date： 2020.7

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：WEB開催  

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Event date： 2019.12

Language：English   Presentation type：Oral presentation (general)  

Venue：淡路夢舞台 国際会議場  

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Event date： 2019.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川市国際会議場  

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Event date： 2019.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川市国際会議場  

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Event date： 2019.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学 教育研究棟Ⅰ１階 D101共用講義室  

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Event date： 2019.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学 教育研究棟一階 D102  

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Event date： 2019.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌グランドホテル  

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Event date： 2019.9

Language：Japanese   Presentation type：Poster presentation  

Venue：国立京都国際会館  

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Event date： 2019.9

Language：English   Presentation type：Oral presentation (general)  

Venue：国立京都国際会館  

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Event date： 2019.8

Language：Japanese   Presentation type：Poster presentation  

Venue：シャトレーゼガトーキングダムサッポロ  

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Event date： 2019.7

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：ホテル椿山荘東京  

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Event date： 2019.5

Language：Japanese   Presentation type：Poster presentation  

Venue：横浜市開港記念会館  

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Event date： 2019.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜市開港記念会館  

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Event date： 2019.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京国際フォーラム  

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Event date： 2019.5

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：東京国際フォーラム  

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Event date： 2019.5

Language：Japanese   Presentation type：Poster presentation  

Venue：東京国際フォーラム  

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Event date： 2019.5

Language：Japanese   Presentation type：Poster presentation  

Venue：東京国際フォーラム  

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Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：たわらや会議室  

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Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学記念ホール  

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Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学記念ホール  

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Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川市国際会議場 大会議室  

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Event date： 2018.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川市国際会議場 大会議室  

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Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪国際会議場  

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Event date： 2018.9

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪国際会議場  

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Event date： 2018.7

Language：Japanese   Presentation type：Poster presentation  

Venue：東京大学伊藤謝恩ホール  

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Event date： 2018.7

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京大学伊藤謝恩ホール  

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Event date： 2018.7

Language：English   Presentation type：Poster presentation  

Venue：東京大学伊藤謝恩ホール  

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Event date： 2018.7

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京大学伊藤謝恩ホール  

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Event date： 2018.6

Language：Japanese   Presentation type：Poster presentation  

Venue：札幌  

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Event date： 2018.6

Language：Japanese   Presentation type：Poster presentation  

Venue：札幌  

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Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌  

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Event date： 2018.6

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：札幌  

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Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌  

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Event date： 2018.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：筑波グランドホテル  

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Event date： 2017.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：神戸ポートアイランド  

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Event date： 2017.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フテラ」  

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Event date： 2017.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フテラ」  

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Event date： 2017.10

Language：English   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部臨床大講堂  

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Event date： 2017.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部臨床大講堂  

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Event date： 2017.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部臨床大講堂  

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Event date： 2017.9

Language：English   Presentation type：Poster presentation  

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Event date： 2017.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：パシフィコ横浜  

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Event date： 2017.7

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：奈良春日野国際フォーラム  

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Event date： 2017.7

Language：Japanese   Presentation type：Oral presentation (keynote)  

Venue：京王プラザホテル(新宿)  

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Event date： 2017.6 - 2017.7

Language：Japanese   Presentation type：Poster presentation  

Venue：旭川市民文化会館  

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Event date： 2017.6 - 2017.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：旭川市民文化会館  

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Event date： 2017.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese   Presentation type：Poster presentation  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル  

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Event date： 2017.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京王プラザホテル  

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Event date： 2016.11 - 2016.12

Language：Japanese   Presentation type：Poster presentation  

Venue：パシフィコ横浜  

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Event date： 2016.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フラテ」  

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Event date： 2016.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フラテ」  

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Event date： 2016.7

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪大学中之島センター  

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Event date： 2016.7

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪大学中之島センター  

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Event date： 2016.5

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：仙台国際センター  

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Event date： 2016.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台国際センター  

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Event date： 2016.5

Language：Japanese   Presentation type：Poster presentation  

Venue：仙台国際センター  

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Event date： 2016.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台国際センター  

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Event date： 2016.5

Language：Japanese   Presentation type：Poster presentation  

Venue：仙台国際センター  

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Event date： 2016.5

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：仙台国際センター  

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Event date： 2016.5

Language：English   Presentation type：Oral presentation (general)  

Venue：仙台国際センター  

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Event date： 2016.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：三浦市民ホール  

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Event date： 2016.2

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：札幌  

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Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学 臨床第一講義室  

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Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学 臨床第一講義室  

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Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学 臨床第一講義室  

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Event date： 2015.10

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2015.10

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.10

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.10

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.10

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：支笏湖畔 丸駒温泉  

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Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フテラ」  

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Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部学友会館「フテラ」  

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Event date： 2015.6

Language：Japanese   Presentation type：Poster presentation  

Venue：米子コンベンションセンター  

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Event date： 2015.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：米子コンベンションセンター  

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Event date： 2015.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：米子コンベンションセンター  

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Event date： 2015.4 - 2015.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.4 - 2015.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.4 - 2015.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.4 - 2015.5

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2015.4 - 2015.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.4 - 2015.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2015.4

Language：English   Presentation type：Poster presentation  

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Event date： 2015.4

Language：English   Presentation type：Poster presentation  

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Event date： 2015.4

Language：English   Presentation type：Symposium, workshop panel (public)  

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Event date： 2015.4

Language：English   Presentation type：Poster presentation  

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Event date： 2014.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川グランドホテル  

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Event date： 2014.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川グランドホテル  

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Event date： 2014.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：旭川グランドホテル  

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Event date： 2014.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：天神の湯（静岡市駿河区）  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.9

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2014.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学  

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Event date： 2014.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌医科大学  

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Event date： 2014.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：スパリゾートハワイアンズ  

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Event date： 2014.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京医科歯科大学  

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Event date： 2014.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京医科歯科大学  

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Event date： 2014.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2014.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：広島国際会議場  

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Event date： 2014.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：広島国際会議場  

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Event date： 2014.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：広島国際会議場  

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Event date： 2014.4

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：広島国際会議場  

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Event date： 2013.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部臨床講義室  

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Event date： 2013.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部臨床講義室  

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Event date： 2013.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北海道大学医学部 フラテ会館  

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Event date： 2013.10

Language：Japanese   Presentation type：Oral presentation (general)