Minoru Tomizawa

Etiological analysis of focal nodular hyperplasia of the liver, with emphasis on similar abnormal vasculatures to nodular regenerative hyperplasia and idiopathic portal hypertension.

Pathological studies were performed on 23 cases of focal nodular hyperplasia (FNH) under the hypothesis that FNH is a hyperplastic lesion caused by abnormal vasculatures of portal tracts within the nodule. For a comparison of the histological features of portal tracts, nodular regenerative hyperplasia (NRH), idiopathic portal hypertension (IPH), chronic hepatitis and so-called normal liver were used as control tissues. Extranodular areas of FNH nodules were also examined. Clinical data were briefly summarized. Most of the portal tracts within FNH nodules showed various abnormal findings, such as dilatation and/or stenosis of portal vein, muscular thickening of arterial wall with dilated or stenotic lumina, lymphocyte infiltration, and bile ductule proliferation. However, portal vein thrombi were not found. These findings were not thought to represent compensatory reaction to portal vein thrombosis. Similar abnormal features were also observed in extranodular areas of FNH although to a milder degree. These abnormal features resembled those of NRH and IPH. Moreover, the characteristic scar-like tissues within FNH nodules were proved to be abnormally large portal tracts including large feeding arteries, portal veins and bile ducts. It has been believed that septa and scar-like tissue within FNH nodules are not portal tracts and that arterial malformation independent of portal tracts are related to the development of FNH. In addition, venous structures within FNH modules have until now not been considered to be portal veins. However, this study revealed that severe anomaly of portal tracts including portal veins and hepatic arterial branches existed in FNH nodules. Moreover, portal tracts in extranodular areas were also abnormal. Clinically, only one patient had a history of oral contraceptives. Based on these findings, congenital anomaly of the portal tracts histologically resembling the abnormal portal tracts of NRH and IPH may be related to the pathogenesis of FNH.

Irradiation with ultrasound of low output intensity increased chemosensitivity of subcutaneous solid tumors to an anti-cancer agent

Ultrasound is a possible mechanical method to deliver small molecules into target cells. In order to evaluate the therapeutic potentials provided by ultrasound irradiation, we compared anti-tumor effects of electroporation- and ultrasound-mediated chemotherapy and efficacy of gene transfer by the two methods. Electric pulses (5 Hz, 100 V/cm, 8 square-wave/100 microsec) or ultrasound (1 MHz, 2 W/cm(2), 5 min) was delivered to subcutaneous solid tumors of murine lymphoma after the tumor-bearing mice received an intraperitoneal injection of bleomycin (BLM) (2.5 mg) or intratumoral injection of plasmid DNA containing the luciferase reporter gene. Administration of BLM alone did not affect the subsequent tumor growth but additive treatment with ultrasound irradiation suppressed the growth to the same level as electroporation. The luciferase activity of the DNA-injected tumors showed that ultrasound irradiation achieved better transfection efficiency than plasmid DNA injection alone but the efficacy was not as great as that by electroporation. The low energy level of ultrasound that is currently used for a diagnostic purpose and physical therapy in clinical fields can thereby increase the in vivo chemosensitivity of treated tumors but further modifications are necessary to achieve better efficacy of the ultrasound-mediated gene transfer.

Interstitial tumour cell invasion in small hepatocellular carcinoma. Evaluation in microscopic and low magnification views.

In order to study the process of hepatocellular carcinoma (HCC) development, and to search for a clue to histologic diagnosis of well‐differentiated HCC (wd‐HCC), interstitial invasion in small HCC was evaluated. The study material consisted of 35 cases of HCC that were smaller than 3 cm that comprised 17 cases of wd‐HCC, 18 cases of moderately or poorly differentiated classical HCC (cl‐HCC), and 20 cases of large regenerative nodules (LRN). Interstitial invasion was microscopically classified into three patterns: (i) crossing type, in which HCC was invading across fibrous septa of tumour nodules; (ii) longitudinal type, in which tumour cells were growing longitudinally within fibrous septa; and (iii) irregular type, in which the portal area was irregularly invaded by HCC. The crossing type was found in two cases (12%) of wd‐HCC and 10 cases (56%) of cl‐HCC while the longitudinal type was observed in 16 cases (94%) of wd‐HCC and eight cases (44%) of cl‐HCC. The irregular type was frequently seen in wd‐HCC (15 cases, 88%), and cl‐HCC (12 cases, 67%). No interstitial invasion was observed in LRN. Interstitial invasion could be recognized even in the low magnification view of histological specimens, with a detection rate of 59% (10 cases) in wd‐HCC and 72% (13 cases) in cl‐HCC.

Growth patterns and interstitial invasion of small hepatocellular carcinoma

Twenty‐five caw of small hepatocellular carcinoma (HCC; dhmeter ≥30mm) were evaluated for overall morphologic features and growth patterns. The tumors often showed a uelldtfferentlated, normotrabecular histologic pattern and insidious interstitial invasion, which resembled benign hepetocytes scattered in connective tissues. As the tumor grew, B less‐differentiated tumor area became predominant. Portal tracts Included in small HCC nodules were quantitatively assessed, revealing that they progressively reduced in number with tumor growth. The tumor margin was often reported to be unclear. The present results indicate that the histologk grade of tumor differentiation, capsular formation, existence of liver cirrhosis and patterns of interstitial invaslon are important factors for determining the nature of the margin. The score of argyrophilic nuclear organizer regions (AgNOR) was examined in 5 cases showing typical interstitial imaslon with the insidious type. In each case, the AgNOR score of the invading tumor cells was lower than that of turnor cells within the HCC nodules, but higher than benign hepatocytes in cirrhotic parenchyma. It clarified that the growth activity of well‐differentiated HCC was rather suppressed upon their interstitial invasion.

Sonoporation: Gene transfer using ultrasound

Genes can be transferred using viral or non-viral vectors. Non-viral methods that use plasmid DNA and short interference RNA (siRNA) have advantages, such as low immunogenicity and low likelihood of genomic integration in the host, when compared to viral methods. Non-viral methods have potential merit, but their gene transfer efficiency is not satisfactory. Therefore, new methods should be developed. Low-frequency ultrasound irradiation causes mechanical perturbation of the cell membrane, allowing the uptake of large molecules in the vicinity of the cavitation bubbles. The collapse of these bubbles generates small transient holes in the cell membrane and induces transient membrane permeabilization. This formation of small pores in the cell membrane using ultrasound allows the transfer of DNA/RNA into the cell. This phenomenon is known as sonoporation and is a gene delivery method that shows great promise as a potential new approach in gene therapy. Microbubbles lower the threshold of cavity formation. Complexes of therapeutic genes and microbubbles improve the transfer efficiency of genes. Diagnostic ultrasound is potentially a suitable sonoporator because it allows the real-time monitoring of irradiated fields.

Roles of fibroblast growth factor 10 (Fgf10) in adipogenesis in vivo.

The development of white adipose tissue (WAT) of Fgf10-/- mouse embryos was greatly impaired. Here, we examined the mechanism of Fgf10 action in adipogenesis in vivo. The proliferative activity in the WAT of Fgf10-/- embryos was greatly decreased. We also examined the expression of transcription factors, C/EBPbeta, C/EBPalpha and PPARgamma, that are important for adipogenesis. Although the expression of C/EBPbeta and PPARgamma in the WAT of Fgf10-/- embryos was greatly decreased, the expression of C/EBPalpha was essentially unchanged. Therefore, we examined their expression in the WAT of C/EBPalpha-/- embryos. Although the expression of C/EBPbeta and PPARgamma in the WAT was greatly decreased, the expression of Fgf10 was essentially unchanged. As these results in vivo appeared to be contradictory to a transcriptional cascade model in vitro that C/EBPbeta induces the expression of PPARgamma and C/EBPalpha reported, we also examined their expression in the WAT of wild type embryos at different developmental stages. The expression of Fgf10 and C/EBPalpha was followed by that of C/EBPbeta and PPARgamma. The present findings indicate that Fgf10 but not C/EBPalpha is required for the proliferation of preadipocytes. In contrast, both Fgf10 and C/EBPalpha acting synergistically in separate, parallel pathways are required for the differentiation. Unexpectedly, the transcriptional cascade of adipogenesis in vivo described here is distinct from the cascade in vitro previously reported.

Survival of Primary Human Hepatocytes and Death of Induced Pluripotent Stem Cells in Media Lacking Glucose and Arginine

Background Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine. Materials and Methods Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM. Results The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells. Conclusion Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.

Niclosamide suppresses hepatoma cell proliferation via the Wnt pathway

Background The Wnt pathway plays an important role in Hepatocarcinogenesis. We analyzed the association of the Wnt pathway with the proliferation of hepatoma cells using Wnt3a and niclosamide, a drug used to treat tapeworm infection. Methods We performed an MTS assay to determine whether Wnt3a stimulated proliferation of Huh-6 and Hep3B human hepatoma cell lines after 72 hours of incubation with Wnt3a in serum-free medium. The cells were subjected to hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) after 48 hours of incubation. RNA was isolated 48 hours after addition of Wnt3a or niclosamide, and cyclin D1 expression levels were analyzed by real-time quantitative polymerase chain reaction. The promoter activity of T-cell factor was analyzed by luciferase assay 48 hours after transfection of TOPflash. Western blot analysis was performed with antibodies against β-catenin, dishevelled 2, and cyclin D1. Results Cell proliferation increased with Wnt3a. Niclosamide suppressed proliferation with or without Wnt3a. Hematoxylin and eosin and TUNEL staining suggested that apoptosis occurred in cells with niclosamide. Cyclin D1 was upregulated in the presence of Wnt3a and downregulated with addition of niclosamide. The promoter activity of T-cell factor increased with Wnt3a, whereas T-cell factor promoter activity decreased with niclosamide. Western blot analysis showed that Wnt3a upregulated β-catenin, dishevelled 2, and cyclin D1, while niclosamide downregulated them. Conclusion Niclosamide is a potential candidate for the treatment of hepatoma.

Circulating microRNA Profiles in Patients with Type-1 Autoimmune Hepatitis

Recent studies have demonstrated that micro (mi)RNA molecules can be detected in the circulation and can serve as potential biomarkers of various diseases. This study used microarray analysis to identify aberrantly expressed circulating miRNAs in patients with type 1 autoimmune hepatitis (AIH) compared with healthy controls. Patients with well-documented and untreated AIH were selected from the National Hospital Organization (NHO)-AIH-liver-network database. They underwent blood sampling and liver biopsy with inflammation grading and fibrosis staging before receiving treatment. To further confirm the microarray data, circulating expression levels of miR-21 and miR-122 were quantified by real-time quantitative polymerase chain reaction in 46 AIH patients, 40 patients with chronic hepatitis C (CHC), and 13 healthy controls. Consistent with the microarray data, serum levels of miR-21 were significantly elevated in AIH patients compared with CHC patients and healthy controls. miR-21 and miR-122 serum levels correlated with alanine aminotransferase levels. Circulating levels of miR-21 and miR-122 were significantly reduced in AIH patients with liver cirrhosis, and were inversely correlated with increased stages of fibrosis. By contrast, levels of circulating miR-21 showed a significant correlation with the histological grades of inflammation in AIH. We postulate that aberrantly expressed serum miRNAs are potential biomarkers of AIH and could be implicated in AIH pathogenesis. Alternations of miR-21 and miR-122 serum levels could reflect their putative roles in the mediation of inflammatory processes in AIH.

Insulin‐like growth factor (IGF)‐II regulates CCAAT/enhancer binding protein α expression via phosphatidyl‐inositol 3 kinase in human hepatoblastoma cell lines

To reveal growth factor and its signal pathway to CCAAT/enhancer binding protein alpha (C/EBPα) in hepatocyte differentiation, we used Huh‐6 and HepG2, human hepatoblastoma (HBL) cell lines that maintain the expression of genes in hepatoblasts and remain at that stage of differentiation. Insulin‐like growth factor (IGF)‐II, hepatocyte growth factor (HGF), and dexamethasone (Dex) stimulated HBL cells for Northern blot analysis. Bromodeoxyuridine (BrdU) up‐take assay and Western blot analysis on albumin was performed to unveil proliferation and differentiation activity of IGF‐II. C/EBPα and phosphorylation of Akt were analyzed by Western blot analysis. LY294002 and wortmannin, specific inhibitors of PI3 kinase, and PD98059, a specific inhibitor of mitogen‐activated protein (MAP) kinase, were used to examine the signaling pathway of C/EBPα upregulated by IGF‐II. Luciferase assay was performed to study the promoter activity of C/EBPα. Actinomycin D was used to analyze half‐life of C/EBPα mRNA. IGF‐II up‐regualted C/EBPα by Northern blot and Western blot while HGF and Dex did not by Northern blot. IGF‐II promoted proliferation and differentiation by BrdU up‐take assay and Western blot analysis on albumin. Akt phosphorylated by IGF‐II, suggested that phosphatidyl‐inositol (PI) 3 kinase mediated the signaling pathway of IGF‐II. LY294002 and wortmannin suppressed expression of C/EBPα. IGF‐II activated the promoter activity and prolonged half‐life of mRNA, suggesting that IGF‐II activated promoter and stabilized mRNA. LY294002 and wortmannin suppressed the promoter activity of C/EBPα while PD98059 did not, suggesting that activation of the promoter was mediated by PI3 kinase. J. Cell. Biochem. 102: 161–170, 2007.