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In this study, we have first described the normal liver structure including the hepatic acinus that is characterized by its structural and functional heterogeneity. Second, we have addressed the pathogenesis of liver fibrosis: the major source of excess extracellular matrix appears to be perisinusoidal stellate cells. The concept of reversibility of liver fibrosis opens the way for new therapeutic perspectives. We have then analyzed the different methods of assessment of liver fibrosis. Liver biopsy is the current reference standard. However, it is invasive and subject to sampling error. Consequently, many attempts are made to develop non-invasive tests: biochemical tests and imaging methods, including functional MR imaging with perfusion, diffusion or spectroscopy, have been proposed. Among the imaging methods, elastography by measuring directly the liver stiffness appears as one of the most promising techniques. Lastly, we have described our research that was focused on MR elastography. Our results show that MR elastography is a feasible, accurate and reproducible method to stage liver fibrosis, and that it is superior to biochemical testing with aspartate-to-platelets ratio index and ultrasound elastography to stage liver fibrosis. Further studies remain to be done to decrease the long examination time of MR elastography and, consequently, to integrate it into a comprehensive hepatic MR protocol.

Bone Morphogenetic proteins (BMPs) belong to transforming growth factor beta (TGF-£]) superfamily. They regulate cell proliferation, cell differentiation, and bone morphogenesis. Previous evidence suggests that BMP-2, as an antagonist of TGF-£], may play an inhibitory role in tissue fibrogenesis. The aim of this study is to examine the expression profile of BMP-2 in fibrotic livers and to test whether BMP-2 gene delivery could alleviate or reverse the liver fibrogenesis models in mice including bile duct ligation (BDL) or carbon tetrachloride (CCl4) model. The results showed that the AST, ALT, and bilirubin levels in sera and the expression of TGF-£], £\-smooth muscle actin, type I collagen in livers were significantly up-regulated by BDL surgery or CCl4 administration. After BDL, the hepatic BMP-2 mRNA and protein levels in mice decreased at 7 and 14 days after surgery. Similarly, the hepatic BMP-2 mRNA and protein levels in mice decreased at day 14 and 28 after CCl4 administration. BMP-2 gene delivery alleviated the inflammation and the liver injury caused by BDL or CCl4 exposure. These findings strongly suggest that BMP-2 is involved in the pathogenesis of liver fibrosis. Moreover, BMP-2 supplementation may facilitate a novel strategy for treatment of liver fibrosis.

Liver fibrosis is a wound healing process in liver with¡@chronic injury and is characterized by the excess production and accumulation of extracellular matrix (ECM) component. Liver injury of any etiology may lead to activation of hepatic stellate cells (HSCs), which are trans-differentiated from lipocyte-like cells to highly proliferative myofibroblast-like cells. Activation of HSCs is considered a crucial event that promotes increased ECM production and consequently hepatic fibrosis. Liver fibros is resulted from a net increased synthesis and decreased degradation of ECM proteins. Pluchea indica (Less) has been reported to have antipyretic, anti-ulcer, anti-inflammatory, anti-oxidant, diuretic and anti-amoebic activities. Our previous studies showed that the aqueous extract of roots from P. indica (PIRAE) showed that it can suppress the growth and migration of HeLa and GBM8401 cancer cell lines, and also significantly reduce serum glutamate pyruvate transaminase (GPT), alpha-smooth muscle actin (£\-SMA) and collagen type I expression in animal model of liver fibrosis induced by thioacetamide (TAA). In this study, we plan to investigate the effects of PIRAE on activation, proliferation and migration of rat culture activated HSCs. The results indicated that protein expression of £\-SMA and collagen type I of HSCs was decreased followed by treatment of either 0.5 or 1.0 mg/ml PIRAE for 48 hours. In addition, the effects of PIRAE on proliferation in culture activated HSCs were assessed by analyses of cell growth curve, MTT, WST-1 and BrdU, respectively. The results showed that PIRAE inhibited HSCs proliferation in a dose- and time-dependent manner. Moreover, wound healing assay and transwell assay showed that PIRAE prevented migration in activated HSCs. In conclusion, PIRAE may suppresse culture activated HSCs proliferation, migration, and activation of culture activated HSCs, as well as accumulation of collagen type I.

Gene of DC2 protein, a novel unknown gene, was identified previously in our laboratory while studying the death progression in the rat brain stem. According to the search results of bioinformatics database, both human DC2 and house mouse DC2 are 149 amino acids long and 16.8 kDa. The entire sequence of human DC2 differs from house mouse DC2 by only a single amino acid substitution. The bioinformatics revealed that human DC2 and house mouse DC2 had three predicted transmembrane regions. These results suggest human DC2 and house mouse are highly homologous. DC2 protein expresses differentially between organs. Human liver is the top fourth DC2-expressed organ, while house mouse liver is ranked 23rd DC2-expressed organ. Shibatani et al (Shibatani et al¡A2005) proposed DC2 protein as a potential subunit of mammalian Oligosaccharyltransferase (OST) after mass spectrometry analysis and suggested DC2 might involve in glycosylation. House mouse liver fibrosis was induced by giving 300mg/L thioacetamide (TAA) in the drinking water for different periods of time, and then gene expression of house mouse DC2 of liver was analyzed. mRNA expression was found in normal house mouse liver and mRNA expression increased gradually after TAA administration. DC2 protein also found in normal house mouse liver and DC2 protein of house mouse liver increased after TAA administration.

Hepatic fibrosis is a serious health problem in many parts of the world. However, its treatment remains severely limited because of inadequate target specificity. HSC are the largest reservoir of vitamin A in the body. They are also the principal players responsible for the pathogenesis of liver fibrosis. Targeting HSC is an effective strategy for treatment of liver fibrosis. The specific association of BMP4 with various liver diseases including liver fibrosis makes it an ideal candidate for targeting HSC cells using siRNA. The objective of this study is to develop and characterize vitamin A (VA)-coupled liposomes for the targeted delivery of BMP4-siRNA to cultured HSC. DOTAP/DOPE liposomes surfaces were prepared by thin film hydration and their surfaces were decorated with VA (1:2 mol/mol). Particle size and zeta potential were determined using ZetaPALS. In addition, the siRNA binding efficiency was determined by ultra-centrifugation and fluorescence assays. The cytotoxicity of VA-conjugated liposomes was evaluated by the WST-1 cytotoxicity assay. Inhibition of BMP4 and α-SMA was determined by real time PCR and ELISA. Their average particle size was in the range of 100-120 nm and they exhibited zeta potential around +45 mV. VA-coated liposomes were mixed with BMP4-siRNA, forming lipoplexes with particle sizes less than 200 nm and zeta potential around +25 mV. The presence of VA did not alter the siRNA binding efficiency, it also had no effect on cytotoxicity, but resulted in enhanced cellular uptake of siRNA as shown by flow cytometry. There was a significant reduction in BMP4 mRNA with VA-coupled liposomes carrying BMP4-siRNA. Moreover, BMP4 gene silencing was accompanied by a significantly reduced the expression of the potent fibrinogenic α-SMA at mRNA and protein levels. In conclusion, VA-coated liposomes were successfully able to target and deliver BMP4-siRNA to HSC. This could offer an interesting perspective for the treatment of liver fibrosis. / February 2016

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19222号 / 医博第4021号 / 新制||医||1010(附属図書館) / 32221 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 長澤 丘司, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

[Truncated abstract] Chronic liver injury results from many etiologies ranging from viral infection to inborn errors of metabolism. A common result of liver injury is activation of hepatic stellate cells and portal fibroblasts to myofibroblasts. In chronic injury, production of extracellular matrix by activated myofibroblasts results in liver fibrosis and ultimately cirrhosis. Kupffer cells and monocytes may play an important role in the pathogenesis of certain liver diseases. Endotoxin-responsive macrophages and recruited monocytes (CD14-positive cells) are potential sources of profibrogenic factors but their potential role in the pathogenesis of liver disease has not previously been examined. The first aim of this thesis described in chapter 3 was to evaluate the hypothesis that CD14-positive macrophages/monocytes are present in the livers of patients with hereditary haemochromatosis (HH), primary biliary cirrhosis (PBC), chronic hepatitis C (HCV) and nonalcoholic steatohepatitis (NASH) and contribute to the pathogenesis of fibrosis as evidenced by co-localization of these cells with activated myofibroblasts. Liver specimens from control subjects and those with HH, PBC, HCV and NASH were immunostained for CD14, CD68 and α-smooth muscle actin and the number of cells expressing these antigens was determined. The total number of hepatic CD68-positive cells was similar in diseased and control livers. The number of CD14-positive cells correlated with advanced fibrosis in HH, PBC, HCV but not in NASH. The number of CD14-positive cells was increased with advanced inflammatory activity in HCV. CD14-positive cells were often associated with α-smooth muscle actin-positive myofibroblasts in fibrous septa. In conclusion, many forms of human chronic liver disease demonstrate increased numbers of CD14-positive macrophages/monocytes which are associated with fibrous septa and myofibroblasts. To determine whether CD14-positive cells contribute to fibrogenesis, experimental models of liver injury were used in chapters 5 and 6. The aim of chapter 5 was to determine whether CD14-positive macrophages/monocytes are detected in a bile duct ligation model of liver injury. To accomplish this aim, a novel antibody to rat CD14 was developed as described in chapter 4. A time-course study was undertaken in rats following bile duct ligation for up to 14 days. An increase in the number of hepatic CD14-positive cells was detected early following bile duct ligation, and was associated with increased gene expression of α-smooth muscle actin and procollagen I. Thus, myofibroblastic transformation in this model was associated with increased numbers of CD14-positive cells suggesting a possible relationship between the two phenomena. In order to specifically evaluate the role of CD14 in myofibroblastic transformation, a final study in CD14 knockout (KO) mice was undertaken in chapter 6

Infection with hepatitis C virus (HCV) can cause liver damage known as fibrosis, which often leads to liver disease and hepatocellular carcinoma. The impairment of circulating, bulk (non-specific and specific) CD8+ T cells within HCV-infection, characterized by an altered phenotype and the increased expression of pro-apoptotic genes, is observed when compared to uninfected controls. The relationship between bulk CD8+ T cell function and the extent of liver damage has not been demonstrated. In this study, widespread immune alterations were observed in untreated HCV infection with advanced liver fibrosis. Untreated HCV-infected individuals with advanced fibrosis possessed a significantly decreased proportion of naïve CD8+ T cells and an increased proportion of late effector memory CD8+ T cells compared to uninfected controls. Upon T cell receptor (TCR) stimulation, these individuals also had an increased intracellular IFN-γ expression for four CD8+ T cell subsets, a decreased CD107a expression for central memory CD8+ T cells, and a decreased perforin induction for naïve and central memory CD8+ T cells. These immune alterations did not reverse 24 weeks after viral cure. This study indicates there is a relationship between the differentiation and function of bulk CD8+ T cells and the extent of liver damage within HCV infection.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease associated with the worldwide spread of obesity. NASH predisposes development of fibrosis and hepatocellular carcinoma (HCC), but has no approved therapy due to incomplete understanding of the pathogenesis. Notch signaling normally specifies cell fate during development, but here we investigate how this pathway becomes dysregulated in NASH and contributes to fibrosis and cancer. In the first study, we show that hepatocyte Notch activity tracks with disease severity and treatment response in NASH patients, and is similarly increased in a mouse model of diet-induced NASH and liver fibrosis. Different genetic models demonstrate causatively that hepatocyte Notch induces liver fibrosis via secretion of the fibrogenic factor Osteopontin that activates hepatic stellate cells (HSCs), while pharmacologic inhibition of hepatocyte Notch could ameliorate NASH-associated fibrosis. In the second study, we research how hepatocyte Notch activation leads to HCC in mice on NASH diet. Transcriptomic analysis reveals nerve growth factor (NGF) as a Notch target gene in hepatocytes, and the abundance of hepatocyte NGF precursor protein (proNGF) is uniquely associated with HCC. We provide evidence that proNGF may facilitate HCC growth and expansion in a non-cell autonomous manner by inducing HSC deactivation and fibrosis remodeling. In summary, hepatocyte Notch maladaptively contributes to fibrogenesis and possibly HCC expansion by directly signaling to HSCs at different stages of NASH progression, and could be an accessible target for treatment of NASH-associated liver pathologies.