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Rogdi was a novel gene with unknown function. According to GeneBank database, the gene is located on chromsome 10q12 and the length of coding regeion is 864 bp that encods 287 animo acids. Earlier studies in our laboratory showed that human ROGDI influenced rate of cell proliferaion in HeLa, Hep3B and NIH3T3 cells. In addition, we found Rogdi protien was up-regulated in fibrotic livers. Following various types of injury to liver, quiescent hepatic stellate cells (HSCs) transform to activated phenotype, leading to exprssion of £\-SMA, increasing rate of cell proliferation and depositing of extracellular matrix. In this study, we found that Rogdi protein was up-regulated in activated HSCs isolated and cultured from rat livers. By either overexpression or RNA interference of Rogdi, we found that Rogdi affected rate of HSCs proliferation, and expressions of £\-SMA and collagen type I. Expression of Rogdi protein was induced after PDGF treatment of rat HSCs. Additionally, we found that Rogdi was involved in MAPK and PI3K/Akt pathways. Furthermore, using wound healing assay and migration assay, Rogdi was found to regulate migration of activated HSCs.

Indiana University-Purdue University Indianapolis (IUPUI) / Liver fibrosis is a common consequence of various chronic liver diseases. Although transforming growth factor β 1 (TGFβ1) expression is known to be associated with liver fibrosis, the reduced clinical efficacy of TGFβ1 inhibition or the inefficiency to completely prevent liver fibrosis in mice with liver-specific knockout of TGF receptor II suggests that other factors can mediate liver fibrogenesis. As a TGFβ superfamily ligand, activin A signaling modulates liver injury by prohibiting hepatocyte proliferation, mediating hepatocyte apoptosis, promoting Kupffer cell activation, and inducing hepatic stellate cell (HSC) activation in vitro. However, the mechanism of action and in vivo functional significance of activin A in liver fibrosis models remain uncertain. Moreover, whether activin B, another ligand structurally related to activin A, is involved in liver fibrogenesis is not yet known. This study aimed to investigate the role of activin A and B in liver fibrosis initiation and progression. The levels of hepatic and circulating activin B and A were analyzed in patients with various chronic liver diseases, including end-stage liver diseases (ESLD), non-alcoholic steatohepatitis (NASH), and alcoholic liver disease (ALD). In addition, their levels were measured in mouse carbon tetrachloride (CCl4), bile duct ligation (BDL), and ALD liver injury models. Mouse primary hepatocytes, RAW264.7 cells, and LX-2 cells were used as in vitro models of hepatocytes, macrophages, and HSCs, respectively. The specificity and potency of anti-activin B monoclonal antibody (mAb) and anti-activin A mAb were evaluated using Smad2/3 luciferase assay. Activin A, activin B, or their combination were immunologically inactivated by the neutralizing mAbs in mice with progressive or established liver fibrosis induced by CCl4 or with developing cholestatic liver fibrosis induced by BDL surgery. In patients with ESLD, NASH, and ALD, increases in hepatic and circulating activin B, but not activin A, were associated with liver fibrosis, irrespective of etiology. In mice with CCl4-, BDL-, or alcohol-induced liver injury, activin B was persistently elevated in the liver and circulation, whereas activin A showed only transient increases. Activin B was expressed and secreted mainly by the hepatocytes and other cells, including cholangiocytes, activated HSCs, and immune cells. Exogenous administration of activin B promoted hepatocyte injury, activated macrophages to release cytokines, and induced a pro-fibrotic expression profile and septa formation in HSCs. Co-treatment of activin A and B interdependently activated the chemokine (C-X-C motif) ligand 1 (CXCL1)/inducible nitric oxide synthase (iNOS) pathway in macrophages and additively upregulated connective tissue growth factor expression in HSCs. Activin B and A had redundant, unique, and interactive effects on the transcripts related to HSC activation. The neutralization of activin B attenuated the development of liver fibrosis and improved liver function in mice with CCl4- or BDL-induced liver fibrosis and largely reversed the already established liver fibrosis in the CCl4 mouse model. These effects were improved by the administration of additional anti-activin A antibody. Combination of both antibodies also inhibited hepatic and circulating inflammatory cytokine production in the BDL mouse model. In conclusion, activin B is a potential circulating biomarker and potent promotor of liver fibrosis. Its levels in the liver and circulation increase significantly in both acute and chronic states of liver injury. Activin B might additively or interdependently cooperate with activin A, which directly acts on multiple liver cell populations during liver injury and fibrosis, as the combination of both proteins increases pro-inflammatory and pro-fibrotic responses in vitro. In addition, the neutralization of both activin A and activin B in vivo enhances the preventive and reversible effects of liver injury and fibrosis compared to that when activin B alone is neutralized. Our data reveal a novel target of liver fibrosis and the mechanism of activin B-mediated initiation of this process by damaging hepatocytes and activating macrophages and HSCs. Our findings show that activin B promotes hepatic fibrogenesis, and that targeting of activin B has anti-inflammatory and anti-fibrotic effects, which ameliorate liver injury by preventing or regressing liver fibrosis. Antagonizing either activin B alone or in combination with activin A prevents and regresses liver fibrosis in multiple animal studies, paving way for future clinical studies.

Abstract: Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFN-γ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFN-γ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFN-γ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in H SC activation. Liver fibrosis was induced in Socs1[superscript -/-]Ifng[superscript -/-] mice with dimethylnitrosamine or carbon tetrachloride. Ifng[superscript -/-] and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1[superscript -/-]Ifng[superscript -/-] mice showed elevated serum ALT levels and increased liver fibrosis com-pared to mice Ifng[superscript -/-]. The latter group showed higher alanine aminotransferase (ALT) levels and fibrosis than C57BL/6 controls. The livers of Socs1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. Socs1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from Socs1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of Socs1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFN-γ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability. / Résumé: Le suppresseur de la signalisation des cytokines 1 (SOCS1) est un régulateur indispensable de la signalisation de l'IFN-γ et a été aussi impliqué dans la régulation de la fibrose hépatique. Cependant, on ne sait pas si les fonctions anti-fibrotiques sont médiées directement dans le foie par SOCS1 ou par la modulation de l'IFN-γ, qui est connu pour son effet atténuateur de la fibrose hépatique. En outre, il est possible que SOCS1 contrôle la fibrose hépatique par la régulation des cellules stellaires hépatiques (CSH), un acteur clé dans la réponse fibrogénique. Alors que les voies d'activation des CSH ont été bien caractérisées, les mécanismes de régulation ne sont pas encore clairs. Les buts de cette étude étaient de dissocier la régulation de la réponse fibrogénique hépatique médiée par SOCS1 et celle dépendante de IFN-γ et d'élucider les fonctions régulatrices de SOCS1 dans l'activation des CSH. La fibrose hépatique a été induite chez des souris Socs1[indice supérieur -/-]Ifng[indice supérieur -/-] par la diméthylnitrosamine ou le tétrachlorure de carbone. Les souris Ifng[indice supérieur -/-] et C57BL6 ont servi comme contrôles. Après les traitements fibrogéniques, les souris Socs1[indice supérieur -/-]Ifng[indice supérieur -/-] ont montré des niveaux sériques élevés d'alanine aminotransférase (ALT) ainsi que l'augmentation de la fibrose du foie par rapport à des souris Ifng[indice supérieur -/-]. Le dernier groupe a montré des niveaux plus élevés d'ALT et de fibrose par rapport aux souris C57BL6 contrôles. Les foies des souris déficientes en Socs1 ont montré une fibrose septale, qui a été associée à une augmentation de l'accumulation des myofibroblastes et à un dépôt abondant du collagène. Les foies déficients en SOCS1 ont montré une expression accrue de gènes codant pour l'actine musculaire lisse, le collagène et les enzymes impliquées dans le remodelage de la matrice extracellulaire, à savoir les métalloprotéinases de la matrice et l'inhibiteur tissulaire des métalloprotéinases. Les CSH primaires de souris déficientes en Socs1 ont montré une prolifération accrue en réponse à des facteurs de croissance tels que le HGF, EGF et le PDGF. Aussi, les foies fibrotiques de souris déficientes en Socs1 ont montré une expression élevée du gène PDGFB. Pris ensemble, ces données indiquent que SOCS1 contrôle la fibrose hépatique indépendamment de l'IFN-γ et qu'une partie de cette régulation peut se produire en régulant la prolifération des HSC et en limitant la disponibilité des facteurs de croissance.

<p>Activin B, a TGFβ ligand, is associated with liver inflammatory response. We aimed to investigate whether it modulates liver fibrogenesis. <b> </b>Liver and serum activin B, along with its analog activin A, were analyzed in patients with liver fibrosis from different etiologies and in mouse acute liver injury and liver fibrosis models. Activin B, activin A, or both was immunologically neutralized in progressive or established carbon tetrachloride-induced mouse liver fibrosis. The direct effects of activin B and A on hepatocytes, macrophages, and hepatic stellate cells (HSCs) were evaluated <i>in vitro</i>. In human patients, increased activin B is associated with liver fibrosis irrespective of the etiologies. In mice, activin B exhibited persistent elevation in liver and circulation following the onset of liver injury, whereas activin A displayed transient increases. Neutralizing activin B largely prevented and remarkably regressed liver fibrosis, which was augmented by co-neutralizing activin A in mice. Mechanistically, activin B promoted hepatocyte injury, activated macrophages to release cytokines, and induced a pro-fibrotic expression profile and septa formation in HSCs, which were magnified by activin A. Furthermore, activin B and A interdependently activated the CXCL1/iNOS pathway in macrophages and additively upregulated CTGF transcript in HSCs <i>in vitro</i>. Consistently, the expression of these genes was prohibited by neutralizing either one of these two ligands in injured livers. Activin B potently drives the initiation and progression of liver fibrogenesis. It additively or interdependently cooperates with activin A, directly acts on multiple liver cell populations, and induces liver fibrogenesis.<b> </b>Antagonizing activin B or both activins B and A prevents and regresses liver fibrosis in mouse CCl₄ model, inspiring the development of a novel therapy of chronic liver diseases.</p>

Plusieurs études ont démontré que les agonistes du PPARgamma, dont la pioglitazone (PGZ), améliorent les paramètres métaboliques et histologiques de la stéatohépatite non-alcoolique (NASH) chez l'homme et la souris, et qu’ils ont des effets bénéfiques sur la fibrose hépatique chez le rat. Les mécanismes d’action sont mal connus. La NASH, caractérisée par de la stéatose, des lésions hépatocytaires, de l’inflammation et une fibrose variable, est considérée comme une complication hépatique du syndrome métabolique. L'obésité, un des facteurs de risque pour le développement de la NASH, est caractérisée par de faibles taux d'adiponectine sérique. Cette adipocytokine, dont l'expression génique est régulée par le PPARgamma, possède des propriétés anti-stéatosique et anti-fibrotique chez la souris. L'activité intracellulaire de l'adiponectine est médiée via ses récepteurs spécifiques qui activent la protéine kinase AMPK et/ou le PPARalpha. Une fois activée, l’AMPK induit les voies cataboliques de production d’énergie (telles que l'oxydation des acide gras) et inhibe les voies consommant de l’ATP (telles que la lipogenèse). L'activation du PPARalpha augmente l'oxydation des acides gras et inhibe la réponse inflammatoire. Le but de notre travail est d’évaluer l'implication de la voie PGZ–adiponectine–AMPK et/ou PPARalpha dans la prévention de la NASH et de la fibrose hépatique. Nous avons tout d’abord évalué l'effet de la PGZ sur la fibrose hépatique chez la souris. Nos observations montrent que, contrairement aux résultats observés chez le rat, la PGZ n’inhibe pas le développement de la fibrose hépatique chez la souris in vivo. Ces résultats ont été confirmés par des études sur les cellules stellaires hépatiques (HSCs), les cellules effectrices de la fibrose, in vitro. Dans une seconde étude, nous avons évalué l'impact de l’AMPK sur la fibrose hépatique in vivo et sur l’activation des HSCs in vitro. Nous avons constaté que l’AMPK jouait un rôle dans le contrôle de la trans-différentiation des HSCs in vitro mais pas dans le développement de la fibrose hépatique chez la souris in vivo. Finalement, nous avons évalué l'hypothèse que l'effet bénéfique de la PGZ sur la NASH résulte de la stimulation de l'AMPK et/ou du PPARalpha par l’adiponectine. Nos résultats ont montrés que cet effet de la PGZ était strictement dépendant de l’adiponectine mais ne semblait pas impliquer l'AMPK ni le PPARalpha. Nous avons également identifié SREBP-1c, régulant la lipogenèse de novo, comme cible thérapeutique potentielle pour le développement de la NASH. Les résultats obtenus dans le cadre de ce travail de thèse fournissent une meilleure compréhension de l’axe PPARgamma–adiponectine–AMPK dans le contrôle du développement de la NASH et de la fibrose hépatique chez la souris. / Several studies have demonstrated that peroxisome proliferator-activated receptor gamma (PPARg) agonists, such as pioglitazone (PGZ), improve metabolic parameters and histology of nonalcoholic steatohepatitis (NASH) development in humans and mice, and have beneficial effects on liver fibrosis in rats. NASH, characterized by steatosis, hepatocellular damage, inflammation and variable fibrosis, is recognised as the hepatic complication of the metabolic syndrome. Obesity, one of the risk factors for NASH development, is characterized by low serum adiponectin levels. This adipocytokine, of which gene expression is regulated by PPARg, demonstrates anti-steatotic and anti-fibrotic properties in mice. Intracellular activity of adiponectin is mediated through its specific receptors which activate AMP-activated protein kinase (AMPK) and PPARalpha. Once activated, AMPK switches on catabolic pathways (such as fatty acid oxidation and glycolysis) and switches off ATP-consuming pathways (such as lipogenesis). Activation of PPARalpha increases fatty acid oxidation and reduces inflammatory reaction. The aim of the present work is to analyse the activation of the axis PGZ-adiponectin-AMPK and/or PPARalpha as a way to control NASH and hepatic fibrosis development. We first evaluated the effect of PGZ on hepatic fibrosis in mice. We observed that, by contrast with results in rats, PGZ did not prevent hepatic fibrosis development in vivo in mice. These results were confirmed by in vitro studies on the key effector cells of fibrogenesis, the hepatic stellate cells (HSCs). We then assessed the impact of AMPK on hepatic fibrosis in vivo and on HSC trans-differentiation/activation phenomenon in vitro. We found that AMPK played a role in the control of HSC trans-differentiation in vitro but was not implicated in the wound-healing fibrosis in vivo in mice. Finally, we tested the hypothesis that the beneficial effect of PGZ on steatohepatitis results from the adiponectin-dependent stimulation of AMPK and/or PPARalpha. We found that this preventive effect was clearly dependent of adiponectin but did not involve AMPK or PPARalpha activation. We have also identified SREBP-1c, implicated in the regulation of de novo lipogenesis, as a potential therapeutic target for the control of the development of NASH. The present thesis provides a better understanding of the axis PPARg–adiponectin–AMPK in the control of NASH and hepatic fibrosis development in mouse.

Chronic immune insult and immunosuppressant-related toxicities have remained an enduring challenge in organ transplantation. Long-term survival of transplant patients has improved marginally in recent decades due to these challenges. To circumvent these issues, transplant investigators have researched immune tolerance mechanisms that demonstrate potential to induce immunosuppression and rejection-free survival in the clinic. One mechanism in particular, the liver tolerance effect, has already demonstrated this experimentally and clinically. Liver transplants in experimental models and human patients have exhibited the ability to become spontaneously accepted without being rejected by the recipient’s immune system. Research in recent decades has revealed that the liver parenchymal and non-parenchymal cell populations harbor potent immunomodulatory properties. In the context of liver transplantation, it has been found that two cell populations in particular, the mesenchyme-derived liver sinusoidal endothelial cells and hepatic stellate cells, mediate the induction of liver transplant tolerance through a mechanism known as mesenchyme-mediated immune control.

Introduction: Hepatic stellate cells (HSC) and myofibroblasts may be relevant stromal drivers of human hepatocellular carcinoma (HCC). It was hypothesised that targeted inhibition of αv integrin-mediated TGF-β activation, by HSC or hepatocytes, may result in reduced peri-tumoural and intra-tumoural extracellular matrix formation, and reduced hepatic carcinogenesis. The role of HSC in acute liver injury is less well characterised. It was anticipated that integrin signalling on HSC and hepatocytes might also be relevant in the acute setting. The emerging technique of intravital microscopy (IVM) allows detailed, real-time investigation of the cellular processes involved in hepatocyte injury, cell death and repair. It was hypothesised that this could be coupled with mouse models of HCC and acute liver injury, to perform sequential imaging under anaesthesia. Aims: (i) To determine the effect of targeted inhibition of αv integrins on HSC and hepatocytes, during hepatocarcinogenesis, in a mouse model of HCC. (ii) To investigate the effect of targeted inhibition of αv and other integrins on HSC, hepatocytes, and liver sinusoidal endothelial cells (LSEC), during acute liver injury, in the mouse model of paracetamol-induced liver injury. (iii) To develop IVM of the liver, via an abdominal imaging window, with optimisation of surgical and imaging techniques, to allow sequential imaging of the same animal. Methods: The diethylnitrosamine (DEN)-induced mouse model of hepatocarcinogenesis was used, and PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were employed to deplete αv integrins on HSC and hepatocytes respectively. Tumours were harvested at 40 weeks post-DEN. Tumour size and number was evaluated in all animals. PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were used in the paracetamol model, to investigate the role of αv integrins in acute liver injury. PDGFRβ-Cre;β8fl/fl and Alb-Cre;β 8fl/fl animals were also tested in this model. The role of integrins in liver sinusoidal endothelial cells (LSEC) during paracetamol-induced liver injury was evaluated using Cdh5-Cre mice. IVM of the liver was performed by surgical implantation of an abdominal imaging window, consisting of a titanium ring and coverslip, secured in place with a purse string suture. Fluorescent reporter mice were used to identify hepatic and vascular architecture, and other label-free microscope technologies were utilised to image collagen, lipid distribution, necrotic areas and blood flow within tissues. Results: In large cohorts of PDGFRβ-Cre;αvfl/fl, Alb-Cre;αvfl/fl, and control animals, there was no difference in mean tumour size or number, at 40 weeks. Targeted inhibition of α v integrins and β 8 integrin on hepatocytes, HSC or LSEC was not protective in paracetamol-induced liver injury. IVM of the liver can be performed on animals with HCC and throughout paracetamol-induced liver injury, to obtain high quality, real-time images of multiple cell lineages and the hepatic microenvironment. Conclusions: The role of TGF-β in HCC pathogenesis is complex and context-dependent. Targeted loss of αv integrin did not result in reduction in tumour burden in this non-cirrhotic model of HCC. IVM of the liver is a powerful tool to quantify inflammatory infiltrates and assessment of vascular remodelling throughout the course of acute liver injury and regeneration, providing insights into the biological processes determining recovery.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20794号 / 医博第4294号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 浅野 雅秀, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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