Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-£]1 (TGF-£]1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-Hepatic injuries due to viral infection and alcoholic abuse frequently lead to activation and proliferation of hepatic stellate cells (HSCs), concomitantly with tissue repairing and remodeling mechanism. Transforming growth factor-£]1 (TGF-£]1) is known to be one of the potent cytokines that directly upregulates synthesis of extracellular matrix, thereby aggravating liver fibrosis. Bone morphogenetic protein-2 (BMP-2), as a member of TGF-£]1 superfamily, has been known to regulate cell proliferation, differentiation, and bone morphogenesis. Our previous study demonstrated that BMP-2 was downregulated in fibrotic liver of mice, supporting its antagonistic role in hepatic fibrogenesis. Downregulation of BMP-2 in fibrotic livers may lose its ability to antagonize the pro-fibrogenic action of TGF-£]1. The purpose of this study was to determine whether mutual regulatory mechanisms exist between BMP-2 and TGF-£]1. Treatment of recombinant protein, TGF-£]1, significantly suppressed BMP-2 expression in hepatocytes clone-9 and HSC-T6 cells. Moreover, treatment of BMP-2 in both cell also attenuated TGF-£]1protein levels in a dose-dependent manner. This finding supports that TGF-£]1 and BMP-2 mutually modulated their expression not only in vivo, but also in vitro. Western blotting analysis showed that TGF-£]1 and BMP-2 exerted different kinase phosphorylation profiles of signaling activities. However, the signal pathways and detail mechanisms of the interactions of BMP-2 and TGF-£]1 in the fibrogenic action will need to further evaluate.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18682号 / 医博第3954号 / 新制||医||1007(附属図書館) / 31615 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 野田 亮, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Non-alcoholic fatty liver disease (NAFLD) is the number one cause of chronic liver disease worldwide, with 25% of these patients developing nonalcoholic steatohepatitis (NASH). NASH is characterized by steatosis, inflammation, cell death, and liver fibrosis and significantly increases the risk of cirrhosis and decompensated liver failure. There are currently no approved drugs on the market for treating NASH leaving a major unmet medical need for drug discovery research. The aim of this dissertation is to better understand the pathophysiology of NASH and elucidate disease driving molecular mechanisms that can be used for drug targeting. Analysis of human liver samples using state of the art mass spectrometry proteomics identified dysregulation of one-carbon metabolism in NASH. This dissertation details the molecular mechanisms for how these proteomic changes can drive NASH pathogenesis and be targeted for therapeutic purposes. Chapter 1 provides an extensive background on NASH prevalence and pathophysiology and the association of one-carbon metabolism and NASH. Chapter 2 presents the identification of reduced glycine N-methyltransferase, a key regulator of one-carbon metabolism, in human NASH patients. Using a diet-induced animal model of NASH and targeted proteomics and metabolomics it was found glycine N-methyltransferase reduction in NASH leads to an accumulation of S-adenosylmethionine, activation of polyamine metabolism, and production of oxidative stress. Oxidative stress is a key component to NASH pathogenesis and this work identifies a novel mechanism for how oxidative stress is produced during NASH. Chapter 3 covers the discovery of increased folate receptor gamma (FOLR3) specifically in the liver of NASH patients. Initially, FOLR3 was predicted to impact one-carbon metabolism through folate metabolism, but molecular characterization found FOLR3 drives liver fibrogenesis independent of one-carbon metabolism. Chapter 3 details the molecular mechanism for how FOLR3 drives liver fibrosis by enhancing transforming growth factor beta activation of hepatic stellate cells through its interaction with serine protease HTRA1. The effect of FOLR3 was then validated in an in vivo model of NASH showing FOLR3 treatment can induce severe liver fibrosis in mice comparable to human liver fibrosis. These findings provide a translational animal model that can be used for NASH drug development and introduce a novel drug target in FOLR3. Chapter 4 discusses the innovation and translational impact of these findings and Chapter 5 summarizes the main results of this dissertation. / Pharmaceutical Sciences

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-29T20:29:44Z No. of bitstreams: 1 Sheilla Andrade deOliveira. Estudo do potencial terapêutico de células mononucleares de medula óssea em lesões hepáticas crônicas em camundongos - UFBA-FIOCRUZ-CPqGM - Tese Doutorado.pdf: 16762445 bytes, checksum: f804f2624774de877295c8841f4ff9c1 (MD5) / Made available in DSpace on 2012-08-29T20:29:44Z (GMT). No. of bitstreams: 1 Sheilla Andrade deOliveira. Estudo do potencial terapêutico de células mononucleares de medula óssea em lesões hepáticas crônicas em camundongos - UFBA-FIOCRUZ-CPqGM - Tese Doutorado.pdf: 16762445 bytes, checksum: f804f2624774de877295c8841f4ff9c1 (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O potencial terapêutico das células-tronco em doenças hepáticas vem sendo investigado. Estudos em modelos animais têm servido de base para a realização de ensaios clínicos utilizando células de medula óssea. Embora os resultados iniciais venham sendo bastante promissores, os mecanismos envolvidos na melhora hepática ainda não estão claros e novas avaliações em modelos experimentais se fazem necessárias. O objetivo da presente investigação foi avaliar a eficácia terapêutica de células mononucleares de medula óssea em doenças hepáticas crônicas. Inicialmente estabeleceu-se cirrose hepática no camundongo C57Bl/6, pela administração de uma solução de 20% de tetracloreto de carbono (CCI4) diluído em azeite de oliva combinado com 5% de etanol (EtOR) na água. Neste estudo observamos que, após seis meses de administração dos agentes hepatotóxicos, ocorre o desenvolvimento de lesões crônicas características de cirrose hepática. O segundo modelo utilizado foi o da infecção crônica pelo Schistosoma mansoni. Uma vez estabelecidas as lesões hepáticas crônicas nos animais, os estímulos "lesivos foram retirados e células mononucleares de medula óssea (3xl07) obtidas de tíbia e fêmures de camundongos transgênicos para proteína fluorescente verde (GFP) foram infundidas. A avaliação da capacidade migratória das células infundidas, bem como das alterações ocorridas após a terapia celular foi avaliada por meio de métodos histológicos, de imunofluorescência, morfológicos e imunológicos no tecido hepático. Após a terapia celular, observamos, nos dois modelos de fibrose hepática analisados, que as células GFP+ infundidas foram capazes de migrar e permanecer nas áreas de lesão do fígado. Inicialmente estas células apresentam formas arredondadas sem adentrar o tecido fibroso. Posteriormente, estas células tendem a invadir o tecido fibroso e assumem formas fusiformes. Uma redução do tecido fibroso foi verificada no 2° mês pós-terapia, em todos os grupos que receberam a terapia celular tanto nas áreas de fibrose septal e portal, quanto nas áreas dos granulomas. Os níveis de TGF-B no tecido hepático estavam diminuídos, após a terapia celular. O número de células ovais, conhecidas como células-tronco do fígado, estava aumentado no parênquima hepático após terapia celular. Os animais esquistossomóticos tratados com células de medula óssea recuperaram parcialmente a produção de albumina, quando comparado aos animais infectados. As observações sobre a terapia celular nos dois modelos de lesão crônica dos fígados avaliados nos permitem concluir que o transplante de células mononucleares de medula óssea diminui as alterações teciduais decorrentes de agressões crônicas ao fígado e reforçam a utilização deste tratamento para pacientes portadores de lesões hepáticas crônicas. / The therapeutic potential of stem cells on chronic liver diseases has been widely investigated. Based on results obtained mainly in studies using experimental models of acute liver injury, phase I clinical trials have already been carried out in patients with chronic liver diseases using therapy with bone marrow cells. Although the initial results were promising, the mechanisms involved on improvement of hepatic function are not clear and new evaluations with experimental models akin to human chronic liver diseases are needed. The object of the present investigation was the evaluation of the therapeutic efficacy of bone marrow mononuclear cells in chronic hepatic disease models. InitialIy, a mouse model of hepatic cirrhosis was induced in C57BI/6 mice by administration of 20% carbon tetrachloride solution (CCI4) diluted in olive oil combined with 5% ethanol (EtOH) in water was established. In this study we observed the development of chronic lesions characteristic of cirrhosis afier at least 6 months of administration of the hepatotoxic agents. The second model used was hepatic fibrosis caused by chronic infection by Schistosoma mansoni. Once the chronic lesions were established, the lesion stimulus was suspended and bone marrow mononuclear cells (3x I 07) obtained from tibiae and femurs of green fluorescence protein (GFP) transgenic mice were administered. The migratory capacity of the administered celIs, as well as the alterations occurring afier the celI therapy, was evaluated by histological, immunofluorescence, morphological and immunological methods in the liver tissue. After cell therapy in both hepatic fibrosis models it was observed that administered GFP+ cells were able to migrate and remain on hepatic lesion areas. Initially those celIs presented an oval shape outside thc fibrous tissue. At latter timepoints, those cells invadcd the fibrous tissue and acquired a fusifonll shape. A reduction of fibrosis was verified after the second month oftherapy in alI groups that received the celI therapy in the granuloma areas, as welI as in areas of septal and frontal fibrosis. The levels of TGF-~ were lower after the cell therapy, with statistical significance only in schistossomotic animaIs. The number of oval cells, known as liver stem cells, was increased in hepatic parenchyma afier cell therapy. The schistossomotic animaIs treated with bone marrow celIs recovered partialIy the production of albumin when compared to infected controls. Based on our observations about cell therapy in both models of chronic liver diseases evaluated we conc1ude that bone marrow mononuclear cell transplantation decreases tissue alterations caused by chronic aggressions to the liver and reinforce the use of this therapy in patients with chronic liver lesions.

Les mécanismes de la fibrogenèse hépatique liée à l'infection chronique par le virus de l'hépatite C (VHC) sont encore mal connus. L'activation de la fibrogenèse semble fortement associée à la réaction inflammatoire locale. Néanmoins le rôle direct du VHC dans le processus fibrogène n'a pas été étudié. Notre hypothèse est que la fibrose peut être au moins en partie directement induite par le VHC, indépendamment de la réponse immune de l'hôte.L'un des aspects inhérents à ce postulat est qu'une relation directe pourrait exister entre les particules du VHC et l'activation des principaux acteurs cellulaires de la fibrogenèse hépatique, les cellules étoilées du foie (CEFs). Ainsi, les objectifs de ce projet ont été d'étudier in vitro, la capacité du VHC à activer les CEFs humaines mais d'abord d'explorer la relation existant entre cette activation et une éventuelle infection de ces cellules par le VHC. Afin d'analyser la permissivité des CEFs à l'infection par le VHC, nous avons combiné plusieurs modèles originaux du VHC, tels que le clone infectieux JFH1, des rétrovirus pseudotypés avec les protéines d'enveloppe du VHC et le réplicon sous-génomique, avec deux modèles cellulaires de CEFs relevant (des cultures primaires humaines et la lignée immortalisée LX2). En conclusion, nous avons démontré que les CEFs humaines sont réfractaires à la fois à l'entrée et à la réplication du VHC. Ces résultats n'écartent cependant pas l'hypothèse d'une interaction directe entre les particules du VHC et la surface des CEFs dans l'activation fibrogénique de ces cellules. Le rôle des protéines d'enveloppe virales sous leur conformation native dans l'activation des CEFs fût étudié en incubant des ppVHC avec les CEFs. Malgré des résultats préliminaires encourageants, la question d'une activation éventuelle des CEFs en culture après contact avec des particules virales du VHC, et ce indépendamment d'une entrée et/ou d'une réplication virale, n'a pu être confirmé et reste encore sans réponse.Un second aspect est que l'expression in vivo de l'ensemble des protéines du VHC dans les hépatocytes pourrait jouer un rôle dans le déclenchement et la progression de la fibrose portale. Nous avons démontré pour la première fois que l'expression hépatocytaire in vivo des protéines du VHC chez des souris transgéniques, les FL-N/35, soumises à un traitement fibrogénique (injection chronique de CCl4) était associée à une fibrose augmentée, et ce de manière indépendante de l'inflammation locale. Cette fibrose augmentée chez les FL-N/35 s'accompagnait d'une production augmentée de d'espèces réactives de l'oxygène intrahépatocytaires, d'une réaction ductulaire caractérisée notamment par une expansion de cellules progénitrices hépatiques (CPHs), et d'une l'inhibition de la prolifération hépatocytaire. On notera également que cette fibrose portale corrélait avec l'expansion des CPHs portales, corollaire implicite de l'inhibition de la prolifération hépatocytaire. Ces observations, également observé chez les patients infectés par le VHC, suggèrent que la RD associée à une altération de la prolifération hépatocytaire jouerait un rôle dans la fibrose portale. Le modèle de souris utilisé présentant une expression intrahépatocytaire des protéines du VHC, nos résultats suggèrent implicitement une perturbation de l'homéostasie hépatocytaire comme point de départ des altérations observées dans cette étude. Afin de caractériser in vivo les altérations de la progression du cycle cellulaire hépatocytaire et d'identifier le mécanisme sous-jacent chez ces souris, un modèle de régénération hépatique, induit par l'injection d'une forte dose de l'hépatotoxique CCl4 a été utilisé. Nos résultats ont mis en évidence une inhibition de la transition G1/S associée à une activation de la voie ATM de réponse aux dommages à l'ADN causés par un stress oxydant exacerbé dans les hépatocytes de souris exprimant les protéines du VHC. / Les mécanismes de la fibrogenèse hépatique liée à l'infection chronique par le virus de l'hépatite C (VHC) sont encore mal connus. L'activation de la fibrogenèse semble fortement associée à la réaction inflammatoire locale. Néanmoins le rôle direct du VHC dans le processus fibrogène n'a pas été étudié. Notre hypothèse est que la fibrose peut être au moins en partie directement induite par le VHC, indépendamment de la réponse immune de l'hôte.L'un des aspects inhérents à ce postulat est qu'une relation directe pourrait exister entre les particules du VHC et l'activation des principaux acteurs cellulaires de la fibrogenèse hépatique, les cellules étoilées du foie (CEFs). Ainsi, les objectifs de ce projet ont été d'étudier in vitro, la capacité du VHC à activer les CEFs humaines mais d'abord d'explorer la relation existant entre cette activation et une éventuelle infection de ces cellules par le VHC. Afin d'analyser la permissivité des CEFs à l'infection par le VHC, nous avons combiné plusieurs modèles originaux du VHC, tels que le clone infectieux JFH1, des rétrovirus pseudotypés avec les protéines d'enveloppe du VHC et le réplicon sous-génomique, avec deux modèles cellulaires de CEFs relevant (des cultures primaires humaines et la lignée immortalisée LX2). En conclusion, nous avons démontré que les CEFs humaines sont réfractaires à la fois à l'entrée et à la réplication du VHC. Ces résultats n'écartent cependant pas l'hypothèse d'une interaction directe entre les particules du VHC et la surface des CEFs dans l'activation fibrogénique de ces cellules. Le rôle des protéines d'enveloppe virales sous leur conformation native dans l'activation des CEFs fût étudié en incubant des ppVHC avec les CEFs. Malgré des résultats préliminaires encourageants, la question d'une activation éventuelle des CEFs en culture après contact avec des particules virales du VHC, et ce indépendamment d'une entrée et/ou d'une réplication virale, n'a pu être confirmé et reste encore sans réponse.Un second aspect est que l'expression in vivo de l'ensemble des protéines du VHC dans les hépatocytes pourrait jouer un rôle dans le déclenchement et la progression de la fibrose portale. Nous avons démontré pour la première fois que l'expression hépatocytaire in vivo des protéines du VHC chez des souris transgéniques, les FL-N/35, soumises à un traitement fibrogénique (injection chronique de CCl4) était associée à une fibrose augmentée, et ce de manière indépendante de l'inflammation locale. Cette fibrose augmentée chez les FL-N/35 s'accompagnait d'une production augmentée de d'espèces réactives de l'oxygène intrahépatocytaires, d'une réaction ductulaire caractérisée notamment par une expansion de cellules progénitrices hépatiques (CPHs), et d'une l'inhibition de la prolifération hépatocytaire. On notera également que cette fibrose portale corrélait avec l'expansion des CPHs portales, corollaire implicite de l'inhibition de la prolifération hépatocytaire. Ces observations, également observé chez les patients infectés par le VHC, suggèrent que la RD associée à une altération de la prolifération hépatocytaire jouerait un rôle dans la fibrose portale. Le modèle de souris utilisé présentant une expression intrahépatocytaire des protéines du VHC, nos résultats suggèrent implicitement une perturbation de l'homéostasie hépatocytaire comme point de départ des altérations observées dans cette étude. Afin de caractériser in vivo les altérations de la progression du cycle cellulaire hépatocytaire et d'identifier le mécanisme sous-jacent chez ces souris, un modèle de régénération hépatique, induit par l'injection d'une forte dose de l'hépatotoxique CCl4 a été utilisé. Nos résultats ont mis en évidence une inhibition de la transition G1/S associée à une activation de la voie ATM de réponse aux dommages à l'ADN causés par un stress oxydant exacerbé dans les hépatocytes de souris exprimant les protéines du VHC.

A fibrose hepática resulta da cronicidade da injúria celular, ocasionando acúmulo dos componentes da matriz extracelular (MEC) pela ativação, principalmente, de células estreladas e fibroblastos portais em miofibroblastos. Estas células se conectam através de junções comunicantes do tipo gap, formadas por proteínas denominadas conexinas (Cx). As junções gap são responsáveis pelo fluxo de moléculas e íons entre as células, desempenhando importante função no controle da homeostasia tecidual. Diversos tipos de conexinas foram descritas nas células hepáticas. Os hepatócitos expressam Cx32 e Cx26, enquanto as demais células não-parenquimatosas expressam Cx43. Alguns estudos analisaram a expressão das conexinas e das junções gap em processos de reparação e fibrogênese em diferentes tecidos, no entanto, poucos avaliaram seu papel na fibrogênese hepática. Sendo assim, o objetivo deste estudo foi avaliar os aspectos morfológicos, histopatológicos e moleculares da fibrose hepática, induzida por tetracloreto de carbono (CCl4), em animais deficientes para as conexinas 43 (Cx43+/-) ou 32 (Cx32-/-). Foram analisados dados biométricos, histopatológicos, ultra-estruturais, imuno-histoquímicos e bioquímicos, além da expressão gênica e protéica das conexinas. Os aspectos moleculares da fibrose hepática foram analisados pela expressão de genes relacionados com a deposição e degradação da matriz extracelular por PCR em tempo real. As análises macroscópicas e de varredura demonstraram um processo de micronodulação da superfície hepática mais acentuado nos camundongos Cx43+/- fibróticos em relação aos animais wild-type (Cx43+/+) fibróticos. Adicionalmente, estes animais apresentaram maior proporção volumétrica de colágeno no tecido hepático; redução na atividade necroinflamatória tecidual; redução nas concentrações séricas de AST e ALT; redução na proliferação celular dos hepatócitos e redução na expressão dos genes: colágeno tipo I, TGFβ-1, MMP-2, MMP-13 e TIMP-1. Por sua vez, os camundongos Cx32-/- fibróticos apresentaram aumento na deposição de colágeno no parênquima hepático; aumento na atividade necroinflamatória tecidual e aumento nos níveis séricos das enzimas hepáticas AST, ALT e fosfatase alcalina em comparação aos animais wild-type (Cx32+/+) fibróticos. Também foram observadas redução na proliferação hepatocelular e maior quantidade de corpúsculos apoptóticos no tecido hepático. Baseando-se em todos os resultados obtidos, observou-se que ambos os modelos animais apresentaram aumento da fibrose hepática, aparentemente ocasionada por diferentes modos de ação. Os animais deficientes em Cx43 apresentaram menor capacidade de degradação do colágeno, ocasionando seu acúmulo no tecido hepático. Por outro lado, os animais deficientes em Cx32 apresentaram maior deposição de colágeno em resposta à injúria hepatocelular mais acentuada, aliada ao desequilíbrio entre as taxas de proliferação celular e apoptose. Em conclusão, os resultados obtidos neste trabalho demonstraram a importante participação das conexinas no controle da fibrogênese hepática, e que podem representar potenciais alvos terapêuticos para o tratamento das doenças hepáticas crônicas em humanos e animais. / Hepatic fibrosis results from chronic cell injury, leading to accumulation of components of extracellular matrix (ECM) through activation mainly of hepatic stellate cells and portal fibroblasts into myofibroblasts. These cells communicate through intercellular gap junctions composed of proteins known as connexins (Cx). Gap junctions are responsible for the exchange of molecules and ions among cells, playing an important role in the control of tissue homeostasis. Several subtypes of connexins were described among hepatic cells. Hepatocytes express Cx32 and Cx26, while the other non-parenchymal cells express Cx43. Some studies analyzed the expression of connexins and gap junctions on processes of healing and fibrogenesis in different tissues; however, few studies evaluated its role on hepatic fibrogenesis. Thus, the objective of this study was to evaluate morphological, histopathological and molecular aspects of hepatic fibrosis induced by carbon tetrachloride (CCl4) in animals with connexin 43 (Cx43+/-) or 32 (Cx32-/-) deficiency. We analyzed biometric, histopathological, ultrastructural, immunohistochemical and biochemical data, besides gene and protein expression of connexins. Molecular aspects of hepatic fibrosis were analyzed with the expression of genes related to deposition and degradation of extracellular matrix by real time PCR. Macroscopic and Scanning Electron Microscopy analyses showed a process of micronodulation of hepatic surface more accentuated on Cx43+/- fibrotic mice when compared to fibrotic wild-type (Cx43+/+) animals. Additionally, these animals presented a higher collagen volumetric proportion on hepatic tissue; reduction of tissue necroinflammatory activity; reduction of serum AST and ALT; reduction of hepatocytes proliferation and reduction of expression type I collagen, TGFβ-1, MMP-2, MMP-13 and TIMP-1 genes. Fibrotic Cx32-/- mice presented an increase of collagen deposition in hepatic parenchyma; increase of tissue necro-inflammatory activity and increase of liver enzymes AST, ALT and alkaline phosphatase when compared to fibrotic wild-type (Cx32+/+) animals. Reduction of hepatocellular proliferation and a higher amount of apoptotic bodies on hepatic tissue were also observed. Based on the results obtained, we observed that both animal models showed an increase of hepatic fibrosis, apparently caused by different modes of action. Cx43 deficient animals showed a reduced capacity to degrade collagen, causing its accumulation in the hepatic tissue. Cx32 deficient animals showed an increased collagen deposition in response to accentuated hepatocellular injury, together to an unbalance between rates of cellular proliferation and apoptosis. In conclusion, results obtained on this study demonstrate an important role of connexins on the control of hepatic fibrogenesis, which could represent potential therapeutical targets for the treatment of chronic liver diseases in humans and animals.

A fibrose hepática resulta da cronicidade da injúria celular, ocasionando acúmulo dos componentes da matriz extracelular (MEC) pela ativação, principalmente, de células estreladas e fibroblastos portais em miofibroblastos. Estas células se conectam através de junções comunicantes do tipo gap, formadas por proteínas denominadas conexinas (Cx). As junções gap são responsáveis pelo fluxo de moléculas e íons entre as células, desempenhando importante função no controle da homeostasia tecidual. Diversos tipos de conexinas foram descritas nas células hepáticas. Os hepatócitos expressam Cx32 e Cx26, enquanto as demais células não-parenquimatosas expressam Cx43. Alguns estudos analisaram a expressão das conexinas e das junções gap em processos de reparação e fibrogênese em diferentes tecidos, no entanto, poucos avaliaram seu papel na fibrogênese hepática. Sendo assim, o objetivo deste estudo foi avaliar os aspectos morfológicos, histopatológicos e moleculares da fibrose hepática, induzida por tetracloreto de carbono (CCl4), em animais deficientes para as conexinas 43 (Cx43+/-) ou 32 (Cx32-/-). Foram analisados dados biométricos, histopatológicos, ultra-estruturais, imuno-histoquímicos e bioquímicos, além da expressão gênica e protéica das conexinas. Os aspectos moleculares da fibrose hepática foram analisados pela expressão de genes relacionados com a deposição e degradação da matriz extracelular por PCR em tempo real. As análises macroscópicas e de varredura demonstraram um processo de micronodulação da superfície hepática mais acentuado nos camundongos Cx43+/- fibróticos em relação aos animais wild-type (Cx43+/+) fibróticos. Adicionalmente, estes animais apresentaram maior proporção volumétrica de colágeno no tecido hepático; redução na atividade necroinflamatória tecidual; redução nas concentrações séricas de AST e ALT; redução na proliferação celular dos hepatócitos e redução na expressão dos genes: colágeno tipo I, TGFβ-1, MMP-2, MMP-13 e TIMP-1. Por sua vez, os camundongos Cx32-/- fibróticos apresentaram aumento na deposição de colágeno no parênquima hepático; aumento na atividade necroinflamatória tecidual e aumento nos níveis séricos das enzimas hepáticas AST, ALT e fosfatase alcalina em comparação aos animais wild-type (Cx32+/+) fibróticos. Também foram observadas redução na proliferação hepatocelular e maior quantidade de corpúsculos apoptóticos no tecido hepático. Baseando-se em todos os resultados obtidos, observou-se que ambos os modelos animais apresentaram aumento da fibrose hepática, aparentemente ocasionada por diferentes modos de ação. Os animais deficientes em Cx43 apresentaram menor capacidade de degradação do colágeno, ocasionando seu acúmulo no tecido hepático. Por outro lado, os animais deficientes em Cx32 apresentaram maior deposição de colágeno em resposta à injúria hepatocelular mais acentuada, aliada ao desequilíbrio entre as taxas de proliferação celular e apoptose. Em conclusão, os resultados obtidos neste trabalho demonstraram a importante participação das conexinas no controle da fibrogênese hepática, e que podem representar potenciais alvos terapêuticos para o tratamento das doenças hepáticas crônicas em humanos e animais. / Hepatic fibrosis results from chronic cell injury, leading to accumulation of components of extracellular matrix (ECM) through activation mainly of hepatic stellate cells and portal fibroblasts into myofibroblasts. These cells communicate through intercellular gap junctions composed of proteins known as connexins (Cx). Gap junctions are responsible for the exchange of molecules and ions among cells, playing an important role in the control of tissue homeostasis. Several subtypes of connexins were described among hepatic cells. Hepatocytes express Cx32 and Cx26, while the other non-parenchymal cells express Cx43. Some studies analyzed the expression of connexins and gap junctions on processes of healing and fibrogenesis in different tissues; however, few studies evaluated its role on hepatic fibrogenesis. Thus, the objective of this study was to evaluate morphological, histopathological and molecular aspects of hepatic fibrosis induced by carbon tetrachloride (CCl4) in animals with connexin 43 (Cx43+/-) or 32 (Cx32-/-) deficiency. We analyzed biometric, histopathological, ultrastructural, immunohistochemical and biochemical data, besides gene and protein expression of connexins. Molecular aspects of hepatic fibrosis were analyzed with the expression of genes related to deposition and degradation of extracellular matrix by real time PCR. Macroscopic and Scanning Electron Microscopy analyses showed a process of micronodulation of hepatic surface more accentuated on Cx43+/- fibrotic mice when compared to fibrotic wild-type (Cx43+/+) animals. Additionally, these animals presented a higher collagen volumetric proportion on hepatic tissue; reduction of tissue necroinflammatory activity; reduction of serum AST and ALT; reduction of hepatocytes proliferation and reduction of expression type I collagen, TGFβ-1, MMP-2, MMP-13 and TIMP-1 genes. Fibrotic Cx32-/- mice presented an increase of collagen deposition in hepatic parenchyma; increase of tissue necro-inflammatory activity and increase of liver enzymes AST, ALT and alkaline phosphatase when compared to fibrotic wild-type (Cx32+/+) animals. Reduction of hepatocellular proliferation and a higher amount of apoptotic bodies on hepatic tissue were also observed. Based on the results obtained, we observed that both animal models showed an increase of hepatic fibrosis, apparently caused by different modes of action. Cx43 deficient animals showed a reduced capacity to degrade collagen, causing its accumulation in the hepatic tissue. Cx32 deficient animals showed an increased collagen deposition in response to accentuated hepatocellular injury, together to an unbalance between rates of cellular proliferation and apoptosis. In conclusion, results obtained on this study demonstrate an important role of connexins on the control of hepatic fibrogenesis, which could represent potential therapeutical targets for the treatment of chronic liver diseases in humans and animals.

La régénération musculaire implique des interactions fonctionnelles entre différents types de cellules mononucléées. Parmi elles, citons les progéniteurs myogéniques (MPs), qui fusionnent pour générer de nouvelles myofibres en réponse à une blessure, et les cellules immunitaires qui envahissent les muscles endommagés. Des dépôts transitoires fibrotiques et d’adipocytes sont observés dans les muscles en régénération qui cependant persistent dans la dystrophie musculaire de Duchenne (DMD) et au cours du vieillissement. Nous avons démontré que les progéniteurs fibro-adipogéniques (FAPs) exprimant le marqueur de surface PDGFRα, contribueraient au développement des dépôts non myogéniques dans les muscles sains. En effet, ces progéniteurs se différencient en adipocytes blancs fonctionnels, bien qu’étant insensibles à l’insuline, et génèrent des myofibroblastes. Quant à la fibrose des muscles DMD, elle se formerait à partir de la différenciation à la fois des MPs et des FAPs. Dans les muscles sains, les FAPs stimulent la myogenèse des MPs au cours de la régénération, alors que les myotubes et les macrophages pro-inflammatoires inhibent l’adipogenèse et la fibrogenèse des FAPs. Pour les progéniteurs âgés ou dystrophiques, les interactions cellulaires entre les FAPs et les MPs sont perturbées. De manière intéressante, la régulation des FAPs DMD ou âgés peut être restaurée en remplaçant les MPs DMD ou âgés par des MPs jeunes et sains. Nos résultats montrent que les muscles humains contiennent des progéniteurs fibro-adipogéniques qui jouent un rôle central dans la régulation de l’homéostasie musculaire en interagissant avec les progéniteurs myogéniques et les macrophages / Muscle regeneration involves functional interactions between different types of mononuclear cells including myogenic progenitors (MPs) and macrophages. Following injury, damaged muscles are invaded by immune cells and MPs fuse to generate new myofibres. Transient fibrotic and adipocyte deposits are observed in regenerating muscles, which however persist in Duchenne muscular dystrophy (DMD) and during aging. We demonstrated that fibro-adipogenic progenitors (FAPs) expressing the PDGFRα surface marker would contribute to the development of non-myogenic deposits in healthy muscles. Indeed, these progenitors differentiate into functional white adipocytes that have the feature to be insulino-resistant, and give rise to myofibroblastes. Intramuscular fibrosis in DMD patients could be formed from both FAPs and MPs differentiation. In healthy muscles, FAPs stimulate myogenesis of MPs during regeneration, while myotubes and pro-inflammatory macrophages inhibit the adipogenesis and fibrogenesis of FAPs. Cellular interactions between FAPs and MPs are disrupted for DMD or aged progenitors. Interestingly, they are restored if aged or DMD FAPs are replaced by healthy and young MPs. Our results show that the human muscles contain fibro-adipogenic progenitors that play a crucial role in the control of muscle homeostasis by interacting with myogenic progenitors and macrophages

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