RESUMO
BACKGROUND AND AIMS: Following liver injury, a fraction of hepatocytes adopt features of biliary epithelial cells (BECs) in a process known as biliary reprogramming. The aim of this study was to elucidate the molecular events accompanying this dramatic shift in cellular identity. APPROACH AND RESULTS: We applied the techniques of bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, and assay for transposase-accessible chromatin with high-throughput sequencing to define the epigenetic and transcriptional changes associated with biliary reprogramming. In addition, we examined the role of TGF-ß signaling by profiling cells undergoing reprogramming in mice with hepatocyte-specific deletion in the downstream TGF-ß signaling component mothers against decapentaplegic homolog 4 (Smad4). Biliary reprogramming followed a stereotyped pattern of altered gene expression consisting of robust induction of biliary genes and weaker repression of hepatocyte genes. These changes in gene expression were accompanied by corresponding modifications at the chromatin level. Although some reprogrammed cells had molecular features of "fully differentiated" BECs, most lacked some biliary characteristics and retained some hepatocyte characteristics. Surprisingly, single-cell analysis of Smad4 mutant mice revealed a dramatic increase in reprogramming. CONCLUSION: Hepatocytes undergo widespread chromatin and transcriptional changes during biliary reprogramming, resulting in epigenetic and gene expression profiles that are similar to, but distinct from, native BECs. Reprogramming involves a progressive accumulation of biliary molecular features without discrete intermediates. Paradoxically, canonical TGF-ß signaling through Smad4 appears to constrain biliary reprogramming, indicating that TGF-ß can either promote or inhibit biliary differentiation depending on which downstream components of the pathway are engaged. This work has implications for the formation of BECs and bile ducts in the adult liver.
Assuntos
Plasticidade Celular/genética , Regeneração Hepática/genética , Fígado/fisiologia , Animais , Ductos Biliares/citologia , Diferenciação Celular/genética , Epigênese Genética , Células Epiteliais/fisiologia , Hepatócitos/fisiologia , Hepatócitos/transplante , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Análise de Célula Única , Proteína Smad4/genéticaRESUMO
Schistosomiasis is a major cause of portal hypertension worldwide. It associates with portal fibrosis that develops during chronic infection. The mechanisms by which the pathogen evokes these host responses remain unclear. We evaluated the hypothesis that schistosome eggs release factors that directly stimulate liver cells to produce osteopontin (OPN), a pro-fibrogenic protein that stimulates hepatic stellate cells to become myofibroblasts. We also investigated the utility of OPN as a biomarker of fibrosis and/or severity of portal hypertension. Cultured cholangiocytes, Kupffer cells and hepatic stellate cells were treated with soluble egg antigen (SEA); OPN production was quantified by quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and ELISA; cell proliferation was assessed by BrdU (5-bromo-2'-deoxyuridine). Mice were infected with Schistosoma mansoni for 6 or 16 weeks to cause early or advanced fibrosis. Liver OPN was evaluated by qRTPCR and immunohistochemistry (IHC) and correlated with liver fibrosis and serum OPN. Livers from patients with schistosomiasis mansoni (early fibrosis n=15; advanced fibrosis n=72) or healthy adults (n=22) were immunostained for OPN and fibrosis markers. Results were correlated with plasma OPN levels and splenic vein pressures. SEA-induced cholangiocyte proliferation and OPN secretion (P<0.001 compared with controls). Cholangiocytes were OPN (+) in Schistosoma-infected mice and humans. Liver and serum OPN levels correlated with fibrosis stage (mice: r=0.861; human r=0.672, P=0.0001) and myofibroblast accumulation (mice: r=0.800; human: r=0.761, P=0.0001). Numbers of OPN (+) bile ductules strongly correlated with splenic vein pressure (r=0.778; P=0.001). S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in liver and blood correlate with fibrosis stage and portal hypertension severity.
Assuntos
Proliferação de Células , Hipertensão Portal/metabolismo , Cirrose Hepática/metabolismo , Osteopontina/metabolismo , Esquistossomose mansoni/metabolismo , Adolescente , Adulto , Animais , Antígenos de Helmintos/farmacologia , Ductos Biliares/citologia , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Linhagem Celular , Células Cultivadas , Feminino , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Interações Hospedeiro-Parasita , Humanos , Hipertensão Portal/genética , Hipertensão Portal/parasitologia , Imuno-Histoquímica , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/parasitologia , Masculino , Camundongos , Pessoa de Meia-Idade , Osteopontina/sangue , Osteopontina/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schistosoma/fisiologia , Esquistossomose mansoni/genética , Esquistossomose mansoni/parasitologia , Adulto JovemRESUMO
This paper has the objective to analyze the cellular aspects of liver regeneration (LR). Upon damage in this organ, the regenerative capacity of hepatocyte is sufficiently able to reestablish the parenchyma as a whole. Taking into account the regenerative capacity of hepatocyte, the need of a progenitor or a liver trunk cell was not obvious. Nowadays it is well-established that precursor cells take part in the liver regenerative process. The liver trunk cell, oval cell, acts as a by-potential precursor, contributing for the liver restoration, mainly when the hepatocytes are unable to proliferate. Another precursor, trunk cell of hematopoetic origin (HSC), takes part in the regenerative process, originating cells of the hepatocytic lineage and colangiocytes, as well as the oval cell. The way the trans-differentiation takes place is not established yet. A number of studies must be undertaken in order to clarify questions, such as the possible occurrence of cellular fusion process between the HSC and the hepatic cells and the possibility of application as a new therapeutic procedure in the treatment of diseases associated with insufficiency of this noble organ.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Hepatócitos/citologia , Regeneração Hepática/fisiologia , Animais , Antígenos de Superfície/metabolismo , Ductos Biliares/citologia , Biomarcadores/metabolismo , Proliferação de Células , Humanos , RatosRESUMO
This paper has the objective to analyze the cellular aspects of liver regeneration (LR). Upon damage in this organ, the regenerative capacity of hepatocyte is sufficiently able to reestablish the parenchyma as a whole. Taking into account the regenerative capacity of hepatocyte, the need of a progenitor or a liver trunk cell was not obvious. Nowadays it is well-established that precursor cells take part in the liver regenerative process. The liver trunk cell, oval cell, acts as a bypotential precursor, contributing for the liver restoration, mainly when the hepatocytes are unable to proliferate. Another precursor, trunk cell of hematopoetic origin (HSC), takes part in the regenerative process, originating cells of the hepatocitic lineage and colangiocytes, as well as the oval cell. The way the trans-differentiation takes place is not established yet. A number of studies must be undertaken in order to clarify questions, such as the possible occurrence of cellular fusion process between the HSC and the hepatic cells and the possibility of application as a new therapeutic procedure in the treatment of diseases associated with insufficiency of this noble organ.
Este artigo tem como objetivo analisar aspectos da regeneração hepática (RH) sob a óptica celular. Em vigência de uma lesão neste órgão a capacidade regenerativa do hepatócito é suficientemente capaz de restabelecer o parênquima como um todo. Levando em conta a elevada capacidade regenerativa do hepatócito, a necessidade de um progenitor ou uma célula tronco hepática não era óbvia. Hoje esta bem estabelecido que células precursoras participam do processo regenerativo hepático. A célula tronco hepática, célula oval, atua como um precursor bipotencial, contribuindo para o restauro do fígado principalmente quando os hepatócitos se encontram impossibilitados de proliferar. Um outro precursor, a célula tronco de origem hematopoética (HSC), participa do processo regenerativo, originando células da linhagem hepatocítica e colangiócitos, assim como a células oval. Ainda não está estabelecido o meio como ocorre o fenômeno de transdiferenciação.Muitos estudos devem ser realizados no intuito de esclarecer questões, tais como a possível ocorrência de processo de fusão celular entre a HSC e as células hepáticas e a possibilidade de ser aplicado como uma nova terapêutica no tratamento de doenças associadas à insuficiência deste nobre órgão.
Assuntos
Humanos , Animais , Ratos , Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Hepatócitos/citologia , Regeneração Hepática/fisiologia , Antígenos de Superfície/metabolismo , Ductos Biliares/citologia , Biomarcadores/metabolismo , Proliferação de CélulasRESUMO
Portal vein perfusion of rat's liver was carried out with Ringer's solution followed by 2.5 per cent glutaraldehyde. Liver samples were postifixed in osmium tetroxide, washed in distilled water, snap-frozen in Freon-12, and fractured at -150§C. After freeze-drying at -80§C., a thin layer of carbon and gold was applied under vacuum. Under the scanning electron microscope, the sinusoidal fenestrations and the relationships of the endothelial cells to hepatocyte microvilli and reticulin fibres were delineated. Images of bile canaliculi and adjoining plasma membranes were obtained. These techniques have great potential for the study of a variety of liver diseases. (AU)