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BACKGROUND: Hepatic progenitor cell (HPC) activity and regenerative process that follows pediatric acute liver failure (PALF) is still not well understood. This clinicopathological study was thus conducted with an aim to study the correlation of liver histology and HPC activity with outcomes in PALF. METHODS: All PALF patients with available hepatic histological specimens were included and specimens were analyzed for hepatocyte loss, HPC activity [using cytokeratin (CK) 7, CK19, sex-determining region Y-related high mobility group box(SOX)9 and epithelial cell adhesion molecule (EpCAM)], hepatocyte proliferation (using Ki67), and hepatocyte senescence (using p53 and p21). RESULTS: Ninety-four children were included: 22 (23.4%) survived with native liver (SNL) (i.e., the good outcome group) while rest (i.e., the poor outcome group) either died [33%, 35.1%] or received liver transplant (LT) [39%, 41.5%]. When compared to subjects with poor outcomes, those in the SNL group exhibited significantly less severe hepatocyte loss, fewer HPC/hpf, more proliferating hepatocytes, and less senescent hepatocytes (p < .05). Increasing severity of hepatocyte loss (adjusted OR: 9.95, 95% CI: 4.22-23.45, p < .001) was identified as an independent predictor of poor outcome. Eighty percent children with >50% native hepatocyte loss had poor outcome within 10 days of hospitalization. CONCLUSION: In PALF, more severe hepatocyte loss, higher number of HPC activation, lesser number of proliferating hepatocytes, and greater number of senescent hepatocytes are associated with a poor outcome. Loss of >50% hepatocytes is an independent predictor of poor outcome in PALF.
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Falência Hepática Aguda , Transplante de Fígado , Humanos , Criança , Fígado/patologia , Falência Hepática Aguda/cirurgia , Hepatócitos/metabolismo , Hepatócitos/patologia , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
INTRODUCTION: Recent studies have highlighted the presence of hepatic progenitor cells (HPCs) in metastatic liver carcinomas. We provide further evidence of this phenomenon, presenting a case of a gastrointestinal stromal tumour (GIST) liver metastasis with evidence of intra- and peritumoral HPC. CASE DESCRIPTION: A 64-year-old man presented with a gastric mass diagnosed as a high-risk KIT-mutated GIST. The patient was treated with imatinib, recurring 5 years later with a liver mass. Liver biopsy disclosed a GIST metastasis, hallmarked by a proliferation of ductular structures without cytological atypia intermingled with the tumour cells, with a CK7/CK19/CD56-positive immunophenotype and rare CD44 positivity. The patient underwent liver resection, and the same ductular structures were present in the tumour interior and at its periphery. CONCLUSION: We document for the time the presence of HPC in the form of ductular structures in a GIST liver metastasis, further supporting their role in the liver metastatic niche.
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Tumores do Estroma Gastrointestinal , Neoplasias Hepáticas , Masculino , Humanos , Pessoa de Meia-Idade , Tumores do Estroma Gastrointestinal/genética , Mesilato de Imatinib/uso terapêutico , Células-Tronco/patologiaRESUMO
Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1-2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.
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Hepatopatias , Regeneração Hepática , Albuminas/metabolismo , Albuminas/farmacologia , Animais , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Descarboxilases de Aminoácido-L-Aromático/farmacologia , Molécula de Adesão da Célula Epitelial/metabolismo , Molécula de Adesão da Célula Epitelial/farmacologia , Receptores ErbB/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Hepatopatias/patologia , Regeneração Hepática/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The malignant transformation of normal resident hepatic stem/progenitor cells has a critical role in hepatocarcinogenesis and the recurrence of hepatocellular carcinoma (HCC). We defined such hepatic progenitor cells as hepatoma-initiating cells. An efficient strategy is required to target and kill the hepatoma-initiating cells. We isolated extracellular microparticles (MPs) derived from apoptotic hepatic progenitor cells (HPCs) and tested their ability to inhibit hepatocarcinogenesis. Extracellular MPs were isolated from HPCs, hepatocytes and liver tumor cells. Their effects on tumor growth were investigated in rat primary HCC models, in which hepatocarcinogenesis is induced by diethylnitrosamine (DEN). The extracellular MPs derived from apoptotic HPCs, apoptotic hepatocytes and apoptotic liver tumor cells were similar in morphology, diameter and zeta potential. However, they had different antitumor effects. In DEN-exposed rats, only the MPs derived from apoptotic HPCs effectively inhibit hepatocarcinogenesis. In vitro and in vivo analyses confirmed that HPCs preferentially take up MPs derived from apoptotic HPCs compared to MPs from other liver cell types. Proteomic analysis of MPs from apoptotic HPCs showed enrichment of proteins involved in cell death pathways. Thus, HPC-derived MPs contain a death signal to induce the killing of hepatoma-initiating cells. Our findings provide evidence that a death signal encapsulated in HPC-derived extracellular microparticles can efficiently clear hepatoma-initiating cells and prevent hepatocarcinogenesis.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Proteômica , Ratos , Células-TroncoRESUMO
Clonorchis sinensis (C. sinensis) infection is a risk factor for cholangiocarcinoma. Whether it also contributes to the development of hepatocellular carcinoma (HCC) is still unclear. This study explored the potential relationship between C. sinensis infection and HCC. A total of 110 Sprague-Dawley rats were divided into four treatment groups, the negative control group (NC) received intragastric (i.g.) administration of saline, while the clonorchiasis group (CS) received i.g. administration of 150 C. sinensis metacercariae. The diethylnitrosamine-induced group (DEN) received intraperitoneal (i.p.) administration of DEN. The clonorchiasis DEN-induced group (CSDEN) received i.g. administration of 150 C. sinensis metacercariae followed by i.p. administration of DEN. Hematoxylin and eosin staining, immunohistochemistry, and Masson's trichrome staining were performed for histopathological analysis of the isolated tissues. RNA-seq technology and RT-PCR were employed for gene expression. In the DEN group, 15 rats survived, of which 9 developed liver cirrhosis and 7 developed HCC. In the CSDEN group, all of the 17 surviving rats developed cirrhosis, and 15 showed development of HCC. The incidence of liver cirrhosis and HCC was significantly higher in the CSDEN group than in the DEN group. KEGG pathway analysis of the differentially expressed genes suggested significant upregulation in inflammation-associated pathways. Immunohistochemistry and RT-PCR results showed significant upregulation of hepatic progenitor cell markers (CK19, SOX9, EpCAM) in the CS group compared to the NC group, as well as in the CSDEN group compared to the DEN group. Our study suggests that C. sinensis infection increases risk of HCC in a rat model by stimulating proliferation of hepatic progenitor cells.
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Neoplasias dos Ductos Biliares , Carcinoma Hepatocelular , Clonorquíase , Clonorchis sinensis , Neoplasias Hepáticas , Ratos , Animais , Clonorquíase/complicações , Clonorquíase/patologia , Ratos Sprague-Dawley , Cirrose Hepática/patologia , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/patologia , Fígado/patologiaRESUMO
BACKGROUND AND AIM: In chronic hepatic diseases where treatment strategies are not available, deposited fibrotic tissues deteriorate the intrinsic regeneration capacity of the liver by creating special restrictions. Thus, if the anti-fibrosis modality is efficiently applied, the regeneration capacity of the liver should be reactivated even in such refractory hepatic diseases. METHODS: Rat liver fibrosis was induced by dimethyl-nitrosamine (DMN). Another liver fibrosis model was established in CCl4 treated Sox9CreERT2ROSA26: YFP mice. To resolve hepatic fibrosis, vitamin A-coupled liposomes containing siRNA HSP47 (VA-liposome siHSP47) were employed. EpCAM + hepatic progenitor cells from GFP rats were transplanted to DMN rat liver to examine their trans-differentiation into hepatic cells after resolution of liver fibrosis. RESULTS: Even under continuous exposure to such strong hepatotoxin as DMN, rats undergoing VA-liposome siHSP47 treatment showed an increment of DNA synthesis of hepatocytes with the concomitant restoration of impaired liver weight and normalization of albumin levels. These results were consistent with the observation that GFP + EpCAM hepatic progenitor cells transplanted to DMN rat liver, trans-differentiated into GFP + mature hepatic cells after VA-liposome siHSP47 treatment. Another rodent model also proved regeneration potential of the fibrotic liver in CCl4 administered Sox9CreERT2ROSA26: YFP mice, VA-liposome siHSP47 treatment-induced restoration of liver weight and trans-differentiation of YEP + Sox9 + cells into YFP + hepatic cells, although because of relatively mild hepatotoxicity of CCl4, undamaged hepatocytes also proliferated. CONCLUSIONS: These results demonstrated that regeneration of chronically damaged liver indeed occurs after anti-fibrosis treatment even under continuous exposure to hepatotoxin, which promises a significant benefit of the anti-fibrosis therapy for refractory liver diseases.
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Lipossomos , Cirrose Hepática , RNA Interferente Pequeno , Vitamina A , Animais , Fibrose , Lipossomos/farmacologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Regeneração Hepática/efeitos dos fármacos , Lesão Pulmonar/patologia , Camundongos , RNA Interferente Pequeno/farmacologia , Ratos , Resultado do Tratamento , Vitamina A/farmacologiaRESUMO
BACKGROUND: Liver fibrosis is a hallmark determinant of morbidity in biliary atresia (BA) even in successfully operated cases. Responsible factors for this rapid progression of fibrosis are not completely defined. Aberrant expression of the transcription factor SOX9 and hepatic progenitor cells (HPCs) proliferation have roles in fibrogenesis in cholestatic disorders. However, they were not investigated sufficiently in BA. We aimed to delineate the relation of SOX9 and HPCs to fibrosis and its progression in BA. METHODS: Forty-eight patients with BA who underwent an initial diagnostic liver biopsy (LB) and consequent intraoperative LB were recruited and compared to 28 cases with non-BA cholestasis that had an LB in their diagnostic workup. Liver fibrosis, tissue SOX9 and HPC expressions were studied in both BA and non-BA-cholestasis cases. Liver fibrosis, SOX9, and HPCs' dynamic changes in BA cases were assessed. Relation of fibrosis and its progression to SOX9 and HPCs in BA was assessed. RESULTS: SOX9 and HPCs in ductular reaction (DR) form were expressed in 100% of BA and their grades increased significantly in the second biopsy. The rapidly progressive fibrosis in BA, represented by fibrosis grade of the intraoperative LB, correlated significantly to SOX9-DR and HPC-DR at the diagnostic (r = 0.420, P = 0.003 and r = 0.405, P = 0.004, respectively) and the intraoperative (r = 0.460, P = 0.001 and r = 0.467, P = 0.001, respectively) biopsy. On the other hand, fibrosis, SOX9-DR, and HPC-DR were significantly lower in non-BA cases at a comparable age (P < 0.001, P = 0.006, and P = 0.014, respectively). CONCLUSIONS: Fibrosis in BA is rapidly progressive within a short time and is significantly correlated to SOX9 and HPCs. Assessment of targeting SOX9 and HPCs on fibrosis progression is warranted.
Assuntos
Atresia Biliar , Colestase , Fatores de Transcrição SOX9/genética , Atresia Biliar/cirurgia , Colestase/patologia , Humanos , Fígado/patologia , Fígado/cirurgia , Cirrose Hepática/patologia , Cirrose Hepática/cirurgiaRESUMO
CONTEXT: Our previous studies indicated that Yiguanjian decoction (YGJ) has an anti-hepatic-fibrosis effect and could regulate macrophage status. OBJECTIVE: To elucidate the mechanism of YGJ in regulating macrophages. MATERIALS AND METHODS: Liver cirrhosis was induced by CCl4 for 12 weeks combined with 2-acetylaminofluorene (2-AAF) for the last 4 weeks in male Wistar rats. YGJ (3.56 mg/kg) orally administered in the last 4 weeks, and SORA (1 mg/kg) as control. In vitro, RAW264.7 cells were treated with lipopolysaccharides (LPSs) to induce macrophage polarization to the M1 phenotype, and they were co-cultured with WB-F344 cells and allocated to M group, YGJ group (2 µg/mL) and WIF-1 group (1 µg/mL) with untreated cells as control. The differentiation direction of WB-F344 cell line was observed in the presence or absence of YGJ. Pathology, fibrosis-related cytokines, macrophage polarization-related components, and Wnt signalling pathway components were detected. RESULTS: In vivo, the expression levels of α-SMA, Col (1), OV6, SOX9, EpCAM and M1 macrophage-related components (STAT1, IRF3, IRF5, IRF8, SOCS3) significantly decreased in the YGJ group compared with those in the 2-AAF/CCl4 group (p < 0.01 or 0.05). In vitro, the expression levels of M1 macrophage-related components, including STAT1, NF-κB, IRF3, IRF5, and SOCS3, in RAW264.7 cells decreased significantly in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). The expression levels of Wnt3A, FZD5, LRP-5/-6, and ß-catenin significantly increased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). In addition, the expression levels of Wnt-4/-5A/-5B, and FZD2 significantly decreased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). CONCLUSION: This study suggests that the anti-cirrhosis effect of YGJ is associated with its ability to inhibit macrophage M1-polarization, which provides a scientific basis for the clinical application of YGJ.
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Medicamentos de Ervas Chinesas/farmacologia , Cirrose Hepática Experimental/tratamento farmacológico , Macrófagos/efeitos dos fármacos , 2-Acetilaminofluoreno , Animais , Tetracloreto de Carbono , Linhagem Celular , Citocinas/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Células RAW 264.7 , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
FoxA2 is an essential transcription factor for liver organogenesis and homeostasis. Although reduced expression of FoxA2 has been associated with chronic liver diseases, hepatic progenitor cells (HPCs) that are activated in these circumstances express FoxA2. However, the functional effects and underlying mechanism of FoxA2 in HPCs are still unknown. As revealed by immunostaining, HPCs expressed FoxA2 in human cirrhotic livers and in the livers of choline-deficient diet supplemented with ethionine (CDE) rats. Knocking down FoxA2 in HPCs isolated from CDE rats significantly increased cell proliferation and aerobic glycolysis. Moreover, gene transcription, protein expression, and the enzyme activities of hexokinase 2 (HK2) were upregulated, and blocking HK2 activities via 2-deoxyglucose markedly reduced cell proliferation and aerobic glycolysis. Kyoto Encyclopedia of Genes and Genomes analysis revealed that FoxA2 knockdown enhanced the transcription of genes involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway and triggered downstream Akt phosphorylation. Blocking the PI3K/Akt pathway by Ly294002 inhibited HK2 activities, aerobic glycolysis, and cell proliferation in FoxA2-knockdown cells. Therefore, FoxA2 plays an important role in the proliferation and inhibition of HPCs by suppressing PI3K/Akt/HK2-regulated aerobic glycolysis.
Assuntos
Glicólise/genética , Fator 3-beta Nuclear de Hepatócito/genética , Hexoquinase/genética , Fígado/metabolismo , Organogênese/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Colina/farmacologia , Deficiência de Colina/genética , Deficiência de Colina/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/crescimento & desenvolvimento , Fosfatidilinositol 3-Quinase/genética , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Células-Tronco/metabolismoRESUMO
The proliferation of hepatic progenitor cells (HPCs) is observed in reactive conditions of the liver and primary liver cancers. Ring1 as a member of polycomb-group proteins which play vital roles in carcinogenesis and stem cell self-renewal was increased in HCC patients and promoted proliferation and survival of cancer cell by degrading p53. However, the mechanisms of Ring1 driving the progression of hepatocarcinogenesis have not been elucidated. In this study, forced expression Ring1 and Ring1 siRNA lentiviral vectors were utilized to stably overexpression and silence Ring1 in HPC cell line (WB-F344), respectively. Our finding indicated that overexpression of Ring1 in HPCs promoted colony formation, cell multiplication, and invasion in vitro, conversely depletion of Ring1 repressed the biological functions of HPCs relative to controls. The expression of ß-catenin was upregulated in the HPCs with overexpression of Ring1, and the correlation analysis also showed that ß-catenin and Ring1 had a significant correlation in the liver cancer tissues and adjacent tissues. The activation of the Wnt/ß-catenin signaling pathway significantly increased the expression of liver cancer stem cells related (LCSCs)-related molecular markers CD90 and EpCAM, which led to the transformation of HPCs into LCSCs. Most importantly, the injection of HPCs with overexpressed Ring1 into the subcutaneous of nude mice leads to the formation of poorly differentiated HCC neoplasm. Our findings elucidate that overexpression of Ring1 the activated Wnt/ß-catenin signaling pathway and drove the transformation of HPCs into cancer stem cell-like cells, suggesting Ring1 has extraordinary potential in early diagnosis of HCC.
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Hepatic ischemia-reperfusion (I/R) is a major complication of liver resection, trauma, and liver transplantation; however, liver repair after I/R in diseased liver has not been studied. The present study sought to determine the manner in which the fibrotic liver repairs itself after I/R. Liver fibrosis was established in mice by CCl4 administration for 6 wk, and then liver I/R was performed to investigate liver injury and subsequent liver repair in fibrotic and control livers. After I/R, fibrotic liver had more injury compared with nonfibrotic, control liver; however, fibrotic liver showed rapid resolution of liver necrosis and reconstruction of liver parenchyma. Marked accumulation of hepatic stellate cells and macrophages were observed specifically in the fibrotic septa in early reparative phase. Fibrotic liver had higher numbers of hepatic stellate cells, macrophages, and hepatic progenitor cells during liver recovery after I/R than did control liver, but hepatocyte proliferation was unchanged. Fibrotic liver also had significantly greater number of phagocytic macrophages than control liver. Clodronate liposome injection into fibrotic mice after I/R caused decreased macrophage accumulation and delay of liver recovery. Conversely, CSF1-Fc injection into normal mice after I/R resulted in increased macrophage accumulation and concomitant decrease in necrotic tissue during liver recovery. In conclusion, fibrotic liver clears necrotic areas and restores normal parenchyma faster than normal liver after I/R. This beneficial response appears to be directly related to the increased numbers of nonparenchymal cells, particularly phagocytic macrophages, in the fibrotic liver.NEW & NOTEWORTHY This study is the first to reveal how diseased liver recovers after ischemia-reperfusion (I/R) injury. Although it was not completely unexpected that fibrotic liver had increased hepatic injury after I/R, a novel finding was that fibrotic liver had accelerated recovery and repair compared with normal liver. Enhanced repair after I/R in fibrotic liver was associated with increased expansion of phagocytic macrophages, hepatic stellate cells, and progenitor cells.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Cirrose Hepática Experimental/fisiopatologia , Regeneração Hepática , Fígado/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Proliferação de Células , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Necrose , Fagocitose , Recuperação de Função Fisiológica , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de TempoRESUMO
Granulocyte colony stimulating factor (G-CSF) has been reported to ameliorate impaired liver function in patients with advanced liver diseases through mobilization and proliferation of hepatic progenitor cells (HPCs). However, the underlying mechanisms remain unknown. We previously showed that G-CSF treatment increased the number of bone marrow (BM)-derived cells migrating to the fibrotic liver following repeated carbon tetrachloride (CCl4 ) injections into mice. In this study, we identified opioid growth factor receptor-like 1 (OGFRL1) as a novel BM cell-derived accelerator of fibrotic liver regeneration in response to G-CSF treatment. Endogenous Ogfrl1 was highly expressed in the hematopoietic organs such as the BM and spleen, whereas the liver contained a relatively small amount of Ogfrl1 mRNA. Among the peripheral blood cells, monocytes were the major sources of OGFRL1. Endogenous Ogfrl1 expression in both the peripheral blood monocytes and the liver was decreased following repeated CCl4 injections. An intrasplenic injection of cells overexpressing OGFRL1 into CCl4 -treated fibrotic mice increased the number of HPC and stimulated proliferation of hepatic parenchymal cells after partial resection of the fibrotic liver. Furthermore, overexpression of OGFRL1 in cultured HPC accelerated their differentiation as estimated by increased expression of liver-specific genes such as hepatocyte nuclear factor 4α, cytochrome P450, and fatty acid binding protein 1, although it did not affect the colony forming ability of HPC. These results indicate a critical role of OGFRL1 in the mobilization and differentiation of HPC in the fibrotic liver, and administration of OGFRL1-expressing cells may serve as a potential regenerative therapy for advanced liver fibrosis. Stem Cells 2019;37:89-101.
Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Cirrose Hepática/genética , Cirrose Hepática/terapia , Regeneração Hepática/genética , Medicina Regenerativa/métodos , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Humanos , Masculino , Camundongos , TransfecçãoRESUMO
The emerging field of regenerative medicine has revealed that the exosome contributes to many aspects of development and disease through intercellular communication between donor and recipient cells. However, the biological functions of exosomes secreted from cells have remained largely unexplored. Here, we report that the human hepatic progenitor cells (CdHs)-derived exosome (EXOhCdHs ) plays a crucial role in maintaining cell viability. The inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease of cell viability. This event provokes inhibition of caspase dependent cell death signaling, leading to a ROS-dependent cell damage response and thus induces promotion of antioxidant gene expression or repair of cell death of hypoxia-exposed cells. Together, these findings show the effect of exosomes in regeneration of liver cells, and offer valuable new insights into liver regeneration.
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Antioxidantes , Exossomos , Hepatócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Criança , Exossomos/química , Exossomos/metabolismo , Feminino , Hepatócitos/metabolismo , Humanos , Células-Tronco/metabolismoRESUMO
BACKGROUND: EDP-305 is a novel and potent farnesoid X receptor (FXR) agonist, with no/minimal cross-reactivity to TGR5 or other nuclear receptors. Herein we report therapeutic efficacy of EDP-305, in direct comparison with the first-in-class FXR agonist obeticholic acid (OCA), in mouse models of liver disease. METHODS: EDP-305 (10 and 30 mg/kg/day) or OCA (30mg/kg/day) was tested in mouse models of pre-established biliary fibrosis (BALBc.Mdr2-/-, n = 9-12/group) and steatohepatitis induced by methionine/choline-deficient diet (MCD, n = 7-12/group). Effects on biliary epithelium were evaluated in vivo and in primary EpCAM + hepatic progenitor cell (HPC) cultures. RESULTS: In a BALBc.Mdr2-/- model, EDP-305 reduced serum transaminases by up to 53% and decreased portal pressure, compared to untreated controls. Periportal bridging fibrosis was suppressed by EDP-305 at both doses, with up to a 39% decrease in collagen deposition in high-dose EDP-305. In MCD-fed mice, EDP-305 treatment reduced serum ALT by 62% compared to controls, and profoundly inhibited perisinusoidal 'chicken wire' fibrosis, with over 80% reduction in collagen deposition. In both models, treatment with 30mg/kg OCA reduced serum transaminases up to 30%, but did not improve fibrosis. The limited impact on fibrosis was mediated by cholestasis-independent worsening of ductular reaction by OCA in both disease models; OCA but not EDP-305 at therapeutic doses promoted ductular proliferation in healthy mice and favoured differentiation of primary HPC towards cholangiocyte lineage in vitro. CONCLUSIONS: EDP-305 potently improved pre-established liver injury and hepatic fibrosis in murine biliary and metabolic models of liver disease, supporting the clinical evaluation of EDP-305 in fibrotic liver diseases including cholangiopathies and non-alcoholic steatohepatitis.
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Ácido Quenodesoxicólico , Fígado , Animais , Ácido Quenodesoxicólico/farmacologia , Fibrose , Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , EsteroidesRESUMO
Performance and advances in liver surgery makes remarkable progress of the understanding of liver regeneration. Liver regeneration after liver resection has been widely researched, and the underlying mechanism mostly concerns proliferation of hepatocytes and the influence by inflammation through activation of Kupffer cells and the other parenchymal cells, the second regenerative pathway by hepatic progenitor cells (HPCs), inducing angiogenesis, remodeling of a extracellular matrix (ECM), and termination mechanisms. New clinical surgeries and the updated multiomics analysis are exploiting the remarkable progress, especially in immune regulation and metabolic process of two emerging hallmarks. This review briefly represents a systemic outline of eight hallmarks, including hepatocyte proliferation, contribution of hepatic progenitor cells, inducing angiogenesis, reprogramming of the extracellular matrix, apoptosis and termination of proliferation, inflammation, immune and metabolic regulation, which are set as organizing characteristics of postoperative liver regeneration and future directions of refining treatment targets.
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Procedimentos Cirúrgicos do Sistema Digestório , Regeneração Hepática/fisiologia , Fígado/cirurgia , Proliferação de Células , Matriz Extracelular/metabolismo , Hepatócitos/fisiologia , Humanos , Células de Kupffer/fisiologia , Fígado/citologia , Fígado/metabolismo , Fígado/fisiopatologia , Neovascularização Fisiológica , Tecido Parenquimatoso/citologia , Tecido Parenquimatoso/fisiologia , Período Pós-Operatório , Células-Tronco/fisiologiaRESUMO
Hepatic stellate cells (HSCs) play an important microenvironmental role in hepatic progenitor cells (HPCs) differentiation fate. To reveal the specific mechanism of HSCs induced by transforming growth factor ß1 (TGF-ß1) signaling in HPCs differentiation process, we used Knockin and knockdown technologies induced by lentivirus to upregulate or downregulate TGF-ß1 level in mouse HSCs (mHSCs) (mHSCs-TGF-ß1 or mHSCs-TGF-ßR1sih3). Primary mouse HPCs (mHPCs) were isolated and were cocultured with mHSCs-TGF-ß1 and mHSCs-TGF-ßR1sih3 for 7 days. Differentiation of mHPCs was detected by quantitative reverse transcriptase polymerase chain reaction analysis and immunofluorence in vitro. mHPCs-E14.5 cell lines and differently treated mHSCs were cotransplanted into mice spleens immediately after establishment of acute liver injury model for animal studies. Engraftment and differentiation of transplanted cells as well as liver function recovery were measured at the seventh day via different methods. mHSCs-TGF-ß1 were transformed into myofibroblasts and highly expressed Jagged1, but that expression was reduced after blockage of TGF-ß1 signaling. mHPCs highly expressed downstream markers of Jagged1/Notch signaling and cholangiocyte markers (CK19, SOX9, and Hes1) after coculture with mHSCs-TGF-ß1 in vitro. In contrast, mature hepatocyte marker (ALB) was upregulated in mHPCs in coculture conditions with mHSCs-TGF-ßR1sih3. At the seventh day of cell transplantation assay, mHPCs-E 14.5 engrafted and differentiated into cholangiocytes after cotransplanting with TGF-ß1-knockin mHSCs, but the cells had a tendency to differentiate into hepatocytes when transplanted with TGF-ßR1-knockdown mHSCs, which corresponded to in vitro studies. HSCs play an important role in regulating HPCs differentiation into cholangiocytes via the TGF-ß1/Jagged1 signaling axis. However, HPCs have a tendency to differentiate into hepatocytes after blockage of TGF-ß1 signaling in HSCs.
Assuntos
Diferenciação Celular , Células Estreladas do Fígado/metabolismo , Proteína Jagged-1/metabolismo , Fígado/citologia , Transdução de Sinais , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Transdiferenciação Celular , Glicogênio/biossíntese , Hepatócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Baço/transplanteRESUMO
Clinical and pharmaceutical applications of primary hepatocytes (PHs) are limited due to inadequate number of donated livers and potential challenges in successful maintenance of PHs in culture. Freshly isolated hepatocytes lose their specific features and rapidly de-differentiate in culture. Bipotent hepatoblasts, as liver precursor cells that can differentiate into both hepatocytes and cholangiocytes (Alb- and Ck19-positive cells, respectively), could be used as an alternative and reliable cell source to produce enough PHs for drug discovery or possible clinical applications. In this study, growth factor-free coculture systems of prenatal or postnatal murine liver stromal cells (pre-LSCs or post-LSCs, respectively) were used as feeder cells to support freshly isolated mice hepatoblasts. DLK1-positive hepatoblasts were isolated from mouse fetuses (E14.5) and cocultured with feeder cells under adherent conditions. The hepatoblasts' bipotent features, proliferation rate, and colony formation capacity were assessed on day 5 and 7 post-seeding. Immunoï¬uorescence staining showed that the hepatoblasts remained double positive for Alb and Ck19 on both Pre- and Post-LSCs, after 5 and 7 days of coculture. Moreover, application of pre-LSCs as feeder cells significantly increased the number of DLK1-positive cells and their proliferation rate (ie, increased the number of Ki-67 positive cells) on day 7, compared to Post-LSCs group. Finally, to address our ultimate goal, which was an extension of hepatoblasts ex vivo maintenance, 3D spheres of isolated hepatoblasts were, cultured in conditioned medium (CM) derived from pre-LSCs until day 30. It was observed that the CM derived from Pre-LSCs could successfully prolong the maintenance of hepatic progenitor cells (HPCs) in 3D suspension culture.
Assuntos
Células Alimentadoras/citologia , Fígado/citologia , Células-Tronco/citologia , Animais , Técnicas de Cocultura , Fígado/embriologia , Camundongos , Células-Tronco/metabolismo , Células Estromais/citologia , Fatores de TempoRESUMO
BACKGROUND & AIMS: Congenital hepatic fibrosis (CHF) is a genetic liver disease resulting in abnormal proliferation of cholangiocytes and progressive hepatic fibrosis. CHF is caused by mutations in the PKHD1 gene and the subsequent dysfunction of the protein it encodes, fibrocystin. However, the underlying molecular mechanism of CHF, which is quite different from liver cirrhosis, remains unclear. This study investigated the molecular mechanism of CHF pathophysiology using a genetically engineered human induced pluripotent stem (iPS) cell model to aid the discovery of novel therapeutic agents for CHF. METHODS: PKHD1-knockout (PKHD1-KO) and heterozygously mutated PKHD1 iPS clones were established by RNA-guided genome editing using the CRISPR/Cas9 system. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts [CCs]) in a 3D-culture system. RESULTS: The CCs were composed of a monolayer of cholangiocyte-like cells. The proliferation of PKHD1-KO CCs was significantly increased by interleukin-8 (IL-8) secreted in an autocrine manner. IL-8 production was significantly elevated in PKHD1-KO CCs due to mitogen-activated protein kinase pathway activation caused by fibrocystin deficiency. The production of connective tissue growth factor (CTGF) was also increased in PKHD1-KO CCs in an IL-8-dependent manner. Furthermore, validation analysis demonstrated that both the serum IL-8 level and the expression of IL-8 and CTGF in the liver samples were significantly increased in patients with CHF, consistent with our in vitro human iPS-disease model of CHF. CONCLUSIONS: Loss of fibrocystin function promotes IL-8-dependent proliferation of, and CTGF production by, human cholangiocytes, suggesting that IL-8 and CTGF are essential for the pathogenesis of CHF. IL-8 and CTGF are candidate molecular targets for the treatment of CHF. LAY SUMMARY: Congenital hepatic fibrosis (CHF) is a genetic liver disease caused by mutations of the PKHD1 gene. Dysfunction of the protein it encodes, fibrocystin, is closely associated with CHF pathogenesis. Using an in vitro human induced pluripotent stem cell model and patient samples, we showed that the loss of fibrocystin function promotes proliferation of cholangiocytes and the production of connective tissue growth factor (CTGF) in an interleukin 8 (IL-8)-dependent manner. These results suggest that IL-8 and CTGF are essential for the pathogenesis of CHF.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Epiteliais/metabolismo , Doenças Genéticas Inatas/metabolismo , Cirrose Hepática/metabolismo , Ductos Biliares/patologia , Proliferação de Células , Edição de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Interleucina-8/metabolismo , Mutagênese Sítio-Dirigida/métodos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Hepatic progenitor cells (HPCs) in adult liver are promising for treatment of liver diseases. A biliary-derived HPC population in adult mice has been characterized by co-expression of stem cell marker Sry (sex determining region Y)-box 9 (SOX9) and biliary marker cytokeratin 7 (CK7). However, isolation of these HPCs in adult healthy liver without any selection procedures remains a big challenge in this field. Here, by establishing a simple and efficient method to isolate and expand the CK7+ SOX9+ HPCs in vitro as clones, we acquired a stable and largely scalable cell source. The CK7+ SOX9+ progenitor cells were then further induced to differentiate into hepatocyte-like cells with expression of mature hepatocyte markers albumin (Alb) and hepatocyte nuclear factor 4 alpha (HNF4α), both in vitro and in vivo in the presence of hepatocyte growth factor (HGF) and fibroblast growth factor 9 (FGF9). Furthermore, we found that the HPCs are highly responsive to transforming growth factor-beta (TGF-ß) signals. Collectively, we identified and harvested a CK7+ SOX9+ progenitor cell population from adult mouse liver by a simple and efficient approach. The exploration of this HPC population offers an alternative strategy of generating hepatocyte-like cells for cell-based therapies of acute and chronic liver disorders.
Assuntos
Hepatócitos/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Queratina-7/genética , Queratina-7/metabolismo , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismoRESUMO
The contribution of glycogen synthase kinase-3ß (GSK-3ß) to cholestatic liver disease (CLD) remains unknown. We investigated the role and mechanism of GSK-3ß in vivo in liver tissues of patients with CLD and the bile duct ligation (BDL) mouse model and in vitro using a hepatic progenitor cell (HPC) and hepatic stellate cell (HSC) coculture system. In liver tissues of patients with CLD, expression of the inactive form of GSK-3ß, phospho-GSK-3ß(Ser9), was increased in HPCs. GSK-3ß inhibition by SB216763 treatment aggravated liver fibrosis and elevated the expression of osteopontin (OPN) in the BDL mouse model. OPN was significantly overexpressed in liver tissues and serum from patients with CLD. In an HPC and HSC coculture system, inhibition of GSK-3ß induced OPN production, which activated HSCs in a cholestatic environment. The expression of activator protein 1 (AP-1), an important downstream transcription factor of GSK-3ß, was significantly increased in liver tissues of patients with CLD and SB216763-treated BDL mice. Finally, OPN expression was directly modulated by AP-1. These observations indicate that GSK-3ß inhibition up-regulates OPN expression via AP-1 activation, which accelerates the progression of cholestatic liver fibrosis in patients with CLD and in BDL mice.-Zhuang, S., Hua, X., He, K., Zhou, T., Zhang, J., Wu, H., Ma, X., Xia, Q., Zhang, J. Inhibition of GSK-3ß induces AP-1-mediated osteopontin expression to promote cholestatic liver fibrosis.