RESUMO
Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 (RAB31) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.
Assuntos
Vesículas Extracelulares , Glicólise , Células Estreladas do Fígado , Cirrose Hepática , Animais , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Vesículas Extracelulares/metabolismo , Camundongos , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Humanos , Modelos Animais de Doenças , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , MasculinoRESUMO
Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB-dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.
Assuntos
Colágeno Tipo IV , Células Endoteliais , Hipertensão Portal , Cirrose Hepática , Fígado , Hipertensão Portal/metabolismo , Hipertensão Portal/patologia , Hipertensão Portal/genética , Animais , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/genética , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/genética , Fígado/patologia , Fígado/metabolismo , Fígado/irrigação sanguínea , Masculino , NF-kappa B/metabolismo , Camundongos Endogâmicos C57BL , Epigênese GenéticaRESUMO
Ductular reactive (DR) cells exacerbate cholestatic liver injury and fibrosis. Herein, we posit that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) emanates from recruited macrophages and restrains DR cell expansion, thereby limiting cholestatic liver injury. Wild type (WT), Trailfl/fl and myeloid-specific Trail deleted (TrailΔmye) C57BL/6 mice were exposed to DDC diet-induced cholestatic liver injury, which induced hepatomegaly and liver injury as compared to control diet-fed mice. However, parameters of liver injury, fibrosis, and inflammation were all increased in the TrailΔmye mice as compared to the WT and Trailfl/fl mice. High dimensional mass cytometry indicated that cholestasis resulted in increased hepatic recruitment of subsets of macrophages and neutrophils in the TrailΔmye mice. Spatial transcriptomics analysis revealed that the PanCK+ cholangiocytes from TrailΔmye mice had increased expression of the known myeloid attractants S100a8, Cxcl5, Cx3cl1, and Cxcl1. Additionally, in situ hybridization of Cxcl1, a potent neutrophil chemoattractant, demonstrated an increased expression in CK19+ cholangiocytes of TrailΔmye mice. Collectively, these data suggest that TRAIL from myeloid cells, particularly macrophages, restrains a subset of DR cells (i.e., Cxcl1 positive cells), limiting liver inflammation and fibrosis. Reprogramming macrophages to express TRAIL may be salutary in cholestasis.
Assuntos
Colestase , Fígado , Animais , Camundongos , Apoptose/genética , Colestase/metabolismo , Fibrose , Ligantes , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cellular senescence and biliary fibrosis are prototypical features of obliterative cholangiopathies, such as primary sclerosing cholangitis (PSC). Telomere dysfunction can lead to senescence either through telomere erosion or damaged telomeres. Our goal was to investigate a mechanistic relationship between telomere damage and biliary fibrosis in PSC. Telomere attrition was observed in the bile ducts of patients with PSC along with a reduction in telomerase reverse transcriptase (TERT) expression, compared with that in normal livers. Similarly, liver tissue from mouse models of biliary fibrosis showed telomere attrition with increased damage at telomeres measured as telomere-associated foci (TAF). Cellular models of senescence induction increased the TAF in cholangiocytes. This coincided with decreased TERT expression and increased senescence, which was rescued by modulating TERT levels. Epigenetic analysis revealed increased acquisition of repressive histone methylation at the TERT promoter, which correlated with decreased TERT transcription. Cholangiocyte-selective deletion of TERT in mice exacerbated fibrosis, whereas androgen therapy toward telomerase rescued liver fibrosis and liver function in a genetic mouse model of PSC. Our results demonstrate a mechanistic role for telomere dysfunction in cellular senescence and fibrosis that characterize PSC. This suggests that PSC may be, in part, a telomere biology disorder, and identifies TERT as a potential therapeutic target.
Assuntos
Colangite Esclerosante , Humanos , Animais , Camundongos , Colangite Esclerosante/genética , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Fígado/metabolismo , Ductos Biliares/metabolismo , Fibrose , TelômeroRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. DESIGN: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. RESULTS: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. CONCLUSION: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Fatores de Transcrição/genética , RNA , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Neoplasias PancreáticasRESUMO
BACKGROUND AIMS: Roux en y anastomosis is a preferred method of biliary reconstruction in liver transplantation that involves living donors or pediatric patients. However, biliary stricture is a frequent and serious complication, accounting for up to 40% of biliary complications in these patients. Previously, we demonstrated that extraluminal delivery of adipose-derived (AD) mesenchymal stromal cells (MSCs) decreased peri-biliary fibrosis and increased neo-angiogenesis in a porcine model of duct-to-duct biliary anastomosis. In this study, we used a porcine model of Roux en y anastomosis to evaluate the beneficial impact of a novel intraluminal MSC delivery system. METHODS: Nine animals were divided into three groups: no stent (group 1), bare stent (group 2) and stent coated with AD-MSCs (group 3). All animals underwent cholecystectomy with roux en y choledochojejunostomy. Two animals per group were followed for 4 weeks and one animal per group was followed for 8 weeks. Cholangiograms and blood were sampled at baseline and the end of study. Biliary tissue was collected and examined by Masson trichrome staining and immunohistochemical staining for MSC markers (CD34 and CD44) and for neo-angiogenesis (CD31). RESULTS: Two of three animals in group 1 developed an anastomotic site stricture. No strictures were observed in the animals of group 2 or group 3. CD34 and CD44 staining showed that AD-MSCs engrafted successfully at the anastomotic site by intraluminal delivery (group 3). Furthermore, biliary tissue from group 3 showed significantly less fibrosis and increased angiogenesis compared with the other groups. CONCLUSIONS: Intraluminal delivery of AD-MSCs resulted in successful biliary engraftment of AD-MSCs as well as reduced peri-biliary fibrosis and increased neo-angiogenesis.
Assuntos
Procedimentos Cirúrgicos do Sistema Biliar , Células-Tronco Mesenquimais , Suínos , Animais , Coledocostomia , Procedimentos Cirúrgicos do Sistema Biliar/métodos , Anastomose em-Y de Roux , Fibrose , Complicações Pós-Operatórias , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The burden of liver diseases is increasing worldwide, with liver transplantation remaining the only treatment option for end-stage liver disease. Regenerative medicine holds great potential as a therapeutic alternative, aiming to repair or replace damaged liver tissue with healthy functional cells. The properties of the cells used are critical for the efficacy of this approach. The advent of liver organoids has not only offered new insights into human physiology and pathophysiology, but also provided an optimal source of cells for regenerative medicine and translational applications. Here, we discuss various historical aspects of 3D organoid culture, how it has been applied to the hepatobiliary system, and how organoid technology intersects with the emerging global field of liver regenerative medicine. We outline the hepatocyte, cholangiocyte, and nonparenchymal organoids systems available and discuss their advantages and limitations for regenerative medicine as well as future directions.
Assuntos
Gastroenterologia , Humanos , Organoides , Fígado , Medicina Regenerativa , HepatócitosRESUMO
BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling in vitro and in vivo. METHODS: Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2fl/fl/Cdh5cre-ERT2) or treatment with the glycolysis inhibitor 3PO were studied in portal hypertension (partial ligation of the inferior vena cava, pIVCL) and early liver fibrosis (CCl4) models. RESULTS: Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin ß1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2fl/fl/Cdh5cre-ERT2 mice showed attenuated neutrophil infiltration and portal hypertension after pIVCL. 3PO treatment attenuated liver fibrosis in a CCl4 model. CONCLUSION: Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease. LAY SUMMARY: Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.
Assuntos
Células Endoteliais , Hipertensão Portal , Actinas/metabolismo , Animais , Quimiocina CXCL1/metabolismo , Cromatina/metabolismo , Células Endoteliais/metabolismo , Epigênese Genética , Glicólise , Histonas/metabolismo , Humanos , Hipertensão Portal/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Mecanotransdução Celular , Camundongos , NF-kappa B/metabolismoRESUMO
Intrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-ß. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-ß receptors I (TßRI) and II (TßRII). While the extracellular domain of PD-L1 (amino acids 19-238) targets TßRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TßRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.
Assuntos
Antígeno B7-H1/metabolismo , Células Estreladas do Fígado/metabolismo , Neoplasias Hepáticas/patologia , Miofibroblastos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Animais , Movimento Celular , Proliferação de Células/fisiologia , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Camundongos , Miofibroblastos/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND & AIMS: Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis. METHODS: We evaluated p300 in vivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. In vitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes. RESULTS: BDL-induced DR and fibrosis were reduced in Krt19-CreERT/p300fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. In vitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes. CONCLUSION: Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases. LAY SUMMARY: We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis.
Assuntos
RNA Longo não Codificante , Animais , Ductos Biliares/patologia , Proliferação de Células , Fibrose , Lipopolissacarídeos , Fígado/patologia , Camundongos , Camundongos Knockout , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory disease of the biliary tract characterized by cellular senescence and periportal fibrogenesis. Specific disease features that are cell intrinsic and either genetically or epigenetically mediated remain unclear due in part to a lack of appropriate, patient-specific, in vitro models. Recently, our group developed systems to create induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs) and biliary epithelial organoids (cholangioids). We use these models to investigate whether PSC cholangiocytes are intrinsically predisposed to cellular senescence. Skin fibroblasts from healthy controls and subjects with PSC were reprogrammed to pluripotency, differentiated to cholangiocytes, and subsequently grown in three-dimensional matrigel-based culture to induce formation of cholangioids. RNA sequencing (RNA-seq) on iDCs showed significant differences in gene expression patterns, including enrichment of pathways associated with cell cycle, senescence, and hepatic fibrosis, that correlate with PSC. These pathways also overlapped with RNA-seq analysis on isolated cholangiocytes from subjects with PSC. Exome sequencing on the subjects with PSC revealed genetic variants of unknown significance in the genes identified in these pathways. Three-dimensional culture revealed smaller size, lack of a central lumen, and increased cellular senescence in PSC-derived cholangioids. Congruent with this, PSC-derived iDCs showed increased secretion of the extracellular matrix molecule fibronectin as well as the inflammatory cytokines interleukin-6, and chemokine (C-C motif) ligand 2. Conditioned media (CM) from PSC-derived iDCs more potently activated hepatic stellate cells compared to control CM. Conclusion: We demonstrated efficient generation of iDCs and cholangioids from patients with PSC that show disease-specific features. PSC cholangiocytes are intrinsically predisposed to cellular senescence. These features are unmasked following biliary differentiation of pluripotent stem cells and have functional consequences in epithelial organoids.