RESUMEN
BACKGROUND & AIMS: Syndromic biliary atresia is a cholangiopathy characterized by fibro-obliterative changes in the extrahepatic bile duct (EHBD) and congenital malformations including laterality defects. The etiology remains elusive and faithful animal models are lacking. Genetic syndromes provide important clues regarding the pathogenic mechanisms underlying the disease. We investigated the role of the gene Pkd1l1 in the pathophysiology of syndromic biliary atresia. METHODS: Constitutive and conditional Pkd1l1 knockout mice were generated to explore genetic pathology as a cause of syndromic biliary atresia. We investigated congenital malformations, EHBD and liver pathology, EHBD gene expression, and biliary epithelial cell turnover. Biliary drainage was functionally assessed with cholangiography. Histology and serum chemistries were assessed after DDC (3,5-diethoxycarbony l-1,4-dihydrocollidine) diet treatment and inhibition of the ciliary signaling effector GLI1. RESULTS: Pkd1l1-deficient mice exhibited congenital anomalies including malrotation and heterotaxy. Pkd1l1-deficient EHBDs were hypertrophic and fibrotic. Pkd1l1-deficient EHBDs were patent but displayed delayed biliary drainage. Pkd1l1-deficient livers exhibited ductular reaction and periportal fibrosis. After DDC treatment, Pkd1l1-deficient mice exhibited EHBD obstruction and advanced liver fibrosis. Pkd1l1-deficient mice had increased expression of fibrosis and extracellular matrix remodeling genes (Tgfα, Cdkn1a, Hb-egf, Fgfr3, Pdgfc, Mmp12, and Mmp15) and decreased expression of genes mediating ciliary signaling (Gli1, Gli2, Ptch1, and Ptch2). Primary cilia were reduced on biliary epithelial cells and altered expression of ciliogenesis genes occurred in Pkd1l1-deficient mice. Small molecule inhibition of the ciliary signaling effector GLI1 with Gant61 recapitulated Pkd1l1-deficiency. CONCLUSIONS: Pkd1l1 loss causes both laterality defects and fibro-proliferative EHBD transformation through disrupted ciliary signaling, phenocopying syndromic biliary atresia. Pkd1l1-deficient mice function as an authentic genetic model for study of the pathogenesis of biliary atresia. IMPACT AND IMPLICATIONS: The syndromic form of biliary atresia is characterized by fibro-obliteration of extrahepatic bile ducts and is often accompanied by laterality defects. The etiology is unknown, but Pkd1l1 was identified as a potential genetic candidate for syndromic biliary atresia. We found that loss of the ciliary gene Pkd1l1 contributes to hepatobiliary pathology in biliary atresia, exhibited by bile duct hypertrophy, reduced biliary drainage, and liver fibrosis in Pkd1l1-deficient mice. Pkd1l1-deficient mice serve as a genetic model of biliary atresia and reveal ciliopathy as an etiology of biliary atresia. This model will help scientists uncover new therapeutic approaches for patients with biliary atresia, while pediatric hepatologists should validate the diagnostic utility of PKD1L1 variants.
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Atresia Biliar , Cilios , Modelos Animales de Enfermedad , Células Epiteliales , Ratones Noqueados , Animales , Ratones , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Atresia Biliar/metabolismo , Atresia Biliar/patología , Atresia Biliar/genética , Cilios/metabolismo , Cilios/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Piridinas , Transducción de SeñalRESUMEN
BACKGROUND AND AIMS: In extrahepatic bile duct (EHBD) cholangiopathies, including primary sclerosing cholangitis, a reactive cholangiocyte phenotype is associated with inflammation and epithelial hyperproliferation. The signaling pathways involved in EHBD injury response are poorly understood. In this study, we investigated the role of Hedgehog (HH) signaling and its downstream effectors in controlling biliary proliferation and inflammation after EHBD injury. APPROACH AND RESULTS: Using mouse bile duct ligation as an acute EHBD injury model, we used inhibitory paradigms to uncover mechanisms promoting the proliferative response. HH signaling was inhibited genetically in Gli1-/- mice or by treating wild-type mice with LDE225. The role of neutrophils was tested using chemical (SB225002) and biological (lymphocyte antigen 6 complex locus G6D [Ly6G] antibodies) inhibitors of neutrophil recruitment. The cellular response was defined through morphometric quantification of proliferating cells and CD45+ and Ly6G+ immune cell populations. Key signaling component expression was measured and localized to specific EHBD cellular compartments by in situ hybridization, reporter strain analysis, and immunohistochemistry. Epithelial cell proliferation peaked 24 h after EHBD injury, preceded stromal cell proliferation, and was associated with neutrophil influx. Indian HH ligand expression in the biliary epithelium rapidly increased after injury. HH-responding cells and neutrophil chemoattractant C-X-C motif chemokine ligand 1 (CXCL1) expression mapped to EHBD stromal cells. Inhibition of HH signaling blocked CXCL1 induction, diminishing neutrophil recruitment and the biliary proliferative response to injury. Directly targeting neutrophils by inhibition of the CXCL1/C-X-C motif chemokine receptor 2/Ly6G signaling axis also decreased biliary proliferation. CONCLUSIONS: HH-regulated CXCL1 orchestrates the early inflammatory response and biliary proliferation after EHBD injury through complex cellular crosstalk.
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Conductos Biliares Extrahepáticos , Quimiocina CXCL1 , Proteínas Hedgehog , Animales , Conductos Biliares Extrahepáticos/metabolismo , Proteínas Hedgehog/metabolismo , Inflamación , Ligandos , Ratones , Receptores de Quimiocina , Proteína con Dedos de Zinc GLI1RESUMEN
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) and extrahepatic bile duct carcinoma (EBDC) are distinct entities with different clinicopathological implications. Therefore, research to differentiate between the two diseases is compulsory. In this study, four biomarkers were selected (Hippocalcin-like 1 (HPCAL1); annexin A10 (ANXA10); MUC5AC; sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1)) and focus was placed on clarifying the diagnostic performance of each biomarker and pioneering novel-combined biomarker panels to discriminate between PDAC and EBDC. PROCEDURES: An immunohistochemical microarray analysis of HPCAL1, ANXA10, MUC5AC, and ATP1B1 was conducted for surgically resected 55 PDACs and 77 EBDCs. The diagnostic performance discriminating between PDAC and EBDC was evaluated using four biomarkers and the combined biomarker panels. RESULTS: PDACs exhibited more positive expressions for HPCAL1, ANXA10, and MUC5AC, whereas EBDCs exhibited more ATP1B1-positive expressions. The PDAC panel with the best diagnostic performance was the profile of (+ in ≥ 2 among HPCAL1, ANXA10, MUC5AC)/ATP1B1-. The immunophenotype pattern of (- in ≥ 1 among HPCAL1, ANXA10, MUC5AC)/ATP1B1+ is the EBDC panel with the most excellent discriminating power. CONCLUSION: The suggested combined biomarker panels demonstrate the distinguishing diagnostic ability between PDAC and EBDC is better than previous studies. Therefore, for differentiation between PDAC and EBDC, these panels are expected to help unravel the clinicopathological enigma as promising biomarker panels in the future.
Asunto(s)
Neoplasias de los Conductos Biliares , Conductos Biliares Extrahepáticos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Extrahepáticos/química , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Humanos , Neoplasias Pancreáticas/patología , Neoplasias PancreáticasRESUMEN
Choledochal cyst (CC) is a cystic disease predominantly involving the extrahepatic biliary tree. Biliary atresia (BA), another disorder of the extrahepatic biliary tree, is sometimes considered to be in the same spectrum as pediatric CC. Recently, the absence and/or the structural abnormality of the primary cilia in the intrahepatic cholangiocytes have been implicated in the pathogenesis of BA. We aimed to evaluate the expression of primary ciliary proteins in the intrahepatic cholangiocytes in cases of pediatric CC and compare it with normal control and BA. We performed immunohistochemistry for primary ciliary proteins (acetylated-α-tubulin and double-cortin domain containing 2) on the liver biopsies of control liver (n=5), pediatric CC (n=13), and BA (n=14). We also compared the expression with various clinical, biochemical, histopathologic (portal fibroinflammation and ductal plate malformation), and immunohistochemical (proliferative index) data. There was significant loss of primary cilia from the intrahepatic cholangiocytes in cases of CC and BA as compared with the normal control by both immunostains (CC: P=0.003 and 0.001, respectively; BA: P=0.001 and 0.001, respectively). There was no significant difference between the CC and BA in terms of ciliary protein loss. The loss of the ciliary proteins occurred irrespective of the proliferative (MIB-1 labeling) index, portal fibroinflammation, or ductal plate malformation. The loss of cilia did not correlate with the clinical follow-up in cases of pediatric CC. The loss of primary cilia from the intrahepatic cholangiocytes may be crucial in the etiopathogenesis of pediatric CC.
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Conductos Biliares Extrahepáticos , Atresia Biliar , Quiste del Colédoco , Cilios , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Atresia Biliar/metabolismo , Atresia Biliar/patología , Niño , Preescolar , Quiste del Colédoco/metabolismo , Quiste del Colédoco/patología , Cilios/metabolismo , Cilios/patología , Femenino , Humanos , Lactante , MasculinoRESUMEN
BACKGROUND & AIMS: Peribiliary glands (PBGs), clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts, have been suggested as biliary epithelial stem/progenitor cell niche; however, evidence to support this claim is limited because of a lack of PBG-specific markers. We therefore sought to identify PBG-specific markers to investigate the potential role of PBGs as stem/progenitor cell niches, as well as an origin of cancer. METHODS: We examined the expression pattern of the Wnt target gene Axin2 in extrahepatic bile ducts. We then applied lineage tracing to investigate whether Axin2-expressing cells from PBGs contribute to biliary regeneration and carcinogenesis using Axin2-CreERT mice. RESULTS: Wnt signaling activation, marked by Axin2, was limited to PBGs located in the periampullary region. Lineage tracing showed that Axin2-expressing periampullary PBG cells are capable of self-renewal and supplying new biliary epithelial cells (BECs) to the luminal surface. Additionally, the expression pattern of Axin2 and the mature ductal cell marker CK19 were mutually exclusive in periampullary region, and fate tracing of CK19+ luminal surface BECs showed gradual replacement by CK19- cells, further supporting the continuous replenishment of new BECs from PBGs to the luminal surface. We also found that Wnt signal enhancer R-spondin3 secreted from Myh11-expressing stromal cells, corresponding to human sphincter of Oddi, maintained the periampullary Wnt signal-activating niche. Notably, introduction of PTEN deletion into Axin2+ PBG cells, but not CK19+ luminal surface BECs, induced ampullary carcinoma whose development was suppressed by Wnt inhibitor. CONCLUSION: A specific cell population receiving Wnt-activating signal in periampullary PBGs functions as biliary epithelial stem/progenitor cells and also the cellular origin of ampullary carcinoma.
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Ampolla Hepatopancreática , Proteína Axina/metabolismo , Carcinoma/patología , Neoplasias del Conducto Colédoco/patología , Células Epiteliales/patología , Células Madre/patología , Vía de Señalización Wnt , Ampolla Hepatopancreática/patología , Animales , Proteína Axina/genética , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Carcinogénesis/genética , Linaje de la Célula , Proliferación Celular , Células Epiteliales/metabolismo , Queratina-19/metabolismo , Ratones , Ratones Endogámicos C57BL , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Fosfohidrolasa PTEN/genética , Esfínter de la Ampolla Hepatopancreática/metabolismo , Células Madre/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismoRESUMEN
INTRODUCTION AND OBJECTIVES: Intrahepatic (I-CCA) and extrahepatic (E-CCA) cholangiocarcinoma (CCA) have different growth patterns and risks for tumor metastasis. Inhibition and/or activation of the chemokine receptor CCR subclasses have been reported to alter tumor cell biology in non-CCA cancers. In this study we documented CCR expression profiles in representative human I-CCA and E-CCA cell lines and the in vitro effects of CCR antagonists and agonists on tumor cell biology. MATERIALS AND METHODS: CCR expression profiles were documented by real-time reverse transcription polymerase chain reaction; cell proliferation by WST-1; spheroid formation by sphere dimensions in anchorage-free medium; cell migration by wound healing and invasion by Transwell invasion chambers. RESULTS: All 10 CCR motifs (CCR1-10) were expressed in the I-CCA, HuCCT1 cell line and six (CCR4, 5, 6, 8, 9 and 10) in the E-CCA, KMBC cell line. In HuCCT1 cells, CCR5 expression was most abundant whereas in KMBC cells, CCR6 followed by CCR5 were most abundant. The CCR5 antagonist Maraviroc significantly inhibited cell proliferation, migration and invasion in HuCCT1 cells, and spheroid formation and invasion in KMBC cells. The CCR5 agonist RANTES had no effect on HuCCT1 cells but increased cell proliferation, migration and invasion of KMBC cells. CONCLUSION: These results suggest that CCR expression profiles differ in I-CCA and E-CCA. They also indicate that CCR5 antagonists and agonists have cell-specific effects but in general, CCR5 inactivation inhibits CCA tumor cell aggressiveness. Additional research is required to determine whether CCR5 inactivation is of value in the treatment of CCA in humans.
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Neoplasias de los Conductos Biliares/genética , Conductos Biliares Extrahepáticos/patología , Conductos Biliares Intrahepáticos/patología , Colangiocarcinoma/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Receptores CCR5/genética , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proliferación Celular/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , ADN de Neoplasias/metabolismo , Humanos , Receptores CCR5/biosíntesis , Transducción de SeñalRESUMEN
PURPOSE: To evaluate the influence of cholecystectomy on the flow dynamic pattern of bile in the extrahepatic bile duct by using cine-dynamic MRCP with spatially-selective inversion-recovery (IR) pulse non-invasively. MATERIALS AND METHODS: 56 patients with cholecystectomy and 48 control subjects without cholecystectomy who underwent cine-dynamic MRCP with spatially-selective IR pulse at 1.5 T or 3 T (TR/TE, 4000 msec/500 msec; echo train spacing, 6.5 msec; echo train length, 172; section thickness, 50 mm; matrix, 320 × 320; field of view, 320 × 320 mm; bandwidth, 488 Hz; and inversion time, 2200 msec). In cine-dynamic MRCP, IR pulse with 20 mm width was placed on the common bile duct (CBD) to evaluate the movement of bile (antegrade and reversed bile flow). Cine-dynamic MRCP imaging was scanned every 15 s (imaging, 4 s; rest, 11 s) during 5 min to acquire a series of single-shot images (a total of 20 images). The frequency that antegrade or reversed bile flow was observed in the extrahepatic bile duct, and 5-point grading score based on the moving distance of antegrade or reversed bile flow were compared between the groups. Both groups were compared using the χ2 and Mann-Whitney U tests (P < 0.05 considered significant). RESULTS: Antegrade bile flow was observed more frequently in the cholecystectomy group than in the non-cholecystectomy group (5.1 times vs. 2.8 times, P = 0.008). Mean grading score of antegrade bile flow was significantly greater in the cholecystectomy group than in the non-cholecystectomy group (mean grade, 0.33 vs 0.21; P = 0.014). Regarding reversed bile flow, there were no significant differences in the frequency and grading score between cholecystectomy group and non-cholecystectomy group. CONCLUSION: Antegrade bile flow was observed more frequently and predominantly in patients after cholecystectomy in cine-dynamic MRCP with spatially-selective IR pulse while reversed bile flow was observed equivalently.
Asunto(s)
Conductos Biliares Extrahepáticos/diagnóstico por imagen , Conductos Biliares Extrahepáticos/metabolismo , Bilis/metabolismo , Pancreatocolangiografía por Resonancia Magnética , Colecistectomía , Adulto , Anciano , Conductos Biliares Extrahepáticos/cirugía , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Distal cholangiocarcinoma (dCCA) is a biliary tract cancer with a dismal prognosis and is often preceded by biliary intraepithelial neoplasia (BilIN), representing the most common biliary non-invasive precursor lesion. BilIN are histologically well defined but have not so far been characterised systematically at the molecular level. The aim of this study was to determine miRNA-regulated genes in cholangiocarcinogenesis via BilIN. We used a clinicopathologically well-characterised cohort of 12 dCCA patients. Matched samples of non-neoplastic biliary epithelia, BilIN and invasive tumour epithelia of each patient were isolated from formalin-fixed paraffin-embedded tissue sections by laser microdissection. The resulting 36 samples were subjected to total RNA extraction and the expression of 798 miRNAs was assessed using the Nanostring® technology. Candidate miRNAs were validated by RT-qPCR and functionally investigated following lentiviral overexpression in dCCA-derived cell lines. Potential direct miRNA target genes were identified by microarray and prediction algorithms and were confirmed by luciferase assay. We identified 49 deregulated miRNAs comparing non-neoplastic and tumour tissue. Clustering of these miRNAs corresponded to the three stages of cholangiocarcinogenesis, supporting the concept of BilIN as a tumour precursor. Two downregulated miRNAs, i.e. miR-451a (-10.9-fold down) and miR-144-3p (-6.3-fold down), stood out by relative decrease. Functional analyses of these candidates revealed a migration inhibitory effect in dCCA cell lines. Activating transcription factor 2 (ATF2) and A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) were identified as direct miR-451a target genes. Specific ATF2 inhibition by pooled siRNAs reproduced the inhibitory impact of miR-451a on cancer cell migration. Thus, our data support the concept of BilIN as a direct precursor of invasive dCCA at the molecular level. In addition, we identified miR-451a and miR-144-3p as putative tumour suppressors attenuating cell migration by inhibiting ATF2 in the process of dCCA tumorigenesis. © The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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Factor de Transcripción Activador 2/metabolismo , Neoplasias de los Conductos Biliares/genética , Biomarcadores de Tumor/genética , Carcinoma in Situ/genética , Colangiocarcinoma/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Biomarcadores de Tumor/metabolismo , Carcinogénesis/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Estudios de Casos y Controles , Movimiento Celular/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Proteínas de la Membrana/metabolismoRESUMEN
Biliary atresia is a neonatal liver disease with extrahepatic bile duct obstruction and progressive liver fibrosis. The etiology and pathogenesis of the disease are unknown. We previously identified a plant toxin, biliatresone, responsible for biliary atresia in naturally-occurring animal models, that causes cholangiocyte destruction in in-vitro models. Decreases in reduced glutathione (GSH) mimic the effects of biliatresone, and agents that replenish cellular GSH ameliorate the effects of the toxin. The goals of this study were to define signaling pathways downstream of biliatresone that lead to cholangiocyte destruction and to determine their relationship to GSH. Using cholangiocyte culture and 3D cholangiocyte spheroid cultures, we found that biliatresone and decreases in GSH upregulated RhoU/Wrch1, a Wnt signaling family member, which then mediated an increase in Hey2 in the NOTCH signaling pathway, causing downregulation of the transcription factor Sox17. When these genes were up- or down-regulated, the biliatresone effect on spheroids was phenocopied, resulting in lumen obstruction. Biopsies of patients with biliary atresia demonstrated increased RhoU/Wrch1 and Hey2 expression in cholangiocytes. We present a novel pathway of cholangiocyte injury in a model of biliary atresia, which is relevant to human BA and may suggest potential future therapeutics.
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Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Atresia Biliar/etiología , Atresia Biliar/metabolismo , Glutatión/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Benzodioxoles/metabolismo , Atresia Biliar/patología , Biomarcadores , Modelos Animales de Enfermedad , Expresión Génica , Modelos Biológicos , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Extramedullary hematopoietic cells are present in the liver of normal neonates in the first few days of life and persist in infants with biliary atresia. Based on a previous report that liver genes are enriched by erythroid pathways, we examined the liver gene expression pattern at diagnosis and found the top 5 enriched pathways are related to erythrocyte pathobiology in children who survived with the native liver beyond 2 years of age. Using immunostaining, anti-CD71 antibodies identified CD71+ erythroid cells among extramedullary hematopoietic cells in the livers at the time of diagnosis. In mechanistic experiments, the preemptive antibody depletion of hepatic CD71+ erythroid cells in neonatal mice rendered them resistant to rhesus rotavirus-induced (RRV-induced) biliary atresia. The depletion of CD71+ erythroid cells increased the number of effector lymphocytes and delayed the RRV infection of livers and extrahepatic bile ducts. In coculture experiments, CD71+ erythroid cells suppressed the activation of hepatic mononuclear cells. These data uncover an immunoregulatory role for CD71+ erythroid cells in the neonatal liver.
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Conductos Biliares Extrahepáticos , Atresia Biliar/metabolismo , Células Eritroides/metabolismo , Hígado/metabolismo , Animales , Animales Recién Nacidos , Conductos Biliares Extrahepáticos/lesiones , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Atresia Biliar/patología , Modelos Animales de Enfermedad , Células Eritroides/patología , Humanos , Hígado/patología , Ratones , Ratones Endogámicos BALB CRESUMEN
Mutations in SPINT2 encoding the epithelial serine protease inhibitor hepatocyte growth factor activator inhibitor-2 (HAI-2) are associated with congenital tufting enteropathy. However, the functions of HAI-2 in vivo are poorly understood. Here we used tamoxifen-induced Cre-LoxP recombination in mice to ablate Spint2. Mice lacking Spint2 died within 6 days after initiating tamoxifen treatment and showed severe epithelial damage in the whole intestinal tracts, and, to a lesser extent, the extrahepatic bile duct. The intestinal epithelium showed enhanced exfoliation, villous atrophy, enterocyte tufts and elongated crypts. Organoid crypt culture indicated that Spint2 ablation induced Epcam cleavage with decreased claudin-7 levels and resulted in organoid rupture. These organoid changes could be rescued by addition of serine protease inhibitors aprotinin, camostat mesilate and matriptase-selective α-ketobenzothiazole as well as by co-deletion of Prss8, encoding the serine protease prostasin. These results indicate that HAI-2 is an essential cellular inhibitor for maintaining intestinal epithelium architecture.
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Molécula de Adhesión Celular Epitelial/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Conductos Biliares Extrahepáticos/metabolismo , Claudinas/metabolismo , Silenciador del Gen , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Organoides/metabolismo , Serina Endopeptidasas/genética , Tamoxifeno/farmacología , TransfecciónRESUMEN
Acute pancreatitis (AP) is a disease defined as acute or chronic inflammatory process of the pancreas characterized by premature activation of digestive enzymes within the pancreatic acinar cells and causing pancreatic auto-digestion. In mammalian tissues, H2S is synthesized endogenously from L-cysteine in regulated enzymatic pathways catalyzed by pyridoxal phosphate-dependent enzymes: cystathionine beta - synthase (CBS), gamma - cystathionase (CTH) and cysteine aminotransferase (CAT) coupled with 3-mercaptopyruvate sulfurtransferase (MPST). In the mitochondria, hydrogen sulfide is oxidized to sulfite, which is then converted to thiosulfate (sulfane sulfur-containing compound) by thiosulfate sulfurtransferase (rhodanese; TST). The activity and the expression of CBS, CTH, MPST, and TST have been determined in vivo in pancreas of control rats, rats with acute pancreatitis and sham group. Levels of low-molecular sulfur compounds such as reduced and oxidized glutathione, cysteine, cystine and cystathionine were also determined. The study showed the significant role of MPST in H2S metabolism in pancreas. Stress caused by the surgery (sham group) and AP cause a decrease in H2S production associated with a decrease of MPST activity and expression. Markedly higher level of cysteine in the AP pancreas may be caused by a reduced rate of cysteine consumption in reaction catalyzed by MPST but it can also be a sign of the processes of proteolysis occurring in the changed tissue.
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Conductos Biliares Extrahepáticos/metabolismo , Sulfuro de Hidrógeno/metabolismo , Páncreas/metabolismo , Conductos Pancreáticos/metabolismo , Pancreatitis/metabolismo , Sulfurtransferasas/metabolismo , Animales , Conductos Biliares Extrahepáticos/cirugía , Cistationina/metabolismo , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Cisteína/metabolismo , Cistina/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Glutatión/metabolismo , Ligadura , Masculino , Mitocondrias/metabolismo , Páncreas/patología , Conductos Pancreáticos/cirugía , Pancreatitis/genética , Pancreatitis/patología , Ratas , Ratas Endogámicas WKY , Sulfurtransferasas/genética , Tiosulfato Azufretransferasa/genética , Tiosulfato Azufretransferasa/metabolismo , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
Peribiliary glands (PBGs) are accessory glands with mucinous and serous acini in the biliary tree. The PBG is composed of a heterogeneous cell population, such as mucus- and pancreatic enzyme-producing epithelial cells, whereas it constitutes niches for multipotential stem/progenitor cells in the human extrahepatic bile duct (EHBD). By contrast, the nature of PBGs in the mouse EHBD remains unclear. Our aim was to establish a method for isolating and characterizing PBG-constituting cells in the mouse EHBD. We found that trophoblast cell surface protein 2 (Trop2) was expressed in the luminal epithelium of mouse EHBD exclusively, but not in the PBG. On the basis of the differential expression profile of Trop2, lumen-forming biliary epithelial cells (LBECs) and PBG-constituting epithelial cells (PBECs) were separately isolated for further characterization. Gene expression analysis revealed that the isolated mouse PBECs expressed several marker genes related to human PBGs. In the colony formation assay, PBECs showed significantly higher colony formation capacity than LBECs. In the organoid formation assay, PBECs formed cystic organoid with LBEC-like phenotype. Interestingly, PBECs proliferated, accompanied by reexpression of Trop2 in vivo after bile duct ligation. Furthermore, the unique expression profile of Trop2 was conserved in human EHBD. Our findings indicate that Trop2 is a useful marker in investigating the pathophysiological roles and characteristics of mouse and human PBGs in biliary diseases.
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Antígenos de Neoplasias/metabolismo , Conductos Biliares Extrahepáticos/citología , Moléculas de Adhesión Celular/metabolismo , Glándulas Endocrinas/citología , Células Madre/citología , Animales , Conductos Biliares Extrahepáticos/metabolismo , Neoplasias del Sistema Biliar/metabolismo , Neoplasias del Sistema Biliar/patología , Proliferación Celular , Células Cultivadas , Glándulas Endocrinas/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fenotipo , Células Madre/metabolismoAsunto(s)
Conductos Biliares Extrahepáticos/cirugía , Colangitis/cirugía , Colecistectomía/métodos , Drenaje/métodos , Enterostomía/métodos , Ictericia Obstructiva/cirugía , Adulto , Conductos Biliares Extrahepáticos/diagnóstico por imagen , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Bilirrubina/sangre , Biomarcadores/sangre , Colangitis/sangre , Colangitis/diagnóstico por imagen , Colangitis/patología , Humanos , Ictericia Obstructiva/sangre , Ictericia Obstructiva/diagnóstico por imagen , Ictericia Obstructiva/patología , Masculino , Tomografía Computarizada por Rayos X , UltrasonografíaRESUMEN
Alu and LINE-1 elements are retrotransposons with a ubiquitous presence in the human genome that can cause genomic instability, specifically relating to telomere length. Genotoxic agents may induce methylation of retrotransposons, in addition to oxidative DNA damage in the form of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Methylation of retrotransposons induced by these agents may contribute to biliary atresia (BA) etiology. Here, we investigated correlations between global methylation, 8-OHdG, and relative telomere length, as well as reporting on Alu and LINE-1 hypomethylation in BA patients. Alu and LINE-1 hypomethylation were found to be associated with elevated risk of BA (OR = 4.07; 95% CI: 2.27-7.32; P < 0.0001 and OR = 3.51; 95% CI: 1.87-6.59; P < 0.0001, respectively). Furthermore, LINE-1 methylation was associated with liver stiffness in BA patients (ß coefficient = -0.17; 95% CI: -0.24 to -0.10; P < 0.0001). Stratified analysis revealed negative correlations between Alu and LINE-1 methylation and 8-OHdG in BA patients (P < 0.0001). In contrast, positive relationships were identified between Alu and LINE-1 methylation and relative telomere length in BA patients (P < 0.0001). These findings suggest that retrotransposon hypomethylation is associated with plasma 8-OHdG and telomere length in BA patients.
Asunto(s)
Elementos Alu , Atresia Biliar/genética , Epigénesis Genética , Elementos de Nucleótido Esparcido Largo , Homeostasis del Telómero , Telómero/química , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Atresia Biliar/metabolismo , Atresia Biliar/patología , Estudios de Casos y Controles , Niño , Daño del ADN , Metilación de ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangre , Desoxiguanosina/genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Estrés Oxidativo , Riesgo , Gemelos MonocigóticosRESUMEN
UNLABELLED: Biliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. The goal of this study was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extrahepatic fibrosis. We treated mouse cholangiocytes in three-dimensional (3D) spheroid culture and neonatal extrahepatic duct explants with biliatresone and compounds that regulate glutathione (GSH). We examined the effects of biliatresone on SOX17 levels and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared with untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in GSH, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone. CONCLUSION: Biliatresone decreases GSH and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability; in extrahepatic bile duct explants, it leads to disruption of the extrahepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia. (Hepatology 2016;64:880-893).
Asunto(s)
Benzodioxoles/toxicidad , Conductos Biliares Extrahepáticos/efectos de los fármacos , Atresia Biliar/inducido químicamente , Glutatión/metabolismo , Proteínas HMGB/metabolismo , Factores de Transcripción SOXF/metabolismo , Animales , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Atresia Biliar/metabolismo , Atresia Biliar/patología , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Cholangiocarcinoma can be classified in intrahepatic cholangiocarcinoma (ICC) and perihilar cholangiocarcinoma (PCC). Moreover, PCC includes two different forms: extrahepatic (EH) PCC, which arises from the perihilar EH large ducts, and intrahepatic (IH) PCC, in which a significant liver mass invades the perihilar bile ducts. In this study, we investigated the molecular profile and molecular prognostic factors in EH-PCC, IH-PCC, and ICC submitted to curative surgery. METHODS: Ninety-one patients with cholangiocarcinoma (38 EH-PCC, 18 IH-PCC, and 35 ICC), who underwent curative surgery in a single tertiary hepatobiliary surgery referral center were assessed for mutational status in 56 cancer-related genes. RESULTS: The most frequently mutated genes in EH-PCC were KRAS (47.4 %), TP53 (23.7 %) and ARID1A (15.8 %); in IH-PCC were KRAS (22.2 %), PBRM1 (16.7 %), and PIK3CA (16.7 %); and in ICC were IDH1 (17.1 %), NRAS (17.1 %), and BAP1 (14.3 %). The presence of mutations in ALK, IDH1, and TP53 genes was significantly associated with poor prognosis in patients with EH-PCC (p < 0.001, p = 0.043, and p = 0.019, respectively). Mutation of the TP53 gene was significantly associated with poor prognosis in patients with IH-PCC (p = 0.049). The presence of mutations in ARID1A, PIK3C2G, STK11, TGFBR2, and TP53 genes was significantly associated with poor prognosis in patients with ICC (p = 0.012, p = 0.030, p = 0.030, p = 0.011, and p = 0.011, respectively). CONCLUSIONS: Mutational gene profiling identified different gene mutations in EH-PCC, IH-PCC, and ICC. Moreover, our study reported specific prognostic genes that can identify patients with poor prognosis after curative surgery who may benefit from traditional or target adjuvant treatments.
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Neoplasias de los Conductos Biliares/patología , Conductos Biliares Extrahepáticos/patología , Conductos Biliares Intrahepáticos/patología , Biomarcadores de Tumor/genética , Colangiocarcinoma/patología , Perfilación de la Expresión Génica , Mutación/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/cirugía , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/cirugía , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/cirugía , Colangiocarcinoma/genética , Colangiocarcinoma/cirugía , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Pronóstico , Estudios Prospectivos , Tasa de SupervivenciaRESUMEN
Perturbations in levels of amino acids (AA) and their derivatives are observed in hepatocellular carcinoma (HCC). Yet, it is unclear whether these alterations precede or are a consequence of the disease, nor whether they pertain to anatomically related cancers of the intrahepatic bile duct (IHBC), and gallbladder and extrahepatic biliary tract (GBTC). Circulating standard AA, biogenic amines and hexoses were measured (Biocrates AbsoluteIDQ-p180Kit) in a case-control study nested within a large prospective cohort (147 HCC, 43 IHBC and 134 GBTC cases). Liver function and hepatitis status biomarkers were determined separately. Multivariable conditional logistic regression was used to calculate odds ratios and 95% confidence intervals (OR; 95%CI) for log-transformed standardised (mean = 0, SD = 1) serum metabolite levels and relevant ratios in relation to HCC, IHBC or GBTC risk. Fourteen metabolites were significantly associated with HCC risk, of which seven metabolites and four ratios were the strongest predictors in continuous models. Leucine, lysine, glutamine and the ratio of branched chain to aromatic AA (Fischer's ratio) were inversely, while phenylalanine, tyrosine and their ratio, glutamate, glutamate/glutamine ratio, kynurenine and its ratio to tryptophan were positively associated with HCC risk. Confounding by hepatitis status and liver enzyme levels was observed. For the other cancers no significant associations were observed. In conclusion, imbalances of specific AA and biogenic amines may be involved in HCC development.
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Aminoácidos/metabolismo , Neoplasias de los Conductos Biliares/metabolismo , Aminas Biogénicas/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias de la Vesícula Biliar/metabolismo , Neoplasias Hepáticas/metabolismo , Anciano , Área Bajo la Curva , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Extrahepáticos/patología , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Estudios Prospectivos , Curva ROCRESUMEN
In order to study molecular similarities and differences of intrahepatic (IH-CCA) and extrahepatic (EH-CCA) cholangiocarcinoma, 24 FFPE tumor samples (13 IH-CCA, 11 EH-CCA) were analyzed for whole genome copy number variations (CNVs) using a new high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay. Common in both tumor subtypes the most frequent losses were detected on chromosome 1p, 3p, 6q and 9 while gains were mostly seen in 1q, 8q as well as complete chromosome 17 and 20. Applying the statistical GISTIC (Genomic Identification of Significant Targets in Cancer) tool we identified potential novel candidate tumor suppressor- (DBC1, FHIT, PPP2R2A) and oncogenes (LYN, FGF19, GRB7, PTPN1) within these regions of chromosomal instability. Next to common aberrations in IH-CCA and EH-CCA, we additionally found significant differences in copy number variations on chromosome 3 and 14. Moreover, due to the fact that mutations in the Isocitrate dehydrogenase (IDH-1 and IDH-2) genes are more frequent in our IH-CCA than in our EH-CCA samples, we suggest that the tumor subtypes have a different molecular profile. In conclusion, new possible target genes within regions of high significant copy number aberrations were detected using a high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay, which opens a future perspective of fast routine copy number and marker gene identification for gene targeted therapy.
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Neoplasias de los Conductos Biliares/genética , Conductos Biliares Extrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/metabolismo , Colangiocarcinoma/genética , Variaciones en el Número de Copia de ADN , Sondas Moleculares/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Extrahepáticos/patología , Conductos Biliares Intrahepáticos/patología , Biomarcadores de Tumor/genética , Colangiocarcinoma/patología , Aberraciones Cromosómicas , Hibridación Genómica Comparativa , Femenino , Genoma Humano , Humanos , Técnicas para Inmunoenzimas , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Mutación/genética , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cholangiocarcinoma is a heterogeneous malignant process, which is further classified into intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC). The poor prognosis of the disease is partly due to the lack of understanding of the disease mechanism. Multiple gene alterations identified by various molecular techniques have been described recently. As a result, multiple targeted therapies for ICC and ECC are being developed. In this study, we identified and compared somatic mutations in ICC and ECC patients using next generation sequencing (NGS) (Ampliseq Cancer Hotspot Panel v2 and Ion Torrent 318v2 chips). Eleven of 16 samples passed internal quality control established for NGS testing. ICC cases (n=3) showed IDH1 (33.3%) and NRAS (33.3%) mutations. Meanwhile, TP53 (75%), KRAS (50%), and BRAF (12.5%) mutations were identified in ECC cases (n=8). Our study confirmed the molecular heterogeneity of ICC and ECC using NGS. This information will be important for individual patients as targeted therapies for ICC and ECC become available in the future.