22 results on '"Fabris, L."'
Search Results
2. REPLY.
- Author
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Lamarca A, Santos-Laso A, Utpatel K, La Casta A, Stock S, Forner A, Adeva J, Folseraas T, Fabris L, Macias RI, Krawczyk M, Krawczyk M, Cardinale V, Braconi C, Alvaro D, Evert M, Banales JM, and Valle JW
- Published
- 2021
- Full Text
- View/download PDF
3. REPLY.
- Author
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Lamarca A, Santos-Laso A, Utpatel K, La Casta A, Stock S, Forner A, Adeva J, Folseraas T, Fabris L, Macias RIR, Krawczyk M, Krawczyk M, Cardinale V, Braconi C, Alvaro D, Evert M, Banales JM, and Valle JW
- Published
- 2021
- Full Text
- View/download PDF
4. Liver Metastases of Intrahepatic Cholangiocarcinoma: Implications for an Updated Staging System.
- Author
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Lamarca A, Santos-Laso A, Utpatel K, La Casta A, Stock S, Forner A, Adeva J, Folseraas T, Fabris L, Macias RIR, Krawczyk M, Krawczyk M, Cardinale V, Braconi C, Alvaro D, Evert M, Banales JM, and Valle JW
- Subjects
- Adult, Aged, Aged, 80 and over, Bile Duct Neoplasms classification, Cholangiocarcinoma classification, Female, Humans, Male, Middle Aged, Neoplasm Invasiveness, Prognosis, SEER Program, Survival Analysis, Bile Duct Neoplasms pathology, Cholangiocarcinoma pathology, Liver Neoplasms secondary, Neoplasm Staging standards
- Abstract
Background and Aims: Intrahepatic cholangiocarcinoma (iCCA) with liver metastases is perceived to have a poor prognosis, but the American Joint Committee on Cancer (AJCC) classifies them as early stage in the absence of lymph nodes or extrahepatic spread., Approach and Results: Patients with iCCA from the European Network for the Study of Cholangiocarcinoma (ENS-CCA) and Surveillance, Epidemiology, and End Results (SEER) registries with survival/staging (AJCC v.7) data were eligible. Modified staging was used (mAJCC v.7): group A: stages I-III (excluding T2bN0); group B: stage IVa (excluding T2bN1M0); group C: liver metastases (T2bN0/1); and group D: stage IVb (extrahepatic metastases). Survival analysis (Kaplan-Meier and Cox regression) was performed in an ENS-CCA training cohort (TC) and findings internally (ENS-CCA iVC) and externally (SEER) validated. The aim was to assess whether liver metastases (group C) had a shorter survival compared to other early stages (group A) to propose a modified version of AJCC v.8 (mAJCC v.8). A total of 574 and 4,171 patients from the ENS-CCA and SEER registries were included. Following the new classification, 19.86% and 17.31% of patients from the ENS-CCA and SEER registries were reclassified into group C, respectively. In the ENS-CCA TC, multivariable Cox regression was adjusted for obesity (p = 0.026) and performance status (P < 0.001); patients in group C (HR, 2.53; 95% CI, 1.18-5.42; P = 0.017) had a higher risk of death (vs. group A). Findings were validated in the ENS-CCA iVC (HR, 2.93; 95% CI, 2.04-4.19; P < 0.001) and in the SEER registry (HR, 1.88; 95% CI, 1.68-2.09; P < 0.001)., Conclusions: iCCA with liver metastases has a worse outcome than other early stages of iCCA. Given that AJCC v.8 does not take this into consideration, a modification of AJCC v.8 (mAJCC v.8), including "liver metastases: multiple liver lesions, with or without vascular invasion" as an "M1a stage," is suggested., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)
- Published
- 2021
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5. The Tumor Microenvironment in Cholangiocarcinoma Progression.
- Author
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Fabris L, Sato K, Alpini G, and Strazzabosco M
- Subjects
- Cancer-Associated Fibroblasts physiology, Disease Progression, Extracellular Vesicles physiology, Humans, Lymphocytes, Tumor-Infiltrating physiology, Tumor-Associated Macrophages physiology, Bile Duct Neoplasms etiology, Cholangiocarcinoma etiology, Tumor Microenvironment physiology
- Published
- 2021
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6. Modulation of the Tryptophan Hydroxylase 1/Monoamine Oxidase-A/5-Hydroxytryptamine/5-Hydroxytryptamine Receptor 2A/2B/2C Axis Regulates Biliary Proliferation and Liver Fibrosis During Cholestasis.
- Author
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Kyritsi K, Chen L, O'Brien A, Francis H, Hein TW, Venter J, Wu N, Ceci L, Zhou T, Zawieja D, Gashev AA, Meng F, Invernizzi P, Fabris L, Wu C, Skill NJ, Saxena R, Liangpunsakul S, Alpini G, and Glaser SS
- Subjects
- ATP Binding Cassette Transporter, Subfamily B physiology, Animals, Cell Proliferation, Cholangitis, Sclerosing etiology, Humans, Male, Mice, Rats, Rats, Sprague-Dawley, Receptor, Serotonin, 5-HT2A physiology, Receptor, Serotonin, 5-HT2B physiology, Receptor, Serotonin, 5-HT2C physiology, Serotonin blood, ATP-Binding Cassette Sub-Family B Member 4, Bile Ducts pathology, Cholestasis pathology, Liver Cirrhosis etiology, Monoamine Oxidase physiology, Receptors, Serotonin physiology, Serotonin physiology, Tryptophan Hydroxylase physiology
- Abstract
Background and Aims: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models., Approach and Results: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2
-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls., Conclusions: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC., (© 2019 by the American Association for the Study of Liver Diseases.)- Published
- 2020
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7. β-Catenin and interleukin-1β-dependent chemokine (C-X-C motif) ligand 10 production drives progression of disease in a mouse model of congenital hepatic fibrosis.
- Author
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Kaffe E, Fiorotto R, Pellegrino F, Mariotti V, Amenduni M, Cadamuro M, Fabris L, Strazzabosco M, and Spirli C
- Subjects
- Animals, Blotting, Western, Disease Models, Animal, Disease Progression, Epithelial Cells metabolism, Flow Cytometry, Immunohistochemistry, Liver metabolism, Liver pathology, Mice, Real-Time Polymerase Chain Reaction, Receptors, CXCR3 metabolism, Signal Transduction, Chemokine CXCL10 metabolism, Genetic Diseases, Inborn metabolism, Interleukin-1beta metabolism, Liver Cirrhosis metabolism, beta Catenin metabolism
- Abstract
Congenital hepatic fibrosis (CHF), a genetic disease caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, encoding for the protein fibrocystin/polyductin complex, is characterized by biliary dysgenesis, progressive portal fibrosis, and a protein kinase A-mediated activating phosphorylation of β-catenin at Ser675. Biliary structures of Pkhd1
del4/del4 mice, a mouse model of CHF, secrete chemokine (C-X-C motif) ligand 10 (CXCL10), a chemokine able to recruit macrophages. The aim of this study was to clarify whether CXCL10 plays a pathogenetic role in disease progression in CHF/Caroli disease and to understand the mechanisms leading to increased CXCL10 secretion. We demonstrate that treatment of Pkhd1del4/del4 mice for 3 months with AMG-487, an inhibitor of CXC chemokine receptor family 3, the cognate receptor of CXCL10, reduces the peribiliary recruitment of alternative activated macrophages (cluster of differentiation 45+ F4/80+ cells), spleen size, liver fibrosis (sirius red), and cyst growth (cytokeratin 19-positive area), consistent with a pathogenetic role of CXCL10. Furthermore, we show that in fibrocystin/polyductin complex-defective cholangiocytes, isolated from Pkhd1del4/del4 mice, CXCL10 production is mediated by Janus kinase/signal transducer and activator of transcription 3 in response to interleukin 1beta (IL-1β) and β-catenin. Specifically, IL-1β promotes signal transducer and activator of transcription 3 phosphorylation, whereas β-catenin promotes its nuclear translocation. Increased pro-IL-1β was regulated by nuclear factor kappa-light-chain-enhancer of activated B cells, and increased secretion of active IL-1β was mediated by the activation of Nod-like receptors, pyrin domain containing 3 inflammasome (increased expression of caspase 1 and Nod-like receptors, pyrin domain containing 3)., Conclusion: In fibrocystin/polyductin complex-defective cholangiocytes, β-catenin and IL-1β are responsible for signal transducer and activator of transcription 3-dependent secretion of CXCL10; in vivo experiments show that the CXCL10/CXC chemokine receptor family 3 axis prevents the recruitment of macrophages, reduces inflammation, and halts the progression of the disease; the increased production of IL-1β highlights the autoinflammatory nature of CHF and may open novel therapeutic avenues. (Hepatology 2018;67:1903-1919)., (© 2017 by the American Association for the Study of Liver Diseases.)- Published
- 2018
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8. Src kinase inhibition reduces inflammatory and cytoskeletal changes in ΔF508 human cholangiocytes and improves cystic fibrosis transmembrane conductance regulator correctors efficacy.
- Author
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Fiorotto R, Amenduni M, Mariotti V, Fabris L, Spirli C, and Strazzabosco M
- Subjects
- Animals, Biliary Tract cytology, Biliary Tract drug effects, Biliary Tract pathology, Cell Culture Techniques, Cystic Fibrosis drug therapy, Cystic Fibrosis Transmembrane Conductance Regulator drug effects, Cytokines metabolism, Cytoskeleton metabolism, Epithelial Cells metabolism, Fluorescent Antibody Technique, Humans, Induced Pluripotent Stem Cells drug effects, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells physiology, Inflammation metabolism, Mice, Microscopy, Confocal, Signal Transduction, src-Family Kinases antagonists & inhibitors, Aminophenols pharmacology, Aminopyridines pharmacology, Benzodioxoles pharmacology, Chloride Channel Agonists pharmacology, Cystic Fibrosis physiopathology, Pyrimidines pharmacology, Quinolones pharmacology, src-Family Kinases metabolism
- Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR), the channel mutated in cystic fibrosis (CF), is expressed by the biliary epithelium (i.e., cholangiocytes) of the liver. Progressive clinical liver disease (CF-associated liver disease; CFLD) occurs in around 10% of CF patients and represents the third leading cause of death. Impaired secretion and inflammation contribute to CFLD; however, the lack of human-derived experimental models has hampered the understanding of CFLD pathophysiology and the search for a cure. We have investigated the cellular mechanisms altered in human CF cholangiocytes using induced pluripotent stem cells (iPSCs) derived from healthy controls and a ΔF508 CFTR patient. We have devised a novel protocol for the differentiation of human iPSC into polarized monolayers of cholangiocytes. Our results show that iPSC-cholangiocytes reproduced the polarity and the secretory function of the biliary epithelium. Protein kinase A/cAMP-mediated fluid secretion was impaired in ΔF508 cholangiocytes and negligibly improved by VX-770 and VX-809, two small molecule drugs used to correct and potentiate ΔF508 CFTR. Moreover, ΔF508 cholangiocytes showed increased phosphorylation of Src kinase and Toll-like receptor 4 and proinflammatory changes, including increased nuclear factor kappa-light-chain-enhancer of activated B cells activation, secretion of proinflammatory chemokines (i.e., monocyte chemotactic protein 1 and interleukin-8), as well as alterations of the F-actin cytoskeleton. Treatment with Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) decreased the inflammatory changes and improved cytoskeletal defects. Inhibition of Src, along with administration of VX-770 and VX-809, successfully restored fluid secretion to normal levels., Conclusion: Our findings have strong translational potential and indicate that targeting Src kinase and decreasing inflammation may increase the efficacy of pharmacological therapies aimed at correcting the basic ΔF508 defect in CF liver patients. These studies also demonstrate the promise of applying iPSC technology in modeling human cholangiopathies. (Hepatology 2018;67:972-988)., (© 2017 by the American Association for the Study of Liver Diseases.)
- Published
- 2018
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9. Macrophage recruitment by fibrocystin-defective biliary epithelial cells promotes portal fibrosis in congenital hepatic fibrosis.
- Author
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Locatelli L, Cadamuro M, Spirlì C, Fiorotto R, Lecchi S, Morell CM, Popov Y, Scirpo R, De Matteis M, Amenduni M, Pietrobattista A, Torre G, Schuppan D, Fabris L, and Strazzabosco M
- Subjects
- Animals, Antigens, Neoplasm metabolism, Clodronic Acid, Collagen metabolism, Disease Models, Animal, Genetic Diseases, Inborn metabolism, Integrins metabolism, Liver Cirrhosis metabolism, Mice, Myofibroblasts physiology, Snail Family Transcription Factors, Transcription Factors metabolism, Transforming Growth Factor beta1 metabolism, Tumor Necrosis Factor-alpha metabolism, Chemokines metabolism, Epithelial Cells metabolism, Genetic Diseases, Inborn immunology, Liver Cirrhosis immunology, Macrophages physiology, Receptors, Cell Surface deficiency
- Abstract
Unlabelled: Congenital hepatic fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure; however, the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1(del4/del4)) mouse, which is orthologous of CHF, we show that Pkhd1(del4/del4) cholangiocytes are characterized by a β-catenin-dependent secretion of a range of chemokines, including chemokine (C-X-C motif) ligands 1, 10, and 12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1(del4/del4) cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvβ6 integrin, an activator of latent local transforming growth factor-β1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts., Conclusion: Fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment, and collagen deposition; these findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis, and macrophage polarization over time., (© 2015 by the American Association for the Study of Liver Diseases.)
- Published
- 2016
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10. Posttranslational regulation of polycystin-2 protein expression as a novel mechanism of cholangiocyte reaction and repair from biliary damage.
- Author
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Spirli C, Villani A, Mariotti V, Fabris L, Fiorotto R, and Strazzabosco M
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Bile Ducts cytology, Cholestasis metabolism, Epithelial Cells physiology, Protein Processing, Post-Translational, TRPP Cation Channels metabolism
- Abstract
Unlabelled: Polycystin-2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2-defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A-dependent activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, hypoxia-inducible factor 1α (HIF-1α)-mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF-1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF-1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2(-/-) knockout, bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF-1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down-regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice and of Mdr2(-/-) mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2., Conclusion: In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down-regulation is associated with activation of ERK1/2 and an increase of HIF-1α-mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases., (© 2015 by the American Association for the Study of Liver Diseases.)
- Published
- 2015
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11. Neural cell adhesion molecule and polysialic acid in ductular reaction: the puzzle is far from completed, but the picture is becoming more clear.
- Author
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Strazzabosco M and Fabris L
- Subjects
- Animals, Male, Liver Regeneration, Neural Cell Adhesion Molecules metabolism, Sialic Acids metabolism
- Published
- 2014
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12. Protein kinase A-dependent pSer(675) -β-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis.
- Author
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Spirli C, Locatelli L, Morell CM, Fiorotto R, Morton SD, Cadamuro M, Fabris L, and Strazzabosco M
- Subjects
- Active Transport, Cell Nucleus, Animals, Bile Ducts cytology, Cell Movement, Cyclic AMP physiology, Disease Models, Animal, Genetic Diseases, Inborn metabolism, Liver Cirrhosis metabolism, Mice, Mice, Inbred C57BL, Neuropeptides physiology, Receptors, Cell Surface physiology, rac1 GTP-Binding Protein physiology, Cyclic AMP-Dependent Protein Kinases physiology, Genetic Diseases, Inborn etiology, Liver Cirrhosis etiology, Signal Transduction, beta Catenin metabolism
- Abstract
Unlabelled: Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β-catenin. In cholangiocytes isolated and cultured from Pkhd1(del4/del4) mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser(675) -phosphorylation of β-catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down-regulation of E-cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho-GTPase, Rac-1, was also significantly activated by cAMP in Pkhd1(del4/del4) cholangiocytes. Rac-1 inhibition blocked cAMP-dependent nuclear translocation and transcriptional activity of pSer(675) -β-catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1(del4/del4) and was inhibited by: (1) PKI, (2) silencing β-catenin (siRNA), and (3) the Rac-1 inhibitor NSC 23766., Conclusion: These data show that in fibrocystin-defective cholangiocytes, cAMP/PKA signaling stimulates pSer(675) -phosphorylation of β-catenin and Rac-1 activity. In the presence of activated Rac-1, pSer(675) -β-catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1(del4/del4) cholangiocytes. β-Catenin-dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target., (© 2013 by the American Association for the Study of Liver Diseases.)
- Published
- 2013
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13. Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma.
- Author
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Cadamuro M, Nardo G, Indraccolo S, Dall'olmo L, Sambado L, Moserle L, Franceschet I, Colledan M, Massani M, Stecca T, Bassi N, Morton S, Spirli C, Fiorotto R, Fabris L, and Strazzabosco M
- Subjects
- Animals, Antineoplastic Agents pharmacology, Benzamides pharmacology, Bile Duct Neoplasms pathology, Cell Line, Tumor, Cell Movement drug effects, Cell Proliferation drug effects, Cells, Cultured, Cholangiocarcinoma pathology, Epithelial-Mesenchymal Transition physiology, Heterografts, Humans, Imatinib Mesylate, In Vitro Techniques, Male, Mice, Mice, SCID, Piperazines pharmacology, Pyrimidines pharmacology, Signal Transduction physiology, Bile Duct Neoplasms physiopathology, Bile Ducts, Intrahepatic, Cell Movement physiology, Cholangiocarcinoma physiopathology, Fibroblasts pathology, Lymphokines physiology, Platelet-Derived Growth Factor physiology, rho GTP-Binding Proteins physiology
- Abstract
Unlabelled: Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration., Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach., (Copyright © 2013 by the American Association for the Study of Liver Diseases.)
- Published
- 2013
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14. Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib.
- Author
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Spirli C, Morell CM, Locatelli L, Okolicsanyi S, Ferrero C, Kim AK, Fabris L, Fiorotto R, and Strazzabosco M
- Subjects
- Animals, Antineoplastic Agents, Hormonal pharmacology, Antineoplastic Agents, Hormonal therapeutic use, Benzenesulfonates therapeutic use, Bile Ducts cytology, Bile Ducts metabolism, Caspase 3 metabolism, Cell Proliferation drug effects, Cells, Cultured, Cysts drug therapy, Cysts pathology, Drug Therapy, Combination, Epithelial Cells drug effects, Epithelial Cells enzymology, Ki-67 Antigen metabolism, Liver Diseases drug therapy, Liver Diseases pathology, Mice, Mice, Knockout, Niacinamide analogs & derivatives, Octreotide pharmacology, Octreotide therapeutic use, Phenylurea Compounds, Phosphorylation, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins B-raf metabolism, Proto-Oncogene Proteins c-raf metabolism, Pyridines therapeutic use, Sorafenib, TRPP Cation Channels genetics, Benzenesulfonates pharmacology, Cyclic AMP-Dependent Protein Kinases metabolism, Cysts enzymology, Liver Diseases enzymology, MAP Kinase Signaling System drug effects, Protein Kinase Inhibitors pharmacology, Pyridines pharmacology, TRPP Cation Channels deficiency
- Abstract
Unlabelled: Mutations in polycystins are a cause of polycystic liver disease. In polycystin-2 (PC2)-defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal-regulated kinase-extracellular signal-regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2-defective mice, we treated PC2 (i.e., Pkd2(flox/-) :pCxCreER(TM) [Pkd2cKO]) mice with sorafenib-tosylate for 8 weeks (20-60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO-cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 μM; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 μM. Raf kinase activity assay showed that whereas B-Raf is inhibited by sorafenib in both wild-type (WT) and Pkd2cKO cells, Raf-1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14-22 amide, myristolated (1 μM) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited., Conclusion: In PC2-defective cells, sorafenib inhibits B-Raf but paradoxically activates Raf-1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf-1 when a PKA-activated Ras promotes Raf-1/B-Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib., (Copyright © 2012 American Association for the Study of Liver Diseases.)
- Published
- 2012
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15. The prognosis of perihilar cholangiocarcinoma after radical treatments.
- Author
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Fabris L and Alvaro D
- Subjects
- Humans, Prognosis, Bile Duct Neoplasms surgery, Bile Ducts, Intrahepatic surgery, Cholangiocarcinoma surgery
- Published
- 2012
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16. Altered store operated calcium entry increases cyclic 3',5'-adenosine monophosphate production and extracellular signal-regulated kinases 1 and 2 phosphorylation in polycystin-2-defective cholangiocytes.
- Author
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Spirli C, Locatelli L, Fiorotto R, Morell CM, Fabris L, Pozzan T, and Strazzabosco M
- Subjects
- Adenylyl Cyclases metabolism, Animals, Calcium Channels, Calcium Signaling physiology, Cells, Cultured, Cyclic AMP-Dependent Protein Kinases metabolism, Homeostasis, Membrane Glycoproteins metabolism, Mice, Mice, Knockout, Models, Animal, Phosphorylation, Signal Transduction physiology, Stromal Interaction Molecule 1, TRPP Cation Channels genetics, TRPP Cation Channels metabolism, Vascular Endothelial Growth Factor A metabolism, Bile Ducts cytology, Bile Ducts metabolism, Calcium metabolism, Cyclic AMP metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, TRPP Cation Channels deficiency
- Abstract
Unlabelled: Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2-defective mice, cyclic 3',5'-adenosine monophosphate/ protein kinase A (cAMP/PKA)-dependent activation of extracellular signal-regulated kinase/ mammalian target of rapamycin (ERK-mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca(2+) and cAMP production and inappropriate ERK signaling in PC2-defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional-KO (knockout) mice (Pkd2(flox/-) :pCxCreER™; hence, called Pkd2KO) and compared to cholangiocytes from wild-type mice (WT). Our results showed that, compared to WT cells, in PC2-defective cholangiocytes (Pkd2KO), cytoplasmic and ER-Ca(2+) (measured with Fura-2 and Mag-Fluo4) levels are decreased and store-operated Ca(2+) entry (SOCE) is inhibited, whereas the expression of Ca(2+) -sensor stromal interaction molecule 1 (STIM1) and store-operated Ca(2+) channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER-Ca(2+) depletion increases cAMP and PKA-dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6)., Conclusion: These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM-dependent activation of AC6 by ER Ca(2+) depletion. In PC2-defective cells, the interaction of STIM-1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store-operated cAMP production., (Copyright © 2011 American Association for the Study of Liver Diseases.)
- Published
- 2012
- Full Text
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17. Nuclear expression of S100A4 calcium-binding protein increases cholangiocarcinoma invasiveness and metastasization.
- Author
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Fabris L, Cadamuro M, Moserle L, Dziura J, Cong X, Sambado L, Nardo G, Sonzogni A, Colledan M, Furlanetto A, Bassi N, Massani M, Cillo U, Mescoli C, Indraccolo S, Rugge M, Okolicsanyi L, and Strazzabosco M
- Subjects
- Aged, Animals, Apoptosis, Bile Duct Neoplasms chemistry, Bile Duct Neoplasms surgery, Cell Movement, Cell Proliferation, Cholangiocarcinoma chemistry, Cholangiocarcinoma surgery, Female, Humans, Male, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 metabolism, Mice, Middle Aged, Neoplasm Invasiveness, Neoplasm Metastasis, Prognosis, S100 Calcium-Binding Protein A4, S100 Proteins analysis, Bile Duct Neoplasms pathology, Bile Ducts, Intrahepatic, Cell Nucleus chemistry, Cholangiocarcinoma pathology, S100 Proteins physiology
- Abstract
Unlabelled: Cholangiocarcinoma (CCA) carries a severe prognosis because of its strong invasiveness and early metastasization. In several patients, otherwise eligible for surgical resection, micrometastasis are already present at the time of surgery. The mechanisms responsible for CCA invasiveness are unclear. S100A4, a member of the S100 family of small Ca(2+)-binding proteins, is expressed in mesenchymal cells, regulates cell motility in several cell types, and is expressed in some epithelial cancers. Thus, we aimed to study the role of S100A4 in CCA invasiveness and metastasization. The expression of S100A4 was studied by immunohistochemistry in 93 human liver samples of CCA patients undergoing surgical resection and correlated with metastases development (67 cases) and patient survival following surgery using log rank tests and multivariate analysis. S100A4 expression was studied in EGI-1 and TFK-1, human CCA cell lines with and without nuclear S100A4 expression, respectively. Metastatic properties of CCA cells were assessed by xenotransplantation in severe combined immunodeficiency (SCID) mice after transduction with lentiviral vectors encoding firefly luciferase gene. Proliferation, motility (wound healing), invasiveness (Boyden chamber), and metalloproteinases (MMPs) secretion were studied in CCA cells, with or without lentiviral silencing of S100A4. Nuclear expression of S100A4 by neoplastic ducts was a strong predictor of metastasization and reduced survival after resection (P < 0.01). EGI-1 CCA cells showed stronger metastatic properties than TFK-1 when xenotransplanted in SCID mice. S100A4-silenced EGI-1 cells showed significantly reduced motility, invasiveness, and MMP-9 secretion in vitro, without changes in cell proliferation., Conclusion: Nuclear S100A4 identifies a subset of CCA patients with a poor prognosis after surgical resection. Nuclear expression of S100A4 increases CCA cells invasiveness and metastasization, indicating that S100A4 may also represent a potential therapeutic target., (Copyright © 2011 American Association for the Study of Liver Diseases.)
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- 2011
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18. Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.
- Author
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Spirli C, Okolicsanyi S, Fiorotto R, Fabris L, Cadamuro M, Lecchi S, Tian X, Somlo S, and Strazzabosco M
- Subjects
- Animals, Cysts pathology, Disease Models, Animal, Extracellular Signal-Regulated MAP Kinases physiology, Hypoxia-Inducible Factor 1, alpha Subunit, Insulin-Like Growth Factor I antagonists & inhibitors, Insulin-Like Growth Factor I physiology, Liver Diseases pathology, Mice, Polycystic Kidney, Autosomal Dominant physiopathology, Sirolimus pharmacology, TOR Serine-Threonine Kinases, Vascular Endothelial Growth Factor A antagonists & inhibitors, Cysts etiology, Intracellular Signaling Peptides and Proteins physiology, Liver Diseases etiology, Protein Serine-Threonine Kinases physiology, TRPP Cation Channels deficiency, Vascular Endothelial Growth Factor A physiology
- Abstract
Unlabelled: Polycystic liver disease may complicate autosomal dominant polycystic kidney disease (ADPKD), a disease caused by mutations in polycystins, which are proteins that regulate signaling, morphogenesis, and differentiation in epithelial cells. The cystic biliary epithelium [liver cystic epithelium (LCE)] secretes vascular endothelial growth factor (VEGF), which promotes liver cyst growth via autocrine and paracrine mechanisms. The expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), and phosphorylated mammalian target of rapamycin (p-mTOR) and the protein kinase A (PKA)-dependent phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) are also up-regulated in LCE. We have hypothesized that mammalian target of rapamycin (mTOR) represents a common pathway for the regulation of hypoxia-inducible factor 1 alpha (HIF1alpha)-dependent VEGF secretion by IGF1 and ERK1/2. Conditional polycystin-2-knockout (Pkd2KO) mice were used for in vivo studies and to isolate cystic cholangiocytes [liver cystic epithelial cells (LCECs)]. The expression of p-mTOR, VEGF, cleaved caspase 3 (CC3), proliferating cell nuclear antigen (PCNA), IGF1, IGF1R, phosphorylated extracellular signal-regulated kinase, p-P70S6K, HIF1alpha, and VEGF in LCE, LCECs, and wild-type cholangiocytes was studied with immunohistochemistry, western blotting, or enzyme-linked immunosorbent assays. The cystic area was measured by computer-assisted morphometry of pancytokeratin-stained sections. Cell proliferation in vitro was studied with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assays. The treatment of Pkd2KO mice with the mTOR inhibitor rapamycin significantly reduced the liver cyst area, liver/body weight ratio, pericystic microvascular density, and PCNA expression while increasing expression of CC3. Rapamycin inhibited IGF1-stimulated HIF1alpha accumulation and VEGF secretion in LCECs. IGF1-stimulated LCEC proliferation was inhibited by rapamycin and SU5416 (a vascular endothelial growth factor receptor 2 inhibitor). Phosphorylation of the mTOR-dependent kinase P70S6K was significantly reduced by PKA inhibitor 14-22 amide and by the mitogen signal-regulated kinase inhibitor U1026., Conclusion: These data demonstrate that PKA-dependent up-regulation of mTOR has a central role in the proliferative, antiapoptotic, and pro-angiogenic effects of IGF1 and VEGF in polycystin-2-defective mice. This study also highlights a mechanistic link between PKA, ERK, mTOR, and HIF1alpha-mediated VEGF secretion and provides a proof of concept for the potential use of mTOR inhibitors in ADPKD and conditions with aberrant cholangiocyte proliferation.
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- 2010
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19. Human leukocyte antigen polymorphisms in Italian primary biliary cirrhosis: a multicenter study of 664 patients and 1992 healthy controls.
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Invernizzi P, Selmi C, Poli F, Frison S, Floreani A, Alvaro D, Almasio P, Rosina F, Marzioni M, Fabris L, Muratori L, Qi L, Seldin MF, Gershwin ME, and Podda M
- Subjects
- Adult, Aged, Aged, 80 and over, Case-Control Studies, Cohort Studies, Female, Gene Frequency, Genetic Predisposition to Disease, Genotype, HLA-DRB1 Chains, Humans, Italy, Liver Cirrhosis, Biliary ethnology, Male, Middle Aged, Models, Genetic, HLA-DR Antigens genetics, Liver Cirrhosis, Biliary genetics, Polymorphism, Genetic genetics
- Abstract
Genetic factors are critical in determining susceptibility to primary biliary cirrhosis (PBC), but there has not been a clear association with human leukocyte antigen (HLA) genes. We performed a multicenter case-control study and analyzed HLA class II DRB1 associations using a large cohort of 664 well-defined cases of PBC and 1992 controls of Italian ancestry. Importantly, healthy controls were rigorously matched not only by age and sex, but also for the geographical origin of the proband four grandparents (Northern, Central, and Southern Italy). After correction for multiple testing, DRB1*08 [odds ratio (OR), 3.3; 95% confidence interval (CI), 2.4-4.5] and DRB1*02 (OR 0.9; 95% CI 0.8-1.2) were significantly associated with PBC, whereas alleles DRB1*11 (OR 0.4; 95% CI 0.3-0.4) and DRB1*13 (OR 0.7; 95% CI 0.6-0.9) were protective. When subjects were stratified according to their grandparental geographical origin, only the associations with DRB1*08 and DRB1*11 were common to all three areas. Associated DRB1 alleles were found only in a minority of patients, whereas an additive genetic model is supported by the gene dosage effect for DRB1*11 allele and the interaction of DRB1*11,*13, and *08. Lastly, no significant associations were detected between specific DRB1 alleles and relevant clinical features represented by the presence of cirrhosis or serum autoantibodies. In conclusion, we confirm the role for HLA to determine PBC susceptibility and suggest that the effect of HLA is limited to patient subgroups. We suggest that a large whole-genome approach is required to identify further genetic elements contributing to the loss of tolerance in this disease.
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- 2008
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20. Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development.
- Author
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Fabris L, Cadamuro M, Libbrecht L, Raynaud P, Spirlì C, Fiorotto R, Okolicsanyi L, Lemaigre F, Strazzabosco M, and Roskams T
- Subjects
- Animals, Bile Ducts embryology, Gestational Age, Hepatocyte Nuclear Factor 6 deficiency, Humans, Mice, Mice, Knockout, Portal System embryology, Portal System pathology, Portal System physiology, Epithelial Cells physiology, Growth Substances physiology, Hepatic Artery cytology, Hepatic Artery physiology, Liver cytology, Liver embryology, Neovascularization, Physiologic
- Abstract
Unlabelled: Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis., Conclusion: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.
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- 2008
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21. Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.
- Author
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Fabris L, Cadamuro M, Fiorotto R, Roskams T, Spirlì C, Melero S, Sonzogni A, Joplin RE, Okolicsanyi L, and Strazzabosco M
- Subjects
- Animals, Cysts etiology, Humans, Immunohistochemistry, Liver Diseases etiology, Mice, Polycystic Kidney, Autosomal Dominant complications, Vascular Endothelial Growth Factor A biosynthesis, Angiogenic Proteins biosynthesis, Bile Ducts cytology, Bile Ducts metabolism, Cysts metabolism, Liver Diseases metabolism
- Abstract
Liver involvement in autosomal dominant polycystic kidney disease (ADPKD) is characterized by altered remodeling of the embryonic ductal plate (DP) with presence of biliary cysts and aberrant portal vasculature. The genetic defect causing ADPKD has been identified, but mechanisms of liver cyst growth remain uncertain. To investigate the possible role of angiogenic mechanisms, we have studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their receptors (VEGFR-1, VEGFR-2, Tie-2) in ADPKD, Caroli's disease, normal and fetal livers. In ADPKD and control livers Ang-1 and Ang-2 gene expression was studied by real-time-PCR. Effects of VEGF on cholangiocyte proliferation were studied by PCNA Western Blot in isolated rat cholangiocytes and by MTS assay in cultured cholangiocytes isolated from ADPKD patients and from an ADPKD mouse model (Pkd2(WS25/-)). Cholangiocytes were strongly positive for VEGF, VEGFR-1, VEGFR-2 and Ang-2 in ADPKD and Caroli, and also for Ang-1 and Tie-2 in ADPKD, similar to fetal ductal plate cells. VEGF stimulated proliferation in both normal and ADPKD cholangiocytes, but the effect was particularly evident in the latter. Ang-1 alone had no effect, but was synergic to VEGF. VEGF expression on cholangiocytes positively correlated with microvascular density. In conclusion, consistent with the immature phenotype of the cystic epithelium, expression of VEGF, VEGFRs, Ang-1 and Tie-2 is strongly upregulated in cholangiocytes from polycystic liver diseases. VEGF and Ang-1 have autocrine proliferative effect on cholangiocyte growth and paracrine effect on portal vasculature, thus promoting the growth of the cysts and their vascular supply. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
- Published
- 2006
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22. Na(+)-dependent and -independent Cl-/HCO-3 exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells.
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Strazzabosco M, Joplin R, Zsembery A, Wallace L, Spirlì C, Fabris L, Granato A, Rossanese A, Poci C, Neuberger JM, Okolicsànyi L, and Crepaldi G
- Subjects
- Bile Ducts, Intrahepatic cytology, Bile Ducts, Intrahepatic drug effects, Cells, Cultured, Chloride-Bicarbonate Antiporters, Cyclic AMP pharmacology, Humans, Hydrogen-Ion Concentration, Ion Transport drug effects, Proton-Translocating ATPases metabolism, Sodium-Hydrogen Exchangers metabolism, Antiporters metabolism, Bicarbonates metabolism, Bile Ducts, Intrahepatic metabolism, Chlorides metabolism, Sodium metabolism
- Abstract
Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.
- Published
- 1997
- Full Text
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