Summary Several cholangiopathies result from a perturbation of developmental processes. Most of these cholangiopathies are characterised by the persistence of biliary structures with foetal ...configuration. Developmental processes are also relevant in acquired liver diseases, as liver repair mechanisms exploit a range of autocrine and paracrine signals transiently expressed in embryonic life. We briefly review the ontogenesis of the intra- and extrahepatic biliary tree, highlighting the morphogens, growth factors, and transcription factors that regulate biliary development, and the relationships between developing bile ducts and other branching biliary structures. Then, we discuss the ontogenetic mechanisms involved in liver repair, and how these mechanisms are recapitulated in ductular reaction, a common reparative response to many forms of biliary and hepatocellular damage. Finally, we discuss the pathogenic aspects of the most important primary cholangiopathies related to altered biliary development, i.e. polycystic and fibropolycystic liver diseases, Alagille syndrome.
In the last 25years, a number of animal models, mainly rodents, have been generated with the goal to mimic cholestatic liver injuries and, thus, to provide in vivo tools to investigate the mechanisms ...of biliary repair and, eventually, to test the efficacy of innovative treatments. Despite fundamental limitations applying to these models, such as the distinct immune system and the different metabolism regulating liver homeostasis in rodents when compared to humans, multiple approaches, such as surgery (bile duct ligation), chemical-induced (3,5-diethoxycarbonyl-1,4-dihydrocollidine, DDC, α-naphthylisothiocyanate, ANIT), viral infections (Rhesus rotavirustype A, RRV-A), and genetic manipulation (Mdr2, Cftr, Pkd1, Pkd2, Prkcsh, Sec63, Pkhd1) have been developed. Overall, they have led to a range of liver phenotypes recapitulating the main features of biliary injury and altered bile acid metabolisms, such as ductular reaction, peribiliary inflammation and fibrosis, obstructive cholestasis and biliary dysgenesis. Although with a limited translability to the human setting, these mouse models have provided us with the ability to probe over time the fundamental mechanisms promoting cholestatic disease progression. Moreover, recent studies from genetically engineered mice have unveiled ‘core’ pathways that make the cholangiocyte a pivotal player in liver repair. In this review, we will highlight the main phenotypic features, the more interesting peculiarities and the different drawbacks of these mouse models. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.
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•Cholestatic liver disorders are chronic diseases, where bile physiology is impaired.•Several animal models were generated to study cholestatic liver injuries.•Despite limitations, these models are useful to unravel cholestasis pathogenesis and cholangiocyte role in biliary repair.
Bile duct epithelial cells, also known as cholangiocytes, regulate the composition of bile and its flow. Acquired, congenital and genetic dysfunctions in these cells give rise to a set of diverse and ...complex diseases, often of unknown aetiology, called cholangiopathies. New knowledge has been steadily acquired about genetic and congenital cholangiopathies, and this has led to a better understanding of the mechanisms of acquired cholangiopathies. This Review focuses on findings from studies on Alagille syndrome, polycystic liver diseases, fibropolycystic liver diseases (Caroli disease and congenital hepatic fibrosis) and cystic fibrosis-related liver disease. In particular, knowledge on the role of Notch signalling in biliary repair and tubulogenesis has been advanced by work on Alagille syndrome, and investigations in polycystic liver diseases have highlighted the role of primary cilia in biliary pathophysiology and the concept of biliary angiogenic signalling and its role in cyst growth and biliary repair. In fibropolycystic liver disease, research has shown that loss of fibrocystin generates a signalling cascade that increases β-catenin signalling, activates the NOD-, LRR- and pyrin domain-containing 3 inflammasome, and promotes production of IL-1β and other chemokines that attract macrophages and orchestrate the process of pericystic and portal fibrosis, which are the main mechanisms of progression in cholangiopathies. In cystic fibrosis-related liver disease, lack of cystic fibrosis transmembrane conductance regulator increases the sensitivity of epithelial Toll-like receptor 4 that sustains the secretion of nuclear factor-κB-dependent cytokines and peribiliary inflammation in response to gut-derived products, providing a model for primary sclerosing cholangitis. These signalling mechanisms may be targeted therapeutically and they offer a possibility for the development of novel treatments for acquired cholangiopathies.
Cholangiocarcinoma(CCA) is a highly aggressive epithelial malignancy still carrying a dismal prognosis, owing to early lymph node metastatic dissemination and striking resistance to conventional ...chemotherapy. Although mechanisms underpinning CCA progression are still a conundrum, it is now increasingly recognized that the desmoplastic microenvironment developing in conjunction with biliary carcinogenesis, recently renamed tumor reactive stroma(TRS), behaves as a paramount tumor-promoting driver. Indeed, once being recruited, activated and dangerously co-opted by neoplastic cells, the cellular components of the TRS(myofibroblasts, macrophages, endothelial cells and mesenchymal stem cells) continuously rekindle malignancy by secreting a huge variety of soluble factors(cyto/chemokines, growth factors, morphogens and proteinases). Furthermore, these factors are long-term stored within an abnormally remodeled extracellular matrix(ECM), which in turn can deleteriously mold cancer cell behavior. In this review, we will highlight evidence for the active role played by reactive stromal cells(as well as by the TRS-associated ECM) in CCA progression, including an overview of the most relevant TRS-derived signals possibly fueling CCA cell aggressiveness. Hopefully, a deeper knowledge of the paracrine communications reciprocally exchanged between cancer and stromal cells will steer the development of innovative, combinatorial therapies, which can finally hinder the progression of CCA, as well as of other cancer types with abundant TRS, such as pancreatic and breast carcinomas.
Liver diseases negatively impact the quality of life and survival of patients, and often require liver transplantation in cases that progress to organ failure. Understanding the cellular and ...molecular mechanisms of liver development and pathogenesis has been a challenging task, in part for the lack of adequate cellular models directly relevant to the human diseases.
Recent technological advances in the stem cell field have shown the potentiality of induced pluripotent stem cells (iPSC) and liver organoids as the next generation tool to model in vitro liver diseases. Hepatocyte-like cells and cholangiocyte are currently being generated from skin fibroblasts and mononuclear blood cells reprogrammed into iPSC and have been successfully used for disease modeling, drug testing and gene editing, with the hope to be able to find application also in regenerative medicine. Protocols to generate other liver cell types are still under development, but the field is advancing rapidly. On the other end, liver cells can now be isolated from liver specimens (liver explants or liver biopsies) and cultured in specific conditions to form polarized 3D organoids. The purpose of this review is to summarize all these recent technological advances and their potential applications but also to analyze the current issues to be addressed before the technology can reach its full potential.
•Lack of human liver cellular models limit the advances in the treatment of liver diseases•iPSC can recapitulate in vitro the different stages of liver development.•Liver organoids are isolated from explanted liver tissue or from needle biopsy.•iPSC and organoids are used for disease modeling, drug testing and gene editing.
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•Cholangiocarcinomas are rich in stroma containing cancer-associated fibroblasts and lymphatic vessels.•PDGF-D released by tumoral ducts attracts and activates liver fibroblasts to ...secrete VEGF-C/VEGF-A.•Lymphangiogenesis and lymphatic invasion are driven by VEGF-A/-C released by liver myofibroblasts.•Targeting liver myofibroblasts in vivo inhibits tumor-associated lymphangiogenesis and lymph node metastases.•These studies identify new possible molecular targets for the treatment of cholangiocarcinoma.
In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma.
Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo.
In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRβ inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases.
PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis.
Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.
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 (Pkhd1del4/del4) mouse, which is orthologous of CHF, we show that Pkhd1del4/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 Pkhd1del4/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. (Hepatology 2016;63:965–982)
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. (Hepatology 2013;53:1042–1053)
In modern hepatology, diseases of the biliary epithelium, currently termed cholangiopathies, represent one of the main gaps in knowledge, both on experimental and clinical grounds, though they ...started to draw attention since the late 80s ....
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