Detection of native, activated Notch receptors in normal human apocrine-bearing skin and in hidradenitis suppurativa.

Notch signalling has generated considerable interest as a pathogenetic factor and a drug target in a range of human diseases. The gamma-secretase complex is crucial in the activation of Notch receptors by cleaving the intracellular domain allowing nuclear translocation. In recent years several mutations in gamma-secretase components have been discovered in patients with familial hidradenitis suppurativa (HS). This has led to hypotheses that impaired Notch signalling could be an important driver for HS in general, not only in the monogenic variants. However, no study has examined in situ Notch activation per se in HS, and some reports with conflicting results have instead been based on expression of Notch receptors or indirect measures of Notch target gene expression. In this study we established immunostaining protocols to identify native, activated Notch receptors in human skin tissue. The ability to detect changes in Notch activation was confirmed with an ex vivo skin organ model in which signal was reduced or obliterated in tissue exposed to a gamma-secretase inhibitor. Using these methods on skin biopsies from healthy volunteers and a general HS cohort we demonstrated for the first time the distribution of active Notch signalling in human apocrine-bearing skin. Quantification of activated NOTCH1 & NOTCH2 revealed similar levels in non-lesional and peri-lesional HS to that of healthy controls, thus ruling out a general defect in Notch activation in HS patients. We did find a variable but significant reduction of activated Notch in epidermis of lesional HS with a distribution that appeared related to the extent of surrounding tissue inflammation.

Expression and role of cocaine-amphetamine regulated transcript (CART) in the proliferation of biliary epithelium.

Cholangiocytes, the epithelial cells that line the biliary tree, can proliferate under the stimulation of several factors through both autocrine and paracrine pathways. The cocaine-amphetamine-regulated-transcript (CART) peptide has several physiological functions, and it is widely expressed in several organs. CART increases the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor (BDNF), whose expression has been correlated to the proliferation rate of cholangiocytes. In the present study, we aimed to evaluate the expression of CART and its role in modulating cholangiocyte proliferation in healthy and bile duct ligated (BDL) rats in vivo, as well as in cultured normal rat cholangiocytes (NRC) in vitro. Liver samples from both healthy and BDL (1 week) rats, were analyzed by immunohistochemistry and immunofluorescence for CART, CK19, TrkB and p75NTR BDNF receptors. PCNA staining was used to evaluate the proliferation of the cholangiocytes, whereas TUNEL assay was used to evaluate biliary apoptosis. NRC treated or not with CART were used to confirm the role of CART on cholangiocytes proliferation and the secretion of BDNF. Cholangiocytes proliferation, apoptosis, CART and TrkB expression were increased in BDL rats, compared to control rats. We found a higher expression of TrkB and p75NTR, which could be correlated with the proliferation rate of biliary tree during BDL. The in vitro study demonstrated increased BDNF secretion by NRC after treatment with CART compared with control cells. As previously reported, proliferating cholangiocytes acquire a neuroendocrine phenotype, modulated by several factors, including neurotrophins. Accordingly, CART may play a key role in the remodeling of biliary epithelium during cholestasis by modulating the secretion of BDNF.

Inhibition of Endothelial NOTCH1 Signaling Attenuates Inflammation by Reducing Cytokine-Mediated Histone Acetylation at Inflammatory Enhancers.

OBJECTIVE: Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. APPROACH AND RESULTS: Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1ß stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers. CONCLUSIONS: Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.

Skeletal muscle myopenia in mice model of bile duct ligation and carbon tetrachloride-induced liver cirrhosis.

Skeletal muscle myopathy is universal in cirrhotic patients, however, little is known about the main mechanisms involved. The study aims to investigate skeletal muscle morphological, histological, and functional modifications in experimental models of cirrhosis and the principal molecular pathways responsible for skeletal muscle myopathy. Cirrhosis was induced by bile duct ligation (BDL) and carbon tetrachloride (CCl4) administration in mice. Control animals (CTR) underwent bile duct exposure or vehicle administration only. At sacrifice, peripheral muscles were dissected and weighed. Contractile properties of extensor digitorum longus (EDL) were studied in vitro. Muscle samples were used for histological and molecular analysis. Quadriceps muscle histology revealed a significant reduction in cross-sectional area of muscle and muscle fibers in cirrhotic mice with respect to CTR. Kinetic properties of EDL in both BDL and CCl4 were reduced with respect to CTR; BDL mice also showed a reduction in muscle force and a decrease in the resistance to fatigue. Increase in myostatin expression associated with a decrease in AKT-mTOR expressions was observed in BDL mice, together with an increase in LC3 protein levels. Upregulation of the proinflammatory citochines TNF-a and IL6 and an increased expression of NF-kB and MuRF-1 were observed in CCl4 mice. In conclusion, skeletal muscle myopenia was present in experimental models of BDL and CCl4-induced cirrhosis. Moreover, reduction in protein synthesis and activation of protein degradation were the main mechanisms responsible for myopenia in BDL mice, while activation of ubiquitin-pathway through inflammatory cytokines seems to be the main potential mechanism involved in CCl4 mice.

Macrophage Activation in Pediatric Nonalcoholic Fatty Liver Disease (NAFLD) Correlates with Hepatic Progenitor Cell Response via Wnt3a Pathway.

Non-alcoholic fatty liver disease is one of the most important causes of liver-related morbidity in children. In non-alcoholic fatty liver disease, the activation of liver resident macrophage pool is a central event in the progression of liver injury. The aims of the present study were to evaluate the polarization of liver macrophages and the possible role of Wnt3a production by macrophages in hepatic progenitor cell response in the progression of pediatric non-alcoholic fatty liver disease. 32 children with biopsy-proven non-alcoholic fatty liver disease were included. 20 out of 32 patients were treated with docosahexaenoic acid for 18 months and biopsies at the baseline and after 18 months were included. Hepatic progenitor cell activation, macrophage subsets and Wnt/ß-catenin pathway were evaluated by immunohistochemistry and immunofluorescence. Our results indicated that in pediatric non-alcoholic fatty liver disease, pro-inflammatory macrophages were the predominant subset. Macrophage polarization was correlated with Non-alcoholic fatty liver disease Activity Score, ductular reaction, and portal fibrosis; docosahexaenoic acid treatment determined a macrophage polarization towards an anti-inflammatory phenotype in correlation with the reduction of serum inflammatory cytokines, with increased macrophage apoptosis, and with the up-regulation of macrophage Wnt3a expression; macrophage Wnt3a expression was correlated with ß-catenin phosphorylation in hepatic progenitor cells and signs of commitment towards hepatocyte fate. In conclusion, macrophage polarization seems to have a key role in the progression of pediatric non-alcoholic fatty liver disease; the modulation of macrophage polarization could drive hepatic progenitor cell response by Wnt3a production.

Peribiliary Glands as a Niche of Extrapancreatic Precursors Yielding Insulin-Producing Cells in Experimental and Human Diabetes.

Peribiliary glands (PBGs) are niches in the biliary tree and containing heterogeneous endodermal stem/progenitors cells that can differentiate, in vitro and in vivo, toward pancreatic islets. The aim of this study was to evaluate, in experimental and human diabetes, proliferation of cells in PBGs and differentiation of the biliary tree stem/progenitor cells (BTSCs) toward insulin-producing cells. Diabetes was generated in mice by intraperitoneal injection of a single dose of 200 mg/kg (N = 12) or 120 mg/kg (N = 12) of streptozotocin. Liver, pancreas, and extrahepatic biliary trees were en bloc dissected and examined. Cells in PBGs proliferated in experimental diabetes, and their proliferation was greatest in the PBGs of the hepatopancreatic ampulla, and inversely correlated with the pancreatic islet area. In rodents, the cell proliferation in PBGs was characterized by the expansion of Sox9-positive stem/progenitor cells that gave rise to insulin-producing cells. Insulin-producing cells were located mostly in PBGs in the portion of the biliary tree closest to the duodenum, and their appearance was associated with upregulation of MafA and Gli1 gene expression. In patients with type 2 diabetes, PBGs at the level of the hepatopancreatic ampulla contained cells showing signs of proliferation and pancreatic fate commitment. In vitro, high glucose concentrations induced the differentiation of human BTSCs cultures toward pancreatic beta cell fates. The cells in PBGs respond to diabetes with proliferation and differentiation towards insulin-producing cells indicating that PBG niches may rescue pancreatic islet impairment in diabetes. These findings offer important implications for the pathophysiology and complications of this disease. Stem Cells 2016;34:1332-1342.

Stem/Progenitor Cell Niches Involved in Hepatic and Biliary Regeneration.

Niches containing stem/progenitor cells are present in different anatomical locations along the human biliary tree and within liver acini. The most primitive stem/progenitors, biliary tree stem/progenitor cells (BTSCs), reside within peribiliary glands located throughout large extrahepatic and intrahepatic bile ducts. BTSCs are multipotent and can differentiate towards hepatic and pancreatic cell fates. These niches' matrix chemistry and other characteristics are undefined. Canals of Hering (bile ductules) are found periportally and contain hepatic stem/progenitor cells (HpSCs), participating in the renewal of small intrahepatic bile ducts and being precursors to hepatocytes and cholangiocytes. The niches also contain precursors to hepatic stellate cells and endothelia, macrophages, and have a matrix chemistry rich in hyaluronans, minimally sulfated proteoglycans, fetal collagens, and laminin. The microenvironment furnishes key signals driving HpSC activation and differentiation. Newly discovered third niches are pericentral within hepatic acini, contain Axin2+ unipotent hepatocytic progenitors linked on their lateral borders to endothelia forming the central vein, and contribute to normal turnover of mature hepatocytes. Their relationship to the other stem/progenitors is undefined. Stem/progenitor niches have important implications in regenerative medicine for the liver and biliary tree and in pathogenic processes leading to diseases of these tissues.

Progenitor cell niches in the human pancreatic duct system and associated pancreatic duct glands: an anatomical and immunophenotyping study.

Pancreatic duct glands (PDGs) are tubule-alveolar glands associated with the pancreatic duct system and can be considered the anatomical counterpart of peribiliary glands (PBGs) found within the biliary tree. Recently, we demonstrated that endodermal precursor niches exist fetally and postnatally and are composed functionally of stem cells and progenitors within PBGs and of committed progenitors within PDGs. Here we have characterized more extensively the anatomy of human PDGs as novel niches containing cells with multiple phenotypes of committed progenitors. Human pancreata (n = 15) were obtained from cadaveric adult donors. Specimens were processed for histology, immunohistochemistry and immunofluorescence. PDGs were found in the walls of larger pancreatic ducts (diameters > 300 µm) and constituted nearly 4% of the duct wall area. All of the cells identified were negative for nuclear expression of Oct4, a pluripotency gene, and so are presumably committed progenitors and not stem cells. In the main pancreatic duct and in large interlobular ducts, Sox9(+) cells represented 5-30% of the cells within PDGs and were located primarily at the bottom of PDGs, whereas rare and scattered Sox9(+) cells were present within the surface epithelium. The expression of PCNA, a marker of cell proliferation, paralleled the distribution of Sox9 expression. Sox9(+) PDG cells proved to be Pdx1(+) /Ngn3(+/-) /Oct4A(-) . Nearly 10% of PDG cells were positive for insulin or glucagon. Intercalated ducts contained Sox9(+) /Pdx1(+) /Ngn3(+) cells, a phenotype that is presumptive of committed endocrine progenitors. Some intercalated ducts appeared in continuity with clusters of insulin-positive cells organized in small pancreatic islet-like structures. In summary, PDGs represent niches of a population of Sox9(+) cells exhibiting a pattern of phenotypic traits implicating a radial axis of maturation from the bottoms of the PDGs to the surface of pancreatic ducts. Our results complete the anatomical background that links biliary and pancreatic tracts and could have important implications for the common patho-physiology of biliary tract and pancreas.

Sensitivity of Human Intrahepatic Cholangiocarcinoma Subtypes to Chemotherapeutics and Molecular Targeted Agents: A Study on Primary Cell Cultures.

We investigated the sensitivity of intrahepatic cholangiocarcinoma (IHCCA) subtypes to chemotherapeutics and molecular targeted agents. Primary cultures of mucin- and mixed-IHCCA were prepared from surgical specimens (N. 18 IHCCA patients) and evaluated for cell proliferation (MTS assay) and apoptosis (Caspase 3) after incubation (72 hours) with increasing concentrations of different drugs. In vivo, subcutaneous human tumor xenografts were evaluated. Primary cultures of mucin- and mixed-IHCCA were characterized by a different pattern of expression of cancer stem cell markers, and by a different drug sensitivity. Gemcitabine and the Gemcitabine-Cisplatin combination were more active in inhibiting cell proliferation in mixed-IHCCA while Cisplatin or Abraxane were more effective against mucin-IHCCA, where Abraxane also enhances apoptosis. 5-Fluoracil showed a slight inhibitory effect on cell proliferation that was more significant in mixed- than mucin-IHCCA primary cultures and, induced apoptosis only in mucin-IHCCA. Among Hg inhibitors, LY2940680 and Vismodegib showed slight effects on proliferation of both IHCCA subtypes. The tyrosine kinase inhibitors, Imatinib Mesylate and Sorafenib showed significant inhibitory effects on proliferation of both mucin- and mixed-IHCCA. The MEK 1/2 inhibitor, Selumetinib, inhibited proliferation of only mucin-IHCCA while the aminopeptidase-N inhibitor, Bestatin was more active against mixed-IHCCA. The c-erbB2 blocking antibody was more active against mixed-IHCCA while, the Wnt inhibitor, LGK974, similarly inhibited proliferation of mucin- and mixed-IHCCA. Either mucin- or mixed-IHCCA showed high sensitivity to nanomolar concentrations of the dual PI3-kinase/mTOR inhibitor, NVP-BEZ235. In vivo, in subcutaneous xenografts, either NVP-BEZ235 or Abraxane, blocked tumor growth. In conclusion, mucin- and mixed-IHCCA are characterized by a different drug sensitivity. Cisplatin, Abraxane and the MEK 1/2 inhibitor, Selumetinib were more active against mucin-IHCCA while, Gemcitabine, Gemcitabine-Cisplatin combination, the c-erbB2 blocking antibody and bestatin worked better against mixed-IHCCA. Remarkably, we identified a dual PI3-kinase/mTOR inhibitor that both in vitro and in vivo, exerts dramatic antiproliferative effects against both mucin- and mixed-IHCCA.

Adult Human Biliary Tree Stem Cells Differentiate to ß-Pancreatic Islet Cells by Treatment with a Recombinant Human Pdx1 Peptide.

Generation of ß-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards ß-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the ß-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage ß-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional ß-pancreatic cells.

Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is characterised by fibro-stenosing strictures involving extrahepatic and/or large intrahepatic bile ducts. Mechanisms leading to bile duct injury are poorly understood. We aimed to study the biliary tree stem cell compartment located in peribiliary glands of extrahepatic and large intrahepatic bile ducts and its role in the pathogenesis of biliary fibrosis in PSC. METHODS: Specimens containing extrahepatic or large intrahepatic bile ducts were obtained from normal liver (n=6), liver explants from patients with PSC (n=11), and primary biliary cirrhosis (n=6). Specimens were processed for histology, immunohistochemistry and immunofluorescence. RESULTS: In PSC samples, progressive hyperplasia and mucinous metaplasia of peribiliary glands were observed in large ducts with fibrosis, but not in inflamed ducts without fibrosis. Peribiliary gland hyperplasia was associated with progressive biliary fibrosis and the occurrence of dysplastic lesions. Hyperplasia of peribiliary glands was determined by the expansion of biliary tree stem cells, which sprouted towards the surface epithelium. In PSC, peribiliary glands and myofibroblasts displayed enhanced expression of Hedgehog pathway components. Peribiliary glands in ducts with onion skin-like fibrosis expressed epithelial-to-mesenchymal transition traits associated with components of Hedgehog pathway, markers of senescence and autophagy. CONCLUSIONS: The biliary tree stem cell compartment is activated in PSC, its activation contributes to biliary fibrosis, and is sustained by the Hedgehog pathway. Our findings suggest a key role for peribiliary glands in the progression of bile duct lesions in PSC and could explain the associated high risk of cholangiocarcinoma.

Profiles of cancer stem cell subpopulations in cholangiocarcinomas.

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13(+) CSCs characterized mixed-intrahepatic, whereas LGR5(+) characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13(+) or CD90(+) CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90(+) and CD13(+) cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell-based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.

Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis.

Biliary hyperplasia and liver fibrosis are common features in cholestatic liver disease. Melatonin is synthesized by the pineal gland as well as the liver. Melatonin inhibits biliary hyperplasia of bile duct-ligated (BDL) rats. Since melatonin synthesis (by the enzyme serotonin N-acetyltransferase, AANAT) from the pineal gland increases after dark exposure, we hypothesized that biliary hyperplasia and liver fibrosis are diminished by continuous darkness via increased melatonin synthesis from the pineal gland. Normal or BDL rats (immediately after surgery) were housed with light-dark cycles or complete dark for 1 wk before evaluation of 1) the expression of AANAT in the pineal gland and melatonin levels in pineal gland tissue supernatants and serum; 2) biliary proliferation and intrahepatic bile duct mass, liver histology, and serum chemistry; 3) secretin-stimulated ductal secretion (functional index of biliary growth); 4) collagen deposition, liver fibrosis markers in liver sections, total liver, and cholangiocytes; and 5) expression of clock genes in cholangiocytes. In BDL rats exposed to dark there was 1) enhanced AANAT expression/melatonin secretion in pineal gland and melatonin serum levels; 2) improved liver morphology, serum chemistry and decreased biliary proliferation and secretin-stimulated choleresis; and 4) decreased fibrosis and expression of fibrosis markers in liver sections, total liver and cholangiocytes and reduced biliary expression of the clock genes PER1, BMAL1, CLOCK, and Cry1. Thus prolonged dark exposure may be a beneficial noninvasive therapeutic approach for the management of biliary disorders.

Inhibition of the liver expression of arylalkylamine N-acetyltransferase increases the expression of angiogenic factors in cholangiocytes.

BACKGROUND AND AIMS: Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl(-)/HCO3 (-) AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by cholangiocytes. METHODS: In this study, we evaluated the effect of local modulation of biliary AANAT expression on the cholangiocytes synthesis of VEGF-A/C. RESULTS: The decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in cholangiocyte lines decreased the expression of VEGF-A/C. CONCLUSIONS: Modulation of melatonin synthesis may affect the expression of VEGF-A/C by cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions.

Expression of brain derivated neurotrophic factor and of its receptors: TrKB and p75NT in normal and bile duct ligated rat liver.

Cholangiocytes are the cells lining the biliary tree from canals of Hering to larger bile ducts. By morphology, they are divided in small and large cholangiocytes, which result heterogeneous at functional and proliferative levels. Proliferating cholangiocytes acquire a neuroendocrine phe- notype, modulated by several factors including neurotrophins. Brain Derivated Neurotrophic Factor (BDNF) is a neurotrophin expressed in the nervous system and also in different types of epithelial and progenitor cells. The aim of the present study is to detect the expression of BDNF and of its two receptors (TrKB and p75NT, or p75NTR) in normal and bile duct ligated (BDL) rat livers. In normal and BDL livers, BDNF and its receptors are expressed by small and large cholan- giocytes and by hepatic progenitors cells. In cholangiocytes, the expression of BDNF and of its receptors changes after different BDL timing. After one or two weeks of BDL, both BDNF and TrKB and p75NT receptors are highly expressed, while after BDL for three weeks BDNF expression is drastically reduced and p75NT receptor prevails on TrKB. The expression of BDNF and of its receptors correlates with the proliferation rate of biliary tree during BDL. Indeed, after one or two weeks of BDL, proliferation prevails on apoptosis, whereas after BDL for three weeks, apoptosis prevails on proliferation. Our morphological results strongly suggest that BDNF plays a role in the remodeling of biliary tree during cholestasis and that it may be involved in the pathophysiology of cholestasic liver diseases.

Role of hepatic progenitor cells in nonalcoholic fatty liver disease development: cellular cross-talks and molecular networks.

Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of diseases ranging from simple fatty liver to nonalcoholic steatohepatitis, (NASH) which may progress to cirrhosis and hepatocellular carcinoma. NASH has been independently correlated with atherosclerosis progression and cardiovascular risk. NASH development is characterized by intricate interactions between resident and recruited cells that enable liver damage progression. The increasing general agreement is that the cross-talk between hepatocytes, hepatic stellate cells (HSCs) and macrophages in NAFLD has a main role in the derangement of lipid homeostasis, insulin resistance, danger recognition, immune tolerance response and fibrogenesis. Moreover, several evidences have suggested that hepatic stem/progenitor cell (HPCs) activation is a component of the adaptive response of the liver to oxidative stress in NAFLD. HPC activation determines the appearance of a ductular reaction. In NASH, ductular reaction is independently correlated with progressive portal fibrosis raising the possibility of a periportal fibrogenetic pathway for fibrogenesis that is parallel to the deposition of subsinusoidal collagen in zone 3 by HSCs. Recent evidences indicated that adipokines, a class of circulating factors, have a key role in the cross-talk among HSCs, HPCs and liver macrophages. This review will be focused on cellular cross-talk and the relative molecular networks which are at the base of NASH progression and fibrosis.

Role of follicle-stimulating hormone on biliary cyst growth in autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the progressive development of renal and hepatic cysts. Follicle-stimulating hormone (FSH) has been demonstrated to be a trophic factor for biliary cells in normal rats and experimental cholestasis induced by bile duct ligation (BDL). AIMS: To assess the effect of FSH on cholangiocyte proliferation during ADPKD using both in vivo and in vitro models. METHODS: Evaluation of FSH receptor (FSHR), FSH, phospho-extracellular-regulated kinase (pERK) and c-myc expression in liver fragments from normal patients and patients with ADPKD. In vitro, we studied proliferating cell nuclear antigen (PCNA) and cAMP levels in a human immortalized, non-malignant cholangiocyte cell line (H69) and in an immortalized cell line obtained from the epithelium lining the hepatic cysts from the patients with ADPKD (LCDE) with or without transient silencing of the FSH gene. RESULTS: Follicle-stimulating hormone is linked to the active proliferation of the cystic wall and to the localization of p-ERK and c-myc. This hormone sustains the biliary growth by activation of the cAMP/ERK signalling pathway. CONCLUSION: These results showed that FSH has an important function in cystic growth acting on the cAMP pathway, demonstrating that it provides a target for medical therapy of hepatic cysts during ADPKD.

Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes in rats.

UNLABELLED: Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3',5'-monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl(-) /HCO 3- AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic bile-duct-ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down-regulation of cAMP-dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N-acetyltransferase (AANAT; the rate-limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl(-) /HCO 3- AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl(-) /HCO 3- AE2 and ablated secretin-stimulated biliary secretion in these cells. CONCLUSION: Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013).

Expression of vascular endothelial growth factors and their receptors by hepatic progenitor cells in human liver diseases.

Hepatic stem/progenitor cells (HPCs) are stem cells residing in the most peripheral branches of the biliary tree; these cells are able to differentiate towards mature hepatocyte or mature cholangiocyte; moreover in normal conditions, they are mostly quiescent cells. HPC activation has been involved in the progression of chronic parenchymal diseases (chronic viral hepatitis) and chronic biliary diseases (such as Primary Biliary Cirrhosis: PBC) and in the occurrence of intrahepatic cholangiocarcinoma. The HPCs participate in the repair of liver damage either through the replacement of dead cells or by driving fundamental repair processes, including fibrosis and angiogenesis. Little information exists regarding the expression of VEGF by HPC in the course of liver non-malignant pathologies. In this study, we evaluated: (I) the presence of HPCs in PBC and HCV-related Cirrhosis (HCV-C) samples, and (II) the expression of VEGFs and VEGF-Rs in PBC and HCV-C samples. Our results showed (I) PBC samples presented a more extensive expansion of HPC population in comparison with those of HCV-C samples; (II) PBC samples showed a more extensive angiogenesis if compared to HCV-C; and (III) PBC samples were characterized by an increased expression of VEGF-A and VEGF-C if compared to HCV-C and the number of HPCs expressing VEGFs was correlated with the extension of ductular reaction and angiogenesis. The role of VEGFs in the expansion of HPC niche could have important implication in the management of fibrogenic processes and carcinogenesis.

Recent advances on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology.

Cholangiocytes are epithelial cells lining the biliary epithelium. Cholangiocytes play several key roles in the modification of ductal bile and are also the target cells in chronic cholestatic liver diseases (i.e., cholangiopathies) such as PSC, PBC, polycystic liver disease (PCLD) and cholangiocarcinoma (CCA). During these pathologies, cholangiocytes (which in normal condition are in a quiescent state) begin to proliferate acquiring phenotypes of neuroendocrine cells, and start secreting different cytokines, growth factors, neuropeptides, and hormones to modulate cholangiocytes proliferation and interaction with the surrounding environment, trying to reestablish the balance between proliferation/loss of cholangiocytes for the maintenance of biliary homeostasis. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology. To clarify the mechanisms of action of these factors we will provide new potential strategies for the management of chronic liver diseases.