Ruolo della melatonina e del pathway su cui essa agisce nella regolazione della crescita dell'epitelio biliare sia in condizioni sperimentali che nelle colangiopatie umane e nel colangiocarcinoma

Cholangiocytes are ephitilial cells which line the biliary tree and that in normal condition are mitotically quiescient. Under experimental conditions, such as bile duct ligation (BDL) or in some human cholangiopathies (eg, primary biliary cirrhosis-PBC) cholangiocytes undergo a marked proliferation. A number of factors including Vascular endothelial growth factor (VEGF) and Follicle-stimulating hormone (FSH) regulate biliary tree growth. Melatonin is secreted by the pineal gland and other tissues where it acts as a local regulator of growth, as in the gastrointestinal tract. Melatonin exerts its effects through two membrane receptors (MT1 and MT2) and is synthesized from tryptophan by four enzymes: tryptophan-hydroxylase (TPH), aromatic-amino acid decarboxylase (AADC), serotonin N-acetyltransferase (AANAT) and acetilserotonin- O-methyltransferase (ASMT). Melatonin acts by modulating the expression of Clock genes in the pineal gland. In the liver melatonin reduces oxidative damage. Little information exists about the effects of melatonin on biliary epithelium, therefore the aim of this study was to evaluate the role of melatonin in the regulation of proliferation of biliary epithelium. To this end, we used normal and BDL rats treated or not with: i) melatonin, ii) Arilalkilamina-N-acetyltransferase (AANAT) mistatch and iii) AANAT Vivo Morpholino for 7days and iiii) nude mice injected with the cholangiocarcinoma cell line (Mz-Cha-1) treated or not with melatonin. On liver samples of the experimental models we evaluated: i) the expression of melatonin, the melatonin receptors (MT1 and MT2) and Clock genes, cholangiocytes proliferation and apoptosis, ii) basal and secretin Cyclic adenosine monophosphate (cAMP) levels and Protein Chinase A (PKA) phosphorylation. In addition, we evaluated the expression of melatonin, its receptors and AANAT also in fragments of normal human liver, cholangiocarcioma and PBC. In vitro, large mouse cholangiocytes (LMC), normal human cholangiocytes (H69) and cholangiocarcinoma cell line (Mz-Cha-1) were treated with melatonin 10-11 M for 48h, plus or minus Luzindole (MT1 antagonist) or cis-4-Phenyl-2-propionamidotetralin(4P-PDOT) (MT1 and MT2 antagonist); Finally, we evaluated the effects of AANAT overexpression in LMC and in Mz-Cha-1 on proliferation, apoptosis and cAMP pathway. The results obtained showed that: cholangiocytes expess AANAT, melatonin and its receptors MT1 and MT2 and Clock genes. Chronic administration of melatonin in vivo decreased cholangiocytes proliferation after BDL, and the down-regulation of AANAT in Normal and BDL AANAT Morpholino treated rats increased proliferation. In vitro, melatonin decreased LMC proliferation, decreased which is blocked by Luzindole but not by (4P-PDOT). Overexpression of AANAT in LMC decreased cholangiocytes proliferation, and the intracellular cAMP levels. The expression of AANAT and melatonin decreased in cholangiocarcinoma, instead increased is the expression of MT1 and MT2. In nude mice injected with CCA cell lines the tumor decreased after treatment with melatonin. Overexpression of AANAT in Mz-Cha-1 decreased proliferation and increased apoptosis. In PBC the expression of AANAT, melatonin and its receptors increased if compared to normal. Based on the results obtained, we can say that melatonin plays an important role in the regulation of cholangiocytes proliferation during cholestatic liver diseases and cholangiocarcinoma

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

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

Peribiliary glands as a niche of extra-pancreatic 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, towards 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) towards 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 hepato-pancreatic 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 up-regulation of MafA and Gli1 gene expression. In patients with type 2 diabetes, PBGs at the level of the hepato-pancreatic ampulla contained cells showing signs of proliferation and pancreatic fate commitment. In vitro, high glucose concentrations induced the differentiation of human BTSCs cultures towards 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 patho-physiology and complications of this disease. This article is protected by copyright. All rights reserved

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 Axin

Adult human biliary tree stem cells differentiate to β-pancreatic islet cells by treatment with a recombinant human Pdx1 peptide

Generation of β-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 β-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 β-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 β-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 β-pancreatic cells

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

Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine/paracrine mechanisms (Alpini et al., 1994). Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic rats by interaction with melatonin recepror 1 (MT1) (Renzi et al., 2011). We will try to localize the key enzyme involved in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT), in cholangiocytes and, possibly, we will try to determine the effect of modulation of AANAT on the autocrine proliferative/secretory responses of cholangiocytes. In liver sections we found that: (i) AANAT is expressed by cholangiocytes and hepatocytes; (ii) the cholangiocytes expression of AANAT decreased in morpholino (AANAT down regulator)-treated rats; and (iii) the decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes is associated with increased biliary proliferation and increased expression of Secretin Receptor (SR) and VEGFA/ C. In vitro, we observed that overexpression of AANAT in large cholangiocyte (LC) decreased proliferation and ablated secretin-stimulated biliary secretion. These results indicate that: in vivo down-regulation of biliary AANAT stimulates cholangiocyte proliferation by an autocrine loop, and in vitro overexpression of AANAT in LC decreases proliferation. Local targeting of AANAT in cholangiocytes may be important for the management of cholangiopathies

Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts

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

Abstract 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

Lactoferrin regulate biliary epithelium growth and the activation of hepatic progenitor cell niche

Lactoferin (Lf) is an iron-binding glycoprotein belonging to the transferrin family and it is present at high levels in breast milk and colostrum. This protein has many known functions and it is a potential antibacterial, antiviral, immunostimulatory, antioxidant, and cancer preventive agent.It has been seen that a 105 kDa Lf receptor (LfR) specifically mediates the effects of Lf in several different cell types (1). In human cholangiopathies, cholangiocytes are able to proliferate and replace the cell loss restoring the integrity of damaged biliary epithelium. However, when cholangiocyte proliferation is severely impaired, the activation of facultative hepatic progenitor cells (HPCs) takes place (2). The aims of our study have been i) to investigate the expression of Lf and LfR in cholangiocytes and in HPCs both in rats and in humans; and ii) to evaluate the in vitro effects of bLf on cholangiocyte proliferation and on HPC activation. Liver specimens have been obtained from normal (N=5) and bile duct ligated (BDL) (N=5) rats; from normal patients (N=5) and from patients with primary biliary cirrhosis (PBC, N=5); Specimens were processed for histology, immunohistochemistry and immunofluorescence. Moreover for the in vitro study small and large cholangioytes from mouse, human non malignant cholangiocytes (H69), and HPCs treated or not with lactoferrin were used. Our results showed that: i) cholangiocytes and hepatic progenitor cells express lactoferrin and its receptor, ii) cholangiocytes and HPC proliferation is enhanced by lactoferrin; iii) the treatment with lactoferrin determine the commitment of HPCs towards cholangiocyte fate; this commitment is characterized by HPC morphological and phenotypical changes. Our current findings suggest that modulation of lactoferrin may be an important therapeutic tool for managing the proliferation of cholangiocyte and the activation of progenitor cell compartment in biliary disorders