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Unlabelled: Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato-pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum-free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2-3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger-2-AE2 and CFTR), or regulatable C-peptide secreting neoislet-like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin-induced diabetic mice, results in functional islets secreting glucose-regulatable human C-peptide., Conclusion: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes., (Copyright © 2011 American Association for the Study of Liver Diseases.)

In bile duct-ligated (BDL) rats, large cholangiocytes proliferate by activation of cAMP-dependent signaling. Melatonin, which is secreted from pineal gland as well as extrapineal tissues, regulates cell mitosis by interacting with melatonin receptors (MT1 and MT2) modulating cAMP and clock genes. In the liver, melatonin suppresses oxidative damage and ameliorates fibrosis. No information exists regarding the role of melatonin in the regulation of biliary hyperplasia. We evaluated the mechanisms of action by which melatonin regulates the growth of cholangiocytes. In normal and BDL rats, we determined the hepatic distribution of MT1, MT2, and the clock genes, CLOCK, BMAL1, CRY1, and PER1. Normal and BDL (immediately after BDL) rats were treated in vivo with melatonin before evaluating 1) serum levels of melatonin, bilirubin, and transaminases; 2) intrahepatic bile duct mass (IBDM) in liver sections; and 3) the expression of MT1 and MT2, clock genes, and PKA phosphorylation. In vitro, large cholangiocytes were stimulated with melatonin in the absence/presence of luzindole (MT1/MT2 antagonist) and 4-phenyl-2-propionamidotetralin (MT2 antagonist) before evaluating cell proliferation, cAMP levels, and PKA phosphorylation. Cholangiocytes express MT1 and MT2, CLOCK, BMAL1, CRY1, and PER1 that were all upregulated following BDL. Administration of melatonin to BDL rats decreased IBDM, serum bilirubin and transaminases levels, the expression of all clock genes, cAMP levels, and PKA phosphorylation in cholangiocytes. In vitro, melatonin decreased the proliferation, cAMP levels, and PKA phosphorylation, decreases that were blocked by luzindole. Melatonin may be important in the management of biliary hyperplasia in human cholangiopathies.

Aim: The aim of this study was to determine whether GSH-C4, a hydrophobic glutathione derivative, affects in vitro and in vivo influenza virus infection by interfering with redox-sensitive intracellular pathways involved in the maturation of viral hemagglutinin (HA)., Results: GSH-C4 strongly inhibited influenza A virus replication in cultured cells and in lethally infected mice, where it also reduced lung damage and mortality. In cell-culture studies, GSH-C4 arrested viral HA folding; the disulfide-rich glycoprotein remained in the endoplasmic reticulum as a reduced monomer instead of undergoing oligomerization and cell plasma-membrane insertion. HA maturation depends on the host-cell oxidoreductase, protein disulfide isomerase (PDI), whose activity in infected cells is probably facilitated by virus-induced glutathione depletion. By correcting this deficit, GSH-C4 increased levels of reduced PDI and inhibited essential disulfide bond formation in HA. Host-cell glycoprotein expression in uninfected cells was unaffected by glutathione, which thus appears to act exclusively on glutathione-depleted cells., Innovation: All currently approved anti-influenza drugs target essential viral structures, and their efficacy is limited by toxicity and by the almost inevitable selection of drug-resistant viral mutants. GSH-C4 inhibits influenza virus replication by modulating redox-sensitive pathways in infected cells, without producing toxicity in uninfected cells or animals. Novel anti-influenza drugs that target intracellular pathways essential for viral replication ("cell-based approach") offer two important potential advantages: they are more difficult for the virus to adapt to and their efficacy should not be dependent on virus type, strain, or antigenic properties., Conclusion: Redox-sensitive host-cell pathways exploited for viral replication are promising targets for effective anti-influenza strategies.

No information exists on the role of neuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, we evaluated the expression and secretion of NPY and its subsequent effects on cholangiocarcinoma growth and invasion. Cholangiocarcinoma cell lines and nonmalignant cholangiocytes were used to assess NPY mRNA expression and protein secretion. NPY expression was assessed by immunohistochemistry in human liver biopsies. Cell proliferation and migration were evaluated in vitro by MTS assays and matrigel invasion chambers, respectively, after treatment with NPY or a neutralizing NPY antibody. The effect of NPY or NPY depletion on tumor growth was assessed in vivo after treatment with NPY or the neutralizing NPY antibody in a xenograft model of cholangiocarcinoma. NPY secretion was upregulated in cholangiocarcinoma compared with normal cholangiocytes. Administration of exogenous NPY decreased proliferation and cell invasion in all cholangiocarcinoma cell lines studied and reduced tumor cell growth in vivo. In vitro, the effects of NPY on proliferation were blocked by specific inhibitors for NPY receptor Y2, but not Y1 or Y5, and were associated with an increase in intracellular d-myo-inositol 1,4,5-trisphosphate and PKCα activation. Blocking of NPY activity using a neutralizing antibody promoted cholangiocarcinoma growth in vitro and in vivo and increased the invasiveness of cholangiocarcinoma in vitro. Increased NPY immunoreactivity in human tumor tissue occurred predominantly in the center of the tumor, with less expression toward the invasion front of the tumor. We demonstrated that NPY expression is upregulated in cholangiocarcinoma, which exerts local control on tumor cell proliferation and invasion. Modulation of NPY secretion may be important for the management of cholangiocarcinoma.

Unlabelled: Small cholangiocytes proliferate via activation of calcium (Ca(2+) )-dependent signaling in response to pathological conditions that trigger the damage of large cyclic adenosine monophosphate-dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca(2+) -dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an α(1) -adrenergic agonist stimulating intracellular D-myo-inositol-1,4,5-triphosphate (IP(3) )/Ca(2+) levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP(3) /Ca(2+) -dependent signaling. Phenylephrine stimulated the production of intracellular IP(3) . The Ca(2+) -dependent transcription factors, nuclear factor of activated T cells 2 (NFAT2) and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca(2+) -dependent DNA-binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with small hairpin RNA. We observed an inhibition of phenylephrine-induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca(2+) -dependent activation of NFAT2 and Sp1., Conclusion: Selective stimulation of Ca(2+) -dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins., (Copyright © 2010 American Association for the Study of Liver Diseases.)

In the design of wearable robots that strictly interact with the human body and, in general, in any robotics application that involves the human component, the possibility of having modular joints able to produce a viscoelastic behaviour is very useful to achieve an efficient and safe human-robot interaction and to give rise to emergent dynamical behaviors. In this paper we propose the design of a compact, passive, rotary viscoelastic joint for assistive wearable robotics applications. The system integrates two functionally distinct sub-modules: one to render a desired torsional stiffness profile and the other to provide a desired torsional damping. Concepts and design choices regarding the overall architecture and the single components are presented and discussed. A viscoelastic model of the system has been developed and the design of the joint is presented., (© 2011 IEEE)

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy.

Background: Ischemic injury by hepatic artery ligation (HAL) during obstructive cholestasis induced by bile duct ligation (BDL) results in bile duct damage, which can be prevented by administration of VEGF-A. The potential regulation of VEGF and VEGF receptor expression and secretion by bile acids in BDL with HAL is unknown., Aims: We evaluated whether taurocholic acid (TC) can prevent HAL-induced cholangiocyte damage via the alteration of VEGFR-2 and/or VEGF-A expression., Methods: Utilizing BDL, BDL+TC, BDL+HAL, BDL+HAL+TC, and BDL+HAL+wortmannin+TC treated rats, we evaluated cholangiocyte apoptosis, proliferation, and secretion as well VEGF-A and VEGFR-2 expression by immunohistochemistry. In vitro, we evaluated the effects of TC on cholangiocyte secretion of VEGF-A and the dependence of TC-induced proliferation on the activity of VEGFR-2., Results: In BDL rats with HAL, chronic feeding of TC prevented HAL-induced loss of bile ducts and HAL-induced decreased cholangiocyte secretion. TC also prevented HAL-inhibited VEGF-A and VEGFR-2 expression in liver sections and HAL-induced circulating VEGF-A levels, which were blocked by wortmannin administration. In vitro, TC stimulated increased VEGF-A secretion by cholangiocytes, which was blocked by wortmannin and stimulated cholangiocyte proliferation that was blocked by VEGFR-2 kinase inhibitor., Conclusion: TC prevented HAL-induced biliary damage by upregulation of VEGF-A expression., (Copyright 2010 Editrice Gastroenterologica Italiana S.r.l. All rights reserved.)

Unlabelled: During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin-dependent proliferation is lacking. SR wild-type (WT) (SR(+/+)) or SR knockout (SR(-/-)) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and SR(-/-) BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin-stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from SR(-/-) BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen-activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth., Conclusion: The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases.

Secretin plays a key role in the regulation of normal cholangiocyte physiology via secretin receptor (SCTR). SCTR expression is upregulated during extrahepatic cholestasis induced by bile duct ligation and closely associated with cholangiocyte proliferative responses. Although well studied in normal cholangiocytes, the role of secretin and the expression of SCTR in the regulation of cholangiocarcinoma proliferation are unknown. In vitro, secretin (10(-7) M) displayed differential effects on normal cholangiocyte [H-69 and human intrahepatic biliary epithelial cell line (HIBEpiC)] and cholangiocarcinoma (Mz-ChA-1, HuH-28, TFK-1, SG231, CCLP1 and HuCC-T1) cell lines as such secretin is mitogenic for normal cholangiocytes and antiproliferative for cholangiocarcinoma. As expected in normal cholangiocytes (HIBEpiC), secretin increased intracellular cyclic adenosine monophosphate (cAMP) levels. However, the effect of secretin on intracellular cAMP levels was suppressed in Mz-ChA-1 cells. Secretin-stimulated intracellular cAMP levels in Mz-ChA-1 were restored by pretreatment with pertussis toxin, suggesting that the receptor coupled to Galpha(i) rather than Galpha(s). SCTR expression was found to be downregulated in 4 of the 6 cholangiocarcinoma cell lines evaluated and in human cholangiocarcinoma biopsy samples. In vivo, secretin significantly inhibited the tumor size and more than doubled tumor latency, which was associated with a decrease in proliferating cell nuclear antigen and an increase in cleaved-caspase 3 expression levels. Our results demonstrate that secretin and/or the modulation of SCTR expression might have potential as a therapeutic tool in the treatment of cholangiocarcinoma.

Over the last years, cholangiocytes, the cells that line the biliary tree, have been considered an important object of study for their biological properties which involves bile formation, proliferation, injury repair, fibrosis and angiogenesis. Cholangiocyte proliferation occurs in all pathologic conditions of liver injury where it is associated with inflammation and regeneration. During these processes, biliary cells start to secrete different cytokines, growth factors, neuropeptides and hormones which represent potential mechanisms for cross talk with other liver cells. Several studies suggest that hormones, and in particular, sex hormones, play a fundamental role in the modulation of the growth of this compartment in the injured liver which functionally conditions the progression of liver disease. Understanding the mechanisms of action and the intracellular pathways of these compounds on cholangiocyte pathophysiology will provide new potential strategies for the management of chronic liver diseases. The purpose of this review is to summarize the recent findings on the role of sex hormones in cholangiocyte proliferation and biology.

Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We have previously shown that serotonin metabolism is dysregulated in cholangiocarcinoma leading to an increased secretion of serotonin, which has growth-promoting effects. Because serotonin and dopamine share the degradation machinery, we evaluated the secretion of dopamine from cholangiocarcinoma and its effects on cell proliferation. Using 4 cholangiocarcinoma cell lines and human biopsy samples, we demonstrated that there was an increase in mRNA and protein expression of the dopamine synthesis enzymes tyrosine hydroxylase and dopa decarboxylase in cholangiocarcinoma. There was increased dopamine secretion from cholangiocarcinoma cell lines compared to H69 and HIBEC cholangiocytes and increased dopamine immunoreactivity in human biopsy samples. Furthermore, administration of dopamine to all cholangiocarcinoma cell lines studied increased proliferation by up to 30%, which could be blocked by the pretreatment of the D2 and D4 dopamine receptor antagonists, whereas blocking dopamine production by alpha-methyldopa administration suppressed growth by up to 25%. Administration of alpha-methyldopa to nude mice also suppressed cholangiocarcinoma tumor growth. The data presented here represent the first evidence that dopamine metabolism is dysregulated in cholangiocarcinoma and that modulation of dopamine synthesis may represent an alternative target for the development of therapeutic strategies.

Large cholangiocytes secrete bicarbonate in response to secretin and proliferate after bile duct ligation by activation of cyclic adenosine 3', 5'-monophosphate signaling. The Ca(2+)-dependent adenylyl cyclase 8 (AC8, expressed by large cholangiocytes) regulates secretin-induced choleresis. Ca(2+)-dependent protein kinase C (PKC) regulates small cholangiocyte function. Because gamma-aminobutyric acid (GABA) affects cell functions by activation of both Ca(2+) signaling and inhibition of AC, we sought to develop an in vivo model characterized by large cholangiocyte damage and proliferation of small ducts. Bile duct ligation rats were treated with GABA for one week, and we evaluated: GABA(A), GABA(B), and GABA(C) receptor expression; intrahepatic bile duct mass (IBDM) and the percentage of apoptotic cholangiocytes; secretin-stimulated choleresis; and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation and activation of Ca(2+-)dependent PKC isoforms and AC8 expression. We found that both small and large cholangiocytes expressed GABA receptors. GABA: (i) induced apoptosis of large cholangiocytes and reduced large IBDM; (ii) decreased secretin-stimulated choleresis; and (iii) reduced ERK1/2 phosphorylation and AC8 expression in large cholangiocytes. Small cholangiocytes: (i) proliferated leading to increased IBDM; (ii) displayed activation of PKCbetaII; and (iii) de novo expressed secretin receptor, cystic fibrosis transmembrane regulator, Cl(-)/HCO(3)(-) anion exchanger 2 and AC8, and responded to secretin. Therefore, in pathologies of large ducts, small ducts replenish the biliary epithelium by amplification of Ca(2+)-dependent signaling and acquisition of large cholangiocyte phenotypes.

Background: Hepatic progenitor cells (HPCs) hold a great potential for therapeutic intervention for currently untreatable liver diseases. However, in human diseases molecular mechanisms involved in proliferation and differentiation of HPCs are poorly understood., Methods and Results: In the present study activated HPCs and their microenvironment (niche) were investigated in acute and chronic human liver disease by gene-expression analysis and immunohistochemistry/immunofluorescence. Cryopreserved liver tissues were used from patients with parenchymal versus biliary diseases: acute necrotising hepatitis (AH), cirrhosis after hepatitis C infection, and primary biliary cirrhosis in order to study differentiation of HPCs towards hepatocytic versus biliary lineage. Keratin 7 positive HPCs/reactive ductules were captured by means of laser capture microdissection and gene-expression profiles were obtained by using a customized PCR array. Gene expression results were confirmed by immunohistochemistry and immunofluorescence double staining. In all disease groups, microdissected HPCs expressed progenitor cell markers such as KRT7, KRT19, NCAM, ABCG2, LIF, KIT, OCT4, CD44 and TERT. In AH, HPCs were most activated and showed a high expression of prominin-1 (CD133) and alpha-fetoprotein, and a strong activation of the Wnt pathway. In contrast to parenchymal diseases, HPCs in primary biliary cirrhosis (biliary differentiation) showed a high activation of Notch signalling., Conclusion: A distinct pattern of HPC surface markers was found between acute and chronic liver diseases. Similar to what is known from animal experiments, strong evidence has been found signifying the role of Wnt signalling in proliferation of human HPCs whereas Notch signalling is involved in biliary differentiation. These pathways can be targeted in future therapies.

The biliary tree system consists of two divisions: intrahepatic bile ducts and extrahepatic bile ducts. The development of the biliary tree, and secondarily the liver, shares a common origin with ventral pancreas. A common progenitor for liver, biliary duct system, and ventral pancreas exists at early stages of development, when the anterior definitive endoderm is forming the foregut. Several studies indicate that the biliary tree contains stem cell compartments for liver, pancreas and the bile duct system and persisting into adulthood. These stem cell compartments are present in the peribiliary glands and possibly give rise to committed progenitors in gallbladder that does not have peribiliary glands. The biliary tree stem/progenitors represent a new source of cells that can be used as tools for regenerative medicine of liver, bile duct and pancreas.

Non-anthropomorphic wearable robots (WRs) give good grounds for expecting advantageous performances over traditional anthropomorphic solutions from both the standpoints of ergonomics and of the dynamical interaction with the human body.

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The d-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKCalpha was visualized by immunofluorescence in cell smears and immunoblotting for PKCalpha in cytosol and membrane fractions. Following knockdown of PKCalpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKCalpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKCalpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKCalpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKCalpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKCalpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKCalpha-dependent ERK1/2 dephosphorylation. Modulation of PKCalpha by histamine receptors may be important in regulating cholangiocarcinoma growth.

Background: Endothelins (ET-1, ET-2, ET-3) are peptides with vasoactive properties interacting with ET(A) and ET(B) receptors. ET-1 inhibits secretin-stimulated ductal secretion (hallmark of cholangiocyte growth) of cholestatic rats by interaction with ET receptors., Aim: The aims of the studies were to evaluate (i) the effect of ET-1 on cholangiocarcinoma growth in Mz-ChA-1 cells and nude mice and (ii) whether ET-1 regulation of cholangiocarcinoma growth is associated with changes in the expression of vascular endothelial growth factor-A (VEGF-A), VEGF-C, VEGF receptor-2 (VEGFR-2) and VEGFR-3., Methods: We determined the expression of ET(A) and ET(B) receptors on normal and malignant (Mz-ChA-1) cholangiocytes and human cholangiocarcinoma tissue and the effect of ET-1 on the proliferation and expression of VEGF-A, VEGF-C (regulators of tumour angiogenesis) and its receptors, VEGFR-2 and VEGFR-3, in Mz-ChA-1 cells. In vivo, Mz-ChA-1 cells were injected into the flanks of athymic mice and injections of ET-1 or saline into the tumours were performed daily. The effect of ET-1 on tumour size, cell proliferation, apoptosis, collagen quantity and the expression of VEGF-A and VEGF-C and VEGFR-2 and VEGFR-3 were measured after 73 days., Results: Higher expression of ET(A) and ET(B) was observed in malignant compared with normal cholangiocytes. ET-1 inhibited proliferation and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression of Mz-ChA-1 cells. Chronic ET-1 treatment decreased tumour volume, tumour cell proliferation and VEGF-A and VEGF-C expression but increased apoptosis and collagen tissue deposition compared with controls., Conclusions: Modulation of VEGF-A and VEGF-C (by ET-1) may be important for managing cholangiocarcinoma growth.

Sex hormones regulate cholangiocyte hyperplasia in bile duct-ligated (BDL) rats. We studied whether follicle-stimulating hormone (FSH) regulates cholangiocyte proliferation. FSH receptor (FSHR) and FSH expression was evaluated in liver sections, purified cholangiocytes, and cholangiocyte cultures (NRICC). In vivo, normal female and male rats were treated with FSH or immediately after BDL with antide (a gonadotropin-releasing hormone antagonist blocking FSH secretion) or a neutralizing FSH antibody for 1 wk. We evaluated 1) cholangiocyte proliferation in sections and cholangiocytes and 2) changes in secretin-stimulated cAMP (functional index of cholangiocyte growth) levels, and ERK1/2 and Elk-1 phosphorylation. NRICC were stimulated with FSH before evaluation of proliferation, cAMP/IP(3) levels, and ERK1/2 and Elk-1 phosphorylation. To determine whether FSH regulates cholangiocyte proliferation by an autocrine mechanism, we evaluated the effects of 1) cholangiocyte supernatant (containing FSH) on NRICC proliferation and 2) FSH silencing in NRICC before measuring proliferation and ERK1/2 and Elk-1 phosphorylation. Cholangiocytes and NRICC express FSHR and FSH and secrete FSH. In vivo administration of FSH to normal rats increased, whereas administration of antide and anti-FSH antibody to BDL rats decreased 1) ductal mass and 2) secretin-stimulated cAMP levels, proliferation, and ERK1/2 and Elk-1 phosphorylation in cholangiocytes compared with controls. In NRICC, FSH increased cholangiocyte proliferation, cAMP levels, and ERK1/2 and Elk-1 phosphorylation. The supernatant of cholangiocytes increased NRICC proliferation, inhibited by preincubation with anti-FSH antibody. Silencing of FSH gene decreases cholangiocyte proliferation and ERK1/2 and Elk-1 phosphorylation. Modulation of cholangiocyte FSH expression may be important for the management of cholangiopathies.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

Unlabelled: Cholangiocytes are the target cells in cholestatic models of ductal hyperplasia including bile duct ligation (BDL). We have shown that: (i) cholangiocytes express VEGFR-2 and VEGFR-3; (ii) VEGF-A and VEGF-C stimulate cholangiocyte proliferation via an autocrine mechanism; and (iii) chronic administration of VEGF-A prevents cholangiocyte damage induced by hepatic artery ligation. Caffeic acid phenethyl ester (CAPE) induces growth inhibition in different cells. Taurocholic acid (TC) protects cholangiocytes against injury induced by parasympathetic or sympathetic denervation. The aims of this study were to determine if: (i) CAPE induces bile duct damage; and (ii) TC prevents CAPE-induced bile duct damage by increasing cholangiocyte VEGF expression., Methods: Normal and BDL rats (immediately after surgery) were fed 1% TC or control diet in the absence/presence of daily IP injections of CAPE (10 mg/Kg BW). One week later, we evaluated: (i) cholangiocyte apoptosis, proliferation and ductal mass in liver sections; (ii) functional activity by measuring secretin-stimulated bile and bicarbonate secretion; and (iii) VEGF-A/C and VEGFR-2/R-3 expression in liver sections. In vitro, BDL cholangiocytes were exposed to CAPE (40 microM) in the absence/presence of TC (40 microM) with and without pretreatment with VEGF receptor inhibitors before evaluating cholangiocyte apoptosis and proliferation., Results: Chronic CAPE administration to BDL rats increased cholangiocyte apoptosis and decreased ductal mass. This effect was associated with reduced expression of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3. In vivo, TC feeding partly prevented CAPE-induced changes in cholangiocyte apoptosis and growth and loss of ductal secretion. The protective effect of TC was associated with enhanced VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3. In vitro, TC partially prevented CAPE-induced increases in apoptosis and decreases in cholangiocyte proliferation. These changes were reversed by pretreatment with VEGF-receptor inhibitors., Conclusion: Manipulation of cholangiocyte VEGF expression by bile acids may be important in preventing the impairment of cholangiocyte proliferation by exogenous agents.

Activated alpha-smooth muscle actin (alpha-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. In the rat, increased GFAP expression in the acute response to injury and down-regulation in the chronic response have been observed, whereas reports concerning GFAP expression in human liver are still conflicting. We investigated the utility of GFAP compared to alpha-SMA as an immunohistochemical marker of early activated HSCs in chronic and posttransplant recurrent hepatitis C and correlated GFAP expression with vascular remodeling and fibrosis progression. With immunohistochemistry and a semiquantitative scoring system, the expression of GFAP and alpha-SMA in HSCs and the microvessel density were analyzed in biopsies from normal livers obtained from cadaveric donors [donor liver (DL); n = 21] and from livers from posttransplant hepatitis C virus recurrent hepatitis (HCV-PTR) patients (n = 19), hepatitis C virus chronic hepatitis (HCV-CH) patients, (n = 12), and hepatitis C virus cirrhosis (HCV-C) patients (n = 16). The percentage of alpha-SMA-positive HSCs was significantly higher in the HCV-PTR, HCV-CH, and HCV-C groups compared to the DL group (P < 0.01). The percentage of GFAP-positive HSCs was significantly higher in the HCV-PTR group compared to the DL, HCV-C (P < 0.01), and HCV-CH (P < 0.05) groups and in the HCV-CH group compared to the DL group (P < 0.01), inversely correlating with the extent of fibrosis and microvessel density (P < 0.01). In the HCV-PTR group, the percentage of GFAP-positive HSCs correlated with fibrosis progression (P < 0.01). In conclusion, GFAP could represent a useful marker of early activation of HSCs in HCV-CH and seems to predict fibrosis progression in HCV-PTR.

Background: Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca2+-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes., Results: Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca2+]i levels and PKCbeta-I phosphorylation but decreased PKCalpha phosphorylation. Administration of an anti-prolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha., Conclusion: We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients.

We evaluated the IGF1 system in cholangiocytes of primay biliary cirrhosis (PBC) patients and investigated the relationships with apoptosis. Biopsies of PBC patients (n=32) and normal subjects (n=5) were investigated by immunohistochemistry for expression in cholangiocytes of IGF1, IGF1-R, pAKT, terminal deoxynucleotide transferase end labeling (TUNEL), Bax (proapoptotic protein), and Bcl2 (antiapoptotic protein). Whereas normal cholangiocytes were almost negative, cholangiocytes of PBC patients showed strong IHC staining for IGF1, IGF1-R, and pAKT, which increases from stage I to stage IV, where >70% of cholangiocytes were positive. Bax/Bcl2 ratio reached the highest value (4.6) in PBC stage III when apoptosis is maximal (24% TUNEL positivity), whereas it declines in stage IV (1.4) when only 7.8% cholangiocytes were TUNEL positive. In PBC stages III and IV, expression of IGF1, IGF1-R, and pAKT in cholangiocytes was directly correlated with the antiapoptotic Bcl2 and inversely correlated with proapoptotic Bax, Bax/Bcl2 ratio, and TUNEL positivity. In conclusion, cholangiocytes of PBC patients showed a marked increase in IGF1, IGF1-R, and pAKT expression involving most cholangiocytes surviving in the terminal ductopenic stage. This was associated and correlated with a balance of pro- and antiapoptotic proteins favoring survival rather than apoptosis, suggesting a major role of IGF1 system in promoting cholangiocyte survival.

As previously showed for the classical self renewing tissues (i.e., bone marrow, gut and skin), rapidly changing concepts about tissue stem cell biology identify the liver as a maturational lineage system capable to generate mature cells (hepatocytes and cholangiocytes) from a resident stem cells compartment localized near the so called Canals of Hering. At present liver transplantation is the only available therapy for end stage liver diseases and there is an ever increasing shortage of donor livers. Thus in the last decades, HPC has becoming the object of many researchers since HPC might offer a new therapeutic approach for controlling the evolution of chronic liver diseases. The aim of the present review is to update readers with the evolving concepts about hepatic stem cells biology, their characterization and isolation methods and finally their therapeutic potential.

The microvascular supply of the biliary tree, the peribiliary plexus (PBP), stems from the hepatic artery branches and flows into the hepatic sinusoids. A detailed three-dimensional study of the PBP has been performed by using the Scanning Electron Microscopy vascular corrosion casts (SEMvcc) technique. Considering that the PBP plays a fundamental role in supporting the secretory and absorptive functions of the biliary epithelium, their organization in either normalcy and pathology is explored. The normal liver shows the PBP arranged around extra- and intrahepatic biliary tree. In the small portal tract PBP was characterized by a single layer of capillaries which progressively continued with the extrahepatic PBP where it showed a more complex vascular network. After common duct ligation (BDL), progressive modifications of bile duct and PBP proliferation are observed. The PBP presents a three-dimensional network arranged around many bile ducts and appears as bundles of vessels, composed by capillaries of homogeneous diameter with a typical round mesh structure. The PBP network is easily distinguishable from the sinusoidal network which appears normal. Considering the enormous extension of the PBP during BDL, the possible role played by the Vascular Endothelial Growth Factor (VEGF) is evaluated. VEGF-A, VEGF-C and their related receptors appeared highly immunopositive in proliferating cholangiocytes of BDL rats. The administration of anti-VEGF-A or anti-VEGF-C antibodies to BDL rats as well as hepatic artery ligation induced a reduced bile duct mass. The administration of rVEGF-A to BDL hepatic artery ligated rats prevented the decrease of cholangiocyte proliferation and VEGF-A expression as compared to BDL control rats. These data suggest the role of arterial blood supply of the biliary tree in conditions of cholangiocyte proliferation, such as it occurs during chronic cholestasis. On the other hand, the role played by VEGF as a tool of cross-talk between cholangiocytes and PBP endothelial cells suggests that manipulation of VEGF release and function could represent a therapeutic strategy for human pathological conditions characterized by damage of hepatic artery or the biliary tree.

Atherosclerosis is an inflammatory process disease that involves the artery wall and that is characterized by the progressive accumulation of lipids. The term arteriosclerosis has been created by Lobstein in 1833. Subsequently, during the 19th century, the contribution of Rokitansky and Virchow was important to elucidate the pathogenesis of arteriosclerosis and the morphologic aspects of the plaque. In the beginning of the 20th century, Aschoff was a leading proponent who regarded the morphologically different intimal lipid deposits of children and adults as early and late stages of one disease and he called them atherosis and atherosclerosis, respectively. The first classification of atherosclerosis was made by the World Health Organization (WHO) in 1958 and it consisted of the following sequence: fatty streak, atheroma, fibrous plaque and complicated lesions. In 1990s, thanks to much more sensitive techniques, the American Heart Association (AHA) proposed a new morphological classification based on eight lesion types designated by Roman numerals which indicate the usual sequence of lesion progression. Finally, Virmani et al. (2000) described a classification with the add of a specific plaque type, not recognized by the AHA classification, called "thin fibrous cap atheroma" which is more likely to rupture. The atherosclerotic process is characterized by typical ultrastructural changes that mainly involve the endothelial and smooth muscle cells. The morphological alterations of the endothelium are associated with dysfunctions leading to a proinflammatory and prothrombotic phenotype. This process seems to be due to turbulent blood flow and low fluid shear stress that normally occurs in particular regions of the vascular tree. Inflammation has a key role in the pathogenesis of atherosclerosis and it is supported by numerous factors such as modified LDL, hypertension, diabetes mellitus, free radicals and, in particular, by infectious agents such as Chlamydia pneumoniae.

The activation of hepatic stellate cells is the conversion of quiescent cells into proliferative, contractile, and fibrogenic myofibroblasts. The alpha-smooth muscle actin is a well known marker of hepatic stellate cells activation. On the other hand, the Glial Fibrillary Acidic Protein expression is a key step in the astrocytes differentiation and constitutes the hallmark response of astrocytes to injury. Glial Fibrillary Acidic Protein expression was reported in quiescent stellate cells in vivo, with an increased expression in the acute response to injury in the rat, and a down-regulation in the chronic one. This study aims to evaluate the morphologic characteristics, distribution and percentage of Glial Fibrillary Acidic Protein expressing cells in normal and injured human livers. Human liver biopsies (n=32) were divided in three groups: 1) donors liver; 2) chronic hepatitis C-virus related; 3) post-viral hepatitis cirrhosis. Samples were immunostained with anti- alpha-smooth muscle actin and anti- Glial Fibrillary Acidic Protein antibody. Subsequently they were semi-quantitatively evaluated. Liver fibrosis was quantified by morphometric analysis. In chronic hepatitis livers and in the cirrhotic ones, hepatic stellate cells became larger with long cytoplasmic processes. The alpha-smooth muscle actin -positive stellate cells increase and expand from areas of piecemeal necrosis to the entire residual parenchyma with the progression of the fibrosis. By contrast Glial Fibrillary Acidic Protein-positive stellate cells are more evenly distributed in the earlier stages and confined to the periphery of the hepatic lobule in the advanced ones. Also some endothelial cells were Glial Fibrillary Acidic Protein positive in vessels of the expanding septa and at the edges of cirrhotic nodules. Glial Fibrillary Acidic Protein expression in the liver is probably linked to the fine modulation of the cytoskeleton during the formation of cytoplasmic processes in both stellate cells and endothelial cells. In the latter Glial Fibrillary Acidic Protein expression might be related to the remodelling of the vascular bed that occurs during the response to injuries. Otherwise in hepatic stellate cells it seems to be a marker related with the acquisition of contractile properties in a subpopulation more closely associated with precocious stages of the fibrosis.

Background: Primary cardiac tumours are rare. Nearly 70% of primary cardiac tumours are benign, the majority of which are represented by myxomas. The most frequent primary cardiac neoplasm is the angiosarcoma that represents 31% of primary cardiac malignant. We report a particular clinical case of cardiac angiosarcoma, its light and transmission electron microscopic aspects and a review of the recent literature., Methods: A 52 years old man died for a severe right ventricle filling deficit caused by an intracavitary tumour originated from the right atrial anterolateral wall. The fragments obtained from autoptic tumoral cardiac tissue were processed for light and electron microscopy. The section were stained with haematoxilineosin, Masson trichromic and Gomori method. An immunohistochemical study for vimentin, Factor VIII related antigen and peroxidase-conjugated lectin from Ulex Europaeus was also performed using the unlabed peroxidase-antiperoxidase method., Results: The hematoxylin-eosin staining showed that the tumoral mass was composed by a well-differentiated histotype characterized by numerous vascular areas in which neoplastic cells were loosely and irregularly arranged to form incomplete vessels or anastomized blood-filled vascular channels. On the other hand, some less-differentiated solid areas were present and irregularly surrounded the differentiated vascular areas. Results of Ulex Europeaeus Agglutinin I labelling were positive in both solid and vascular areas of the tumour although the positive reaction was less evident in the solid zones Factor VIII related antigen positive cells were less numerous and mainly found in vascular areas. The observation by electron microscopy showed the lack of evident pinocytotic vesicles, the presence of thin and delicate cytoplasmatic processes, Weibel-Palade bodies, and also the disarrangement of the extracellular fibrous matrix., Conclusion: The light microscopy observation and immunohistochemical study underscore that is not easy to obtain information about the level of differentiation of this tumour. The presence of blood-filled lumina and the identification of typical markers of endothelial cells seems to indicate a well-differentiated nature. However, the ultrastructural findings seem to indicate a less differentiated nature.

Background: The alpha isotype of actin expressed by hepatic stellate cells reflects their activation to myofibroblast-like cell and has been directly related to experimental liver fibrogenesis, and indirectly to human fibrosis in chronic liver disease., Aims: To evaluate the changes in distribution and percentage of alpha-smooth muscle actin-positive hepatic stellate cells and the correlation with the degree of the fibrosis in cirrhotic livers, as well as in patients with recurrent HCV chronic hepatitis after liver transplantation., Methods: Human liver biopsies were divided in four groups: (1) normal livers obtained from cadaveric liver donors (n=35), (2) cirrhosis post-HBV hepatitis (n=11), (3) cirrhosis post-HCV hepatitis (n=10), and (4) post-transplant recurrent HCV chronic hepatitis (n=13). Samples were stained with anti-alpha-smooth muscle actin antibody by immunoperoxidase method and semi-quantitatively evaluated. Liver fibrosis was assessed from specimens stained with Masson's trichrome and quantified by computer image analysis., Results: The percentage of alpha-smooth muscle actin-positive hepatic stellate cells was significantly higher in the HBV cirrhosis, HCV cirrhosis and post-transplant HCV recurrent hepatitis groups (36.1+/-15.2, 23.8+/-19.7 and 27.8+/-16.4%, respectively) compared to the liver donor group (2.9+/-4.0%). The alpha-smooth muscle actin-positive hepatic stellate cells to fibrous tissue ratio were significantly higher in the post-transplant recurrent HCV hepatitis group (2.36+/-1.12) compared to both the donor livers and the HCV cirrhosis groups (0.74+/-1.09 and 1.03+/-0.91, respectively). The alpha-smooth muscle actin-positive hepatic stellate cell percentage and fibrosis correlated positively in the post-transplant recurrent HCV hepatitis group and negatively in the HCV cirrhosis group. No difference in the immunohistochemical and morphometrical variables was found between the HCV cirrhosis and HBV cirrhosis groups., Conclusions: These results indirectly confirm that, in vivo, alpha-smooth muscle actin expression is a reliable marker of hepatic stellate cells activation which precedes fibrous tissue deposition even in the setting of recurrent HCV chronic hepatitis after liver transplantation, and it could be useful to identify the earliest stages of hepatic fibrosis and monitoring the efficacy of the therapy. In the presence of advanced cirrhosis other factors, rather than alpha-smooth muscle actin-positive hepatic stellate cells, may sustain fibrosis deposition.

Hepatic stellate cells have been considered the most important cell-type involved in hepatic fibrogenesis. Proliferation and differentiation of hepatic stellate cells into myofibroblast-like cells has been related to the development of liver fibrosis. The alpha-actin expressed by hepatic stellate cells was considered a marker of their activation to myofibroblast-like cell. The aim of this study is to evaluate the changes in morphology, distribution, percentage and alpha-smooth muscle actin expression of hepatic stellate cells in normal and cirrhotic livers, and to correlate activated hepatic stellate cells with the progression of fibrosis. Human liver biopsies (n=121) were divided in five groups: 1) normal livers (controls); 2) cirrhosis post-HCV hepatitis; 3) cirrhosis post-HBV hepatitis; 4) non viral related cirrhosis; 5) recurrent HCV hepatitis after orthotopic liver transplantation. Samples immunostained with anti alpha-smooth muscle actin antibody by immunoperoxidase method were semi-quantitatively evaluated. Liver fibrosis was quantified by computer image analysis on specimens stained with Masson's trichrome. In normal adult livers stellate cells were very rarely stained for alpha-smooth muscle actin. In cirrhotic livers, a strongly enhanced percentage of stellate cells expressing alpha-smooth muscle actin was detected in cirrhotic fragments with respect to the control group, with a significant correlation between alpha-smooth muscle actin positive stellate cells and the volume fraction of fibrosis. Moreover, liver biopsies of recurrent hepatitis revealed an increased number of activated stellate cells compared to normal livers, and intermediate volume fraction of fibrosis. These results confirmed that a direct correlation existed between activated stellate cells and the progression of fibrosis. Alpha-smooth muscle actin confirmed to be a reliable marker of hepatic stellate cells activation also in precocious stages of the disease.