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Massimiliano Cadamuro,
Giorgia Nardo,
Stefano Indraccolo,
Luigi Dall'olmo,
Luisa Sambado,
Lidia Moserle,
Irene Franceschet,
Michele Colledan,
Marco Massani,
Tommaso Stecca,
Nicolò Bassi,
Stuart Morton,
Carlo Spirli, Romina Fiorotto,
Luca Fabris,
Mario Strazzabosco
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ABSTRACT: Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAF) are pivotal players in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied: 1) the expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells; 2) the lineage tracking of an EGFP-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice; 3) the expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro; 4) the secretion of PDGFs by CCA cells; 5) the role of PDGF-D in fibroblast recruitment in vitro; 6) the downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers but not α-SMA. Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAF, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whilst CAF expressed PDGFRβ. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium, and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and JNK. Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration. Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAF. Instead, CCA cells recruit CAF by secreting PDGF-D, which stimulates fibroblast migration via PDGFRβ and Rho GTPase and JNK activation. Targeting tumor/stroma interactions with inhibitors of PDGF-D pathway may offer a novel therapeutic approach. (HEPATOLOGY 2013.).
Hepatology 03/2013; · 11.66 Impact Factor
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ABSTRACT: BACKGROUND AND AIMS: Repair from biliary damages requires the biliary specification of hepatic progenitor cells and the remodeling of ductular reactive structures into branching biliary tubules. We hypothesized that the morphogenetic role of Notch signaling is maintained during the repair process and have addressed this hypothesis using pharmacologic and genetic models of defective Notch signaling. METHODS AND RESULTS: Treatment with DDC (3,5-diethoxycarbonyl1,4-dihydrocollidine) or ANIT (alpha-naphthyl-isothiocyanate) was used to induce biliary damage in wild-type mice and in mice with a liver-specific defect in the Notch-2 receptor (Notch-2-cko) or in RPB-Jk. Hepatic progenitor cells, ductular reaction and mature ductules were quantified using K19 and SOX-9. In DDC-treated wild-type mice, pharmacologic Notch inhibition with dibenzazepine decreased the number of both ductular reaction and hepatic progenitor cells. Notch-2-cko mice treated with DDC or ANIT accumulated hepatic progenitor cells that failed to progress into mature ducts. In RBP-Jκ-cko mice, mature ducts and hepatic progenitor cells were both significantly reduced with respect to similarly treated wild-type mice. The mouse progenitor cell line BMOL cultured on Matrigel, formed a tubular network allowing the study of tubule formation in vitro; γ-secretase inhibitor treatment and siRNAs silencing of Notch-1, Notch2 or Jagged1 significantly reduced both the length and the number of tubular branches. CONCLUSIONS: These data demonstrate that Notch signaling plays an essential role in biliary repair. Lack of Notch-2 prevents biliary tubule formation, both in vivo and in vitro. Lack of RBP-Jk inhibits the generation of biliary-committed precursors and tubule formation.
Journal of Hepatology 03/2013; · 9.26 Impact Factor
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ABSTRACT: Mutations in polycystins are a cause of polycystic liver disease. In polycystin-2 (PC2)-defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal-regulated kinase-extracellular signal-regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2-defective mice, we treated PC2 (i.e., Pkd2(flox/-) :pCxCreER(TM) [Pkd2cKO]) mice with sorafenib-tosylate for 8 weeks (20-60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO-cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 μM; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 μM. Raf kinase activity assay showed that whereas B-Raf is inhibited by sorafenib in both wild-type (WT) and Pkd2cKO cells, Raf-1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14-22 amide, myristolated (1 μM) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited. Conclusion: In PC2-defective cells, sorafenib inhibits B-Raf but paradoxically activates Raf-1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf-1 when a PKA-activated Ras promotes Raf-1/B-Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib. (HEPATOLOGY 2012).
Hepatology 05/2012; · 11.66 Impact Factor
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ABSTRACT: Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2-defective mice, cyclic 3',5'-adenosine monophosphate/ protein kinase A (cAMP/PKA)-dependent activation of extracellular signal-regulated kinase/ mammalian target of rapamycin (ERK-mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca(2+) and cAMP production and inappropriate ERK signaling in PC2-defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional-KO (knockout) mice (Pkd2(flox/-) :pCxCreER™; hence, called Pkd2KO) and compared to cholangiocytes from wild-type mice (WT). Our results showed that, compared to WT cells, in PC2-defective cholangiocytes (Pkd2KO), cytoplasmic and ER-Ca(2+) (measured with Fura-2 and Mag-Fluo4) levels are decreased and store-operated Ca(2+) entry (SOCE) is inhibited, whereas the expression of Ca(2+) -sensor stromal interaction molecule 1 (STIM1) and store-operated Ca(2+) channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER-Ca(2+) depletion increases cAMP and PKA-dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6). CONCLUSION: These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM-dependent activation of AC6 by ER Ca(2+) depletion. In PC2-defective cells, the interaction of STIM-1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store-operated cAMP production.
Hepatology 03/2012; 55(3):856-68. · 11.66 Impact Factor
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ABSTRACT: Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins.
Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftrtm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor κB (NF-κB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs.
DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF-κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-κB.
CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-κB. These findings might be used to develop therapies for CF-associated cholangiopathy.
Gastroenterology 06/2011; 141(4):1498-508, 1508.e1-5. · 11.68 Impact Factor
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ABSTRACT: Polycystic liver disease may complicate autosomal dominant polycystic kidney disease (ADPKD), a disease caused by mutations in polycystins, which are proteins that regulate signaling, morphogenesis, and differentiation in epithelial cells. The cystic biliary epithelium [liver cystic epithelium (LCE)] secretes vascular endothelial growth factor (VEGF), which promotes liver cyst growth via autocrine and paracrine mechanisms. The expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), and phosphorylated mammalian target of rapamycin (p-mTOR) and the protein kinase A (PKA)-dependent phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) are also up-regulated in LCE. We have hypothesized that mammalian target of rapamycin (mTOR) represents a common pathway for the regulation of hypoxia-inducible factor 1 alpha (HIF1alpha)-dependent VEGF secretion by IGF1 and ERK1/2. Conditional polycystin-2-knockout (Pkd2KO) mice were used for in vivo studies and to isolate cystic cholangiocytes [liver cystic epithelial cells (LCECs)]. The expression of p-mTOR, VEGF, cleaved caspase 3 (CC3), proliferating cell nuclear antigen (PCNA), IGF1, IGF1R, phosphorylated extracellular signal-regulated kinase, p-P70S6K, HIF1alpha, and VEGF in LCE, LCECs, and wild-type cholangiocytes was studied with immunohistochemistry, western blotting, or enzyme-linked immunosorbent assays. The cystic area was measured by computer-assisted morphometry of pancytokeratin-stained sections. Cell proliferation in vitro was studied with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assays. The treatment of Pkd2KO mice with the mTOR inhibitor rapamycin significantly reduced the liver cyst area, liver/body weight ratio, pericystic microvascular density, and PCNA expression while increasing expression of CC3. Rapamycin inhibited IGF1-stimulated HIF1alpha accumulation and VEGF secretion in LCECs. IGF1-stimulated LCEC proliferation was inhibited by rapamycin and SU5416 (a vascular endothelial growth factor receptor 2 inhibitor). Phosphorylation of the mTOR-dependent kinase P70S6K was significantly reduced by PKA inhibitor 14-22 amide and by the mitogen signal-regulated kinase inhibitor U1026. CONCLUSION: These data demonstrate that PKA-dependent up-regulation of mTOR has a central role in the proliferative, antiapoptotic, and pro-angiogenic effects of IGF1 and VEGF in polycystin-2-defective mice. This study also highlights a mechanistic link between PKA, ERK, mTOR, and HIF1alpha-mediated VEGF secretion and provides a proof of concept for the potential use of mTOR inhibitors in ADPKD and conditions with aberrant cholangiocyte proliferation.
Hepatology 05/2010; 51(5):1778-88. · 11.66 Impact Factor
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ABSTRACT: Polycystic liver diseases are hereditary disorders that affect the biliary epithelium, often in conjunction with the renal
tubule epithelium. Characterized by the progressive formation of cysts throughout the liver and kidney, they can often lead
to severe life-threatening complications. Polycystins and fibrocystin, the defective proteins in the dominant and in the recessive
form of the disease, respectively, are mainly expressed in the primary (nonmotile) cilia of cholangiocytes, the epithelial
cells that line the intrahepatic biliary tree. Important clues for understanding the pathogenesis of cystic diseases come
from understanding the biology and pathobiology of cholangiocytes. In this chapter, cholangiocyte function and morphology
is first briefly described, with particular emphasis on the regulation of their secretory properties and the complex intercellular
signaling. Then, we discuss a number of possible mechanisms leading to cyst formation and progressive growth of the cysts.
In both autosomal dominant and recessive forms, liver cysts arise from an aberrant development of intrahepatic bile duct epithelium.
During cyst expansion, different factors, including excessive fluid secretion, extracellular matrix remodeling, increased
proliferation of the epithelial cells lining the cyst, and aberrant hypervascularization around the cyst wall, variably take
part in promoting progressive cyst growth. Many of these factors act via autocrine mechanisms. Each of them represents a possible
target for therapies aimed at reducing the growth of liver cysts.
Key WordsCholangiocytes-ADPKD-ARPKD-Polycystin-1-Polycystin-2-Fibrocystin-VEGF-Cilium-Ductal plate malformation
12/2009: pages 23-43;
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ABSTRACT: Severe polycystic liver disease can complicate adult dominant polycystic kidney disease, a genetic disease caused by defects in polycystin-1 (Pkd1) or polycystin-2 (Pkd2). Liver cyst epithelial cells (LCECs) express vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2. We investigated the effects of VEGF on liver cyst growth and autocrine VEGF signaling in mice with Pkd1 and Pkd2 conditional knockouts.
We studied mice in which Pkd1 or Pkd2 were conditionally inactivated following exposure to tamoxifen; these mice were called Pkd1(flox/-):pCxCreER (Pkd1KO) and Pkd2(flox/-):pCxCreER (Pkd2KO).
Pkd1KO and Pkd2KO mice developed liver defects; their LCECs expressed VEGF, VEGFR-2, hypoxia-inducible factor (HIF)-1alpha, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), and proliferating cell nuclear antigen (PCNA). In Pkd2KO but not Pkd1KO mice, exposure to the VEGFR-2 inhibitor SU5416 significantly reduced liver cyst development, liver/body weight ratio, and expression of pERK and PCNA. VEGF secretion and phosphorylation of ERK1/2 and VEGFR-2 were significantly increased in cultured LCECs from Pkd2KO compared with Pkd1KO mice. Inhibition of protein kinase A (PKA) reduced VEGF secretion and pERK1/2 expression. Addition of VEGF to LCECs from Pkd2KO mice increased phosphorylated VEGFR-2 and phosphorylated mitogen signal-regulated kinase (MEK) expression and induced phosphorylation of ERK1/2; this was inhibited by SU5416. Expression of HIF-1alpha increased in parallel with secretion of VEGF following LCEC stimulation. VEGF-induced cell proliferation was inhibited by the MEK inhibitor U1026 and by ERK1/2 small interfering RNA.
The PKA-ERK1/2-VEGF signaling pathway promotes growth of liver cysts in mice. In Pkd2-defective LCECs, PKA-dependent ERK1/2 signaling controls HIF-1alpha-dependent VEGF secretion and VEGFR-2 signaling. Autocrine and paracrine VEGF signaling promotes the growth of liver cysts in Pkd2KO mice. VEGF inhibitors might be used to treat patients with polycystic liver disease.
Gastroenterology 09/2009; 138(1):360-371.e7. · 11.68 Impact Factor
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ABSTRACT: cAMP is generated by adenylyl cyclases (ACs) a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli and are differentially modulated in cholestasis. Methods: Small (SDC) and large (LDC) cholangiocytes were isolated from controls and from lipopolysaccharide-treated (LPS) or α-naphthylisothiocyanate-treated (ANIT) rats. ACs isoforms expression was assessed by real-time PCR. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO 3 − /CO 2, cholinergic and β-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or sAC with siRNA. Results: Gene expression of the Ca 2+ -insensitive isoforms (AC4, AC7) was higher in SDC, while that of the Ca 2+ -inhibitable (AC5, AC6, AC9), the Ca 2+ /calmodulin stimulated AC8, and the soluble sAC, was higher in LDC. Ca 2+ /calmodulin-inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin-inibitors was cAMP-dependent and GABA-inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO 3 − ] i , was cAMP-dependent and inhibited by sAC-inhibitior and by sAC gene silencing. Treatment with LPS or ANIT increased expression of AC7 and sAC, while decreasing that of the others ACs. Conclusion: These studies demonstrate a previously unrecognized role of AC in biliary pathophysiology. In fact: 1) ACs isoforms are differentially expressed in cholangiocyte subpopulations, 2) AC8, AC9, and sAC mediate cholangiocyte secretion in response to secretin, β-adrenergic agonists, or changes in [HCO 3 − ] i , respectively, 3) ACs gene expression is modulated in experimental cholestasis.
Hepatology 08/2009; 50(1):244-252. · 11.66 Impact Factor
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Emina Halilbasic, Romina Fiorotto,
Peter Fickert,
Hanns-Ulrich Marschall,
Tarek Moustafa,
Carlo Spirli,
Andrea Fuchsbichler,
Judith Gumhold,
Dagmar Silbert,
Kurt Zatloukal,
Cord Langner,
Uday Maitra,
Helmut Denk,
Alan F Hofmann,
Mario Strazzabosco,
Michael Trauner
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ABSTRACT: 24-norursodeoxycholic acid (norUDCA), a side chain-modified ursodeoxycholic acid derivative, has dramatic therapeutic effects in experimental cholestasis and may be a promising agent for the treatment of cholestatic liver diseases. We aimed to better understand the physiologic and therapeutic properties of norUDCA and to test if they are related to its side chain length and/or relative resistance to amidation. For this purpose, Mdr2(-/-) mice, a model for sclerosing cholangitis, received either a standard diet or a norUDCA-, tauro norursodeoxycholic acid (tauro- norUDCA)-, or di norursodeoxycholic acid (di norUDCA)-enriched diet. Bile composition, serum biochemistry, liver histology, fibrosis, and expression of key detoxification and transport systems were investigated. Direct choleretic effects were addressed in isolated bile duct units. The role of Cftr for norUDCA-induced choleresis was explored in Cftr(-/-) mice. norUDCA had pharmacologic features that were not shared by its derivatives, including the increase in hepatic and serum bile acid levels and a strong stimulation of biliary HCO(3)(-)-output. norUDCA directly stimulated fluid secretion in isolated bile duct units in a HCO(3)(-)-dependent fashion to a higher extent than the other bile acids. Notably, the norUDCA significantly stimulated HCO(3)(-)-output also in Cftr(-/-) mice. In Mdr2(-/-) mice, cholangitis and fibrosis strongly improved with norUDCA, remained unchanged with tauro- norUDCA, and worsened with di norUDCA. Expression of Mrp4, Cyp2b10, and Sult2a1 was increased by norUDCA and di norUDCA, but was unaffected by tauro- norUDCA. CONCLUSION:The relative resistance of norUDCA to amidation may explain its unique physiologic and pharmacologic properties. These include the ability to undergo cholehepatic shunting and to directly stimulate cholangiocyte secretion, both resulting in a HCO(3)(-)-rich hypercholeresis that protects the liver from cholestatic injury.
Hepatology 07/2009; 49(6):1972-81. · 11.66 Impact Factor
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ABSTRACT: Cyclic adenosine monophosphate (cAMP) is generated by adenylyl cyclases (ACs), a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli, and are differentially modulated in cholestasis. Small duct and large duct cholangiocytes were isolated from controls and from lipopolysaccharide-treated or alpha-naphthylisothiocyanate-treated rats. AC isoform expression was assessed via real-time polymerase chain reaction. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO(3)(-)/CO(2), cholinergic agonists, and beta-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or soluble adenylyl cyclase (sAC) with small interfering RNA. Gene expression of the Ca(2+)-insensitive isoforms (AC4, AC7) was higher in small duct cholangiocytes, whereas that of the Ca(2+)-inhibitable (AC5, AC6, AC9), the Ca(2+)/calmodulin-stimulated AC8, and the soluble sAC was higher in large duct cholangiocytes. Ca(2+)/calmodulin inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin inibitors was cAMP-dependent and gamma-aminobutyric acid-inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO(3)(-)](i) was cAMP-dependent and inhibited by sAC inhibitor and sAC gene silencing. Treatment with lipopolysaccharide or alpha-naphthylisothiocyanate increased expression of AC7 and sAC but decreased expression of the other ACs. Conclusion: These studies demonstrate a previously unrecognized role of ACs in biliary pathophysiology. In fact: (1) AC isoforms are differentially expressed in cholangiocyte subpopulations; (2) AC8, AC9, and sAC mediate cholangiocyte secretion in response to secretin, beta-adrenergic agonists, or changes in [HCO(3)(-)](i), respectively; and (3) AC gene expression is modulated in experimental cholestasis.
Hepatology 04/2009; 50(1):244-52. · 11.66 Impact Factor
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ABSTRACT: The pathogenicity of the Vibrio cholerae strains belonging to serogroup O1 and O139 is due to the production of virulence factors such as cholera toxin (CT) and the toxin-coregulated pilus (TCP). The remaining serogroups, which mostly lack CT and TCP, are more frequently isolated from aquatic environmental sources than from clinical samples; nevertheless, these strains have been reported to cause human disease, such as sporadic outbreaks of watery diarrhoea and inflammatory enterocolitis. This evidence suggested the possibility that other virulence factor(s) than cholera toxin might be crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea, but their nature remains unknown. VCC, the hemolysin produced by virtually all Vibrio cholerae strains, has been proposed as a possible candidate, though a clear-cut demonstration attesting VCC as crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea is still lacking.
Electrophysiological parameters and paracellular permeability of stripped human healthy colon tissues, obtained at subtotal colectomy, mounted in Ussing chamber were studied in the presence or absence of VCC purified from culture supernatants of V. cholerae O1 El Tor strain. Short circuit current (I(SC)) and transepithelial resistance (R(T)) were measured by a computerized voltage clamp system. The exposure of sigmoid colon specimens to 1 nM VCC resulted in an increase of I(SC) by 20.7%, with respect to the basal values, while R(T) was reduced by 12.3%. Moreover, increase in I(SC) was abolished by bilateral Cl(-) reduction.
Our results demonstrate that VCC, by forming anion channels on the apical membrane of enterocytes, triggers an outward transcellular flux of chloride. Such an ion movement, associated with the outward movement of Na(+) and water, might be responsible for the diarrhoea caused by the non-toxigenic strains of Vibrio cholerae.
PLoS ONE 02/2009; 4(3):e5074. · 4.09 Impact Factor
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ABSTRACT: Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis. CONCLUSION: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.
Hepatology 03/2008; 47(2):719-28. · 11.66 Impact Factor
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ABSTRACT: Cholangiopathies are characterized by impaired cholangiocyte secretion. Ursodeoxycholic acid (UDCA) is widely used for cholangiopathy treatment, but its effects on cholangiocyte secretory functions remain unclear and are the subject of this study.
Polarized mouse cholangiocytes in tubular (isolated bile-duct units [IBDU]) or monolayer configuration were obtained from wild-type (WT) and B6-129-Cftr(tm1Kth) and Cftr(tm1Unc) mice that are defective in CFTR, an adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl(-) channel expressed in cholangiocytes. Fluid secretion was assessed by video-optical planimetry, Cl(-) and Ca(2+) efflux by microfluorimetry (6-methoxy-N-ethylquinolinium chloride, fura-2, and fluo-4), adenosine triphosphate (ATP) secretion by luciferin-luciferase assay, and protein kinase C (PKC) by Western blot.
UDCA stimulated fluid secretion and Cl(-) efflux in WT-IBDU but not in CFTR-KO-IBDU or in WT-IBDU exposed to CFTR inhibitors. UDCA did not affect intracellular cAMP levels but increased [Ca(2+)]i in WT and not in CFTR-KO cholangiocytes. UDCA stimulated apical ATP secretion in WT but not in CFTR-KO cholangiocytes. UDCA-stimulated [Ca(2+)]i increase was inhibited by suramin, a purinergic 2Y-receptor inhibitor. UDCA stimulated the translocation of PKC-alpha and PKC-epsilon to the plasma membrane. UDCA-stimulated secretion was inhibited by 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and by phospholipase C and PKC inhibitors. UDCA increased ATP output in isolated perfused livers from WT but not from CFTR-KO mice.
Our data indicate that UDCA stimulates a CFTR-dependent apical ATP release in cholangiocytes. Secreted ATP activates purinergic 2Y receptors, and, through [Ca(2+)]i increase and PKC activation stimulates Cl(-) efflux and fluid secretion. These data support the concept that CFTR plays a role in modulating purinergic signaling in secretory epithelia and suggest a novel mechanism explaining the choleretic effect of UDCA.
Gastroenterology 11/2007; 133(5):1603-13. · 11.68 Impact Factor
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Luca Fabris,
Massimiliano Cadamuro,
Maria Guido,
Carlo Spirli, Romina Fiorotto,
Michele Colledan,
Giuliano Torre,
Daniele Alberti,
Aurelio Sonzogni,
Lajos Okolicsanyi,
Mario Strazzabosco
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ABSTRACT: Patients with Alagille syndrome (AGS), a genetic disorder of Notch signaling, suffer from severe ductopenia and cholestasis, but progression to biliary cirrhosis is rare. Instead, in biliary atresia (BA) severe cholestasis is associated with a pronounced "ductular reaction" and rapid progression to biliary cirrhosis. Given the role of Notch in biliary development, we hypothesized that defective Notch signaling would influence the reparative mechanisms in cholestatic cholangiopathies. Thus we compared phenotype and relative abundance of the epithelial components of the hepatic reparative complex in AGS (n = 10) and BA (n = 30) using immunohistochemistry and computer-assisted morphometry. BA was characterized by an increase in reactive ductular and hepatic progenitor cells, whereas in AGS, a striking increase in intermediate hepatobiliary cells contrasted with the near absence of reactive ductular cells and hepatic progenitor cells. Hepatocellular mitoinhibition index (p21(waf1)/Ki67) was similar in AGS and BA. Fibrosis was more severe in BA, where portal septa thickness positively correlated with reactive ductular cells and hepatic progenitor cells. AGS hepatobiliary cells failed to express hepatic nuclear factor (HNF) 1beta, a biliary-specific transcription factor. These data indicate that Notch signaling plays a role in liver repair mechanisms in postnatal life: its defect results in absent reactive ductular cells and accumulation of hepatobiliary cells lacking HNF1beta, thus being unable to switch to a biliary phenotype.
American Journal Of Pathology 09/2007; 171(2):641-53. · 4.89 Impact Factor
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ABSTRACT: Amiodarone (AMI) is a potent antiarrhythmic agent; however, its clinical use is limited due to numerous side effects. In order to investigate the structure--cytotoxicity relationship, AMI analogues were synthesized, and then, using rabbit alveolar macrophages, were tested for viability and for the ability to interfere with the degradation of surfactant protein A (SP-A) and with the accumulation of an acidotropic dye. Our data revealed that modification of the diethylamino-beta-ethoxy group of the AMI molecule may affect viability, the ability to degrade SP-A and vacuolation differently. In particular, PIPAM (2d), an analogue with a piperidyl moiety, acts toward the cells in a similar manner to AMI, but is less toxic. Thus, it would be possible to reduce the cytotoxicity of AMI by modifying its chemical structure.
European Journal of Medicinal Chemistry 07/2007; 42(6):861-7. · 3.35 Impact Factor
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ABSTRACT: Liver involvement in autosomal dominant polycystic kidney disease (ADPKD) is characterized by altered remodeling of the embryonic ductal plate (DP) with presence of biliary cysts and aberrant portal vasculature. The genetic defect causing ADPKD has been identified, but mechanisms of liver cyst growth remain uncertain. To investigate the possible role of angiogenic mechanisms, we have studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their receptors (VEGFR-1, VEGFR-2, Tie-2) in ADPKD, Caroli's disease, normal and fetal livers. In ADPKD and control livers Ang-1 and Ang-2 gene expression was studied by real-time-PCR. Effects of VEGF on cholangiocyte proliferation were studied by PCNA Western Blot in isolated rat cholangiocytes and by MTS assay in cultured cholangiocytes isolated from ADPKD patients and from an ADPKD mouse model (Pkd2(WS25/-)). Cholangiocytes were strongly positive for VEGF, VEGFR-1, VEGFR-2 and Ang-2 in ADPKD and Caroli, and also for Ang-1 and Tie-2 in ADPKD, similar to fetal ductal plate cells. VEGF stimulated proliferation in both normal and ADPKD cholangiocytes, but the effect was particularly evident in the latter. Ang-1 alone had no effect, but was synergic to VEGF. VEGF expression on cholangiocytes positively correlated with microvascular density. In conclusion, consistent with the immature phenotype of the cystic epithelium, expression of VEGF, VEGFRs, Ang-1 and Tie-2 is strongly upregulated in cholangiocytes from polycystic liver diseases. VEGF and Ang-1 have autocrine proliferative effect on cholangiocyte growth and paracrine effect on portal vasculature, thus promoting the growth of the cysts and their vascular supply. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
Hepatology 06/2006; 43(5):1001-12. · 11.66 Impact Factor
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Carlo Spirlì, Romina Fiorotto,
Lei Song,
Joseph Santos-Sacchi,
Lajos Okolicsanyi,
Sara Masier,
Loretta Rocchi,
Maria Pia Vairetti,
Marina De Bernard,
Saida Melero,
Tullio Pozzan,
Mario Strazzabosco
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ABSTRACT: Progressive liver disease is a severe complication of cystic fibrosis, a genetic disease characterized by impaired epithelial adenosine 3',5'-cyclic monophosphate-dependent secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In the liver, CFTR is expressed in cholangiocytes and regulates the fluid and electrolyte content of the bile. Glibenclamide, a sulfonylurea and a known CFTR inhibitor, paradoxically stimulates cholangiocyte secretion. We studied the molecular mechanisms underlying this effect and whether glibenclamide could restore cholangiocyte secretion in cystic fibrosis.
NRC-1 cells, freshly isolated rat cholangiocytes, isolated rat biliary ducts, and isolated biliary ducts from CFTR-defective mice (Cftr tm1Unc ) were used to study fluid secretion (by video-optical planimetry), glibenclamide-induced secretion (by high-performance liquid chromatography in cell culture medium), intracellular pH and intracellular Ca 2+ concentration transients [2'7'-bis(2-carboxyethyl)-5,6,carboxyfluorescein-acetoxymethylester and Fura-2 f-AM (5-Oxazolecarboxylic acid, 2-(6-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-, (acetyloxy)methyl ester) microfluorometry], gene expression (by reverse-transcription polymerase chain reaction), and changes in membrane capacitance (by patch-clamp experiments).
Stimulation of cholangiocyte secretion by glibenclamide and tolbutamide required Cl - and was mediated by the sulfonylurea receptor 2B. Glibenclamide-induced secretion was blocked by inhibitors of exocytosis (colchicine, wortmannin, LY294002, and N -ethylmaleimide) and by inhibitors of secretory granule acidification (vanadate, bafilomycin A1, and niflumic acid) but was Ca 2+ and depolarization independent; membrane capacitance measurements were consistent with stimulation of vesicular transport and fusion. Glibenclamide, unlike secretin and forskolin, was able to stimulate secretion in Cftr tm1Unc mice, thus indicating that this secretory mechanism was preserved.
The ability of glibenclamide to stimulate secretion in CFTR-defective mice makes sulfonylureas a model class of compounds to design drugs useful in the treatment of cystic fibrosis with liver impairment and possibly of other cholestatic diseases.
Gastroenterology 08/2005; 129(1):220-33. · 11.68 Impact Factor
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ABSTRACT: The biliary epithelium is involved both in bile production and in the inflammatory/reparative response to liver damage. Recent data indicate that inflammatory aggression to intrahepatic bile ducts results in chronic progressive cholestasis.
To understand the effects of nitric oxide on cholangiocyte secretion and biliary tract pathophysiology we have investigated: (1) the effects of proinflammatory cytokines on NO production and expression of the inducible nitric oxide synthase (NOS2), (2) the effects of NO on cAMP-dependent secretory mechanisms, and (3) the immunohistochemical expression of NOS2 in a number of human chronic liver diseases.
Our results show that: (1) tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, synergically stimulate NO production in cultured cholangiocytes through an increase in NOS2 gene and protein expression; (2) micromolar concentrations of NO inhibit forskolin-stimulated cAMP production by adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP)-dependent fluid secretion, and cAMP-dependent Cl(-) and HCO(3)(-) transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and anion exchanger isoform 2, respectively; (3) cholestatic effects of NO and of proinflammatory cytokines are prevented by NOS-2 inhibitors and by agents (manganese(III)-tetrakis(4-benzoic acid)porphyrin [MnTBAP], urate, trolox) able to block the formation of reactive nitrogen oxide species (RNOS); (4) NOS2 expression is increased significantly in the biliary epithelium of patients with primary sclerosing cholangitis (PSC).
Our findings show that proinflammatory cytokines stimulate the biliary epithelium to generate NO, via NOS2 induction, and that NO causes ductular cholestasis by a RNOS-mediated inhibition of AC and of cAMP-dependent HCO(3)(-) and Cl(-) secretory mechanisms. This pathogenetic sequence may contribute to ductal cholestasis in inflammatory cholangiopathies.
Gastroenterology 03/2003; 124(3):737-53. · 11.68 Impact Factor
