Romina Fiorotto

Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.

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, Carolis 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 (Pkd2WS25/−). 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. (HEPATOLOGY 2006;43:1001–1012.)

Side Chain Structure Determines Unique Physiologic and Therapeutic Properties of norUrsodeoxycholic Acid in Mdr2−/− Mice

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 HCO3− ‐output. norUDCA directly stimulated fluid secretion in isolated bile duct units in a HCO3− ‐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 HCO3− ‐rich hypercholeresis that protects the liver from cholestatic injury. (HEPATOLOGY 2009;49:1972–1981.)

ERK1/2-Dependent Vascular Endothelial Growth Factor Signaling Sustains Cyst Growth in Polycystin-2 Defective Mice

BACKGROUND & AIMS 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. METHODS 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). RESULTS 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. CONCLUSIONS 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.

Loss of CFTR Affects Biliary Epithelium Innate Immunity and Causes TLR4–NF-κB—Mediated Inflammatory Response in Mice

BACKGROUND & AIMS 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.

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 (HIF1α)–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, HIF1α, 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 HIF1α 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 HIF1α‐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 2010.)

Platelet‐derived growth factor‐D and Rho GTPases regulate recruitment of cancer‐associated fibroblasts in cholangiocarcinoma

Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer‐associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial‐mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)‐expressing human male CCA cell line (EGI‐1) after xenotransplantation into severe‐combined‐immunodeficient mice, (3) expression of platelet‐derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF‐D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF‐D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α‐SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α‐SMA‐expressing CAFs, which were negative for EGFP and the human Y‐probe, but positive for the murine Y‐probe. CCA cells were strongly immunoreactive for PDGF‐A and ‐D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF‐D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF‐D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF‐D expression in CCA cells. In fibroblasts, PDGF‐D activated the Rac1 and Cdc42 Rho GTPases and c‐Jun N‐terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF‐D‐induced fibroblast migration. Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF‐D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF‐D pathway may offer a novel therapeutic approach. (Hepatology 2013;53:1042–1053)

DIFERENTIALLY EXPRESSED ADENYLYL CYCLASE ISOFORMS MEDIATE SECRETORY FUNCTIONS IN CHOLANGIOCYTE SUBPOPULATION

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 α‐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, HCO3−/CO2, cholinergic agonists, and β‐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 Ca2+‐insensitive isoforms (AC4, AC7) was higher in small duct cholangiocytes, whereas that of the Ca2+‐inhibitable (AC5, AC6, AC9), the Ca2+/calmodulin‐stimulated AC8, and the soluble sAC was higher in large duct cholangiocytes. Ca2+/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 γ‐aminobutyric acid–inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO3−]i was cAMP‐dependent and inhibited by sAC inhibitor and sAC gene silencing. Treatment with lipopolysaccharide or α‐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, β‐adrenergic agonists, or changes in [HCO3−]i, respectively; and (3) AC gene expression is modulated in experimental cholestasis. (HEPATOLOGY 2009)

Notch signaling regulates tubular morphogenesis during repair from biliary damage in mice

BACKGROUND & 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 Treatment with DDC (3,5-diethoxycarbonyl-1,4-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. RESULTS 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-Jk-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, Notch-2 or Jagged-1 significantly reduced both the length and 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.

Altered store operated calcium entry increases cyclic 3′,5′‐adenosine monophosphate production and extracellular signal‐regulated kinases 1 and 2 phosphorylation in polycystin‐2‐defective cholangiocytes

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 Ca2+ and cAMP production and inappropriate ERK signaling in PC2‐defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional‐KO (knockout) mice (Pkd2flox/−: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‐Ca2+ (measured with Fura‐2 and Mag‐Fluo4) levels are decreased and store‐operated Ca2+ entry (SOCE) is inhibited, whereas the expression of Ca2+‐sensor stromal interaction molecule 1 (STIM1) and store‐operated Ca2+ channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER‐Ca2+ 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 Ca2+ 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 2012)

Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development

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 2008.)