Mario Strazzabosco

Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse cholangiopathy model by promoting biliary HCO⁻₃ output

Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. The nuclear farnesoid X receptor (FXR) and the membrane G protein‐coupled receptor, TGR5, regulate bile acid (BA) homeostasis and inflammation. Therefore, we hypothesized that activation of FXR and/or TGR5 could ameliorate liver injury in Mdr2−/− (Abcb4−/−) mice, a model of chronic cholangiopathy. Hepatic inflammation, fibrosis, as well as bile secretion and key genes of BA homeostasis were addressed in Mdr2−/− mice fed either a chow diet or a diet supplemented with the FXR agonist, INT‐747, the TGR5 agonist, INT‐777, or the dual FXR/TGR5 agonist, INT‐767 (0.03% w/w). Only the dual FXR/TGR5 agonist, INT‐767, significantly improved serum liver enzymes, hepatic inflammation, and biliary fibrosis in Mdr2−/− mice, whereas INT‐747 and INT‐777 had no hepatoprotective effects. In line with this, INT‐767 significantly induced bile flow and biliary HCO  3− output, as well as gene expression of carbonic anhydrase 14, an important enzyme able to enhance HCO  3− transport, in an Fxr‐dependent manner. In addition, INT‐767 dramatically reduced bile acid synthesis via the induction of ileal Fgf15 and hepatic Shp gene expression, thus resulting in significantly reduced biliary bile acid output in Mdr2−/− mice. Conclusion: This study shows that FXR activation improves liver injury in a mouse model of chronic cholangiopathy by reduction of biliary BA output and promotion of HCO  3− ‐rich bile secretion. (HEPATOLOGY 2011;54:1303–1312)

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.)

Characterization and Isolation of Ductular Cells Coexpressing Neural Cell Adhesion Molecule and Bcl-2 from Primary Cholangiopathies and Ductal Plate Malformations

It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10-16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies.

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.)

Intracellular pH regulation in isolated rat bile duct epithelial cells.

To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport.

Development of the bile ducts: Essentials for the clinical hepatologist

Several cholangiopathies result from a perturbation of developmental processes. Most of these cholangiopathies are characterised by the persistence of biliary structures with foetal configuration. Developmental processes are also relevant in acquired liver diseases, as liver repair mechanisms exploit a range of autocrine and paracrine signals transiently expressed in embryonic life. We briefly review the ontogenesis of the intra- and extrahepatic biliary tree, highlighting the morphogens, growth factors, and transcription factors that regulate biliary development, and the relationships between developing bile ducts and other branching biliary structures. Then, we discuss the ontogenetic mechanisms involved in liver repair, and how these mechanisms are recapitulated in ductular reaction, a common reparative response to many forms of biliary and hepatocellular damage. Finally, we discuss the pathogenic aspects of the most important primary cholangiopathies related to altered biliary development, i.e. polycystic and fibropolycystic liver diseases, Alagille syndrome.

Emerging roles of Notch signaling in liver disease

This review critically discusses the most recent advances in the role of Notch signaling in liver development, homeostasis, and disease. It is now clear that the significance of Notch in determining mammalian cell fates and functions extends beyond development, and Notch is a major regular of organ homeostasis. Moreover, Notch signaling is reactivated upon injury and regulates the complex interactions between the distinct liver cell types involved in the repair process. Notch is also involved in the regulation of liver metabolism, inflammation, and cancer. The net effects of Notch signaling are highly variable and finely regulated at multiple levels, but also depend on the specific cellular context in which Notch is activated. Persistent activation of Notch signaling is associated with liver malignancies, such as hepatocellular carcinoma with stem cell features and intrahepatic cholangiocarcinoma. The complexity of the pathway provides several possible targets for agents able to inhibit Notch. However, further cell‐ and context‐specific in‐depth understanding of Notch signaling in liver homeostasis and disease will be essential to translate these concepts into clinical practice and be able to predict benefits and risks of evolving therapies. (Hepatology 2015;61:382–392)

Functional Anatomy of Normal Bile Ducts

The biliary tree is a complex network of conduits that begins with the canals of Hering and progressively merges into a system of interlobular, septal, and major ducts which then coalesce to form the extrahepatic bile ducts, which finally deliver bile to the gallbladder and to the intestine. The biliary epithelium shows a morphological heterogeneity that is strictly associated with a variety of functions performed at the different levels of the biliary tree. In addition to funneling bile into the intestine, cholangiocytes (the epithelial cells lining the bile ducts) are actively involved in bile production by performing both absorbitive and secretory functions. More recently, other important biological properties restricted to cholangiocytes lining the smaller bile ducts have been outlined, with regard to their plasticity (i.e., the ability to undergo limited phenotypic changes), reactivity (i.e., the ability to participate in the inflammatory reaction to liver damage), and ability to behave as liver progenitor cells. Functional interactions with other branching systems, such as nerve and vascular structures, are crucial in the modulation of the different cholangiocyte functions. Anat Rec, 291:653–660, 2008.

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.

Ca2+-activated Cl− channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion

This study addresses the mechanisms by which a defect in CFTR impairs pancreatic duct bicarbonate secretion in cystic fibrosis. We used control (PANC‐1) and CFTR‐deficient (CFPAC‐1; ΔF508 mutation) cell lines and measured HCO3− extrusion by the rate of recovery of intracellular pH after an alkaline load and recorded whole cell membrane currents using patch clamp techniques. 1) In PANC‐1 cells, cAMP causes parallel activation of Cl− channels and of HCO3− extrusion by DIDS‐sensitive and Na+‐independent Cl‐/HCO3− exchange, both effects being inhibited by Cl− channel blockers NPPB and glibenclamide. 2) In CFPAC‐1 cells, cAMP fails to stimulate Cl−/ HCO3− exchange and Cl− channels, except after promoting surface expression of ΔF508‐CFTR by glycerol treatment. Instead, raising intracellular Ca2+ concentration to 1 μmol/l or stimulating purinergic receptors with ATP (10 and 100 μmol/l) leads to parallel activation of Cl− channels and HCO3− extrusion. 3) K+ channel function is required for coupling cAMP‐ and Ca2+‐dependent Cl− channel activation to effective stimulation of Cl−/HCO3− exchange in control and CF cells, respectively. It is concluded that stimulation of pancreatic duct bicarbonate secretion via Cl−/ HCO3− exchange is directly correlated to activation of apical membrane Cl− channels. Reduced bicarbonate secretion in cystic fibrosis results from defective cAMP‐activated Cl− channels. This defect is partially compensated for by an increased sensitivity of CF cells to purinergic stimulation and by alternative activation of Ca2+‐dependent Cl− channels, mechanisms of interest with respect to possible treatment of cystic fibrosis and of related chronic pancreatic diseases.—Zsembery, A., Strazzabosco, M., Graf, J. Ca2+‐activated Cl− channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion. FASEB J. 14, 2345–2356 (2000)