Leptin Enhances Hepatic Fibrosis and Inflammation in a Mouse Model of Cholestasis.

Leptin is an adipokine with roles in food intake and energy metabolism through its actions on neurons in the hypothalamus. The role of leptin in obesity and cardiovascular disorders is well documented. However, its influence on liver conditions such as cholestasis is poorly understood. The effects of exogenous leptin and leptin-neutralizing antibody on biliary hyperplasia, hepatic fibrosis, and inflammation in the multidrug resistance protein 2 knockout (Mdr2KO) mouse model of cholestasis were assessed by quantifying markers specific for cholangiocytes, activated hepatic stellate cells (HSCs), and cytokines. Serum and hepatic leptin were increased in Mdr2KO mice compared with FVB/NJ (FVBN) controls, and exogenous leptin enhanced biliary hyperplasia and liver fibrosis in Mdr2KO and FVBN mice. Leptin administration increased hepatic expression of C-C motif chemokine ligand 2 and IL-6 in Mdr2KO mice. In contrast, leptin-neutralizing antibody reduced intrahepatic bile duct mass and decreased HSC activation in Mdr2KO mice compared with FVBN controls. Sex-related differences were noted, with female Mdr2KO mice having more leptin than males. In cholangiocytes and LX2 cells in vitro, leptin increased phosphorylated Akt and stimulated cell proliferation. Leptin receptor siRNA and inhibitors of Akt phosphorylation impaired leptin-induced cell proliferation and proinflammatory cytokines. The current data suggest that leptin is abnormally increased in cholestatic mice, and excess leptin increases ductular reaction, hepatic fibrosis, and inflammation via leptin receptor-mediated phosphorylation of Akt in cholangiocytes and HSCs.

Modulation of the Tryptophan Hydroxylase 1/Monoamine Oxidase-A/5-Hydroxytryptamine/5-Hydroxytryptamine Receptor 2A/2B/2C Axis Regulates Biliary Proliferation and Liver Fibrosis During Cholestasis.

BACKGROUND AND AIMS: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. APPROACH AND RESULTS: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. CONCLUSIONS: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

Correction to: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA.

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell-derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)-/- mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2-/- mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2-/- mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Primary biliary cholangitis (PBC) primarily targets cholangiocytes and is characterized by liver fibrosis and biliary proliferation. Activation of the secretin (Sct)/secretin receptor (SR) axis, expressed only by cholangiocytes, increases biliary proliferation, liver fibrosis, and bicarbonate secretion. We evaluated the effectiveness of SR antagonist treatment for early-stage PBC. Male and female dominant-negative TGF-ß receptor II (dnTGF-ßRII) (model of PBC) and wild-type mice at 12 wk of age were treated with saline or the SR antagonist, Sec 5-27, for 1 wk. dnTGF-ßRII mice expressed features of early-stage PBC along with enhanced Sct/SR axis activation and Sct secretion. dnTGF-ßRII mice had increased biliary proliferation or senescence, inflammation, and liver fibrosis. In dnTGF-ßRII mice, there was increased microRNA-125b/TGF-ß1/TGF-ß receptor 1/VEGF-A signaling. Human early-stage PBC patients had an increase in hepatobiliary Sct and SR expression and serum Sct levels. Increased biliary Sct/SR signaling promotes biliary and hepatic damage during early-stage PBC.-Kennedy, L., Francis, H., Invernizzi, P., Venter, J., Wu, N., Carbone, M., Gershwin, M. E., Bernuzzi, F., Franchitto, A., Alvaro, D., Marzioni, M., Onori, P., Gaudio, E., Sybenga, A., Fabris, L., Meng, F., Glaser, S., Alpini, G. Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes.

α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP-/-) mouse model. α-CGRP-/- and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP-/- and BDL α-CGRP-/- mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP-/- mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

The Secretin/Secretin Receptor Axis Modulates Ductular Reaction and Liver Fibrosis through Changes in Transforming Growth Factor-ß1-Mediated Biliary Senescence.

Activation of the secretin (Sct)/secretin receptor (SR) axis stimulates ductular reaction and liver fibrosis, which are hallmarks of cholangiopathies. Our aim was to define the role of Sct-regulated cellular senescence, and we demonstrated that both ductular reaction and liver fibrosis are significantly reduced in Sct-/-, SR-/-, and Sct-/-/SR-/- bile duct ligated (BDL) mice compared with BDL wild-type mice. The reduction in hepatic fibrosis in Sct-/-, SR-/-, and Sct-/-/SR-/- BDL mice was accompanied by reduced transforming growth factor-ß1 levels in serum and cholangiocyte supernatant, as well as decreased expression of markers of cellular senescence in cholangiocytes in contrast to enhanced cellular senescence in hepatic stellate cells compared with BDL wild-type mice. Secretin directly stimulated the senescence of cholangiocytes and regulated, by a paracrine mechanism, the senescence of hepatic stellate cells and liver fibrosis via modulation of transforming growth factor-ß1 biliary secretion. Targeting senescent cholangiocytes may represent a novel therapeutic approach for ameliorating hepatic fibrosis during cholestatic liver injury.

Knockout of secretin receptor reduces biliary damage and liver fibrosis in Mdr2-/- mice by diminishing senescence of cholangiocytes.

Secretin receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2-/- mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR-/-, Mdr2-/-, and SR-/-/Mdr2-/- mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-ß-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-ß1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2-/- mice was reduced in SR-/-/Mdr2-/- mice. Enhanced senescence levels in cholangiocytes from Mdr2-/- mice were reversed to normal in SR-/-/Mdr2-/- mice. However, senescence was decreased in HSCs from Mdr2-/- mice but returned to normal values in SR-/-/Mdr2-/- mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-ß1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-ß1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.

miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation.

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24-dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.

Knockdown of Hepatic Gonadotropin-Releasing Hormone by Vivo-Morpholino Decreases Liver Fibrosis in Multidrug Resistance Gene 2 Knockout Mice by Down-Regulation of miR-200b.

Hepatic fibrosis occurs during the progression of primary sclerosing cholangitis (PSC) and is characterized by accumulation of extracellular matrix proteins. Proliferating cholangiocytes and activated hepatic stellate cells (HSCs) participate in the promotion of liver fibrosis during cholestasis. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone synthesized by hypothalamic neurons and the biliary epithelium and exerts its biological effects on cholangiocytes by interaction with the receptor subtype (GnRHR1) expressed by cholangiocytes and HSCs. Previously, we demonstrated that administration of GnRH to normal rats increased intrahepatic biliary mass (IBDM) and hepatic fibrosis. Also, miR-200b is associated with the progression of hepatic fibrosis; however, the role of the GnRH/GnRHR1/miR-200b axis in the development of hepatic fibrosis in PSC is unknown. Herein, using the mouse model of PSC (multidrug resistance gene 2 knockout), the hepatic knockdown of GnRH decreased IBDM and liver fibrosis. In vivo and in vitro administration of GnRH increased the expression of miR-200b and fibrosis markers. The GnRH/GnRHR1 axis and miR-200b were up-regulated in human PSC samples. Cetrorelix, a GnRHR1 antagonist, inhibited the expression of fibrotic genes in vitro and decreased IBDM and hepatic fibrosis in vivo. Inhibition of miR-200b decreased the expression of fibrosis genes in vitro in cholangiocyte and HSC lines. Targeting the GnRH/GnRHR1/miR-200b axis may be key for the management of hepatic fibrosis during the progression of PSC.

Regulation of Cellular Senescence by miR-34a in Alcoholic Liver Injury.

Alcoholic liver disease remains a major cause of liver-related morbidity and mortality, which ranges from alcoholic steatohepatitis to fibrosis/cirrhosis and hepatocellular carcinoma, and the related mechanisms are understood poorly. In this study, we aimed to investigate the role of miR-34a in alcohol-induced cellular senescence and liver fibrosis. We found that hepatic miR-34a expression was upregulated in ethanol-fed mice and heavy drinkers with steatohepatitis compared with respective controls. Mice treated with miR-34a Vivo-Morpholino developed less severe liver fibrosis than wild-type mice after 5 weeks of ethanol feeding. Further mechanism exploration showed that inhibition of miR-34a increased cellular senescence of hepatic stellate cells (HSCs) in ethanol-fed mice, although it decreased senescence in total liver and hepatocytes, which was verified by the changes of senescence-associated ß-galactosidase and gene expression. Furthermore, enhanced cellular senescence was observed in liver tissues from steatohepatitis patients compared with healthy controls. In addition, the expression of transforming growth factor-ß1, drosophila mothers against decapentaplegic protein 2 (Smad2), and Smad3 was decreased after inhibition of miR-34a in ethanol-fed mice. Our in vitro experiments showed that silencing of miR-34a partially blocked activation of HSCs by lipopolysaccharide and enhanced senescence of HSCs. Furthermore, inhibition of miR-34a decreased lipopolysaccharide-induced fibrotic gene expression in cultured hepatocytes. In conclusion, our data suggest that miR-34a functions as a profibrotic factor that promotes alcohol-induced liver fibrosis by reducing HSC senescence and increasing the senescence of hepatocytes.

Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct-ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R-/- ) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2-/- ) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2-/- mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2-/- mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-ß1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R-/- mice with BDL surgery or Mdr2-/- mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. CONCLUSION: Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528-541).

Forkhead box A2 regulates biliary heterogeneity and senescence during cholestatic liver injury in mice‡.

Biliary-committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real-time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2-/- ) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post-BDL showed significant changes compared to vehicle-treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2-/- mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. CONCLUSION: The definitive endoderm marker and the positive regulator of biliary development, FoxA2, mediates the therapeutic effect of biliary-committed progenitor cells during cholestatic liver injury. (Hepatology 2017;65:544-559).

Prolonged darkness reduces liver fibrosis in a mouse model of primary sclerosing cholangitis by miR-200b down-regulation.

Melatonin therapy or prolonged exposure to complete darkness reduces biliary hyperplasia and liver fibrosis in bile-duct-ligated (BDL) rats; however, no information exists in primary sclerosing cholangitis (PSC). Thus, we aimed to determine the therapeutic effects of prolonged dark therapy or melatonin administration on hepatic fibrosis in the multidrug resistance gene 2-knockout (Mdr2-/-) mouse model of PSC. Melatonin levels, biliary mass, liver fibrosis, angiogenesis and miR-200b expression were evaluated in wild-type and Mdr2-/- mice exposed to darkness or melatonin treatment or in male patients with PSC and healthy controls. Mdr2-/- mice were also treated with miR-200b inhibitor or control before evaluating biliary mass, liver fibrosis, and angiogenesis. After overexpression of arylalkylamine N-acetyltransferase (AANAT; the enzyme regulating melatonin synthesis) or inhibition of miR-200b in cholangiocytes and hepatic stellate cells in vitro, we evaluated angiogenesis and fibrosis gene expression. After exposure to darkness or administration of melatonin, Mdr2-/- mice show elevated serum melatonin levels and inhibition of biliary mass, along with reduction of liver fibrosis and angiogenesis. MicroRNA PCR analysis demonstrated that miR-200b expression increased in Mdr2-/- mice and patients with PSC compared with controls and decreased in Mdr2-/- mice subjected to dark exposure or melatonin treatment. Inhibition of miR-200b in Mdr2-/- ablates biliary proliferation, liver fibrosis, and angiogenesis. In vitro, overexpression of AANAT or inhibition of miR-200b in cholangiocytes and hepatic stellate cells decreased the expression of miR-200b, angiogenesis, and fibrosis genes. Dark therapy or targeting melatonin/miR-200b axis may be important in the management of biliary damage and liver fibrosis in cholangiopathies including PSC.-Wu, N., Meng, F., Zhou, T., Han, Y., Kennedy, L., Venter, J., Francis, H., DeMorrow, S., Onori, P., Invernizzi, P., Bernuzzi, F., Mancinelli, R., Gaudio, E., Franchitto, A., Glaser, S., Alpini G. Prolonged darkness reduces liver fibrosis in a mouse model of primary sclerosing cholangitis by miR-200b down-regulation.

α7-nAChR Knockout Mice Decreases Biliary Hyperplasia and Liver Fibrosis in Cholestatic Bile Duct-Ligated Mice.

α7-nAChR is a nicotinic acetylcholine receptor [specifically expressed on hepatic stellate cells (HSCs), Kupffer cells, and cholangiocytes] that regulates inflammation and apoptosis in the liver. Thus, targeting α7-nAChR may be therapeutic in biliary diseases. Bile duct ligation (BDL) was performed on wild-type (WT) and α7-nAChR-/- mice. We first evaluated the expression of α7-nAChR by immunohistochemistry (IHC) in liver sections. IHC was also performed to assess intrahepatic bile duct mass (IBDM), and Sirius Red staining was performed to quantify the amount of collagen deposition. Immunofluorescence was performed to assess colocalization of α7-nAChR with bile ducts (costained with CK-19) and HSCs (costained with desmin). The mRNA expression of α7-nAChR, Ki-67/PCNA (proliferation), fibrosis genes (TGF-ß1, fibronectin-1, Col1α1, and α-SMA), and inflammatory markers (IL-6, IL-1ß, and TNF-α) was measured by real-time PCR. Biliary TGF-ß1 and hepatic CD68 (Kupffer cell marker) expression was assessed using IHC. α7-nAChR immunoreactivity was observed in both bile ducts and HSCs and increased following BDL. α7-nAChR-/- BDL mice exhibited decreased (i) bile duct mass, liver fibrosis, and inflammation, and (ii) immunoreactivity of TGF-ß1 as well as expression of fibrosis genes compared to WT BDL mice. α7-nAChR activation triggers biliary proliferation and liver fibrosis and may be a therapeutic target in managing extrahepatic biliary obstruction.

The Hippo signaling functions through the Notch signaling to regulate intrahepatic bile duct development in mammals.

The Hippo signaling pathway and the Notch signaling pathway are evolutionary conserved signaling cascades that have important roles in embryonic development of many organs. In murine liver, disruption of either pathway impairs intrahepatic bile duct development. Recent studies suggested that the Notch signaling receptor Notch2 is a direct transcriptional target of the Hippo signaling pathway effector YAP, and the Notch signaling is a major mediator of the Hippo signaling in maintaining biliary cell characteristics in adult mice. However, it remains to be determined whether the Hippo signaling pathway functions through the Notch signaling in intrahepatic bile duct development. We found that loss of the Hippo signaling pathway tumor suppressor Nf2 resulted in increased expression levels of the Notch signaling pathway receptor Notch2 in cholangiocytes but not in hepatocytes. When knocking down Notch2 on the background of Nf2 deficiency in mouse livers, the excessive bile duct development induced by Nf2 deficiency was suppressed by heterozygous and homozygous deletion of Notch2 in a dose-dependent manner. These results implicated that Notch signaling is one of the downstream effectors of the Hippo signaling pathway in regulating intrahepatic bile duct development.

Melatonin inhibits hypothalamic gonadotropin-releasing hormone release and reduces biliary hyperplasia and fibrosis in cholestatic rats.

Melatonin is a hormone produced by the pineal gland with increased circulating levels shown to inhibit biliary hyperplasia and fibrosis during cholestatic liver injury. Melatonin also has the capability to suppress the release of hypothalamic gonadotropin-releasing hormone (GnRH), a hormone that promotes cholangiocyte proliferation when serum levels are elevated. However, the interplay and contribution of neural melatonin and GnRH to cholangiocyte proliferation and fibrosis in bile duct-ligated (BDL) rats have not been investigated. To test this, cranial levels of melatonin were increased by implanting osmotic minipumps that performed an intracerebroventricular (ICV) infusion of melatonin or saline for 7 days starting at the time of BDL. Hypothalamic GnRH mRNA and cholangiocyte secretion of GnRH and melatonin were assessed. Cholangiocyte proliferation and fibrosis were measured. Primary human hepatic stellate cells (HSCs) were treated with cholangiocyte supernatants, GnRH, or the GnRH receptor antagonist cetrorelix acetate, and cell proliferation and fibrosis gene expression were assessed. Melatonin infusion reduced hypothalamic GnRH mRNA expression and led to decreased GnRH and increased melatonin secretion from cholangiocytes. Infusion of melatonin was found to reduce hepatic injury, cholangiocyte proliferation, and fibrosis during BDL-induced liver injury. HSCs supplemented with BDL cholangiocyte supernatant had increased proliferation, and this increase was reversed when HSCs were supplemented with supernatants from melatonin-infused rats. GnRH stimulated fibrosis gene expression in HSCs, and this was reversed by cetrorelix acetate cotreatment. Increasing bioavailability of melatonin in the brain may improve outcomes during cholestatic liver disease.NEW & NOTEWORTHY We have previously demonstrated that GnRH is expressed in cholangiocytes and promotes their proliferation during cholestasis. In addition, dark therapy, which increases melatonin, reduced cholangiocyte proliferation and fibrosis during cholestasis. This study expands these findings by investigating neural GnRH regulation by melatonin during BDL-induced cholestasis by infusing melatonin into the brain. Melatonin infusion reduced cholangiocyte proliferation and fibrosis, and these effects are due to GNRH receptor 1-dependent paracrine signaling between cholangiocytes and hepatic stellate cells.

The secretin/secretin receptor axis modulates liver fibrosis through changes in transforming growth factor-ß1 biliary secretion in mice.

UNLABELLED: The secretin/secretin receptor (SR) axis is up-regulated by proliferating cholangiocytes during cholestasis. Secretin stimulates biliary proliferation by down-regulation of let-7a and subsequent up-regulation of the growth-promoting factor, nerve growth factor (NGF). It is not known whether the secretin/SR axis plays a role in subepithelial fibrosis observed during cholestasis. Our aim was to determine the role of the secretin/SR axis in activation of biliary fibrosis in animal models and human primary sclerosing cholangitis (PSC). Studies were performed in wild-type (WT) mice with bile duct ligation (BDL), BDL SR(-/-) mice, or Mdr2(-/-) mouse models of cholestatic liver injury. In selected studies, the SR antagonist (Sec 5-27) was used to block the secretin/SR axis. Biliary proliferation and fibrosis were evaluated as well as secretion of secretin (by cholangiocytes and S cells), expression of markers of fibrosis, transforming growth factor-ß1 (TGF-ß1), transforming growth factor-ß1 receptor (TGF-ß1R), let-7a, and downstream expression of NGF. Correlative studies were performed in human control and PSC liver tissue biopsies, serum, and bile. SR antagonist reduced biliary proliferation and hepatic fibrosis in BDL WT and Mdr2(-/-) mice. There was decreased expression of let-7a in BDL and Mdr2(-/-) cholangiocytes that was associated with increased NGF expression. Inhibition of let-7a accelerated liver fibrosis was attributed to cholestasis. There was increased expression of TGF-ß1 and TGF-ß1R. Significantly higher expression of secretin, SR, and TGF-ß1 was observed in PSC patient liver samples compared to healthy controls. In addition, there was higher expression of fibrosis genes and remarkably decreased expression of let-7a and increased expression of NGF compared to the control. CONCLUSION: The secretin/SR axis plays a key role in regulating the biliary contribution to cholestasis-induced hepatic fibrosis. (Hepatology 2016;64:865-879).

Vasopressin regulates the growth of the biliary epithelium in polycystic liver disease.

The neurohypophysial hormone arginine vasopressin (AVP) acts by three distinct receptor subtypes: V1a, V1b, and V2. In the liver, AVP is involved in ureogenesis, glycogenolysis, neoglucogenesis and regeneration. No data exist about the presence of AVP in the biliary epithelium. Cholangiocytes are the target cells in a number of animal models of cholestasis, including bile duct ligation (BDL), and in several human pathologies, such as polycystic liver disease characterized by the presence of cysts that bud from the biliary epithelium. In vivo, liver fragments from normal and BDL mice and rats as well as liver samples from normal and ADPKD patients were collected to evaluate: (i) intrahepatic bile duct mass by immunohistochemistry for cytokeratin-19; and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real-time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from the cystic epithelium) were stimulated with vasopressin in the absence/presence of AVP antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT assay and cAMP levels. Cholangiocytes express V2 receptor that was upregulated following BDL and in ADPKD liver samples. Administration of AVP increased proliferation and cAMP levels of small cholangiocytes and LCDE cells. We found no effect in the proliferation of large mouse cholangiocytes and H69 cells. Increases were blocked by preincubation with the AVP antagonists. These results showed that AVP and its receptors may be important in the modulation of the proliferation rate of the biliary epithelium.

Knockout of microRNA-21 reduces biliary hyperplasia and liver fibrosis in cholestatic bile duct ligated mice.

Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to have a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR-21-/- mice underwent Sham or bile duct ligation (BDL) for 1 week, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and small mothers against decapentaplegic 7 (Smad-7) expression. In vitro, immortalized murine biliary cell lines (IMCLs) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. In addition, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers transforming growth factor-ß1 and α-smooth muscle actin. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared with control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury, miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis.