Prolonged administration of a secretin receptor antagonist inhibits biliary senescence and liver fibrosis in Mdr2 -/- mice.

BACKGROUND AND AIMS: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 -/- [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2 -/- mice. APPROACH AND RESULTS: In vivo studies were performed in male wild-type and Mdr2 -/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 -/- mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-ß1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 -/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 -/- compared with wild-type mice, which was reversed by long-term SCT 5-27 treatment. In vitro , SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-ß1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. CONCLUSIONS: Modulating the SCT/SR axis may be critical for managing PSC.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential.

BACKGROUND & AIMS: Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. METHODS: Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers. RESULTS: In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. In vitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. In vivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver. CONCLUSIONS: A cell population with clonal growth and organoid formation capability, which has liver differentiation potency in vitro and in vivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver. IMPACT AND IMPLICATIONS: Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Melatonin receptor 1A, but not 1B, knockout decreases biliary damage and liver fibrosis during cholestatic liver injury.

BACKGROUND AND AIMS: Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein-coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFß receptor type I (TGFßRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. APPROACH AND RESULTS: Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2-/- ) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2-/- mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin's interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFßR1 signaling, which was reduced by loss of MT1. CONCLUSIONS: Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFßR1 activation. Blocking GPR50/TGFßR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.

Effect of Calcium-Sulphate-Bicarbonate Water in a Murine Model of Non-Alcoholic Fatty Liver Disease: A Histopathology Study.

The progression of nonalcoholic fatty liver disease (NAFLD) is associated with alterations of the gut-liver axis. The activation of toll-like receptor 4 (TLR4) pathways by endotoxins, such as lipopolysaccharide (LPS), contributes to liver injury. The aim of the present study was to evaluate the possible beneficial effects of a calcium-sulphate-bicarbonate natural mineral water on the gut-liver axis by evaluating liver and terminal ileum histopathology in a murine model of NAFLD. NAFLD was induced in mice by administrating a methionine-choline-deficient (MCD) diet. The following experimental groups were evaluated: controls (N = 10); MCD+Tap water (MCD; N = 10); MCD+Calcium-sulphate-bicarbonate water (MCD/Wcsb; N = 10). Mice were euthanised after 4 and 8 weeks. Liver and terminal ileum samples were collected. Samples were studied by histomorphology, immunohistochemistry, and immunofluorescence. In mice subjected to the MCD diet, treatment with mineral water improved inflammation and fibrosis, and was associated with a reduced number of activated hepatic stellate cells when compared to MCD mice not treated with mineral water. Moreover, MCD/Wcsb mice showed lower liver LPS localization and less activation of TLR4 pathways compared to the MCD. Finally, Wcsb treatment was associated with improved histopathology and higher occludin positivity in intestinal mucosa. In conclusion, calcium-sulphate-bicarbonate water may exert modulatory activity on the gut-liver axis in MCD mice, suggesting potential beneficial effects on NAFLD.

Knockout of the Tachykinin Receptor 1 in the Mdr2-/- (Abcb4-/-) Mouse Model of Primary Sclerosing Cholangitis Reduces Biliary Damage and Liver Fibrosis.

Activation of the substance P (SP)/neurokinin 1 receptor (NK1R) axis triggers biliary damage/senescence and liver fibrosis in bile duct ligated and Mdr2-/- (alias Abcb4-/-) mice through enhanced transforming growth factor-ß1 (TGF-ß1) biliary secretion. Recent evidence indicates a role for miR-31 (MIR31) in TGF-ß1-induced liver fibrosis. We aimed to define the role of the SP/NK1R/TGF-ß1/miR-31 axis in regulating biliary proliferation and liver fibrosis during cholestasis. Thus, we generated a novel model with double knockout of Mdr2-/- and NK1R-/ (alias Tacr1-/-) to further address the role of the SP/NK1R axis during chronic cholestasis. In vivo studies were performed in the following 12-week-old male mice: (i) NK1R-/-; (ii) Mdr2-/-; and (iii) NK1R-/-/Mdr2-/- (Tacr1-/-/Abcb4-/-) and their corresponding wild-type controls. Liver tissues and cholangiocytes were collected, and liver damage, changes in biliary mass/senescence, and inflammation as well as liver fibrosis were evaluated by both immunohistochemistry in liver sections and real-time PCR. miR-31 expression was measured by real-time PCR in isolated cholangiocytes. Decreased ductular reaction, liver fibrosis, biliary senescence, and biliary inflammation were observed in NK1R-/-/Mdr2-/- mice compared with Mdr2-/- mice. Elevated expression of miR-31 was observed in Mdr2-/- mice, which was reduced in NK1R-/-/Mdr2-/- mice. Targeting the SP/NK1R and/or miR-31 may be a potential approach in treating human cholangiopathies, including primary sclerosing cholangitis.

Peribiliary Gland Niche Participates in Biliary Tree Regeneration in Mouse and in Human Primary Sclerosing Cholangitis.

BACKGROUND AND AIMS: Mechanisms underlying the repair of extrahepatic biliary tree (EHBT) after injury have been scarcely explored. The aims of this study were to evaluate, by using a lineage tracing approach, the contribution of peribiliary gland (PBG) niche in the regeneration of EHBT after damage and to evaluate, in vivo and in vitro, the signaling pathways involved. APPROACH AND RESULTS: Bile duct injury was induced by the administration of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 14 days to Krt19Cre TdTomatoLSL mice. Human biliary tree stem/progenitor cells (BTSC) within PBGs were isolated from EHBT obtained from liver donors. Hepatic duct samples (n = 10) were obtained from patients affected by primary sclerosing cholangitis (PSC). Samples were analyzed by histology, immunohistochemistry, western blotting, and polymerase chain reaction. DDC administration causes hyperplasia of PBGs and periductal fibrosis in EHBT. A PBG cell population (Cytokeratin19- /SOX9+ ) is involved in the renewal of surface epithelium in injured EHBT. The Wnt signaling pathway triggers human BTSC proliferation in vitro and influences PBG hyperplasia in vivo in the DDC-mediated mouse biliary injury model. The Notch signaling pathway activation induces BTSC differentiation in vitro toward mature cholangiocytes and is associated with PBG activation in the DDC model. In human PSC, inflammatory and stromal cells trigger PBG activation through the up-regulation of the Wnt and Notch signaling pathways. CONCLUSIONS: We demonstrated the involvement of PBG cells in regenerating the injured biliary epithelium and identified the signaling pathways driving BTSC activation. These results could have relevant implications on the pathophysiology and treatment of cholangiopathies.

The Contribution of the Adipose Tissue-Liver Axis in Pediatric Patients with Nonalcoholic Fatty Liver Disease after Laparoscopic Sleeve Gastrectomy.

OBJECTIVE: To evaluate the histopathologic modifications in liver and visceral adipose tissue (VAT), and to correlate these changes with clinical measures, adipokine production, and proinflammatory cytokines in a population of adolescents with obesity with nonalcoholic fatty liver disease (NAFLD) who underwent laparoscopic sleeve gastrectomy (LSG). STUDY DESIGN: Twenty adolescents with obesity who underwent LSG and with biopsy-proven NAFLD were included. Patients underwent clinical evaluation and blood tests at baseline and 1 year after the surgical procedure. Liver and VAT specimens were processed for routine histology, immunohistochemistry, and immunofluorescence. RESULTS: In adolescents with obesity and NAFLD, hepatic histologic alterations were uncorrelated with VAT inflammation. LSG induced in both liver and VAT tissue histopathology amelioration and macrophage profile modification that were correlated with body mass index and improvement in insulin resistance. The adipokine profile in liver and VAT was associated with weight loss and histologic improvement after LSG. Serum proinflammatory cytokines were correlated with liver and VAT histopathology and IL-1ß and IL-6 levels were independently predicted by liver necroinflammatory grade. CONCLUSIONS: This study suggests a unique adipose tissue/fatty liver crosstalk in pediatric patients. LSG induces a similar pattern of histologic improvement in the liver and in VAT. Besides VAT, our results strengthen the role of the liver in adipocytokine production and its contribution to systemic inflammation in pediatric patients with NAFLD.

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.

Melatonin and circadian rhythms in liver diseases: Functional roles and potential therapies.

Circadian rhythms and clock gene expressions are regulated by the suprachiasmatic nucleus in the hypothalamus, and melatonin is produced in the pineal gland. Although the brain detects the light through retinas and regulates rhythms and melatonin secretion throughout the body, the liver has independent circadian rhythms and expressions as well as melatonin production. Previous studies indicate the association between circadian rhythms with various liver diseases, and disruption of rhythms or clock gene expression may promote liver steatosis, inflammation, or cancer development. It is well known that melatonin has strong antioxidant effects. Alcohol drinking or excess fatty acid accumulation produces reactive oxygen species and oxidative stress in the liver leading to liver injuries. Melatonin administration protects these oxidative stress-induced liver damage and improves liver conditions. Recent studies have demonstrated that melatonin administration is not limited to antioxidant effects and it has various other effects contributing to the management of liver conditions. Accumulating evidence suggests that restoring circadian rhythms or expressions as well as melatonin supplementation may be promising therapeutic strategies for liver diseases. This review summarizes recent findings for the functional roles and therapeutic potentials of circadian rhythms and melatonin in liver diseases.

Viral Hepatitis and Iron Dysregulation: Molecular Pathways and the Role of Lactoferrin.

The liver is a frontline immune site specifically designed to check and detect potential pathogens from the bloodstream to maintain a general state of immune hyporesponsiveness. One of the main functions of the liver is the regulation of iron homeostasis. The liver detects changes in systemic iron requirements and can regulate its concentration. Pathological states lead to the dysregulation of iron homeostasis which, in turn, can promote infectious and inflammatory processes. In this context, hepatic viruses deviate hepatocytes' iron metabolism in order to better replicate. Indeed, some viruses are able to alter the expression of iron-related proteins or exploit host receptors to enter inside host cells. Lactoferrin (Lf), a multifunctional iron-binding glycoprotein belonging to the innate immunity, is endowed with potent antiviral activity, mainly related to its ability to block viral entry into host cells by interacting with viral and/or cell surface receptors. Moreover, Lf can act as an iron scavenger by both direct iron-chelation or the modulation of the main iron-related proteins. In this review, the complex interplay between viral hepatitis, iron homeostasis, and inflammation as well as the role of Lf are outlined.

Hepatic Stem/Progenitor Cell Activation Differs between Primary Sclerosing and Primary Biliary Cholangitis.

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are human primary cholangiopathies characterized by the damage of mature cholangiocytes and by the appearance of ductular reaction (DR) as the results of hepatic progenitor cell activation. This study evaluated the differences in progenitor cell niche activation between these two cholangiopathies. Liver tissue was obtained from healthy liver donors (n = 5) and from patients with PSC (n = 20) or PBC (n = 20). DR, progenitor cell phenotype, and signaling pathways were investigated by IHC analysis and immunofluorescence. Our results indicated that DR was more extended, appeared earlier, and had a higher proliferation index in PBC compared with PSC. In PBC, DR was strongly correlated with clinical prognostic scores. A higher percentage of sex determining region Y-box (SOX)9+ and cytokeratin 19+ cells but fewer features of hepatocyte fate characterized progenitor cell activation in PBC versus PSC. Lower levels of laminin and neurogenic locus notch homolog protein 1 but higher expression of wingless-related integration site (WNT) family pathway components characterize progenitor cell niche in PSC compared with PBC. In conclusion, progenitor cell activation differs between PSC and PBC and is characterized by a divergent fate commitment and different signaling pathway predominance. In PBC, DR represents a relevant histologic prognostic marker.

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.

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.

Laparoscopic Sleeve Gastrectomy Improves Nonalcoholic Fatty Liver Disease-Related Liver Damage in Adolescents by Reshaping Cellular Interactions and Hepatic Adipocytokine Production.

OBJECTIVES: To investigate whether the modulation of local cellular cross-talks and the modification of hepatic adipocytokine expression could mechanistically explain the improvement of liver histopathology after laparoscopic sleeve gastrectomy (LSG) in adolescents with nonalcoholic fatty liver disease (NAFLD). STUDY DESIGN: Twenty obese (body mass index of ≥35 kg/m2) adolescents who underwent LSG and with biopsy-proven NAFLD were included. At baseline (T0) and 1 year after treatment, patients underwent clinical evaluation, blood tests, and liver biopsy. Hepatic progenitor cells, hepatic stellate cells (HSCs), macrophages, and adipocytokines were evaluated by immunohistochemistry and immunofluorescence. RESULTS: Liver biopsy samples after LSG demonstrated a significant improvement of NAFLD Activity Score and fibrosis. Immunohistochemistry indicated a significant reduction of hepatocyte cell cycle arrest, ductular reaction, activated HSC, and macrophage number after LSG compared with T0. The activation state of HSC was accompanied by modification in the expression of the autophagy marker LC3. Hepatocyte expression of adiponectin was significant higher after LSG than into T0. Moreover, LSG caused decreased resistin expression in Sox9+ hepatic progenitor cells compared with T0. The number of S100A9+ macrophages was also reduced by LSG correlating with resistin expression. Finally, serum levels of proinflammatory cytokines significantly correlated with macrophages and activated HSC numbers. CONCLUSIONS: The histologic improvement induced by LSG is associated with the reduced activation of local cellular compartments (hepatic progenitor cells, HSCs, and macrophages), thus, strengthening the role of cellular interactions and hepatic adipocytokine production in the pathogenesis of NAFLD.

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.

Role of lactoferrin and its receptors on biliary epithelium.

Human lactoferrin is an iron-binding glycoprotein present at high concentrations in breast milk and colostrum. It is produced by many exocrine glands and widely distributed in a variety of body fluids. This protein has antimicrobial, immunomodulatory, antioxidant, and anticancer properties. Two important hLf receptors have been identified: LDL receptor related protein (LRP1), a low specificity receptor, and intelectin-1 (ITLN1), a high specificity receptor. No data are present on the role of hLf on the biliary epithelium. Our aims have been to evaluate the expression of Lf and its receptors in human and murine cholangiocytes and its effect on proliferation. Immunohistochemistry and immunofluorescence (IF) were conducted on human healthy and primary biliary cholangitis (PBC) liver samples as well as on liver samples obtained from normal and bile duct ligated (BDL) mice to evaluate the expression of Lf, LRP1 and ITLN1. Cell proliferation in vitro studies were performed on human cholangiocyte cell lines via 3-(4,5-dimetiltiazol-2-il)-2,5-diphenyltetrazolium assay as well as IF to evaluate proliferating cell nuclear antigen (PCNA) expression. Our results show that mouse and human cholangiocytes express Lf, LRP1 and ITLN1, at higher extent in cholangiocytes from BDL and PBC samples. Furthermore, the in vitro addition of bovine Lf (bLf) has a proliferative effect on human cholangiocyte cell line. The results support a proliferative role of hLf on the biliary epithelium; this pro-proliferative effect of hLf and bLf on cholangiocytes could be particularly relevant in human cholangiopathies such as PBC, characterized by cholangiocyte death and ductopenia.

Contribution of Resident Stem Cells to Liver and Biliary Tree Regeneration in Human Diseases.

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression.

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).