The journal retracts the article titled “The Effects of Taurocholic Acid on Biliary Damage and Liver Fibrosis Are Mediated by Calcitonin-Gene-Related Peptide Signaling” [...]

BACKGROUND AND AIMS:NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling.APPROACH AND RESULTS:4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro.CONCLUSION:Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Background Alcohol-related liver disease (ALD) is characterized by ductular reaction (DR), liver inflammation, steatosis, fibrosis, and cirrhosis. The secretin (Sct)/secretin receptor (SR) axis (expressed only by cholangiocytes) regulates liver phenotypes in cholestasis. We evaluated the role of Sct signaling on ALD phenotypes. Methods We used male wild-type and Sct −/− mice fed a control diet (CD) or ethanol (EtOH) for 8 wk. Changes in liver phenotypes were measured in mice, female/male healthy controls, and patients with alcoholic cirrhosis. Since Cyp4a10 and Cyp4a11/22 regulate EtOH liver metabolism, we measured their expression in mouse/human liver. We evaluated: (i) the immunoreactivity of the lipogenesis enzyme elongation of very-long-chain fatty acids 1 (Elovl, mainly expressed by hepatocytes) in mouse/human liver sections by immunostaining; (ii) the expression of miR-125b (that is downregulated in cholestasis by Sct) in mouse liver by q PCR; and (iii) total bile acid (BA) levels in mouse liver by enzymatic assay, and the mRNA expression of genes regulating BA synthesis (cholesterol 7a-hydroxylase, Cyp27a1, 12a-hydroxylase, Cyp8b1, and oxysterol 7a-hydroxylase, Cyp7b11) and transport (bile salt export pump, Bsep, Na + -taurocholate cotransporting polypeptide, NTCP, and the organic solute transporter alpha (OSTa) in mouse liver by q PCR. Results In EtOH-fed WT mice there was increased biliary and liver damage compared to control mice, but decreased miR-125b expression, phenotypes that were blunted in EtOH-fed Sct −/− mice. The expression of Cyp4a10 increased in cholangiocytes and hepatocytes from EtOH-fed WT compared to control mice but decreased in EtOH-fed Sct −/− mice. There was increased immunoreactivity of Cyp4a11/22 in patients with alcoholic cirrhosis compared to controls. The expression of miR-125b decreased in EtOH-fed WT mice but returned at normal values in EtOH-fed Sct −/− mice. Elovl1 immunoreactivity increased in patients with alcoholic cirrhosis compared to controls. There was no difference in BA levels between WT mice fed CD or EtOH; BA levels decreased in EtOH-fed Sct −/− compared to EtOH-fed WT mice. There was increased expression of Cyp27a1, Cyp8b1, Cyp7b1, Bsep, NTCP and Osta in total liver from EtOH-fed WT compared to control mice, which decreased in EtOH-fed Sct −/− compared to EtOH-fed WT mice. Conclusions Targeting Sct/SR signaling may be important for modulating ALD phenotypes.

ABSTRACTOrganoids are novelin vitromodels to study intercellular crosstalk between the different types of cells in the pathophysiology of disease. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), we developed scaffold-free multi-cellular 3D cholangiocyte organoids (3D-CHO) using ‘primary’ liver cell lines derived from normal and PSC patients. Human liver samples from healthy donors and late-stage PSC patients were used to isolate ‘primary’ cholangiocytes (EPCAM+/CK-19+), liver endothelial cells (LECs, CD31+), and hepatic stellate cells (HSCs, CD31−/CD68−/Desmin+/Vitamin A+). 3D-CHOs were formed using cholangiocytes:HSCs:LECs and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence (IF), qRT-PCR, and transmission electron microscopy. Gene expressions for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, SCTR), fibrosis (ACTA2, COL1A1, DESMIN, TGFβ1), angiogenesis (PECAM, VEGF, CDH5, vWF), and inflammation (IL-6, TNF-α) confirmed PSC phenotypes of 3D-CHOs. Since cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal-IF demonstrated that neurokinin-1 receptor (NK-1R, expressed by cholangiocytes and upregulated in PSC), was localized within CK-19+cholangiocytes. Moreover, 3D-CHOs from PSC patients confirmed PSC phenotypes with upregulated NK-1R, tachykinin precursor 1, and downregulated membrane metalloendopeptidase. Our viable scaffold-free multiple-cell 3D-CHOs showed superiority as anin vitromodel in mimicking PSCin vivophenotypes compared to 2D cell culture, which can be used in PSC disease-related research.

Cholestatic liver diseases are named primarily due to the blockage of bile flow and buildup of bile acids in the liver. Cholestasis can occur in cholangiopathies, fatty liver diseases, and during COVID-19 infection. Most literature evaluates damage occurring to the intrahepatic biliary tree during cholestasis; however, there may be associations between liver damage and gallbladder damage. Gallbladder damage can manifest as acute or chronic inflammation, perforation, polyps, cancer, and most commonly gallstones. Considering the gallbladder is an extension of the intrahepatic biliary network, and both tissues are lined by biliary epithelial cells that share common mechanisms and properties, it is worth further evaluation to understand the association between bile duct and gallbladder damage. In this comprehensive article, we discuss background information of the biliary tree and gallbladder, from function, damage, and therapeutic approaches. We then discuss published findings that identify gallbladder disorders in various liver diseases. Lastly, we provide the clinical aspect of gallbladder disorders in liver diseases and ways to enhance diagnostic and therapeutic approaches for congruent diagnosis. © 2023 American Physiological Society. Compr Physiol 13:4909-4943, 2023.

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 chol-angiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. (c) 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

BackgroundPrimary biliary cholangitis (PBC) is characterized by increased biliary damage, inflammation and liver fibrosis. Early stage PBC is marked by biliary proliferation, whereas late stage PBC shows ductopenia. We have shown that dominant‐negative transforming growth factor b receptor II (dnTGFβRII) mice at 12 wk of age mimic early stage PBC. We have found that melatonin therapy or prolonged exposure to complete darkness reduces biliary hyperplasia and liver fibrosis in models of cholestasis. However, the impact of melatonin or dark therapy on PBC‐related injury is unknown. The aim of our study was to evaluate the effects of melatonin or dark therapy in a mouse model of early stage PBC.MethodsWe used background‐matched, male wild‐type (WT) and dnTGFβRII at 12 wk of age that were given drinking water containing melatonin (0.03%) or placed in complete darkness for 1 wk along with the related controls. Liver damage was evaluated by H&E. Intrahepatic bile duct mass (IBDM) was measured by CK‐19 staining. Biliary senescence was evaluated by staining for p16 and p21, and SA‐β‐galactosidase activity. Biliary and liver inflammation were determined by IL‐6 and F4/80 (Kupffer cell marker) staining. Liver fibrosis was determined by Sirius Red staining and immunofluorescence for collagen type‐1a. Hepatic stellate cell (HSC) activation was shown by SYP‐9 and α‐SMA staining. Human control and early stage PBC serum samples were obtained, and serum melatonin levels were measured by EIA.ResultsdnTGFβRII at 12 wk of age (early stage PBC mouse model) treated with melatonin or complete darkness had decreased (i) IBDM, (ii) biliary senescence, (iii) liver fibrosis, (iv) biliary and liver inflammation, and (v) HSC activation/liver fibrosis. Human early stage PBC samples had decreased serum melatonin levels.ConclusionInhibition of melatonin signaling perpetuates biliary damage associated with PBC. Restoration of melatonin‐dependent signaling via melatonin treatment or dark therapy may be therapeutic for patients with early stage PBC.Support or Funding InformationNIH NIDDK R01, VA MeritThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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-β1/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-β1/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.

Fibroblast growth factor 1 (FGF1) belongs to a family of growth factors involved in cellular growth and division. MicroRNA 16 (miR‐16) is a regulator of gene expression, which is dysregulated during liver injury and insult. However, the role of FGF1 in the progression of biliary proliferation, senescence, fibrosis, inflammation, angiogenesis, and its potential interaction with miR‐16, are unknown. In vivo studies were performed in male bile duct–ligated (BDL, 12‐week‐old) mice, multidrug resistance 2 knockout (Mdr2−/−) mice (10‐week‐old), and their corresponding controls, treated with recombinant human FGF1 (rhFGF1), fibroblast growth factor receptor (FGFR) antagonist (AZD4547), or anti‐FGF1 monoclonal antibody (mAb). In vitro, the human cholangiocyte cell line (H69) and human hepatic stellate cells (HSCs) were used to determine the expression of proliferation, fibrosis, angiogenesis, and inflammatory genes following rhFGF1 treatment. PSC patient and control livers were used to evaluate FGF1 and miR‐16 expression. Intrahepatic bile duct mass (IBDM), along with hepatic fibrosis and inflammation, increased in BDL mice treated with rhFGF1, with a corresponding decrease in miR‐16, while treatment with AZD4547 or anti‐FGF1 mAb decreased hepatic fibrosis, IBDM, and inflammation in BDL and Mdr2−/− mice. In vitro, H69 and HSCs treated with rhFGF1 had increased expression of proliferation, fibrosis, and inflammatory markers. PSC samples also showed increased FGF1 and FGFRs with corresponding decreases in miR‐16 compared with healthy controls. Conclusion: Our study demonstrates that suppression of FGF1 and miR‐16 signaling decreases the presence of hepatic fibrosis, biliary proliferation, inflammation, senescence, and angiogenesis. Targeting the FGF1 and miR‐16 axis may provide therapeutic options in treating cholangiopathies such as PSC.

Bile ducts are heterogenous in structure and function, and primary sclerosing cholangitis (PSC) damages specific bile ducts leading to ductular reaction (DR), mast cell (MC) infiltration, increased histamine release, inflammation, and fibrosis. Bile duct ligation (BDL) induces large duct damage via cyclic adenosine monophosphate (cAMP)/extracellular signal-related protein kinase (ERK) signaling, and large cholangiocytes express H2 histamine receptor (H2HR). We evaluated how MCs interact with large cholangiocytes during cholestasis. Male wild-type (WT) and MC-deficient (KitW-sh ) mice 10-12 weeks of age were subjected to BDL for 7 days. Select KitW-sh mice were injected with MCs pretreated with control or H2HR antagonist (ranitidine, 25 μm, 48 h) via tail vein injection. In vitro, MC migration toward small mouse cholangiocytes (SMCCs) and large mouse cholangiocytes (LMCCs) treated with lipopolysaccharide or histamine (±ranitidine) was measured. LMCCs were stimulated with MC supernatants pretreated with control, α-methyl-dl-histidine (to block histamine release), or ranitidine. Liver damage, large duct DR/senescence, inflammation, fibrosis, and cAMP/ERK immunoreactivity increased in BDL WT and KitW-sh +MC mice but decreased in BDL KitW-sh and KitW-sh +MC-H2HR mice. In vitro, MCs migrate toward damaged LMCCs (but not SMCCs) blocked by inhibition of H2HR. Loss of MC histamine or MC-H2HR decreases LMCC proliferation, senescence, H2HR, and cAMP/ERK levels. Human PSC livers have increased MC number found near DR, senescent ducts, and H2HR-positive ducts. Conclusion: Infiltrating MCs preferentially interact with large ducts via H2HR signaling promoting biliary and liver damage. Mediation of MCs may be a therapeutic strategy for PSC.

Primary Sclerosing Cholangitis (PSC) is a cholestatic liver disease characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N‐acetyltransferase (AANAT) in the pineal gland, as well as extrapineal sites such as cholangiocytes. We previously found that: (i) the MT1 receptor is primarily expressed in cholangiocytes with low and absent expression in hepatic stellate cells and hepatocytes, respectively; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases biliary proliferation and liver fibrosis in bile duct ligated rats by downregulation of MT1 and clock genes (PER1, CRY1, CLOCK and BMAL1). We aimed to evaluate the beneficial effects of long‐term melatonin treatment and MT1 signaling on biliary phenotypes, liver fibrosis and portal inflammation in Mdr2‐/‐ mice (a model of PSC).