Serum miRNA profiles are altered in patients with primary sclerosing cholangitis receiving high-dose ursodeoxycholic acid.

Background & Aims: Primary sclerosing cholangitis (PSC) is a chronic, progressive cholestatic liver disease that can lead to end-stage liver disease and cholangiocarcinoma. High-dose ursodeoxycholic acid (hd-UDCA, 28-30 mg/kg/day) was evaluated in a previous multicentre, randomised placebo-controlled trial; however, the study was discontinued early because of increased liver-related serious adverse events (SAEs), despite improvement in serum liver biochemical tests. We investigated longitudinal changes in serum miRNA and cytokine profiles over time among patients treated with either hd-UDCA or placebo in this trial as potential biomarkers for PSC and response to hd-UDCA, as well as to understand the toxicity associated with hd-UDCA treatment. Methods: Thirty-eight patients with PSC were enrolled in a multicentred, randomised, double-blinded trial of hd-UDCA vs. placebo. Results: Significant alterations in serum miRNA profiles were found over time in both patients treated with hd-UDCA or placebo. Additionally, there were striking differences between miRNA profiles in patients treated with hd-UDCA compared with placebo. In patients treated with placebo, the changes in concentration of serum miRNAs miR-26a, miR-199b-5p, miR-373, and miR-663 suggest alterations of inflammatory and cell proliferative processes consistent with disease progression. However, patients treated with hd-UDCA exhibited a more pronounced differential expression of serum miRNAs, suggesting that hd-UDCA induces significant cellular miRNA changes and tissue injury. Pathway enrichment analysis for UDCA-associated miRNAs suggested unique dysregulation of cell cycle and inflammatory response pathways. Conclusions: Patients with PSC have distinct miRNAs in the serum and bile, although the implications of these unique patterns have not been studied longitudinally or in relation to adverse events related to hd-UDCA. Our study demonstrates marked changes in miRNA serum profiles with hd-UDCA treatment and suggests mechanisms for the increased liver toxicity with therapy. Impact and implications: Using serum samples from patients with PSC enrolled in a clinical trial comparing hd-UDCA with placebo, our study found distinct miRNA changes in patients with PSC who are treated with hd-UDCA over a period of time. Our study also noted distinct miRNA patterns in patients who developed SAEs during the study period.

The Epigenetic Reader, Bromodomain Containing 2, Mediates Cholangiocyte Senescence via Interaction With ETS Proto-Oncogene 1.

BACKGROUND & AIMS: We reported that cholangiocyte senescence, regulated by the transcription factor ETS proto-oncogene 1 (ETS1), is a pathogenic feature of primary sclerosing cholangitis (PSC). Furthermore, histone 3 lysine 27 is acetylated at senescence-associated loci. The epigenetic readers, bromodomain and extra-terminal domain (BET) proteins, bind acetylated histones, recruit transcription factors, and drive gene expression. Thus, we tested the hypothesis that BET proteins interact with ETS1 to drive gene expression and cholangiocyte senescence. METHODS: We performed immunofluorescence for BET proteins (BRD2 and 4) in liver tissue from liver tissue from PSC patients and a mouse PSC model. Using normal human cholangiocytes (NHCs), NHCs experimentally induced to senescence (NHCsen), and PSC patient-derived cholangiocytes (PSCDCs), we assessed senescence, fibroinflammatory secretome, and apoptosis after BET inhibition or RNA interference depletion. We assessed BET interaction with ETS1 in NHCsen and tissues from PSC patient, and the effects of BET inhibitors on liver fibrosis, senescence, and inflammatory gene expression in mouse models. RESULTS: Tissue from patients with PSC and a mouse PSC model exhibited increased cholangiocyte BRD2 and 4 protein (∼5×) compared with controls without disease. NHCsen exhibited increased BRD2 and 4 (∼2×), whereas PSCDCs exhibited increased BRD2 protein (∼2×) relative to NHC. BET inhibition in NHCsen and PSCDCs reduced senescence markers and inhibited the fibroinflammatory secretome. ETS1 interacted with BRD2 in NHCsen, and BRD2 depletion diminished NHCsen p21 expression. BET inhibitors reduced senescence, fibroinflammatory gene expression, and fibrosis in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed and Mdr2-/- mouse models. CONCLUSION: Our data suggest that BRD2 is an essential mediator of the senescent cholangiocyte phenotype and is a potential therapeutic target for patients with PSC.

Genetics, pathobiology and therapeutic opportunities of polycystic liver disease.

Polycystic liver diseases (PLDs) are inherited genetic disorders characterized by progressive development of intrahepatic, fluid-filled biliary cysts (more than ten), which constitute the main cause of morbidity and markedly affect the quality of life. Liver cysts arise in patients with autosomal dominant PLD (ADPLD) or in co-occurrence with renal cysts in patients with autosomal dominant or autosomal recessive polycystic kidney disease (ADPKD and ARPKD, respectively). Hepatic cystogenesis is a heterogeneous process, with several risk factors increasing the odds of developing larger cysts. Depending on the causative gene, PLDs can arise exclusively in the liver or in parallel with renal cysts. Current therapeutic strategies, mainly based on surgical procedures and/or chronic administration of somatostatin analogues, show modest benefits, with liver transplantation as the only potentially curative option. Increasing research has shed light on the genetic landscape of PLDs and consequent cholangiocyte abnormalities, which can pave the way for discovering new targets for therapy and the design of novel potential treatments for patients. Herein, we provide a critical and comprehensive overview of the latest advances in the field of PLDs, mainly focusing on genetics, pathobiology, risk factors and next-generation therapeutic strategies, highlighting future directions in basic, translational and clinical research.

Cellular senescence in the cholangiopathies: a driver of immunopathology and a novel therapeutic target.

The cholangiopathies are a group of liver diseases that affect cholangiocytes, the epithelial cells that line the bile ducts. Biliary atresia (BA), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC) are three cholangiopathies with significant immune-mediated pathogenesis where chronic inflammation and fibrosis lead to obliteration of bile ducts and eventual liver cirrhosis. Cellular senescence is a state of cell cycle arrest in which cells become resistant to apoptosis and profusely secrete a bioactive secretome. Recent evidence indicates that cholangiocyte senescence contributes to the pathogenesis of BA, PBC, and PSC. This review explores the role of cholangiocyte senescence in BA, PBC, and PSC, ascertains how cholangiocyte senescence may promote a senescence-associated immunopathology in these cholangiopathies, and provides the rationale for therapeutically targeting senescence as a treatment option for BA, PBC, and PSC.

Cellular senescence in the cholangiopathies.

PURPOSE OF REVIEW: Cellular senescence (i.e. permanent withdrawal from the cell cycle) is increasingly recognized as a pathologic feature in a variety of inflammatory liver diseases, including primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC) and additional cholangiopathies. Herein, we provide an update on the interplay between cholangiocytes, cellular senescence and the cholangiopathies. RECENT FINDINGS: The themes covered by this review include novel models for studying the role of senescent cholangiocytes and the cholangiopathies, identification and modulation of key pathways or molecules regulating cholangiocyte senescence, and discovery of druggable targets to advance therapeutic options for the cholangiopathies. Most recent studies focused on PSC; however, the concepts and findings may be applied to additional cholangiopathies. SUMMARY: Cholangiopathies present unique and divergent clinicopathological features, causes and genetic backgrounds, but share several common disease processes. Cholangiocyte senescence in the cholestatic cholangiopathies, primarily PSC and PBC, is regarded as a key pathogenetic process. Importantly, senescent cholangiocytes exhibit phenotypic features including the senescence-associated secretory phenotype (SASP) and resistance to apoptosis that provide new directions for basic research and new prognostic and therapeutic approaches for clinical practice.

Polycystic Liver Disease: Advances in Understanding and Treatment.

Polycystic liver disease (PLD) is a group of genetic disorders characterized by progressive development of cholangiocyte-derived fluid-filled hepatic cysts. PLD is the most common manifestation of autosomal dominant and autosomal recessive polycystic kidney diseases and rarely occurs as autosomal dominant PLD. The mechanisms of PLD are a sequence of the primary (mutations in PLD-causative genes), secondary (initiation of cyst formation), and tertiary (progression of hepatic cystogenesis) interconnected molecular and cellular events in cholangiocytes. Nonsurgical, surgical, and limited pharmacological treatment options are currently available for clinical management of PLD. Substantial evidence suggests that pharmacological targeting of the signaling pathways and intracellular processes involved in the progression of hepatic cystogenesis is beneficial for PLD. Many of these targets have been evaluated in preclinical and clinical trials. In this review, we discuss the genetic, molecular, and cellular mechanisms of PLD and clinical and preclinical treatment strategies.

Autophagy promotes hepatic cystogenesis in polycystic liver disease by depletion of cholangiocyte ciliogenic proteins.

BACKGROUNDS AND AIMS: Polycystic liver disease (PLD) is characterized by defective cholangiocyte cilia that regulate progressive growth of hepatic cysts. Because formation of primary cilia is influenced by autophagy through degradation of proteins involved in ciliogenesis, we hypothesized that ciliary defects in PLD cholangiocytes (PLDCs) originate from autophagy-mediated depletion of ciliogenic proteins ADP-ribosylation factor-like protein 3 (ARL3) and ADP-ribosylation factor-like protein 13B (ARL13B) and ARL-dependent mislocation of a ciliary-localized bile acid receptor, Takeda G-protein-coupled receptor 5 (TGR5), the activation of which enhances hepatic cystogenesis (HCG). The aims here were to determine whether: (1) ciliogenesis is impaired in PLDC, is associated with increased autophagy, and involves autophagy-mediated depletion of ARL3 and ARL13B; (2) depletion of ARL3 and ARL13B in PLDC cilia impacts ciliary localization of TGR5; and (3) pharmacological inhibition of autophagy re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL3B, and TGR5 and reduces HCG. APPROACH AND RESULTS: By using liver tissue from healthy persons and patients with PLD, in vitro and in vivo models of PLD, and in vitro models of ciliogenesis, we demonstrated that, in PLDCs: ciliogenesis is impaired; autophagy is enhanced; ARL3 and ARL13B are ubiquitinated by HDAC6, depleted in cilia, and present in autophagosomes; depletion of ARL3 and ARL13B impacts ciliary localization of TGR5; and pharmacological inhibition of autophagy with mefloquine and verteporfin re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL13B, and TGR5 and reduces HCG. CONCLUSIONS: The intersection between autophagy, defective cholangiocyte cilia, and enhanced HCG contributes to PLD progression and can be considered a target for therapeutic interventions.

Genetic or pharmacological reduction of cholangiocyte senescence improves inflammation and fibrosis in the Mdr2 -/- mouse.

BACKGROUND & AIMS: Cholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2 -/-). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of cholangiocyte senescence would ameliorate disease in Mdr2 -/- mice. METHODS: We used 2 novel genetic murine models to reduce cholangiocyte senescence: (i) p16Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)xMdr2 -/-, in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16-expressing cells; and (ii) mice deficient in both p16 and Mdr2. Mdr2 -/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1ß, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining. RESULTS: AP20187 treatment reduced p16 and p21 expression by ~35% and ~70% (p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1ß, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% (p >0.05). Similarly, p16 -/- xMdr2 -/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1ß (60%), and MCP-1 (65%) and reduced fibrosis (~50%) (p >0.05) compared with Mdr2 -/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1ß (50%), MCP-1 (70%), and fibrosis (60%) (p >0.05). CONCLUSIONS: Our data support a pathophysiological role of cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent cholangiocytes is a plausible therapeutic approach. LAY SUMMARY: Primary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease.

Targeting UBC9-mediated protein hyper-SUMOylation in cystic cholangiocytes halts polycystic liver disease in experimental models.

BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Herein, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting. METHODS: Levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated in vitro, in vivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry. RESULTS: Most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdh1-mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. In vitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUMO1-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered proteasome hyperactivity in cystic cholangiocytes, leading to activation of the unfolded protein response and stress-related apoptosis. CONCLUSIONS: Cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy. LAY SUMMARY: Protein SUMOylation is a dynamic post-translational event implicated in numerous cellular processes. This study revealed dysregulated protein SUMOylation in polycystic liver disease, which promotes hepatic cystogenesis. Administration of S-adenosylmethionine (SAMe), a natural UBC9-dependent SUMOylation inhibitor, halted polycystic liver disease in experimental models, thus representing a potential therapeutic agent for patients.

Early Cholangiocarcinoma Detection With Magnetic Resonance Imaging Versus Ultrasound in Primary Sclerosing Cholangitis.

BACKGROUND AND AIMS: Early detection of perihilar cholangiocarcinoma (CCA) among patients with primary sclerosing cholangitis (PSC) is important to identify more people eligible for curative therapy. While many recommend CCA screening, there are divergent opinions and limited data regarding the use of ultrasound or magnetic resonance imaging (MRI) for early CCA detection, and it is unknown whether there is benefit in testing asymptomatic individuals. Our aims were to assess the diagnostic performances and prognostic implications of ultrasound and MRI-based CCA detection. APPROACH AND RESULTS: This is a multicenter review of 266 adults with PSC (CCA, n = 120) who underwent both an ultrasound and MRI within 3 months. Images were re-examined by radiologists who were blinded to the clinical information. Respectively, MRI had a higher area under the curve compared with ultrasound for CCA detection: 0.87 versus 0.70 for the entire cohort; 0.81 versus 0.59 for asymptomatic individuals; and 0.88 versus 0.71 for those listed for CCA transplant protocol. The absence of symptoms at CCA diagnosis was associated with improved 5-year outcomes including overall survival (82% vs. 46%, log-rank P < 0.01) and recurrence-free survival following liver transplant (89% vs. 65%, log-rank P = 0.04). Among those with asymptomatic CCA, MRI detection (compared with ultrasound) was associated with reduction in both mortality (hazard ratio, 0.10; 95% confidence interval, 0.01-0.96) and CCA progression after transplant listing (hazard ratio, 0.10; 95% confidence interval, 0.01-0.90). These benefits continued among patients who had annual monitoring and PSC for more than 1 year before CCA was diagnosed. CONCLUSIONS: MRI is superior to ultrasound for the detection of early-stage CCA in patients with PSC. Identification of CCA before the onset of symptoms with MRI is associated with improved outcomes.

Liver Stiffness Measured by Either Magnetic Resonance or Transient Elastography Is Associated With Liver Fibrosis and Is an Independent Predictor of Outcomes Among Patients With Primary Biliary Cholangitis.

GOALS: We aimed to describe the diagnostic and prognostic performance of transient elastography (TE) and magnetic resonance elastography (MRE) in patients with primary biliary cholangitis (PBC). BACKGROUND: The diagnostic performance of TE and MRE in detecting advanced fibrosis in PBC and in predicting outcomes independent of existing serologic prognostic markers is incompletely understood. MATERIALS AND METHODS: Five hundred thirty-eight consecutive patients with PBC at 3 centers with liver stiffness (LS) measurements by TE (n=286) or MRE (n=332) were reviewed. LS cutoffs for predicting fibrosis stages were determined by receiver operating characteristic curves among those with a liver biopsy (TE, n=63; MRE, n=98). Cox proportional hazard regression modeling was used to identify associations between covariates and hepatic decompensation. RESULTS: The optimal LS thresholds for predicting histologic stage F4 were 14.40 kPa (area under the curve=0.94) for TE and 4.60 kPa (area under the curve=0.82) for MRE. Both TE and MRE outperformed biochemical markers for the prediction of histologic advanced fibrosis. Optimal LS thresholds to predict hepatic decompensation were 10.20 kPa on TE and 4.30 kPa on MRE. LS by TE and MRE (respectively) remained predictors of hepatic decompensation after adjusting for ursodeoxycholic acid responsiveness [hazard ratio (HR), 1.14; 95% confidence interval (CI), 1.05-1.24 and HR, 1.68; 95% CI, 1.28-2.19] and the GLOBE score (HR, 1.13; 95% CI, 1.07-1.19 and HR, 2.09; 95% CI, 1.57-2.78). CONCLUSION: LS measurement with either TE or MRE can accurately detect advanced fibrosis and offers additional prognostic value beyond existing serologic predictive tools.

Pansomatostatin Agonist Pasireotide Long-Acting Release for Patients with Autosomal Dominant Polycystic Kidney or Liver Disease with Severe Liver Involvement: A Randomized Clinical Trial.

BACKGROUND AND OBJECTIVES: We assessed safety and efficacy of another somatostatin receptor analog, pasireotide long-acting release, in severe polycystic liver disease and autosomal dominant polycystic kidney disease. Pasireotide long-acting release, with its broader binding profile and higher affinity to known somatostatin receptors, has potential for greater efficacy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Individuals with severe polycystic liver disease were assigned in a 2:1 ratio in a 1-year, double-blind, randomized trial to receive pasireotide long-acting release or placebo. Primary outcome was change in total liver volume; secondary outcomes were change in total kidney volume, eGFR, and quality of life. RESULTS: Of 48 subjects randomized, 41 completed total liver volume measurements (n=29 pasireotide long-acting release and n=12 placebo). From baseline, there were -99±189 ml/m absolute and -3%±7% change in annualized change in height-adjusted total liver volume (from 2582±1381 to 2479±1317 ml/m) in the pasireotide long-acting release group compared with 136±117 ml/m absolute and 6%±7% increase (from 2387±759 to 2533±770 ml/m) in placebo (P<0.001 for both). Total kidney volumes decreased by -12±34 ml/m and -1%±4% in pasireotide long-acting release compared with 21±21 ml/m and 4%±5% increase in the placebo group (P=0.05 for both). Changes in eGFR were similar between groups. Among the n=48 randomized, adverse events included hyperglycemia (26 of 33 [79%] in pasireotide long-acting release versus four of 15 [27%] in the placebo group; P<0.001), and among the 47 without diabetes at baseline, 19 of 32 (59%) in the pasireotide long-acting release group versus one of 15 (7%) in the placebo group developed diabetes (P=0.001). CONCLUSIONS: Another somatostatin analog, pasireotide long-acting release, slowed progressive increase in both total liver volume/total kidney volume growth rates without affecting GFR decline. Participants experienced higher frequency of adverse events (hyperglycemia and diabetes). CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Pasireotide LAR in Severe Polycystic Liver Disease, NCT01670110 PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2020_08_28_CJN13661119.mp3.

Polarized human cholangiocytes release distinct populations of apical and basolateral small extracellular vesicles.

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2-interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.

Senescent cholangiocytes release extracellular vesicles that alter target cell phenotype via the epidermal growth factor receptor.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by peribiliary inflammation and fibrosis. Cholangiocyte senescence is a prominent feature of PSC. Here, we hypothesize that extracellular vesicles (EVs) from senescent cholangiocytes influence the phenotype of target cells. METHODS: EVs were isolated from normal human cholangiocytes (NHCs), cholangiocytes from PSC patients and NHCs experimentally induced to senescence. NHCs, malignant human cholangiocytes (MHCs) and monocytes were exposed to 108 EVs from each donor cell population and assessed for proliferation, MAPK activation and migration. Additionally, we isolated EVs from plasma of wild-type and Mdr2-/- mice (a murine model of PSC), and assessed mouse monocyte activation. RESULTS: EVs exhibited the size and protein markers of exosomes. The number of EVs released from senescent human cholangiocytes was increased; similarly, the EVs in plasma from Mdr2-/- mice were increased. Additionally, EVs from senescent cholangiocytes were enriched in multiple growth factors, including EGF. NHCs exposed to EVs from senescent cholangiocytes showed increased NRAS and ERK1/2 activation. Moreover, EVs from senescent cholangiocytes promoted proliferation of NHCs and MHCs, findings that were blocked by erlotinib, an EGF receptor inhibitor. Furthermore, EVs from senescent cholangiocytes induced EGF-dependent Interleukin 1-beta and Tumour necrosis factor expression and migration of human monocytes; similarly, Mdr2-/- mouse plasma EVs induced activation of mouse monocytes. CONCLUSIONS: The data continue to support the importance of cholangiocyte senescence in PSC pathogenesis, directly implicate EVs in cholangiocyte proliferation, malignant progression and immune cell activation and migration, and identify novel therapeutic approaches for PSC.

Proteostasis disturbances and endoplasmic reticulum stress contribute to polycystic liver disease: New therapeutic targets.

BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD-causative genes, encoding for endoplasmic reticulum (ER)-resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. METHODS: ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4-phenylbutyric acid (4-PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. RESULTS: The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4-PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4-PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. CONCLUSIONS: Restoration of proteostasis in cystic cholangiocytes with 4-PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.

Ileo-colonic delivery of conjugated bile acids improves glucose homeostasis via colonic GLP-1-producing enteroendocrine cells in human obesity and diabetes.

BACKGROUND: The bile acid (BA) pathway plays a role in regulation of food intake and glucose metabolism, based mainly on findings in animal models. Our aim was to determine whether the BA pathway is altered and correctable in human obesity and diabetes. METHODS: We conducted 3 investigations: 1) BA receptor pathways were studied in NCI-H716 enteroendocrine cell (EEC) line, whole human colonic mucosal tissue and in human colonic EEC isolated by Fluorescence-activated Cell Sorting (ex vivo) from endoscopically-obtained biopsies colon mucosa; 2) We characterized the BA pathway in 307 participants by measuring during fasting and postprandial levels of FGF19, 7αC4 and serum BA; 3) In a placebo-controlled, double-blind, randomised, 28-day trial, we studied the effect of ileo-colonic delivery of conjugated BAs (IC-CBAS) on glucose metabolism, incretins, and lipids, in participants with obesity and diabetes. FINDINGS: Human colonic GLP-1-producing EECs express TGR5, and upon treatment with bile acids in vitro, human EEC differentially expressed GLP-1 at the protein and mRNA level. In Ussing Chamber, GLP-1 release was stimulated by Taurocholic acid in either the apical or basolateral compartment. FGF19 was decreased in obesity and diabetes compared to controls. When compared to placebo, IC-CBAS significantly decreased postprandial glucose, fructosamine, fasting insulin, fasting LDL, and postprandial FGF19 and increased postprandial GLP-1 and C-peptide. Increase in faecal BA was associated with weight loss and with decreased fructosamine. INTERPRETATIONS: In humans, BA signalling machinery is expressed in colonic EECs, deficient in obesity and diabetes, and when stimulated with IC-CBAS, improved glucose homeostasis. ClinicalTrials.gov number, NCT02871882, NCT02033876. FUNDING: Research support and drug was provided by Satiogen Pharmaceuticals (San Diego, CA). AA, MC, and NFL report grants (AA- C-Sig P30DK84567, K23 DK114460; MC- NIH R01 DK67071; NFL- R01 DK057993) from the NIH. JR was supported by an Early Career Grant from Society for Endocrinology.

An update on primary sclerosing cholangitis epidemiology, outcomes and quantification of alkaline phosphatase variability in a population-based cohort.

BACKGROUND: Contemporary primary sclerosing cholangitis (PSC) population-based cohorts describing the epidemiology, natural history, and long-term fluctuations in serum alkaline phosphatase (SAP) and their prognostic relevance are lacking. Therefore, we investigated the incidence and natural history of PSC and quantified SAP fluctuations among those with PSC in Olmsted County, Minnesota over the last 41 years. METHODS: The Rochester Epidemiology Project was used to identify 56 subjects diagnosed with PSC between 1976 and 2017 in Olmsted County. The primary endpoint (n = 19) included liver transplantation, hepatic decompensation, and cholangiocarcinoma. RESULTS: The age- and sex-adjusted incidence of PSC (per 100,000 person years) nearly doubled from 2001 to 2017 compared to 1976-2000 (1.47; 95% CI 0.99-1.96 versus 0.79; 95% CI 0.42-1.16, p = 0.02). This increase paralleled a rise in patients with markers of a milder phenotype at the time of diagnosis: normal SAP (26.32% versus 0%, p < 0.01) and lower Mayo PSC risk score [0.36 (- 0.57 to 1.55) versus - 0.50 (- 1.25 to 0.35), p = 0.03]. Intra-individual SAP fluctuates with a median coefficient of variation of 36.20%. SAP normalization and dropping below 1.5 × upper limit of normal (ULN) occurs at a rate of 5% and 10% per year, respectively. SAP less than 1.5 × ULN was associated with a lower risk of PSC-related complications (hazard ratio 0.11; 95% CI 0.03-0.42). CONCLUSIONS: The patients with PSC are increasingly being diagnosed with a milder phenotype. While a lower SAP is associated with improved outcomes, the high intra-individual variation of SAP levels calls into question the practice of using a single SAP value as a surrogate endpoint in clinical trials.

The Spectrum of Reactive Cholangiocytes in Primary Sclerosing Cholangitis.

Cholangiocytes are the target of a group of chronic liver diseases termed the "cholangiopathies," in which cholangiocytes react to exogenous and endogenous insults, leading to disease initiation and progression. In primary sclerosing cholangitis (PSC), the focus of this review, the cholangiocyte response to genetic or environmental insults can lead to a heterogeneous response; that is, a subpopulation acquires a ductular reactive and proliferative phenotype, while another subpopulation undergoes senescence and growth arrest. Both ductular reactive cholangiocytes and senescent cholangiocytes can modify the periductal microenvironment through their ability to secrete various cytokines, chemokines, and growth factors, initiating and perpetuating inflammatory and profibrotic responses. This review discusses the similarities and differences, the interrelationships, and the potential pathogenic roles of these reactive proliferative and senescent cholangiocyte subpopulations in PSC.

Primary Sclerosing Cholangitis Risk Estimate Tool (PREsTo) Predicts Outcomes of the Disease: A Derivation and Validation Study Using Machine Learning.

Improved methods are needed to risk stratify and predict outcomes in patients with primary sclerosing cholangitis (PSC). Therefore, we sought to derive and validate a prediction model and compare its performance to existing surrogate markers. The model was derived using 509 subjects from a multicenter North American cohort and validated in an international multicenter cohort (n = 278). Gradient boosting, a machine-based learning technique, was used to create the model. The endpoint was hepatic decompensation (ascites, variceal hemorrhage, or encephalopathy). Subjects with advanced PSC or cholangiocarcinoma (CCA) at baseline were excluded. The PSC risk estimate tool (PREsTo) consists of nine variables: bilirubin, albumin, serum alkaline phosphatase (SAP) times the upper limit of normal (ULN), platelets, aspartate aminotransferase (AST), hemoglobin, sodium, patient age, and number of years since PSC was diagnosed. Validation in an independent cohort confirms that PREsTo accurately predicts decompensation (C-statistic, 0.90; 95% confidence interval [CI], 0.84-0.95) and performed well compared to Model for End-Stage Liver Disease (MELD) score (C-statistic, 0.72; 95% CI, 0.57-0.84), Mayo PSC risk score (C-statistic, 0.85; 95% CI, 0.77-0.92), and SAP <1.5 × ULN (C-statistic, 0.65; 95% CI, 0.55-0.73). PREsTo continued to be accurate among individuals with a bilirubin <2.0 mg/dL (C-statistic, 0.90; 95% CI, 0.82-0.96) and when the score was reapplied at a later course in the disease (C-statistic, 0.82; 95% CI, 0.64-0.95). Conclusion: PREsTo accurately predicts hepatic decompensation (HD) in PSC and exceeds the performance among other widely available, noninvasive prognostic scoring systems.