Publications by authors named "Ronald Oude Elferink"

162 Publications

Long-Term Effects of Biliverdin Reductase a Deficiency in Mice: Impact on Redox Status and Metabolism.

Antioxidants (Basel) 2021 Dec 20;10(12). Epub 2021 Dec 20.

Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Amsterdam University Medical Centers, Location AMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

Accumulation of neurotoxic bilirubin due to a transient neonatal or persistent inherited deficiency of bilirubin glucuronidation activity can cause irreversible brain damage and death. Strategies to inhibit bilirubin production and prevent neurotoxicity in neonatal and adult settings seem promising. We evaluated the impact of deficiency in neonatal and aged mice, in a background of unconjugated hyperbilirubinemia, by abolishing bilirubin production. We also investigated the disposal of biliverdin during fetal development. In mice, deficiency appeared sufficient to prevent lethality and to normalize bilirubin level in adults. Although biliverdin accumulated in -deficient fetuses, both and pups were healthy and reached adulthood having normal liver, brain, and spleen histology, albeit with increased iron levels in the latter. During aging, both and mice presented normal levels of relevant hematological and metabolic parameters. Interestingly, the oxidative status in erythrocytes from 9-months-old and mice was significantly reduced. In addition, triglycerides levels in these 9-months-old mice were significantly higher than WT controls, while tested normal. The normal parameters observed in mice fed chow diet indicate that inhibition to treat unconjugated hyperbilirubinemia seems safe and effective.
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http://dx.doi.org/10.3390/antiox10122029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8698966PMC
December 2021

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

J Hepatol 2022 Feb 27;76(2):319-331. Epub 2021 Oct 27.

Department of Gastroenterology and Hepatology and Tytgat Institute for Liver and Intestinal Research, AGEM, Amsterdam University Medical Centers, location AMC, Amsterdam, The Netherlands. Electronic address:

Background & Aims: Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC.

Methods: Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum.

Results: Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression.

Conclusion: Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'.

Lay Summary: We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.
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http://dx.doi.org/10.1016/j.jhep.2021.10.009DOI Listing
February 2022

Inhibition of autotaxin by bile salts and bile salt-like molecules increases its expression by feedback regulation.

Biochim Biophys Acta Mol Basis Dis 2021 11 10;1867(11):166239. Epub 2021 Aug 10.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AGEM, Amsterdam, the Netherlands. Electronic address:

Background: Autotaxin is an enzyme that converts lysophospholipid into lysophosphatidic acid (LPA), a highly potent signaling molecule through a range of LPA receptors. It is therefore important to investigate which factors play a role in regulating ATX expression. Since we have reported that ATX levels increase dramatically in patients with various forms of cholestasis, we embarked on a study to reveal factors that influence the enzyme activity ATX as well as its expression level in vitro and in vivo.

Methods: Bile from cholestatic patients was fractionated by HPLC and analyzed for modulation of ATX activity. ATX expression was measured in fibroblasts upon stimulation or inhibition of LPA signaling.

Results: Surprisingly, ATX activity was stimulated by most forms of its product LPA, but it was inhibited by bile salts and bile salt-like molecules, particularly by 3-OH sulfated bile salts and sulfated progesterone metabolites that are known to accumulate during chronic cholestasis and cholestasis of pregnancy, respectively. Activation of fibroblasts by LPA decreased ATX expression by 72%. Conversely, inhibition of LPA signaling increased ATX expression 3-fold, indicating strong feedback regulation by LPA signaling. In fibroblasts, we could verify that inhibition of ATX activity by bile salts induces its expression. Furthermore, induction of cholestasis in mice causes increased plasma ATX activity.

Conclusions: Multiple biliary compounds that accumulate in the systemic circulation during cholestasis inhibit ATX activity and thereby increase ATX expression through feedback regulation. This mechanism may contribute to increased serum ATX activity in patients with cholestasis.
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http://dx.doi.org/10.1016/j.bbadis.2021.166239DOI Listing
November 2021

Erratum to: "Hepatobiliary acid-base homeostasis: Insights from analogous secretory epithelia" (J Hepatol 2021; 74: 428-441).

J Hepatol 2021 Aug 13;75(2):497. Epub 2021 May 13.

Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2021.05.002DOI Listing
August 2021

Molecular regulation of the hepatic bile acid uptake transporter and HBV entry receptor NTCP.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 08 29;1866(8):158960. Epub 2021 Apr 29.

Tytgat Institute for Liver and Intestinal Research, Amsterdam University Medical Centers Amsterdam, the Netherlands; Amsterdam Gastroenterology, Endocrinology, Metabolism (AGEM), Amsterdam, the Netherlands; Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Transporters expressed by hepatocytes and enterocytes play a critical role in maintaining the enterohepatic circulation of bile acids. The sodium taurocholate cotransporting polypeptide (NTCP), exclusively expressed at the basolateral side of hepatocytes, mediates the uptake of conjugated bile acids. In conditions where bile flow is impaired (cholestasis), pharmacological inhibition of NTCP-mediated bile acid influx is suggested to reduce hepatocellular damage due to bile acid overload. Furthermore, NTCP has been shown to play an important role in hepatitis B virus (HBV) and hepatitis Delta virus (HDV) infection by functioning as receptor for viral entry into hepatocytes. This review provides a summary of current molecular insight into the regulation of NTCP expression at the plasma membrane, hepatic bile acid transport, and NTCP-mediated viral infection.
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http://dx.doi.org/10.1016/j.bbalip.2021.158960DOI Listing
August 2021

Molecular regulation of the hepatic bile acid uptake transporter and HBV entry receptor NTCP.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 08 29;1866(8):158960. Epub 2021 Apr 29.

Tytgat Institute for Liver and Intestinal Research, Amsterdam University Medical Centers Amsterdam, the Netherlands; Amsterdam Gastroenterology, Endocrinology, Metabolism (AGEM), Amsterdam, the Netherlands; Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Transporters expressed by hepatocytes and enterocytes play a critical role in maintaining the enterohepatic circulation of bile acids. The sodium taurocholate cotransporting polypeptide (NTCP), exclusively expressed at the basolateral side of hepatocytes, mediates the uptake of conjugated bile acids. In conditions where bile flow is impaired (cholestasis), pharmacological inhibition of NTCP-mediated bile acid influx is suggested to reduce hepatocellular damage due to bile acid overload. Furthermore, NTCP has been shown to play an important role in hepatitis B virus (HBV) and hepatitis Delta virus (HDV) infection by functioning as receptor for viral entry into hepatocytes. This review provides a summary of current molecular insight into the regulation of NTCP expression at the plasma membrane, hepatic bile acid transport, and NTCP-mediated viral infection.
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http://dx.doi.org/10.1016/j.bbalip.2021.158960DOI Listing
August 2021

Reduced spontaneous itch in mouse models of cholestasis.

Sci Rep 2021 03 17;11(1):6127. Epub 2021 Mar 17.

Amsterdam UMC, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism Research Institute, University of Amsterdam, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.

Pruritus is one of the most distressing symptoms in cholestatic patients. Plasma autotaxin (ATX) activity correlates with the severity of pruritus in cholestatic patients, but the pathophysiology is unclear. To study pruritus in mice, we measured scratch activity in cholestatic Atp8b1 mutant mice, a model for Progressive Familial Intrahepatic Cholestasis type 1, and wild type mice (WT) with alpha-naphthylisothiocyanate (ANIT)-induced cholestasis. To induce cholestasis, Atp8b1 mutant mice received a diet containing 0.1% cholic acid (CA) and WT mice were treated with ANIT. In these mice ATX was also overexpressed by transduction with AAV-ATX. Scratch activity was measured using an unbiased, electronic assay. Marked cholestasis was accomplished in both Atp8b1 mutant mice on a CA-supplemented diet and in ANIT-treatment in WT mice, but scratch activity was decreased rather than increased while plasma ATX activity was increased. Plasma ATX activity was further increased up to fivefold with AAV-ATX, but this did not induce scratch activity. In contrast to several reports two cholestatic mouse models did not display increased scratch activity as a measure of itch perception. Increasing plasma ATX activity by overexpression also did not lead to increased scratch activity in mice. This questions whether mice are suitable to study cholestatic itch.
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http://dx.doi.org/10.1038/s41598-021-85660-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969945PMC
March 2021

Efficacy of AAV8-h with Rapamycin in neonatal, suckling, and juvenile rats to model treatment in pediatric CNs patients.

Mol Ther Methods Clin Dev 2021 Mar 3;20:287-297. Epub 2020 Dec 3.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AGEM, Meibergdreef 69-71, 1105 BK Amsterdam, the Netherlands.

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (h) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-h, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs.
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http://dx.doi.org/10.1016/j.omtm.2020.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809245PMC
March 2021

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation.

Biochim Biophys Acta Bioenerg 2021 04 5;1862(4):148367. Epub 2021 Jan 5.

Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands; Amsterdam Gastroenterology Endocrinology Metabolism (AGEM) Research Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD] ratio, which was corroborated by using the NADH/NAD redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.
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http://dx.doi.org/10.1016/j.bbabio.2020.148367DOI Listing
April 2021

Gene Therapy for Progressive Familial Intrahepatic Cholestasis: Current Progress and Future Prospects.

Int J Mol Sci 2020 Dec 29;22(1). Epub 2020 Dec 29.

Tytgat Institute for Liver and Intestinal Research and Department of Gastroenterology and Hepatology, AGEM, Amsterdam UMC, University of Amsterdam, 1105 BK Amsterdam, The Netherlands.

Progressive Familial Intrahepatic Cholestasis (PFIC) are inherited severe liver disorders presenting early in life, with high serum bile salt and bilirubin levels. Six types have been reported, two of these are caused by deficiency of an ABC transporter; ABCB11 (bile salt export pump) in type 2; ABCB4 (phosphatidylcholine floppase) in type 3. In addition, ABCB11 function is affected in 3 other types of PFIC. A lack of effective treatment makes a liver transplantation necessary in most patients. In view of long-term adverse effects, for instance due to life-long immune suppression needed to prevent organ rejection, gene therapy could be a preferable approach, as supported by proof of concept in animal models for PFIC3. This review discusses the feasibility of gene therapy as an alternative for liver transplantation for all forms of PFIC based on their pathological mechanism. Using presently available gene therapy vectors, major hurdles need to be overcome to make gene therapy for all types of PFIC a reality.
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http://dx.doi.org/10.3390/ijms22010273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796371PMC
December 2020

Hepatobiliary acid-base homeostasis: Insights from analogous secretory epithelia.

J Hepatol 2021 02 24;74(2):428-441. Epub 2020 Oct 24.

Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands. Electronic address:

Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical stress. Bicarbonate is a key component of human bile and impaired biliary bicarbonate secretion is associated with liver damage. Major efforts have been undertaken to gain insight into acid-base homeostasis in cholangiocytes and more can be learned from analogous secretory epithelia. Extrahepatic examples include salivary and pancreatic duct cells, duodenocytes, airway and renal epithelial cells. The cellular machinery involved in acid-base homeostasis includes carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors. This pH-regulatory system is orchestrated by protein-protein interactions, the establishment of an electrochemical gradient across the plasma membrane and bicarbonate sensing of the intra- and extracellular compartment. In this review, we discuss conserved principles identified in analogous secretory epithelia in the light of current knowledge on cholangiocyte physiology. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system.
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http://dx.doi.org/10.1016/j.jhep.2020.10.010DOI Listing
February 2021

The extracellular lactate-to-pyruvate ratio modulates the sensitivity to oxidative stress-induced apoptosis via the cytosolic NADH/NAD redox state.

Apoptosis 2021 02 23;26(1-2):38-51. Epub 2020 Nov 23.

Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

The advantages of the Warburg effect on tumor growth and progression are well recognized. However, the relevance of the Warburg effect for the inherent resistance to apoptosis of cancer cells has received much less attention. Here, we show here that the Warburg effect modulates the extracellular lactate-to-pyruvate ratio, which profoundly regulates the sensitivity towards apoptosis induced by oxidative stress in several cell lines. To induce oxidative stress, we used the rapid apoptosis inducer Raptinal. We observed that medium conditioned by HepG2 cells has a high lactate-to-pyruvate ratio and confers resistance to Raptinal-induced apoptosis. In addition, imposing a high extracellular lactate-to-pyruvate ratio in media reduces the cytosolic NADH/NAD redox state and protects against Raptinal-induced apoptosis. Conversely, a low extracellular lactate-to-pyruvate ratio oxidizes the cytosolic NADH/NAD redox state and sensitizes HepG2 cells to oxidative stress-induced apoptosis. Mechanistically, a high extracellular lactate-to-pyruvate ratio decreases the activation of JNK and Bax under oxidative stress, thereby inhibiting the intrinsic apoptotic pathway. Our observations demonstrate that the Warburg effect of cancer cells generates an anti-apoptotic extracellular environment by elevating the extracellular lactate-to-pyruvate ratio which desensitizes cancer cells towards apoptotic insults. Consequently, our study suggests that the Warburg effect can be targeted to reverse the lactate-to-pyruvate ratios in the tumor microenvironment and thereby re-sensitize cancer cells to oxidative stress-inducing therapies.
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http://dx.doi.org/10.1007/s10495-020-01648-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902596PMC
February 2021

Targeting the Four Pillars of Enterohepatic Bile Salt Cycling; Lessons From Genetics and Pharmacology.

Hepatology 2021 06 24;73(6):2577-2585. Epub 2021 May 24.

Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

Bile salts play a pivotal role in lipid homeostasis, are sensed by specialized receptors, and have been implicated in various disorders affecting the gut or liver. They may play a role either as culprit or as potential panacea. Four very efficient transporters mediate most of the hepatic and intestinal bile salt uptake and efflux, and are each essential for the efficient enterohepatic circulation of bile salts. Starting from the intestinal lumen, conjugated bile salts cross the otherwise impermeable lipid bilayer of (primarily terminal ileal) enterocytes through the apical sodium-dependent bile acid transporter (gene SLC10A2) and leave the enterocyte through the basolateral heteromeric organic solute transporter, which consists of an alpha and beta subunit (encoded by SLC51A and SLC51B). The Na -taurocholate cotransporting polypeptide (gene SLC10A1) efficiently clears the portal circulation of bile salts, and the apical bile salt export pump (gene ABCB11) pumps the bile salts out of the hepatocyte into primary bile, against a very steep concentration gradient. Recently, individuals lacking either functional Na -taurocholate cotransporting polypeptide or organic solute transporter have been described, completing the quartet of bile acid transport deficiencies, as apical sodium-dependent bile acid transporter and bile salt export pump deficiencies were already known for years. Novel pathophysiological insights have been obtained from knockout mice lacking functional expression of these genes and from pharmacological transporter inhibition in mice or humans. Conclusion: We provide a concise overview of the four main bile salt transport pathways and of their status as possible targets of interventions in cholestatic or metabolic disorders.
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http://dx.doi.org/10.1002/hep.31651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252069PMC
June 2021

Fibrates for Itch (FITCH) in Fibrosing Cholangiopathies: A Double-Blind, Randomized, Placebo-Controlled Trial.

Gastroenterology 2021 02 5;160(3):734-743.e6. Epub 2020 Oct 5.

Department of Gastroenterology & Hepatology and Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology & Metabolism, Amsterdam University Medical Centers, location AMC, Amsterdam, the Netherlands. Electronic address:

Background And Aims: Pruritus may seriously impair quality of life in patients with cholestatic diseases such as primary or secondary sclerosing cholangitis (PSC, SSC) and primary biliary cholangitis (PBC). Pharmacologic strategies show limited efficacy and can provoke serious side effects. We hypothesized that bezafibrate, a broad peroxisome proliferator-activated receptor (PPAR) agonist, relieves cholestasis-associated itch by alleviating hepatobiliary injury. The aim of this investigator-initiated FITCH trial (Fibrates for cholestatic ITCH) was to assess effects of bezafibrate on pruritus in patients with PSC, PBC, and SSC.

Methods: Patients with moderate to severe pruritus (≥5 of 10 on visual analog scale [VAS]) due to PSC, PBC, or SSC were recruited for this double-blind, randomized, placebo-controlled trial between 2016 and 2019. Patients received once-daily bezafibrate (400 mg) or placebo for 21 days. The primary end point was ≥50% reduction of pruritus (VAS; intention-to-treat).

Results: Of 74 randomized patients, 70 completed the trial (95%; 44 PSC, 24 PBC, 2 SSC). For the primary end point, bezafibrate led in 45% (41% PSC, 55% PBC) and placebo in 11% to ≥50% reduction of severe or moderate pruritus (P = .003). For secondary end points, bezafibrate reduced morning (P = .01 vs placebo) and evening (P = .007) intensity of pruritus (VAS) and improved the validated 5D-Itch questionnaire (P = .002 vs placebo). Bezafibrate also reduced serum alkaline phosphatase (-35%, P = .03 vs placebo) correlating with improved pruritus (VAS, P = .01) suggesting reduced biliary damage. Serum bile acids and autotaxin activity remained unchanged. Serum creatinine levels tended to mildly increase (3% bezafibrate, 5% placebo, P = .14).

Conclusions: Bezafibrate is superior to placebo in improving moderate to severe pruritus in patients with PSC and PBC.

Trial Registration: Netherlands Trial Register, ID: NTR5436 (August 3, 2015), ClinicalTrials.gov ID: NCT02701166 (March 2, 2016).
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http://dx.doi.org/10.1053/j.gastro.2020.10.001DOI Listing
February 2021

Fruticuline A, a chemically-defined diterpene, exerts antineoplastic effects in vitro and in vivo by multiple mechanisms.

Sci Rep 2020 10 5;10(1):16477. Epub 2020 Oct 5.

Department of Pharmacology, Biological Sciences Sector, Federal University of Parana - UFPR, PO Box 19031, Curitiba, PR, 81531-980, Brazil.

Natural products have been recognized as important bioactive compounds on the basis of their wide biological properties. Here we investigated the antitumor effect and molecular mechanisms of the diterpene Fruticuline A (fruti) from Salvia lachnostachys, in human cancer cell lineages and Solid Ehrlich Carcinoma in mice. Fruti reduced MCF-7 and HepG2 proliferation by the reduction of Cyclin D1 levels and decreased NF-κB gene levels in both cell types. Furthermore, fruti also induced apoptosis in HepG2 cells, reduced Bcl-2 gene expression and induced necroptosis by increasing Ripk in MCF-7 cells. In mice, fruti prevented tumor development and reduced Cyclin D1, Bcl-2 and Rela gene levels, and reduced the p-NF-κB/NF-κB ratio in tumor tissue. Furthermore, fruti induced necrosis and apoptosis, increased N-acetyl-β-D-glucosaminidase and TNF-α levels and reduced IL-10 and Vegf levels in tumor tissue. Collectively, fruti exerts antitumor effects through the inhibition of the NF-κB pathway, reducing Cyclin D1 and Bcl-2 levels. In vitro the apoptosis and necroptosis pathways are involved in the cellular death, whereas in vivo, cells undergo necrosis by increased tumor inflammation and reduction of angiogenesis. Thus, fruticuline A acts in tumor cells by multiple mechanisms and represents a promising molecule for drug development in cancer treatment.
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http://dx.doi.org/10.1038/s41598-020-73432-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536426PMC
October 2020

Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells.

Toxicol Appl Pharmacol 2020 07 16;399:115055. Epub 2020 May 16.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands. Electronic address:

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.
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http://dx.doi.org/10.1016/j.taap.2020.115055DOI Listing
July 2020

Reply.

Hepatology 2020 11 16;72(5):1885-1886. Epub 2020 Oct 16.

Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

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http://dx.doi.org/10.1002/hep.31291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702153PMC
November 2020

Inhibition of Hepatic Bile Acid Uptake by Myrcludex B Promotes Glucagon-Like Peptide-1 Release and Reduces Obesity.

Cell Mol Gastroenterol Hepatol 2020 21;10(3):451-466. Epub 2020 Apr 21.

Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Department of Gastroenterology and Hepatology, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Background & Aims: Bile acids are important metabolic signaling molecules. Bile acid receptor activation promotes body weight loss and improves glycemic control. The incretin hormone GLP-1 and thyroid hormone activation of T4 to T3 have been suggested as important contributors. Here, we identify the hepatic bile acid uptake transporter Na taurocholate co-transporting polypeptide (NTCP) as target to prolong postprandial bile acid signaling.

Methods: Organic anion transporting polypeptide (OATP)1a/1b KO mice with or without reconstitution with human OATP1B1 in the liver were treated with the NTCP inhibitor Myrcludex B for 3.5 weeks after the onset of obesity induced by high fat diet-feeding. Furthermore, radiolabeled T4 was injected to determine the role of NTCP and OATPs in thyroid hormone clearance from plasma.

Results: Inhibition of NTCP by Myrcludex B in obese Oatp1a/1b KO mice inhibited hepatic clearance of bile acids from portal and systemic blood, stimulated GLP-1 secretion, reduced body weight, and decreased (hepatic) adiposity. NTCP inhibition did not affect hepatic T4 uptake nor lead to increased thyroid hormone activation. Myrcludex B treatment increased fecal energy output, explaining body weight reductions amongst unaltered food intake and energy expenditure.

Conclusions: Pharmacologically targeting hepatic bile acid uptake to increase bile acid signaling is a novel approach to treat obesity and induce GLP1- secretion.
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http://dx.doi.org/10.1016/j.jcmgh.2020.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363705PMC
September 2021

Genome-wide expression profiling reveals increased stability and mitochondrial energy metabolism of the human liver cell line HepaRG-CAR.

Cytotechnology 2020 Jun 4;72(3):377-395. Epub 2020 Mar 4.

Tytgat Institute for Liver and Intestinal Research, AG&M, Amsterdam UMC, University of Amsterdam, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.

Human liver cell line HepaRG is a well-known source of human hepatocyte-like cells which, however, displays limited biotransformation and a tendency to transform after 20 passages. The new HepaRG-CAR cell line overexpressing constitutive androstane receptor (CAR, NR1I3), a regulator of detoxification and energy metabolism outperforms the parental HepaRG cell line in various liver functions. To further characterize this cell line and assess its stability we compared HepaRG-CAR with HepaRG cells at different passages for their expression profile, ammonia and lactate metabolism, bile acid and reactive oxygen species (ROS) production. Transcriptomic profiling of HepaRG-CAR vs. HepaRG early-passage revealed downregulation of hypoxia, glycolysis and proliferation and upregulation of oxidative phosphorylation genesets. In addition CAR overexpression downregulated the mTORC1 signaling pathway, which, as mediator of proliferation and metabolic reprogramming, may play an important role in the establishment of the HepaRG-CAR phenotype. The ammonia and lactate metabolism and bile acid production of HepaRG-CAR cells was stable for 10 additional passages compared to HepaRG cells. Interestingly, bile acid production was 4.5-fold higher in HepaRG-CAR vs. HepaRG cells, whereas lactate and ROS production were 2.7- and 2.0-fold lower, respectively. Principal component analysis showed clustering of HepaRG-CAR (early- and late-passage) and HepaRG early-passage and not with HepaRG late-passage indicating that passaging exerted larger effect on the transcriptional profile of HepaRG than HepaRG-CAR cells. In conclusion, overexpression of CAR in HepaRG cells improves their bile acid production, mitochondrial energy metabolism, and stability, with the latter possibly due to reduced ROS production, resulting in an optimized source of human hepatocytes.
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http://dx.doi.org/10.1007/s10616-020-00384-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225227PMC
June 2020

Glycochenodeoxycholate Promotes Liver Fibrosis in Mice with Hepatocellular Cholestasis.

Cells 2020 01 23;9(2). Epub 2020 Jan 23.

Tytgat Institute for Liver and Intestinal Research, Department of Gastroenterology and Hepatology, Amsterdam Gastroenterology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Hydrophobic bile salts are considered to promote liver fibrosis in cholestasis. However, evidence for this widely accepted hypothesis remains scarce. In established animal models of cholestasis, e.g., by knockout, cholestasis and fibrosis are both secondary to biliary damage. Therefore, to test the specific contribution of accumulating bile salts to liver fibrosis in cholestatic disease, we applied the unique model of inducible hepatocellular cholestasis in cholate-fed mice. Glycochenodeoxycholate (GCDCA) was supplemented to humanize the murine bile salt pool, as confirmed by HPLC. Biomarkers of cholestasis and liver fibrosis were quantified. Hepatic stellate cells (HSC) isolated from wild-type mice were stimulated with bile salts. Proliferation, cell accumulation, and collagen deposition of HSC were determined. In cholestatic mice, increased hepatic expression of αSMA and collagen1a mRNA and excess hepatic collagen deposition indicated development of liver fibrosis only upon GCDCA supplementation. In vitro, numbers of myofibroblasts and deposition of collagen were increased after incubation with hydrophobic but not hydrophilic bile salts, and associated with EGFR and MEK1/2 activation. We concluded that chronic hepatocellular cholestasis alone, independently of biliary damage, induces liver fibrosis in mice in presence of the human bile salt GCDCA. Bile salts may have direct pro-fibrotic effects on HSC, putatively involving EGFR and MEK1/2 signaling.
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http://dx.doi.org/10.3390/cells9020281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072501PMC
January 2020

Glyco-conjugated bile acids drive the initial metaplastic gland formation from multi-layered glands through crypt-fission in a murine model.

PLoS One 2019 26;14(7):e0220050. Epub 2019 Jul 26.

Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, Amsterdam, The Netherlands.

Bile acid reflux is known to be associated with the development of Barrett's esophagus and esophageal adenocarcinoma (EAC), yet the role of specific bile acids and the mechanism behind the metaplastic changes is unclear. Here, we demonstrate that multi-layered glandular structures at the squamo-columnar junction in mice contain multiple cell lineages, which resemble the human esophageal submucosal gland ducts. Exposing mice to patient's refluxates induced expansion of multi-layered glandular structures and development of columnar metaplasia at the squamo-columnar junction. The glycine conjugated bile acids induced an intestinal type of metaplasia more typical for Barrett's esophagus. Through lineage tracing, we excluded the involvement of K5+, DCLK1+, and LGR5+ progenitor cells as the primary source in the development of the glandular metaplastic epithelium. We show that the mechanism behind development of metaplasia involves crypt fission and may be independent of stem cell proliferation. Our findings support the hypothesis that in humans, BE arises from non-squamous cells residing in submucosal gland ducts and that induction of intestinal type of metaplasia is most effectively induced by glycine-conjugated bile acids. These novel insights may lead to more effective strategies to prevent development of Barrett's esophagus and esophageal adenocarcinoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220050PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660124PMC
February 2020

NTCP deficiency in mice protects against obesity and hepatosteatosis.

JCI Insight 2019 06 25;5. Epub 2019 Jun 25.

Tytgat Institute for Liver and Intestinal Research, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, Netherlands.

Bile acids play a major role in the regulation of lipid and energy metabolism. Here we propose the hepatic bile acid uptake transporter Na+ taurocholate co-transporting polypeptide (NTCP) as a target to prolong postprandial bile acid elevations in plasma. Reducing hepatic clearance of bile acids from plasma by genetic deletion of NTCP moderately increased plasma bile acid levels, reduced diet-induced obesity, attenuated hepatic steatosis, and lowered plasma cholesterol levels. NTCP-G protein-coupled bile acid receptor (TGR5) double knockout mice were equally protected against diet-induced-obesity as NTCP single knockout mice. NTCP knockout mice displayed decreased intestinal fat absorption and a trend towards higher fecal energy output. Furthermore, NTCP deficiency was associated with an increased uncoupled respiration in brown adipose tissue, leading to increased energy expenditure. We conclude that targeting NTCP-mediated bile acid uptake can be a novel approach to treat obesity and obesity-related hepatosteatosis by simultaneously dampening intestinal fat absorption and increasing energy expenditure.
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http://dx.doi.org/10.1172/jci.insight.127197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675549PMC
June 2019

Blocking Sodium-Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice.

Hepatology 2020 01 13;71(1):247-258. Epub 2019 Aug 13.

Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium-taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B-mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP-binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8 mice. Stimulatory effects of NTCP inhibition were maintained in Sr-b1 mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high-density lipoprotein-derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.
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http://dx.doi.org/10.1002/hep.30792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003915PMC
January 2020

Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice.

J Hepatol 2019 07 29;71(1):153-162. Epub 2019 Mar 29.

Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Meibergdreef 9, Amsterdam, The Netherlands. Electronic address:

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3), for which there are limited therapeutic options, often leads to end-stage liver disease before adulthood due to impaired ABCB4-dependent phospholipid transport to bile. Using adeno-associated virus serotype 8 (AAV8)-mediated gene therapy, we aimed to restore the phospholipid content in bile to levels that prevent liver damage, thereby enabling stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3.

Methods: Ten-week-old Abcb4 mice received a single dose of AAV8-hABCB4 (n = 10) or AAV8-GFP (n = 7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by immunohistochemistry.

Results: Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration. AAV8-hABCB4 expression restored biliary phospholipid excretion, increasing the phospholipid and cholesterol content in bile to levels that ameliorate liver damage. This resulted in normalization of the plasma cholestatic markers, alkaline phosphatase and bilirubin. In addition, AAV8-hABCB4 prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study.

Conclusion: Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Translational studies that verify the clinical feasibility of this approach are warranted.

Lay Summary: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. The proliferation of liver cells was also reduced, which contributes to long-term correction of the phenotype. Further studies are required to evaluate whether this approach can be applied to patients with PFIC3.
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http://dx.doi.org/10.1016/j.jhep.2019.03.021DOI Listing
July 2019

Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions.

Mitochondrion 2019 07 22;47:298-308. Epub 2019 Feb 22.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands; Amsterdam UMC, University of Amsterdam, Surgical Laboratory, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Electronic address:

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.
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http://dx.doi.org/10.1016/j.mito.2019.02.005DOI Listing
July 2019

Therapeutic plasma exchange as a novel treatment for severe intrahepatic cholestasis of pregnancy: Case series and mechanism of action.

J Clin Apher 2018 Dec 15;33(6):638-644. Epub 2018 Oct 15.

Department of Women and Children's Health, King's College London, London, United Kingdom.

Introduction: Intrahepatic cholestasis of pregnancy is characterised by pruritus and elevated serum bile acids. The pruritus can be severe, and pharmacological options achieve inconsistent symptomatic improvement. Raised bile acids are linearly associated with adverse fetal outcomes, with existing management of limited benefit. We hypothesised that therapeutic plasma exchange removes pruritogens and lowers total bile acid concentrations, and improves symptoms and biochemical abnormalities in severe cases that have not responded to other treatments.

Methods: Four women with severe pruritus and hypercholanemia were managed with therapeutic plasma exchange. Serial blood biochemistry and visual analogue scores of itch severity were obtained. Blood and waste plasma samples were collected before and after exchange; individual bile acids and sulfated progesterone metabolites were measured with HPLC-MS, autotaxin activity and cytokine profiles with enzymatic methods. Results were analysed using segmental linear regression to describe longitudinal trends, and ratio t tests.

Results: Total bile acids and visual analogue itch scores demonstrated trends to transiently improve following plasma exchange, with temporary symptomatic benefit reported. Individual bile acids (excluding the drug ursodeoxycholic acid), and the sulfated metabolites of progesterone reduced following exchange (P = .03 and P = .04, respectively), whilst analysis of waste plasma demonstrated removal of autotaxin and cytokines.

Conclusions: Therapeutic plasma exchange can lower potentially harmful bile acids and improve itch, likely secondary to the demonstrated removal of pruritogens. However, the limited current experience and potential complications, along with minimal sustained symptomatic benefit, restrict its current use to women with the most severe disease for whom other treatment options have been exhausted.
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http://dx.doi.org/10.1002/jca.21654DOI Listing
December 2018

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity.

Cell Physiol Biochem 2018 15;48(5):2189-2204. Epub 2018 Aug 15.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (AMC), Amsterdam, the Netherlands.

Background/aims: For applicability of cell-based therapies aimed at the treatment of liver failure, such as bioartificial livers (BALs) and hepatocyte transplantation, it is essential that the applied hepatocytes tolerate exposure to the patient plasma. However, plasma from both healthy donors and acute liver failure (ALF) patients is detrimental to hepatocytes and hepatic cell lines, such as HepaRG. We aimed to elucidate the underlying mechanisms of plasma-induced toxicity against HepaRG cells in order to ultimately develop methods to reduce this toxicity and render HepaRG-BAL treatment more effective.

Methods: Differentiated HepaRG cells cultured in monolayers and laboratory-scale BALs were exposed to culture medium, healthy human plasma, healthy porcine plasma and ALF porcine plasma. Healthy human plasma was fractionated based on size- and polarity, albumin depleted and heat treated to characterize the toxic fraction. The cells were assessed for viability by total protein content and trypan blue staining. Their hepatic differentiation was assessed on transcript level through qRT-PCR and microarray analysis, and on functional level for Cytochrome P450 3A4 activity and ammonia elimination. Mitochondrial damage was assessed by JC-1 staining and mitochondrial gene transcription.

Results: Sixteen hours of healthy human plasma exposure did not affect viability, however, hepatic gene-transcript levels decreased dramatically and dose-dependently within four hours of exposure. These changes were associated with early NF-kB signaling and a shift from mitochondrial energy metabolism towards glycolysis. Healthy human plasma-toxicity was associated with the dose-dependent presence of heat-resistant, albumin-bound and (partly) hydrophobic toxic compound(s). HepaRG cells cultured in BALs were partially protected from plasma-toxicity, which was mainly attributable to medium perfusion and/or 3D configuration applied during BAL culturing. The detrimental human plasma effects were reversible in BAL-cultured cells. Porcine ALF-plasma elicited mitotoxicity additional to the basal detrimental effect of porcine healthy plasma, which were only partially reversible.

Conclusion: A specific fraction of human plasma reduces hepatic differentiation of HepaRG cultures, in association with early NF-κB activation. In addition, ALF-plasma elicits mitotoxic effects. These findings allow for a targeted approach in preventing plasma-induced cell damage.
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http://dx.doi.org/10.1159/000492560DOI Listing
September 2018

Role of the placenta in serum autotaxin elevation during maternal cholestasis.

Am J Physiol Gastrointest Liver Physiol 2018 09 21;315(3):G399-G407. Epub 2018 Jun 21.

Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca , Salamanca , Spain.

Intrahepatic cholestasis of pregnancy (ICP) is frequently accompanied by pruritus, whose etiology has been associated with an enhanced production of lysophosphatidic acid (LPA) by the combined action of phospholipase A1/A2 (PLA1/PLA2) and autotaxin (ATX). Here, we have investigated whether the placenta is involved in LPA release to maternal circulation during ICP. Serum levels of ATX and LPA (determined by ELISA) were elevated in women with ICP, and a correlation between both parameters was found. No relationship between serum levels of ATX or LPA and bile acids was found. Expression levels of ATX and PLA2 were determined by RT-qPCR and Western blot. Placenta ATX but not PLA2 was significantly upregulated in ICP, and a tendency to increase was found at the protein level. A correlation between serum ATX and placental ATX mRNA levels was found. In human placenta at term, ATX was clearly detected (by immunofluorescence) in Hofbauer cells, but only faintly in trophoblast cells. In pregnant rats, the expression of Atx and Pla2 in placenta was lower than in liver. When obstructive cholestasis was imposed by bile duct ligation from day 14 of gestation until term, placenta Atx and Pla2 expression was markedly enhanced, and overexpression was confirmed at the protein level for Pla2, whereas Atx protein was not detected. In conclusion, the placenta substantially participates in LPA production during gestation. This contribution is markedly higher during maternal cholestasis and hence, may be involved in ICP-associated pruritus. NEW & NOTEWORTHY Fetal placental macrophages and, to a lesser extent, trophoblast cells express high levels of autotaxin at term. An increased expression of mRNA and protein autotaxin, the key secretory enzyme responsible for the production of lysophosphatidic acid in serum, has been observed in placentas of women with cholestasis of pregnancy, which supports that the placenta can contribute to an increased production of this pruritogenic compound in women suffering from this liver disease.
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http://dx.doi.org/10.1152/ajpgi.00112.2018DOI Listing
September 2018

Pilot study with IBAT inhibitor A4250 for the treatment of cholestatic pruritus in primary biliary cholangitis.

Sci Rep 2018 04 27;8(1):6658. Epub 2018 Apr 27.

Sahlgrenska Academy, Institute of Medicine, Department of Molecular and Clinical Medicine and Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden.

Pruritus is a common complication of cholestatic liver diseases. Inhibition of the ileal bile acid transporter (IBAT/ASBT) may emerge as treatment option. Our aim was to assess tolerability and effect on pruritus of the selective IBAT inhibitor A4250 in patients with primary biliary cholangitis (PBC). Ten patients with PBC and bile acid sequestrant treatment of cholestatic pruritus were after a two-week wash out of the bile acid sequestrant treated with either 0.75 mg (n = 4) or 1.5 mg (n = 5) of A4250 for four weeks. Patients' pruritus was assessed by Visual Analogue Scale (VAS), 5-D itch scale and the pruritus module of the PBC40 questionnaire. Plasma bile acids and 7α-hydroxy-4-cholesten-3-one were measured by UPLC-MS/MS, plasma fibroblast growth factor 19 by ELISA, and serum autotaxin activity by homemade assay. All nine patients exposed to A4250 reported a remarkable improvement in pruritus, until none or mild according to 5-D itch, VAS and PBC40 pruritus. Five patients finished the study prematurely due to abdominal pain (5/5) and diarrhoea (4/5). The high incidence of probably bile acid malabsorption-related diarrhoea and abdominal pain in the bile acid sequestrant pre-treated population indicates that the start dose of A4250 may have been too high for adult patients.
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http://dx.doi.org/10.1038/s41598-018-25214-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923243PMC
April 2018

Intestinal Farnesoid X Receptor Activation by Pharmacologic Inhibition of the Organic Solute Transporter α-β.

Cell Mol Gastroenterol Hepatol 2018 Mar 28;5(3):223-237. Epub 2017 Nov 28.

Tytgat Institute for Liver and Intestinal Research, Department of Gastroenterology and Hepatology, Amsterdam Gastroenterology and Metabolism, Academic Medical Center, Amsterdam, The Netherlands.

Background & Aims: The organic solute transporter α-β (OSTα-OSTβ) mainly facilitates transport of bile acids across the basolateral membrane of ileal enterocytes. Therefore, inhibition of OSTα-OSTβ might have similar beneficial metabolic effects as intestine-specific agonists of the major nuclear receptor for bile acids, the farnesoid X receptor (FXR). However, no OSTα-OSTβ inhibitors have yet been identified.

Methods: Here, we developed a screen to identify specific inhibitors of OSTα-OSTβ using a genetically encoded Förster Resonance Energy Transfer (FRET)-bile acid sensor that enables rapid visualization of bile acid efflux in living cells.

Results: As proof of concept, we screened 1280 Food and Drug Administration-approved drugs of the Prestwick chemical library. Clofazimine was the most specific hit for OSTα-OSTβ and reduced transcellular transport of taurocholate across Madin-Darby canine kidney epithelial cell monolayers expressing apical sodium bile acid transporter and OSTα-OSTβ in a dose-dependent manner. Moreover, pharmacologic inhibition of OSTα-OSTβ also moderately increased intracellular taurocholate levels and increased activation of intestinal FXR target genes. Oral administration of clofazimine in mice (transiently) increased intestinal FXR target gene expression, confirming OSTα-OSTβ inhibition in vivo.

Conclusions: This study identifies clofazimine as an inhibitor of OSTα-OSTβ in vitro and in vivo, validates OSTα-OSTβ as a drug target to enhance intestinal bile acid signaling, and confirmed the applicability of the Förster Resonance Energy Transfer-bile acid sensor to screen for inhibitors of bile acid efflux pathways.
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http://dx.doi.org/10.1016/j.jcmgh.2017.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904037PMC
March 2018
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