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Acetaminophen (APAP) intoxication is the foremost cause of drug-induced liver failure in developed countries. The only pharmacologic treatment option, N-acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti-MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP-intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti-MT antibodies. Anti-MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS-stimulated bone marrow-derived macrophages. Importantly, NAC and anti-MT antibodies were equally effective whereas administration of anti-MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP-induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono- or add-on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. HCC typically develops on a background of chronic inflammation and fibrosis with tumor associated macrophages (TAMs) playing an important role in chronic inflammation-induced HCC and progression. However, the liver harbors unique macrophages, resident liver Kupffer cells (KCs) and monocyte-derived macrophages (Mo-Mφ), and their contribution to HCC and to the population of TAMs is incompletely known. Here, we characterized the tumor microenvironment and the proportion and transcriptional profile of hepatic macrophages (Mφ) in two commonly used HCC mouse models. A gradually increased expression of inflammatory, immune regulatory, fibrotic and cell proliferation pathways and markers was observed during diethylnitrosamine (DEN)- and non-alcoholic steatohepatitis (NASH)-induced HCC development. The transcriptional phenotypes of isolated hepatic Mφ subsets were clearly distinct and shifted during HCC development, with mixed pro-inflammatory and tumor-promoting expression profiles. There were marked differences between the models as well, with Mφ in NASH-HCC exhibiting a more immunomodulatory phenotype, in conjunction with an upregulation of lipid metabolism genes. Our data show that at least some infiltrated macrophages display expression of pro-tumoral markers, and that Kupffer cells are part of the population of TAMs and enhance tumor progression. These insights are useful to further unravel sequential pathogenic events during hepatocarcinogenesis and direct future development of new treatment strategies for HCC.

Cholestatic liver disease denotes any situation associated with impaired bile flow concomitant with a noxious bile acid accumulation in the liver and/or systemic circulation. Cholestatic liver disease can be subdivided into different types according to its clinical phenotype, such as biliary atresia, drug‐induced cholestasis, gallstone liver disease, intrahepatic cholestasis of pregnancy, primary biliary cholangitis and primary sclerosing cholangitis. Considerable effort has been devoted to elucidating underlying mechanisms of cholestatic liver injuries and explore novel therapeutic and diagnostic strategies using animal models. Animal models employed according to their appropriate applicability domain herein play a crucial role. This review provides an overview of currently available in vivo animal models, fit‐for‐purpose in modelling different types of cholestatic liver diseases. Moreover, a practical guide and workflow is provided which can be used for translational research purposes, including all advantages and disadvantages of currently available in vivo animal models.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. For advanced HCC, there is still an unmet need for more effective therapeutic strategies. HCC is typically associated with hypoxia and the hypoxia-inducible factor (HIF) regulatory pathway plays an important role in HCC development and progression. Therefore, we investigated the therapeutic potential of isoform-specific HIF-1α and HIF-2α antisense oligonucleotides (ASOs), along with their effect on the inflammatory and fibrotic component of the tumor microenvironment (TME), in an experimental HCC mouse model. Based on its efficacy and safety, a dosage regimen of 20 mg/kg intraperitoneal injection of HIFα ASO twice per week was selected for further investigation in a preventive and therapeutic setting in a N,N-diethylnitrous amide (DEN)-induced HCC mouse model. DEN administration resulted in 100% tumor formation and HIFα ASO administration led to effective and selective hepatic downregulation of its target genes. HIFα ASO treatment had no effect on tumor numbers, but even enhanced the increased hepatic expression of HCC tumor markers, α-fetoprotein and glypican-3, compared to scrambled control ASO treatment in HCC mice. Especially HIF-1α ASO treatment resulted in an enhanced increase of monocytes and monocyte-derived macrophages in the liver and an enhanced hepatic upregulation of inflammatory markers. Both HIFα ASOs aggravated liver fibrosis in HCC mice compared to scrambled ASO treatment. The observed effects of our dosing regimen for HIF-1α and HIF-2α ASO treatment in the DEN-induced HCC mouse model discourage the use of HIFα isoforms as targets for the treatment of HCC.

Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated with new input information.

Made available in DSpace on 2022-04-29T08:36:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fonds Wetenschappelijk Onderzoek Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer‐related death globally. HCC is a complex multistep disease and usually emerges in the setting of chronic liver diseases. The molecular pathogenesis of HCC varies according to the etiology, mainly caused by chronic hepatitis B and C virus infections, chronic alcohol consumption, aflatoxin‐contaminated food, and non‐alcoholic fatty liver disease associated with metabolic syndrome or diabetes mellitus. The establishment of HCC models has become essential for both basic and translational research to improve our understanding of the pathophysiology and unravel new molecular drivers of this disease. The ideal model should recapitulate key events observed during hepatocarcinogenesis and HCC progression in view of establishing effective diagnostic and therapeutic strategies to be translated into clinical practice. Despite considerable efforts currently devoted to liver cancer research, only a few anti‐HCC drugs are available, and patient prognosis and survival are still poor. The present paper provides a state-of‐the‐art overview of in vivo and in vitro models used for translational modeling of HCC with a specific focus on their key molecular hallmarks. Department of Pathology School of Veterinary Medicine and Animal Science University of São Paulo (USP) Department of Structural and Functional Biology Biosciences Institute São Paulo State University (UNESP) Department of Pathology Botucatu Medical School São Paulo State University (UNESP) Department of Pharmaceutical and Pharmacological Sciences Vrije Universiteit Brussel Gut‐Liver Immunopharmacology Unit Basic and Applied Medical Sciences Liver Research Center Ghent Faculty of Medicine and Health Sciences Ghent University Hepatology Research Unit Internal Medicine and Paediatrics Liver Research Center Ghent Faculty of Medicine and Health Sciences Ghent University Department of Gastroenterology Clinics Hospital School of Medicine University of São Paulo (HC‐FMUSP) Department of Immunology Institute of Biomedical Sciences IV University of São Paulo (USP) Department of Structural and Functional Biology Biosciences Institute São Paulo State University (UNESP) Department of Pathology Botucatu Medical School São Paulo State University (UNESP) CAPES: 001 FAPESP: 16/12015‐0 FAPESP: 16/14420‐0 FAPESP: 18/10953‐9 Fonds Wetenschappelijk Onderzoek: 18/10953‐9 CNPq: 310557/2019‐4 Fonds Wetenschappelijk Onderzoek: G009514N Fonds Wetenschappelijk Onderzoek: G010214N

The provided dataset describes the differential gene expression profile of human hepatoma HepaRG cells cultured in monolayer configuration upon treatment with chemical compounds with cholestatic potential, including food additives sunset yellow and tartrazine and cosmetic ingredient triclosan, while being exposed to a highly concentrated bile acid mixture. Whole genome microarray Affymetrix Human U133 plus 2.0 was used to obtain the raw data followed by normalization, summarization and background adjustments by means of Robust Multichip Average Express software. Raw data of the different conditions were included as .CEL files in the Gene Expression Omnibus with accession number GSE169072. These data may serve as the basis for further refinement studies to establish an adequate transcriptomic signature of chemical-induced cholestasis fit-for-purpose in screening the cholestatic liability of different types of chemical compounds. (C) 2021 The Authors. Published by Elsevier Inc.

Patients with chronic liver disease from different aetiologies show a light serum Cu isotopic composition compared to the reference population, with the enrichment in the 63Cu isotope correlating with the severity of the disease. However, the mechanisms underlying Cu isotope fractionation at the onset and during progression of the disease are still unclear. In this work, a common bile duct ligation (CBDL) murine model was used to investigate the effect of cholestasis-induced liver disease on the Cu isotopic composition. Wild type male and female mice underwent surgical ligation of the common bile duct and were sacrificed 2, 4 and 6 weeks, and 4, 6 and 8 weeks after the surgical intervention, respectively. The age- and gender-matched control mice underwent sham surgery. Disease progression was evaluated using serum bilirubin levels, hepatic pro-inflammatory chemokine levels and Metavir fibrosis score. CBDL-operated mice show an overall body enrichment in the light isotope 63Cu. The Cu isotopic composition of organs, bone and serum becomes gradually lighter compared to the sham-operated mice with increasing severity of the disease. The light Cu isotopic composition of the CBDL-operated mice might result from an altered Cu intake and/or excretion. As the intestinal uptake of dietary Cu is largely mediated by transporters of Cu(i), mRNA and protein expression levels of two major metal transporters (CTR1 and DMT1) and Cu reductases (STEAP proteins and duodenal cytochrome B) were examined in the duodenal tissues as potential factors inducing Cu isotope fractionation. However, no significant differences in protein expression levels were observed between the CBDL- and sham-operated mice.

Recently, our research group reported on the development of 3β-[18F]Fluorocholic acid (3β-[18F]FCA), a 18F labeled bile acid to detect drug interference with the bile acid transporters (drug-induced cholestasis). It was hypothesized that 3β-[18F]FCA could also be used as a non-invasive tool to monitor (regional) liver function in vivo in different liver diseases through altered expression of bile acid transporters. Methods Hepatobiliary transport of 3β-[18F]FCA was evaluated in four murine liver disease models. Acute liver injury was induced by oral gavage of an acetaminophen (APAP) overdose (300 mg/kg). Chronic cholangiopathy and non-alcoholic steatohepatitis (NASH) were induced by feeding mice 3,5-diethoxycarbonyl- 1,4-dihydrocollidine (DDC) diet or methionine and choline deficient (MCD) diet, respectively. Hepatocellular carcinoma (HCC) was evoked by intraperitoneal injection of 35 mg/kg diethylnitrosamine (DEN) once a week for 23 weeks. Gene expression of the murine bile acid transporters was determined by RT-qPCR. Results Hepatobiliary transport of 3β-[18F]FCA was not significantly altered after an APAP overdose. Mice fed the DDC or MCD diet showed impaired transport of 3β-[18F]FCA compared to baseline, which was associated with altered expression of the bile acid transporters ntcp, oatp4 and mrp2. After recovery from DDC- and MCD-induced liver injury, 3β-[18F]FCA parameters returned to baseline. Global hepatobiliary transport of 3β-[18F]FCA in HCC bearing mice was not significantly different compared to control mice. However, HCC lesions showed reduced hepatic uptake of the tracer (tumor-to-background: 0.45 ± 0.13), which was in line with decreased in expression of basolateral bile acid uptake transporters nctp and oatp4 in tumor tissue. Conclusion 3β-[18F]FCA is a useful tool to assess and longitudinally follow-up liver function in several mouse models for liver diseases that are associated with altered expression of the bile acid transporters. These results point towards the (pre)clinical utility of 3β-[18F]FCA as a PET tracer to monitor altered liver functionality in patients with chronic liver diseases.

The prevalence of obesity and metabolic diseases such as type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) has risen dramatically over the past decades. At present, bariatric surgery is the most effective treatment for this global health problem, through effects on food intake, gut hormone secretion, metabolic signaling pathways, and adipose tissue dysfunction. The liver occupies a central role in carbohydrate, protein, and lipid metabolism. Notably, a reduction in hepatic fat content and an improvement in hepatic insulin resistance are among the earliest beneficial effects of bariatric surgery, which has therefore emerged as an attractive treatment option for NAFLD. However, as the scope and popularity of weight loss surgery have expanded, new questions have arisen regarding its safety in patients with liver cirrhosis, the outcome of liver transplantation in patients with a history of bariatric surgery, and over incidental reports of liver failure following surgery. Studies in humans and rodents have also linked bariatric surgery to an increased risk of developing alcohol use disorder, a major risk factor for liver disease. This review integrates data from clinical and translational research to delineate both the beneficial impact of bariatric surgery on the liver and the potential risks involved.

Cholestasis is a major pathological manifestation, often resulting in detrimental liver conditions, which occurs in a variety of indications collectively termed cholestatic liver diseases. The frequent asymptomatic character and complexity of cholestasis, together with the lack of a straightforward biomarker, hampers early detection and treatment of the condition. The 'omics' era, however, has resulted in a plethora of cholestatic indicators, yet a single clinically applicable biomarker for a given cholestatic disease remains missing. The criteria to fulfil as an ideal biomarker as well as the challenging molecular pathways in cholestatic liver diseases advocate for a scenario in which multiple biomarkers, originating from different domains, will be assessed concomitantly. This review gives an overview of classical clinical and novel molecular biomarkers in cholestasis, focusing on their benefits and drawbacks. ispartof: BIOMARKERS IN MEDICINE vol:15 issue:6 pages:437-454 ispartof: location:England status: published

Liver disease, cirrhosis and portal hypertension can be complicated by pulmonary vascular disease, which may affect prognosis and influence liver transplantation (LT) candidacy. Pulmonary vascular complications comprise hepatopulmonary syndrome (HPS) and portopulmonary hypertension (POPH). Although these two conditions develop on a same background and share a common trigger, pulmonary responses are distinct and occur at different anatomical sites of the pulmonary circulation. HPS affects 10-30% of patients referred for LT, and is characterized by gas exchange abnormalities due to pulmonary vasodilation and right-to-left shunting. POPH occurs in 5%, and is defined by pulmonary arterial hypertension due to increased pulmonary vascular resistance, which leads to hemodynamic failure. Even though HPS and POPH may have a substantial negative impact on survival, both entities are clinically underrecognized and frequently misdiagnosed. Without intervention, the 5-year survival rate is 23% in HPS and 14% in POPH. Their presence should be actively sought by organized screening in patients presenting with dyspnea and in all patients on the waitlist for LT, also because clinical symptoms are commonly non-specific or even absent. LT may lead to resolution, however, advanced stages of either HPS or POPH may jeopardize safe and successful LT. This implicates the need of proper identification of HPS and POPH cases, as well as the need to be able to successfully ‘bridge’ patients to LT by medical intervention. A review article on this topic has been published in this journal in 2007 (1). This updated review focuses on recent advances in the diagnosis and management of these 2 liver-induced pulmonary vascular disorders and incorporates results from our recent work.

Background & Aims Nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The intrauterine and early life environment can have an important impact on long-term metabolic health. We investigated the impact of maternal prepregnancy obesity, (pre)gestational diabetes, breastfeeding, and birth anthropometrics/preterm birth on the development of NAFLD in children and adolescents. Methods A comprehensive search was performed in MEDLINE, PubMed Central, EMBASE, and grey literature databases through August 2020. The primary outcome was the prevalence of pediatric NAFLD, whereas the histologic severity of steatohepatitis and/or fibrosis were secondary outcomes. Study selection, data extraction, and quality assessment were performed by 2 independent reviewers. Results Our systematic review included 33 articles. Study heterogeneity regarding patient populations, diagnostic tools, and overall quality was considerable. Eight studies determined the impact of maternal prepregnancy overweight/obesity and identified this as a possible modifiable risk factor for pediatric NAFLD. Conversely, 8 studies investigated (pre)gestational diabetes, yet the evidence on its impact is conflicting. Breastfeeding was associated with a reduced risk for NAFLD, steatohepatitis, and fibrosis, especially in studies that evaluated longer periods of breastfeeding. Being born preterm or small for gestational age has an unclear impact on the development of NAFLD, although an early catch-up growth might drive NAFLD. Conclusions In a systematic review, we found that maternal prepregnancy overweight and obesity were associated with an increased risk of pediatric NAFLD. Breastfeeding might be protective against the development of NAFLD when the duration of breastfeeding is sufficiently long (≥6 months).

Background The poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment. Methods The in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, the human THP1 monocyte cell line and mouse macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20–25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage, daily or twice daily, in preventive and therapeutic studies. The liver damage was evaluated for inflammation, angiogenesis, fibrosis and HCC development via histology, RT-qPCR, multiplex analyses and ROS levels. Results A concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100 % tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment. Conclusions NOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of chimeric mice respond to hypercaloric diets. As early as 4 weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatohepatitis selectively in the human graft, followed by pericellular fibrosis after 8 weeks of hypercaloric feeding. The PNPLA3 148M variant, either from a homozygous 148M human donor or overexpressed in a homozygous 148I donor background, caused microvesicular and more severe steatosis. In these livers hepatocytes displayed frequent ballooning degeneration, resulting in more active steatohepatitis than in 148I livers. We conclude that PNPLA3 148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetics associated with advanced fatty liver diseases.

Background and aims Obesity, diabetes and associated non-alcoholic steatohepatitis (NASH) are rising risk factors for hepatocellular carcinoma (HCC). Macrophages are important immune cells involved in inflammation and tumour development. Macrophage inositol-requiring enzyme 1 alpha (IRE1α), an ER-stress protein, has been shown to be involved in macrophage cytokine production, and myeloid-specific IRE1α knock-out (myeloid IRE1α-KO) mice showed reduced weight gain during high-fat diet feeding. However, the effect of myeloid IRE1α on NASH and subsequent HCC development has not been examined. Here, we characterized the transcriptional profile of the hepatic macrophage population in a diabetes-NASH-HCC mouse model, and investigated the effect of myeloid-specific IRE1α deletion on the phenotype of hepatic macrophage subsets and experimental NASH-HCC development. Methods Mice with non-functional myeloid IRE1α were created by crossing Ire1a floxed mice with Lysm-Cre mice. Two-day old myeloid IRE1α-KO and wild type (WT) mice were subcutaneously injected with streptozotocin (STZ), and male mice were fed a high-fat, -sucrose, -cholesterol diet (Western diet, WD) from the age of 4 weeks until 21 weeks. Control myeloid IRE1α-KO and WT mice received a PBS injection and were fed a matched control diet. These mice were evaluated for obesity, diabetes, NASH and HCC. The hepatic macrophage population was evaluated by flow cytometry and RNA sequencing on FACS-isolated macrophage subsets. Results STZ-injection and WD feeding resulted in an impaired glucose tolerance, advanced NASH with fibrosis, and HCC development. Myeloid IRE1α-KO STZ mice showed lower fasting glucose levels at the start of WD feeding, and an improved glucose tolerance and attenuated HCC development after 17 weeks of WD feeding despite a similar degree of liver steatosis and inflammation compared to WT mice. Transcriptomic analysis of WT liver Kupffer cells, macrophages and monocytes revealed phenotypical changes in those cell subsets during NASH-HCC development. Isolated liver Kupffer cells and macrophages from mice with a myeloid IRE1α deletion showed downregulated pathways involved in immune system activation and metabolic pathways (only in Kupffer cells), whereas pathways involved in cell division and metabolism were upregulated in monocytes. These transcriptional differences were attenuated during NASH-HCC development. Conclusion Our results show that myeloid-specific IRE1α deletion results in an altered transcriptional profile of hepatic macrophages and dampens diabetes-induced NASH-HCC development, possibly by attenuated diabetes induction.

In parallel to the obesity pandemic, nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in adults worldwide [1]. Especially its inflammatory form, no...

Chronic liver disease (CLD) is a growing health concern which accounts for two million deaths per year. Obesity, alcohol overconsumption, and progressive cholestasis are commonly characterized by persistent low-grade inflammation and advancing fibrosis, which form the basis for development of end-stage liver disease complications, including hepatocellular carcinoma. CLD pathophysiology extends to the intestinal tract and is characterized by intestinal dysbiosis, bile acid dysregulation, and gut barrier disruption. In addition, macrophages are key players in CLD progression and intestinal barrier breakdown. Emerging studies are unveiling macrophage heterogeneity and driving factors of their plasticity in health and disease. To date, in-depth investigation of how gut–liver axis disruption impacts the hepatic and intestinal macrophage pool in CLD pathogenesis is scarce. In this review, we give an overview of the role of intestinal and hepatic macrophages in homeostasis and gut–liver axis disruption in progressive stages of CLD.

Hepatic encephalopathy (HE) is a neurological complication of hepatic dysfunction and portosystemic shunting. It is highly prevalent in patients with cirrhosis and is associated with poor outcomes. New insights into the role of peripheral origins in HE have led to the development of innovative treatment strategies like faecal microbiota transplantation. However, this broadening of view has not been applied fully to perturbations in the central nervous system. The old paradigm that HE is the clinical manifestation of ammonia-induced astrocyte dysfunction and its secondary neuronal consequences requires updating. In this review, we will use the holistic concept of the neurogliovascular unit to describe central nervous system disturbances in HE, an approach that has proven instrumental in other neurological disorders. We will describe HE as a global dysfunction of the neurogliovascular unit, where blood flow and nutrient supply to the brain, as well as the function of the blood-brain barrier, are impaired. This leads to an accumulation of neurotoxic substances, chief among them ammonia and inflammatory mediators, causing dysfunction of astrocytes and microglia. Finally, glymphatic dysfunction impairs the clearance of these neurotoxins, further aggravating their effect on the brain. Taking a broader view of central nervous system alterations in liver disease could serve as the basis for further research into the specific brain pathophysiology of HE, as well as the development of therapeutic strategies specifically aimed at counteracting the often irreversible central nervous system damage seen in these patients. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL).

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and is strongly associated with obesity, dyslipidemia and insulin resistance. NAFLD often presents as simple steatosis (NAFL) but can progress to non-alcoholic steatohepatitis (NASH) and fibrosis. Current non-invasive biomarkers are not tailored to identify significant (⩾F2) fibrosis, although recent guidelines recommend a stringent follow-up of this patient population. We and others have reported on the role of pathological angiogenesis in the pathogenesis of NAFLD, highlighting pro-angiogenic factors as potential diagnostic markers.To investigate the applicability of angiogenic and endothelial dysfunction markers as non-invasive diagnostic tools for NASH or NASH-associated fibrosis in obese patients.In a prospective cross-sectional study, male patients undergoing bariatric surgery (n=61) and control patients (n=35) were recruited. Serum protein levels and visceral adipose tissue gene expression of endothelial dysfunction and angiogenic markers were analyzed by multiplex bead-based assay and quantitative RT-PCR, respectively. For validation, we recruited a second cohort of patients undergoing bariatric surgery (n=40) and a cohort of NAFLD patients from our outpatient clinic (n=30).We identified serum vascular cell adhesion molecule-1 (VCAM-1) as an independent predictor for ⩾F2 fibrosis (median 14.0 vs 8.7 ng mlVCAM-1 levels are able to accurately predict significant (⩾F2) fibrosis in NAFLD patients.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1β production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFβ-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. Methods Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. Results ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFβ1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. Conclusions Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was deployed for quantitative mapping of the Cu distribution in cryo-sections of liver tissue from mice with cholestatic liver disease induced via common bile duct ligation (CBDL). Cu distribution maps of the liver sections were obtained from the CBDL-operated mice sacrificed at different time points (2, 4 and 6 weeks) after the surgical intervention and compared with those of the corresponding control (sham-operated) mice. Cu quantification was accomplished versus matrix-matched thin sections of spiked liver tissue homogenates and versus spiked gelatin droplet standards. No statistical differences were obtained between the results using the two calibration approaches and thus, both were considered suitable for quantitative Cu bioimaging of liver cryo-sections. On the basis of practical considerations, i.e. simplicity, low cost and availability of the material, spiked gelatin droplet standards are the preferred choice for quantitative determination of the Cu distribution in liver tissue cryo-sections. An inhomogeneous hepatic Cu distribution was observed in the CBDL mice, in contrast to the homogeneous hepatic Cu distribution established for the sham-operated mice. The Cu levels increased with the progression of the disease and a strong accumulation was observed in some necrotic areas. High-resolution LA-ICP-MS bioimaging, using a circular spot size of 2 μm, was suitable for the visualization of the Cu distribution in liver tissue on a (sub-)cellular level. In addition to the quantitative Cu mapping, the spatial distribution of Zn was also monitored in the liver cryo-sections of the control and the 2, 4 and 6 week CBDL mice, but in all cases, Zn was homogeneously distributed across the tissue.

Background & Aims Peroxisome proliferator-activated receptors (PPARs) are essential regulators of whole-body metabolism, but also modulate inflammation in immune cells, notably macrophages. We compared the effects of selective PPAR agonists to those of the pan-PPAR agonist lanifibranor in non-alcoholic fatty liver disease (NAFLD), and studied isoform-specific effects on hepatic macrophage biology. Methods Lanifibranor or selective PPARα (fenofibrate), PPARγ (pioglitazone) and PPARδ (GW501516) agonists were therapeutically administered in choline-deficient, amino acid-defined high-fat diet (CDAA-HFD)- and Western diet (WD)-fed mouse models of NAFLD. Acute liver injury was induced by carbon tetrachloride (CCl4). The role of PPARs on macrophage functionality was studied in isolated hepatic macrophages, bone marrow-derived macrophages stimulated with palmitic acid, and circulating monocytes from patients with NAFLD. Results Lanifibranor improved all histological features of steatohepatitis in CDAA-HFD-fed mice, including liver fibrosis, thereby combining and exceeding specific effects of the single PPAR agonists. Its potent anti-steatotic efficacy was confirmed in a 3D liver biochip model with primary cells. Infiltrating hepatic monocyte-derived macrophages were reduced following PPAR agonist administration, especially with lanifibranor, even after short-term treatment, paralleling improved steatosis and hepatitis. Lanifibranor similarly decreased steatosis, liver injury and monocyte infiltration in the WD model. In the acute CCl4 model, neither single nor pan-PPAR agonists directly affected monocyte recruitment. Hepatic macrophages isolated from WD-fed mice displayed a metabolically activated phenotype. Lanifibranor attenuated the accompanying inflammatory activation in both murine palmitic acid-stimulated bone marrow-derived macrophages, as well as patient-derived circulating monocytes, in a PPARδ-dependent fashion. Conclusion Pan-PPAR agonists combine the beneficial effects of selective PPAR agonists and may counteract inflammation and disease progression more potently. PPARδ agonism and lanifibranor directly modulate macrophage activation, but not infiltration, thereby synergizing with beneficial metabolic effects of PPARα/γ agonists. Lay summary Peroxisome proliferated-activated receptors (PPARs) are essential regulators of metabolism and inflammation. We demonstrated that the pan-PPAR agonist lanifibranor ameliorated all aspects of non-alcoholic fatty liver disease in independent experimental mouse models. Non-alcoholic fatty liver disease and fatty acids induce a specific polarization status in macrophages, which was altered by lanifibranor to increase expression of lipid handling genes, thereby decreasing inflammation. PPAR isoforms have differential therapeutic effects on fat-laden hepatocytes, activated hepatic stellate cells and inflammatory macrophages, supporting the clinical development of pan-PPAR agonists.

Hepatocyte death during acetaminophen (APAP) intoxication elicits a reactive inflammatory response, with hepatic recruitment of neutrophils and monocytes, which further aggravates liver injury. Neutrophil elastase (NE), secreted by activated neutrophils, carries degradative and cytotoxic functions and maintains a proinflammatory state. We investigated NE as a therapeutic target in acetaminophen-induced liver injury (AILI). C57BL/6 mice were administered a toxic dose of APAP, 2 h prior to receiving the NE inhibitor sivelestat, N-acetylcysteine (NAC), or a combination therapy, and were euthanized after 24 and 48 h. Upon APAP overdose, neutrophils and monocytes infiltrate the injured liver, accompanied by increased levels of NE. Combination therapy of NAC and sivelestat significantly limits liver damage, as evidenced by lower serum transaminase levels and less hepatic necrosis compared to mice that received APAP only, and this to a greater extent than NAC monotherapy. Lower hepatic expression of proinflammatory markers was observed in the combination treatment group, and flow cytometry revealed significantly less monocyte influx in livers from mice treated with the combination therapy, compared to untreated mice and mice treated with NAC only. The potential of NE to induce leukocyte migration was confirmed in vitro. Importantly, sivelestat did not impair hepatic repair. In conclusion, combination of NE inhibition with sivelestat and NAC dampens the inflammatory response and reduces liver damage following APAP overdose. This strategy exceeds the standard of care and might represent a novel therapeutic option for AILI.

Cholestasis underlies one of the major manifestations of drug-induced liver injury. Drug-induced cholestatic liver toxicity is a complex process, as it can be triggered by a variety of factors that induce 2 types of biological responses, namely a deteriorative response, caused by bile acid accumulation, and an adaptive response, aimed at removing the accumulated bile acids. Several key events in both types of responses have been characterized in the past few years. In parallel, many efforts have focused on the development and further optimization of experimental cell culture models to predict the occurrence of drug-induced cholestatic liver toxicity in vivo. In this paper, a state-of-the-art overview of mechanisms and in vitro models of drug-induced cholestatic liver injury is provided.

Cholestatic liver disease covers a range of biliary disorders marked by an impaired bile duct flow. Various conditions can result in bile obstruction including choledocholithiasis, surgical trauma, and autoimmune disorders. Cholestatic liver disease can be mild but generally progresses to more severe conditions with increased hepatobiliary injury, cholangitis, and ultimately liver fibrosis and cirrhosis. An extensively used experimental model to investigate the pathophysiology of biliary cirrhosis and potential novel therapies is the common bile duct ligation in mice and rats. Common bile duct ligation induces the different stages of cholestatic-induced liver disease being cholestasis, subsequently accompanied by inflammation and finally liver fibrosis and cirrhosis. In this protocol, an outline of the surgical procedures to conduct common bile duct ligation in mice is provided. The major steps include the isolation of the common bile duct, followed by ligation and dissection.

Angiogenesis contributes to the development of nonalcoholic steatohepatitis (NASH) and promotes inflammation, fibrosis, and progression to hepatocellular carcinoma (HCC). Angiopoietin-2 (Ang-2) is a key regulator of angiogenesis. We aimed to investigate the role of Ang-2 and its potential as a therapeutic target in NASH using human samples, in vivo mouse models, and in vitro assays. Serum Ang-2 levels were determined in 104 obese patients undergoing bariatric surgery and concomitant liver biopsy. The effect of the Ang-2/Tie2 receptor inhibiting peptibody L1-10 was evaluated in the methionine-choline deficient (MCD) and streptozotocin-western diet nonalcoholic fatty liver disease mouse models, and in vitro on endothelial cells and bone marrow-derived macrophages. The hepatic vasculature was visualized with mu CT scans and scanning electron microscopy of vascular casts. Serum Ang-2 levels were increased in patients with histological NASH compared with patients with simple steatosis and correlated with hepatic CD34 immunoreactivity as a marker of hepatic angiogenesis. Serum and hepatic Ang-2 levels were similarly increased in mice with steatohepatitis. Both preventive and therapeutic L1-10 treatment reduced hepatocyte ballooning and fibrosis in MCD diet-fed mice and was associated with reduced hepatic angiogenesis and normalization of the vascular micro-architecture. Liver-isolated endothelial cells and monocytes from MCD-fed L1-10-treated mice showed reduced expression of leukocyte adhesion and inflammatory markers, respectively, compared with cells from untreated MCD diet-fed mice. In the streptozotocin-western diet model, therapeutic Ang-2 inhibition was able to reverse NASH and attenuate HCC progression. In vitro, L1-10 treatment mitigated increased cytokine production in lipopolysaccharide-stimulated endothelial cells but not in macrophages. Conclusion: Our findings provide evidence for Ang-2 inhibition as a therapeutic strategy to target pathological angiogenesis in NASH.

Bile acid accumulation and subsequent liver damage is a frequent adverse effect induced by drugs. Considerable efforts have therefore been focused on the introduction and characterization of tools that allow reliable prediction of this type of drug-induced liver injury. Among those are the cholestatic index and transcriptomic profiling, which are typically assessed in in vitro settings. The present study was set up to test the applicability of both tools to non-pharmaceutical compounds with cholestatic potential, including the industrial compound bis(2-ethylhexyl)phthalate, the cosmetic ingredients triclosan and octynoic acid, the herbicides paraquat and quizalofop-para-ethyl, and the food additives sunset yellow and tartrazine, in a human hepatoma cell culture model of cholestatic liver injury. The cholestatic index method showed cholestatic liability of sunset yellow, tartrazine and triclosan. Of those, tartrazine induced transcriptional changes reminiscent of the transcriptional profile of cholestatic drugs. Furthermore, a number of genes were found to be uniquely modulated by tartrazine, in accordance with the cholestatic drugs atazanavir, cyclosporin A and nefazodone, which may have potential as novel transcriptomic biomarkers of chemical-induced cholestatic liver injury. In conclusion, unambiguous identification of the non-pharmaceutical compounds tested in this study as inducers of cholestasis could not be achieved.

Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.

Overnutrition and a sedentary lifestyle have resulted in the expansion of human obesity and associated metabolic complications. Nonalcoholic fatty liver disease has become the most common chronic liver disease in Western developed countries and can range from simple hepatic steatosis to a combination of steatosis, inflammation, and ballooning degeneration (nonalcoholic steatohepatitis). Obesity and its related liver disease are both risk factors for hepatocellular carcinoma, the incidence of which is expected to increase rapidly. The pathogenesis of nonalcoholic fatty liver disease and its progression to nonalcoholic steatohepatitis and hepatocellular carcinoma involve a deregulated lipid metabolism and a disruption of immune homeostasis and tissue integrity and are associated with a state of chronic inflammation. Macrophages are immune cells essential for maintenance of organ function and homeostasis but can also contribute to tissue damage and maintain a proinflammatory response. Their function depends on their origin, and tissue and can be converted based on local environmental cues. Resident liver macrophages, Kupffer cells, which function as sentinels, provide a first defense and are assisted by infiltrating monocytes in cases of hepatic insult. Until now, the contribution of tissue-residing and infiltrating macrophages to the onset and progression of nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and hepatocellular carcinoma has been only partially unraveled. This review summarizes the current knowledge on the contribution of macrophage subsets to obesity-driven fatty liver disease and its complications and sheds light on still unexplored areas.

Due to the usually late diagnosis and lack of effective therapies, hepatocellular carcinoma (HCC), which poses a growing global health problem, is characterized by a poor prognosis. Angiogenesis plays an important role in HCC progression, and vascular endothelial growth factor (VEGF) and angiopoietins (Angs) are key drivers of HCC angiogenesis. VEGF-targeting strategies already represent an important component of today’s systemic treatment landscape of HCC, whereas targeting the Ang/Tie2 signaling pathway may harbor future potential in this context due to reported beneficial anticancer effects when targeting this pathway. In addition, a better understanding of the relation between Angs and HCC angiogenesis and progression may reveal their potential as predictive factors for post-treatment disease progression and prognosis. In this review, we give a comprehensive overview of the complex role of Ang/Tie2 signaling in HCC, pinpointing its potential value as biomarker and target for HCC treatments, aiding HCC diagnosis and therapy.

The transcriptomic dataset (whole genome microarray Affymetrix Human U133 plus 2.0 and Affymetrix Mouse Genome 430 2.0) presented in this paper describes the differential gene expression profile of a human in vitro model of drug-induced cholestasis and a well-known mouse in vivo model of cholestasis. The in vitro model consists of human hepatoma HepaRG cells in monolayer configuration exposed to 3 different cholestatic drugs with or without bile acids. For in vivo modelling of cholestasis, mice were subjected to bile duct ligation surgery. Consecutive normalization, summarization and background adjustments have been made by means of Robust Multichip Average Express software. (C) 2020 The Author(s). Published by Elsevier Inc.

Hepatopulmonary syndrome (HPS) is a severe complication of cirrhosis with increased risk of mortality. Pulmonary microvascular alterations are key features of HPS, but underlying mechanisms are incompletely understood, and studies on HPS are limited to rats. Placental growth factor (PlGF), a pro-angiogenic molecule that is selectively involved in pathological angiogenesis, may play an important role in HPS development, however, its role has never been investigated. In this study, we validated an HPS model by common bile duct ligation (CBDL) in mice, investigated the kinetic changes in pulmonary angiogenesis and inflammation during HPS development, and provide evidence for a novel therapeutic strategy by targeting pathological angiogenesis. Mice with CBDL developed hypoxemia and intrapulmonary shunting on a background of liver fibrosis. Pulmonary alterations included increased levels of pro-angiogenic and inflammatory markers, which was confirmed in serum of human HPS patients. Increased PlGF production in HPS mice originated from alveolar type II cells and lung macrophages, demonstrated by immunofluorescent stainings. Dysfunctional vessel formation in CBDL mice was visualized by microscopy on vascular corrosion casts. Both prophylactic and therapeutic anti-PlGF (αPlGF) antibody treatment impeded HPS development, as demonstrated by significantly less intrapulmonary shunting and improved gas exchange. αPlGF treatment decreased endothelial cell dysfunction in vivo and in vitro, and was accompanied by reduced pulmonary inflammation. Importantly, αPlGF therapy did not affect liver alterations, supporting αPlGF's capability to directly target the pulmonary compartment. Conclusion: CBDL in mice induces HPS, which is mediated by PlGF production. αPlGF treatment improves experimental HPS by counteracting pulmonary angiogenesis and might be an attractive therapeutic strategy for human HPS. This article is protected by copyright. All rights reserved.

Kupffer cells (KCs) and monocyte-derived macrophages are implicated in non-alcoholic steatohepatitis (NASH) pathogenesis but their functions remain unclear due to the lack of specific markers to distinguish between the different cell types. Additionally, it is unclear if multiple subsets of KCs are present during NASH. Here, we characterized the liver macrophage subsets during methionine/choline deficient (MCD) diet-induced NASH and recovery. We observed a significant reduced contribution of Ly6CloClec4F+Tim4+KCs to the hepatic macrophage pool in MCD fed mice, which normalized during recovery. Ly6CloClec4F-Tim4- monocyte-derived macrophages increased during MCD feeding and returned to baseline during recovery. Ly6CloClec4F+Tim4- monocyte-derived KCs developed during initial recovery but did not self-renew as their numbers were reduced after full recovery. Initial recovery from MCD diet feeding was further characterized by increased proportions of Ki-67+ proliferating KCs. In conclusion, the hepatic macrophage pool undergoes substantial albeit transient changes during NASH and recovery, with the KC pool being maintained by proliferation and differentiation of short-lived monocyte-derived KCs.

Metallothioneins (MTs) are small molecular weight stress response proteins that play a central role as reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been considered to be an intracellular protein with roles to play in the management of heavy metals, as a regulator of cellular redox potential, and as a buffer of free radicals. Our recent studies have highlighted immunomodulatory role of MT in inflammatory diseases and also in the progression of metastatic cell movement. Hence, manipulation and detection of MT is essential for its possible use as a diagnostic and in therapeutic interventions of chronic inflammation. This review describes procedures used to detect MT using techniques such as western immunoblot, competition ELISA, flow cytometry and immunohistochemistry. Additionally, it also describes the use of a colorimetric cell proliferation assay (CellTiter 96 AQueous One Solution/MTS) to study the proliferative effect of MT. © 2017 by John Wiley & Sons, Inc.

Introduction : Drug-induced cholestasis is a liver disorder that might be caused by interference of drugs with the hepatobiliary bile acid transporters. It is important to identify this interference early on in drug development. In this work, Positron Emission Tomography (PET)-imaging with a F-18 labeled bile acid analogue was introduced to detect disturbed hepatobiliary transport of bile acids. Methods : 3 beta-[F-18]fluorocholic acid ([F-18]FCA) was prepared by nucleophilic substitution of a mesylated precursor with [F-18]fluoride, followed by deprotection with sodium hydroxide. Transport of [F-18]FCA was assessed in vitro using CHO-NTCP, HEK-OATP1B1, HEK-OATP1B3 transfected cells and BSEP & MRP2 membrane vesicles. Investigation of [F-18]FCA metabolites was performed with primary mouse hepatocytes. Hepatobiliary transport of [F-18]FCA was evaluated in vivo in wild-type, rifampicin and bosentan pretreated FVB-mice by dynamic mu PET scanning. Results : Radiosynthesis of [F-18] FCA was achieved in a moderate radiochemical yield (8.11-1.94%; non-decay corrected; n = 10) and high radiochemical purity (>99%). FCA was transported by the basolateral bile acid uptake transporters NTCP, OATP1B1 and OATP1B3. For canalicular efflux, BSEP and MRP2 are the relevant bile acid transporters. [F-18]FCA was found to be metabolically stable. In vivo, [F-18]FCA showed fast hepatic uptake (4.5-0.5 min to reach 71.8-1.2% maximum % ID) and subsequent efflux to the gallbladder and intestines (93.3-6.0% ID after 1 hour). Hepatobiliary transport of [F-18]FCA was significantly inhibited by both rifampicin and bosentan. Conclusion : A F-18 labeled bile acid analogue, [F-18]FCA, has been developed that shows transport by NTCP, OATP, MRP2 and BSEP. [F-18]FCA can be used as a probe to monitor disturbed hepatobiliary transport in vivo and accumulation of bile acids in blood and liver during drug development.

The role of endoplasmic reticulum stress and the unfolded protein response (UPR) in cholestatic liver disease and fibrosis is not fully unraveled. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been shown to reduce endoplasmic reticulum (ER) stress and counteract apoptosis in different pathologies. We aimed to investigate the therapeutic potential of TUDCA in experimental secondary biliary liver fibrosis in mice, induced by common bile duct ligation. The kinetics of the hepatic UPR and apoptosis during the development of biliary fibrosis was studied by measuring markers at six different timepoints post-surgery by qPCR and Western blot. Next, we investigated the therapeutic potential of TUDCA, 10 mg/kg/day in drinking water, on liver damage (AST/ALT levels) and fibrosis (Sirius red-staining), in both a preventive and therapeutic setting. Common bile duct ligation resulted in the increased protein expression of CCAAT/enhancer-binding protein homologous protein (CHOP) at all timepoints, along with upregulation of pro-apoptotic caspase 3 and 12, tumor necrosis factor receptor superfamily, member 1A (TNFRsf1a) and Fas-Associated protein with Death Domain (FADD) expression. Treatment with TUDCA led to a significant reduction of liver fibrosis, accompanied by a slight reduction of liver damage, decreased hepatic protein expression of CHOP and reduced gene and protein expression of pro-apoptotic markers. These data indicate that TUDCA exerts a beneficial effect on liver fibrosis in a model of cholestatic liver disease, and suggest that this effect might, at least in part, be attributed to decreased hepatic UPR signaling and apoptotic cell death.

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that have a wide range of functions in cellular homeostasis and immunity. MTs can be induced by a variety of conditions including metals, glucocorticoids, endotoxin, acute phase cytokines, stress, and irradiation. In addition to their important immunomodulatory functions, MTs can protect essential cellular compartments from toxicants, serve as a reservoir of essential heavy metals, and regulate cellular redox potential. Many of the roles of MTs in the neuroinflammation, intestinal inflammation, and stress response have been investigated and were the subject of a session at the 6th International Congress on Stress Proteins in Biology and Medicine in Sheffield, UK. Like the rest of the cell stress response, there are therapeutic opportunities that arise from an understanding of MTs, and these proteins also provide potential insights into the world of the heat shock protein.

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients.

Bile acids regulate the expression of intestinal bile acid transporters and are natural ligands for nuclear receptors controlling inflammation. Accumulating evidence suggests that signaling through these receptors is impaired in inflammatory bowel disease. We investigated whether tauroursodeoxycholic acid (TUDCA), a secondary bile acid with cytoprotective properties, regulates ileal nuclear receptor and bile acid transporter expression and assessed its therapeutic potential in an experimental model of Crohn's disease (CD). Gene expression of the nuclear receptors farnesoid X receptor, pregnane X receptor and vitamin D receptor and the bile acid transporters apical sodium-dependent bile acid transporter and organic solute transporter α and β was analyzed in Caco-2 cell monolayers exposed to tumor necrosis factor (TNF)α, in ileal tissue of TNF ΔARE/WT mice and in inflamed ileal biopsies from CD patients by quantitative real-time polymerase chain reaction. TNF ΔARE/WT mice and wild-type littermates were treated with TUDCA or placebo for 11 weeks and ileal histopathology and expression of the aforementioned genes were determined. Exposing Caco-2 cell monolayers to TNFα impaired the mRNA expression of nuclear receptors and bile acid transporters, whereas co-incubation with TUDCA antagonized their downregulation. TNF ΔARE/WT mice displayed altered ileal bile acid homeostasis that mimicked the situation in human CD ileitis. Administration of TUDCA attenuated ileitis and alleviated the downregulation of nuclear receptors and bile acid transporters in these mice. These results show that TUDCA protects bile acid homeostasis under inflammatory conditions and suppresses CD-like ileitis. Together with previous observations showing similar efficacy in experimental colitis, we conclude that TUDCA could be a promising therapeutic agent for inflammatory bowel disease, warranting a clinical trial.