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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

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.

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).

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.

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.

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.

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.

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.

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.

Objective To examine the influence of a detomidine constant rate infusion (CRI) on cardiovascular function, isoflurane requirements and recovery quality in horses undergoing elective surgery. Study design Prospective, randomized, blinded, clinical trial. Animals Twenty adult healthy horses. Methods After sedation (detomidine, 10 μg kg−1 intravenously [IV]) and induction of anaesthesia (midazolam 0.06 mg kg−1, ketamine 2.2 mg kg−1 IV), anaesthesia was maintained with isoflurane in oxygen/air (inspiratory oxygen fraction 55%). When indicated, the lungs were mechanically ventilated. Dobutamine was administered when MAP < 70 mmHg. The horses were randomly allocated to one of two groups and throughout anaesthesia, received either a detomidine (5 μg kg−1 hour−1) (D) or saline (S) CRI, with the anaesthetist unaware of the treatment. Monitoring included end-tidal isoflurane concentration, arterial pH, PaCO2, PaO2, dobutamine administration rate, heart rate (HR), arterial pressure, cardiac index (CI), systemic vascular resistance (SVR), stroke index and oxygen delivery index ( D ˙ O2I). For recovery from anaesthesia, all horses received 2.5 μg kg−1 detomidine IV. Recovery quality and duration were recorded in each horse. For statistical analysis, anova , Pearson chi-square and Wilcoxon rank sum tests were used as relevant. Results Heart rate (p = 0.0176) and D ˙ O2I (p = 0.0084) were lower and SVR higher (p = 0.0126) in group D, compared to group S. Heart rate (p = 0.0011) and pH (p = 0.0187) increased over time. Significant differences in isoflurane requirements were not detected. Recovery quality and duration were comparable between treatments. Conclusions and clinical relevance A detomidine CRI produced cardiovascular effects typical for a2-agonists, without affecting isoflurane requirements, recovery duration or recovery quality.

Intestinal inflammation is associated with enhanced mucosal hypoxia, which contributes to the ongoing inflammatory process and hampers appropriate mucosal healing. We questioned whether local treatment with an oxygen (O(2))-carrying and -releasing molecule (oxygenated perfluorodecalin, O(2)-PFD) could positively influence the course of experimental colitis. The impact of intrarectal (IR) treatment with O(2)-PFD was tested using the murine dextran sodium sulfate (DSS)-induced model of distal colitis, both in preventive and therapeutic settings. Colonic mucosal hypoxia was visualized by pimonidazole staining. Colonic permeability was evaluated with FITC-dextran. In the preventive study, mice treated with O(2)-PFD were protected against DSS colitis compared with saline-treated mice, as demonstrated by reduced shortening of colon length, reduced colonic tumor necrosis factor-alpha levels and a lower histological inflammation score (P

Objective To evaluate the effects of a constant rate infusion (CRI) of romifidine on the requirement of isoflurane, cardiovascular performance and recovery in anaesthetized horses undergoing arthroscopic surgery. Study design Randomized blinded prospective clinical trial. Animals Thirty horses scheduled for routine arthroscopy. Methods After premedication (acepromazine 0.02 mg kg −1 , romifidine 80 μg kg −1 , methadone 0.1 mg kg −1 ) and induction (midazolam 0.06 mg kg −1 ketamine 2.2 mg kg −1 ), anaesthesia was maintained with isoflurane in oxygen. Horses were assigned randomly to receive a CRI of saline (group S) or 40 μg kg −1 hour −1 romifidine (group R). The influences of time and treatment on anaesthetic and cardiovascular parameters were evaluated using an analysis of variance. Body weight ( t ‐test), duration of anaesthesia ( t ‐test) and recovery score (Wilcoxon Rank Sum Test) were compared between groups. Significance was set at p Results All but one horse were positioned in the dorsal recumbent position and ventilated from the start of anaesthesia. End tidal isoflurane concentrations were similar in both groups at similar time points and over the whole anaesthetic period. Cardiac output was significantly lower in horses of the R group, but there were no significant differences between groups in cardiac index, body weight or age. All other cardiovascular parameters were similar in both groups. Quality of recovery did not differ significantly between groups, but more horses in group R stood without ataxia at the first attempt. One horse from group S had a problematic recovery. Conclusions and clinical relevance No inhalation anaesthetic sparing effect or side effects were observed by using a 40 μg kg −1 hour −1 romifidine CRI in isoflurane anaesthetized horses under clinical conditions. Cardiovascular performance remained acceptable. Further studies are needed to identify the effective dose of romifidine that will induce an inhalation anaesthetic sparing effect in anaesthetized horses.

Objective To investigate the influence of two ventilation modes on the performance of an anaesthetic conserving device (AnaConDa) in piglets. Study design Prospective randomized experimental trial. Animals Eight female piglets weighing 24.7 ± 2.2 kg. Methods Anaesthesia was maintained with sevoflurane (in 60% oxygen) delivered from the AnaConDa placed between endotracheal tube (ETT) and Y‐piece. Anaesthetic depth was guided using standard clinical parameters. Ventilation mode was volume controlled (VC) during the first and pressure support (PS) during the second period of anaesthesia in four piglets (group 1); the order was reversed in group 2. Anaesthetic gases were sampled before (at the proximal end of the ETT) and after the AnaConDa (Y‐piece). Data were analysed using a model I anova , with treatment and group as fixed categorical effects. Using a paired t ‐test, partial pressures of carbon dioxide (P e ′CO 2 ) on both sides of the device were compared. Results Although the mean administration rate of sevoflurane was comparable in both groups (3.8 ± 1.8 mL hour −1 ), E ′Sevo was higher in group 1, more specifically during the first period ( p = 0.035). Less sevoflurane escaped during VC (14.0 ± 3.4%) compared with PS ventilation (17.2 ± 5.7%) ( p = 0.001). P e ′CO 2 was lower at the Y‐piece (6.4 ± 0.8 kPa, 48 ± 6 mmHg) compared with the ETT (9.3 ± 1.4 kPa, 70 ± 11 mmHg) in both groups and ventilation modes. On average, inspiratory CO 2 tension was 2.0 ±1.0 kPa (15 ± 8 mmHg). Respiration rate was comparable in all piglets while tidal volume ( V ˙ T ) and peak inspiratory pressure were lower during VC compared with PS ( p p = 0.015 respectively). Conclusions and clinical relevance The observed differences in E ′Sevo concentration and sevoflurane breakthrough were probably related to differences in V ˙ T . The observed high F i CO 2 indicated an excessive dead space with the AnaConDa for these piglets.

Background: Postoperative ileus (POI) is a frequent and often fatal complication of colic surgery. Reliably effective treatments are not available. Objectives: To determine risk factors and protective factors associated with POI, and to assess the effect of lidocaine IV on short-term survival. Animals: One hundred and twenty-six horses that underwent small intestinal colic surgery and that survived for at least 24 hours postoperatively. Methods: Retrospective cross-sectional study. The association of 31 pre-, intra-, and postoperative variables with POI and the association of lidocaine treatment with short-term survival were investigated. Associations were evaluated with univariable logistic regression models, followed by multivariable analysis. Results: Significant associations of high heart rate (odds ratio [OR] = 1.05, 95% confidence interval [CI] 1.03–1.08), the presence of more than 8 L of reflux at admission (OR = 3.02, 95% CI 1.13–8.02) and the performance of a small intestinal resection (OR = 2.46, 95% CI 1.15–5.27) with an increased probability of POI were demonstrated. Prophylactic lidocaine treatment was significantly associated with a reduced incidence of POI (OR = 0.25, 95% CI 0.11–0.56). Lidocaine treatment was also significantly associated with enhanced short-term survival (OR = 0.30, 95% CI 0.09–0.98). Conclusions and Clinical Importance: The variables associated with an increased risk of POI can be useful in identifying horses at risk of POI and in providing a more accurate prognosis. The results are supportive for lidocaine IV as an effective prokinetic treatment after small intestinal colic surgery.

The Ly49E NK receptor is a unique inhibitory receptor, presenting with a high degree of conservation among mouse strains and expression on both NK cells and intraepithelial-localised T cells. Amongst intraepithelial-localised T cells, the Ly49E receptor is abundantly expressed on CD8 alpha alpha-expressing innate-like intestinal intraepithelial lymphocytes (iIELs), which contribute to front-line defense at the mucosal barrier. Inflammatory bowel diseases (IBDs), encompassing Crohn's disease and ulcerative colitis, have previously been suggested to have an autoreactive origin and to evolve from a dysbalance between regulatory and effector functions in the intestinal immune system. Here, we made use of Ly49E-deficient mice to characterize the role of Ly49E receptor expression on CD8 alpha alpha-expressing iIELs in the development and progression of IBD. For this purpose we used the dextran sodium sulphate (DSS)- and trinitrobenzenesulfonic-acid (TNBS)-induced colitis models, and the TNF Delta ARE ileitis model. We show that Ly49E is expressed on a high proportion of CD8 alpha alpha-positive iIELs, with higher expression in the colon as compared to the small intestine. However, Ly49E expression on small intestinal and colonic iIELs does not influence the development or progression of inflammatory bowel diseases.

Artemisinins are antimalarial drugs that exert potent anticancer activity. We evaluated the effects of artesunate, a semisynthetic derivative of artemisinin, on tumor growth, angiogenesis, the unfolded protein response, and chemoresistance in hepatocellular carcinoma.The effect of artesunate was examined in HepG2 and BWTG3 cells under normoxic and hypoxic conditions and in a diethylnitrosamine-induced mouse model. Histology was performed with hematoxylin/eosin and reticulin staining. The expression of chemoresistance-related transporters and angiogenic and unfolded protein response factors was determined. Cytotoxicity was assessed by alanine and aspartate transaminase, lactate dehydrogenase, water-soluble tetrazolium salt, and caspase-3 activity assays. Small animal imaging was performed using dynamic contrast-enhanced MRI and choline PET to assess tumor progression.Artesunate dose dependently reduced cell viability (from 50 μmol/l; P0.05) and increased caspase-3 activity (P0.05) in HepG2 and BWTG3 cells. These effects were enhanced by hypoxia (from 12.5 μmol/l; P0.01). Moreover, artesunate downregulated vascular endothelial growth factor and placental growth factor expression in vitro (both P0.05) and in vivo (both P0.01). In mice, artesunate decreased vessel density and tumor burden (both P0.05). These in-vivo effects were enhanced by combination with sorafenib (P0.05 and P=0.07, respectively), without apparent hepatotoxicity. Furthermore, artesunate modulated the unfolded protein response in vitro and in vivo, increasing proapoptotic signaling, and did not induce doxorubicin chemoresistance.These findings indicate that artesunate could offer a new approach to the therapy of hepatocellular carcinoma. Clinical trials with artesunate as monotherapy or in combination with current hypoxia-inducing approaches are necessary.

Objective To examine the influence of direct current shock application in anaesthetized horses with atrial fibrillation (AF) and to study the effects of cardioversion to sinus rhythm (SR). Study design Prospective clinical study. Animals Eight horses successfully treated for AF (transvenous electrical cardioversion after amiodarone pre-treatment). Methods Cardioversion catheters and a pacing catheter were placed under sedation [detomidine 10 μg kg−1 intravenously (IV)]. After additional sedation (5–10 μg kg−1 detomidine, 0.1 mg kg−1 methadone IV), anaesthesia was induced with ketamine, 2.2 mg kg−1 and midazolam, 0.06 mg kg−1 (IV) in a sling and maintained with isoflurane in oxygen. Flunixin meglumine, 1.1 mg kg−1, was administered IV. Shocks were delivered as biphasic truncated exponential waves, synchronized with the R-wave of the electrocardiogram. Monitoring included pulse oximetry, electrocardiography, capnography, inhalational anaesthetic agent concentration, arterial blood pressure, LiDCO and PulseCO cardiac index (CI) and arterial blood gases. Values before and after the first unsuccessful shock and before and after cardioversion to SR were compared. Results Values before the first shock were comparable to reported values in healthy, isoflurane anaesthetized horses. Reliable CI measurements could not be obtained using the PulseCO technique. Intermittent positive pressure ventilation was required in most horses (bradypnea and/or PaCO2 >8 kPa, 60 mmHg), while dobutamine was administered in two horses (0.3–0.5 μg kg−1 minute−1). After the 1st unsuccessful shock application, systolic arterial pressure (SAP) was decreased (p = 0.025), other recorded values were not influenced (CI measurements not available for this analysis). SR was associated with increases in CI (p = 0.039) and stroke index (p = 0.002) and a decrease in SAP (p = 0.030). Conclusions and clinical relevance Despite the presence of AF, cardiovascular function was well maintained during anaesthesia and was not affected by shock application. Cardiac index and stroke index increased and SAP decreased after cardioversion.

Reasons for performing stud-v: There is a lack of evidence-based data on the prevalence, outcome and risk factors of distal limb cast sores, and no objective tool has been described for the early detection of cast sores. Objectives: To investigate the prevalence, location, outcome and risk factors of cast sores after application of a distal limb cast and to determine whether static thermography of the cast is a valuable tool for the assessment of sores. Methods: A prospective study was conducted on horses treated with a distal limb cast. At each cast removal, cast sores were graded as superficial sores (SS), deep dermal sores (DS) or full thickness skin ulcerations (FS). In several cases, a thermographic evaluation of the cast was performed immediately prior to removal and differences in temperature (Delta T) between the coolest point of the cast and 2 cast regions predisposed for sore development (dorsoproximal mc/mtIII and palmar/plantar fetlock) were calculated. Results: Mean +/- s.d. total casting time of 70 horses was 31 +/- 18 days. Overall, 57 legs (81%) developed at least SS. Twenty-four legs (34%) ultimately developed DS and one horse had an FS. Multivariable analysis showed that the severity of sores was positively associated with increasing age (OR: 1.111, P = 0.028), a normal (vs. swollen) limb (OR: 3.387, P = 0.023) and an increase in total casting time (OR per week: 1.363, P = 0.002). The thermographic evaluation (35 casts) revealed that the severity of sores was positively associated with increasing Delta T (OR: 2.100, P = 0.000-5). The optimal cut-off values for the presence of SS and DS were set at, respectively, Delta T = 2.3 and 4.3 degrees C. Conclusion and potential relevance: Distal limb cast is a safe coaptation technique with increasing risk of developing sores with time. Thermography is a valuable and rapid clinical tool to monitor the development of cast sores.

ABOMASAL fistulation has been described in association with umbilical hernia ([Fubini and Smith 1984][1]), umbilical infection ([Newcomb and Morton 1970][2]), perforated abomasal ulcers ([Hemmingsen 1967][3]) and abomasopexy as treatment for abomasal displacement ([Parker and Fubini 1987][4], [Sams