The Space of Disse: The Liver Hub in Health and Disease

Since it was first described by the German anatomist and histologist, Joseph Hugo Vincenz Disse, the structure and functions of the space of Disse, a thin perisinusoidal area between the endothelial cells and hepatocytes filled with blood plasma, have acquired great importance in liver disease. The space of Disse is home for the hepatic stellate cells (HSCs), the major fibrogenic players in the liver. Quiescent HSCs (qHSCs) store vitamin A, and upon activation they lose their retinol reservoir and become activated. Activated HSCs (aHSCs) are responsible for secretion of extracellular matrix (ECM) into the space of Disse. This early event in hepatic injury is accompanied by loss of the pores—known as fenestrations—of the endothelial cells, triggering loss of balance between the blood flow and the hepatocyte, and underlies the link between fibrosis and organ dysfunction. If the imbalance persists, the expansion of the fibrotic scar followed by the vascularized septae leads to cirrhosis and/or end-stage hepatocellular carcinoma (HCC). Thus, researchers have been focused on finding therapeutic targets that reduce fibrosis. The space of Disse provides the perfect microenvironment for the stem cells niche in the liver and the interchange of nutrients between cells. In the present review article, we focused on the space of Disse, its components and its leading role in liver disease development

A Shortcut from Metabolic-Associated Fatty Liver Disease (MAFLD) to Hepatocellular Carcinoma (HCC): c-MYC a Promising Target for Preventative Strategies and Individualized Therapy

Background: Metabolic-associated fatty liver disease (MAFLD) has risen as one of the leading etiologies for hepatocellular carcinoma (HCC). Oncogenes have been suggested to be responsible for the high risk of MAFLD-related HCC. We analyzed the impact of the proto-oncogene c-MYC in the development of human and murine MAFLD and MAFLD-associated HCC. Methods: alb-myctg mice were studied at baseline conditions and after administration of Western diet (WD) in comparison to WT littermates. c-MYC expression was analyzed in biopsies of patients with MAFLD and MAFLD-associated HCC by immunohistochemistry. Results: Mild obesity, spontaneous hyperlipidaemia, glucose intolerance and insulin resistance were characteristic of 36-week-old alb-myctg mice. Middle-aged alb-myctg exhibited liver steatosis and increased triglyceride content. Liver injury and inflammation were associated with elevated ALT, an upregulation of ER-stress response and increased ROS production, collagen deposition and compensatory proliferation. At 52 weeks, 20% of transgenic mice developed HCC. WD feeding exacerbated metabolic abnormalities, steatohepatitis, fibrogenesis and tumor prevalence. Therapeutic use of metformin partly attenuated the spontaneous MAFLD phenotype of alb-myctg mice. Importantly, upregulation and nuclear localization of c-MYC were characteristic of patients with MAFLD and MAFLD-related HCC. Conclusions: A novel function of c-MYC in MAFLD progression was identified opening new avenues for preventative strategies

Ethanol and Arachidonic Acid Synergize to Activate Kupffer Cells and Modulate the Fibrogenic Response via Tumor Necrosis Factor , Reduced Glutathione, and Transforming Growth Factor beta–Dependent Mechanisms

Abbreviations: alpha-SMA, alpha-smooth muscle actin; AA, arachidonic acid; DPI, diphenyleneiodonium; ECM, extracellular matrix; GSH, reduced glutathione; GSSG, oxidized glutathione; HSC, hepatic stellate cells; HSCcontrol, control stellate cells; HSCethanol, stellate cells from ethanol-fed rats; KC, Kupffer cells; KCcontrol, control Kupffer cells; KCethanol, Kupffer cells from ethanol-fed rats; PUFA, polyunsaturated fatty acids; NADPH, nicotinamide adenine dinucleotide phosphate reduced form; ROS, reactive oxygen species; SEM, standard error of the mean; TGF- , transforming growth factor beta; TNF- , tumor necrosis factor alpha. From the Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY. Both authors contributed equally to the experimental part of this work Received June 7, 2008; accepted August 4, 2008. Supported by a Postdoctoral Fellowship from the Spanish Ministry of Education and Science Ref. No. EX2006-0070 (F.J.C) and by the US Public Health Service Grant 5R01DK069286-03 from the National Institute of Diabetes and Digestive and Kidney Diseases (N.N.). Address reprint requests to: Natalia Nieto, Department of Medicine, Division of Liver Diseases, Mount Sinai School of Medicine, Box 1123, 1425 Madison Avenue, Room 11-76, New York, NY 10029. E-mail: [email protected]; fax: 212-849-2574. Copyright © 2008 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.22592 Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article.Because of the contribution of ethanol and polyunsaturated fatty acids (PUFAs) to alcoholic liver disease, we investigated whether chronic ethanol administration and arachidonic acid (AA) could synergistically mediate Kupffer cell (KC) activation and modulate the stellate cell (HSC) fibrogenic response. Results: (1) the effects of ethanol and AA on KC and HSC were as follows: Cell proliferation, lipid peroxidation, H(2)O(2), O(2).(-), nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity, and tumor necrosis factor alpha (TNF-alpha) were higher in KC(ethanol) than in KC(control), and were enhanced by AA; HSC(ethanol) proliferated faster, increased collagen, and showed higher GSH than HSC(control), with modest effects by AA. (2) AA effects on the control co-culture: We previously reported the ability of KC to induce a pro-fibrogenic response in HSC via reactive oxygen species (ROS)-dependent mechanisms; we now show that AA further increases cell proliferation and collagen in the control co-culture. The latter was prevented by vitamin E (an antioxidant) and by diphenyleneiodonium (a NADPH oxidase inhibitor). (3) Ethanol effects on the co-cultures: Co-culture with KC(control) or KC(ethanol) induced HSC(control) and HSC(ethanol) proliferation; however, the pro-fibrogenic response in HSC(ethanol) was suppressed because of up-regulation of TNF-alpha and GSH, which was prevented by a TNF-alpha neutralizing antibody (Ab) and by L-buthionine-sulfoximine, a GSH-depleting agent. (4) Ethanol plus AA effects on the co-cultures: AA lowered TNF-alpha in the HSC(control) co-cultures, allowing for enhanced collagen deposition; furthermore, AA restored the pro-fibrogenic response in the HSC(ethanol) co-cultures by counteracting the up-regulation of TNF-alpha and GSH with a significant increase in GSSG and in pro-fibrogenic transforming growth factor beta (TGF-beta). Conclusion: These results unveil synergism between ethanol and AA to the mechanism whereby KC mediate ECM remodeling and suggest that even if chronic ethanol consumption sensitizes HSC to up-regulate anti-fibrogenic signals, their effects are blunted by a second "hit" such as AA.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Kupffer cells and alcoholic liver disease

Department of Medicine. Division of Liver Diseases. Mount Sinai School of Medicine. New York, USALiver disease is a major cause of illness and death worldwide. A central component in the complex network leading to the development of alcoholic liver disease is the activation of Kupffer cells by endotoxin and other soluble mediators. Alcohol consumption induces a state of "leaky gut increasing plasma and liver endotoxin levels. When Kupffer cells become activated, they interact with a complex of proteins located on the extracellular membrane signaling to produce a wide array of soluble factors, including cytokines, chemokines, growth factors, cyclooxygenase and lipoxygenase metabolites, and reactive oxygen species such as superoxide anion, hydrogen peroxide, and nitric oxide, all of which provide physiologically diverse and pivotal paracrine effects on all other liver cell types and, ultimately, liver injury. Kupffer cells are also central to the liver homeostatic response to injury as upon cellular degenerative changes, they immediately respond to the insult and release mediators to orchestrate inflammatory and reparative responses. Thus, the homeostatic responses are initiated by Kupffer cell-derived mediators at the cellular level and underlie the liver s defense and reparative mechanisms against injury. In order to understand better the role of Kupffer cells in the onset of liver injury, animal models in which Kupffer cells are inactivated, and cell culture settings (e.g. co-cultures) are being used with promising results that advance our understanding of alcoholic liver disease.National Institute of Diabetes and Digestive and Kidney Diseases (US Public Health Service)Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Obesity under the moonlight of c-MYC

The moonlighting protein c-Myc is a master regulator of multiple biological processes including cell proliferation, differentiation, angiogenesis, apoptosis and metabolism. It is constitutively and aberrantly expressed in more than 70% of human cancers. Overwhelming evidence suggests that c-Myc dysregulation is involved in several inflammatory, autoimmune, metabolic and other non-cancerous diseases. In this review, we addressed the role of c-Myc in obesity. Obesity is a systemic disease, accompanied by multi-organ dysfunction apart from white adipose tissue (WAT), such as the liver, the pancreas, and the intestine. c-Myc plays a big diversity of functions regulating cellular proliferation, the maturation of progenitor cells, fatty acids (FAs) metabolism, and extracellular matrix (ECM) remodeling. Moreover, c-Myc drives the expression of a wide range of metabolic genes, modulates the inflammatory response, induces insulin resistance (IR), and contributes to the regulation of intestinal dysbiosis. Altogether, c-Myc is an interesting diagnostic tool and/or therapeutic target in order to mitigate obesity and its consequences.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Combined Activities of JNK1 and JNK2 in Hepatocytes Protect Against Toxic Liver Injury

Instituciones participantes: 1Department of Internal Medicine III, University Hospital, RWTH Aachen; 2Department of Gastroenterology and Hepatology, University Hospital Duisburg-Essen, Essen, Germany; 3Nutrition, Metabolism & Genomics group, Wageningen University, Division of Human Nutrition, Wageningen, The Netherlands; 4Norwich Medical School, University of East Anglia, Norwich, United Kingdom; 5Proteomics Facility, University Hospital, RWTH Aachen; 6Institute of Pathology, University Hospital, RWTH Aachen, Germany; and 7Howard Hughes Medical Institute and University of Massachusetts Medical School, Worcester, MassachusettsBACKGROUND & AIMS: c-Jun N-terminal kinase (JNK) 1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated via phosphorylation in response to acetaminophen- or carbon tetrachloride (CCl4)- induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissue from patients and in mice with genetic deletion of JNK in hepatocytes. METHODS: We studied liver sections from patients with DILI (due to acetaminophen, phenprocoumon, nonsteroidal antiinflammatory drugs, or autoimmune hepatitis) or patients without acute liver failure (controls) collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and Western blotting. Mice with hepatocyte-specific deletion of Jnk1 (Jnk1Dhepa) or combination of Jnk1 and Jnk2 (JnkDhepa), as well as Jnk1-floxed C57BL/6 (control) mice, were given injections of CCl4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. RESULTS: Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to JnkDhepa mice produced a greater level of liver injury than that observed in Jnk1Dhepa or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl4 also induced stronger hepatic injury in JnkDhepa mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared with Jnk1Dhepa or control mice. Hepatocytes from JnkDhepa mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of adenosine monophosphate-activated protein kinase, total protein adenosine monophosphate-activated protein kinase levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected JnkDhepa and control mice from liver injury. CONCLUSIONS: In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl4- and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

TNFR1 determines progression of chronic liver injury in the IKKc/Nemo genetic model

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-jB essential modulator in hepatocytes (IKKc/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKKc/Nemo-deleted livers. We crossed hepatocyte-specific IKKc/Nemo knockout mice (NemoDhepa) with constitutive TNFR1 / and TRAIL / mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKKc/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury–a finding that has direct implications for treating chronic liver disease.Aachen UniversityDeutsche ForschungsgemeinschaftDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Arachidonic acid stimulates TNFalfa production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York; and 2 Department of Medicine III, University Hospital RWTH Aachen, Aachen, GermanyKupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.Ministerio de Educación y CienciaNational Institute of Diabetes and Digestive and Kidney Diseases (US Public Health Service)National Institute on Alcohol Abuse and Alcoholism (USA)Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Inactivation of caspase 8 in liver parenchymal cells confers protection against murine obstructive cholestasis

2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. En esta publicación han participado: 1Department of Internal Medicine III, University Hospital, RWTH, Aachen, Germany; 2Department of Immunology, Ophtalmology & ORL,Complutense University School of Medicine, Madrid, Spain; 3 12 de Octubre Health Research Institute (imas12), Madrid, Spain; 4Department of Hepatobiliary Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing Jiangsu, China; 5Department of Anesthesiology and Pain Management, Shanghai East Hospital, Tongji University, Shanghai, China; 6Institute of Pathology, Braunschweig Hospital, Braunschweig, GermanyBackground & Aims: Caspase 8 (CASP8) is the apical initiator caspase in death receptor-mediated apoptosis. Strong evidence for a link between death receptor signaling pathways and cholestasis has recently emerged. Herein, we investigated the role of CASP8-dependent and independent pathways during experimental cholestasis. Methods: Liver injury was characterized in a cohort of human sera (n = 28) and biopsies from patients with stage IV primary biliary cholangitis. In parallel, mice with either specific deletion of Casp8 in liver parenchymal cells (Casp8Dhepa) or hepatocytes (Casp8Dhep), and mice with constitutive Ripk3 (Ripk3 / ) deletion, were subjected to surgical ligation of the common bile duct (BDL) from 2 to 28 days. Floxed (Casp8fl/fl) and Ripk3+/+ mice were used as controls. Moreover, the pan-caspase inhibitor IDN-7314 was used, and cell death mechanisms were studied in primary isolated hepatocytes. Results: Overexpression of activated caspase 3, CASP8 and RIPK3 was characteristic of liver explants from patients with primary biliary cholangitis. Twenty-eight days after BDL, Casp8Dhepa mice showed decreased necrotic foci, serum aminotransferase levels and apoptosis along with diminished compensatory proliferation and ductular reaction. These results correlated with a decreased inflammatory profile and ameliorated liver fibrogenesis. A similar phenotype was observed in Ripk3-/- mice. IDN-7314 treatment decreased CASP8 levels but failed to prevent BDL-induced cholestasis, independently of CASP8 in hepatocytes. Conclusion: These findings show that intervention against CASP8 in liver parenchymal cells – specifically in cholangiocytes– might be a beneficial option for treating obstructive cholestasis, while broad pan-caspase inhibition might trigger undesirable side effects. Lay summary: Loss of caspase 8 – a protein involved in programmed cell death – in liver parenchymal cells protects against experimental cholestasis. Therefore, specific pharmacological intervention against caspase 8 might be a valid alternative for the treatment of obstructive cholestasis in the clinic, whereas broad pan-caspase inhibiting drugs might trigger undesirable side effects.IZKFMinisterio de Economía, Comercio y EmpresaDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu