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1.
Am J Physiol Cell Physiol ; 326(5): C1556-C1562, 2024 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-38618702

RESUMEN

Healthy livers contain 80% of body resident macrophages known as Kupffer cells. In diseased livers, the number of Kupffer cells usually drops but is compensated by infiltration of monocyte-derived macrophages, some of which can differentiate into Kupffer-like cells. Early studies suggest that Kupffer cells play important roles in both promoting liver injury and liver regeneration. Yet, the distinction between the functionalities of resident and infiltrating macrophages is not always made. By using more specific macrophage markers and targeted cell depletion and single-cell RNA sequencing, recent studies revealed several subsets of monocyte-derived macrophages that play important functions in inducing liver damage and inflammation as well as in liver repair and regeneration. In this review, we discuss the different roles that hepatic macrophages play in promoting necrotic liver lesion resolution and dead cell clearance, as well as the targeting of these cells as potential tools for the development of novel therapies for acute liver failure and acute-on-chronic liver failure.


Asunto(s)
Macrófagos del Hígado , Regeneración Hepática , Hígado , Necrosis , Humanos , Animales , Hígado/patología , Hígado/metabolismo , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/inmunología
2.
Liver Int ; 43(5): 975-988, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36719059

RESUMEN

BACKGROUND: It is unclear whether the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 C-to-G single nucleotide polymorphism, resulting in the substitution of isoleucine to methionine at position 148 (I148M), impedes regression of hepatic steatosis when treating non-alcoholic fatty liver disease (NAFLD). OBJECTIVES: Investigate if carriage of the PNPLA3 148M allele affects the anti-steatotic efficacy of all possible anti-NAFLD interventions, identify gaps in current knowledge and provide guidance for individual treatment. METHODS: Research available in public databases was searched up to 13 November 2022. Studies were included if a treatment in NAFLD patients decreased hepatic steatosis in the pooled patient group or a PNPLA3 I148M polymorphism subgroup (II/IM/MM). The risk of bias was assessed using the Cochrane Risk-Of-Bias 2 Tool and the Newcastle-Ottawa Scale. RESULTS: Moderate evidence indicates that NAFLD patients homozygous for the PNPLA3 148M allele benefit less or not at all from omega-3 carboxylic acids to decrease liver fat, while the PNPLA3 148I allele shows moderate benefit. Low evidence suggests that interventions employing lifestyle changes are more effective to reduce liver fat in NAFLD patients homozygous for the PNPLA3 148M allele compared to patients with wild-type PNPLA3. CONCLUSIONS: NAFLD patients homozygous for the PNPLA3 148M allele might not benefit from omega-3 carboxylic acids to reduce hepatic steatosis in contrast to patients with wild-type PNPLA3. Instead, patients with two PNPLA3 148M alleles should be especially advised to adopt lifestyle changes. Genotyping for PNPLA3 I148M should be encouraged in therapeutic studies for NAFLD. REGISTRATION NUMBER (PROSPERO): CRD42022375028.


Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/genética , Polimorfismo de Nucleótido Simple , Homocigoto , Ácidos Carboxílicos , Predisposición Genética a la Enfermedad
3.
J Hepatol ; 75(3): 690-705, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33989701

RESUMEN

There is an unmet need for functional primary human hepatocytes to support the pharmaceutical and (bio)medical demand. The unique discovery, a decade ago, that somatic cells can be drawn out of their apparent biological lockdown to reacquire a pluripotent state has revealed a completely new avenue of possibilities for generating surrogate human hepatocytes. Since then, the number of papers reporting the direct conversion of somatic cells into induced hepatocytes (iHeps) has burgeoned. A hepatic cell fate can be established via the ectopic expression of native liver-enriched transcription factors in somatic cells, thereby bypassing the need for an intermediate (pluripotent) stem cell state. That said, understanding and eventually controlling the processes that give rise to functional iHeps remains challenging. In this review, we provide an overview of the state-of-the-art reprogramming cocktails and techniques, as well as their corresponding conversion efficiencies. Special attention is paid to the role of liver-enriched transcription factors as hepatogenic reprogramming tools and small molecules as facilitators of hepatic transdifferentiation. To conclude, we formulate recommendations to optimise, standardise and enrich the in vitro production of iHeps to reach clinical standards, and propose minimal criteria for their characterisation.


Asunto(s)
Células Madre Adultas/fisiología , Transdiferenciación Celular/fisiología , Hepatocitos/fisiología , Células Madre Adultas/metabolismo , Hepatocitos/metabolismo , Humanos
4.
J Hepatol ; 75(1): 163-176, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33610678

RESUMEN

BACKGROUND & AIMS: Interleukin (IL)-20 and IL-22 belong to the IL-10 family. IL-10 is a well-documented anti-inflammatory cytokine while IL-22 is well known for epithelial protection and its antibacterial function, showing great therapeutic potential for organ damage; however, the function of IL-20 remains largely unknown. METHODS: Il20 knockout (Il20-/-) mice and wild-type littermates were generated and injected with Concanavalin A (ConA) and Klebsiella pneumoniae (K.P.) to induce acute hepatitis and bacterial infection, respectively. RESULTS: Il20-/- mice were resistant to acute hepatitis and exhibited selectively elevated levels of the hepatoprotective cytokine IL-6. Such selective inhibition of IL-6 by IL-20 was due to IL-20 targeting hepatocytes that produce high levels of IL-6 but a limited number of other cytokines. Mechanistically, IL-20 upregulated NAD(P)H: quinone oxidoreductase 1 (NQO1) expression and subsequently promoted the protein degradation of transcription factor IκBζ, resulting in selective downregulation of the IκBζ-dependent gene Il6 as well several other IκBζ-dependent genes including lipocalin-2 (Lcn2). Given the important role of IL-6 and LCN2 in limiting bacterial infection, we examined the effect of IL-20 on bacterial infection and found Il20-/- mice were resistant to K.P. infection and exhibited elevated levels of hepatic IκBζ-dependent antibacterial genes. Moreover, IL-20 upregulated hepatic NQO1 by binding to IL-22R1/IL-20R2 and activating ERK/p38MAPK/NRF2 signaling pathways. Finally, the levels of hepatic IL1B, IL20, and IκBζ target genes were elevated, and correlated with each other, in patients with severe alcoholic hepatitis. CONCLUSIONS: IL-20 selectively inhibits hepatic IL-6 production rather than exerting IL-10-like broad anti-inflammatory properties. Unlike IL-22, IL-20 aggravates acute hepatitis and bacterial infection. Thus, anti-IL-20 therapy could be a promising option to control acute hepatitis and bacterial infection. LAY SUMMARY: Several interleukin (IL)-20 family cytokines have been shown to play important roles in controllimg inflammatory responses, infection and tissue damage, but the role of IL-20 remains unclear. Herein, we elucidated the role of IL-20 in liver disease and bacterial infection. We show that IL-20 can aggravate hepatitis and bacterial infection; thus, targeting IL-20 holds promise for the treatment of patients with liver disease.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Infecciones Bacterianas , Hepatitis Alcohólica , Hepatitis , Interleucina-1beta/metabolismo , Interleucinas/metabolismo , Animales , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Descubrimiento de Drogas , Regulación de la Expresión Génica/efectos de los fármacos , Hepatitis/tratamiento farmacológico , Hepatitis/inmunología , Hepatitis/metabolismo , Hepatitis Alcohólica/inmunología , Hepatitis Alcohólica/metabolismo , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Proteolisis , Regulación hacia Arriba
5.
Hepatology ; 71(4): 1391-1407, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31469200

RESUMEN

BACKGROUND AND AIMS: Hepatic cardiomyopathy, a special type of heart failure, develops in up to 50% of patients with cirrhosis and is a major determinant of survival. However, there is no reliable model of hepatic cardiomyopathy in mice. We aimed to characterize the detailed hemodynamics of mice with bile duct ligation (BDL)-induced liver fibrosis, by monitoring echocardiography and intracardiac pressure-volume relationships and myocardial structural alterations. Treatment of mice with a selective cannabinoid-2 receptor (CB2 -R) agonist, known to attenuate inflammation and fibrosis, was used to explore the impact of liver inflammation and fibrosis on cardiac function. APPROACH AND RESULTS: BDL induced massive inflammation (increased leukocyte infiltration, inflammatory cytokines, and chemokines), oxidative stress, microvascular dysfunction, and fibrosis in the liver. These pathological changes were accompanied by impaired diastolic, systolic, and macrovascular functions; cardiac inflammation (increased macrophage inflammatory protein 1, interleukin-1, P-selectin, cluster of differentiation 45-positive cells); and oxidative stress (increased malondialdehyde, 3-nitrotyrosine, and nicotinamide adenine dinucleotide phosphate oxidases). CB2 -R up-regulation was observed in both livers and hearts of mice exposed to BDL. CB2 -R activation markedly improved hepatic inflammation, impaired microcirculation, and fibrosis. CB2 -R activation also decreased serum tumor necrosis factor-alpha levels and improved cardiac dysfunction, myocardial inflammation, and oxidative stress, underlining the importance of inflammatory mediators in the pathology of hepatic cardiomyopathy. CONCLUSIONS: We propose BDL-induced cardiomyopathy in mice as a model for hepatic/cirrhotic cardiomyopathy. This cardiomyopathy, similar to cirrhotic cardiomyopathy in humans, is characterized by systemic hypotension and impaired macrovascular and microvascular function accompanied by both systolic and diastolic dysfunction. Our results indicate that the liver-heart inflammatory axis has a pivotal pathophysiological role in the development of hepatic cardiomyopathy. Thus, controlling liver and/or myocardial inflammation (e.g., with selective CB2 -R agonists) may delay or prevent the development of cardiomyopathy in severe liver disease.


Asunto(s)
Cardiomiopatías/etiología , Insuficiencia Cardíaca/etiología , Cirrosis Hepática/complicaciones , Receptor Cannabinoide CB2/metabolismo , Animales , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/patología , Hepatitis/metabolismo , Hepatitis/patología , Inflamación/metabolismo , Inflamación/patología , Hígado , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocarditis/metabolismo , Miocarditis/patología , Miocardio/metabolismo , Miocardio/patología , Receptor Cannabinoide CB2/agonistas , Transducción de Señal
6.
Hepatology ; 72(3): 873-891, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32463484

RESUMEN

BACKGROUND AND AIMS: Neutrophil infiltration is a hallmark of nonalcoholic steatohepatitis (NASH), but how this occurs during the progression from steatosis to NASH remains obscure. Human NASH features hepatic neutrophil infiltration and up-regulation of major neutrophil-recruiting chemokines (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1] and interleukin [IL]-8). However, mice fed a high-fat diet (HFD) only develop fatty liver without significant neutrophil infiltration or elevation of chemokines. The aim of this study was to determine why mice are resistant to NASH development and the involvement of p38 mitogen-activated protein kinase (p38) activated by neutrophil-derived oxidative stress in the pathogenesis of NASH. APPROACH AND RESULTS: Inflamed human hepatocytes attracted neutrophils more effectively than inflamed mouse hepatocytes because of the greater induction of CXCL1 and IL-8 in human hepatocytes. Hepatic overexpression of Cxcl1 and/or IL-8 promoted steatosis-to-NASH progression in HFD-fed mice by inducing liver inflammation, injury, and p38 activation. Pharmacological inhibition of p38α/ß or hepatocyte-specific deletion of p38a (a predominant form in the liver) attenuated liver injury and fibrosis in the HFD+Cxcl1 -induced NASH model that is associated with strong hepatic p38α activation. In contrast, hepatocyte-specific deletion of p38a in HFD-induced fatty liver where p38α activation is relatively weak exacerbated steatosis and liver injury. Mechanistically, weak p38α activation in fatty liver up-regulated the genes involved in fatty acid ß-oxidation through peroxisome proliferator-activated receptor alpha phosphorylation, thereby reducing steatosis. Conversely, strong p38α activation in NASH promoted caspase-3 cleavage, CCAAT-enhancer-binding proteins homologous protein expression, and B cell lymphoma 2 phosphorylation, thereby exacerbating hepatocyte death. CONCLUSIONS: Genetic ablation of hepatic p38a increases simple steatosis but ameliorates oxidative stress-driven NASH, indicating that p38α plays distinct roles depending on the disease stages, which may set the stage for investigating p38α as a therapeutic target for the treatment of NASH.


Asunto(s)
Hígado Graso , Hepatocitos/inmunología , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Animales , Quimiocina CXCL1/inmunología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Hígado Graso/inmunología , Hígado Graso/metabolismo , Eliminación de Gen , Humanos , Interleucina-8/inmunología , Ratones , Infiltración Neutrófila/inmunología , Enfermedad del Hígado Graso no Alcohólico/inmunología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Estrés Oxidativo , Índice de Severidad de la Enfermedad
7.
Biomacromolecules ; 22(12): 4919-4932, 2021 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-34723502

RESUMEN

Implementation of hydrogel precursors in two-photon polymerization (2PP) technology provides promising opportunities in the tissue engineering field thanks to their soft characteristics and similarity to extracellular matrix. Most of the hydrogels, however, are prone to post-fabrication deformations, leading to a mismatch between the computer-aided design and the printed structure. In the present work, we have developed novel synthetic hydrogel precursors to overcome the limitations associated with 2PP processing of conventional hydrogel precursors such as post-processing deformations and a narrow processing window. The precursors are based on a poly(ethylene glycol) backbone containing urethane linkers and are, on average, functionalized with six acrylate terminal groups (three on each terminal group). As a benchmark material, we exploited a precursor with an identical backbone and urethane linkers, albeit functionalized with two acrylate groups, that were reported as state-of-the-art. An in-depth characterization of the hexafunctional precursors revealed a reduced swelling ratio (<0.7) and higher stiffness (>36 MPa Young's modulus) compared to their difunctional analogs. The superior physical properties of the newly developed hydrogels lead to 2PP-based fabrication of stable microstructures with excellent shape fidelity at laser scanning speeds up to at least 90 mm s-1, in contrast with the distorted structures of conventional difunctional precursors. The hydrogel films and microscaffolds revealed a good cell interactivity after functionalization of their surface with a gelatin methacrylamide-based coating. The proposed synthesis strategy provides a one-pot and scalable synthesis of hydrogel building blocks that can overcome the current limitations associated with 2PP fabrication of hydrogel microstructures.


Asunto(s)
Hidrogeles , Microtecnología , Ingeniería de Tejidos , Diseño de Equipo/métodos , Gelatina/química , Hidrogeles/química , Industria Manufacturera , Polimerizacion , Ingeniería de Tejidos/métodos
8.
Cell Biol Toxicol ; 37(2): 293-311, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-32613381

RESUMEN

Non-alcoholic steatohepatitis (NASH) is a highly prevalent, chronic liver disease characterized by hepatic lipid accumulation, inflammation, and concomitant fibrosis. Up to date, no anti-NASH drugs have been approved. In this study, we reproduced key NASH characteristics in vitro by exposing primary human hepatocytes (PHH), human skin stem cell-derived hepatic cells (hSKP-HPC), HepaRG and HepG2 cell lines, as well as LX-2 cells to multiple factors that play a role in the onset of NASH. The obtained in vitro disease models showed intracellular lipid accumulation, secretion of inflammatory chemokines, induced ATP content, apoptosis, and increased pro-fibrotic gene expression. These cell systems were then used to evaluate the anti-NASH properties of eight peroxisome proliferator-activated receptor (PPAR) agonists (bezafibrate, elafibranor, fenofibrate, lanifibranor, pemafibrate, pioglitazone, rosiglitazone, and saroglitazar). PPAR agonists differently attenuated lipid accumulation, inflammatory chemokine secretion, and pro-fibrotic gene expression.Based on the obtained readouts, a scoring system was developed to grade the anti-NASH potencies. The in vitro scoring system, based on a battery of the most performant models, namely PHH, hSKP-HPC, and LX-2 cultures, showed that elafibranor, followed by saroglitazar and pioglitazone, induced the strongest anti-NASH effects. These data corroborate available clinical data and show the relevance of these in vitro models for the preclinical investigation of anti-NASH compounds.


Asunto(s)
Hígado/patología , Modelos Biológicos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Receptores Activados del Proliferador del Peroxisoma/agonistas , Quimiocinas/metabolismo , Niño , Preescolar , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Mediadores de Inflamación/metabolismo , Lipogénesis , Enfermedad del Hígado Graso no Alcohólico/patología , Piel/citología , Factores de Transcripción/metabolismo
9.
Arch Toxicol ; 95(7): 2235-2253, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34027561

RESUMEN

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease that affects about a quarter of the world population. MAFLD encompasses different disease stadia ranging from isolated liver steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Although MAFLD is considered as the hepatic manifestation of the metabolic syndrome, multiple concomitant disease-potentiating factors can accelerate disease progression. Among these risk factors are diet, lifestyle, genetic traits, intake of steatogenic drugs, male gender and particular infections. Although infections often outweigh the development of fatty liver disease, pre-existing MAFLD could be triggered to progress towards more severe disease stadia. These combined disease cases might be underreported because of the high prevalence of both MAFLD and infectious diseases that can promote or exacerbate fatty liver disease development. In this review, we portray the molecular and cellular mechanisms by which the most relevant viral, bacterial and parasitic infections influence the progression of fatty liver disease and steatohepatitis. We focus in particular on how infectious diseases, including coronavirus disease-19, hepatitis C, acquired immunodeficiency syndrome, peptic ulcer and periodontitis, exacerbate MAFLD. We specifically underscore the synergistic effects of these infections with other MAFLD-promoting factors.


Asunto(s)
Infecciones Bacterianas/complicaciones , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedades Parasitarias/complicaciones , Brote de los Síntomas , Virosis/complicaciones , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Infecciones Bacterianas/microbiología , COVID-19/complicaciones , Hepatitis Viral Humana/complicaciones , Humanos , Hígado/fisiopatología , Síndrome Metabólico , Enfermedad del Hígado Graso no Alcohólico/microbiología , Enfermedad del Hígado Graso no Alcohólico/parasitología , Enfermedad del Hígado Graso no Alcohólico/virología , Enfermedades Parasitarias/parasitología , Úlcera Péptica , Periodontitis , Factores de Riesgo , Virosis/virología
10.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-34298927

RESUMEN

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1ß, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFß. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.


Asunto(s)
Citocinas/genética , Regulación de la Expresión Génica/genética , Inflamación/genética , Células Madre Mesenquimatosas/metabolismo , Transducción de Señal/genética , Receptores Toll-Like/genética , Transcripción Genética/genética , Proliferación Celular/genética , Regulación hacia Abajo/genética , Humanos , Grasa Subcutánea/metabolismo , Regulación hacia Arriba/genética
11.
Toxicol Appl Pharmacol ; 379: 114666, 2019 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-31323262

RESUMEN

Cholestasis is a liver disease associated with retention of bile in the liver, which leads to local hepatic inflammation and severe liver damage. In order to investigate the mode of action of drug-induced cholestasis, in vitro models have shown to be able to recapitulate important elements of this disease. In this study, we applied untargeted metabolomics to investigate the metabolic perturbances in HepaRG® cells exposed for 24 h and 72 h to bosentan, a cholestatic reference toxicant. Intracellular profiles were extracted and analysed with liquid chromatography and accurate-mass spectrometry. Metabolites of interest were selected using partial least-squares discriminant analysis and random forest classifier models. The observed metabolic patterns associated with cholestasis in vitro were complex. Acute (24 h) exposure revealed metabolites related to apoptosis, such as ceramide and triglyceride accumulation, in combination with phosphatidylethanolamine, choline and carnitine depletion. Metabolomic alterations during exposure to lower dosages and a prolonged exposure (72 h) included carnitine upregulation and changes in the polyamine metabolism. These metabolites were linked to changes in phospholipid metabolism, mitochondrial pathways and energy homeostasis. The metabolic changes confirmed the mitotoxic effects of bosentan and revealed the potential involvement of phospholipid metabolism as part of the mode of action of drug-induced cholestasis.


Asunto(s)
Colestasis/metabolismo , Hígado/metabolismo , Bosentán/farmacología , Línea Celular , Ceramidas/metabolismo , Colestasis/inducido químicamente , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Hígado/efectos de los fármacos , Espectrometría de Masas , Metabolómica , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Fosfolípidos/metabolismo , Triglicéridos/metabolismo
12.
Pharmacol Res ; 144: 377-389, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31028903

RESUMEN

Non-alcoholic steatohepatitis (NASH) is characterized by hepatocellular steatosis with concomitant hepatic inflammation. Despite its pandemic proportions, no anti-NASH drugs have been approved yet. This is partially because drug development is decelerated due to the lack of adequate tools to assess the efficacy of potential new drug candidates. The present study describes the development and application of a new preclinical model for NASH using hepatic cells generated from human skin-derived precursors. Exposure of these cells to lipogenic (insulin, glucose, fatty acids) and pro-inflammatory factors (IL-1ß, TNF-α, TGF-ß) resulted in a characteristic NASH response, as indicated by intracellular lipid accumulation, modulation of NASH-specific gene expression, increased caspase-3/7 activity and the expression and/or secretion of inflammatory markers, including CCL2, CCL5, CCL7, CCL8, CXCL5, CXCL8, IL1a, IL6 and IL11. The human relevance of the proposed NASH model was verified by transcriptomics analyses that revealed commonly modulated genes and the identification of the same gene classes between the in vitro system and patients suffering from NASH. The application potential of this in vitro model was demonstrated by testing elafibranor, a promising anti-NASH compound currently under clinical phase III trial evaluation. Elafibranor attenuated in vitro key features of NASH, and dramatically lowered lipid load as well as the expression and secretion of inflammatory chemokines, which in vivo are responsible for the recruitment of immune cells. This reduction in inflammatory response was NFκB-mediated. In summary, this human-relevant, in vitro system proved to be a sensitive testing tool for the investigation of novel anti-NASH compounds.


Asunto(s)
Chalconas/farmacología , Hepatocitos/efectos de los fármacos , Inflamación/tratamiento farmacológico , Lipogénesis/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Propionatos/farmacología , Células Cultivadas , Hepatocitos/citología , Hepatocitos/patología , Humanos , Inflamación/complicaciones , Inflamación/patología , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/patología , Piel/citología , Piel/efectos de los fármacos , Piel/patología , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/patología
13.
Arch Toxicol ; 93(7): 1789-1805, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31037322

RESUMEN

Stem cells are characterized by their self-renewal capacity and their ability to differentiate into multiple cell types of the human body. Using directed differentiation strategies, stem cells can now be converted into hepatocyte-like cells (HLCs) and therefore, represent a unique cell source for toxicological applications in vitro. However, the acquired hepatic functionality of stem cell-derived HLCs is still significantly inferior to primary human hepatocytes. One of the main reasons for this is that most in vitro models use traditional two-dimensional (2D) setups where the flat substrata cannot properly mimic the physiology of the human liver. Therefore, 2D-setups are progressively being replaced by more advanced culture systems, which attempt to replicate the natural liver microenvironment, in which stem cells can better differentiate towards HLCs. This review highlights the most recent cell culture systems, including scaffold-free and scaffold-based three-dimensional (3D) technologies and microfluidics that can be employed for culture and hepatic differentiation of stem cells intended for hepatotoxicity testing. These methodologies have shown to improve in vitro liver cell functionality according to the in vivo liver physiology and allow to establish stem cell-based hepatic in vitro platforms for the accurate evaluation of xenobiotics.


Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Diferenciación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Células Madre/efectos de los fármacos , Xenobióticos/toxicidad , Técnicas de Cultivo de Célula , Hepatocitos/citología , Humanos , Células Madre/citología
14.
Pharmacol Res ; 134: 257-267, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29964161

RESUMEN

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by excessive triglyceride accumulation in the liver accompanied by inflammation, cell stress and apoptosis. It is the tipping point to the life-threatening stages of non-alcoholic fatty liver disease (NAFLD). Despite the high prevalence of NASH, up to five percent of the global population, there are currently no approved drugs to treat this disease. Animal models, mostly based on specific diets and genetic modifications, are often employed in anti-NASH drug development. However, due to interspecies differences and artificial pathogenic conditions, they do not represent the human situation accurately and are inadequate for testing the efficacy and safety of potential new drugs. Human-based in vitro models provide a more legitimate representation of the human NASH pathophysiology and can be used to investigate the dysregulation of cellular functions associated with the disease. Also in silico methodologies and pathway-based approaches using human datasets, may contribute to a more accurate representation of NASH, thereby facilitating the quest for new anti-NASH drugs. In this review, we describe the molecular components of NASH and how human-based tools can contribute to unraveling the pathogenesis of this disease and be used in anti-NASH drug development. We also propose a roadmap for the development and application of human-based approaches for future investigation of NASH.


Asunto(s)
Hepatocitos/metabolismo , Mediadores de Inflamación/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Triglicéridos/metabolismo , Animales , Apoptosis , Células Cultivadas , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de Señal
15.
Arch Toxicol ; 92(10): 3007-3029, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30155722

RESUMEN

Omics technologies, and in particular metabolomics, have received an increasing attention during the assessment of hepatotoxicity in vitro. However, at present, a consensus on good metabolomics practices has yet to be reached. Therefore, in this review, a range of experimental approaches, applied methodologies, and data processing workflows are compared and critically evaluated. Experimental designs among the studies are similar, reporting the use of primary hepatocytes or hepatic cell lines as the most frequently used cell sources. Experiments are usually conducted in short time-frames (< 48 h) at sub-toxic dosages. Applied sample preparations are protein precipitation or Bligh-and-Dyer extraction. Most analytical platforms rely on chromatographic separations with mass spectrometric detection using high-resolution instruments. Untargeted metabolomics was typically used to allow the simultaneous detection of several classes of the metabolome, including endogenous metabolites that are not initially linked to toxicity. This non-biased detection platform is a valuable tool for generating hypothesis-based mechanistic research. The most frequently reported metabolites that are altered under toxicological impulses are alanine, lactate, and proline, which are often correlated. Other unspecific biomarkers of hepatotoxicity in vitro are the down-regulation of choline, glutathione, and 3-phospho-glycerate. Disruptions on the Krebs cycle are associated with increased glutamate, tryptophan, and valine. Phospholipid alterations are described in steatosis, lipo-apoptosis, and oxidative stress. Although there is a growing trend towards quality control, data analysis procedures do often not follow good contemporary metabolomics practices, which include feature filtering, false-discovery rate correction, and reporting the confidence of metabolite annotation. The currently annotated biomarkers can be used to identify hepatotoxicity in general and provide, to a certain extent, a tool for mechanistic distinction.


Asunto(s)
Biomarcadores/análisis , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/efectos de los fármacos , Metabolómica/métodos , Pruebas de Toxicidad/métodos , Animales , Biomarcadores/metabolismo , Células Cultivadas , Fraccionamiento Químico , Técnicas de Química Analítica/métodos , Interpretación Estadística de Datos , Humanos , Hígado/efectos de los fármacos , Metabolómica/estadística & datos numéricos , Distribución Aleatoria , Pruebas de Toxicidad/normas , Pruebas de Toxicidad/estadística & datos numéricos
17.
Arch Toxicol ; 92(6): 1939-1952, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29761207

RESUMEN

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.


Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Bosentán/toxicidad , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Ácidos y Sales Biliares/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , Hígado/metabolismo , Metabolómica , Estrés Oxidativo/genética , Proteómica , Receptores Citoplasmáticos y Nucleares/genética
18.
Arch Toxicol ; 90(3): 677-89, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25716160

RESUMEN

Steatosis, also known as fatty liver disease (FLD), is a disorder in which the lipid metabolism of the liver is disturbed, leading to the abnormal retention of lipids in hepatocytes. FLD can be induced by several drugs, and although it is mostly asymptomatic, it can lead to steatohepatitis, which is associated with liver inflammation and damage. Drug-induced liver injury is currently the major cause of postmarketing withdrawal of pharmaceuticals and discontinuation of the development of new chemical entities. Therefore, the potential induction of steatosis must be evaluated during preclinical drug development. However, robust human-relevant in vitro models are lacking. In the present study, we explore the applicability of hepatic cells (hSKP-HPCs) derived from postnatal skin precursors, a stem cell population residing in human dermis, to investigate the steatosis-inducing effects of sodium valproate (Na-VPA). Exposure of hSKP-HPC to sub-cytotoxic concentrations of this reference steatogenic compound showed an increased intracellular accumulation of lipid droplets, and the modulation of key factors involved in lipid metabolism. Using a toxicogenomics approach, we further compared Na-VPA-treated hSKP-HPC and Na-VPA-treated primary human hepatocytes to liver samples from patients suffering from mild and advanced steatosis. Our data show that in hSKP-HPC exposed to Na-VPA and liver samples of patients suffering from mild steatosis, but not in primary human hepatocytes, "liver steatosis" was efficiently identified as a toxicological response. These findings illustrate the potential of hSKP-HPC as a human-relevant in vitro model to identify hepatosteatotic effects of chemical compounds.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Pruebas de Toxicidad/métodos , Ácido Valproico/toxicidad , Células Cultivadas , Citometría de Flujo/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Piel/citología , Células Madre/citología , Células Madre/efectos de los fármacos , Toxicogenética
19.
Arch Toxicol ; 90(6): 1529-1539, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-27129694

RESUMEN

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.


Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Pruebas de Toxicidad/métodos , Alternativas a las Pruebas en Animales/legislación & jurisprudencia , Alternativas a las Pruebas en Animales/organización & administración , Animales , Biomarcadores/análisis , Células Cultivadas , Seguridad de Productos para el Consumidor , Unión Europea , Regulación Gubernamental , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro
20.
Stem Cells ; 32(8): 2215-28, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24585677

RESUMEN

Human skin-derived precursors (hSKPs) are multipotent somatic stem cells that persist within the dermis throughout adulthood and harbor potential clinical applicability. In this study, we investigated their immunogenicity and immunosuppressive features, both in vitro and in vivo. As such, this study provides a solid basis for developing their future clinical applications. We found that hSKPs express HLA-ABC molecules, but not HLA-DR, rendering them poorly immunogenic. Using a coculture set-up, we could further demonstrate that hSKPs inhibit the proliferation of allogeneic activated T cells and alter their cytokine secretion profile, in a dose-dependent manner. Cotransplantation of hSKP and human peripheral blood leukocytes (PBL) into severe combined immune-deficient mice also showed a significant impairment of the graft-versus-host response 1 week post-transplantation and a drastic increase in survival time of 60%. From a mechanistic point of view, we found that hSKPs require cell contact as well as secretion of soluble inhibitory factors in order to modulate the immune response. The expression/secretion levels of these factors further increases upon inflammation or in the presence of activated T cells. As such, we believe that these features could be beneficial in a later allogeneic clinical setting, because rejection of engrafted allogeneic hSKP might be delayed or even avoided due to their own promotion of a tolerogenic microenvironment.


Asunto(s)
Aloinjertos/inmunología , Células Madre Multipotentes/inmunología , Piel/citología , Piel/inmunología , Animales , Técnicas de Cocultivo , Dinoprostona/biosíntesis , Citometría de Flujo , Antígenos HLA/biosíntesis , Factor de Crecimiento de Hepatocito/biosíntesis , Humanos , Inmunohistoquímica , Factor Inhibidor de Leucemia/biosíntesis , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones SCID , Células Madre Multipotentes/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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