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1.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298967

RESUMO

Pathological fibrosis of the liver is a landmark feature in chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Diagnosis and assessment of progress or treatment efficacy today requires biopsy of the liver, which is a challenge in, e.g., longitudinal interventional studies. Molecular imaging techniques such as positron emission tomography (PET) have the potential to enable minimally invasive assessment of liver fibrosis. This review will summarize and discuss the current status of the development of innovative imaging markers for processes relevant for fibrogenesis in liver, e.g., certain immune cells, activated fibroblasts, and collagen depositions.


Assuntos
Imagem Molecular/tendências , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Alarminas/metabolismo , Animais , Aquaporinas/análise , Colágeno/análise , Meios de Contraste , Citocinas/metabolismo , Técnicas de Imagem por Elasticidade/métodos , Endopeptidases/análise , Ácidos Graxos/metabolismo , Fibroblastos/química , Fibroblastos/ultraestrutura , Radioisótopos de Flúor , Radioisótopos de Gálio , Células Estreladas do Fígado/química , Células Estreladas do Fígado/ultraestrutura , Hepatócitos/metabolismo , Humanos , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Proteínas de Membrana/análise , Camundongos , Imagem Molecular/métodos , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Ratos , Receptores CCR2/análise , Triglicerídeos/metabolismo
2.
Biochim Biophys Acta Gen Subj ; 1865(4): 129559, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32084396

RESUMO

BACKGROUND: Extracellular vesicles (EVs) are a diverse group of membrane-bound nanovesicles potentially released by every cell. With the liver's unique ensemble of cells and its fundamental physiological tasks, elucidating the role of EV-mediated hepatic cellular crosstalk and their role in different pathologies has been gaining the attention of many scientists. SCOPE OF REVIEW: The present review shifts the perspective into practice: we aim to critically discuss the methods used to purify and to biochemically analyse EVs from specific liver resident cells, including hepatocytes, hepatic stellate cells, cholangiocytes, liver sinusoidal endothelial cells, Kupffer cells, liver stem cells. The review offers a reference guide to current approaches. MAJOR CONCLUSIONS: Strategies for EV isolation and characterization are as varied as the research groups performing them. We present main advantages and disadvantages for the methods, highlighting common causes for concern, such as FBS handling, reporting of cell viability, EV yield and storage, differences in differential centrifugations, suboptimal method descriptions, and method transferability. We both looked at how adaptable the research between human and rodent cells in vitro is, and also assessed how well either of them translates to ex vivo settings. GENERAL SIGNIFICANCE: We reviewed methodological practices for the isolation and analysis of liver-derived EVs, making a cell type specific user guide that shows where to start, what has worked so far and to what extent. We critically discussed room for improvement, placing a particular focus on working towards a potential standardization of methods.


Assuntos
Vesículas Extracelulares/química , Fígado/citologia , Animais , Centrifugação/métodos , Células Endoteliais/química , Células Endoteliais/citologia , Células Estreladas do Fígado/química , Células Estreladas do Fígado/citologia , Hepatócitos/química , Hepatócitos/citologia , Humanos , Células de Kupffer/química , Células de Kupffer/citologia , Fígado/química , Células-Tronco/química , Células-Tronco/citologia
3.
FEBS Open Bio ; 10(6): 1044-1055, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32237041

RESUMO

The activation of hepatic stellate cells (HSCs) is associated with liver fibrosis, the pathological feature of most forms of chronic hepatic damage, and is accompanied by abnormal deposition of the extracellular matrix (ECM). During the pathological process, acid-sensing ion channel 1a (ASIC1a), which is responsible for Ca2+ transportation, is involved in the activation of HSCs. It has previously been identified that ASIC1a is related to pyroptosis in articular chondrocytes. However, it remains unclear whether ASIC1a restrains pyroptosis during liver fibrosis. Here, we determined that the levels of pyroptosis-associated speck-like protein, gasdermin D, caspase-1, nucleotide-binding oligomerization domain (NOD)-like receptor 3, and apoptosis-associated speck-like protein (ASC) decreased, while the level of α-smooth muscle actin and collagen-I increased upon introduction of ASIC1a into an acid-induced model. Inhibition or silencing of ASIC1a and the use of Ca2+ -free medium were able to promote the pyroptosis of activated HSCs, which reduced their deposition. In summary, our study indicates that ASIC1a inhibits pyroptosis of HSCs and that inhibition of ASIC1a may be able to promote pyroptosis to relieve liver fibrosis.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Células Estreladas do Fígado/imunologia , Cirrose Hepática/imunologia , Piroptose/imunologia , Canais Iônicos Sensíveis a Ácido/genética , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cálcio/metabolismo , Caspase 1/metabolismo , Cátions Bivalentes/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Células Estreladas do Fígado/química , Células Estreladas do Fígado/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/citologia , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
J Cell Physiol ; 235(9): 6167-6182, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31975386

RESUMO

Hepatocellular carcinoma (HCC) is a major health problem worldwide and in the United States as its incidence has increased substantially within the past two decades. HCC therapy remains a challenge, primarily due to underlying liver disorders such as cirrhosis that determines treatment approach and efficacy. Activated hepatic stellate cells (A-HSCs) are the key cell types involved in hepatic fibrosis/cirrhosis. A-HSCs are important constituents of HCC tumor microenvironment (TME) and support tumor growth, chemotherapy resistance, cancer cell migration, and escaping immune surveillance. This makes A-HSCs an important therapeutic target in hepatic fibrosis/cirrhosis as well as in HCC. Although many studies have reported the role of A-HSCs in cancer generation and investigated the therapeutic potential of A-HSCs reversion in cancer arrest, not much is known about inactivated or quiescent HSCs (Q-HSCs) in cancer growth or arrest. Here we report that Q-HSCs resist cancer cell growth by inducing cytotoxicity and enhancing chemotherapy sensitivity. We observed that the conditioned media from Q-HSCs (Q-HSCCM) induces cancer cell death through a caspase-independent mechanism that involves an increase in apoptosis-inducing factor expression, nuclear localization, DNA fragmentation, and cell death. We further observed that Q-HSCCM enhanced the efficiency of doxorubicin, as measured by cell viability assay. Exosomes present in the conditioned media were not involved in the mechanism, which suggests the role of other factors (proteins, metabolites, or microRNA) secreted by the cells. Identification and characterization of these factors are important in the development of effective HCC therapy.


Assuntos
Fator de Indução de Apoptose/genética , Carcinoma Hepatocelular/tratamento farmacológico , Células Estreladas do Fígado/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Caspases/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células Estreladas do Fígado/química , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Microambiente Tumoral
5.
Contrast Media Mol Imaging ; 2019: 6298128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31866798

RESUMO

Purpose: Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin α v ß 3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin α v ß 3 imaging agent [18F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging. Procedures: Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [18F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin α v ß 3, smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis. Results: The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [18F]FtRGD correlated well with integrin α v ß 3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [18F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49. Conclusions: The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin α v ß 3 and extracellular matrix protein expression. [18F]FtRGD showed very good correlation between liver uptake and integrin α v ß 3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Células Estreladas do Fígado/ultraestrutura , Integrina alfaVbeta3/análise , Cirrose Hepática/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Actinas/biossíntese , Actinas/genética , Animais , Deficiência de Colina/complicações , Colágeno/biossíntese , Colágeno/genética , Progressão da Doença , Diagnóstico Precoce , Radioisótopos de Flúor , Regulação da Expressão Gênica , Células Estreladas do Fígado/química , Hidroxiprolina/análise , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/genética , Fígado/química , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Tamanho do Órgão , RNA Mensageiro/biossíntese , Compostos Radiofarmacêuticos , Índice de Gravidade de Doença , Triglicerídeos/análise
6.
BMC Complement Altern Med ; 19(1): 222, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438947

RESUMO

BACKGROUND: Chronic hepatic diseases are serious problems worldwide, which may lead to the development of fibrosis and eventually cirrhosis. Despite the significant number of people affected by hepatic fibrosis, no effective treatment is available. In the liver, hepatic stellate cells are the major fibrogenic cell type that play a relevant function in chronic liver diseases. Thus, the characterization of components that control the fibrogenesis in the hepatic stellate cells is relevant in supporting the development of innovative therapies to treat and/or control liver fibrosis. The present study investigated the effects of Baccharis dracunculifolia D.C. and Plectranthus barbatus Andrews medicinal plant extracts in LX-2 transdifferentiation. METHODS: LX-2 is a human immortalized hepatic stellate cell that can transdifferentiate in vitro from a quiescent-like phenotype to a more proliferative and activated behavior, and it provides a useful platform to assess antifibrotic drugs. Then, the antifibrotic effects of hydroalcoholic extracts of Baccharis dracunculifolia and Plectranthus barbatus medicinal plants on LX-2 were evaluated. RESULTS: The results in our cellular analyses, under the investigated concentrations of the plant extracts, indicate no deleterious effects on LX-2 metabolism, such as toxicity, genotoxicity, or apoptosis. Moreover, the extracts induced changes in actin filament distribution of activated LX-2, despite not affecting the cellular markers of transdifferentiation. Consistent effects in cellular retinoid metabolism were observed, supporting the presumed activity of the plant extracts in hepatic lipids metabolism, which corroborated the traditional knowledge about their uses for liver dysfunction. CONCLUSION: The combined results suggested a potential hepatoprotective effect of the investigated plant extracts reinforcing their safe use as coadjuvants in treating imbalanced liver lipid metabolism.


Assuntos
Baccharis , Células Estreladas do Fígado , Extratos Vegetais/farmacologia , Plectranthus , Substâncias Protetoras/farmacologia , Retinoides/metabolismo , Linhagem Celular , Sobrevivência Celular , Transdiferenciação Celular/efeitos dos fármacos , Células Estreladas do Fígado/química , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Plantas Medicinais , Retinoides/análise
7.
Theranostics ; 9(12): 3622-3638, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281502

RESUMO

Long non-coding RNAs (lncRNAs) are involved in numerous biological functions and pathological processes. However, the clinical significance of lncRNAs and their functions in liver fibrosis remain largely unclear. Methods: The transcript of lncRNA SCARNA10 in serum and liver samples from patients with advanced hepatic fibrosis, liver tissues from two fibrosis mouse models, and cultured hepatic stellate cells (HSCs) was determined by real-time RT-PCR. The effects of lentivirus-mediated knockdown or over-expression of SCARNA10 in liver fibrosis were examined in vitro and in vivo. Moreover, the effects and mechanisms of down-regulation or over-expression of SCARNA10 on the expression of the genes involved in TGFß pathway were determined. Results: It was found lncRNA ENSMUST00000158992, named as Scarna10, was remarkably up-regulated in mouse fibrotic livers according to the microarray data. We observed that the transcript of SCARNA10 was increased in the serum and liver from patients with advanced hepatic fibrosis. Furthermore, we found that SCARNA10 promoted liver fibrosis both in vitro and in vivo through inducing hepatocytes (HCs) apoptosis and HSCs activation. Mechanistically, RNA immunoprecipitation (RIP) assays demonstrated that SCARNA10 physically associated with polycomb repressive complex 2 (PRC2). Additionally, our results demonstrated that SCARNA10 functioned as a novel positive regulator of TGFß signaling in hepatic fibrogenesis by inhibiting the binding of PRC2 to the promoters of the genes associated with ECM and TGFß pathway, thus promoting the transcription of these genes. Conclusions: Our study identified a crucial role of SCARNA10 in liver fibrosis, providing a proof of this molecule as a potential diagnostic marker and a possible therapeutic target against liver fibrosis.


Assuntos
Biomarcadores/análise , Células Estreladas do Fígado/química , Cirrose Hepática/fisiopatologia , Fígado/patologia , RNA Longo não Codificante/análise , RNA Longo não Codificante/metabolismo , Soro/química , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Cirrose Hepática/patologia , Camundongos , Modelos Biológicos , RNA Longo não Codificante/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Nat Prod Res ; 33(17): 2507-2514, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29607746

RESUMO

In this study, the vitellogenin (Vtg) modulation by genistein and polychlorinated biphenyl-126 (PCB-126) exposure in zebrafishes has been investigated. Both PCB-126 and genistein have been identified as aquatic pollutants and can further increase estrogenicity of waterways. Vtg is egg yolk precursor protein release by the hepatocytes during vitellogenesis. This process occurs normally in the hepatocytes in response to the activation with the estrogens such as 17-ß-estradiol. Our immunohistochemical findings showed a Vtg expression that increases at 12 h and at 72 h in the liver of treated fishes with both PCB-126 and genistein, individually and in combination. Furthermore, for the first time, also hepatic stellate cells (HSC) in the liver parenchyma were strongly positive for vitellogenin.


Assuntos
Genisteína/farmacologia , Bifenilos Policlorados/toxicidade , Vitelogeninas/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Estradiol/farmacologia , Antagonistas de Estrogênios/toxicidade , Estrogênios/farmacologia , Células Estreladas do Fígado/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/metabolismo , Vitelogeninas/metabolismo
9.
Semin Cell Dev Biol ; 73: 13-30, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28688930

RESUMO

With the arrival of atomic force microscopy (AFM) about thirty years ago, this new imaging tool opened up a new area for the exploration of biological samples, ranging from the tissue and cellular level down to the supramolecular scale. Commercial instruments of this new imaging technique began to appear in the five years following its discovery in 1986 by Binnig, Quate & Gerber. From that point onwards the AFM has attracted many liver biologists, and the number of publications describing structure-function relationships on the diverse set of liver cells has grown steadily ever since. It is therefore timely to reflect on the achievements of AFM in disclosing the cellular architecture of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, stellate cells and liver-associated natural killer cells. In this thematic paper, we present new data and provide an in-depth overview of the current AFM literature on liver cell biology. We furthermore include a future outlook on how this scanning probe imaging tool and its latest developments can contribute to clarify various structural and functional aspects of cells in liver health and disease.


Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/ultraestrutura , Células Matadoras Naturais/ultraestrutura , Células de Kupffer/metabolismo , Células de Kupffer/ultraestrutura , Microscopia de Força Atômica , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Endoteliais/química , Células Estreladas do Fígado/química , Humanos , Células Matadoras Naturais/química , Células Matadoras Naturais/metabolismo , Células de Kupffer/química , Modelos Estruturais , Relação Estrutura-Atividade
10.
Cell Mol Biol (Noisy-le-grand) ; 62(3): 51-64, 2016 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-27064874

RESUMO

Proteins N-glycosylation is significantly increased in the activated human hepatic stellate cells (HSCs) stimulated by transforming growth factor-ß1 (TGF-ß1) compared to the quiescent HSCs according to our previous study. However, little is known about the alteration of N-glycoprotein profiles in the activated HSCs. Profiles of N-glycopeptides / N-glycoproteins / N-glycosites in LX-2 cells, with and without activation by TGF-ß1, were identified and compared using hydrazide chemistry enrichment coupled with liquid chromatography - mass spectrometry analysis. Western blot and immunohistochemistry were further used for validation. A total of 103 non-redundant N-glycopeptides, with 107 glycosylation sites from 86 N-glycoproteins, were identified in activated and quiescent LX-2 cells respectively. Among these, 23 proteins were known N-glycoproteins, and 58 were newly identified N-glycoproteins. In addition, 43 proteins (e.g., pigment epithelium-derived factor and clathrin heavy chain 1) were solely identified or up-regulated in the activated LX-2 cells, which participated in focal adhesion and glycosaminoglycan degradation pathways and were involved in interaction clusters of cytoskeletal proteins (e.g., myosin light chains and keratins). The increased expression of glucosamine (N-acetyl)-6-sulfatase and phospholipase C beta 2 and the decreased expression of zinc finger and BTB domain-containing protein 1 were validated in the activated compared to the quiescent LX-2 cells. In conclusion, increased expression of N-glycoproteins and N-glycosites play important roles in cellular contractility, signal transduction, and responses to stimuli in the activated HSCs, which might provide useful information for discovering novel molecular mechanism of HSC activation and therapeutic targets in liver fibrosis.


Assuntos
Glicoproteínas/metabolismo , Células Estreladas do Fígado/metabolismo , Mapas de Interação de Proteínas , Fator de Crescimento Transformador beta1/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Glicoproteínas/análise , Células Estreladas do Fígado/química , Células Estreladas do Fígado/citologia , Humanos , Cirrose Hepática/metabolismo
11.
J Pharm Sci ; 105(5): 1779-1789, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27020986

RESUMO

Xenobiotic transporters play key roles in disposition of certain therapeutic agents, although limited information is available on their roles other than pharmacokinetic issues. Here, suppressive effect of multispecific organic cation transporter OCTN1/SLC22A4 on liver fibrosis was proposed in liver injury models. After injection of hepatotoxins such as dimethylnitrosamine (DMN) or concanavalin A, hepatic fibrosis, and oxidative stress, evaluated in terms of Sirius red and 4-hydroxy-2-nonenal staining, respectively, were more severe in liver of octn1/slc22a4 gene knockout (octn1(-/-)) mice than that in wild-type mice. DMN treatment markedly increased α-smooth muscle actin and F4/80, markers of activated stellate and Kupffer cells, respectively, in liver of octn1(-/-), but had less effect in wild-type mice. Thus, octn1/slc22a4 gene deletion results in more severe hepatic fibrosis, oxidative stress, and inflammation. DMN-treated wild-type mice showed increased Octn1 staining and hepatic concentration of its food-derived antioxidant ergothioneine (ERGO). The upregulated Octn1 was co-localized with α-smooth muscle actin. Functional expression of Octn1 was demonstrated in activated human hepatic stellate cell lines, LI90 and LX-2. Provision of ERGO-rich feed ameliorated DMN-induced liver fibrosis and oxidative stress. Overall, Octn1 is upregulated in activated stellate cells, resulting in increased delivery of its substrate antioxidant ERGO and a protective effect against liver fibrosis.


Assuntos
Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/prevenção & controle , Proteínas de Membrana/deficiência , Xenobióticos/metabolismo , Animais , Proteínas de Transporte/análise , Linhagem Celular , Feminino , Células HEK293 , Células Estreladas do Fígado/química , Humanos , Proteínas de Membrana/análise , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte de Cátions Orgânicos , Simportadores
12.
Gut ; 65(7): 1175-85, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26078290

RESUMO

INTRODUCTION: CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. OBJECTIVE: To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. DESIGN: CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. RESULTS: Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-ß at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. CONCLUSIONS: Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/metabolismo , Fígado/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Actinas/análise , Indutores da Angiogênese/farmacologia , Animais , Antígenos CD/análise , Antígenos de Neoplasias/análise , Becaplermina , Tetracloreto de Carbono , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Doença Crônica , Colágeno/metabolismo , Desmina/análise , Fibrose , Expressão Gênica , Células Estreladas do Fígado/química , Humanos , Inflamação/genética , Fígado/química , Cirrose Hepática/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Vimentina/análise
13.
Fetal Pediatr Pathol ; 34(4): 216-22, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26030091

RESUMO

Congenital bilateral diaphragm agenesis is a very rare condition. We describe limited (abdomen only) autopsy findings of a case of bilateral diaphragm agenesis in a 27-week male fetus with unusual findings of fibrosis of the pancreatic head and ectopic liver nodules in a mass at the upper abdomen that may represent a possible diaphragm anlage. We have correlated our observations with data from experimental and embryological studies to suggest possible mechanisms for the malformations that were present and their implications for our understanding of pancreas, liver and diaphragm development in the human fetus.


Assuntos
Anormalidades Múltiplas/embriologia , Coristoma/embriologia , Diafragma/anormalidades , Hérnia Diafragmática/embriologia , Fígado , Pâncreas/anormalidades , Cavidade Abdominal/embriologia , Anormalidades Múltiplas/patologia , Adulto , Coristoma/patologia , Diafragma/embriologia , Diafragma/patologia , Evolução Fatal , Feminino , Fibrose , Idade Gestacional , Células Estreladas do Fígado/química , Células Estreladas do Fígado/patologia , Hérnia Diafragmática/patologia , Humanos , Recém-Nascido Prematuro , Fígado/embriologia , Fígado/patologia , Masculino , Pâncreas/embriologia , Pâncreas/patologia , Poli-Hidrâmnios/etiologia , Gravidez , Tórax/embriologia
14.
Proteomics ; 15(19): 3283-95, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26058380

RESUMO

Glycan-binding proteins (GBPs) play an important role in cell adhesion, bacterial/viral infection, and cellular signaling pathways. However, little is known about the precision alteration of GBPs referred to pathological changes in hepatic stellate cells (HSCs) during liver fibrosis. Here, the carbohydrate microarrays were used to probe the alteration of GBPs in the activated HSCs and quiescent HSCs. As a result, 12 carbohydrates (e.g. Gal, GalNAc, and Man-9Glycan) showed increased signal, while seven carbohydrates (e.g. NeuAc, Lac, and GlcNAc-O-Ser) showed decreased signal in activated HSCs. Three carbohydrates (Gal, GalNAc, and NeuAc) were selected and subsequently used to validate the results of the carbohydrate microarrays as well as assess the distribution and localization of their binding proteins in HSCs and liver tissues by cy/histochemistry; the results showed that GBPs mainly distributed in the cytoplasma membrane and perinuclear region of cytoplasm. The immunocytochemistry was further used to verify some GBPs really exist in Golgi apparatus of the cells. The precision alteration and localization of GBPs referred to pathological changes in HSCs may provide pivotal information to help understand the biological functions of glycans how to exert through their recognition by a wide variety of GBPs. This study could lead to the development of new anti-fibrotic strategies.


Assuntos
Células Estreladas do Fígado/metabolismo , Lectinas/metabolismo , Cirrose Hepática/metabolismo , Polissacarídeos/metabolismo , Células Cultivadas , Células Estreladas do Fígado/química , Humanos , Imuno-Histoquímica , Lectinas/análise , Cirrose Hepática/fisiopatologia , Transporte Proteico
15.
Anal Chim Acta ; 859: 59-65, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25622606

RESUMO

A ratiometric fluorescent probe for H2S was developed based on a coumarin- benzopyrylium platform. The ratiometric sensing is realized by a selective conversion of acyl azide to the corresponding amide, which subsequently undergoes an intramolecular spirocyclization to alter the large π-conjugated system of CB fluorophore. Compared with the traditional azide-based H2S probes, the proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid response (∼1min) towards H2S, which is superior to most of the azide-based H2S probes. Preliminary fluorescence imaging experiments show that probe 1 has potential to track H2S in living cells.


Assuntos
Benzopiranos/química , Cumarínicos/química , Corantes Fluorescentes/química , Gasotransmissores/análise , Sulfeto de Hidrogênio/análise , Linhagem Celular , Sobrevivência Celular , Células Estreladas do Fígado/química , Células Estreladas do Fígado/citologia , Humanos , Imagem Óptica
16.
Proteomics ; 14(17-18): 1977-86, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24995952

RESUMO

Activated hepatic stellate cell (HSC) is the main myofibroblast cell in the liver fibrosis (LF). An important characteristic of the recovery of LF is not only the apoptosis of activated HSCs but also reversal of myofibroblast-like phenotype to a quiescent-like phenotype. Understanding the changes of secreted proteins in the reversion of activated HSCs may provide the broader view of cellular regulatory networks and discover candidate markers or targets for therapeutic strategies of LF. In this study, stable isotope labeling with amino acids (SILAC) combined with linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT MS) was performed on in vitro activated HSCs and reverted HSCs to obtain a proteomic view of secretory proteins. In total, 330 proteins showed significant differences in reverted HSCs. Among these, 109 upregulated proteins were mainly involved in amino acid metabolism pathway and glucose metabolism pathway using GeneGO/MetaCore software, while 221 downregulated proteins are closely associated with HSCs activation, such as cytoskeleton remodeling, chemokines, and cell adhesion. Additionally, a set of novel proteins associated with HSCs activation and reversion were validated by Western blotting in the cell secretion and in the sera of LF, including vitronectin, laminin beta 1, and ubiquitin conjugation factor E4B. Our study provided the valuable insight into the mechanisms in the reversion of activated HSCs and identified some potential biomarkers of LF in clinical studies. All MS data have been deposited in the ProteomeXchange with identifier PXD000773 (http://proteomecentral.proteomexchange.org/dataset/PXD000773).


Assuntos
Células Estreladas do Fígado/metabolismo , Marcação por Isótopo/métodos , Proteoma/análise , Proteoma/química , Proteômica/métodos , Western Blotting , Linhagem Celular , Células Estreladas do Fígado/química , Humanos , Espectrometria de Massas/métodos , Proteínas/análise , Proteínas/química , Proteínas/classificação , Reprodutibilidade dos Testes
17.
World J Gastroenterol ; 20(22): 7067-74, 2014 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-24966580

RESUMO

Recently, there have been reports from liver biopsies that showed the progression of liver fibrosis in liver transplant patients after the cessation of immunosuppression. Herein, we focused on activated hepatic stellate cells expressing alpha smooth muscle actin (α-SMA) to understand the correlation between immunosuppressant medication and liver fibrosis. The study enrolled two pediatric patients who underwent living donor liver transplantation and ceased immunosuppressant therapy. The number of α-SMA-positive cells in the specimens obtained by liver biopsy from these two patients showed a three-fold increase compared with the number from four transplanted pediatric patients who were continuing immunosuppressant therapy. In addition, the α-SMA-positive area evaluated using the WinRooF image processing software program continued to increase over time in three adult transplanted patients with liver fibrosis, and the α-SMA-positive area was increasing even during the pre-fibrotic stage in these adult cases, according to a retrospective review. Therefore, α-SMA could be a useful marker for the detection of early stage fibrosis.


Assuntos
Actinas/análise , Atresia Biliar/cirurgia , Células Estreladas do Fígado/química , Cirrose Hepática/metabolismo , Transplante de Fígado/métodos , Doadores Vivos , Transplantados , Atresia Biliar/diagnóstico , Biomarcadores/análise , Biópsia , Criança , Pré-Escolar , Esquema de Medicação , Diagnóstico Precoce , Feminino , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Interpretação de Imagem Assistida por Computador , Imuno-Histoquímica , Imunossupressores/administração & dosagem , Lactente , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Masculino , Valor Preditivo dos Testes , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
18.
BMC Gastroenterol ; 14: 39, 2014 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-24559185

RESUMO

BACKGROUND: The relationship between resistin and non-alcoholic steatohepatitis (NASH) is not clear, some studies claimed that serum resistin levels were associated with neither the presence of NASH nor its severity, others declared that serum resistin was related with inflammation and fibrosis in NASH. Our animal study verified that the distribution of resistin in the liver is correlated with inflammation in NASH. However, there is no pertinent study in humans. METHODS: Thirty patients with NASH, 28 simple steatosis, and 43 controls were recruited. Blood was collected for resistin, liver chemistries, fasting insulin and some metabolic parameters. Liver histology was scored according to NAFLD activity scoring system. Hepatic resistin expression was examined by real-time polymerase chain reaction, immunohistochemistry. Resistin protein expression was confirmed by western blotting in 13 patients with concomitant NAFLD and gallstone. RESULTS: Serum resistin was significantly elevated in both NASH and simple steatotic subjects compared with controls (all P < 0.05). Hepatic resistin was significantly increased in NASH patients in both mRNA and protein levels than those in simple steatosis and control subjects (all P < 0.05). Both serum and hepatic resistin had a correlation with obesity, but not with insulin resistance. The distribution of resistin positive cells was predominantly in perisinusoidal cells (such as Kupffer cells and hepatic stellate cells) in human NASH. Multivariate analysis revealed that waist-hip ratio, higher serum triglyceride, and hyperresistinemia were independent factors related to higher grade of steatosis; whereas hepatic resistin and serum cytokeratin predict NASH and severity of liver fibrosis. CONCLUSIONS: Hepatic resistin overexpression in NASH patients is associated with the severity of liver inflammation and fibrosis. Liver-derived resistin may be involved in the pathogenesis of human NASH.


Assuntos
Fígado Gorduroso/sangue , Fígado Gorduroso/patologia , RNA Mensageiro/análise , Resistina/sangue , Adulto , Estudos de Casos e Controles , Fígado Gorduroso/metabolismo , Feminino , Células Estreladas do Fígado/química , Humanos , Resistência à Insulina , Queratinas/sangue , Células de Kupffer/química , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica , Obesidade/metabolismo , Estudos Prospectivos , Resistina/análise , Resistina/genética , Triglicerídeos/sangue , Regulação para Cima , Relação Cintura-Quadril
19.
Zhongguo Zhong Yao Za Zhi ; 38(13): 2206-9, 2013 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-24079255

RESUMO

OBJECTIVE: To observe the effect of neferine on Collagen-I, TIMP-1 and MMP-2 expressions and protein secretion of hepatic stellate cells. METHOD: The hepatic stellate cell line HSC-T6 was cultured in vitro, and then randomly divided into 5 groups: the control group, the platelet-derived growth factor (PDGF) group and PDGF + neferine (2, 6, 10 micromol x L(-1)) groups. All of the groups were cultured for 48 h, and their cells were collected to extract mRNA and detect Collagen-I, TIMP-1 and MMP-2 expressions with RT-PCR. Their cell supernatants were also collected to determine the protein content of three factors with ELISA. RESULT: Compared with the control group, PDGF could remarkably increase the Collagen-I, TIMP-1 and MMP-2 expressions and protein secretion of hepatic stellate cells. Compared with the PDGF group, PDGF + neferine (6, 10 micromol x L(-1)) groups showed a notable decrease in the Collagen-I and mRNA expression and protein secretion along with the increase in the concentration, whereas the PDGF + neferine (2 micromol x L(-1)) group showed no significant change in the Collagen-I and mRNA expression and protein secretion. Compared with the PDGF group, three PDGF + neferine groups showed no notable change in MMP-2 expression and protein secretion. CONCLUSION: Neferine can inhibit the Collagen-I, TIMP-1 and mRNA protein expression and protein secretion of PDGF-induced HSCs along with the increase in the concentration, but with not remarkable effect on the MMP-2 expression and secretion.


Assuntos
Benzilisoquinolinas/farmacologia , Colágeno Tipo I/análise , Medicamentos de Ervas Chinesas/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Metaloproteinase 2 da Matriz/análise , Inibidor Tecidual de Metaloproteinase-1/análise , Animais , Células Cultivadas , Colágeno Tipo I/genética , Células Estreladas do Fígado/química , Metaloproteinase 2 da Matriz/genética , Ratos , Inibidor Tecidual de Metaloproteinase-1/genética
20.
Anat Rec (Hoboken) ; 295(10): 1660-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22907891

RESUMO

We performed a systematic characterization of the hepatic vitamin A storage in mammals and birds of the Svalbard Archipelago and Greenland. The liver of top predators, including polar bear, Arctic fox, bearded seal, and glaucous gull, contained about 10-20 times more vitamin A than the liver of all other arctic animals studied, as well as their genetically related continental top predators. The values are also high compared to normal human and experimental animals like mouse and rat. This massive amount of hepatic vitamin A was located in large autofluorescent lipid droplets in hepatic stellate cells (HSCs; also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, or Ito cells). The droplets made up most of the cells' cytoplasm. The development of such an efficient vitamin A-storing mechanism in HSCs may have contributed to the survival of top predators in the extreme environment of the arctic. These animals demonstrated no signs of hypervitaminosis A. We suggest that HSCs have capacity to take-up and store large amounts of vitamin A, which may play a pivotal role in maintenance of the food web, food chain, biodiversity, and eventually ecology of the arctic.


Assuntos
Células Estreladas do Fígado/química , Células Estreladas do Fígado/metabolismo , Comportamento Predatório/fisiologia , Vitamina A/química , Vitamina A/metabolismo , Animais , Regiões Árticas , Charadriiformes/metabolismo , Rena/metabolismo , Focas Verdadeiras/metabolismo , Especificidade da Espécie , Sobrevida/fisiologia , Ursidae/metabolismo
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