RESUMO
The disposal of apoptotic bodies by professional phagocytes is crucial to effective inflammation resolution. Our ability to improve the disposal of apoptotic bodies by professional phagocytes is impaired by a limited understanding of the molecular mechanisms that regulate the engulfment and digestion of the efferocytic cargo. Macrophages are professional phagocytes necessary for liver inflammation, fibrosis, and resolution, switching their phenotype from proinflammatory to restorative. Using sterile liver injury models, we show that the STAT3-IL-10-IL-6 axis is a positive regulator of macrophage efferocytosis, survival, and phenotypic conversion, directly linking debris engulfment to tissue repair.
Assuntos
Interleucina-10/metabolismo , Interleucina-6/metabolismo , Cirrose Hepática/patologia , Fígado/lesões , Macrófagos/imunologia , Fagocitose/imunologia , Fator de Transcrição STAT3/metabolismo , Transferência Adotiva , Animais , Apoptose/imunologia , Humanos , Fígado/patologia , Macrófagos/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose/imunologia , Regeneração/fisiologia , Peixe-Zebra/embriologiaRESUMO
The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ativadores de Enzimas/isolamento & purificação , Cirrose Hepática/tratamento farmacológico , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Biópsia , Células Cultivadas , Ativadores de Enzimas/síntese química , Ativadores de Enzimas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Cirrose Hepática/patologiaRESUMO
Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl(4)-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B(hi) F4/80(int) Ly-6C(lo) macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter-diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C(hi) monocytes, a common origin with profibrotic Ly-6C(hi) macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C(lo) subset, compared with Ly-6C(hi) macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.
Assuntos
Antígenos Ly/imunologia , Intoxicação por Tetracloreto de Carbono/imunologia , Regulação da Expressão Gênica/imunologia , Cirrose Hepática/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Animais , Antígenos Ly/genética , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Tetracloreto de Carbono/toxicidade , Intoxicação por Tetracloreto de Carbono/genética , Intoxicação por Tetracloreto de Carbono/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/imunologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/patologia , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Transgênicos , Monócitos/patologiaRESUMO
UNLABELLED: Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP-12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl(4) ) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP-12 elastin degradation was defined mechanistically using CD11b-DTR and MMP-12 knockout mice. In a CCl(4) model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP-12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP-12 was bound in greater quantities to its inhibitor TIMP-1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP-12. Exposure of CCl(4) in MMP-12(-/-) mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP-12(-/-) mice compared with WT. CONCLUSION: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage-derived MMP-12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies.
Assuntos
Elastina/metabolismo , Cirrose Hepática Experimental/metabolismo , Macrófagos/enzimologia , Metaloproteinase 12 da Matriz/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Fígado/metabolismo , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Inibidor Tecidual de Metaloproteinase-1/genética , Tropoelastina/biossínteseRESUMO
UNLABELLED: There is increasing evidence that the physical environment is a critical mediator of tumor behavior. Hepatocellular carcinoma (HCC) develops within an altered biomechanical environment, and increasing matrix stiffness is a strong predictor of HCC development. The aim of this study was to establish whether changes in matrix stiffness, which are characteristic of inflammation and fibrosis, regulate HCC cell proliferation and chemotherapeutic response. Using an in vitro system of "mechanically tunable" matrix-coated polyacrylamide gels, matrix stiffness was modeled across a pathophysiologically relevant range, corresponding to values encountered in normal and fibrotic livers. Increasing matrix stiffness was found to promote HCC cell proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7-fold and 12.2-fold higher, respectively, when the cells were cultured on stiff (12 kPa) versus soft (1 kPa) supports. This was associated with stiffness-dependent regulation of basal and hepatocyte growth factor-stimulated mitogenic signaling through extracellular signal-regulated kinase, protein kinase B (PKB/Akt), and signal transducer and activator of transcription 3. ß1-Integrin and focal adhesion kinase were found to modulate stiffness-dependent HCC cell proliferation. Following treatment with cisplatin, we observed reduced apoptosis in HCC cells cultured on stiff versus soft (physiological) supports. Interestingly, however, surviving cells from soft supports had significantly higher clonogenic capacity than surviving cells from a stiff microenvironment. This was associated with enhanced expression of cancer stem cell markers, including clusters of differentiation 44 (CD44), CD133, c-kit, cysteine-X-cysteine receptor 4, octamer-4 (CXCR4), and NANOG. CONCLUSION: Increasing matrix stiffness promotes proliferation and chemotherapeutic resistance, whereas a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC. This has implications for both the treatment of primary HCC and the prevention of tumor outgrowth from disseminated tumor cells. (HEPATOLOGY 2011;).
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
The pathological mechanisms that distinguish simple steatosis from steatohepatitis (or NASH, with consequent risk of cirrhosis and hepatocellular cancer) remain incompletely defined. Whereas both a methionine- and choline-deficient diet (MCDD) and a choline-deficient diet (CDD) lead to hepatic triglyceride accumulation, MCDD alone is associated with hepatic insulin resistance and inflammation (steatohepatitis). We used metabolic tracer techniques, including stable isotope ([¹³C4]palmitate) dilution and mass isotopomer distribution analysis (MIDA) of [¹³C2]acetate, to define differences in intrahepatic fatty acid metabolism that could explain the contrasting effect of MCDD and CDD on NASH in C57Bl6 mice. Compared with control-supplemented (CS) diet, liver triglyceride pool sizes were similarly elevated in CDD and MCDD groups (24.37 ± 2.4, 45.94 ± 3.9, and 43.30 ± 3.5 µmol/liver for CS, CDD, and MCDD, respectively), but intrahepatic neutrophil infiltration and plasma alanine aminotransferase (31 ± 3, 48 ± 4, 231 ± 79 U/l, P < 0.05) were elevated only in MCDD mice. However, despite loss of peripheral fat in MCDD mice, neither the rate of appearance of palmitate (27.2 ± 3.5, 26.3 ± 2.3, and 28.3 ± 3.5 µmol·kg⻹·min⻹) nor the contribution of circulating fatty acids to the liver triglyceride pool differed between groups. Unlike CDD, MCDD had a defect in hepatic triglyceride export that was confirmed using intravenous tyloxapol (142 ± 21, 122 ± 15, and 80 ± 7 mg·kg⻹·h⻹, P < 0.05). Moreover, hepatic de novo lipogenesis was significantly elevated in the MCDD group only (1.4 ± 0.3, 2.3 ± 0.4, and 3.4 ± 0.4 µmol/day, P < 0.01). These findings suggest that important alterations in hepatic fatty acid metabolism may promote the development of steatohepatitis. Similar mechanisms may predispose to hepatocyte damage in human NASH.
Assuntos
Deficiência de Colina/metabolismo , Ácidos Graxos/metabolismo , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Metionina/deficiência , Tecido Adiposo/metabolismo , Animais , Dieta , Ingestão de Alimentos/fisiologia , Fígado Gorduroso/patologia , Cromatografia Gasosa-Espectrometria de Massas , Hepatite/metabolismo , Hepatócitos/patologia , Imuno-Histoquímica , Cinética , Lipogênese/fisiologia , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/fisiologia , Ácidos Palmíticos/metabolismo , Triglicerídeos/metabolismoRESUMO
We have compared the distribution of endogenous heterochromatin protein 1 (HP1) proteins (alpha, beta and gamma) in different epithelial lines, pluripotent stem cells and embryonic fibroblasts. In parallel, we have interrogated assembly and dynamics of newly expressed HP1-GFP proteins in cells lacking both HP1alpha and HP1beta alleles, blocked at the G1-S boundary, or cultured in the presence of HDAC and HAT inhibitors. The results reveal a range of cell type and differentiation state-specific patterns that do not correlate with 'fast' or 'slow' subunit exchange in heterochromatin. Furthermore, our observations show that targeting of HP1gamma to heterochromatic sites depends on HP1alpha and H1beta and that, on an architectural level, HP1alpha is the most polymorphic variant of the HP1 family. These data provide evidence for HP1 plasticity under shifting microenvironmental conditions and offer a new conceptual framework for understanding chromatin dynamics at the molecular level.