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1.
Gastroenterology ; 159(4): 1487-1503.e17, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32574624

RESUMO

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4µ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.


Assuntos
Endorribonucleases/fisiologia , Vesículas Extracelulares/patologia , Hepatócitos/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Ceramidas/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo
2.
Am J Pathol ; 190(6): 1284-1297, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32240619

RESUMO

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr-/-) mice were crossbred with ATP binding cassette subfamily B member 4-deficient (Abcb4-/-, alias Mdr2-/-) mice. Male and female wild-type, Tr-/-, Mdr2-/-, and Tr-/-Mdr2-/- mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2-/- and Tr-/-Mdr2-/- mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2-/- mice, compared with extensive bridging fibrosis in Tr-/-Mdr2-/- mice. DR cell population was greatly expanded in the Tr-/-Mdr2-/- versus Mdr2-/- mice. The expanded DR cell population in Tr-/-Mdr2-/- mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr-/-Mdr2-/- versus Mdr2-/- mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2-/- mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2-/- model of cholestasis.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Cirrose Hepática/genética , Fígado/metabolismo , Macrófagos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Colestase/genética , Colestase/metabolismo , Colestase/patologia , Modelos Animais de Doenças , Feminino , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/fisiologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
3.
Hepatology ; 72(3): 1013-1028, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32128842

RESUMO

BACKGROUND AND AIMS: In cholestatic liver diseases, ductular reactive (DR) cells extend into the hepatic parenchyma and promote inflammation and fibrosis. We have previously observed that multidrug-resistant 2 (Mdr2-/- ) double knockout (DKO) mice lacking tumor necrosis factor-related apoptosis-inducing ligand receptor (Tr-/- ) display a more extensive ductular reaction and hepatic fibrosis compared to Mdr2-/- mice. This observation suggests that the magnitude of the DR-cell population may be regulated by apoptosis. APPROACH AND RESULTS: To examine this concept, we cultured epithelial cell adhesion molecule-positive reactive cholangioids (ERCs) obtained from wild-type (WT), Tr-/- , Mdr2-/- and DKO mice. Single-cell transcriptomics and immunostaining of both WT and DKO ERCs confirmed their DR-cell phenotype. Moreover, DKO ERCs displayed a unique translational cluster with expression of chemokines, indicating a reactive state. Incubation with the myeloid cell leukemia 1 (MCL1) inhibitor S63845, a proapoptotic BH3-mimetic therapy, significantly decreased DKO and Mdr2-/- ERC viability compared to WT. Intravenous administration of S63845 significantly reduced the DR-cell population and markers of inflammation and liver fibrosis in Mdr2-/- and DKO mice. Furthermore, DKO mice treated with S63845 displayed a significant decrease in hepatic B lymphocytes compared to untreated mice as assessed by high-definition mass cytometry by time-of-flight. Coculture of bone marrow-derived macrophages with ERCs from DKO mouse livers up-regulated expression of the B cell-directed chemokine (C-C motif) ligand 5. Finally, DR cells were noted to be primed for apoptosis with Bcl-2 homologous antagonist/killer activation in vitro and in vivo in primary sclerosing cholangitis liver specimens. CONCLUSIONS: DR cells appear to play a key role in recruiting immune cells to the liver to actively create an inflammatory and profibrogenic microenvironment. Pharmacologic targeting of MCL1 in a mouse model of chronic cholestasis reduces DR-cell and B-cell populations and hepatic fibrosis.


Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B , Ductos Biliares Intra-Hepáticos/patologia , Colestase , Células Epiteliais , Proteína de Sequência 1 de Leucemia de Células Mieloides , Pirimidinas/farmacologia , Tiofenos/farmacologia , Animais , Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Colestase/tratamento farmacológico , Colestase/metabolismo , Colestase/patologia , Modelos Animais de Doenças , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/prevenção & controle , Camundongos , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
Molecules ; 26(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466232

RESUMO

S100B is a calcium-binding protein that governs calcium-mediated responses in a variety of cells-especially neuronal and glial cells. It is also extensively investigated as a potential biomarker for several disease conditions, especially neurodegenerative ones. In order to establish S100B as a viable pharmaceutical target, it is critical to understand its mechanistic role in signaling pathways and its interacting partners. In this report, we provide evidence to support a calcium-regulated interaction between S100B and the neuronal calcium sensor protein, neurocalcin delta both in vitro and in living cells. Membrane overlay assays were used to test the interaction between purified proteins in vitro and bimolecular fluorescence complementation assays, for interactions in living cells. Added calcium is essential for interaction in vitro; however, in living cells, calcium elevation causes translocation of the NCALD-S100B complex to the membrane-rich, perinuclear trans-Golgi network in COS7 cells, suggesting that the response is independent of specialized structures/molecules found in neuronal/glial cells. Similar results are also observed with hippocalcin, a closely related paralog; however, the interaction appears less robust in vitro. The N-terminal region of NCALD and HPCA appear to be critical for interaction with S100B based on in vitro experiments. The possible physiological significance of this interaction is discussed.


Assuntos
Cálcio/metabolismo , Neurocalcina/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Humanos , Transporte Proteico , Transdução de Sinais
5.
Am J Physiol Gastrointest Liver Physiol ; 319(3): G333-G344, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32683952

RESUMO

Sulfatase 2 (SULF2) is a heparan sulfate editing enzyme that regulates the milieu of growth factors and cytokines involved in a variety of cellular processes. We used a murine model of diet-induced steatohepatitis to assess the effect of SULF2 downregulation on the development of nonalcoholic steatohepatitis (NASH) and liver fibrosis. Wild-type B6;129 mice (WT) and Sulf2-knockout B6;129P2-SULF2Gt(PST111)Byg mice (Sulf2-KO) were fed a fast-food diet (FFD) rich in saturated fats, cholesterol, and fructose or a standard chow diet (SC) ad libitum for 9 mo. WT mice on FFD showed a threefold increase in hepatic Sulf2 mRNA expression, and a 2.2-fold increase in hepatic SULF2 protein expression compared with WT mice on SC. Knockout of Sulf2 led to a significant decrease in diet-mediated weight gain and dyslipidemia compared with WT mice on FFD. Knockout of Sulf2 also abrogated diet-induced steatohepatitis and hepatic fibrosis compared with WT mice on FFD. Furthermore, expression levels of the profibrogenic receptors TGFßR2 and PDGFRß were significantly decreased in Sulf2-KO mice compared with WT mice on FFD. Together, our data suggest that knockout of Sulf2 significantly downregulates dyslipidemia, steatohepatitis, and hepatic fibrosis in a diet-induced mouse model of NAFLD, suggesting that targeting of SULF2 signaling may be a potential therapeutic mechanism in NASH.NEW & NOTEWORTHY We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.


Assuntos
Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Sulfatases/genética , Animais , Dieta Ocidental , Regulação para Baixo , Dislipidemias/genética , Fast Foods , Feminino , Resistência à Insulina , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , RNA Interferente Pequeno/genética , Aumento de Peso/genética
6.
Am J Physiol Cell Physiol ; 317(4): C788-C799, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31365294

RESUMO

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/ß inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.


Assuntos
Alarminas/metabolismo , Vesículas Extracelulares/metabolismo , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Animais , Comunicação Celular/fisiologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Proteômica/métodos , Proteínas S100/metabolismo
7.
J Hepatol ; 71(6): 1193-1205, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31433301

RESUMO

BACKGROUND & AIMS: Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin ß1 (ITGß1), which promotes monocyte adhesion and liver inflammation in murine NASH. METHODS: Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGß1 neutralizing antibody (ITGß1Ab) or control IgG isotype. RESULTS: Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGß1. Furthermore, we showed that LPC treatment in hepatocytes activates ITGß1 and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGß1Ab. FFC-fed, ITGß1Ab-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGß1Ab reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGß1Ab treatment significantly ameliorated liver injury and fibrosis. CONCLUSIONS: Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGß1-dependent mechanism. ITGß1Ab ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGß1 is a potential anti-inflammatory therapeutic strategy in human NASH. LAY SUMMARY: Herein, we report that a cell adhesion molecule termed integrin ß1 (ITGß1) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGß1 is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGß1 reduces liver inflammation, injury and fibrosis. Hence, ITGß1 inhibition may serve as a new therapeutic strategy for NASH.


Assuntos
Anticorpos Neutralizantes , Adesão Celular/imunologia , Hepatócitos/imunologia , Integrina beta1/imunologia , Lisofosfatidilcolinas/farmacologia , Macrófagos/imunologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Vesículas Extracelulares/imunologia , Hepatócitos/metabolismo , Humanos , Cirrose Hepática/prevenção & controle , Camundongos , Monócitos/imunologia , Hepatopatia Gordurosa não Alcoólica/terapia
8.
J Hepatol ; 69(3): 676-686, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29802947

RESUMO

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.


Assuntos
Quimiocina CCL2/metabolismo , Colangite Esclerosante , Imidazóis/farmacologia , Cirrose Hepática , Macrófagos , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Animais , Antagonistas dos Receptores CCR5/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Colangite Esclerosante/tratamento farmacológico , Colangite Esclerosante/imunologia , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Sulfóxidos , Resultado do Tratamento
9.
Hepatology ; 65(4): 1249-1266, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27981602

RESUMO

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c-MYC interacts directly with both proteins. Furthermore, c-MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c-MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down-regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c-MYC, MAFG, and c-MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E-box element, and repress E-box promoter activity. PHB1 promoter contains a repressive E-box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c-MYC, c-MAF, and MAFG in cancer cells and human HCC/CCA. All 8-month-old liver-specific Phb1 knockout mice developed HCC, and one developed CCA. Five-month-old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. CONCLUSION: We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266).


Assuntos
Neoplasias dos Ductos Biliares/patologia , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/patologia , Neoplasias Hepáticas/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Neoplasias dos Ductos Biliares/metabolismo , Biópsia por Agulha , Western Blotting , Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Colangiocarcinoma/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Elementos E-Box/genética , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase/métodos , Proibitinas , RNA Mensageiro/análise , Distribuição Aleatória , Sensibilidade e Especificidade
10.
J Biol Chem ; 291(15): 8031-47, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-26826125

RESUMO

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares/patologia , Colangiocarcinoma/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Camundongos , Camundongos SCID , Fosfoproteínas/análise , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento de Fibroblastos/análise , Receptores de Fatores de Crescimento de Fibroblastos/genética , Fatores de Transcrição , Proteínas de Sinalização YAP
11.
Lab Invest ; 97(11): 1385-1396, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28892096

RESUMO

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 µm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP; increased IL-6, p21, SA-ß-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles (EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to generate normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.


Assuntos
Ductos Biliares/patologia , Colangite Esclerosante/patologia , Esferoides Celulares/patologia , Autoantígenos/metabolismo , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Ductos Biliares/ultraestrutura , Biomarcadores/metabolismo , Linhagem Celular , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Colangite Esclerosante/imunologia , Colangite Esclerosante/metabolismo , Técnicas de Cocultura , Meios de Cultivo Condicionados , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Vesículas Extracelulares/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Queratina-19/metabolismo , Queratina-7/metabolismo , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Corpos Multivesiculares/efeitos dos fármacos , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/patologia , Corpos Multivesiculares/ultraestrutura , Oxidantes/toxicidade , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/ultraestrutura
12.
Am J Physiol Gastrointest Liver Physiol ; 312(6): G666-G680, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28232454

RESUMO

The sequence of events that lead to inflammation and fibrosing nonalcoholic steatohepatitis (NASH) is incompletely understood. Hence, we investigated the chronology of whole body, tissue, and cellular events that occur during the evolution of diet-induced NASH. Male C57Bl/6 mice were assigned to a fast-food (FF; high calorie, high cholesterol, high fructose) or standard-chow (SC) diet over a period of 36 wk. Liver histology, body composition, mitochondrial respiration, metabolic rate, gene expression, and hepatic lipid content were analyzed. Insulin resistance [homeostasis model assessment-insulin resistance (HOMA-IR)] increased 10-fold after 4 wk. Fibrosing NASH was fully established by 16 wk. Total hepatic lipids increased by 4 wk and remained two- to threefold increased throughout. Hepatic triglycerides declined from sixfold increase at 8 wk to threefold increase by 36 wk. In contrast, hepatic cholesterol levels steadily increased from baseline at 8 wk to twofold by 36 wk. The hepatic immune cell population altered over time with macrophages persisting beyond 16 wk. Mitochondrial oxygen flux rates of FF mice diet were uniformly lower with all the tested substrates (13-276 pmol·s-1·ml-1 per unit citrate synthase) than SC mice (17-394 pmol·s-1·ml-1 per unit citrate synthase) and was accompanied by decreased mitochondrial:nuclear gene copy number ratios after 4 wk. Metabolic rate was lower in FF mice. Mitochondrial glutathione was significantly decreased at 24 wk in FF mice. Expression of dismutases and catalase was also decreased in FF mice. The evolution of NASH in the FF diet-induced model is multiphasic, particularly in terms of hepatic lipid composition. Insulin resistance precedes hepatic inflammation and fibrosis. Mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent. Collectively, these observations provide a unique overview of the sequence of changes that coevolve with the histological evolution of NASH.NEW & NOTEWORTHY This study demonstrates in a first of kind longitudinal analysis, the evolution of nonalcoholic steatohepatitis (NASH) on a fast-food diet-induced model. Key findings include 1) hepatic lipid composition changes in a multiphasic fashion as NASH evolves; 2) insulin resistance precedes hepatic inflammation and fibrosis, answering a longstanding chicken-and-egg question regarding the relationship of insulin resistance to liver histology in NASH; and 3) mitochondrial dysfunction and depletion occur after the histological features of NASH are apparent.


Assuntos
Cirrose Hepática/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Adiposidade , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Metabolismo Energético , Humanos , Mediadores da Inflamação/sangue , Insulina/sangue , Resistência à Insulina , Lipídeos/sangue , Fígado/fisiologia , Fígado/fisiopatologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Tamanho do Órgão , Especificidade da Espécie , Fatores de Tempo , Aumento de Peso
13.
Gastroenterology ; 150(4): 956-67, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26764184

RESUMO

BACKGROUND & AIMS: Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. METHODS: Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction. RESULTS: Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1ß and interleukin 6 messenger RNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis. CONCLUSIONS: Lipids, which stimulate DR5, induce release of hepatocyte EVs, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EVs by hepatocytes might be developed for the treatment of patients with NASH.


Assuntos
Vesículas Extracelulares/efeitos dos fármacos , Hepatite/metabolismo , Hepatócitos/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Fígado/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ácido Palmítico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Caspases/metabolismo , Linhagem Celular Tumoral , Vesículas Extracelulares/metabolismo , Células HEK293 , Hepatite/tratamento farmacológico , Hepatite/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Ratos Sprague-Dawley , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transfecção , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
14.
Hepatology ; 64(2): 439-55, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26969892

RESUMO

UNLABELLED: c-Myc induction drives cholestatic liver injury and cholangiocarcinoma (CCA) in mice, and induction of Maf proteins (MafG and c-Maf) contributes to cholestatic liver injury, whereas S-adenosylmethionine (SAMe) administration is protective. Here, we determined whether there is interplay between c-Myc, Maf proteins, and methionine adenosyltransferase α1 (MATα1), which is responsible for SAMe biosynthesis in the liver. We used bile duct ligation (BDL) and lithocholic acid (LCA) treatment in mice as chronic cholestasis models, a murine CCA model, human CCA cell lines KMCH and Huh-28, human liver cancer HepG2, and human CCA specimens to study gene and protein expression, protein-protein interactions, molecular mechanisms, and functional outcomes. We found that c-Myc, MATα1 (encoded by MAT1A), MafG, and c-Maf interact with one another directly. MAT1A expression fell in hepatocytes and bile duct epithelial cells during chronic cholestasis and in murine and human CCA. The opposite occurred with c-Myc, MafG, and c-Maf expression. MATα1 interacts mainly with Mnt in normal liver, but this switches to c-Maf, MafG, and c-Myc in cholestatic livers and CCA. Promoter regions of these genes have E-boxes that are bound by MATα1 and Mnt in normal liver and benign bile duct epithelial cells that switched to c-Myc, c-Maf, and MafG in cholestasis and CCA cells. E-box positively regulates c-Myc, MafG, and c-Maf, but it negatively regulates MAT1A. MATα1 represses, whereas c-Myc, MafG, and c-Maf enhance, E-box-driven promoter activity. Knocking down MAT1A or overexpressing MafG or c-Maf enhanced CCA growth and invasion in vivo. CONCLUSION: There is a novel interplay between MATα1, c-Myc, and Maf proteins, and their deregulation during chronic cholestasis may facilitate CCA oncogenesis. (Hepatology 2016;64:439-455).


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Metionina Adenosiltransferase/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , alfa-Amilases Salivares/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Metilação de DNA , Elementos E-Box , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Fator de Transcrição MafG/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Repressoras/metabolismo
15.
J Hepatol ; 65(2): 334-43, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27151182

RESUMO

BACKGROUND & AIMS: Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34(+) hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. METHODS: The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah(-/-) mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. RESULTS: Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah(-/-) mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. CONCLUSIONS: Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. LAY SUMMARY: Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.


Assuntos
Células Matadoras Naturais , Monócitos , Animais , Hepatite B , Hepatócitos , Humanos , Camundongos , Camundongos SCID
16.
Anal Biochem ; 494: 93-100, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26617128

RESUMO

In proteins of the neuronal calcium sensor (NCS) family, changes in structure as well as function are brought about by the binding of calcium. In this article, we demonstrate that these structural changes, solely due to calcium binding, can be assessed through electrophoresis in native gels. The results demonstrate that the NCS proteins undergo ligand-dependent conformational changes that are detectable in native gels as a gradual decrease in mobility with increasing calcium but not other tested divalent cations such as magnesium, strontium, and barium. Surprisingly, such a gradual change over the entire tested range is exhibited only by the NCS proteins but not by other tested calcium-binding proteins such as calmodulin and S100B, indicating that the change in mobility may be linked to a unique NCS family feature--the calcium-myristoyl switch. Even within the NCS family, the changes in mobility are characteristic of the protein, indicating that the technique is sensitive to the individual features of the protein. Thus, electrophoretic mobility on native gels provides a simple and elegant method to investigate calcium (small ligand)-induced structural changes at least in the superfamily of NCS proteins.


Assuntos
Cálcio/metabolismo , Eletroforese em Gel de Poliacrilamida Nativa , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Cálcio/química , Escherichia coli/metabolismo , Proteínas Sensoras de Cálcio Neuronal/química , Proteínas Sensoras de Cálcio Neuronal/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
17.
Protein Expr Purif ; 123: 35-41, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27001424

RESUMO

Hippocalcin is a 193 aa protein that is a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. Mice that lack the function of this protein are compromised in the long term potentiation aspect of memory generation. Recently, mutations in the gene have been linked with dystonia in human. The protein has no intrinsic enzyme activity but is known to bind to variety of target proteins. Very little information is available on how the protein executes its critical role in signaling pathways, except that it is regulated by binding of calcium. Further delineation of its function requires large amounts of pure protein. In this report, we present a single-step purification procedure that yields high quantities of the bacterially expressed, recombinant protein. The procedure may be adapted to purify the protein from inclusion bodies or cytosol in its myristoylated or non-myristoylated forms. MALDI-MS (in source decay) analyses demonstrates that the myristoylation occurs at the glycine residue. The protein is also biologically active as measured through tryptophan fluorescence, mobility shift and guanylate cyclase activity assays. Thus, further analyses of hippocalcin, both structural and functional, need no longer be limited by protein availability.


Assuntos
Escherichia coli/genética , Hipocalcina/genética , Hipocalcina/isolamento & purificação , Animais , Cromatografia Líquida , Expressão Gênica , Vetores Genéticos/genética , Hipocalcina/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
J Hepatol ; 62(5): 1156-63, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25445398

RESUMO

BACKGROUND & AIMS: Low-grade chronic inflammation is a cardinal feature of the metabolic syndrome, yet its pathogenesis is not well defined. The purpose of this study was to examine the role of TRAIL receptor (TR) signaling in the pathogenesis of obesity-associated inflammation using mice with the genetic deletion of TR. METHODS: TR knockout (TR(-/-)) mice and their littermate wild-type (WT) mice were fed a diet high in saturated fat, cholesterol and fructose (FFC) or chow. Metabolic phenotyping, liver injury, and liver and adipose tissue inflammation were assessed. Chemotaxis and activation of mouse bone marrow-derived macrophages (BMDMϕ) was measured. RESULTS: Genetic deletion of TR completely repressed weight gain, adiposity and insulin resistance in FFC-fed mice. Moreover, TR(-/-) mice suppressed steatohepatitis, with essentially normal serum ALT, hepatocyte apoptosis and liver triglyceride accumulation. Gene array data implicated inhibition of macrophage-associated hepatic inflammation in the absence of the TR. In keeping with this, there was diminished accumulation and activation of inflammatory macrophages in liver and adipose tissue. TR(-/-) BMDMϕ manifest reduced chemotaxis and diminished activation of nuclear factor-κ B signaling upon activation by palmitate and lipopolysaccharide. CONCLUSIONS: These data advance the concept that macrophage-associated hepatic and adipose tissue inflammation of nutrient excess requires TR signaling.


Assuntos
Tecido Adiposo , Inflamação , Fígado , Macrófagos , Obesidade , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Quimiotaxia , Dieta Hiperlipídica/métodos , Modelos Animais de Doenças , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Fígado/metabolismo , Fígado/patologia , Ativação de Macrófagos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Obesidade/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais
19.
Artigo em Inglês | MEDLINE | ID: mdl-38908410

RESUMO

PURPOSE: To study the late urinary toxicity in patients with prostate cancer with prior transurethral resection of prostate (TURP) and treated with hypofractionated prostate radiotherapy. METHODS: Patients diagnosed with prostate cancer, with a prior TURP, and treated with moderate or extreme hypofractionated intensity modulated radiotherapy (MHRT or SBRT), were included in this study. Severity and duration of urinary symptoms observed during serial follow up after at least three months from radiotherapy were graded per CTCAE v5.0 using information from prospectively maintained institutional database. Impact of hypofractionation and other potential contributory factors on cumulative grade 2+ late urinary toxicity was analysed with univariable and multivariable binary logistic regression. RESULTS: Total 203 eligible patients were included (MHRT=114, 64-68Gy/25#; SBRT=89, 35-37.5Gy/5#). Median time from TURP to radiotherapy was 10 months (IQR 7-16), similar for MHRT and SBRT. Overall, mean cavity volume was 1.17cc (IQR 0.5-1.35), while in MHRT and SBRT groups was 1.03 cc (IQR 0.4-1.15) and 1.27 cc (IQR 0.5-1.4), respectively. At a median follow up of 37 months, cumulative grade 3 and grade 2 late urinary toxicity was 8.4% (n=17) and 23.2% (n=47) respectively. Grade 3 symptoms were observed at median 29 months (IQR 19-62) after radiotherapy completion, lasting for a median duration of 8 months (IQR 2-14). Hematuria (6.4%) and urinary obstruction (3.4%) were the chief grade 3 symptoms. Multivariable analysis for age, diabetes, pelvic radiotherapy, fraction size, prostate volume, TURP to radiotherapy duration, and TURP cavity volume showed no significant association with late grade 2+ urinary toxicity. CONCLUSION: In this large cohort of patients with prior TURP and treated with hypofractionated prostate radiotherapy, incidence of severe late urinary adverse effects was <10%, mainly hematuria or urinary obstruction. Most of these were temporary, and no significant contributory factors were identified for late urinary morbidity after TURP and radiotherapy.

20.
Sci Rep ; 14(1): 2145, 2024 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-38273071

RESUMO

Ductular reactive (DR) cells exacerbate cholestatic liver injury and fibrosis. Herein, we posit that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) emanates from recruited macrophages and restrains DR cell expansion, thereby limiting cholestatic liver injury. Wild type (WT), Trailfl/fl and myeloid-specific Trail deleted (TrailΔmye) C57BL/6 mice were exposed to DDC diet-induced cholestatic liver injury, which induced hepatomegaly and liver injury as compared to control diet-fed mice. However, parameters of liver injury, fibrosis, and inflammation were all increased in the TrailΔmye mice as compared to the WT and Trailfl/fl mice. High dimensional mass cytometry indicated that cholestasis resulted in increased hepatic recruitment of subsets of macrophages and neutrophils in the TrailΔmye mice. Spatial transcriptomics analysis revealed that the PanCK+ cholangiocytes from TrailΔmye mice had increased expression of the known myeloid attractants S100a8, Cxcl5, Cx3cl1, and Cxcl1. Additionally, in situ hybridization of Cxcl1, a potent neutrophil chemoattractant, demonstrated an increased expression in CK19+ cholangiocytes of TrailΔmye mice. Collectively, these data suggest that TRAIL from myeloid cells, particularly macrophages, restrains a subset of DR cells (i.e., Cxcl1 positive cells), limiting liver inflammation and fibrosis. Reprogramming macrophages to express TRAIL may be salutary in cholestasis.


Assuntos
Colestase , Fígado , Animais , Camundongos , Apoptose/genética , Colestase/metabolismo , Fibrose , Ligantes , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismo
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