RESUMO
BACKGROUND AND AIMS: Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary sclerosing cholangitis (PSC) typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile-duct-ligated (BDL) MC-deficient mice (B6.Cg-KitW-sh /HNihrJaeBsmJ [KitW-sh ]), ductular reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection exacerbates liver damage in normal mice. APPROACH AND RESULTS: In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared with control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and histamine in KitW-sh mice. Inhibition of MC-FXR before injection reduced these parameters. BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15, and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following FXR inhibition. BDL and multidrug resistance 2/ATP-binding cassette family 2 member 4 knockout (Mdr2-/- ) mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2-/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation. CONCLUSIONS: Our studies demonstrate that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling.
Assuntos
Colangite Esclerosante/complicações , Colestase/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mastócitos/imunologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ductos Biliares/imunologia , Ductos Biliares/patologia , Colangite Esclerosante/imunologia , Colangite Esclerosante/patologia , Colestase/patologia , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Mastócitos/metabolismo , CamundongosRESUMO
BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. APPROACH AND RESULTS: Wild-type (WT) and KitW-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and KitW-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in KitW-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in KitW-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in KitW-sh WD mice and was found to target ALDH1A3. CONCLUSIONS: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.
Assuntos
Sistema Biliar/patologia , Dieta Ocidental/efeitos adversos , Cirrose Hepática/imunologia , Mastócitos/imunologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aldeído Oxirredutases/genética , Animais , Sistema Biliar/imunologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/imunologia , Hepatócitos/patologia , Humanos , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Mastócitos/metabolismo , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Adulto JovemRESUMO
BACKGROUND AND AIMS: Following liver injury, mast cells (MCs) migrate into the liver and are activated in patients with cholestasis. Inhibition of MC mediators decreases ductular reaction (DR) and liver fibrosis. Transforming growth factor beta 1 (TGF-ß1) contributes to fibrosis and promotes liver disease. Our aim was to demonstrate that reintroduction of MCs induces cholestatic injury through TGF-ß1. APPROACH AND RESULTS: Wild-type, KitW-sh (MC-deficient), and multidrug resistance transporter 2/ABC transporter B family member 2 knockout mice lacking l-histidine decarboxylase were injected with vehicle or PKH26-tagged murine MCs pretreated with 0.01% dimethyl sulfoxide (DMSO) or the TGF-ß1 receptor inhibitor (TGF-ßRi), LY2109761 (10 µM) 3 days before sacrifice. Hepatic damage was assessed by hematoxylin and eosin (H&E) and serum chemistry. Injected MCs were detected in liver, spleen, and lung by immunofluorescence (IF). DR was measured by cytokeratin 19 (CK-19) immunohistochemistry and F4/80 staining coupled with real-time quantitative PCR (qPCR) for interleukin (IL)-1ß, IL-33, and F4/80; biliary senescence was evaluated by IF or qPCR for p16, p18, and p21. Fibrosis was evaluated by sirius red/fast green staining and IF for synaptophysin 9 (SYP-9), desmin, and alpha smooth muscle actin (α-SMA). TGF-ß1 secretion/expression was measured by enzyme immunoassay and qPCR. Angiogenesis was detected by IF for von Willebrand factor and vascular endothelial growth factor C qPCR. In vitro, MC-TGF-ß1 expression/secretion were measured after TGF-ßRi treatment; conditioned medium was collected. Cholangiocytes and hepatic stellate cells (HSCs) were treated with MC-conditioned medium, and biliary proliferation/senescence was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and qPCR; HSC activation evaluated for α-SMA, SYP-9, and collagen type-1a expression. MC injection recapitulates cholestatic liver injury characterized by increased DR, fibrosis/TGF-ß1 secretion, and angiogenesis. Injection of MC-TGF-ßRi reversed these parameters. In vitro, MCs induce biliary proliferation/senescence and HSC activation that was reversed with MCs lacking TGF-ß1. CONCLUSIONS: Our study demonstrates that reintroduction of MCs mimics cholestatic liver injury and that MC-derived TGF-ß1 may be a target in chronic cholestatic liver disease.
Assuntos
Actinas/metabolismo , Colestase Intra-Hepática/metabolismo , Cirrose Hepática , Fígado/patologia , Mastócitos , Fator de Crescimento Transformador beta1 , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Ensaios de Migração Celular , Proliferação de Células , Senescência Celular , Descoberta de Drogas , Células Estreladas do Fígado , Histamina/sangue , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo , Regulação para CimaRESUMO
Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor (H2HR), which is increased in patients with primary sclerosing cholangitis (PSC). Ranitidine decreases liver damage in Mdr2-/- (ATP binding cassette subfamily B member 4 null) mice. We targeted hepatic H2HR in Mdr2-/- mice using vivo-morpholino. Wild-type and Mdr2-/- mice were treated with mismatch or H2HR vivo-morpholino by tail vein injection for 1 week. Liver damage, mast cell (MC) activation, biliary H2HR, and histamine serum levels were studied. MC markers were determined by quantitative real-time PCR for chymase and c-kit. Intrahepatic biliary mass was detected by cytokeratin-19 and F4/80 to evaluate inflammation. Biliary senescence was determined by immunofluorescence and senescence-associated ß-galactosidase staining. Hepatic fibrosis was evaluated by staining for desmin, Sirius Red/Fast Green, and vimentin. Immunofluorescence for transforming growth factor-ß1, vascular endothelial growth factor-A/C, and cAMP/ERK expression was performed. Transforming growth factor-ß1 and vascular endothelial growth factor-A secretion was measured in serum and/or cholangiocyte supernatant. Treatment with H2HR vivo-morpholino in Mdr2-/--mice decreased hepatic damage; H2HR protein expression and MC presence or activation; large intrahepatic bile duct mass, inflammation and senescence; and fibrosis, angiogenesis, and cAMP/phospho-ERK expression. Inhibition of H2HR signaling ameliorates large ductal PSC-induced damage. The H2HR axis may be targeted in treating PSC.
Assuntos
Ductos Biliares/metabolismo , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Receptores Histamínicos H2/metabolismo , Animais , Ductos Biliares/patologia , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Morfolinos/farmacologia , Receptores Histamínicos H2/genéticaRESUMO
Primary sclerosing cholangitis (PSC) is characterized by biliary damage and fibrosis. Multidrug resistance-2 gene knockout (Mdr2-/-) mice and PSC patients have increased histamine (HA) levels (synthesized by l-histidine decarboxylase, HDC) and HA receptor (HR) expression. Cholestatic HDC-/- mice display ameliorated biliary damage and hepatic fibrosis. The current study evaluated the effects of knockout of HDC-/- in Mdr2-/- mice (DKO) on biliary damage and hepatic fibrosis. WT, Mdr2-/- mice, and homozygous DKO mice were used. Selected DKO mice were treated with HA. We evaluated liver damage along with HDC expression and HA serum levels. Changes in ductular reaction were evaluated along with liver fibrosis, inflammation and bile acid signaling pathways. The expression of H1HR/PKC-α/TGF-ß1 and H2HR/pERK/VEGF-C was determined. In vitro, cholangiocyte lines were treated with HA with/without H1/H2 inhibitors before measuring: H1/H2HR, TGF-ß1, and VEGF-C expression. Knockout of HDC ameliorates hepatic damage, ductular reaction, fibrosis, inflammation, bile acid signaling and H1HR/PKC-α/TGF-ß1 and H2HR/pERK/VEGF-C signaling. Reactivation of the HDC/HA axis increased these parameters. In vitro, stimulation with HA increased HR expression and PKC-α, TGF-ß1, and VEGF-C expression, which was reduced with HR inhibitors. Our data demonstrate the key role for the HDC/HA axis in the management of PSC progression.
Assuntos
Colangite Esclerosante , Histamina/metabolismo , Histidina Descarboxilase , Cirrose Hepática , Transdução de Sinais/genética , Animais , Colangite Esclerosante/enzimologia , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/enzimologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Feeding a high-fat diet (HFD) coupled with sugar, mimicking a Western diet, causes fatty liver disease in mice. Histamine induces biliary proliferation and fibrosis and regulates leptin signaling. Wild-type (WT) and l-histidine decarboxylase (Hdc-/-) mice were fed a control diet or an HFD coupled with a high fructose corn syrup equivalent. Hematoxylin and eosin and Oil Red O staining were performed to determine steatosis. Biliary mass and cholangiocyte proliferation were evaluated by immunohistochemistry. Senescence and fibrosis were measured by quantitative PCR and immunohistochemistry. Hepatic stellate cell activation was detected by immunofluorescence. Histamine and leptin levels were measured by enzyme immunoassay. Leptin receptor (Ob-R) was evaluated by quantitative PCR. The HDC/histamine/histamine receptor axis, ductular reaction, and biliary senescence were evaluated in patients with nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, or end-stage liver disease. Hdc-/- HFD mice had increased steatosis compared with WT HFD mice. WT HFD mice had increased biliary mass, biliary proliferation, senescence, fibrosis, and hepatic stellate cell activation, which were reduced in Hdc-/- HFD mice. In Hdc-/- HFD mice, serum leptin levels increased, whereas biliary Ob-R expression decreased. Nonalcoholic steatohepatitis patients had increased HDC/histamine/histamine receptor signaling. Hdc-/- HFD mice are susceptible to obesity via dysregulated leptin/Ob-R signaling, whereas the lack of HDC protects from HFD-induced fibrosis and cholangiocyte damage. HDC/histamine/leptin signaling may be important in managing obesity-induced biliary damage.
Assuntos
Dieta Hiperlipídica , Histamina/metabolismo , Histidina Descarboxilase/metabolismo , Leptina/metabolismo , Cirrose Hepática/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Adulto , Idoso , Animais , Feminino , Histidina Descarboxilase/genética , Humanos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais/fisiologiaRESUMO
Primary sclerosing cholangitis (PSC) patients are at risk of developing cholangiocarcinoma (CCA). We have shown that (1) histamine increases biliary hyperplasia through H1/H2 histamine receptors (HRs) and (2) histamine levels increase and mast cells (MCs) infiltrate during PSC and CCA. We examined the effects of chronic treatment with H1/H2HR antagonists on PSC and CCA. Wild-type and multidrug-resistant knockout (Mdr2-/- ) mice were treated by osmotic minipumps with saline, mepyramine, or ranitidine (10 mg/kg body weight/day) or a combination of mepyramine/ranitidine for 4 weeks. Liver damage was assessed by hematoxylin and eosin. We evaluated (1) H1/H2HR expression, (2) MC presence, (3) L-histidine decarboxylase/histamine axis, (4) cholangiocyte proliferation/bile duct mass, and (5) fibrosis/hepatic stellate cell activation. Nu/nu mice were implanted with Mz-ChA-1 cells into the hind flanks and treated with saline, mepyramine, or ranitidine. Tumor growth was measured, and (1) H1/H2HR expression, (2) proliferation, (3) MC activation, (4) angiogenesis, and (5) epithelial-mesenchymal transition (EMT) were evaluated. In vitro, human hepatic stellate cells were evaluated for H1HR and H2HR expression. Cultured cholangiocytes and CCA lines were treated with saline, mepyramine, or ranitidine (25 µM) before evaluating proliferation, angiogenesis, EMT, and potential signaling mechanisms. H1/H2HR and MC presence increased in human PSC and CCA. In H1/H2HR antagonist (alone or in combination)-treated Mdr2-/- mice, liver and biliary damage and fibrosis decreased compared to saline treatment. H1/H2HR antagonists decreased tumor growth, serum histamine, angiogenesis, and EMT. In vitro, H1/H2HR blockers reduced biliary proliferation, and CCA cells had decreased proliferation, angiogenesis, EMT, and migration. Conclusion: Inhibition of H1/H2HR reverses PSC-associated damage and decreases CCA growth, angiogenesis, and EMT; because PSC patients are at risk of developing CCA, using HR blockers may be therapeutic for these diseases. (Hepatology 2018).
Assuntos
Colangiocarcinoma/prevenção & controle , Colangite Esclerosante/tratamento farmacológico , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Antagonistas dos Receptores H2 da Histamina/uso terapêutico , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Colangiocarcinoma/etiologia , Colangite Esclerosante/complicações , Avaliação Pré-Clínica de Medicamentos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H1/farmacologia , Antagonistas dos Receptores H2 da Histamina/farmacologia , Humanos , Fígado/efeitos dos fármacos , Masculino , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Neovascularização Patológica/prevenção & controle , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. MCs infiltrate Mdr2-/- mice liver (model of primary sclerosing cholangitis (PSC)). MC-derived histamine increases inflammation, hepatic stellate cell (HSC) activation and fibrosis. The objective was to determine the effects of UDCA treatment on MC infiltration, biliary damage, inflammation and fibrosis in Mdr2-/- mice and human PSC. Wild-type and Mdr2-/- mice were fed bile acid control diet or UDCA (0.5% wt/wt). Human samples were collected from control and PSC patients treated with placebo or UDCA (15 mg/kg/BW). MC infiltration was measured by immunhistochemistry and quantitative polymerase chain reaction (qPCR) for c-Kit, chymase, and tryptase. The HDC/histamine/histamine receptor (HR)-axis was evaluated by EIA and qPCR. Intrahepatic bile duct mass (IBDM) and biliary proliferation was evaluated by CK-19 and Ki-67 staining. Fibrosis was detected by immunostaining and qPCR for fibrotic markers. Inflammatory components were measured by qPCR. HSC activation was measured by SYP-9 staining. Inflammation was detected by qPCR for CD68. In vitro, MCs were treated with UDCA (40 µM) prior to HA secretion evaluation and coculturing with cholangiocytes or HSCs. BrDU incorporation and fibrosis by qPCR was performed. UDCA reduced MC number, the HDC/histamine/HR-axis, IBDM, HSC activation, inflammation, and fibrosis in Mdr2-/- mice and PSC patients. In vitro, UDCA decreases MC-histamine release, which was restored by blocking ASBT and FXRß. Proliferation and fibrosis decreased after treatment with UDCA-treated MCs. We conclude that UDCA acts on MCs reducing histamine levels and decreases the inflammatory/hyperplastic/fibrotic reaction seen in PSC. Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. Following liver injury like primary sclerosing cholangitis in mice and humans, MCs infiltrate. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.
Assuntos
Colagogos e Coleréticos/farmacologia , Colangite Esclerosante/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Ácido Ursodesoxicólico/farmacologia , Animais , Estudos de Casos e Controles , Colagogos e Coleréticos/uso terapêutico , Células Estreladas do Fígado/efeitos dos fármacos , Histamina/metabolismo , Humanos , Hepatopatias/prevenção & controle , Camundongos Knockout , Ácido Ursodesoxicólico/uso terapêuticoRESUMO
Activated mast cells (MCs) release histamine (HA) and MCs infiltrate the liver following bile duct ligation (BDL), increasing intrahepatic bile duct mass (IBDM) and fibrosis. We evaluated the effects of BDL in MC-deficient (KitW-sh ) mice. Wild-type (WT) and KitW-sh mice were subjected to sham or BDL for up to 7 days and KitW-sh mice were injected with cultured mast cells or 1× phosphate-buffered saline (PBS) before collecting serum, liver, and cholangiocytes. Liver damage was assessed by hematoxylin and eosin and alanine aminotransferase levels. IBDM was detected by cytokeratin-19 expression and proliferation by Ki-67 immunohistochemistry (IHC). Fibrosis was detected by IHC, hydroxyproline content, and by qPCR for fibrotic markers. Hepatic stellate cell (HSC) activation and transforming growth factor-beta 1 (TGF-ß1) expression/secretion were evaluated. Histidine decarboxylase (HDC) and histamine receptor (HR) expression were detected by qPCR and HA secretion by enzymatic immunoassay. To evaluate vascular cells, von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-C expression were measured. In vitro, cultured HSCs were stimulated with cholangiocyte supernatants and alpha-smooth muscle actin levels were measured. BDL-induced liver damage was reduced in BDL KitW-sh mice, whereas injection of MCs did not mimic BDL-induced damage. In BDL KitW-sh mice, IBDM, proliferation, HSC activation/fibrosis, and TGF-ß1 expression/secretion were decreased. The HDC/HA/HR axis was ablated in sham and BDL KitW-sh mice. vWF and VEGF-C expression decreased in BDL KitW-sh mice. In KitW-sh mice injected with MCs, IBDM, proliferation, fibrosis, and vascular cell activation increased. Stimulation with cholangiocyte supernatants from BDL WT or KitW-sh mice injected with MCs increased HSC activation, which decreased with supernatants from BDL KitW-sh mice. CONCLUSION: MCs promote hyperplasia, fibrosis, and vascular cell activation. Knockout of MCs decreases BDL-induced damage. Modulation of MCs may be important in developing therapeutics for cholangiopathies. (Hepatology 2017;65:1991-2004).
Assuntos
Doenças Biliares/patologia , Cirrose Hepática/patologia , Fígado/lesões , Mastócitos/transplante , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Ductos Biliares Intra-Hepáticos/cirurgia , Doenças Biliares/fisiopatologia , Biópsia por Agulha , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hiperplasia/patologia , Imuno-Histoquímica , Ligadura/métodos , Fígado/patologia , Masculino , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Valores de ReferênciaRESUMO
Nonalcoholic fatty liver disease (NAFLD) is a disease of increasing interest, as its prevalence is on the rise. NAFLD has been linked to metabolic syndrome, which is becoming more common due to the Western diet. Because NAFLD can lead to cirrhosis and related complications including hepatocellular carcinoma, the increasing prevalence is concerning, and medical therapy aimed at treating NAFLD is of great interest. Researchers studying the effects of medical therapy on NAFLD use dietary mouse models. The two main types of mouse model diets are the methionine- and choline-deficient (MCD) diet and the Western-like diet (WD). Although both induce NAFLD, the mechanisms are very different. We reviewed several studies conducted within the last 5 years that used MCD diet or WD mouse models in order to mimic this disease in a way most similar to humans. The MCD diet inconsistently induces NAFLD and fibrosis and does not completely induce metabolic syndrome. Thus, the clinical significance of the MCD diet is questionable. In contrast, WD mouse models consisting of high fat, cholesterol, and a combination of high-fructose corn syrup, sucrose, fructose, or glucose not only lead to metabolic syndrome but also induce NAFLD with fibrosis, making these choices most suitable for research.
Assuntos
Deficiência de Colina/metabolismo , Dieta Ocidental/efeitos adversos , Modelos Animais de Doenças , Metionina/deficiência , Hepatopatia Gordurosa não Alcoólica/etiologia , Animais , Deficiência de Colina/complicações , Deficiência de Colina/patologia , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
The depth of our knowledge regarding mast cells has widened exponentially in the last 20 years. Once thought to be only important for allergy-mediated events, mast cells are now recognized to be important regulators of a number of pathological processes. The revelation that mast cells can influence organs, tissues, and cells has increased interest in mast cell research during liver disease. The purpose of this review is to refresh the reader's knowledge of the development, type, and location of mast cells and to review recent work that demonstrates the role of hepatic mast cells during diseased states. This review focuses primarily on liver diseases and mast cells during autoimmune disease, hepatitis, fatty liver disease, liver cancer, and aging in the liver. Overall, these studies demonstrate the potential role of mast cells in disease progression.
Assuntos
Doenças Autoimunes/patologia , Hepatócitos/patologia , Hepatopatias/patologia , Fígado/patologia , Mastócitos/patologia , Animais , Doenças Autoimunes/imunologia , Hepatócitos/imunologia , Humanos , Fígado/imunologia , Hepatopatias/imunologiaRESUMO
UNLABELLED: Hepatic fibrosis is marked by activation of hepatic stellate cells (HSCs). Cholestatic injury precedes liver fibrosis, and cholangiocytes interact with HSCs promoting fibrosis. Mast cells (MCs) infiltrate following liver injury and release histamine, increasing biliary proliferation. We evaluated if inhibition of MC-derived histamine decreases biliary proliferation and fibrosis. Wild-type and multidrug resistance 2 knockout mice (9-11 weeks) were treated with cromolyn sodium for 1 week to block MC-derived histamine. Biliary mass and proliferation were evaluated by immunohistochemistry for cytokeratin 19 and Ki-67. Bile flow, bicarbonate excretion, and total bile acids were measured in all mice. Fibrosis was evaluated by sirius red/fast green staining and by quantitative polymerase chain reaction for alpha-smooth muscle actin, fibronectin, collagen type 1a, and transforming growth factor-beta 1. HSC activation was evaluated by quantitative polymerase chain reaction in total liver and immunofluorescent staining in tissues for synaptophysin 9. Histamine serum secretion was measured by enzymatic immunoassay. Mouse liver and human liver samples from control or primary sclerosing cholangitis patients were evaluated for MC markers by quantitative polymerase chain reaction and immunohistochemistry. In vitro, cultured MCs were transfected with histidine decarboxylase short hairpin RNA to decrease histamine secretion and subsequently cocultured with cholangiocytes or HSCs prior to measuring fibrosis markers, proliferation, and transforming growth factor-beta 1 secretion. Treatment with cromolyn sodium decreased biliary proliferation, fibrosis, histamine secretion, and bile flow in multidrug resistance 2 knockout mice. Primary sclerosing cholangitis mice and patients have increased MCs. Knockdown of MC histidine decarboxylase decreased cholangiocyte and HSC proliferation/activation. CONCLUSION: MCs are recruited to proliferating cholangiocytes and promote fibrosis. Inhibition of MC-derived histamine decreases fibrosis, and regulation of MC mediators may be therapeutic for primary sclerosing cholangitis. (Hepatology 2016;64:1202-1216).
Assuntos
Colangite Esclerosante/patologia , Liberação de Histamina , Mastócitos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Animais , Sistema Biliar/patologia , Proliferação de Células , Masculino , Camundongos , Camundongos Knockout , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decreases biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 h and up to 14 days. MC infiltration was evaluated by toluidine blue. BDL rats were perfused using standard collagenase perfusion. Following enzymatic digestion, tissue was passed through a fine gauge needle. Suspensions were incubated with MAb AA4, washed and incubated with goat anti-mouse-coated Dynal beads. MCs were stained with toluidine blue, and in isolated MCs the expression of FCÉRI and MC proteases was measured. The expression of histidine decarboxylase, histamine receptors, VEGF receptors, and TIE 1 and 2 was evaluated by qPCR. Histamine and VEGF-A secretion was measured in MC supernatants. MC purity was evaluated by CK-19, CK-8, albumin, VAP-1, and α-SMA expression. In vitro, cholangiocytes and HSCs were treated with isolated MC supernatants from BDL rats treated with either NaCl or cromolyn sodium (to block MC histamine release) and biliary proliferation and hepatic fibrosis were measured. MCs infiltrate the liver and surround bile ducts starting at day 2. We isolated a virtually pure preparation of mature, functional MCs. TEM images reveal distinct secretory granules and isolated MCs secrete histamine. MCs express FCÉRI, chymase, tryptase, RMCP-I, and RMCP-II, but were virtually void of other cell markers. Biliary proliferation and fibrosis increased following treatment with MC supernatants from BDL rats+NaCl and these parameters decreased in cells treated with MC supernatants from BDL+cromolyn sodium. In conclusion, we have isolated and characterized MCs from cholestatic livers. MCs regulate cholestatic liver injury and hepatic fibrosis. This tool provides a better understanding of the paracrine influence of mast cells on biliary/liver pathologies.
Assuntos
Separação Celular/métodos , Colestase/imunologia , Mastócitos/fisiologia , Animais , Fígado/citologia , Fígado/imunologia , Masculino , Ratos Endogâmicos F344RESUMO
OBJECTIVE: Although significant research has detailed angiogenesis during development and cancer, little is known about cardiac angiogenesis, yet it is critical for survival following pathological insult. The transcription factor c-Myc is a target of anticancer therapies because of its mitogenic and proangiogenic induction. In the current study, we investigate its role in cardiac angiogenesis in a cell-dependent and gene-specific context. METHODS AND RESULTS: Angiogenesis assays using c-Myc-deficient cardiac endothelial cells and fibroblasts demonstrate that c-Myc is essential to vessel formation, and fibroblast-mediated vessel formation is dependent on c-Myc expression in fibroblasts. Gene analyses revealed that c-Myc-mediated gene expression is unique in cardiac angiogenesis and varies in a cell-dependent manner. In vitro 3-dimensional cultures demonstrated c-Myc's role in the expression of secreted angiogenic factors, while also providing evidence for c-Myc-mediated cell-cell interactions. Additional in vivo vascular analyses support c-Myc's critical role in capillary formation and vessel patterning during development and also in response to a pathological stimulus where its expression in myocytes is required for angiogenic remodeling. CONCLUSIONS: These data demonstrate that proper c-Myc expression in cardiac fibroblasts and myocytes is essential to cardiac angiogenesis. These results have the potential for novel therapeutic applications involving the angiogenic response during cardiac remodeling.
Assuntos
Vasos Coronários/citologia , Neovascularização Fisiológica/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA/genética , Transdução de Sinais , Animais , Comunicação Celular , Células Cultivadas , Vasos Coronários/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis. METHODS: FVB/NJ and Mdr2-/- mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber. RESULTS: Biliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2-/- mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2-/- mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation. CONCLUSION: Cholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Colangite Esclerosante/terapia , Cirrose Hepática/terapia , Mastócitos/patologia , Morfolinos/uso terapêutico , Fator de Células-Tronco/genética , Animais , Sistema Biliar/citologia , Sistema Biliar/metabolismo , Sistema Biliar/patologia , Movimento Celular , Senescência Celular , Colangite Esclerosante/genética , Colangite Esclerosante/patologia , Regulação para Baixo , Feminino , Deleção de Genes , Humanos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Morfolinos/genética , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The functions of the liver are very diverse. From detoxifying blood to storing glucose in the form of glycogen and producing bile to facilitate fat digestion, the liver is a very active and important organ. The liver is comprised of many varied cell types whose functions are equally diverse. Cholangiocytes line the biliary tree and aid in transporting and adjusting the composition of bile as it travels to the gallbladder. Hepatic stellate cells and portal fibroblasts are located in different areas within the liver architecture, but both contribute to the development of fibrosis upon activation after liver injury. Vascular cells, including those that constitute the peribiliary vascular plexus, are involved in functions other than blood delivery to and from the liver, such as supporting the growth of the biliary tree during development. Mast cells are normally found in healthy livers but in very low numbers. However, after injury, mast cell numbers greatly increase as they infiltrate and release factors that exacerbate the fibrotic response. While not an all-inclusive list, these cells have individual roles within the liver, but they are also able to communicate with each other by cellular crosstalk. In this review, we examine some of these pathways that can lead to an increase in the homeostatic dysfunction seen in liver injury.
RESUMO
Cholangiocarcinoma (CCA) is an aggressive malignancy that arises from damaged epithelial cells, cholangiocytes, and possibly de-differentiated hepatocytes. CCA has a poor overall survival rate and limited therapeutic options. Based on this data, it is imperative that new diagnostic and therapeutic interventions be developed. Recent work has attempted to understand the pathological mechanisms driving CCA progression. Specifically, recent publications have delved into the role of cancer stem cells (CSCs), mesenchymal stem cells (MSCs), and microRNAs (miRNAs) during CCA pathology. CSCs are a specific subset of cells within the tumor environment that are derived from a cell with stem-like properties and have been shown to influence recurrence and chemoresistance during CCA. MSCs are known for their anti-inflammatory activity and have been postulated to influence malignancy during CCA, but little is known about their exact functions. miRNAs exert various functions via gene regulation at both the transcriptional and the translational levels, giving miRNAs diverse roles in CCA progression. Additionally, current miRNA-based therapeutic approaches are in clinical trials for various liver diseases, giving hope for similar approaches for CCA. However, the interactions among these three factors in the context of CCA are unknown. In this review, we focus on recently published data (within the last 3 years) that discuss the role of CSCs, MSCs, and miRNAs and their possible interactions during CCA pathogenesis.