RESUMEN
Myocardial infarction (MI) is characterized by a significant loss of cardiomyocytes (CMs), and it is suggested that reactive oxygen species (ROS) are involved in cell cycle arrest, leading to impaired CM renewal. Thioredoxin-1 (Trx-1) scavenges ROS and may play a role in restoring CM renewal. However, the truncated form of Trx-1, Trx-80, can compromise its efficacy by exerting antagonistic effects. Therefore, a Trx-1 mimetic peptide called CB3 was tested as an alternative way to restore CMs. This study aimed to investigate the effects of Trx-1, Trx-80, and CB3 on mice with experimental MI and study the underlying mechanism of CB3 on CMs. Mouse cardiac parameters were quantified by echocardiography, and infarction size and fibrosis determined using Trichrome and Picro-Sirius Red staining. The study found that Trx-1 and CB3 improved mouse cardiac function, reduced the size of cardiac infarct and fibrosis, and decreased the expression of cardiac inflammatory markers. Furthermore, CB3 polarized macrophages into M2 phenotype, reduced apoptosis and oxidative stress after MI, and increased CM proliferation in cell culture and in vivo. CB3 effectively protected against myocardial infarction and could represent a new class of compounds for treating MI.
Asunto(s)
Infarto del Miocardio , Tiorredoxinas , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/metabolismo , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Péptidos/farmacología , Péptidos/uso terapéutico , Péptidos/metabolismo , Apoptosis , Fibrosis , Remodelación Ventricular , Miocardio/metabolismo , Modelos Animales de EnfermedadRESUMEN
ETHNOPHARMACOLOGICAL EVIDENCE: Thymus atlanticus (TA) is used in traditional medicine in Morocco to treat chronic inflammatory diseases, after local and oral treatment. AIM OF STUDY: This study aimed to investigate the in vitro and in vivo anti-inflammatory and anticoagulant activities of an aqueous extract (AE) and polyphenol fraction (PF) derived from TA. MATERIALS AND METHODS: The effect of AE and PF on monocyte chemoattractant protein-1 (MCP-1) production by naïve and LPS-stimulated peritoneal macrophages isolated from C57Bl/6 mice was assessed by ELISA assay. The effect of chronic administration of the extracts at three different doses by oral rout for 2 weeks on blood coagulation and inflammation induced by carrageenan in Wistar rats was evaluated. In addition, the in vitro anticoagulant effect was tested on blood plasma collected from healthy rats using the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests. The acute toxicity of AE was investigated. Phytochemical analysis was carried out by HPLC. RESULTS: Analysis by HPLC indicated rosmarinic acid as the main phenolic acid in TA extracts. Compared to control macrophages, MCP-1 level was lower in medium supplemented with AE at 50 and 500 µg/mL and PF at 500 µg/mL, but higher in medium with PF at 50 µg/mL. Rosmarinic and chicoric acids, served as controls, significantly decreased MCP-1 production. Chronic oral administration of TA extracts prevented inflammation induced by carrageenan and induced a significant prolongation of blood coagulation time, in a dose dependant manner, in Wistar rats. The results of the in vitro assay showed that the coagulation time was significantly prolonged in plasma incubated with extracts in APTT, PT and TT tests. Lethal dose 50 of AE in mice was 27.90 ± 1.19 g/kg. CONCLUSION: This study indicated TA as an herb with anti-inflammatory and anticoagulant proprieties and supports the traditional use of this plant for the treatment of inflammatory diseases.
Asunto(s)
Antiinflamatorios/uso terapéutico , Anticoagulantes/uso terapéutico , Extractos Vegetales/uso terapéutico , Polifenoles/uso terapéutico , Thymus (Planta) , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/toxicidad , Anticoagulantes/farmacología , Anticoagulantes/toxicidad , Coagulación Sanguínea/efectos de los fármacos , Carragenina , Quimiocina CCL2/inmunología , Edema/inducido químicamente , Edema/tratamiento farmacológico , Femenino , Dosificación Letal Mediana , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Masculino , Medicina Tradicional , Ratones Endogámicos C57BL , Marruecos , Extractos Vegetales/farmacología , Extractos Vegetales/toxicidad , Polifenoles/farmacología , Polifenoles/toxicidad , Ratas WistarRESUMEN
Aims: Oxidative stress and inflammation play a pathogenic role in atherosclerosis. Thioredoxin-1 (Trx-1) is an anti-oxidative, anti-inflammatory protein with atheroprotective effects. However, in vivo cleavage of Trx-1 generates a truncated pro-inflammatory protein, Trx-80, which compromises the therapeutic use of Trx-1. Here we analysed whether the thioredoxin-mimetic peptide (TxMP), CB3 might exert anti-oxidative, anti-inflammatory, and atheroprotective effects in ApoE2.Ki mice. Methods and results: We synthesized a small TxMP, Ac-Cys-Pro-Cys-amide, CB3 and characterized its antioxidant and anti-inflammatory effects on cultured peritoneal murine macrophages. CB3 significantly and dose-dependently reduced the level of reactive oxygen species in lipopolysaccharides (LPS)-activated macrophages. In addition, it efficiently lowered LPS-induced inflammatory process through NF-κB inhibition, as evidenced by the reduced secretion of monocyte chemoattractant protein-1, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α by macrophages. Nevertheless, CB3 did not affect cholesterol accumulation in macrophages. A daily-administered dose of 10 µg/g body weight CB3 to ApoE2.Ki mice on high fat diet did not affect plasma of total cholesterol and triglycerides levels but significantly reduced the plasma levels of pro-inflammatory cytokines (IL-33 and TNF-α) and oxidative markers. In contrast, it significantly induced the plasma levels of anti-inflammatory proteins (adiponectin, IL-10). In addition, CB3 reduced the number of pro-inflammatory M1 macrophages in spleen and decreased the ratio of M1/M2 macrophages in atherosclerotic lesion areas. Finally, CB3 significantly reduced the surface area of aortic lesions. Conclusions: Our results clearly showed that similar to the full length Trx-1, CB3 exerts protective effects, by reducing inflammation and oxidative stress in macrophages and in ApoE2.Ki mice. The atheroprotective effect of CB3 opens promising therapeutic approaches for treatment of atherosclerosis.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Dieta Alta en Grasa , Mediadores de Inflamación/metabolismo , Imitación Molecular , Oligopéptidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Placa Aterosclerótica , Compuestos de Sulfhidrilo/farmacología , Animales , Antiinflamatorios/síntesis química , Antioxidantes/síntesis química , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Oligopéptidos/síntesis química , Transducción de Señal , Compuestos de Sulfhidrilo/síntesis química , Tiorredoxinas/metabolismoRESUMEN
BACKGROUND: Thioredoxin (TRX)-1, a ubiquitous 12-kDa protein, exerts antioxidant and anti-inflammatory effects. In contrast, the truncated form, called TRX80, produced by macrophages induces upregulation of proinflammatory cytokines. TRX80 also promotes the differentiation of mouse peritoneal and human macrophages toward a proinflammatory M1 phenotype. METHODS: TRX1 and TRX80 plasma levels were determined with a specific ELISA. A disintegrin and metalloproteinase domain-containing protein (ADAM)-10, ADAM-17, and ADAM-10 activities were measured with SensoLyte 520 ADAM10 Activity Assay Kit, Fluorimetric, and InnoZyme TACE Activity Kit, respectively. Western immunoblots were performed with specific antibodies to ADAM-10 or ADAM-17. Angiogenesis study was evaluated in vitro with human microvascular endothelial cells-1 and in vivo with the Matrigel plug angiogenesis assay in mice. The expression of macrophage phenotype markers was investigated with real-time polymerase chain reaction. Phosphorylation of Akt, mechanistic target of rapamycin, and 70S6K was determined with specific antibodies. The effect of TRX80 on NLRP3 inflammasome activity was evaluated by measuring the level of interleukin-1ß and -18 in the supernatants of activated macrophages with ELISA. Hearts were used for lesion surface evaluation and immunohistochemical studies, and whole descending aorta were stained with Oil Red O. For transgenic mice generation, the human scavenger receptor (SR-A) promoter/enhancer was used to drive macrophage-specific expression of human TRX80 in mice. RESULTS: In this study, we observed a significant increase of plasma levels of TRX80 in old subjects compared with healthy young subjects. In parallel, an increase in expression and activity of ADAM-10 and ADAM-17 in old peripheral blood mononuclear cells compared with those of young subjects was observed. Furthermore, TRX80 was found to colocalize with tumor necrosis factor-α, a macrophage M1 marker, in human atherosclerotic plaque. In addition, TRX80 induced the expression of murine M1 macrophage markers through Akt2/mechanistic target of rapamycin-C1/70S6K pathway and activated the inflammasome NLRP3, leading to the release of interleukin-1ß and -18, potent atherogenic cytokines. Moreover, TRX80 exerts a powerful angiogenic effect in both in vitro and in vivo mouse studies. Finally, transgenic mice that overexpress human TRX80 specifically in macrophages of apoE-/- mice have a significant increase of aortic atherosclerotic lesions. CONCLUSIONS: TRX80 showed an age-dependent increase in human plasma. In mouse models, TRX80 was associated with a proinflammatory status and increased atherosclerosis.
Asunto(s)
Envejecimiento , Aterosclerosis/patología , Fragmentos de Péptidos/sangre , Tiorredoxinas/sangre , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Adulto , Anciano , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Inflamación , Interleucina-18/sangre , Interleucina-1beta/sangre , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacologíaRESUMEN
Intraperitoneal injection of arglabin (2.5 ng/g of body weight, twice daily, 13 weeks) into female human apolipoprotein E2 gene knock-in (ApoE2Ki) mice fed a high-fat Western-type diet (HFD) reduced plasma levels of glucose and insulin by â¼20.0% ± 3.5% and by 50.0% ± 2.0%, respectively, in comparison with vehicle-treated mice. Immunohistochemical analysis revealed the absence of active caspase-3 in islet sections from ApoE2Ki mice fed a HFD and treated with arglabin. In addition, arglabin reduced interleukin-1ß (IL-1ß) production in a concentration-dependent manner in Langerhans islets isolated from ApoE2Ki mice treated with lipopolysaccharide (LPS) and with cholesterol crystals. This inhibitory effect is specific for the inflammasome NOD-like receptor family, pyrin domain-containing 3 (NLRP3) because IL-1ß production was abolished in Langerhans islets isolated from Nlrp3(-/-) mice. In the insulin-secreting INS-1 cells, arglabin inhibited, in a concentration-dependent manner, the maturation of pro-IL-1ß into biologically active IL-1ß probably through the inhibition of the maturation of procaspase-1 into active capsase-1. Moreover, arglabin reduced the susceptibility of INS-1 cells to apoptosis by increasing Bcl-2 levels. Similarly, autophagy activation by rapamycin decreased apoptosis susceptibility while autophagy inhibition by 3-methyladenin treatment promoted apoptosis. Arglabin further increased the expression of the autophagic markers Bcl2-interacting protein (Beclin-1) and microtubule-associated protein 1 light chain 3 II (LC3-II) in a concentration-dependent manner. Thus, arglabin reduces NLRP3-dependent inflammation as well as apoptosis in pancreatic ß-cells in vivo and in the INS-1 cell line in vitro, whereas it increases autophagy in cultured INS-1 cells, indicating survival-promoting properties of the compound in these cells. Hence, arglabin may represent a new promising compound to treat inflammation and type 2 diabetes mellitus development.
Asunto(s)
Apolipoproteína E2/genética , Apoptosis/efectos de los fármacos , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa/efectos adversos , Inflamasomas/antagonistas & inhibidores , Células Secretoras de Insulina/efectos de los fármacos , Sesquiterpenos/farmacología , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Caspasa 1/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Activación Enzimática/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Sustitución del Gen , Humanos , Inflamación/tratamiento farmacológico , Insulina/sangre , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Interleucina-1beta/biosíntesis , Ratones , Ratas , Sesquiterpenos/uso terapéutico , Sesquiterpenos de Guayano , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND: This study was designed to evaluate the effect of arglabin on the NLRP3 inflammasome inhibition and atherosclerotic lesion in ApoE2Ki mice fed a high-fat Western-type diet. METHODS AND RESULTS: Arglabin was purified, and its chemical identity was confirmed by mass spectrometry. It inhibited, in a concentration-dependent manner, interleukin (IL)-1ß and IL-18, but not IL-6 and IL-12, production in lipopolysaccharide and cholesterol crystal-activated cultured mouse peritoneal macrophages, with a maximum effect at ≈50 nmol/L and EC50 values for both cytokines of ≈ 10 nmol/L. Lipopolysaccharide and cholesterol crystals did not induce IL-1ß and IL-18 production in Nlrp3(-/-) macrophages. In addition, arglabin activated autophagy as evidenced by the increase in LC3-II protein. Intraperitoneal injection of arglabin (2.5 ng/g body weight twice daily for 13 weeks) into female ApoE2.Ki mice fed a high-fat diet resulted in a decreased IL-1ß plasma level compared with vehicle-treated mice (5.2±1.0 versus 11.7±1.1 pg/mL). Surprisingly, arglabin also reduced plasma levels of total cholesterol and triglycerides to 41% and 42%, respectively. Moreover, arglabin oriented the proinflammatory M1 macrophages into the anti-inflammatory M2 phenotype in spleen and arterial lesions. Finally, arglabin treatment markedly reduced the median lesion areas in the sinus and whole aorta to 54% (P=0.02) and 41% (P=0.02), respectively. CONCLUSIONS: Arglabin reduces inflammation and plasma lipids, increases autophagy, and orients tissue macrophages into an anti-inflammatory phenotype in ApoE2.Ki mice fed a high-fat diet. Consequently, a marked reduction in atherosclerotic lesions was observed. Thus, arglabin may represent a promising new drug to treat inflammation and atherosclerosis.
Asunto(s)
Apolipoproteína E2/deficiencia , Aterosclerosis/tratamiento farmacológico , Proteínas Portadoras/antagonistas & inhibidores , Dieta Alta en Grasa/efectos adversos , Inflamasomas/antagonistas & inhibidores , Sesquiterpenos/uso terapéutico , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Aterosclerosis/sangre , Aterosclerosis/etiología , Femenino , Inflamasomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Sesquiterpenos/farmacología , Sesquiterpenos de Guayano , Resultado del TratamientoRESUMEN
IL-1ß production is critically regulated by cytosolic molecular complexes, termed inflammasomes. Different inflammasome complexes have been described to date. While all inflammasomes recognize certain pathogens, it is the distinctive feature of NLRP3 inflammasome to be activated by many and diverse stimuli making NLRP3 the most versatile, and importantly also the most clinically implicated inflammasome. However, NLRP3 activation has remained the most enigmatic. It is not plausible that the intracellular NLRP3 receptor is able to detect all of its many and diverse triggers through direct interactions; instead, it is discussed that NLRP3 is responding to certain generic cellular stress-signals induced by the multitude of molecules that trigger its activation. An ever increasing number of studies link the sensing of cellular stress signals to a direct pathophysiological role of NLRP3 activation in a wide range of autoinflammatory and autoimmune disorders, and thus provide a novel mechanistic rational, on how molecules trigger and support sterile inflammatory diseases. A vast interest has created to unravel how NLRP3 becomes activated, since mechanistic insight is the prerequisite for a knowledge-based development of therapeutic intervention strategies that specifically target the NLRP3 triggered IL-1ß production. In this review, we have updated knowledge on NLRP3 inflammasome assembly and activation and on the pyrin domain in NLRP3 that could represent a drug target to treat sterile inflammatory diseases. We have reported mutations in NLRP3 that were found to be associated with certain diseases. In addition, we have reviewed the functional link between NLRP3 inflammasome, the regulator of cellular redox status Trx/TXNIP complex, endoplasmic reticulum stress and the pathogenesis of diseases such as type 2 diabetes. Finally, we have provided data on NLRP3 inflammasome, as a critical regulator involved in the pathogenesis of obesity and cardiovascular diseases.
Asunto(s)
Enfermedades Cardiovasculares/genética , Proteínas Portadoras/genética , Diabetes Mellitus Tipo 2/genética , Obesidad/genética , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Proteínas Portadoras/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Inflamación , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mutación , Proteína con Dominio Pirina 3 de la Familia NLR , Obesidad/metabolismo , Obesidad/patología , Estrés Oxidativo , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Vascular cells are particularly susceptible to oxidative stress that is believed to play a key role in the pathogenesis of cardiovascular disorders. Thioredoxin-1 (Trx-1) is an oxidative stress-limiting protein with anti-inflammatory and anti-apoptotic properties. In contrast, its truncated form (Trx-80) exerts pro-inflammatory effects. Here we analyzed whether Trx-80 might exert atherogenic effects by promoting macrophage differentiation into the M1 pro-inflammatory phenotype. Trx-80 at 1 µg/ml significantly attenuated the polarization of anti-inflammatory M2 macrophages induced by exposure to either IL-4 at 15 ng/ml or IL-4/IL-13 (10 ng/ml each) in vitro, as evidenced by the expression of the characteristic markers, CD206 and IL-10. By contrast, in LPS-challenged macrophages, Trx-80 significantly potentiated the differentiation into inflammatory M1 macrophages as indicated by the expression of the M1 cytokines, TNF-α and MCP-1. When Trx-80 was administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/g body weight (b.w.) challenged either with LPS at 30 µg/30 g (b.w.) or IL-4 at 500 ng/30 g (b.w.), it significantly induced the M1 phenotype but inhibited differentiation of M2 macrophages in thymus and liver. When ApoE2.Ki mice were challenged once weekly with LPS for 5 weeks, they showed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. Such effect was potentiated when mice received daily, in addition to LPS, the Trx-80. Moreover, the Trx-80 treatment led to a significantly increased aortic lesion area. The ability of Trx-80 to promote differentiation of macrophages into the classical proinflammatory phenotype may explain its atherogenic effects in cardiovascular diseases.
Asunto(s)
Aterosclerosis/fisiopatología , Inflamación/fisiopatología , Macrófagos/fisiología , Fragmentos de Péptidos/fisiología , Tiorredoxinas/fisiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína E2/genética , Apolipoproteína E2/metabolismo , Aterosclerosis/etiología , Aterosclerosis/patología , Biomarcadores/metabolismo , Diferenciación Celular , Humanos , Inflamación/etiología , Inflamación/patología , Lectinas Tipo C/metabolismo , Macrófagos/clasificación , Macrófagos/patología , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Fenotipo , Receptores de Superficie Celular/metabolismoRESUMEN
OBJECTIVE: Oxidative stress is believed to play a key role in cardiovascular disorders. Thioredoxin (Trx) is an oxidative stress-limiting protein with anti-inflammatory and antiapoptotic properties. Here, we analyzed whether Trx-1 might exert atheroprotective effects by promoting macrophage differentiation into the M2 anti-inflammatory phenotype. METHODS AND RESULTS: Trx-1 at 1 µg/mL induced downregulation of p16(INK4a) and significantly promoted the polarization of anti-inflammatory M2 macrophages in macrophages exposed to interleukin (IL)-4 at 15 ng/mL or IL-4/IL-13 (10 ng/mL each) in vitro, as evidenced by the expression of the CD206 and IL-10 markers. In addition, Trx-1 induced downregulation of nuclear translocation of activator protein-1 and Ref-1, and significantly reduced the lipopolysaccharide-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-α and monocyte chemoattractant protein-1. Consistently, Trx-1 administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/30 g body weight challenged either with lipopolysaccharide at 30 µg/30 g body weight or with IL-4 at 500 ng/30 g body weight significantly induced the M2 phenotype while inhibiting differentiation of macrophages into the M1 phenotype in liver and thymus. ApoE2.Ki mice challenged once weekly with lipopolysaccharide for 5 weeks developed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. In contrast, however, daily injections of Trx-1 shifted the phenotype pattern of lesional macrophages in these animals to predominantly M2 over M1, and the aortic lesion area was significantly reduced (from 100%±18% to 62.8%±9.8%; n=8; P<0.01). Consistently, Trx-1 colocalized with M2 but not with M1 macrophage markers in human atherosclerotic vessel specimens. CONCLUSIONS: The ability of Trx-1 to promote differentiation of macrophages into an alternative, anti-inflammatory phenotype may explain its protective effects in cardiovascular diseases. These data provide novel insight into the link between oxidative stress and cardiovascular diseases.
Asunto(s)
Antiinflamatorios/farmacología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Diferenciación Celular/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Tiorredoxinas/farmacología , Animales , Enfermedades de la Aorta/inducido químicamente , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteína E2/genética , Apolipoproteína E2/metabolismo , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Citocinas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Lectinas Tipo C/metabolismo , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/farmacología , Factores de Tiempo , Factor de Transcripción AP-1/metabolismoRESUMEN
Hepatic fibrosis, the major complication of virtually all types of chronic liver damage, usually begins in portal areas, and its severity has been correlated to liver progenitor cells (LPC) expansion from periportal areas, even if the primary targets of injury are intralobular hepatocytes. The aim of this study was to determine the potential fibrogenic role of LPC, using a new experimental model in which rat liver fibrosis was induced by chronic carbon tetrachloride (CCl(4)) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl(4) alone caused a significant increase in serum transaminase activity as well as liver fibrosis initiating around central veins and leading to formation of incomplete centro-central septa with sparse fibrogenic cells expressing α-smooth muscle actin (αSMA). In AAF/CCl(4)-treated animals, the fibrogenic response was profoundly worsened, with formation of multiple porto-central bridging septa leading to cirrhosis, whereas hepatocellular necrosis and inflammation were similar to those observed in CCl(4)-treated animals. Enhanced fibrosis in AAF/CCl(4) group was accompanied by ductule forming LPC expanding from portal areas, αSMA-positive cells accumulation in the fibrotic areas and increased expression of hepatic collagen type 1, 3 and 4 mRNA. Moreover, CK19-positive LPC expressed the most potent fibrogenic cytokine transforming growth factor-ß (TGFß) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial-mesenchymal transition. In this new experimental model, LPC, by expressing TGFß, contributed to the accumulation of αSMA-positive myofibroblasts in the ductular reaction leading to enhanced fibrosis but also to disease progression and to a fibrotic pattern similar to that observed in humans.
Asunto(s)
Cirrosis Hepática Experimental/etiología , Hígado/patología , Células Madre/fisiología , 2-Acetilaminofluoreno/toxicidad , Actinas/análisis , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Tetracloruro de Carbono/toxicidad , Transición Epitelial-Mesenquimal , Queratina-19/análisis , Masculino , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in ß-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1ß, TNF-α, lymphotoxin-ß, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-ß and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.
Asunto(s)
Hígado Graso/prevención & control , Hepatitis/prevención & control , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Cirrosis Hepática Experimental/prevención & control , Hígado/metabolismo , Animales , Tetracloruro de Carbono , Proliferación Celular , Deficiencia de Colina/complicaciones , Progresión de la Enfermedad , Etionina , Hígado Graso/etiología , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Regulación de la Expresión Génica , Hepatitis/etiología , Hepatitis/genética , Hepatitis/metabolismo , Hepatitis/patología , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Metabolismo de los Lípidos , Hígado/patología , Cirrosis Hepática Experimental/etiología , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miofibroblastos/metabolismo , Miofibroblastos/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células Madre/metabolismo , Células Madre/patología , Tioacetamida , Factores de Tiempo , Tirosina Quinasa del Receptor AxlRESUMEN
The protein product of the growth arrest-specific gene 6 (Gas6) is a secreted ligand for tyrosine kinase receptors, among which Axl is the most widely distributed and displays the highest affinity for Gas6. The Gas6/Axl signaling pathway has been increasingly implicated in growth and survival processes occurring during development and tissue repair. In liver, after an acute or chronic injury, repair involves macrophages and hepatic stellate cells (HSC) activated into myofibroblastic cells (HSC/MFB), which produce cytokines and matrix proteins. We investigated the expression and the role of Gas6 and its receptor Axl in liver repair. Three days after CCl4-induced liver injury in the rat, we detected the expression of Gas6 in ED1-positive macrophages as well as in desmin-positive HSC, which accumulated in injured areas. Axl, the high-affinity receptor for Gas6, was detected in macrophages, HSC, and HSC/MFB. In vitro, expression of gamma-carboxylated Gas6 was strongly induced in HSC along with their transformation into myofibroblasts, and it exerted an anti-apoptotic effect on both HSC and HSC/MFB mediated by the Axl/PI3-kinase/Akt pathway. In conclusion, Gas6 is a survival factor for these cells and we suggest that Gas6, secreted by macrophages and HSC/MFB in vivo after liver injury, promotes HSC and HSC/MFB survival and might support transient HSC/MFB accumulation during liver healing.
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Apoptosis/fisiología , Expresión Génica , Hepatocitos/patología , Péptidos y Proteínas de Señalización Intercelular/genética , Hepatopatías/patología , ARN/genética , Animales , Tetracloruro de Carbono/toxicidad , Supervivencia Celular , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hepatopatías/metabolismo , Masculino , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND & AIMS: The growth arrest-specific gene 6 (Gas6) protein is a vitamin K-dependent protein that binds to the Axl subfamily of tyrosine kinase receptors and exerts antiapoptotic and proliferative effects. Because Gas6 plays a role in development and tissue remodelling, we studied its expression as well as that of its high-affinity receptor Axl in a well-characterized model of hepatic regeneration from precursor oval cells. METHODS: Hepatic regeneration was induced by treating rats with acetylaminofluorene followed by partial hepatectomy. RESULTS: Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 and 14 after hepatectomy and declined thereafter. Oval cells expressed Gas6 protein and messenger RNA (mRNA). Axl mRNA hepatic levels paralleled the number of oval cells, and immunohistochemistry showed Axl expression in these cells. WB-F344 cells, a hepatocytic precursor cell line, also expressed Gas6 and Axl. Addition of Gas6 significantly increased the number of WB-F344 cells cultured with or without serum. Gas6 did not increase cell entry in the S phase of the cell cycle but inhibited 15-d-prostaglandin J2-induced WB-F344 cell apoptosis. CONCLUSIONS: Our data demonstrate an expression of Gas6 and of its receptor Axl by oval cells during hepatic regeneration. Because the Gas6/Axl couple protects from apoptosis a hepatocytic precursor cell line, these results strongly suggest that the Gas6/Axl couple favors oval cell accumulation in regenerating liver by an autocrine/paracrine mechanism.
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Péptidos y Proteínas de Señalización Intercelular/genética , Regeneración Hepática/fisiología , Hígado/citología , Hígado/fisiología , Proteínas Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Apoptosis/fisiología , Comunicación Autocrina/fisiología , Células Cultivadas , Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Proteínas Oncogénicas/metabolismo , Comunicación Paracrina/fisiología , Proteínas Proto-Oncogénicas , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células Madre/fisiología , Timidina/farmacocinética , Tritio , Tirosina Quinasa del Receptor AxlRESUMEN
Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.
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Quimiocinas CXC/metabolismo , Regeneración Hepática , Hígado/citología , Receptores CXCR4/metabolismo , 2-Acetilaminofluoreno/farmacología , Animales , Quimiocina CXCL12 , Quimiocinas CXC/genética , Hepatectomía , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Polisacáridos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Wistar , Receptores CXCR4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismoRESUMEN
Differentiation of hepatic precursor cells in the biliary lineage has rarely been investigated, owing to the lack of convenient in vitro models. In this study, we used sodium butyrate and culture on Matrigel to promote differentiation of WB-F344 rat liver epithelial cells along the biliary phenotype. This differentiation was assessed by following the expression of phenotypic markers at the protein or mRNA level. Sodium butyrate induced cytokeratin 19 expression and gamma-glutamyltranspeptidase activity, together with a large increase in gamma-glutamyltranspeptidase mRNA IV, a transcript expressed at high levels in biliary cells. We also observed an increase in aquaporin-1 and beta4 integrin mRNAs, encoding two proteins expressed in adult biliary cells. Culture on Matrigel increased cytokeratin 19, gamma-glutamyltranspeptidase, and BDS7 expression in WB-F344 cells which still expressed aquaporin-1 and beta4 integrin. These results show that WB-F344 cells are able to differentiate in vitro along the biliary pathway, making them a candidate model for analyzing the molecular events associated with the hepatoblast-biliary cell transition.