RESUMEN
The endothelial-mesenchymal transition (EndMT) plays a key role in the development of cardiac fibrosis (CF) after acute myocardial infarction (AMI). The results of our previous study showed that amphiregulin (AR) expression was enhanced after MI. However, the role of AR on EndMT post MI remains unknown. This study aimed to elucidate the impact of AR on EndMT post MI and the associated molecular mechanisms. AR expression was markedly enhanced in infarct border area post MI, and endothelial cells were one of the primary cell sources of AR secretion. Stimulation with AR promoted endothelial cell proliferation, invasion, migration, collagen synthesis and EndMT. In addition, EGFR and downstream gene expression was significantly enhanced. In vivo, EndMT was significantly inhibited after lentivirus-AR-shRNA was delivered to the myocardium post MI. In addition, silencing AR ameliorated cardiac function by decreasing the extent of CF. Furthermore, the levels of EGFR pathway components in endothelial cells extracted from infarct border myocardium were all significantly decreased in lentivirus-AR-shRNA-treated MI mice. Our results demonstrate that AR induces CF post MI by enhancing EndMT in endothelial cells. Thus, targeting the regulation of AR may provide a potentially novel therapeutic option for CF after MI.
Asunto(s)
Anfirregulina/genética , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Infarto del Miocardio/genética , Miocardio/metabolismo , Actinas/genética , Actinas/metabolismo , Anfirregulina/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Supervivencia Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III , Vasos Coronarios/cirugía , Modelos Animales de Enfermedad , Fibrosis Endomiocárdica , Células Endoteliales/patología , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patología , Protocadherinas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Vimentina/genética , Vimentina/metabolismoRESUMEN
Cardiac fibrosis (CF), a main process of ventricular remodeling after myocardial infarction (MI), plays a crucial role in the pathogenesis of heart failure (HF) post-MI. It is known that amphiregulin (AR) is involved in fibrosis of several organs. However, the expression of AR and its role post-MI are yet to be determined. This study aimed to investigate the impact of AR on CF post-MI and related mechanisms. Significantly upregulated AR expression was evidenced in the infarct border zone of MI mice in vivo and the AR secretion was enhanced in macrophages, but not in cardiac fibroblasts. In vitro, treatment with AR increased cardiac fibroblast migration, proliferation and collagen synthesis, and upregulated the expression of epidermal growth factor receptor (EGFR) and the downstream genes such as Akt, ERK1/2 and Samd2/3 on cardiac fibroblasts. All these effects could be abrogated by pretreatment with a specific EGFR inhibitor. To verify the functions of AR in MI hearts, lentivirus-AR-shRNA and negative control vectors were delivered into the infarct border zone. After 28 days, knock-down of AR increased the survival rate and improved cardiac function, while decreasing the extent of myocardial fibrosis of MI mice. Moreover, EGFR and the downstream genes were significantly downregulated in lentivirus-AR-shRNA treated MI mice. Our results thus indicate that AR plays an important role in promoting CF after MI partly though activating the EGFR pathway. Targeting AR might be a novel therapeutic option for attenuating CF and improve cardiac function after MI.
Asunto(s)
Anfirregulina/metabolismo , Receptores ErbB/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Anfirregulina/genética , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Colágeno/metabolismo , Modelos Animales de Enfermedad , Receptores ErbB/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Función Ventricular Izquierda/genética , Remodelación Ventricular/genéticaRESUMEN
BACKGROUND/AIMS: Reperfusion after an ischaemic insult might cause infarct extension. Mesenchymal stem cell (MSC)-derived exosomes could attenuate myocardial remodelling in animal models of myocardial ischaemia reperfusion injury (MIRI), and the present study aimed to explore the related mechanisms. METHODS: In vitro, rat H9C2 cardiomyocytes (H9C2s) were exposed to H2O2. Cell viability was detected by the CCK-8 assay, apoptosis was detected by Annexin V-PE/7-AAD staining, ROS production was detected by fluorescence microscopy and flow cytometry, and apoptosis-related proteins and signalling pathway-related proteins were detected by western blot analysis. Autophagic flux was measured using the tandem fluorescent mRFG-GFP-LC3 assay. MSC-derived exosomes were extracted using the total exosome isolation reagent. Apoptosis, myocardial infarction size, heart function and myocardial LC3B expression were examined in an in vivo I/R model by the TUNEL assay, TTC/Evan blue staining, echocardiography and immunohistochemicalstaining, respectively. RESULTS: In vitro, H2O2 dose-dependently increased ROS production and cell apoptosis in H9C2s and blocked autophagic flux after 3 h of exposure; autophagy gradually decreased thereafter, and the lowest level was detected at 12 h after exposure. MSC-derived exosomes reduced H2O2-induced ROS production and cell apoptosis and enhanced autophagy at 12 h after exposure. In H9C2 cells exposed to H2O2 for 12 h, treatment with exosomes enhanced autophagy via the AMPK/mTOR and Akt/mTOR pathways. Likewise, in vivo exosome injections in rats that underwent I/R injury significantly reduced apoptosis and the myocardial infarct size and upregulated myocardial LC3B expression as well as improved heart function. CONCLUSIONS: Our results indicate that MSC-derived exosomes could reduce MIRI by inducing cardiomyocyte autophagy via AMPK/mTOR and Akt/mTOR pathways.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Exosomas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Células de la Médula Ósea/citología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Corazón/diagnóstico por imagen , Peróxido de Hidrógeno/toxicidad , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown. METHODS: MSCs, CSCs, and macrophages were obtained from bone marrow, hearts, and peritoneal cavity of mice, respectively. dMSCs were harvested after hypoxia for 24 h, and incubated with macrophages (2:1) for another 2 days with or without lipopolysaccharide (LPS, 50 ng/mL) and sorted by flow cytometry to obtain pMΦ. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Expression of inflammation and growth factors was assessed by real-time polymerase chain reaction (RT-PCR) in MΦ, LPS-pMΦ, and pMΦ cells. RESULTS: pMΦ expressed higher levels of interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß)and lower levels of tumor necrosis factor-α(TNF-α)and IL-6 than LPS-pMΦ, higher levels of growth factors and of GATA-4 and c-TnI at the 7th day, which were similar to those in MSCs. CSCs cultured with supernatant fluids of pMΦ exhibited higher proliferative, anti-hypoxic, and differentiation activities. CONCLUSION: The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Furthermore, some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of myocardial infarction in the future.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Corazón/fisiología , Macrófagos Peritoneales/patología , Células Madre Mesenquimatosas/patología , Fagocitosis , Células Madre/fisiología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Citocinas/genética , Citocinas/metabolismo , Corazón/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
Advanced glycation end products (AGEs) are major inflammatory mediators in diabetes, affecting atherosclerosis progression via macrophages. Metformin slows diabetic atherosclerosis progression through mechanisms that remain to be fully elucidated. The present study of murine bone marrow derived macrophages showed that (1) AGEs enhanced proinflammatory cytokines (interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α)) mRNA expression, RAGE expression, and NFκB activation; (2) metformin pretreatment inhibited AGEs effects and AGEs-induced cluster designation 86 (CD86) (M1 marker) expression, while promoting CD206 (M2 marker) surface expression and anti-inflammatory cytokine (IL-10) mRNA expression; and (3) the AMPK inhibitor, Compound C, attenuated metformin effects. In conclusion, metformin inhibits AGEs-induced inflammatory response in murine macrophages partly through AMPK activation and RAGE/NFκB pathway suppression.
Asunto(s)
Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Productos Finales de Glicación Avanzada/efectos de los fármacos , Hipoglucemiantes/farmacología , Macrófagos/efectos de los fármacos , Metformina/farmacología , FN-kappa B/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Receptor para Productos Finales de Glicación Avanzada/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Citocinas/efectos de los fármacos , Citocinas/genética , Productos Finales de Glicación Avanzada/metabolismo , Inflamación , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/genética , Interleucina-6/genética , Macrófagos/metabolismo , Masculino , Ratones , FN-kappa B/metabolismo , ARN Mensajero/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Classically activated macrophages (M1) are associated with inflammation in diabetic patients. Inflammation is a known risk factor in diabetes. The present study tested the hypothesis that pioglitazone (PIO) alleviates inflammation in diabetic mice fed a high-fat diet by inhibiting advanced glycation end-product (AGE)-induced classical macrophage activation. It was found that AGE treatment promoted the transcription of pro-inflammatory molecules and M1 surface markers, whereas PIO increased the expression of anti-inflammatory genes and decreased the expression of pro-inflammatory mediators in bone marrow-derived macrophages (BMDMs) in a dose-dependent manner. Furthermore, pretreatment with PIO abrogated the effects of AGE on pro-inflammatory markers and partly inhibited AGE-induced nuclear factor-κB (NF-κB) activation. PIO treatment partly reduced the inflammatory phenotype in diabetic ApoE(-/-) mice, and significantly reduced NF-κB activation in plaques. Therefore, we conclude that PIO blocks classical activation of macrophages and attenuates inflammation in mouse models of diabetes.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Activación de Macrófagos/efectos de los fármacos , Tiazolidinedionas/administración & dosificación , Animales , Antiinflamatorios/administración & dosificación , Médula Ósea/efectos de los fármacos , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Dieta Alta en Grasa , Productos Finales de Glicación Avanzada/genética , Humanos , Inflamación/complicaciones , Inflamación/genética , Inflamación/patología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , FN-kappa B/genética , PioglitazonaRESUMEN
Atherosclerotic lesions are accelerated in patients with diabetes. M1 (classically activated in contrast to M2 alternatively activated) macrophages play key roles in the progression of atherosclerosis. Since advanced glycation end products (AGEs) are major pathogenic factors and active inflammation inducers in diabetes mellitus, this study assessed the effects of AGEs on macrophage polarization. The present study showed that AGEs significantly promoted macrophages to express IL-6 and TNF-α. M1 macrophage markers such as iNOS and surface markers including CD11c and CD86 were significantly upregulated while M2 macrophage markers such as Arg1 and CD206 remained unchanged after AGEs stimulation. AGEs significantly increased RAGE expression in macrophages and activated NF-κB pathway, and the aforementioned effects were partly abolished by administration of anti-RAGE antibody or NF-κB inhibitor PDTC. In conclusion, our results suggest that AGEs enhance macrophage differentiation into proinflammatory M1 phenotype at least partly via RAGE/NF-κB pathway activation.