RESUMEN
The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular (125) I-LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE(-/-) mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age-dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.
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Autofagia/genética , Endotelio Vascular/metabolismo , Homeostasis/fisiología , Metabolismo de los Lípidos/fisiología , Lipoproteínas LDL/metabolismo , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Homeostasis/genética , Metabolismo de los Lípidos/genética , Lipoproteínas LDL/genética , Ratones Noqueados , Oxidación-Reducción , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
OBJECTIVE: Monocyte-derived microparticles (mono-MPs) are emerging as critical transducers of inflammatory signals, and have been suggested to link cardiovascular risk factors to vascular injury. Since adiponectin has been proposed to exert multiple anti-inflammatory and vasculoprotective effects, we hypothesized that it might serve to limit the production and/or action of mono-MPs. METHODS: Flow cytometry and western blot studies were conducted on THP-1 cells, THP-1-derived MPs, human umbilical vein endothelial cells (HUVECs), peripheral blood CD14+ monocytes and mice to evaluate the effects of adiponectin on mono-MPs. RESULTS: Adiponectin attenuated lipopolysaccharide (LPS)-evoked MP release from THP-1 monocytes (30% difference) and peripheral blood monocytes (both P < 0.05) as well as dampened LPS-induced mono-MP generation in vivo. Furthermore, peritoneal monocytes from Adipoq(-/-) mice generated significantly greater MPs than those from Adipoq(+/+) littermates in the absence (2.3 fold difference, P < 0.05) and presence (1.6 fold difference, P < 0.05) of LPS. LPS-induced MP expression of NLRP3 inflammasome and its key components, namely cleaved ASC, caspase-1 and IL-1ß (pro- and cleaved), were markedly attenuated by adiponectin. HUVECs incubated with MPs from LPS-treated THP-1 cells exhibited increased VCAM-1 levels and adhesion to THP-1 cells. Adiponectin abrogated these effects. From a mechanistic standpoint, the effects of adiponectin on MP release and molecular signaling occurred at least in part through the AMPK, Akt and NFκB pathways. CONCLUSION: Adiponectin exerts novel effects to limit the production and action of mono-MPs, underscoring yet another pleiotropic effect of this adipokine.
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Proteínas Quinasas Activadas por AMP/genética , Adiponectina/genética , Aterosclerosis/genética , Regulación de la Expresión Génica , FN-kappa B/genética , Proteínas Proto-Oncogénicas c-akt/genética , ARN/genética , Proteínas Quinasas Activadas por AMP/biosíntesis , Adiponectina/biosíntesis , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Humanos , Inflamasomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Monocitos/metabolismo , Monocitos/ultraestructura , FN-kappa B/biosíntesis , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Riesgo , Transducción de SeñalRESUMEN
Autophagy regulates cellular homeostasis and integrates the cellular pro-survival machinery. We investigated the role of autophagy in the natural history of murine abdominal aortic aneurysms (AAA). ApoE(-/-) mice were implanted with saline- or angiotensin II (Ang-II)-filled miniosmotic pumps then treated with either the autophagy inhibitor chloroquine (CQ; 50 mg·(kg body mass)(-1)·day(-1), by intraperitoneal injection) or saline. Ang-II-elicited aneurysmal expansion of the suprarenal aorta coupled with thrombus formation were apparent 8 weeks later. CQ had no impact on the incidence (50% for Ang-II compared with 46.2% for Ang-II + CQ; P = NS) and categorical distribution of aneurysms. The markedly reduced survival rate observed with Ang-II (57.1% for Ang-II compared with 100% for saline; P < 0.05) was unaffected by CQ (61.5% for Ang-II + CQ; P = NS compared with Ang-II). CQ did not affect the mean maximum suprarenal aortic diameter (1.91 ± 0.19 mm for Ang-II compared with 1.97 ± 0.21 mm for Ang-II + CQ; P = NS). Elastin fragmentation, collagen accumulation, and smooth muscle attrition, which were higher in Ang-II-treated mice, were unaffected by CQ treatment. Long-term CQ administration does not affect the natural history and prognosis of experimental AAA, suggesting that global loss of autophagy is unlikely to be a causal factor in the development of aortic aneurysms. Manipulation of autophagy as a mechanism to reduce AAA may need re-evaluation.
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Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Cloroquina/farmacología , Angiotensina II , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Autofagia/efectos de los fármacos , Colágeno/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Elastina/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de TiempoRESUMEN
Obesity is a multifactorial chronic disease characterized by an accumulation of visceral and subcutaneous fat, which leads to a predisposition toward cardiometabolic diseases. A plethora of mechanisms, including abnormalities in lipid metabolism, insulin resistance, inflammation, endothelial dysfunction, adipokine imbalance, and inflammasome activation have been suggested to underlie the relationship between obesity and atherosclerosis. More recent data point toward an emerging role of impaired autophagy and altered gut microbiome homeostasis as potentially contributing factors. This review provides an overview of this area.
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Tejido Adiposo/metabolismo , Aterosclerosis/etiología , Metabolismo Energético , Obesidad/complicaciones , Aterosclerosis/metabolismo , Homeostasis , Humanos , Obesidad/metabolismoRESUMEN
Pulmonary fibrosis is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix components. Although the origin of fibroblasts is multifactorial, recent data implicate endothelial-to-mesenchymal transition as an important source of fibroblasts. We report herein that loss of the essential autophagy gene ATG7 in endothelial cells (ECs) leads to impaired autophagic flux accompanied by marked changes in EC architecture, loss of endothelial, and gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition. Loss of ATG7 also up-regulates TGFß signaling and key pro-fibrotic genes in vitro. In vivo, EC-specific ATG7 knock-out mice exhibit a basal reduction in endothelial-specific markers and demonstrate an increased susceptibility to bleomycin-induced pulmonary fibrosis and collagen accumulation. Our findings help define the role of endothelial autophagy as a potential therapeutic target to limit organ fibrosis, a condition for which presently there are no effective available treatments.
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Células Endoteliales/citología , Mesodermo/citología , Proteínas Asociadas a Microtúbulos/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia , Bleomicina/toxicidad , Línea Celular , Técnica del Anticuerpo Fluorescente , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Fibrosis Pulmonar/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Enzimas Activadoras de Ubiquitina/genéticaRESUMEN
OBJECTIVE: Autophagy is an evolutionary conserved adaptive response that is believed to promote cell survival in response to stressful stimuli via recycling of precursors derived from the degradation of endogenous cellular components. The autophagic molecular machinery is controlled by a large family of autophagy-related genes (ATGs) and downstream regulators. We sought to define the autophagy gene fingerprint associated with human ischemia and reperfusion (IR) injury using an intraoperative model developed by Sellke and colleagues. METHODS: Right atrial appendages, collected from human hearts before and after cardioplegic arrest and after reperfusion, were submitted for polymerase chain reaction (PCR) array, quantitative real-time PCR, and immunoblot analysis for autophagy proteins and their associated upstream regulators. RESULTS: Perioperative IR significantly upregulated 11 (13.1%) and downregulated 3 (3.6%) of 84 ATGs. Specifically, there were increases in the autophagy machinery components ATG4A, ATG4C, and ATG4D; tumor necrosis factor-related apoptosis-inducing ligand, MAPK8 and BCL2L1; and chaperone-mediated autophagy activity with increased heat shock protein (HSP) A8, HSP90AA1, and a-synuclein. Autophagy activity was confirmed through observations of higher LC3-I levels and an increase in the LC3-II/LC3-I ratio. Autophagy activation coincided with increased AMPK activation and decreased protein levels of the mammalian target of rapamycin, the latter a key negative regulator of autophagy. CONCLUSIONS: We provide the first human cardiac fingerprint of autophagy gene expression in response to IR. These findings may inform on appropriate cell- and gene-based therapeutic approaches to limit aberrant cardiac injury.
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Apéndice Atrial/patología , Autofagia/genética , Perfilación de la Expresión Génica , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Anciano , Apoptosis/genética , Apéndice Atrial/metabolismo , Apéndice Atrial/cirugía , Puente Cardiopulmonar , Puente de Arteria Coronaria , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Paro Cardíaco Inducido , Humanos , Masculino , Persona de Mediana Edad , Daño por Reperfusión Miocárdica/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Excessive production of reactive oxygen species (ROS), in part via upregulation of DNA damage pathways, is a central mechanism governing pathologic activation of vascular smooth muscle cells (VSMCs). We hypothesized that the breast cancer 1, early onset (BRCA1) gene that is involved in cellular resistance to DNA damage limits ROS production and oxidative stress in VSMCs. METHODS: We evaluated basal and H2O2-stimulated expression of BRCA1 in human aortic smooth muscle cells (HASMCs). In vitro gain-of-function experiments were performed in BRCA1 adenovirus (Ad-BRCA1)-transfected HASMCs. ROS production and expression of Nox1 and its key regulatory subunit p47phox, key components of the ROS-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, were evaluated. In vivo gain-of-function experiments were performed in spontaneously hypertensive (SHR) rats treated with Ad-BRCA1 (5 × 10(10) IU/rat). Blood pressure, vascular ROS generation, Nox1, and p47phox expression were measured. RESULTS: BRCA1 was constitutively expressed in murine, rat, and human smooth muscle cells (SMCs). H2O2 significantly reduced BRCA1 expression with a resultant increase in ROS generation. BRCA1-overexpressing HASMCs were protected against H2O2-induced ROS generation, in part, via downregulation of the ROS-producing NADPH oxidase subunits Nox1 and p47phox. Ad-BRCA1 treatment in SHR rats was associated with a sustained increase in aortic BRCA1 expression, lower aortic ROS production, reduced γH2A.X levels, greater RAD51 foci, and decreases in blood pressure. CONCLUSIONS: BRCA1 is a novel and previously unrecognized target that may shield VSMCs from oxidative stress by inhibiting NADPH Nox1-dependent ROS production. Gene- and/or cell-based approaches that improve BRCA1 bioavailability may represent a new approach in the treatment of diverse vascular diseases associated with an aberrant VSMC phenotype.
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Proteína BRCA1/metabolismo , Hipertensión/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Proteína BRCA1/genética , Presión Sanguínea , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Terapia Genética/métodos , Histonas/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Hipertensión/genética , Hipertensión/fisiopatología , Hipertensión/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasas/metabolismo , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosfoproteínas/metabolismo , Recombinasa Rad51/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , TransfecciónRESUMEN
OBJECTIVE: Neointimal hyperplasia secondary to vascular smooth muscle cell (VSMC) activation limits the long-term patency of saphenous vein grafts (SVGs). We compared markers of vascular injury and VSMC activation in SVGs harvested using the pedicled 'no-touch' (NT) vs the conventional (CON) technique. METHODS: Patients undergoing coronary artery bypass surgery were enrolled in the PATENT SVG trial (clinicaltrials.gov NCT01488084). Patients were randomly allocated to have SVGs harvested with the NT technique from one leg and the CON method from the other. SVG segments underwent morphometry, histological and electron microscopy assessments and transcript measurements of VSMC activation and differentiation markers. Leg wound functional recovery and harvest site complications were assessed using a quality-of-life questionnaire. RESULTS: A total of 17 patients (65.3 ± 7.3 years) were enrolled. SVGs harvested using the NT vs CON technique exhibited preserved intimal, medial and adventitial architecture. CON harvest was associated with greater medial Kruppel-like factor 4 transcript levels (0.26 ± 0.05 vs 0.11 ± 0.02, P < 0.05). CON samples had significantly lower medial serum response factor (0.53 ± 0.11 vs 1.44 ± 0.50, P < 0.05) and myocardin (0.59 ± 0.08 vs 1.33 ± 0.33, P < 0.05) transcript levels. MicroRNA-145, an inhibitor of VSMC activation and differentiation, was higher in the NT vs CON samples (1.84 ± 1.03 vs 0.50 ± 0.19, P < 0.05). Leg assessment scores were worse in the NT legs at 3 months, but similar to CON scores at 12 months. CONCLUSIONS: SVGs harvested using the 'NT' technique exhibit an early molecular and morphological pattern consistent with decreased VSMC activation compared with CON harvesting. Functional leg recovery was similar in both groups at 12 months. Larger studies are required to corroborate these findings.
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Puente de Arteria Coronaria/métodos , Músculo Liso Vascular/patología , Vena Safena/patología , Vena Safena/trasplante , Recolección de Tejidos y Órganos/métodos , Anciano , Femenino , Humanos , Factor 4 Similar a Kruppel , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Vena Safena/química , Vena Safena/citología , Colgajos Quirúrgicos/patologíaRESUMEN
BACKGROUND: Endothelial dysfunction is an early event in the development and progression of a wide range of cardiovascular diseases. Various human studies have identified that measures of endothelial dysfunction may offer prognostic information with respect to vascular events. Microparticles (MPs) are a heterogeneous population of small membrane fragments shed from various cell types. The endothelium is one of the primary targets of circulating MPs, and MPs isolated from blood have been considered biomarkers of vascular injury and inflammation. CONTENT: This review summarizes current knowledge of the potential functional role of circulating MPs in promoting endothelial dysfunction. Cells exposed to different stimuli such as shear stress, physiological agonists, proapoptotic stimulation, or damage release MPs, which contribute to endothelial dysfunction and the development of cardiovascular diseases. Numerous studies indicate that MPs may trigger endothelial dysfunction by disrupting production of nitric oxide release from vascular endothelial cells and subsequently modifying vascular tone. Circulating MPs affect both proinflammatory and proatherosclerotic processes in endothelial cells. In addition, MPs can promote coagulation and inflammation or alter angiogenesis and apoptosis in endothelial cells. SUMMARY: MPs play an important role in promoting endothelial dysfunction and may prove to be true biomarkers of disease state and progression.
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Enfermedades Cardiovasculares/etiología , Micropartículas Derivadas de Células/fisiología , Endotelio Vascular/metabolismo , Apoptosis , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/fisiopatología , Proliferación Celular , Supervivencia Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Humanos , Inflamación/etiología , Inflamación/metabolismo , Neovascularización Fisiológica , Óxido Nítrico/metabolismo , Estrés OxidativoRESUMEN
OBJECTIVE: BRCA1, a tumor suppressor gene implicated in breast and ovarian cancers, exerts multiple effects on DNA repair and affords resistance against cellular stress responses. We hypothesized that BRCA1 limits endothelial cell apoptosis and dysfunction, and via this mechanism attenuates atherosclerosis. METHODS: Loss and gain of function were achieved in cultured endothelial cells by silencing and overexpressing BRCA1, respectively. In vivo loss and gain of function were performed by generating endothelial cell-specific knockout (EC-BRCA1(-/-)) mice and administering a BRCA1 adenovirus. Well-established cell and animal models of angiogenesis and atherosclerosis were used. RESULTS: BRCA1 is basally expressed in endothelial cells. BRCA1 overexpression protected and BRCA1 silencing exaggerated inflammation- and doxorubicin-induced endothelial cell apoptosis. Key indices of endothelial function were modulated in a manner consistent with an effect of BRCA1 to limit endothelial cell apoptosis and improve endothelial function. BRCA1 overexpression strongly attenuated the production of reactive oxygen species and upregulated endothelial nitric oxide synthase, phosphorylated endothelial nitric oxide synthase, phosphorylated Akt, and vascular endothelial growth factor-a expression. BRCA1 overexpression also improved capillary density and promoted blood flow restoration in mice subjected to hind-limb ischemia. BRCA1-overexpressing ApoE(-/-) mice fed a Western diet developed significantly less aortic plaque lesions, exhibited reduced macrophage infiltration, and generated less reactive oxygen species. Lung sections and aortic segments from EC-BRCA1(-/-) mice demonstrated greater inflammation-associated apoptosis and impaired endothelial function, respectively. BRCA1 expression was attenuated in the plaque region of human atherosclerotic carotid artery samples compared with the adjacent plaque-free area. CONCLUSIONS: These data collectively highlight a previously unrecognized role of BRCA1 as a gatekeeper of inflammation-induced endothelial cell function and a target to limit atherosclerosis. Translational studies evaluating endothelial function and atherosclerosis in individuals with BRCA1 mutations are suggested.
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Aterosclerosis/prevención & control , Proteína BRCA1/metabolismo , Células Endoteliales/metabolismo , Terapia Genética/métodos , Isquemia/terapia , Músculo Esquelético/irrigación sanguínea , Adenoviridae/genética , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apoptosis , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Proteína BRCA1/genética , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Vectores Genéticos , Miembro Posterior , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Recuperación de la Función , Flujo Sanguíneo Regional , Factores de Tiempo , Transfección , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Alterations in cardiac energy and substrate metabolism play a critical role in the development and clinical course of heart failure. We hypothesized that the cardioprotective role of the breast cancer 1, early onset (BRCA1) gene might be mediated in part by alterations in cardiac bioenergetics. METHODS: We generated cardiomyocyte-specific BRCA1 homozygous and heterozygous knockout mice using the Cre-loxP technology and evaluated the key molecules and pathways involved in glucose metabolism, fatty acid metabolism, and mitochondrial bioenergetics. RESULTS: Cardiomyocyte-specific BRCA1-deficient mice showed reduced cardiac expression of glucose and fatty acid transporters, reduced acetyl-coenzyme A carboxylase 2 and malonyl-coenzyme A decarboxylase (key enzymes that control malonyl coenzyme A, which in turn controls fatty acid oxidation), and reduced carnitine palmitoyltransferase I, a rate-limiting enzyme for mitochondrial fatty acid uptake. Peroxisome proliferator-activated receptor α and γ and carnitine palmitoyltransferase I levels were also downregulated in these hearts. Rates of glucose and fatty acid oxidation were reduced in the hearts of heterozygous cardiomyocyte-restricted BRCA1-deficient mice, resulting in a decrease in the rate of adenosine triphosphate production. This decrease in metabolism and adenosine triphosphate production occurred despite an increase in 5'-adenosine monophosphate-activated protein kinase and AKT activation in the heart. CONCLUSIONS: Cardiomyocyte-specific loss of BRCA1 alters critical pathways of fatty acid and glucose metabolism, leading to an energy starved heart. BRCA1-based cell or gene therapy might serve as a novel target to improve cardiac bioenergetics in patients with heart failure.
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Proteína BRCA1/metabolismo , Daño del ADN , Reparación del ADN , Metabolismo Energético , Miocitos Cardíacos/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Carboxiliasas/metabolismo , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Glucosa/metabolismo , Heterocigoto , Homocigoto , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: MicroRNA are essential posttranscriptional modulators of gene expression implicated in various chronic diseases. Because microRNA-145 is highly expressed in vascular smooth muscle cells (VSMC) and regulates VSMC fate and plasticity, we hypothesized that it may be a novel regulator of atherosclerosis and plaque stability. METHODS AND RESULTS: Apolipoprotein E knockout mice (ApoE(-/-)) mice were treated with either a microRNA-145 lentivirus under the control of the smooth muscle cell (SMC)-specific promoter SM22α or a SM22α control lentivirus before commencing the Western diet for 12 weeks. The SMC-targeted microRNA-145 treatment markedly reduced plaque size in aortic sinuses, ascending aortas, and brachiocephalic arteries. It also significantly increased fibrous cap area, reduced necrotic core area, and increased plaque collagen content. Cellular plaque composition analyses revealed significantly less macrophages in ApoE(-/-) mice treated with the SMC-specific microRNA-145. These mice also demonstrated marked increases in calponin levels and α-smooth muscle actin-positive SMC areas in their atherosclerotic lesions. Furthermore, lentiviral delivery of microRNA-145 resulted in reduced KLF4 and elevated myocardin expression in aortas from ApoE(-/-) mice, consistent with an effect of microRNA-145 to promote a contractile phenotype in VSMC. CONCLUSIONS: VSMC-specific overexpression of microRNA-145 is a novel in vivo therapeutic target to limit atherosclerotic plaque morphology and cellular composition, shifting the balance toward plaque stability vs plaque rupture.
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Aterosclerosis/prevención & control , Terapia Genética , Vectores Genéticos/uso terapéutico , MicroARNs/fisiología , Actinas/genética , Animales , Aorta/citología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Aterosclerosis/genética , Aterosclerosis/patología , Arterias Carótidas/metabolismo , Células Cultivadas , Dieta Aterogénica , Genes Reporteros , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Lentivirus/genética , Lípidos/sangre , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/fisiología , Transducción GenéticaRESUMEN
OBJECTIVE: Bone morphogenetic protein-2 (BMP-2) is a major regulator of aortic valve calcification. MicroRNAs (miRNAs) are essential post-transcriptional modulators of gene expression and miRNA-141 is a known repressor of BMP-2-mediated osteogenesis. We hypothesized that miRNA-141 is a key regulator of aortic valve calcification. METHODS: Porcine valvular interstitial cells were isolated, transfected with miRNA-141 or control, and stimulated with transforming growth factor-ß. The BMP-2, extracellular signal-regulated kinase 1/2, and runt-related transcription factor 2 levels were determined by immunoblotting and reverse transcriptase polymerase chain reaction. To determine the role of miRNA-141 in bicuspid aortic valve disease, human bicuspid (n = 19) and tricuspid (n = 17) aortic valve leaflets obtained intraoperatively were submitted for GenoExplorer human microRNA array, immunoblotting, and histologic and immunohistochemical analyses. RESULTS: Stimulation of porcine aortic valvular interstitial cells with transforming growth factor-ß induced morphologic alterations consistent with myofibroblastic transformation, BMP-2 signaling, and calcification. Transfection with miRNA-141 restored transforming growth factor-ß-induced valvular interstitial cell activation, BMP-2 signaling, and alkaline phosphatase activity (3.55 ± 0.18 vs 4.01 ± 0.21, P < .05), suggesting upstream regulation by miRNA-141. miRNA microarray demonstrated differential expression of 35 of 1583 miRNA sequences in the bicuspid versus tricuspid aortic valve leaflets, with a 14.5-fold decrease in miRNA-141 in the bicuspid versus tricuspid leaflets (P < .05). This was associated with significantly increased BMP-2 protein expression in bicuspid aortic valve compared with the tricuspid aortic valve leaflets (P < .001). CONCLUSIONS: We report a completely novel role of miRNA-141 as a regulator of BMP-2-dependent aortic valvular calcification and demonstrate marked attenuation of miRNA-141 expression in patients with bicuspid aortic valve-associated aortic stenosis. Therapeutic targeting of miRNA-141 could serve as a novel strategy to limit progressive calcification in aortic stenosis.
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Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/metabolismo , Calcinosis/metabolismo , MicroARNs/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Proteína Morfogenética Ósea 2/metabolismo , Distribución de Chi-Cuadrado , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Porcinos , Transfección , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.
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Apoptosis/efectos de los fármacos , Proteína BRCA2/genética , Doxorrubicina/toxicidad , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/patología , Miocitos Cardíacos/patología , Animales , Antibióticos Antineoplásicos/toxicidad , Proteína BRCA2/deficiencia , Insuficiencia Cardíaca/epidemiología , Ratones , Ratones Noqueados , Miocardio/patología , Fenotipo , Factores de Riesgo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The tumour suppressor BRCA1 is mutated in familial breast and ovarian cancer but its role in protecting other tissues from DNA damage has not been explored. Here we show a new role for BRCA1 as a gatekeeper of cardiac function and survival. In mice, loss of BRCA1 in cardiomyocytes results in adverse cardiac remodelling, poor ventricular function and higher mortality in response to ischaemic or genotoxic stress. Mechanistically, loss of cardiomyocyte BRCA1 results in impaired DNA double-strand break repair and activated p53-mediated pro-apoptotic signalling culminating in increased cardiomyocyte apoptosis, whereas deletion of the p53 gene rescues BRCA1-deficient mice from cardiac failure. In human adult and fetal cardiac tissues, ischaemia induces double-strand breaks and upregulates BRCA1 expression. These data reveal BRCA1 as a novel and essential adaptive response molecule shielding cardiomyocytes from DNA damage, apoptosis and heart dysfunction. BRCA1 mutation carriers, in addition to risk of breast and ovarian cancer, may be at a previously unrecognized risk of cardiac failure.
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Apoptosis/genética , Proteína BRCA1/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Daño por Reperfusión/metabolismo , Proteína p53 Supresora de Tumor/genética , Adaptación Fisiológica , Adulto , Animales , Proteína BRCA1/deficiencia , Neoplasias de la Mama/genética , Roturas del ADN de Doble Cadena , Femenino , Eliminación de Gen , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación , Miocardio/patología , Miocitos Cardíacos/citología , Neoplasias Ováricas/genética , Daño por Reperfusión/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/deficiencia , Remodelación Ventricular/genéticaRESUMEN
P53 protein levels are elevated by trastuzumab and the biologically similar rat ERBB2/HER2/NEU antibody; and that this coincides with enhanced apoptosis, increased cleaved caspase-3 levels and diminished cardiac function. We also demonstrate that MDM2 may be a regulatory target of anti-ERBB2 thereby implicating the MDM2/p53 axis as a potential molecular component for the undesirable cardiac outcomes noted with trastuzumab. Finally, we show that these MDM2/p53-mediated events are independent of both the ERK1/2 and Akt systems. In conclusion, our findings suggest that the adverse cardiac events observed with trastuzumab may stem from its negative regulation of MDM2 events which impairs p53 degradation resultantly promoting apoptosis leading to cardiac dysfunction. These observations may have important therapeutic implications since they suggest that anticancer agents that inhibit MDM2 and its downstream actions may curb tumor progression at the expense of increasing cardiac stress.
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Anticuerpos Monoclonales/efectos adversos , Antineoplásicos/efectos adversos , Apoptosis/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Receptor ErbB-2/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados , Corazón/efectos de los fármacos , Corazón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Trastuzumab , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
BACKGROUND: Adropin is a recently identified protein that has been implicated in the maintenance of energy homeostasis and insulin resistance. Because vascular function and insulin sensitivity are closely related, we hypothesized that adropin may also exert direct effects on the endothelium. METHODS AND RESULTS: In vitro cell culture models were partnered with an in vivo murine injury model to determine the potential vascular effects of adropin. Adropin was expressed in human umbilical vein and coronary artery endothelial cells (ECs). Adropin-treated endothelial cells exhibited greater proliferation, migration and capillary-like tube formation and less permeability and tumor necrosis factor-α-induced apoptosis. In keeping with a vascular protective effect, adropin stimulated Akt Ser(473) and endothelial nitric oxide (NO) synthase Ser(1177) phosphorylation. The former was abrogated in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002, whereas the latter was attenuated by LY294002 and by mitogen-activated protein kinase kinase 1 inhibition with PD98059. Together, these findings suggest that adropin regulates NO bioavailability and events via the phosphatidylinositol 3-kinase-Akt and extracellular signal regulated kinase 1/2 signaling pathways. Adropin markedly upregulated vascular endothelial growth factor receptor-2 (VEGFR2) transcript and protein levels, and in VEGFR2-silenced endothelial cells, adropin failed to induce phosphorylation of endothelial NO synthase, Akt, and extracellular signal regulated kinase 1/2, supporting VEGFR2 as an upstream target of adropin-mediated endothelial NO synthase activation. Last, adropin improved murine limb perfusion and elevated capillary density following induction of hindlimb ischemia. CONCLUSIONS: We report a potential endothelial protective role of adropin that is likely mediated via upregulation of endothelial NO synthase expression through the VEGFR2-phosphatidylinositol 3-kinase-Akt and VEGFR2-extracellular signal regulated kinase 1/2 pathways. Adropin represents a novel target to limit diseases characterized by endothelial dysfunction in addition to its favorable metabolic profile.
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Movimiento Celular , Proliferación Celular , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Proteínas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Silenciador del Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Morfolinas/farmacología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Altered macrophage kinetics is a pivotal mechanism of visceral obesity-induced inflammation and cardiometabolic risk. Because monocytes can differentiate into either proatherogenic M1 macrophages or anti-inflammatory M2 macrophages, approaches that limit M1 while promoting M2 differentiation represent a unique therapeutic strategy. We hypothesized that adiponectin may prime human monocytes toward the M2 phenotype. Adiponectin promoted the alternative activation of human monocytes into anti-inflammatory M2 macrophages as opposed to the classically activated M1 phenotype. Adiponectin-treated cells displayed increased M2 markers, including the mannose receptor (MR) and alternative macrophage activation-associated CC chemokine-1. Incubation of M1 macrophages with adiponectin-treated M2-derived culture supernatant resulted in a pronounced inhibition of tumor necrosis factor-alpha and monocyte chemotactic protein-1 secretion. Activation of human monocytes into M2 macrophages by adiponectin was mediated, in addition to AMP-activated protein kinase and peroxisome proliferator-activated receptor (PPAR)-gamma, via PPAR-alpha. Furthermore, macrophages isolated from adiponectin knockout mice demonstrated diminished levels of M2 markers such as MR, which were restored with adiponectin treatment. We report a novel immunoregulatory mechanism through which adiponectin primes human monocyte differentiation into anti-inflammatory M2 macrophages. Conditions associated with low adiponectin levels, such as visceral obesity and insulin resistance, may promote atherosclerosis, in part through aberrant macrophage kinetics.
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Adiponectina/metabolismo , Diferenciación Celular/fisiología , Activación de Macrófagos , Macrófagos/metabolismo , Monocitos/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Adiponectina/genética , Adiponectina/farmacología , Análisis de Varianza , Animales , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , PPAR gamma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Conventional haemodialysis (CHD) is associated with a reduction in vascular smooth muscle cells (VSMCs) proliferation and an increase in apoptosis. METHODS: VSMC proliferation, migration, apoptosis and Runx2 expression were assessed under normal conditions (n = 4) and before and after conversion from CHD to NHD (n = 15). RESULTS: Compared to normal, CHD is associated with a reduction in VSMC proliferation [0.49 +/- 0.07 (CHD) versus 1.34 +/- 0.02 RFU, P < 0.01], an augmented caspase-3 activity [0.30 +/- 0.02 (CHD) versus 0.22 +/- 0.02 RFU, P = 0.014] and a 1.4 +/- 0.3 fold increase in Runx2 expression. After conversion to NHD, VSMC proliferation was higher than during CHD [from 0.49 +/- 0.07 (CHD) to 0.68 +/- 0.09 RFU, P = 0.006] and approached that of controls (1.34 +/- 0.02, P > 0.05). Caspase-3 activity was restored to similar values as controls [0.26 +/- 0.02 (NHD) versus 0.22 +/- 0.04 (normal), P > 0.05]. Runx2 expression decreased to similar levels as normal controls. NHD enhanced dialysis dose delivery, lowered blood pressure, plasma parathyroid hormone levels and normalized plasma phosphate (from 1.7 +/- 0.1 to 1.2 +/- 0.1 mmol/L, P < 0.01). The reduction in plasma phosphate correlated with the change in VSMC proliferation (r = -0.71, P = 0.007). CONCLUSIONS: We demonstrate that NHD is associated with restoration of abnormal VSMC biology in ESRD. Given the increasing importance of VSMCs in the pathogenesis of atherosclerosis and medial calcification, these data may have important implications for vascular risk in ESRD patients.