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[Figure: see text].
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Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Inflamação/metabolismo , Estresse Oxidativo/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Remodelação Vascular/fisiologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismoRESUMO
Abdominal aortic aneurysm (AAA) is a common life-threatening condition characterized by exacerbated inflammation and the generation of reactive oxygen species. Pharmacological treatments to slow AAA progression or to prevent its rupture remain a challenge. Targeting phosphodiesterase 4 (PDE4) has been verified as an effective therapeutic strategy for an array of inflammatory conditions; however, no studies have assessed yet PDE4 in AAA. Here, we used angiotensin II (AngII)-infused apolipoprotein E deficient mice to study the involvement of the PDE4 subfamily in aneurysmal disease. PDE4B but not PDE4D was upregulated in inflammatory cells from both experimental and human AAA. The administration of the PDE4 selective inhibitor rolipram (3 mg/kg/day) to AngII-challenged mice (1000 ng/kg bodyweight/min) protected against AAA formation, limiting the progressive increase in the aortic diameter without affecting the blood pressure. The drug strongly attenuated the rise in vascular oxidative stress (superoxide anion) induced by AngII, and decreased the expression of inflammatory markers, as well as the recruitment of macrophages (MAC3+), lymphocytes (CD3+), and neutrophils (ELANE+) into the vessel wall. Rolipram also normalized the vascular MMP2 expression and MMP activity, preserving the elastin integrity and improving the vascular remodelling. These results point to PDE4B as a new therapeutic target for AAA.
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Lysyl oxidase (LOX) is an enzyme critically involved in collagen maturation, whose activity releases H2O2 as a by-product. Previous studies demonstrated that LOX over-expression enhances reactive oxygen species (ROS) production and exacerbates cardiac remodeling induced by pressure overload. However, whether LOX influences acute myocardial infarction and post-infarct left ventricular remodeling and the contribution of LOX to myocardial oxidative stress following ischemia-reperfusion have not been analyzed. Isolated hearts from transgenic mice over-expressing human LOX in the heart (TgLOX) and wild-type (WT) littermates were subjected to global ischemia and reperfusion. Although under basal conditions LOX transgenesis is associated with higher cardiac superoxide levels than WT mice, no differences in ROS production were detected in ischemic hearts and a comparable acute ischemia-reperfusion injury was observed (infarct size: 56.24 ± 9.44 vs. 48.63 ± 2.99% of cardiac weight in WT and TgLOX, respectively). Further, similar changes in cardiac dimensions and function were observed in TgLOX and WT mice 28 days after myocardial infarction induced by transient left anterior descending (LAD) coronary artery occlusion, and no differences in scar area were detected (20.29 ± 3.10 vs. 21.83 ± 2.83% of left ventricle). Our data evidence that, although LOX transgenesis induces baseline myocardial oxidative stress, neither ROS production, infarct size, nor post-infarction cardiac remodeling were exacerbated following myocardial ischemia-reperfusion.
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Abstract: Connexin 43 (Cx43) is essential for cardiac electrical coupling, but its effects on myocardial fibrosis is controversial. Here, we analyzed the role of Cx43 in myocardial fibrosis caused by angiotensin II (AngII) using Cx43fl/fl and Cx43Cre-ER(T)/fl inducible knock-out (Cx43 content: 50%) mice treated with vehicle or 4-hydroxytamoxifen (4-OHT) to induce a Cre-ER(T)-mediated global deletion of the Cx43 floxed allele. Myocardial collagen content was enhanced by AngII in all groups (n = 8-10/group, p < 0.05). However, animals with partial Cx43 deficiency (vehicle-treated Cx43Cre-ER(T)/fl) had a significantly higher AngII-induced collagen accumulation that reverted when treated with 4-OHT, which abolished Cx43 expression. The exaggerated fibrotic response to AngII in partially deficient Cx43Cre-ER(T)/fl mice was associated with enhanced p38 MAPK activation and was not evident in Cx43 heterozygous (Cx43+/-) mice. In contrast, normalization of interstitial collagen in 4-OHT-treated Cx43Cre-ER(T)/fl animals correlated with enhanced MMP-9 activity, IL-6 and NOX2 mRNA expression, and macrophage content, and with reduced ï¡-SMA and SM22ï¡ in isolated fibroblasts. In conclusion, our data demonstrates an exaggerated, p38 MAPK-dependent, fibrotic response to AngII in partially deficient Cx43Cre-ER(T)/fl mice, and a paradoxical normalization of collagen deposition in animals with an almost complete Cx43 ablation, an effect associated with increased MMP-9 activity and inflammatory response and reduced fibroblasts differentiation.
Assuntos
Angiotensina II/efeitos adversos , Cardiomiopatias/metabolismo , Diferenciação Celular , Conexina 43/deficiência , Fibroblastos/metabolismo , Miocárdio/metabolismo , Angiotensina II/farmacologia , Animais , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/genética , Cardiomiopatias/patologia , Conexina 43/metabolismo , Fibrose , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/patologiaRESUMO
Lysyl oxidases (LOX and LOX-likes (LOXLs) isoenzymes) belong to a family of copper-dependent enzymes classically involved in the covalent cross-linking of collagen and elastin, a pivotal process that ensures extracellular matrix (ECM) stability and provides the tensile and elastic characteristics of connective tissues. Besides this structural role, in the last years, novel biological properties have been attributed to these enzymes, which can critically influence cardiovascular function. LOX and LOXLs control cell proliferation, migration, adhesion, differentiation, oxidative stress, and transcriptional regulation and, thereby, their dysregulation has been linked to a myriad of cardiovascular pathologies. Lysyl oxidase could modulate virtually all stages of the atherosclerotic process, from endothelial dysfunction and plaque progression to calcification and rupture of advanced and complicated plaques, and contributes to vascular stiffness in hypertension. The alteration of LOX/LOXLs expression underlies the development of other vascular pathologies characterized by a destructive remodeling of the ECM, such as aneurysm and artery dissections, and contributes to the adverse myocardial remodeling and dysfunction in hypertension, myocardial infarction, and obesity. This review examines the most recent advances in the study of LOX and LOXLs biology and their pathophysiological role in cardiovascular diseases with special emphasis on their potential as therapeutic targets.
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Anti-Hipertensivos/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Sistema Cardiovascular/enzimologia , Proteína-Lisina 6-Oxidase/metabolismo , Animais , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Humanos , Proteína-Lisina 6-Oxidase/deficiência , Proteína-Lisina 6-Oxidase/genéticaRESUMO
Lysyl oxidase (LOX) plays a critical role in extracellular matrix maturation and limits VSMC proliferation and vascular remodeling. We have investigated whether this anti-proliferative effect relies on the extracellular catalytically active LOX or on its biologically active propeptide (LOX-PP). High expression levels of both LOX and LOX-PP were detected in the vascular wall from transgenic mice over-expressing the full-length human LOX cDNA under the control of SM22α promoter (TgLOX), which targets the transgene to VSMC without affecting the expression of mouse LOX isoenzymes. TgLOX VSMC also secrete high amounts of both mature LOX and LOX-PP. Wild-type (WT) mouse VSMC exposed to VSMC supernatants from transgenic animals showed reduced proliferative rates (low [3H]-thymidine uptake and expression of PCNA) than those incubated with conditioned media from WT cells, effect that was abrogated by ß-aminopropionitrile (BAPN), an inhibitor of LOX activity. Lentiviral over-expression of LOX, but not LOX-PP, decreased human VSMC proliferation, effect that was also prevented by BAPN. LOX transgenesis neither impacted local nor systemic inflammatory response induced by carotid artery ligation. Interestingly, in this model, BAPN normalized the reduced neointimal thickening observed in TgLOX mice. Therefore, extracellular enzymatically active LOX is required to limit both VSMC proliferation and vascular remodeling.
Assuntos
Músculo Liso Vascular/citologia , Neointima/metabolismo , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Aminopropionitrilo/farmacologia , Animais , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
Introducción: La lisil oxidasa (LOX) contribuye al ensamblaje de las fibras de colágeno y elastina de la matriz extracelular (MEC). Hemos determinado las consecuencias de la sobreexpresión vascular de LOX sobre la estructura de la MEC y su contribución al estrés oxidativo. Métodos: Los estudios se desarrollaron en ratones que sobreexpresan la LOX (Tg) específicamente en células musculares lisas vasculares (CMLV). Se realizaron análisis por PCR a tiempo real, tinción de rojo sirio, producción de H2O2 y actividad NADPH oxidasa. Se caracterizaron las fenestras de la lámina elástica interna mediante microscopia confocal. Resultados: Las CMLV de ratones transgénicos presentaron niveles de actividad LOX superiores a los de animales control. En consonancia, las células transgénicas depositaron más fibras de elastina organizada y sus sobrenadantes indujeron un mayor ensamblaje de colágeno en ensayos in vitro. El nivel de colágeno maduro fue superior en la pared vascular de ratones Tg, que presentaban una menor área de las fenestras y un aumento de la expresión de la fibulina-5. La producción vascular de H2O2 y la actividad NADPH oxidasa fueron superiores en los ratones transgénicos. La incubación de CMLV con catalasa atenuó el incremento en la deposición de fibras de elastina madura inducido por la transgénesis de LOX. Conclusiones: La sobreexpresión de la LOX en CMLV se asocia a una alteración de la estructura vascular del colágeno y la elastina. La LOX podría constituir una nueva fuente de estrés oxidativo que participaría en la alteración estructural de la MEC y podría contribuir al remodelado vascular (AU)
Introduction: Lysyl oxidase (LOX) participates in the assembly of collagen and elastin fibres. The impact of vascular LOX over-expression on extracellular matrix (ECM) structure and its contribution to oxidative stress has been analysed. Methods: Studies were conducted on mice over-expressing LOX (Tg), specifically in smooth muscle cells (VSMC). Gene expression was assessed by real-time PCR analysis. Sirius Red staining, H2O2 production and NADPH oxidase activity were analysed in different vascular beds. The size and number of fenestra of the internal elastic lamina were determined by confocal microscopy. Results: LOX activity was up-regulated in VSMC of transgenic mice compared with cells from control animals. At the same time, transgenic cells deposited more organised elastin fibres and their supernatants induced a stronger collagen assembly in in vitro assays. Vascular collagen cross-linking was also higher in Tg mice, which showed a decrease in the size of fenestrae and an enhanced expression of Fibulin-5. Interestingly, higher H2O2 production and NADPH oxidase activity was detected in the vascular wall from transgenic mice. The H2O2 scavenger catalase attenuated the stronger deposition of mature elastin fibres induced by LOX transgenesis. Conclusions: LOX over-expression in VSMC was associated with a change in the structure of collagen and elastin fibres. LOX could constitute a novel source of oxidative stress that might participate in elastin changes and contribute to vascular remodeling (AU)
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Animais , Ratos , Proteína-Lisina 6-Oxidase , Estresse Oxidativo/fisiologia , Miócitos de Músculo Liso/fisiologia , Matriz Extracelular/fisiologia , Colágeno , Elastina , Técnicas In Vitro , Remodelação Vascular/fisiologia , Transgenes/fisiologiaRESUMO
INTRODUCTION: Lysyl oxidase (LOX) participates in the assembly of collagen and elastin fibres. The impact of vascular LOX over-expression on extracellular matrix (ECM) structure and its contribution to oxidative stress has been analysed. METHODS: Studies were conducted on mice over-expressing LOX (Tg), specifically in smooth muscle cells (VSMC). Gene expression was assessed by real-time PCR analysis. Sirius Red staining, H2O2 production and NADPH oxidase activity were analysed in different vascular beds. The size and number of fenestra of the internal elastic lamina were determined by confocal microscopy. RESULTS: LOX activity was up-regulated in VSMC of transgenic mice compared with cells from control animals. At the same time, transgenic cells deposited more organised elastin fibres and their supernatants induced a stronger collagen assembly in in vitro assays. Vascular collagen cross-linking was also higher in Tg mice, which showed a decrease in the size of fenestrae and an enhanced expression of Fibulin-5. Interestingly, higher H2O2 production and NADPH oxidase activity was detected in the vascular wall from transgenic mice. The H2O2 scavenger catalase attenuated the stronger deposition of mature elastin fibres induced by LOX transgenesis. CONCLUSIONS: LOX over-expression in VSMC was associated with a change in the structure of collagen and elastin fibres. LOX could constitute a novel source of oxidative stress that might participate in elastin changes and contribute to vascular remodelling.
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Proteínas da Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Estresse Oxidativo/fisiologia , Proteína-Lisina 6-Oxidase/genética , Animais , Colágeno/metabolismo , Elastina/metabolismo , Matriz Extracelular/genética , Regulação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Remodelação Vascular/genética , Remodelação Vascular/fisiologiaRESUMO
Lysyl oxidase (LOX) controls matrix remodeling, a key process that underlies cardiovascular diseases and heart failure; however, a lack of suitable animal models has limited our knowledge with regard to the contribution of LOX to cardiac dysfunction. Here, we assessed the impact of LOX overexpression on ventricular function and cardiac hypertrophy in a transgenic LOX (TgLOX) mouse model with a strong cardiac expression of human LOX. TgLOX mice exhibited high expression of the transgene in cardiomyocytes and cardiofibroblasts, which are associated with enhanced LOX activity and H2O2 production and with cardiofibroblast reprogramming. LOX overexpression promoted an age-associated concentric remodeling of the left ventricle and impaired diastolic function. Furthermore, LOX transgenesis aggravated angiotensin II (Ang II)-induced cardiac hypertrophy and dysfunction, which triggered a greater fibrotic response that was characterized by stronger collagen deposition and cross-linking and high expression of fibrotic markers. In addition, LOX transgenesis increased the Ang II-induced myocardial inflammatory infiltrate, exacerbated expression of proinflammatory markers, and decreased that of cardioprotective factors. Mechanistically, LOX overexpression enhanced oxidative stress and potentiated the Ang II-mediated cardiac activation of p38 MAPK while reducing AMPK activation. Our findings suggest that LOX induces an age-dependent disturbance of diastolic function and aggravates Ang II-induced hypertrophy, which provides novel insights into the role of LOX in cardiac performance.-Galán, M., Varona, S., Guadall, A., Orriols, M., Navas, M., Aguiló, S., de Diego, A., Navarro, M. A., García-Dorado, D., Rodríguez-Sinovas, A., Martínez-González, J., Rodriguez, C. Lysyl oxidase overexpression accelerates cardiac remodeling and aggravates angiotensin II-induced hypertrophy.
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Angiotensina II/farmacologia , Cardiomegalia/induzido quimicamente , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteína-Lisina 6-Oxidase/metabolismo , Remodelação Ventricular/fisiologia , Animais , Cardiomegalia/enzimologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Inflamação , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Proteína-Lisina 6-Oxidase/genética , Transdução de SinaisRESUMO
AIMS: Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H2O2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. RESULTS: Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with ß-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H2O2 and O2.- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H2O2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. INNOVATION: We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. CONCLUSION: LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27, 379-397.
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Proteínas da Matriz Extracelular/metabolismo , Hipertensão/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Transdução de Sinais , Rigidez Vascular , Animais , Modelos Animais de Doenças , Elastina/química , Proteínas da Matriz Extracelular/genética , Hipertensão/genética , Masculino , Camundongos , Estresse Oxidativo , Proteína-Lisina 6-Oxidase/genética , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Introducción: La fibulina-5 (FBLN5) es una proteína elastogénica implicada en el remodelado de la matriz extracelular (MEX), un proceso fundamental en el aneurisma de aorta abdominal (AAA). Sin embargo, no se ha determinado la posible contribución de la FBLN5 al AAA. Métodos: Se realizaron análisis por PCR a tiempo real, Western blot, transducción lentiviral, transfección transitoria e inmunoprecipitación de cromatina (ChIP) en aorta abdominal de pacientes con AAA o donantes y en células musculares lisas de aorta humana (CMLV). Resultados: La expresión vascular de la FBLN5 disminuye en la aorta abdominal de pacientes con AAA frente a donantes sanos. El nivel de ARNm y proteína de la FBLN5 y su secreción al espacio extracelular se redujeron en CMLV expuestas a estímulos inflamatorios. Este efecto se produce a través de un mecanismo transcripcional en el que está implicada una región proximal del promotor de la FBLN5 que contiene un elemento de respuesta a SOX. De hecho, la expresión de SOX9 se inhibe en CMLV tratadas con LPS y TNFα y disminuye en el AAA, en el que correlaciona con la de la FBLN5. Además, la sobreexpresión de SOX9 contrarrestó la disminución de la expresión y actividad transcripcional de la FBLN5 inducida por el TNFα. Finalmente, observamos que SOX9 interacciona con el promotor de la FBLN5 y que esta unión se reduce en respuesta a TNFα. Conclusiones: La inhibición de la FBLN5 en el AAA humano podría contribuir al remodelado destructivo de la matriz extracelular inducido por el componente inflamatorio de la patología
Introduction: Fibulin-5 (FBLN5) is an elastogenic protein critically involved in extracellular matrix (ECM) remodelling, a key process in abdominal aortic aneurysm (AAA). However, the possible contribution of FBLN5 to AAA development has not been addressed. Methods: Expression levels were determined by real-time PCR and Western blot in human abdominal aorta from patients with AAA or healthy donors, as well as in human aortic vascular smooth muscle cells (VSMC). Lentiviral transduction, transient transfections, and chromatin immunoprecipitation (ChIP) assays were also performed. Results: The expression of FBLN5 in human AAA was significantly lower than in healthy donors. FBLN5 mRNA and protein levels and their secretion to the extracellular environment were down-regulated in VSMC exposed to inflammatory stimuli. Interestingly, FBLN5 transcriptional activity was inhibited by TNFα and lipopolysaccharide (LPS), and depends on a SOX response element. In fact, SOX9 expression was reduced in VMSC induced by inflammatory mediators and in human AAA, and correlated with that of FBLN5. Furthermore, SOX9 over-expression limited the reduction of FBLN5 expression induced by cytokines in VSMC. Finally, it was observed that SOX9 interacts with FBLN5 promoter, and that this binding was reduced upon TNFα exposure. Conclusions: FBLN5 downregulation in human AAA could contribute to extracellular matrix remodelling induced by the inflammatory component of the disease
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Humanos , Inflamação/fisiopatologia , Aneurisma da Aorta Abdominal/fisiopatologia , Mediadores da Inflamação/análise , Matriz Extracelular , Fatores de Transcrição SOX9/fisiologia , Imuno-Histoquímica/métodos , Reação em Cadeia da Polimerase , LentivirusRESUMO
INTRODUCTION: Fibulin-5 (FBLN5) is an elastogenic protein critically involved in extracellular matrix (ECM) remodelling, a key process in abdominal aortic aneurysm (AAA). However, the possible contribution of FBLN5 to AAA development has not been addressed. METHODS: Expression levels were determined by real-time PCR and Western blot in human abdominal aorta from patients with AAA or healthy donors, as well as in human aortic vascular smooth muscle cells (VSMC). Lentiviral transduction, transient transfections, and chromatin immunoprecipitation (ChIP) assays were also performed. RESULTS: The expression of FBLN5 in human AAA was significantly lower than in healthy donors. FBLN5 mRNA and protein levels and their secretion to the extracellular environment were down-regulated in VSMC exposed to inflammatory stimuli. Interestingly, FBLN5 transcriptional activity was inhibited by TNFα and lipopolysaccharide (LPS), and depends on a SOX response element. In fact, SOX9 expression was reduced in VMSC induced by inflammatory mediators and in human AAA, and correlated with that of FBLN5. Furthermore, SOX9 over-expression limited the reduction of FBLN5 expression induced by cytokines in VSMC. Finally, it was observed that SOX9 interacts with FBLN5 promoter, and that this binding was reduced upon TNFα exposure. CONCLUSIONS: FBLN5 downregulation in human AAA could contribute to extracellular matrix remodelling induced by the inflammatory component of the disease.
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Aneurisma da Aorta Abdominal/patologia , Proteínas da Matriz Extracelular/genética , Inflamação/patologia , Fatores de Transcrição SOX9/genética , Aorta/metabolismo , Aneurisma da Aorta Abdominal/genética , Western Blotting , Estudos de Casos e Controles , Imunoprecipitação da Cromatina/métodos , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS: Destructive remodelling of extracellular matrix (ECM) and inflammation lead to dilation and ultimately abdominal aortic aneurysm (AAA). Fibulin-5 (FBLN5) mediates cell-ECM interactions and elastic fibre assembly and is critical for ECM remodelling. We aimed to characterize FBLN5 regulation in human AAA and analyse the underlying mechanisms. METHODS AND RESULTS: FBLN5 expression was significantly decreased in human aneurysmatic aortas compared with healthy vessels. Local FBLN5 knockdown promoted aortic dilation and enhanced vascular expression of inflammatory markers in Ang II-infused C57BL/6J mice. Inflammatory stimuli down-regulated FBLN5 expression and transcriptional activity in human aortic vascular smooth muscle cells (VSMC). Further, aortic FBLN5 expression was reduced in LPS-challenged mice. A SOX response element was critical for FBLN5 promoter activity. The SOX9 expression pattern in human AAA parallels that of FBLN5, and like FBLN5, it was reduced in TNFα-stimulated VSMC. Interestingly, SOX9 over-expression prevented the cytokine-mediated reduction of FBLN5 expression and transcription. The inhibition of Class I histone deacetylases (HDACs) by MS-275 or gene silencing attenuated the inflammation-mediated decrease of FBLN5 expression in VSMC and in the vascular wall. Consistently, HDAC inhibition counteracted the reduction of SOX9 expression induced by inflammatory stimuli and prevented the TNFα-mediated decrease in the binding of SOX9 to FBLN5 promoter normalizing FBLN5 expression. CONCLUSION: We evidence the deregulation of FBLN5 in human AAA and identify a SOX9/HDAC-dependent mechanism involved in the down-regulation of FBLN5 by inflammation. HDAC inhibitors or pharmacological approaches that aimed to preserve FBLN5 could be useful to prevent the disorganization of ECM induced by inflammation in AAA.
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Aneurisma da Aorta Abdominal/metabolismo , Epigênese Genética , Proteínas da Matriz Extracelular/metabolismo , Mediadores da Inflamação/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Angiotensina II , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Sítios de Ligação , Estudos de Casos e Controles , Células Cultivadas , Dilatação Patológica , Modelos Animais de Doenças , Regulação para Baixo , Epigênese Genética/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Regiões Promotoras Genéticas , Interferência de RNA , Proteínas Recombinantes/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Clinical management of abdominal aortic aneurysm (AAA) is currently limited to elective surgical repair because an effective pharmacotherapy is still awaited. Inhibition of histone deacetylase (HDAC) activity could be a promising therapeutic option in cardiovascular diseases. We aimed to characterise HDAC expression in human AAA and to evaluate the therapeutic potential of class I and IIa HDAC inhibitors in the AAA model of angiotensin II (Ang II)-infused apolipoprotein-E-deficient (ApoE(-/-)) mice. Real-time PCR, western blot and immunohistochemistry evidenced an increased expression of HDACs 1, 2 (both class I), 4 and 7 (both class IIa) in abdominal aorta samples from patients undergoing AAA open repair (n=22) compared with those from donors (n=14). Aortic aneurysms from Ang-II-infused ApoE(-/-) mice exhibited a similar HDAC expression profile. In these animals, treatment with a class I HDAC inhibitor (MS-275) or a class IIa inhibitor (MC-1568) improved survival, reduced the incidence and severity of AAA and limited aneurysmal expansion evaluated by Doppler ultrasonography. These beneficial effects were more potent in MC-1568-treated mice. The disorganisation of elastin and collagen fibres and lymphocyte and macrophage infiltration were effectively reduced by both inhibitors. Additionally, HDAC inhibition attenuated the exacerbated expression of pro-inflammatory markers and the increase in metalloproteinase-2 and -9 activity induced by Ang II in this model. Therefore, our data evidence that HDAC expression is deregulated in human AAA and that class-selective HDAC inhibitors limit aneurysm expansion in an AAA mouse model. New-generation HDAC inhibitors represent a promising therapeutic approach to overcome human aneurysm progression.
Assuntos
Aneurisma da Aorta Abdominal/tratamento farmacológico , Aneurisma da Aorta Abdominal/enzimologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/biossíntese , Angiotensina II , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Biomarcadores/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Elastina/metabolismo , Indução Enzimática/efeitos dos fármacos , Feminino , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Inflamação/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Regulação para Cima/efeitos dos fármacosRESUMO
Extracellular matrix (ECM) remodelling of the adipose tissue plays a pivotal role in the pathophysiology of obesity. The lysyl oxidase (LOX) family of amine oxidases, including LOX and LOX-like (LOXL) isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its involvement in adipose tissue dysfunction in obesity is unclear. In this study, we observed that LOX is the main isoenzyme expressed in human adipose tissue and that its expression is strongly upregulated in samples from obese individuals that had been referred to bariatric surgery. LOX expression was also induced in the adipose tissue from male Wistar rats fed a high-fat diet (HFD). Interestingly, treatment with ß-aminopropionitrile (BAPN), a specific and irreversible inhibitor of LOX activity, attenuated the increase in body weight and fat mass that was observed in obese animals and shifted adipocyte size toward smaller adipocytes. BAPN also ameliorated the increase in collagen content that was observed in adipose tissue from obese animals and improved several metabolic parameters - it ameliorated glucose and insulin levels, decreased homeostasis model assessment (HOMA) index and reduced plasma triglyceride levels. Furthermore, in white adipose tissue from obese animals, BAPN prevented the downregulation of adiponectin and glucose transporter 4 (GLUT4), as well as the increase in suppressor of cytokine signaling 3 (SOCS3) and dipeptidyl peptidase 4 (DPP4) levels, triggered by the HFD. Likewise, in the TNFα-induced insulin-resistant 3T3-L1 adipocyte model, BAPN prevented the downregulation of adiponectin and GLUT4 and the increase in SOCS3 levels, and consequently normalised insulin-stimulated glucose uptake. Therefore, our data provide evidence that LOX plays a pathologically relevant role in the metabolic dysfunction induced by obesity and emphasise the interest of novel pharmacological interventions that target adipose tissue fibrosis and LOX activity for the clinical management of this disease.
Assuntos
Aminopropionitrilo/farmacologia , Aminopropionitrilo/uso terapêutico , Metaboloma/efeitos dos fármacos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Aumento de Peso/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adiponectina/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Tamanho Celular/efeitos dos fármacos , Colágeno/metabolismo , Dieta Hiperlipídica , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Fibrose , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos , Modelos Biológicos , Proteína-Lisina 6-Oxidase/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that seems to play a critical role in vascular remodelling. However, the lack of viable LOX-deficient animal models has been an obstacle to deep in LOX biology. In this study we have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated by conventional methods (TgLOX). Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. Interestingly, cell proliferation was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of Myh10 (marker of SMC phenotypic switching), PCNA (marker of cell proliferation) and MCP-1, and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA and MCP-1 immunostaining. Our results give evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.