RESUMEN
Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14(+) Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14(+) Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca(2+)-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca(2+) ionophore. Exposure of Mac to increasing concentrations of IFNγ (5-100 ng/ml) or oxidized LDL (5-100 µg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca(2+) concentration. Immunohistochemical staining revealed that Nox5 is present in CD68(+) Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis.
Asunto(s)
Aterosclerosis/enzimología , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Monocitos/enzimología , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Humanos , NADPH Oxidasa 5RESUMEN
High glucose induces vascular smooth muscle cell (SMC) dysfunction by generating oxidative stress attributable, in part, to the up-regulated NADPH oxidases (Nox). We have attempted to elucidate the high-glucose-generated molecular signals that mediate this effect and hypothesize that products of high-glucose-induced lipid peroxidation regulate Nox by activating peroxisome proliferator-activated receptors (PPARs). Human aortic SMCs were exposed to glucose (5.5-25 mM) or 4-hydroxynonenal (1-25 µM, 4-HNE). Lucigenin assay, real-time polymerase chain reaction, western blot, and promoter analyses were employed to investigate Nox. We found that high glucose generated an increase in Nox activity and expression. It also promoted oxidative stress that consequently induced lipid peroxidation, which resulted in the production of 4-HNE. Pharmacological inhibition of Nox activity significantly reduced the formation of high-glucose-induced 4-HNE. Exposure of SMCs to non-cytotoxic concentrations (1-10 µM) of 4-HNE alone mimicked the effect of high glucose incubation, whereas scavenging of 4-HNE by N-acetyl L-cysteine completely abolished both the effects of high glucose and 4-HNE. The latter exerted its effect by activating PPARα and PPARß/δ, but not PPARγ, as assessed pharmacologically by the inhibitory effect of selective antagonists and following the silencing of the expression of these receptors. These new data indicate that 4-HNE, generated following Nox activation, functions as an endogenous activator of PPARα and PPARß/δ. The newly discovered "lipid peroxidation products-PPARs-Nox axis" represents a novel mechanism of Nox regulation and an additional therapeutic target for oxidative stress in diabetes.
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Aldehídos/metabolismo , Glucosa/metabolismo , Músculo Liso Vascular/citología , NADPH Oxidasas/metabolismo , PPAR alfa/metabolismo , PPAR delta/metabolismo , PPAR-beta/metabolismo , Aorta/citología , Aorta/metabolismo , Línea Celular , Proliferación Celular , Activación Enzimática , Humanos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasas/genética , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
In atherosclerosis, oxidative stress-induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by up-regulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer-binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to pro-inflammatory conditions. Human aortic SMCs were treated with interferon-γ (IFN-γ) for up to 24 hrs. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN-γ dose-dependently induced Nox activity and expression, nuclear translocation and up-regulation of C/EBPα, C/EBPß and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPß or C/EBPδ reduced significantly but differentially the IFN-γ-induced up-regulation of Nox activity, gene and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4 and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPß or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPß and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN-γ-exposed SMCs. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP-related signalling pathways may be used to counteract the adverse effects of oxidative stress.
Asunto(s)
Aorta/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Músculo Liso Vascular/metabolismo , NADPH Oxidasas/genética , Regiones Promotoras Genéticas/genética , Aorta/citología , Aorta/efectos de los fármacos , Western Blotting , Proteínas Potenciadoras de Unión a CCAAT/genética , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Impedancia Eléctrica , Humanos , Interferón gamma/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transcripción Genética , Activación TranscripcionalRESUMEN
Dysregulated epigenetic mechanisms promote transcriptomic and phenotypic alterations in cardiovascular diseases. The role of histone methylation-related pathways in atherosclerosis is largely unknown. We hypothesize that lysine-specific demethylase 1A (LSD1/KDM1A) regulates key molecular effectors and pathways linked to atherosclerotic plaque formation. Human non-atherosclerotic and atherosclerotic tissue specimens, ApoE-/- mice, and in vitro polarized macrophages (Mac) were examined. Male ApoE-/- mice fed a normal/atherogenic diet were randomized to receive GSK2879552, a highly specific LSD1 inhibitor, or its vehicle, for 4 weeks. The mRNA and protein expression levels of LSD1/KDM1A were significantly elevated in atherosclerotic human carotid arteries, atherosclerotic aortas of ApoE-/- mice, and M1-Mac. Treatment of ApoE-/- mice with GSK2879552 significantly reduced the extent of atherosclerotic lesions and the aortic expression of NADPH oxidase subunits (Nox1/2/4, p22phox) and 4-hydroxynonenal-protein adducts. Concomitantly, the markers of immune cell infiltration and vascular inflammation were significantly decreased. LSD1 blockade down-regulated the expression of genes associated with Mac pro-inflammatory phenotype. Nox subunit transcript levels were significantly elevated in HEK293 reporter cells overexpressing LSD1. In experimental atherosclerosis, LSD1 mediates the up-regulation of molecular effectors connected to oxidative stress and inflammation. Together, these data indicate that LSD1-pharmacological interventions are novel targets for supportive therapeutic strategies in atherosclerosis.
RESUMEN
The study aim was to investigate NOS3 VNTR, NOS3 G894T, EDN1 C8002T, ACE I/D, AGT M235T and AGTR1 A1166C in nonobese and obese T2DM patients, and their interaction with the incidence of microangiopathy. T2DM subjects (n=250; 166 nonobese, and 84 obese) were genotyped for the gene variants by PCR/RFLP. The interaction of these polymorphisms with obesity and their contribution to microangiopathy were analyzed by multivariate regression analysis. A higher frequency of NOS3 4a allele was found in obese (P=0.027) vs. nonobese subjects. ACE D (P=0.009) and AGT 235T (P=0.026) alleles were associated with the reduced risk of diabetic nephropathy in nonobese and obese patients, respectively. In obese subjects, NOS3 4a (P=0.011) had a converse effect to NOS3 894T (P=0.043), and EDN1 8002T (P=0.035) on the prevalence of combined microangiopathy (neuropathy/retinopathy/nephropathy) vs. microangiopathy-negative subjects. The study indicates association of RAS variants with obesity and nephropathy, and an opposite effect of NOS3 VNTR and NOS3 G894T on the occurrence of combined microangiopathy.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Angiopatías Diabéticas/genética , Endotelina-1/genética , Óxido Nítrico Sintasa de Tipo III/genética , Obesidad/complicaciones , Proteínas ras/genética , Adulto , Anciano , Anciano de 80 o más Años , Angiopatías Diabéticas/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Receptor de Angiotensina Tipo 1/genéticaRESUMEN
Accumulating evidence implicates the histone acetylation-based epigenetic mechanisms in the pathoetiology of diabetes-associated micro-/macrovascular complications. Diabetic kidney disease (DKD) is a progressive chronic inflammatory microvascular disorder ultimately leading to glomerulosclerosis and kidney failure. We hypothesized that histone acetyltransferase p300/CBP may be involved in mediating diabetes-accelerated renal damage. In this study, we aimed at investigating the potential role of p300/CBP in the up-regulation of renal NADPH oxidase (Nox), reactive oxygen species (ROS) production, inflammation, and fibrosis in diabetic mice. Diabetic C57BL/6J mice were randomized to receive 10 mg/kg C646, a selective p300/CBP inhibitor, or its vehicle for 4 weeks. We found that in the kidney of C646-treated diabetic mice, the level of H3K27ac, an epigenetic mark of active gene expression, was significantly reduced. Pharmacological inhibition of p300/CBP significantly down-regulated the diabetes-induced enhanced expression of Nox subtypes, pro-inflammatory, and pro-fibrotic molecules in the kidney of mice, and the glomerular ROS overproduction. Our study provides evidence that the activation of p300/CBP enhances ROS production, potentially generated by up-regulated Nox, inflammation, and the production of extracellular matrix proteins in the diabetic kidney. The data suggest that p300/CBP-pharmacological inhibitors may be attractive tools to modulate diabetes-associated pathological processes to efficiently reduce the burden of DKD.
RESUMEN
Excessive production of reactive oxygen species (ROS) and the ensuing oxidative stress are instrumental in all phases of atherosclerosis. Despite the major achievements in understanding the regulatory pathways and molecular sources of ROS in the vasculature, the specific detection and quantification of ROS in experimental models of disease remain a challenge. We aimed to develop a reliable and straightforward imaging procedure to interrogate the ROS overproduction in the vasculature and in various organs/tissues in atherosclerosis. To this purpose, the cell-impermeant ROS Brite™ 700 (RB700) probe that produces bright near-infrared fluorescence upon ROS oxidation was encapsulated into VCAM-1-targeted, sterically stabilized liposomes (VLp). Cultured human endothelial cells (EC) and macrophages (Mac) were used for in vitro experiments. C57BL6/J and ApoE-/- mice were randomized to receive normal or high-fat, cholesterol-rich diet for 10 or 32 weeks. The mice received a retroorbital injection with fluorescent tagged VLp incorporating RB700 (VLp-RB700). After two hours, the specific signals of the oxidized RB700 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DSPE), inserted into liposome bilayers, were measured ex vivo in the mouse aorta and various organs by high-resolution fluorescent imaging. VLp-RB700 was efficiently taken up by cultured human EC and Mac, as confirmed by fluorescence microscopy and spectrofluorimetry. After systemic administration in atherosclerotic ApoE-/- mice, VLp-RB700 were efficiently concentrated at the sites of aortic lesions, as indicated by the augmented NBD fluorescence. Significant increases in oxidized RB700 signal were detected in the aorta and in the liver and kidney of atherosclerotic ApoE-/- mice. RB700 encapsulation into sterically stabilized VCAM-1-sensitive Lp could be a novel strategy for the qualitative and quantitative detection of ROS in the vasculature and various organs and tissues in animal models of disease. The accurate and precise detection of ROS in experimental models of disease could ease the translation of the results to human pathologies.
Asunto(s)
Aorta/patología , Aterosclerosis/patología , Colorantes Fluorescentes/química , Imagen Óptica , Especies Reactivas de Oxígeno/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Apolipoproteínas E/deficiencia , Muerte Celular , Fluorescencia , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/química , Microscopía Intravital , Hierro/química , Liposomas , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Oxidación-Reducción , Estrés Oxidativo , Espectroscopía Infrarroja Corta , Células THP-1 , Tirosina/análogos & derivados , Tirosina/metabolismo , Regulación hacia ArribaRESUMEN
Emerging evidence demonstrates the involvement of endothelin-1 (ET-1) in the pathophysiology of cardiovascular disorders associated with diabetes mellitus. The molecular mechanisms accountable for the increased production of ET-1 are not completely defined. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is an essential pathogenic mechanism leading to endothelial cell dysfunction. Our aim has been to investigate the role of JAK/STAT in the regulation of ET-1 synthesis in human endothelial cells (EAhy926 cells line). EAhy926 cells were exposed to normal (5 mM) or high (25 mM) glucose concentrations in the presence/absence of various JAK/STAT inhibitors. Using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gene reporter assay, we found that JAK/STAT inhibitors (STAT1 decoy oligodeoxynucleotides, AG490, S3I201, WP1066) significantly diminished the high-glucose-dependent up-regulation of ET-1 mRNA, peptide synthesis, and promoter activity. In silico analysis of the human ET-1 promoter revealed the presence of typical STAT1-gamma-activated sequence (STAT1-GAS) elements. Transient overexpression of STAT1 indicated an up-regulation of ET-1 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1 proteins with the predicted GAS sites. Regulation of ET-1 synthesis by the JAK/STAT pathway thus represents a novel mechanism by which high glucose induces endothelial cell dysfunction in diabetes. Since the JAK/STAT system is an important regulator of the response of endothelial cells to injury, the modulation of this system and the subsequent decrease in ET-1 level may represent a key pharmacological target in diabetes-associated cardiovascular disorders.
Asunto(s)
Células Endoteliales/metabolismo , Endotelina-1/biosíntesis , Hiperglucemia/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/fisiología , Células Cultivadas , Angiopatías Diabéticas/genética , Angiopatías Diabéticas/metabolismo , Angiopatías Diabéticas/fisiopatología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Endotelina-1/genética , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Hiperglucemia/fisiopatología , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Factores de Transcripción STAT/antagonistas & inhibidores , Factores de Transcripción STAT/genética , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are instrumental in all inflammatory phases of atherosclerosis. Dysregulated histone deacetylase (HDAC)-related epigenetic pathways have been mechanistically linked to alterations in gene expression in experimental models of cardiovascular disorders. Hitherto, the relation between HDAC and Nox in atherosclerosis is not known. We aimed at uncovering whether HDAC plays a role in mediating Nox up-regulation, oxidative stress, inflammation, and atherosclerotic lesion progression. Human non-atherosclerotic and atherosclerotic arterial samples, ApoE-/- mice, and in vitro polarized monocyte-derived M1/M2-macrophages (Mac) were examined. Male ApoE-/- mice, maintained on normal or high-fat, cholesterol-rich diet, were randomized to receive 10â¯mg/kg suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, or its vehicle, for 4 weeks. In the human/animal studies, real-time PCR, Western blot, lipid staining, lucigenin-enhanced chemiluminescence assay, and enzyme-linked immunosorbent assay were employed. The protein levels of class I, class IIa, class IIb, and class IV HDAC isoenzymes were significantly elevated both in human atherosclerotic tissue samples and in atherosclerotic aorta of ApoE-/- mice. Treatment of ApoE-/- mice with SAHA reduced significantly the extent of atherosclerotic lesions, and the aortic expression of Nox subtypes, NADPH-stimulated ROS production, oxidative stress and pro-inflammatory markers. Significantly up-regulated HDAC and Nox subtypes were detected in inflammatory M1-Mac. In these cells, SAHA reduced the Nox1/2/4 transcript levels. Collectively, HDAC inhibition reduced atherosclerotic lesion progression in ApoE-/- mice, possibly by intertwined mechanisms involving negative regulation of Nox expression and inflammation. The data propose that HDAC-oriented pharmacological interventions could represent an effective therapeutic strategy in atherosclerosis.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , NADPH Oxidasas/genética , Estrés Oxidativo/efectos de los fármacos , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Biopsia , LDL-Colesterol/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Epigénesis Genética , Humanos , Masculino , Ratones , Ratones Noqueados , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/etiología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages express functional NADPH oxidase 5 (Nox5) that contributes to oxidative stress in atherogenesis. The mechanisms of Nox5 regulation are not entirely elucidated. The aim of this study was to investigate the expression pattern of key histone acetyltransferase subtypes (p300, HAT1) in human atherosclerosis and to determine their role in mediating the upregulation of Nox5 in macrophages under inflammatory conditions. Human nonatherosclerotic and atherosclerotic tissue samples were collected in order to determine the expression of p300 and HAT1 isoforms, H3K27ac, and Nox5. In vitro determinations were done on human macrophages exposed to lipopolysaccharide in the absence or presence of histone acetyltransferase inhibitors. Western blot, immunohistochemistry, immunofluorescence, real-time PCR, transfection, and chromatin immunoprecipitation assay were employed. The protein levels of p300 and HAT1 isoforms, H3K27ac, and Nox5 were found significantly elevated in human atherosclerotic specimens. Immunohistochemistry/immunofluorescence staining revealed that p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins were colocalized in the area of CD45+/CD68+ immune cells and lipid-rich deposits within human atherosclerotic plaques. Lipopolysaccharide induced the levels of HAT1, H3K27ac, H3K9ac, and Nox5 and the recruitment of p300 and HAT1 at the sites of active transcription within Nox5 gene promoter in cultured human macrophages. Pharmacological inhibition of histone acetyltransferase significantly reduced the Nox5 gene and protein expression in lipopolysaccharide-challenged macrophages. The overexpression of p300 or HAT1 enhanced the Nox5 gene promoter activity. The histone acetyltransferase system is altered in human atherosclerosis. Under inflammatory conditions, HAT subtypes control Nox5 overexpression in cultured human macrophages. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Histona Acetiltransferasas/metabolismo , Macrófagos/enzimología , NADPH Oxidasa 5/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Proteína p300 Asociada a E1A/genética , Epigénesis Genética , Histona Acetiltransferasas/genética , Histonas/biosíntesis , Histonas/genética , Histonas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/patología , NADPH Oxidasa 5/biosíntesis , NADPH Oxidasa 5/genética , Células THP-1 , Transfección , Regulación hacia ArribaRESUMEN
Reactive oxygen species (ROS) generated by up-regulated NADPH oxidase (Nox) contribute to structural-functional alterations of the vascular wall in diabetes. Epigenetic mechanisms, such as histone acetylation, emerged as important regulators of gene expression in cardiovascular disorders. Since their role in diabetes is still elusive we hypothesized that histone deacetylase (HDAC)-dependent mechanisms could mediate vascular Nox overexpression in diabetic conditions. Non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were randomized to receive vehicle or suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor. In vitro studies were performed on a human aortic smooth muscle cell (SMC) line. Aortic SMCs typically express Nox1, Nox4, and Nox5 subtypes. HDAC1 and HDAC2 proteins along with Nox1, Nox2, and Nox4 levels were found significantly elevated in the aortas of diabetic mice compared to non-diabetic animals. Treatment of diabetic mice with SAHA mitigated the aortic expression of Nox1, Nox2, and Nox4 subtypes and NADPH-stimulated ROS production. High concentrations of glucose increased HDAC1 and HDAC2 protein levels in cultured SMCs. SAHA significantly reduced the high glucose-induced Nox1/4/5 expression, ROS production, and the formation malondialdehyde-protein adducts in SMCs. Overexpression of HDAC2 up-regulated the Nox1/4/5 gene promoter activities in SMCs. Physical interactions of HDAC1/2 and p300 proteins with Nox1/4/5 promoters were detected at the sites of active transcription. High glucose induced histone H3K27 acetylation enrichment at the promoters of Nox1/4/5 genes in SMCs. The novel data of this study indicate that HDACs mediate vascular Nox up-regulation in diabetes. HDAC inhibition reduces vascular ROS production in experimental diabetes, possibly by a mechanism involving negative regulation of Nox expression.
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Diabetes Mellitus Experimental/genética , NADPH Oxidasa 1/genética , NADPH Oxidasa 4/genética , NADPH Oxidasa 5/genética , Animales , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/patología , Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Histona Desacetilasas/genética , Humanos , Ratones , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Oxidación-Reducción , Regiones Promotoras Genéticas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genéticaRESUMEN
Endothelin-1 (ET-1) plays an important role in the pathophysiology of diabetes-associated cardiovascular disorders. The molecular mechanisms leading to ET-1 upregulation in diabetes are not entirely defined. c-Src tyrosine kinase regulates important pathophysiological aspects of vascular response to insults. In this study, we aimed to elucidate whether high glucose-activated c-Src signaling plays a role in the regulation of ET-1 expression. Human endothelial cells EAhy926 (ECs) were exposed to normal or high levels of glucose for 24h. Male C57BL/6J mice were rendered diabetic with streptozotocin and then treated with a specific c-Src inhibitor (Src I1) or c-Src siRNA. Real-time PCR, Western blot, and ELISA, were used to investigate ET-1 regulation. The c-Src activity and expression were selectively downregulated by pharmacological inhibition and siRNA-mediated gene silencing, respectively. High glucose dose-dependently up-regulated c-Src phosphorylation and ET-1 gene and protein expression levels in human ECs. Chemical inhibition or silencing of c-Src significantly decreased the high-glucose augmented ET-1 expression in cultured ECs. In vivo studies showed significant elevations in the aortic ET-1 mRNA expression and plasma ET-1 concentration in diabetic mice compared to non-diabetic animals. Treatment with Src I1, as well as in vivo silencing of c-Src, significantly reduced the upregulated ET-1 expression in diabetic mice. These data provide new insights into the regulation of ET-1 expression in endothelial cells in diabetes. Pharmacological targeting of c-Src activity and/or expression may represent a potential therapeutic strategy to reduce ET-1 level and to counteract diabetes-induced deleterious vascular effects.
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Endotelina-1/genética , Endotelina-1/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Familia-src Quinasas/metabolismo , Animales , Proteína Tirosina Quinasa CSK , Línea Celular , Diabetes Mellitus Experimental/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Silenciador del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/deficiencia , Familia-src Quinasas/genéticaRESUMEN
NADPH oxidases (Nox) represent a family of hetero-oligomeric enzymes whose exclusive biological function is the generation of reactive oxygen species (ROS). Nox-derived ROS are essential modulators of signal transduction pathways that control key physiological activities such as cell growth, proliferation, migration, differentiation, and apoptosis, immune responses, and biochemical pathways. Enhanced formation of Nox-derived ROS, which is generally associated with the up-regulation of different Nox subtypes, has been established in various pathologies, namely cardiovascular diseases, diabetes, obesity, cancer, and neurodegeneration. The detrimental effects of Nox-derived ROS are related to alterations in cell signalling and/or direct irreversible oxidative damage of nucleic acids, proteins, carbohydrates, and lipids. Thus, understanding of transcriptional regulation mechanisms of Nox enzymes have been extensively investigated in an attempt to find ways to counteract the excessive formation of Nox-derived ROS in various pathological states. Despite the numerous existing data, the molecular pathways responsible for Nox up-regulation are not completely understood. This review article summarizes some of the recent advances and concepts related to the regulation of Nox expression in the vascular pathophysiology. It highlights the role of transcription factors and epigenetic mechanisms in this process. Identification of the signalling molecules involved in Nox up-regulation, which is associated with the onset and development of cardiovascular dysfunction may contribute to the development of novel strategies for the treatment of cardiovascular diseases.
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Epigénesis Genética , NADPH Oxidasas/metabolismo , Factores de Transcripción/metabolismo , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Humanos , NADPH Oxidasas/genética , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
High glucose-induced endothelial dysfunction is partially mediated by the down-stream pathophysiological effects triggered by increased expression of endothelin-1 (ET-1). The molecular control mechanisms of ET-1 synthesis are yet to be discovered. Members of the CCAAT/enhancer-binding proteins (C/EBP) family are important regulators of key metabolic processes, cellular differentiation and proinflammatory genes. In this study, we aimed at elucidating the role of C/EBP in mediating the high glucose effect on ET-1 expression in human endothelial cells (EC). Human umbilical vein cells (EAhy926) and primary cultures of human aortic EC were exposed to high levels of glucose (16.5-25 mM). Real-time PCR, Western blot, enzyme-linked immunosorbent assay, ET-1 promoter-luciferase reporter analysis, and chromatin immunoprecipitation assays were employed to investigate ET-1 regulation. High glucose activated C/EBPα, C/EBPß, and C/EBPδ in a dose-dependent manner. It also promoted significant increases in ET-1 gene and peptide expression. Chemical inhibition of JNK, p38MAPK and ERK1/2 diminished significantly the high glucose-induced nuclear translocation of C/EBP and ET-1 expression. Silencing of C/EBPα, C/EBPß or C/EBPδ greatly reduced the high glucose-induced upregulation of ET-1 mRNA, pre-pro-ET-1, and ET-1 secretion. The expression of various C/EBP isoforms was selectively downregulated by siRNA-mediated gene silencing. In silico analysis indicated the existence of typical C/EBP elements within human ET-1 gene promoter. Transient overexpression of C/EBPα, C/EBPß or C/EBPδ upregulated the luciferase level controlled by the ET-1 gene promoter. The direct interaction of C/EBPα, C/EBPß or C/EBPδ proteins with the ET-1 promoter in high glucose-exposed EC was confirmed by chromatin immunoprecipitation assay. High glucose-induced ET-1 expression is mediated through multiple mechanisms. We present evidence that members of the C/EBP proinflammatory transcription factors are important regulators of ET-1 in high glucose-exposed human endothelial cells. High glucose-induced activation of C/EBP-related signaling pathways may induce excessive ET-1 synthesis, thus promoting vasoconstriction and dysfunction of the vascular wall cells in diabetes.