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Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.
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Dieta Alta en Grasa , Epigénesis Genética , Mitocondrias , ARN Mitocondrial , Espermatozoides , Animales , Femenino , Humanos , Masculino , Ratones , Índice de Masa Corporal , Dieta Alta en Grasa/efectos adversos , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Epidídimo/citología , Epigénesis Genética/genética , Fertilización/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones Endogámicos C57BL , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Obesidad/genética , Obesidad/metabolismo , Obesidad/etiología , Oocitos/metabolismo , Sobrepeso/genética , Sobrepeso/metabolismo , Herencia Paterna/genética , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Espermatozoides/metabolismo , Testículo/citología , Transcripción GenéticaRESUMEN
Activation of NADPH oxidase (NOX) enzymes and the generation of reactive oxygen species and oxidative stress regulate vascular and renal function and contribute to the pathogenesis of hypertension. The present study examined the role of NOXA1/NOX1 function in vascular reactivity of renal and mesenteric resistance arteries/arterioles of wild-type and Noxa1-/- mice. A major finding was that renal blood flow is less sensitive to acute stimulation by angiotensin II (ANG II) in Noxa1-/- mice compared with wild-type mice, with a direct action on resistance arterioles independent of nitric oxide (NO) bioavailability. These functional results were reinforced by immunofluorescence evidence of NOXA1/NOX1 protein presence in renal arteries, afferent arterioles, and glomeruli as well as their upregulation by ANG II. In contrast, the renal vascular response to the thromboxane mimetic U46619 was effectively blunted by NO and was similar in both mouse genotypes and thus independent of NOXA1/NOX1 signaling. However, phenylephrine- and ANG II-induced contraction of isolated mesenteric arteries was less pronounced and buffering of vasoconstriction after acetylcholine and nitroprusside stimulation was reduced in Noxa1-/- mice, suggesting endothelial NO-dependent mechanisms. An involvement of NOXA1/NOX1/O2â¢- signaling in response to ANG II was demonstrated with the specific NOXA1/NOX1 assembly inhibitor C25 and the nonspecific NOX inhibitor diphenyleneiodonium chloride in cultured vascular smooth muscle cells and isolated mesenteric resistance arteries. Collectively, our data indicate that the NOX1/NOXA1/O2â¢- pathway contributes to acute vasoconstriction induced by ANG II in renal and mesenteric vascular beds and may contribute to ANG II-induced hypertension.NEW & NOTEWORTHY Renal reactivity to angiotensin II (ANG II) is mediated by superoxide signaling produced by NADPH oxidase (NOX)A1/NOX1. Acute vasoconstriction of renal arteries by ANG was blunted in Noxa1-/- compared with wild-type mice. NOXA1/NOX1/O2â¢- signaling was also observed in ANG II stimulation of vascular smooth muscle cells and isolated mesenteric resistance arteries, indicating that it contributes to ANG II-induced hypertension. A NOXA1/NOX1 assembly inhibitor (C25) has been characterized that inhibits superoxide production and ameliorates the effects of ANG II.
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Hipertensión , Superóxidos , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Angiotensina II/farmacología , Angiotensina II/metabolismo , Riñón/metabolismo , NADPH Oxidasas/metabolismo , Superóxidos/metabolismoRESUMEN
The activity of the epithelial Na+ channel (ENaC) in principal cells of the distal nephron fine-tunes renal Na+ excretion. The renin-angiotensin-aldosterone system modulates ENaC activity to control blood pressure, in part, by influencing Na+ excretion. NADPH oxidase activator 1-dependent NADPH oxidase 1 (NOXA1/NOX1) signaling may play a key role in angiotensin II (ANG II)-dependent activation of ENaC. The present study aimed to explore the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC in renal principal cells. Patch-clamp electrophysiology and principal cell-specific Noxa1 knockout (PC-Noxa1 KO) mice were used to determine the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC. The activity of ENaC in the luminal plasma membrane of principal cells was quantified in freshly isolated split-opened tubules using voltage-clamp electrophysiology. ANG II significantly increased ENaC activity. This effect was robust and observed in response to both acute (40 min) and more chronic (48-72 h) ANG II treatment of isolated tubules and mice, respectively. Inhibition of ANG II type 1 receptors with losartan abolished ANG II-dependent stimulation of ENaC. Similarly, treatment with ML171, a specific inhibitor of NOX1, abolished stimulation of ENaC by ANG II. Treatment with ANG II failed to increase ENaC activity in principal cells in tubules isolated from the PC-Noxa1 KO mouse. Tubules from wild-type littermate controls, though, retained their ability to respond to ANG II with an increase in ENaC activity. These results indicate that NOXA1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells. As such, NOXA1/NOX1 signaling in the distal nephron plays a central role in Na+ homeostasis and control of blood pressure, particularly as it relates to regulation by the renin-ANG II axis.NEW & NOTEWORTHY Activity of the epithelial Na+ channel (ENaC) in the distal nephron fine-tunes renal Na+ excretion. Angiotensin II (ANG II) has been reported to enhance ENaC activity. Emerging evidence suggests that NADPH oxidase (NOX) signaling plays an important role in the stimulation of ENaC by ANG II in principal cells. The present findings indicate that NOX activator 1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells.
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Angiotensina II , Canales Epiteliales de Sodio , Animales , Ratones , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Angiotensina II/farmacología , Angiotensina II/metabolismo , NADPH Oxidasa 1/metabolismo , Sodio/metabolismo , Ratones Noqueados , NADPH Oxidasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Studies have linked exposure to ultrafine particulate matter (PM) and adverse cardiovascular events. PM-induced oxidative stress is believed to be a key mechanism underlying observed adverse vascular effects. Advanced age is one factor known to decrease antioxidant defenses and confer susceptibility to the detrimental vascular effects seen following PM exposure. The present study was designed to investigate the vasomotor responses following ultrafine PM exposure in wild type (WT) and superoxide dismutase 2-deficient (SOD2+/-) mice that possess decreased antioxidant defense. Thoracic aortic rings isolated from young and aged WT and SOD2+/- mice were exposed to ultrafine PM in a tissue bath system. Aortic rings were then constricted with increasing concentrations of phenylephrine, followed by relaxation with rising amounts of nitroglycerin (NTG). Data demonstrated that ultrafine PM decreased the relaxation response in both young WT and young SOD2+/- mouse aortas, and relaxation was significantly reduced in young SOD2+/- compared to WT mice. Ultrafine PM significantly diminished the NTG-induced relaxation response in aged compared to young mouse aortas. After ultrafine PM exposure, the relaxation response did not differ markedly between aged WT and aged SOD2+/- mice. Data demonstrated that the greater vascular effect in aortic rings in aged mice ex vivo after ultrafine PM exposure may be attributed to ultrafine PM-induced oxidative stress and loss of antioxidant defenses in aged vascular tissue. Consistent with this conclusion is the attenuation of NTG-induced relaxation response in young SOD2+/- mice. ABBREVIATIONS: H2O2: hydrogen peroxide; NTG: nitroglycerin; PAH: polycyclic aromatic hydrocarbons; PE: l-phenylephrine; PM: particulate matter; ROS: reactive oxygen species; SOD2: superoxide dismutase 2 deficient; WT: wild type.
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Enfermedades de la Aorta/metabolismo , Material Particulado/toxicidad , Sistema Vasomotor/fisiología , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Aorta/metabolismo , Aorta/fisiopatología , Enfermedades de la Aorta/diagnóstico por imagen , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/fisiopatología , Ratones , Ratones Endogámicos C57BL , Superóxido Dismutasa-1/deficiencia , Superóxido Dismutasa-1/metabolismo , Sistema Vasomotor/efectos de los fármacosRESUMEN
We recently reported that increased NADPH oxidase 4 (NOX4) expression and activity during aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. The goal of the present study was to elucidate the molecular mechanism(s) for these effects and determine the importance of NOX4 modulation of proinflammatory gene expression in mouse vascular smooth muscle cells (VSMCs). A novel peptide-mediated siRNA transfection approach was used to inhibit Nox4 expression with minimal cellular toxicity. Using melittin-derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs. 85% with Lipofectamine) and decreased toxicity (p<0.001 vs. Lipofectamine in MTT and p<0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGFß1 significantly upregulates Nox4 mRNA (p<0.01) and protein (p<0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGFß1-induced upregulation of Nox4 mRNA (p<0.01) and protein (p<0.0001) levels and decreased hydrogen peroxide production (p<0.0001). Expression of pro-inflammatory genes Ccl2, Ccl5, Il6, and Vcam1 was significantly upregulated in VSMCs in several settings cells isolated from aged vs. young wild-type mice, in atherosclerotic arteries of Apoe-/- mice, and atherosclerotic human carotid arteries and correlated with NOX4 expression. p5RHH-mediated Nox4 siRNA transfection significantly attenuated the expression of these pro-inflammatory genes in TGFß1-treated mouse VSMCs, with the highest degree of inhibition in the expression of Il6. p5RHH peptide-mediated knockdown of TGFß-activated kinase 1 (TAK1, also known as Map3k7), Jun, and Rela, but not Nfkb2, downregulated TGFß1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGFß1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis.
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Envejecimiento/genética , Aterosclerosis/genética , NADPH Oxidasas/genética , Vasculitis/etiología , Envejecimiento/metabolismo , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Supervivencia Celular/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Peróxido de Hidrógeno/metabolismo , Mediadores de Inflamación/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Vasculitis/metabolismo , Vasculitis/patologíaRESUMEN
The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.
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Introduction: Aging increases the risk of atherosclerotic vascular disease and its complications. Macrophages are pivotal in the pathogenesis of vascular aging, driving inflammation and atherosclerosis progression. NOX4 (NADPH oxidase 4) expression increases with age, correlating with mitochondrial dysfunction, inflammation, and atherosclerosis. We hypothesized that the NOX4-dependent mitochondrial oxidative stress promotes aging-associated atherosclerosis progression by causing metabolic dysfunction and inflammatory phenotype switch in macrophages. Methods: We studied atherosclerotic lesion morphology and macrophage phenotype in young (5-month-old) and aged (16-month-old) Nox4 -/-/Apoe -/- and Apoe -/- mice fed Western diet. Results: Young Nox4-/-/Apoe-/- and Apoe-/- mice had comparable aortic and brachiocephalic artery atherosclerotic lesion cross-sectional areas. Aged mice showed significantly increased lesion area compared with young mice. Aged Nox4-/-/Apoe-/- had significantly lower lesion areas than Apoe-/- mice. Compared with Apoe-/- mice, atherosclerotic lesions in aged Nox4-/-/Apoe-/- showed reduced cellular and mitochondrial ROS and oxidative DNA damage, lower necrotic core area, higher collagen content, and decreased inflammatory cytokine expression. Immunofluorescence and flow cytometry analysis revealed that aged Apoe-/- mice had a higher percentage of classically activated pro-inflammatory macrophages (CD38+CD80+) in the lesions. Aged Nox4-/-/Apoe-/- mice had a significantly higher proportion of alternatively activated pro-resolving macrophages (EGR2+/CD163+CD206+) in the lesions, with an increased CD38+/EGR2+ cell ratio compared with Apoe-/- mice. Mitochondrial respiration assessment revealed impaired oxidative phosphorylation and increased glycolytic ATP production in macrophages from aged Apoe-/- mice. In contrast, macrophages from Nox4-/-/Apoe-/- mice were less glycolytic and more aerobic, with preserved basal and maximal respiration and mitochondrial ATP production. Macrophages from Nox4-/-/Apoe-/- mice also had lower mitochondrial ROS levels and reduced IL1ß secretion; flow cytometry analysis showed fewer CD38+ cells after IFNγ+LPS treatment and more EGR2+ cells after IL4 treatment than in Apoe-/- macrophages. In aged Apoe-/- mice, inhibition of NOX4 activity using GKT137831 significantly reduced macrophage mitochondrial ROS and improved mitochondrial function, resulting in decreased CD68+CD80+ and increased CD163+CD206+ lesion macrophage proportion and attenuated atherosclerosis. Discussion: Our findings suggest that increased NOX4 in aging drives macrophage mitochondrial dysfunction, glycolytic metabolic switch, and pro-inflammatory phenotype, advancing atherosclerosis. Inhibiting NOX4 or mitochondrial dysfunction could alleviate vascular inflammation and atherosclerosis, preserving plaque integrity.
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Envejecimiento , Aterosclerosis , Macrófagos , Mitocondrias , NADPH Oxidasa 4 , Fenotipo , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/etiología , Aterosclerosis/inmunología , Mitocondrias/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Envejecimiento/inmunología , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , Progresión de la Enfermedad , Ratones Noqueados , Estrés Oxidativo , Inflamación/inmunología , Inflamación/metabolismo , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Masculino , Modelos Animales de Enfermedad , Apolipoproteínas E/genética , Apolipoproteínas E/deficiencia , Ratones Noqueados para ApoE , Reprogramación MetabólicaRESUMEN
OBJECTIVE: Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models. METHODS AND RESULTS: Pulse wave velocity, determined using Doppler, increased with age in superoxide dismutase 2 (SOD2)+/- but not in wild-type, p47phox-/- and SOD1+/- mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2+/- versus wild-type (16-month-old) mice. Aortic SMCs from aged SOD2+/- mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity, and increased sensitivity to staurosporine-induced apoptosis versus aged wild-type and young (4-month-old) SOD2+/- mice. Smooth muscle α-actin levels were increased with age in SOD2+/- versus wild-type SMCs. Aged SOD2+/- SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-α-induced Jun N-terminal kinase 1 activation. Aged SOD2+/- SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/- SMCs. CONCLUSION: Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part by inducing vascular wall remodeling, intrinsic changes in SMC stiffness, and aortic SMC apoptosis.
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Envejecimiento/metabolismo , Enfermedades de la Aorta/metabolismo , Mitocondrias/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo , Actinas/metabolismo , Factores de Edad , Envejecimiento/patología , Animales , Aorta/metabolismo , Aorta/fisiopatología , Enfermedades de la Aorta/diagnóstico por imagen , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/fisiopatología , Apoptosis , Células Cultivadas , Colágeno Tipo I/metabolismo , Adaptabilidad , Modelos Animales de Enfermedad , Elastina/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Genotipo , Peróxido de Hidrógeno/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/patología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Músculo Liso Vascular/diagnóstico por imagen , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/patología , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Flujo Pulsátil , Volumen Sistólico , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Superóxidos/metabolismo , Transfección , Ultrasonografía Doppler de Pulso , Vasodilatación , Función Ventricular Izquierda , Presión VentricularRESUMEN
In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).
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Etilnitrosourea/farmacología , Genoma , Mutágenos/farmacología , Mutación/efectos de los fármacos , Animales , Cruzamientos Genéticos , Criopreservación , Femenino , Miembro Anterior/anomalías , Inmunidad/genética , Inmunidad/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mutagénesis , Mutación/genética , Mutación/inmunología , FenotipoRESUMEN
Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and its prevalence increases with age. The irregular and rapid contraction of the atria can lead to ineffective blood pumping, local blood stasis, blood clots, ischemic stroke, and heart failure. NADPH oxidases (NOX) and mitochondria are the main sources of reactive oxygen species in the heart, and dysregulated activation of NOX and mitochondrial dysfunction are associated with AF pathogenesis. NOX- and mitochondria-derived oxidative stress contribute to the onset of paroxysmal AF by inducing electrophysiological changes in atrial myocytes and structural remodeling in the atria. Because high atrial activity causes cardiac myocytes to expend extremely high energy to maintain excitation-contraction coupling during persistent AF, mitochondria, the primary energy source, undergo metabolic stress, affecting their morphology, Ca2+ handling, and ATP generation. In this review, we discuss the role of oxidative stress in activating AF-triggered activities, regulating intracellular Ca2+ handling, and functional and anatomical reentry mechanisms, all of which are associated with AF initiation, perpetuation, and progression. Changes in the extracellular matrix, inflammation, ion channel expression and function, myofibril structure, and mitochondrial function occur during the early transitional stages of AF, opening a window of opportunity to target NOX and mitochondria-derived oxidative stress using isoform-specific NOX inhibitors and mitochondrial ROS scavengers, as well as drugs that improve mitochondrial dynamics and metabolism to treat persistent AF and its transition to permanent AF.
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Rapid over-activation of ß-adrenergic receptors (ß-AR) following acute stress initiates cardiac inflammation and injury by activating interleukin-18 (IL-18), however, the process of inflammation cascades has not been fully illustrated. The present study aimed to determine the mechanisms of cardiac inflammatory amplification following acute sympathetic activation. With bioinformatics analysis, galectin-3 was identified as a potential key downstream effector of ß-AR and IL-18 activation. The serum level of galectin-3 was positively correlated with norepinephrine or IL-18 in patients with chest pain. In the heart of mice treated with ß-AR agonist isoproterenol (ISO, 5 mg kg-1), galectin-3 expression was upregulated markedly later than IL-18 activation, and Nlrp3-/- and Il18-/- mice did not show ISO-induced galectin-3 upregulation. It was further revealed that cardiomyocyte-derived IL-18 induced galectin-3 expression in macrophages following ISO treatment. Moreover, galectin-3 deficiency suppressed ISO-induced cardiac inflammation and fibrosis without blocking ISO-induced IL-18 increase. Treatment with a galectin-3 inhibitor, but not a ß-blocker, one day after ISO treatment effectively attenuated cardiac inflammation and injury. In conclusion, galectin-3 is upregulated to exaggerate cardiac inflammation and injury following acute ß-AR activation, a galectin-3 inhibitor effectively blocks cardiac injury one day after ß-AR insult.
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Galectina 3 , Interleucina-18 , Animales , Ratones , Galectina 3/genética , Galectina 3/metabolismo , Galectina 3/farmacología , Adrenérgicos/metabolismo , Adrenérgicos/farmacología , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Arritmias Cardíacas , Fibrosis , Inflamación/metabolismoRESUMEN
In acute sympathetic stress, catecholamine overload can lead to stress cardiomyopathy. We tested the hypothesis that cardiomyocyte NOX4 (NADPH oxidase 4)-dependent mitochondrial oxidative stress mediates inflammation and diastolic dysfunction in stress cardiomyopathy. Isoproterenol (ISO; 5 mg/kg) injection induced sympathetic stress in wild-type and cardiomyocyte (CM)-specific Nox4 knockout (Nox4CM-/-) mice. Wild-type mice treated with ISO showed higher CM NOX4 expression, H2O2 levels, inflammasome activation, and IL18, IL6, CCL2, and TNFα levels than Nox4CM-/- mice. Spectral flow cytometry and t-SNE analysis of cardiac cell suspensions showed significant increases in pro-inflammatory and pro-fibrotic embryonic-derived resident (CCR2-MHCIIhiCX3CR1hi) macrophages in wild-type mice 3 days after ISO treatment, whereas Nox4CM-/- mice had a higher proportion of embryonic-derived resident tissue-repair (CCR2-MHCIIloCX3CR1lo) macrophages. A significant increase in cardiac fibroblast activation and interstitial collagen deposition and a restrictive pattern of diastolic dysfunction with increased filling pressure was observed in wild-type hearts compared with Nox4CM-/- 7 days post-ISO. A selective NOX4 inhibitor, GKT137831, reduced myocardial mitochondrial ROS, macrophage infiltration, and fibrosis in ISO-injected wild-type mice, and preserved diastolic function. Our data suggest sympathetic overstimulation induces resident macrophage (CCR2-MHCII+) activation and myocardial inflammation, resulting in fibrosis and impaired diastolic function mediated by CM NOX4-dependent ROS.
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Miocitos Cardíacos , Cardiomiopatía de Takotsubo , Animales , Ratones , Fibrosis , Peróxido de Hidrógeno/metabolismo , Inflamación/metabolismo , Miocitos Cardíacos/metabolismo , NADPH Oxidasa 4/genética , NADPH Oxidasa 4/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cardiomiopatía de Takotsubo/metabolismo , Cardiomiopatía de Takotsubo/patologíaRESUMEN
Reactive oxygen species (ROS) and telomere dysfunction are both associated with aging and the development of age-related diseases. Although there is evidence for a direct relationship between ROS and telomere dysfunction as well as an independent association of oxidative stress and telomere attrition with age-related disorders, there has not been sufficient exploration of how the interaction between oxidative stress and telomere function may contribute to the pathophysiology of cardiovascular diseases (CVD). To better understand the complex relationships between oxidative stress, telomerase biology and pathophysiology, we examined the telomere biology of aortic smooth muscle cells (ASMCs) isolated from mutant mouse models of oxidative stress. We discovered that telomere lengths were significantly shorter in ASMCs isolated from superoxide dismutase 2 heterozygous (Sod2+/-) mice, which exhibit increased arterial stiffness with aging, and the observed telomere attrition occurred over time. Furthermore, the telomere erosion occurred even though telomerase activity increased. In contrast, telomeres remained stable in wild-type and superoxide dismutase 1 heterozygous (Sod1+/-) mice, which do not exhibit CVD phenotypes. The data indicate that mitochondrial oxidative stress, in particular elevated superoxide levels and decreased hydrogen peroxide levels, induces telomere erosion in the ASMCs of the Sod2+/- mice. This reduction in telomere length occurs despite an increase in telomerase activity and correlates with the onset of disease phenotype. Our results suggest that the oxidative stress caused by imbalance in mitochondrial ROS, from deficient SOD2 activity as a model for mitochondrial dysfunction results in telomere dysfunction, which may contribute to pathogenesis of CVD.
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Enfermedades Cardiovasculares , Telomerasa , Animales , Enfermedades Cardiovasculares/patología , Ratones , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa-1/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genéticaRESUMEN
Diastolic dysfunction (DD) underlies heart failure with preserved ejection fraction (HFpEF), a clinical syndrome associated with aging that is becoming more prevalent. Despite extensive clinical studies, no effective treatment exists for HFpEF. Recent findings suggest that oxidative stress contributes to the pathophysiology of DD, but molecular mechanisms underpinning redox-sensitive cardiac remodeling in DD remain obscure. Using transgenic mice with mitochondria-targeted NOX4 overexpression (Nox4TG618) as a model, we demonstrate that NOX4-dependent mitochondrial oxidative stress induces DD in mice as measured by increased E/E', isovolumic relaxation time, Tau Glantz and reduced dP/dtmin while EF is preserved. In Nox4TG618 mice, fragmentation of cardiomyocyte mitochondria, increased DRP1 phosphorylation, decreased expression of MFN2, and a higher percentage of apoptotic cells in the myocardium are associated with lower ATP-driven and maximal mitochondrial oxygen consumption rates, a decrease in respiratory reserve, and a decrease in citrate synthase and Complex I activities. Transgenic mice have an increased concentration of TGFß and osteopontin in LV lysates, as well as MCP-1 in plasma, which correlates with a higher percentage of LV myocardial periostin- and ACTA2-positive cells compared with wild-type mice. Accordingly, the levels of ECM as measured by Picrosirius Red staining as well as interstitial deposition of collagen I are elevated in the myocardium of Nox4TG618 mice. The LV tissue of Nox4TG618 mice also exhibited increased ICaL current, calpain 2 expression, and altered/disrupted Z-disc structure. As it pertains to human pathology, similar changes were found in samples of LV from patients with DD. Finally, treatment with GKT137831, a specific NOX1 and NOX4 inhibitor, or overexpression of mCAT attenuated myocardial fibrosis and prevented DD in the Nox4TG618 mice. Together, our results indicate that mitochondrial oxidative stress contributes to DD by causing mitochondrial dysfunction, impaired mitochondrial dynamics, increased synthesis of pro-inflammatory and pro-fibrotic cytokines, activation of fibroblasts, and the accumulation of extracellular matrix, which leads to interstitial fibrosis and passive stiffness of the myocardium. Further, mitochondrial oxidative stress increases cardiomyocyte Ca2+ influx, which worsens CM relaxation and raises the LV filling pressure in conjunction with structural proteolytic damage.
RESUMEN
Aims: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are implicated in the pathophysiology of hypertension in chronic kidney disease patients. Genetic deletion of NOX activator 1 (Noxa1) subunit of NOX1 decreases ROS under pathophysiological conditions. Here, we investigated the role of NOXA1-dependent NOX1 activity in the pathogenesis of angiotensin II (Ang II)-induced hypertension (AIH) and possible involvement of abnormal renal function. Results: NOXA1 is present in epithelial cells of Henle's thick ascending limb and distal nephron. Telemetry showed lower basal systolic blood pressure (BP) in Noxa1-/-versus wild-type mice. Ang II infusion for 1 and 14 days increased NOXA1/NOX1 expression and ROS in kidney of male but not female wild-type mice. Mean BP increased 30 mmHg in wild-type males, with smaller increases in Noxa1-deficient males and wild-type or Noxa1-/- females. In response to an acute salt load, Na+ excretion was similar in wild-type and Noxa1-/- mice before and 14 days after Ang II infusion. However, Na+ excretion was delayed after 1-2 days of Ang II in male wild-type versus Noxa1-/- mice. Ang II increased epithelial Na+ channel (ENaC) levels and activation in the collecting duct principal epithelial cells of wild-type but not Noxa1-/- mice. Aldosterone induced ROS levels and Noxa1 and Scnn1a expression and ENaC activity in a mouse renal epithelial cell line, responses abolished by Noxa1 small-interfering RNA. Innovation and Conclusion: Ang II activation of renal NOXA1/NOX1-dependent ROS enhances tubular ENaC expression and Na+ reabsorption, leading to increased BP. Attenuation of AIH in females is attributed to weaker NOXA1/NOX1-dependent ROS signaling and efficient natriuresis. Antioxid. Redox Signal. 36, 550-566.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Angiotensina II , Canales Epiteliales de Sodio , Hipertensión , NADPH Oxidasa 1 , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Angiotensina II/farmacología , Animales , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Femenino , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Riñón/metabolismo , Masculino , Ratones , NADPH Oxidasa 1/genética , NADPH Oxidasa 1/metabolismo , Sodio/metabolismoRESUMEN
Loss-of-function (LOF) variants in SCN1B, encoding the voltage-gated sodium channel ß1/ß1B subunits, are linked to neurological and cardiovascular diseases. Scn1b-null mice have spontaneous seizures and ventricular arrhythmias and die by approximately 21 days after birth. ß1/ß1B Subunits play critical roles in regulating the excitability of ventricular cardiomyocytes and maintaining ventricular rhythmicity. However, whether they also regulate atrial excitability is unknown. We used neonatal Scn1b-null mice to model the effects of SCN1B LOF on atrial physiology in pediatric patients. Scn1b deletion resulted in altered expression of genes associated with atrial dysfunction. Scn1b-null hearts had a significant accumulation of atrial collagen, increased susceptibility to pacing induced atrial fibrillation (AF), sinoatrial node (SAN) dysfunction, and increased numbers of cholinergic neurons in ganglia that innervate the SAN. Atropine reduced the incidence of AF in null animals. Action potential duration was prolonged in null atrial myocytes, with increased late sodium current density and reduced L-type calcium current density. Scn1b LOF results in altered atrial structure and AF, demonstrating the critical role played by Scn1b in atrial physiology during early postnatal mouse development. Our results suggest that SCN1B LOF variants may significantly impact the developing pediatric heart.
Asunto(s)
Fibrilación Atrial , Potenciales de Acción , Animales , Fibrilación Atrial/genética , Humanos , Ratones , Ratones Noqueados , Nodo Sinoatrial/metabolismo , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genética , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/metabolismoRESUMEN
The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.
Asunto(s)
Aterosclerosis/etiología , Regulación de la Expresión Génica , Receptores de Hialuranos/genética , Ácido Hialurónico/genética , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasas/fisiología , Animales , Aorta , Aterosclerosis/enzimología , Aterosclerosis/metabolismo , Técnicas In Vitro , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/enzimología , Especies Reactivas de Oxígeno , Trombina/farmacologíaRESUMEN
BACKGROUND: Despite a concerted effort by many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homolog of p67phox, in VSMC NADPH oxidase function and atherosclerosis. METHODS AND RESULTS: The presence of NoxA1 in mouse aortic VSMCs was confirmed by reverse-transcription polymerase chain reaction and sequencing. NoxA1/p47phox interaction after thrombin treatment was observed by immunoprecipitation/Western analysis of lysates from p47phox(-/-) VSMCs transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with adenoviral NoxA1 significantly enhanced thrombin-induced reactive oxygen species generation in wild-type but not in p47phox(-/-) and Nox1(-/-) VSMCs. Thrombin-induced reactive oxygen species production and VSMC proliferation were significantly reduced after downregulation of NoxA1 with shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of the redox-sensitive protein kinases (Janus kinase 2, Akt, and p38 mitogen-activated protein kinase) in VSMCs. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMCs and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE(-/-) mice compared with age-matched wild-type mice. Furthermore, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions. CONCLUSIONS: NoxA1 is the functional homolog of p67phox in VSMCs that regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases.
Asunto(s)
Aterosclerosis/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/patología , Arterias Carótidas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Proteínas/genética , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Despite decades of investigation, the roles of reactive oxygen species (ROS) in atherosclerosis and aging have yet to be defined. We have developed mouse models that allow this question to be addressed experimentally. Given the increase in risk factors for atherosclerosis--particularly related to marked increases in obesity and diabetes in industrialized countries--a better understanding of the molecular mechanisms that define both atherosclerosis and aging is needed.
Asunto(s)
Envejecimiento/metabolismo , Arterias/metabolismo , Miocardio/metabolismo , Estrés Oxidativo , Envejecimiento/patología , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Arterias/patología , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Dieta Aterogénica , Humanos , Ratones , Ratones Noqueados , Modelos Animales , Modelos Cardiovasculares , Miocardio/patología , Especies Reactivas de Oxígeno/metabolismo , Factores de RiesgoRESUMEN
Increased production of reactive oxygen species in mitochondria, accumulation of mitochondrial DNA damage, and progressive respiratory chain dysfunction are associated with atherosclerosis or cardiomyopathy in human investigations and animal models of oxidative stress. Moreover, major precursors of atherosclerosis-hypercholesterolemia, hyperglycemia, hypertriglyceridemia, and even the process of aging-all induce mitochondrial dysfunction. Chronic overproduction of mitochondrial reactive oxygen species leads to destruction of pancreatic beta-cells, increased oxidation of low-density lipoprotein and dysfunction of endothelial cells-factors that promote atherosclerosis. An additional mechanism by which impaired mitochondrial integrity predisposes to clinical manifestations of vascular diseases relates to vascular cell growth. Mitochondrial function is required for normal vascular cell growth and function. Mitochondrial dysfunction can result in apoptosis, favoring plaque rupture. Subclinical episodes of plaque rupture accelerate the progression of hemodynamically significant atherosclerotic lesions. Flow-limiting plaque rupture can result in myocardial infarction, stroke, and ischemic/reperfusion damage. Much of what is known on reactive oxygen species generation and modulation comes from studies in cultured cells and animal models. In this review, we have focused on linking this large body of literature to the clinical syndromes that predispose humans to atherosclerosis and its complications.