RESUMO
The endothelium is exposed to various flow patterns such as vasoprotective unidirectional laminar shear stress (LSS) and atherogenic oscillatory shear stress (OSS). A software-controlled, valve-operated OsciFlow device with parallel chambers was used to apply LSS and OSS to endothelial cells. Although LSS inhibited superoxide over time, OSS time-dependently increased superoxide production from endothelial cells. Immunocytochemical staining revealed that, at resting state, p47phox colocalizes with NOX2, whereas NOXO1 colocalizes with NOX1. RNAi of p47phox had no effects on superoxide or NO production in response to OSS but significantly reduced NO production in LSS, implicating a p47phox-bound NADPH oxidase (NOX) in mediating basal NO production. Indeed, RNAi of p47phox inhibited endothelial nitric oxide synthase (eNOS) serine 1179 phosphorylation, whereas PEG-catalase scavenging of intracellular hydrogen peroxide or RNAi of NOX2 produced similar results, indicating a role of NOX2/p47phox-derived hydrogen peroxide in mediating the basal activity of NO production from eNOS. In contrast, RNAi of NOXO1 resulted in no significant changes in NO and superoxide levels in response to LSS but significantly reduced superoxide while increasing NO in response to OSS. Furthermore, we identified, for the first time, that OSS uncouples eNOS, which was corrected by RNAi of NOXO1. In summary, LSS activates the NOX2-p47phox complex to activate eNOS phosphorylation and NO production. OSS instead activates the NOX1-NOXO1 complex to uncouple eNOS. These results demonstrate differential roles of NOXs in modulating the redox state in response to different shear stresses, which may promote the development of novel therapeutic agents to mimic the protective effects of LSS while inhibiting the injurious effects of OSS.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Relógios Biológicos/fisiologia , Células Endoteliais/metabolismo , NADPH Oxidases/metabolismo , Estresse Mecânico , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Bovinos , Células Cultivadas , Células Endoteliais/citologia , NADPH Oxidases/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Fosforilação , Superóxidos/metabolismoRESUMO
Reperfusion injury of the heart is a severe complication of angioplasty treatment of acute myocardial ischemia, for which no therapeutics are currently available. The present study aimed to identify whether and how a novel protein, netrin-1, induces cardioprotection in vivo during ischemia/reperfusion (I/R) injury. Wild type (WT) C57BL6/J mice were subjected to a 30 min coronary occlusion followed by a 24h reperfusion with vehicle (normal saline), netrin-1, UO126 (MEK1/2 inhibitor), PTIO (nitric oxide/NO scavenger), netrin-1/UO126 or netrin-1/PTIO intraventricularly. Some were injected of netrin-1 via tail vein. Netrin-1 at 5µg/kg induced a substantial reduction in infarct size (19.7 ± 5.0% from 41.3 ± 1.8% in the controls), and markedly improved cardiac function as measured by ejection fraction and fractional shortening from echocardiography. Experiments with mice deficient in netrin-1 receptor DCC (deleted in colorectal cancer, DCC+/-), or reperfusion with netrin-1/UO126 or netrin-1/PTIO, attenuated the protective effects of netrin-1, implicating intermediate roles of DCC, ERK1/2 and NO. Netrin-1 induced phosphorylation of ERK1/2 and eNOS was abolished in DCC+/-mice. Electron spin resonance (ESR) determination of NO production from isolated left ventricles demonstrated that netrin-1 improves NO bioavailability, which was attenuated by UO126 or in DCC+/-mice, suggesting upstream roles of DCC and ERK1/2 in NO production. Netrin-1 further reduced mitochondrial swelling and mitochondrial superoxide production, which was absent when co-treated with PTIO or UO126, or in DCC+/-mice, indicating critical roles of DCC, ERK1/2 and NO in preserving mitochondrial integrity. In a permanent coronary ligation model of myocardial infarction (MI) to assess post-MI remodeling, netrin-1 abolished the marked increase in autophagy. In summary, our data demonstrate robust cardioprotective effect of netrin-1 in vivo, as shown by reduced infarct size and improved cardiac function. Mechanistically, this protection is mediated by netrin-1 receptor DCC, and NO dependent preservation of mitochondria. This work clearly establishes a therapeutic potential of netrin-1 for acute treatment of MI, perhaps also for chronic post-MI remodeling. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.
Assuntos
Autofagia , Testes de Função Cardíaca , Mitocôndrias/metabolismo , Fatores de Crescimento Neural/metabolismo , Óxido Nítrico/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Cardiotônicos/metabolismo , Receptor DCC , Regulação para Baixo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Netrina-1 , Óxido Nítrico Sintase Tipo III/metabolismo , Superóxidos/metabolismo , Remodelação VascularRESUMO
Despite an established role of mitochondrial dysfunction in cardiac ischemia/reperfusion (I/R) injury, the upstream activators have remained incompletely defined. We have recently identified an innovative role of exogenously applied netrin-1 in cardioprotection, which is mediated by increased nitric oxide (NO) bioavailability. Here, we tested the hypothesis that this "pharmacological" treatment of netrin-1 preserves mitochondrial function via novel mechanisms that are NO dependent. Freshly isolated C57BL6 mouse hearts were perfused using a Langendorff system, and subjected to a 20min global ischemia/60min reperfusion, in the presence or absence of netrin-1. I/R induced marked increases in infarct size, total superoxide and hydrogen peroxide production, activity and protein abundance of NADPH oxidase (NOX) isoform 4 (NOX4), as well as impaired mitochondrial integrity and function, all of which were attenuated by netrin-1. This protective effect of netrin-1 is attributed to cGMP, a downstream effector of NO. The protein levels of NOX1 and NOX2 were however unaffected, and infarct size from NOX1 and NOX2 knockouts was not different from wild type animals. Scavenging of NO with PTIO reversed inhibitory effects of netrin-1 on NOX4, while NO donor attenuated NOX4 protein abundance. In vivo NOX4 RNAi, or sepiapterin perfusion, resulted in recoupling of NOS, decreased infarct size, and blockade of dysfunctional mitochondrial swelling and mitochondrial superoxide production. These data demonstrate that netrin-1 induces cardioprotection through inhibition of NOX4 activity, which leads to recoupling of NOS, augmented NO bioavailability, reduction in oxidative stress, and ultimately preservation of mitochondrial function. The NO-dependent NOX4 inhibition connects with our previously established pathway of DCC/ERK1/2/eNOS/NO/DCC feed-forward mechanism, to maintain NOS in the coupling state to attenuate oxidative stress to preserve mitochondrial function. These findings may promote development of novel therapeutics for cardiac I/R injury. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".
Assuntos
Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , NADPH Oxidases/metabolismo , Fatores de Crescimento Neural/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Proteínas Supressoras de Tumor/farmacologia , Animais , Cardiotônicos/administração & dosagem , Cardiotônicos/farmacologia , Modelos Animais de Doenças , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/terapia , NADPH Oxidase 4 , NADPH Oxidases/genética , Fatores de Crescimento Neural/administração & dosagem , Netrina-1 , Estresse Oxidativo , Pterinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Superóxidos/metabolismo , Proteínas Supressoras de Tumor/administração & dosagemRESUMO
Rupture of abdominal aortic aneurysm (AAA) is a lethal event. No oral medicine has been available to prevent or treat AAA. We have recently identified a novel mechanism of eNOS uncoupling by which AAA develops, in angiotensin II (Ang II) infused hyperphenylalaninemia 1 (hph-1) mice. Using this unique model we investigated effects on AAA formation of the L-type calcium channel blocker nifedipine, in view of the unclear relationship between hypertension and AAA, and unclear mechanisms of aneurysm protective effects of some blood pressure lowering drugs. Six-month old hph-1 mice were infused with Ang II (0.7 mg/kg/day) for 2 weeks, and fed nifedipine chow at two different doses (5 and 20 mg/kg/day). While the high dose of nifedipine reduced blood pressure, the lower dose had no effect. Interestingly, the incidence rate of AAA dropped from 71% to 7 and 12.5% for low and high dose nifedipine, respectively. Expansion of abdominal aorta, determined by ultrasound imaging, was abolished by both doses of nifedipine, which recoupled eNOS completely to improve NO bioavailability. Both also abrogated aortic superoxide production. Of note, Ang II activation of NADPH oxidase in vascular smooth muscle cells and endothelial cells, known to uncouple eNOS, was also attenuated by nifedipine. Although low dose was a sub-pressor while the high dose reduced blood pressure via inhibition of calcium channels, both doses were highly effective in preventing AAA by preserving eNOS coupling activity to eliminate sustained oxidative stress from uncoupled eNOS. These data demonstrate that oral treatment of nifedipine is highly effective in preserving eNOS function to attenuate AAA formation. Nifedipine may be used for AAA prevention either at low dose in AAA risk group, or at high dose in patients with co-existing hypertension.
Assuntos
Anti-Hipertensivos/administração & dosagem , Aneurisma da Aorta Abdominal/tratamento farmacológico , Nifedipino/administração & dosagem , Óxido Nítrico Sintase Tipo III/metabolismo , Angiotensina II/genética , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Camundongos , Camundongos Transgênicos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Estresse Oxidativo/efeitos dos fármacos , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/genética , Fenilcetonúrias/patologiaRESUMO
Oxidative stress has been implicated in cardiac arrhythmia, although a causal relationship remains undefined. We have recently demonstrated a marked up-regulation of NADPH oxidase isoform 4 (NOX4) in patients with atrial fibrillation, which is accompanied by overproduction of reactive oxygen species (ROS). In this study, we investigated the impact on the cardiac phenotype of NOX4 overexpression in zebrafish. One-cell stage embryos were injected with NOX4 RNA prior to video recording of a GFP-labeled (myl7:GFP zebrafish line) beating heart in real time at 24-31 h post-fertilization. Intriguingly, NOX4 embryos developed cardiac arrhythmia that is characterized by irregular heartbeats. When quantitatively analyzed by an established LQ-1 program, the NOX4 embryos displayed much more variable beat-to-beat intervals (mean S.D. of beat-to-beat intervals was 0.027 s/beat in control embryos versus 0.038 s/beat in NOX4 embryos). Both the phenotype and the increased ROS in NOX4 embryos were attenuated by NOX4 morpholino co-injection, treatments of the embryos with polyethylene glycol-conjugated superoxide dismutase, or NOX4 inhibitors fulvene-5, 6-dimethylamino-fulvene, and proton sponge blue. Injection of NOX4-P437H mutant RNA had no effect on the cardiac phenotype or ROS production. In addition, phosphorylation of calcium/calmodulin-dependent protein kinase II was increased in NOX4 embryos but diminished by polyethylene glycol-conjugated superoxide dismutase, whereas its inhibitor KN93 or AIP abolished the arrhythmic phenotype. Taken together, our data for the first time uncover a novel pathway that underlies the development of cardiac arrhythmia, namely NOX4 activation, subsequent NOX4-specific NADPH-driven ROS production, and redox-sensitive CaMKII activation. These findings may ultimately lead to novel therapeutics targeting cardiac arrhythmia.
Assuntos
Arritmias Cardíacas/enzimologia , Contração Miocárdica , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Arritmias Cardíacas/embriologia , Arritmias Cardíacas/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , NADPH Oxidases/genética , Fenótipo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Rupture of abdominal aortic aneurysm (AAA) is unpredictable and lethal. A clinically valid biomarker to monitor the disease has not been available. Based on our recent discoveries that uncoupled endothelial nitric oxide synthase (eNOS)/tetrahydrobiopterin deficiency plays a causal role in various models of AAA, the present study examined the relationship between circulating and tissue levels of tetrahydrobiopterin (H4B) in angiotensin II-infused hyperphenylalaninemia (hph-1) and apoE null mice. For apoE null mice, tissue and plasma H4B levels decreased time dependently, to 2.69 ± 0.15 and 1.99 ± 0.06 pmol/mg, respectively (from 4.86 ± 0.32 and 3.31 ± 0.13 pmol/mg at baseline) by week 3, when aneurysms developed. For hph-1 mice, tissue and plasma H4B levels decreased significantly to 1.02 ± 0.10 and 0.98 ± 0.09 pmol/mg, respectively (from 1.84 ± 0.18 and 1.48 ± 0.12 pmol/mg at baseline), by week 1, when aneurysms developed. Oral folic acid administration, which has been shown to improve aortic H4B levels to completely prevent or markedly decrease the incidence of AAA, significantly increased tissue and plasma H4B levels in both animal models starting at week 1. The two H4B measurements at all conditions showed significant linear correlation, suggesting that plasma H4B accurately predicts its tissue levels when H4B is either reduced or enhanced. Together, these data demonstrate that H4B levels decrease with AAA development and increase with folic acid treatment in two different murine models of AAA and that plasma H4B levels accurately reflect H4B levels in the tissue, suggesting that circulating H4B levels may be used clinically as a novel and powerful biomarker for the development and response to treatment of AAA.
Assuntos
Aneurisma da Aorta Abdominal/sangue , Biomarcadores/sangue , Biopterinas/análogos & derivados , Angiotensina II/administração & dosagem , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/diagnóstico , Apolipoproteínas E/genética , Biopterinas/sangue , Ácido Fólico/farmacologia , Hematínicos/farmacologia , Masculino , Camundongos , Camundongos Knockout , Fenilalanina/sangue , Fenilalanina/genética , Vasoconstritores/administração & dosagemRESUMO
We have previously shown that circulating levels of tetrahydrobiopterin (H4B) function as a robust biomarker for aortic aneurysms in several independent animal models. In the present study, we examined diagnostic and predictive values of circulating H4B levels in human patients of thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) for the first time, while clinically applicable biomarkers for aortic aneurysms have never been previously available. Ninety-five patients scheduled for TAA repair surgeries and 53 control subjects were recruited at University of California Los Angeles (UCLA) Ronald Regan Medical Center, while 44 control subjects and 29 AAA patients were recruited through National Institute of Health (NIH) National Disease Research Interchange (NDRI) program. We had intriguing observations that circulating H4B levels were substantially lower in TAA and AAA patients, linearly correlated with aortic H4B levels (blood: R = 0.8071, p < 0.0001, n = 75; plasma: R = 0.7983, p < 0.0001, n = 75), and associated with incidence of TAA (blood: adjusted OR 0.495; 95% CI 0.379-0.647; p < 0.001; plasma: adjusted OR 0.501; 95% CI 0.385-0.652; p < 0.001) or AAA (blood: adjusted OR 0.329; 95% CI 0.125-0.868; p = 0.025) after adjustment for other factors. Blood or plasma H4B levels below 0.2 pmol/µg serve as an important threshold for prediction of aortic aneurysms independent of age and gender (for TAA risk - blood: adjusted OR 419.67; 95% CI 59.191-2975.540; p < 0.001; plasma: adjusted OR 206.11; 95% CI 40.956-1037.279; p < 0.001). This threshold was also significantly associated with incidence of AAA (p < 0.001 by Chi-square analysis). In addition, we observed previously unrecognized inverse association of Statin use with TAA, and an association of AAA with arrhythmia. Taken together, our data strongly demonstrate for the first time that circulating H4B levels can serve as a first-in-class, sensitive, robust and independent biomarker for clinical diagnosis and prediction of TAA and AAA in human patients, which can be rapidly translated to bedside to fundamentally improve clinical management of the devastating human disease of aortic aneurysms.
Assuntos
Aneurisma da Aorta Abdominal , Aneurisma da Aorta Torácica , Inibidores de Hidroximetilglutaril-CoA Redutases , Animais , Aneurisma da Aorta Abdominal/diagnóstico , Aneurisma da Aorta Torácica/complicações , Aneurisma da Aorta Torácica/diagnóstico , Biomarcadores , Biopterinas/análogos & derivados , HumanosRESUMO
In the present study we aimed to identify novel mechanisms and therapeutics for thoracic aortic aneurysm (TAA) in Fbn1C1039G/+ Marfan Syndrome (MFS) mice. The expression of mature/active TGFß and its downstream effector NOX4 were upregulated while tetrahydrobiopterin (H4B) salvage enzyme dihydrofolate reductase (DHFR) was downregulated in Fbn1C1039G/+ mice. In vivo treatment with anti-TGFß completely attenuated NOX4 expression, restored DHFR protein abundance, reduced ROS production, recoupled eNOS and attenuated aneurysm formation. Intriguingly, oral administration with folic acid (FA) to recouple eNOS markedly alleviated expansion of aortic roots and abdominal aortas in Fbn1C1039G/+ mice, which was attributed to substantially upregulated DHFR expression and activity in the endothelium to restore tissue and circulating levels of H4B. Notably, circulating H4B levels were accurately predictive of tissue H4B bioavailability, and negatively associated with expansion of aortic roots, indicating a novel biomarker role of circulating H4B for TAA. Furthermore, FA diet abrogated TGFß and NOX4 expression, disrupting the feed-forward loop to inactivate TGFß/NOX4/DHFR/eNOS uncoupling axis in vivo and in vitro, while PTIO, a NO scavenger, reversed this effect in cultured human aortic endothelial cells (HAECs). Besides, expression of the rate limiting H4B synthetic enzyme GTP cyclohydrolase 1 (GTPCHI), was downregulated in Fbn1C1039G/+ mice at baseline. In cultured HAECs, RNAi inhibition of fibrillin resulted in reduced GTPCHI expression, while this response was abrogated by anti-TGFß, indicating TGFß-dependent downregulation of GTPCHI in response to fibrillin deficiency. Taken together, our data for the first time reveal that uncoupled eNOS plays a central role in TAA formation, while anti-TGFß and FA diet robustly abolish aneurysm formation via inactivation of a novel TGFß/NOX4/DHFR/eNOS uncoupling/TGFß feed-forward pathway. Correction of fibrillin deficiency is additionally beneficial via preservation of GTPCHI function.
Assuntos
Aneurisma Aórtico , Tetra-Hidrofolato Desidrogenase , Animais , Células Endoteliais , Ácido Fólico , Camundongos , NADPH Oxidase 4/genética , Tetra-Hidrofolato Desidrogenase/genética , Fator de Crescimento Transformador betaRESUMO
Hypertension and abdominal aortic aneurysm (AAA) are severe cardiovascular diseases with incompletely defined molecular mechanisms. In the current study we generated dihydrofolate reductase (DHFR) knockout mice for the first time to examine its potential contribution to the development of hypertension and AAA, as well as the underlying molecular mechanisms. Whereas the homozygote knockout mice were embryonically lethal, the heterozygote knockout mice had global reduction in DHFR protein expression and activity. Angiotensin II infusion into these animals resulted in substantially exaggerated elevation in blood pressure and development of AAA, which was accompanied by excessive eNOS uncoupling activity (featured by significantly impaired tetrahydrobiopterin and nitric oxide bioavailability), vascular remodeling (MMP2 activation, medial elastin breakdown and adventitial fibrosis) and inflammation (macrophage infiltration). Importantly, scavenging of mitochondrial reactive oxygen species with Mito-Tempo in vivo completely abrogated development of hypertension and AAA in DHFR knockout mice, indicating a novel role of mitochondria in mediating hypertension and AAA downstream of DHFR deficiency-dependent eNOS uncoupling. These data for the first time demonstrate that targeting DHFR-deficiency driven mitochondrial dysfunction may represent an innovative therapeutic option for the treatment of AAA and hypertension.
Assuntos
Anemia Megaloblástica/complicações , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/metabolismo , Hipertensão/etiologia , Hipertensão/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Tetra-Hidrofolato Desidrogenase/deficiência , Angiotensina II/metabolismo , Animais , Aneurisma da Aorta Abdominal/patologia , Pressão Sanguínea , Modelos Animais de Doenças , Loci Gênicos , Hipertensão/diagnóstico , Hipertensão/fisiopatologia , Macrófagos/metabolismo , Macrófagos/patologia , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , UltrassonografiaRESUMO
We have shown that hydrogen peroxide (H2O2) downregulates tetrahydrobiopterin salvage enzyme DHFR (dihydrofolate reductase) to result in eNOS (endothelial NO synthase) uncoupling and elevated blood pressure. Here, we aimed to delineate molecular mechanisms underlying H2O2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression and effects on blood pressure regulation of targeting these novel mechanisms. H2O2 dose and time dependently attenuated DHFR mRNA and protein expression and enzymatic activity in endothelial cells. Deletion of E2F-binding sites, but not those of Sp1 (specificity protein 1), abolished H2O2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H2O2 on DHFR promoter activity. H2O2 treatment diminished mRNA and protein expression of E2F1/2/3a, whereas overexpression of E2F isoforms increased DHFR protein levels. Chromatin immunoprecipitation assay indicated direct binding of E2F1/2/3a to the DHFR promoter, which was weakened by H2O2. E2F1 RNA interference attenuated DHFR protein levels, whereas its overexpression elevated tetrahydrobiopterin levels and tetrahydrobiopterin/dihydrobiopterin ratios in vitro and in vivo. In Ang II (angiotensin II)-infused mice, adenovirus-mediated overexpression of E2F1 markedly abrogated blood pressure to control levels, by restoring endothelial DHFR function to improve NO bioavailability and vasorelaxation. Bioinformatic analyses confirmed a positive correlation between E2F1 and DHFR in human endothelial cells and arteries, and downregulation of both by oxidized phospholipids. In summary, endothelial DHFR is downregulated by H2O2 transcriptionally via an E2F-dependent mechanism, and that specifically targeting E2F1/2/3a to restore DHFR and eNOS function may serve as a novel therapeutic option for the treatment of hypertension.
Assuntos
Pressão Sanguínea , Fator de Transcrição E2F1 , Células Endoteliais , Peróxido de Hidrogênio , Hipertensão , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Regulação para Baixo , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F3/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/fisiologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
Restenosis after angioplasty is a serious clinical problem that can result in re-occlusion of the coronary artery. Although current drug-eluting stents have proved to be more effective in reducing restenosis, they have drawbacks of inhibiting reendothelialization to promote thrombosis. New treatment options are in urgent need. We have shown that netrin-1, an axon-guiding protein, promotes angiogenesis and cardioprotection via production of nitric oxide (NO). The present study examined whether and how netrin-1 attenuates neointimal formation in a femoral wire injury model. Infusion of netrin-1 into C57BL/6 mice markedly attenuated neointimal formation following wire injury of femoral arteries, measured by intimal to media ratio (from 1.94 ± 0.55 to 0.45 ± 0.86 at 4 weeks). Proliferation of VSMC in situ was largely reduced. This protective effect was absent in DCC+/- animals. NO production was increased by netrin-1 in both intact and injured femoral arteries, indicating netrin-1 stimulation of endogenous NO production from intact endothelium and remaining endothelial cells post-injury. VSMC migration was abrogated by netrin-1 via a NO/cGMP/p38 MAPK pathway, while timely EPC homing was induced. Injection of netrin-1 preconditioned wild-type EPCs, but not EPCs of DCC+/- animals, substantially attenuated neointimal formation. EPC proliferation, NO production, and resistance to oxidative stress induced apoptosis were augmented by netrin-1 treatment. In conclusion, our data for the first time demonstrate that netrin-1 is highly effective in reducing neointimal formation following vascular endothelial injury, which is dependent on DCC, and attributed to inhibition of VSMC proliferation and migration, as well as improved EPC function. These data may support usage of netrin-1 and netrin-1 preconditioned EPCs as novel therapies for post angioplasty restenosis. KEY MESSAGE: Netrin-1 attenuates neointimal formation following post endothelial injury via DCC and NO. Netrin-1 inhibits VSMC proliferation in situ following endothelial injury. Netrin-1 inhibits VSMC migration via a NO/cGMP/p38 MAPK pathway. Netrin-1 augments proliferation of endothelial progenitor cells (EPCs) and EPC eNOS/NO activation. Netrin-1 enhances resistance of EPCs to oxidative stress, improving re-endothelialization following injury.
Assuntos
Células Progenitoras Endoteliais/efeitos dos fármacos , Neointima/tratamento farmacológico , Netrina-1/uso terapêutico , Óxido Nítrico/metabolismo , Substâncias Protetoras/uso terapêutico , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Receptor DCC/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neointima/metabolismo , Neointima/patologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Oxidative stress plays an important role in the formation of abdominal aortic aneurysm (AAA), and we have recently established a causal role of uncoupled eNOS in this severe human disease. We have also shown that activation of NADPH oxidase (NOX) lies upstream of uncoupled eNOS. Therefore, identification of the specific NOX isoforms that are required for eNOS uncoupling and AAA formation would ultimately lead to novel therapies for AAA. In the present study, we used the Ang II infused hph-1 mice to examine the roles of NOX isoforms in the development of AAA. We generated double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4. After two weeks of Ang II infusion, the incidence rate of AAA substantially dropped from 76.5% in Ang II infused hph-1 mice (n=34) to 11.1%, 15.0%, 9.5% and 0% in hph-1-NOX1 (n=27), hph-1-NOX2 (n=40), hph-1-p47phox (n=21), and hph-1-NOX4 (n=33) double mutant mice, respectively. The size of abdominal aortas of the four double mutant mice, determined by ultrasound analyses, was significantly smaller than the hph-1 mice. Aortic nitric oxide and H4B bioavailabilities were markedly improved in the double mutants, while superoxide production and eNOS uncoupling activity were substantially diminished. These effects seemed attributed to an endothelial specific restoration of dihydrofolate reductase expression and activity, deficiency of which has been shown to induce eNOS uncoupling and AAA formation in both Ang II-infused hph-1 and apoE null animals. In addition, over-expression of human NOX4 N129S or T555S mutant newly identified in aneurysm patients increased hydrogen peroxide production, further implicating a relationship between NOX and human aneurysm. Taken together, these data indicate that NOX isoforms 1, 2 or 4 lies upstream of dihydrofolate reductase deficiency and eNOS uncoupling to induce AAA formation. These findings may promote development of novel therapeutics for the treatment of the disease by inhibiting NOX signaling.
Assuntos
Aneurisma da Aorta Abdominal/genética , NADPH Oxidase 4/genética , NADPH Oxidases/genética , Óxido Nítrico Sintase Tipo III/genética , Complexo Repressor Polycomb 1/genética , Angiotensina II/metabolismo , Animais , Aneurisma da Aorta Abdominal/fisiopatologia , Apolipoproteínas E/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Mutação , Estresse Oxidativo/genética , Isoformas de Proteínas/genética , Superóxidos/metabolismo , Tetra-Hidrofolato Desidrogenase/deficiência , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
We have previously shown that eNOS uncoupling mediates abdominal aortic aneurysm (AAA) formation in hph-1 mice. In the present study we examined whether recoupling of eNOS prevents AAA formation in a well-established model of Angiotensin II-infused apolipoprotein E (apoE) null mice by targeting some common pathologies of AAA. Infusion of Ang II resulted in a 92% incidence rate of AAA in the apoE null animals. In a separate group, animals were treated orally with folic acid (FA), which is known to recouple eNOS through augmentation of dihydrofolate reductase (DHFR) function. This resulted in a reduction of AAA rate to 19.5%. Imaging with ultrasound showed that FA markedly inhibited expansion of abdominal aorta. FA also abolished elastin breakdown and macrophage infiltration in the AAA animals. The eNOS uncoupling activity, assessed by L-NAME-sensitive superoxide production, was minimal at baseline but greatly exaggerated with Ang II infusion, which was completely attenuated by FA. This was accompanied by markedly improved tetrahydrobiopterin and nitric oxide bioavailability. Furthermore, the expression and activity of DHFR was decreased in Ang II-infused apoE null mice specifically in the endothelial cells, while FA administration resulted in its recovery. Taken together, these data further establish a significant role of uncoupled eNOS in mediating AAA formation, and a universal efficacy of FA in preventing AAA formation via restoration of DHFR to restore eNOS function.
Assuntos
Angiotensina II/farmacologia , Aneurisma da Aorta Abdominal/prevenção & controle , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Ácido Fólico/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Administração Oral , Animais , Aorta Abdominal/diagnóstico por imagem , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Ácido Fólico/administração & dosagem , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Radiografia , Superóxidos/metabolismo , Ultrassonografia , Remodelação Vascular/efeitos dos fármacosRESUMO
Obesity is associated with vascular diseases that are often attributed to vascular oxidative stress. We tested the hypothesis that vascular oxidative stress could induce obesity. We previously developed mice that overexpress p22phox in vascular smooth muscle, tg(sm/p22phox), which have increased vascular ROS production. At baseline, tg(sm/p22phox) mice have a modest increase in body weight. With high-fat feeding, tg(sm/p22phox) mice developed exaggerated obesity and increased fat mass. Body weight increased from 32.16 ± 2.34 g to 43.03 ± 1.44 g in tg(sm/p22phox) mice (vs. 30.81 ± 0.71 g to 37.89 ± 1.16 g in the WT mice). This was associated with development of glucose intolerance, reduced HDL cholesterol, and increased levels of leptin and MCP-1. Tg(sm/p22phox) mice displayed impaired spontaneous activity and increased mitochondrial ROS production and mitochondrial dysfunction in skeletal muscle. In mice with vascular smooth muscle-targeted deletion of p22phox (p22phox(loxp/loxp)/tg(smmhc/cre) mice), high-fat feeding did not induce weight gain or leptin resistance. These mice also had reduced T-cell infiltration of perivascular fat. In conclusion, these data indicate that vascular oxidative stress induces obesity and metabolic syndrome, accompanied by and likely due to exercise intolerance, vascular inflammation, and augmented adipogenesis. These data indicate that vascular ROS may play a causal role in the development of obesity and metabolic syndrome.