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[Figure: see text].
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Biopterinas/análogos & derivados , Presión Sanguínea/fisiología , Células Endoteliales/metabolismo , Desarrollo Fetal/fisiología , GTP Ciclohidrolasa/metabolismo , Placenta/metabolismo , Útero/metabolismo , Animales , Biopterinas/genética , Biopterinas/metabolismo , Endotelio Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , GTP Ciclohidrolasa/genética , Humanos , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , EmbarazoRESUMEN
INTRODUCTION: GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo. METHODS AND RESULTS: Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2-3 of the Gch1 gene. Gch1(fl/fl) mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1(-/-) embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1(-/-) embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l-DOPA. CONCLUSION: These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4.
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Biopterinas/análogos & derivados , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , GTP Ciclohidrolasa/metabolismo , Animales , Biopterinas/metabolismo , Cromatografía Líquida de Alta Presión , Embrión de Mamíferos/embriología , Femenino , GTP Ciclohidrolasa/genética , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Levodopa/metabolismo , Masculino , Espectrometría de Masas , Metabolómica , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) function and NO generation. Augmentation of BH4 levels can prevent eNOS uncoupling and can improve endothelial dysfunction in vascular disease states. However, the physiological requirement for de novo endothelial cell BH4 biosynthesis in eNOS function remains unclear. We generated a novel mouse model with endothelial cell-specific deletion of GCH1, encoding GTP cyclohydrolase 1, an essential enzyme for BH4 biosynthesis, to test the cell-autonomous requirement for endothelial BH4 biosynthesis in vivo. Mice with a floxed GCH1 allele (GCH1(fl/fl)) were crossed with Tie2cre mice to delete GCH1 in endothelial cells. GCH1(fl/fl)Tie2cre mice demonstrated virtually absent endothelial NO bioactivity and significantly greater O2 (â¢-) production. GCH1(fl/fl)Tie2cre aortas and mesenteric arteries had enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. Endothelium-dependent vasodilatations in GCH1(fl/fl)Tie2cre aortas were, in part, mediated by eNOS-derived hydrogen peroxide (H2O2), which mediated vasodilatation through soluble guanylate cyclase. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin restored normal endothelial function and abolished eNOS-derived H2O2 production in GCH1(fl/fl)Tie2cre aortas. GCH1(fl/fl)Tie2cre mice had higher systemic blood pressure than wild-type littermates, which was normalized by NOS inhibitor, NG-nitro-L-arginine methyl ester. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure and identify endothelial cell BH4 as a pivotal regulator of NO versus H2O2 as alternative eNOS-derived endothelial-derived relaxing factors.
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Biopterinas/análogos & derivados , Presión Sanguínea/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , GTP Ciclohidrolasa/fisiología , Acetilcolina/farmacología , Animales , Biopterinas/genética , Biopterinas/fisiología , Presión Sanguínea/genética , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Femenino , GTP Ciclohidrolasa/deficiencia , GTP Ciclohidrolasa/genética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oligopéptidos/farmacología , Oxígeno/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Vasodilatadores/farmacologíaRESUMEN
AIMS: Predicting the likely success of primary PCI to salvage potential infarcted myocardium is desirable. We compared early invasive parameters of coronary microcirculation function with the levels of circulating endothelin (ET-1) and 6-month ejection fraction after STEMI. METHODS AND RESULTS: Forty-four STEMI patients underwent assessment of coronary flow reserve (CFR) and index of myocardial resistance (IMR) on completion of PPCI and one day later. Cardiac magnetic resonance (CMR) at 24 h and 6 months assessed ejection fraction, oedema, late gadolinium enhancement, and salvage. In patients with depressed EF, there was no difference in IMR or CFR measured immediately after PPCI compared with those with preserved EF. However, by Day 1, CFR was significantly lower in those with depressed EF [2.0(1.5-2.3) vs. 2.6(2.1-3.3), P = 0.008]. In multivariable models, higher CFR post-PPCI [EST: +8.9 (SE 3.7) per 1 CFR unit, P = 0.03] and greater increase in CFR between post-PPCI and Day 1 [EST: +8.5 (SE 3.4) per 1 CFR unit, P = 0.01] were associated with higher salvage index. Circulating endothelin levels were significantly elevated in the low EF group at both 6 and 24 h, and 24 h levels correlated with CFR. CONCLUSION: Changes of the coronary microcirculation in the first day after PPCI are associated with 6-month ejection fraction and myocardial salvage. Depressed CFR at 24 h is associated with CMR imaging indices of MVO and haemorrhage and elevated endothelin levels.
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Circulación Coronaria/fisiología , Infarto del Miocardio/fisiopatología , Intervención Coronaria Percutánea , Oclusión Coronaria/fisiopatología , Vasos Coronarios/fisiología , Endotelina-1/metabolismo , Femenino , Hemorragia/fisiopatología , Humanos , Angiografía por Resonancia Magnética , Masculino , Microcirculación/fisiología , Microvasos/fisiología , Persona de Mediana Edad , Infarto del Miocardio/terapia , Estudios Prospectivos , Recuperación de la Función , Volumen Sistólico/fisiología , Resistencia Vascular/fisiologíaRESUMEN
AIMS: Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. METHODS AND RESULTS: Endothelial cell repopulation was assessed en face in stented arteries in ApoE(-/-) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm(2), P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm(2), P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(-/-) mice had less neointima formation compared with ApoE(-/-) littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm(2), P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm(2), P = 0.043). CONCLUSION: Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.
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Aterosclerosis/patología , Células Endoteliales/patología , Endotelio Vascular/patología , Stents , Animales , Aspirina/farmacología , Stents Liberadores de Fármacos , Fibrinolíticos/farmacología , Masculino , Ratones , Ratones Endogámicos , Neointima/patología , Paclitaxel/farmacología , Moduladores de Tubulina/farmacologíaRESUMEN
RATIONALE: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthases (NOS). Oral BH4 supplementation preserves cardiac function in animal models of cardiac disease; however, the mechanisms underlying these findings are not completely understood. OBJECTIVE: To study the effect of myocardial transgenic overexpression of the rate-limiting enzyme in BH4 biosynthesis, GTP cyclohydrolase 1 (GCH1), on NOS activity, myocardial function, and Ca2+ handling. METHODS AND RESULTS: GCH1overexpression significantly increased the biopterins level in left ventricular (LV) myocytes but not in the nonmyocyte component of the LV myocardium or in plasma. The ratio between BH4 and its oxidized products was lower in mGCH1-Tg, indicating that a large proportion of the myocardial biopterin pool was oxidized; nevertheless, myocardial NOS1 activity was increased in mGCH1-Tg, and superoxide release was significantly reduced. Isolated hearts and field-stimulated LV myocytes (3 Hz, 35°C) overexpressing GCH1 showed a faster relaxation and a PKA-mediated increase in the PLB Ser16 phosphorylated fraction and in the rate of decay of the [Ca2+]i transient. RyR2 S-nitrosylation and diastolic Ca2+ leak were larger in mGCH1-Tg and ICa density was lower; nevertheless the amplitude of the [Ca2+]i transient and contraction did not differ between genotypes, because of an increase in the SR fractional release of Ca2+ in mGCH1-Tg myocytes. Xanthine oxidoreductase inhibition abolished the difference in superoxide production but did not affect myocardial function in either group. By contrast, NOS1 inhibition abolished the differences in ICa density, Ser16 PLB phosphorylation, [Ca2+]i decay, and myocardial relaxation between genotypes. CONCLUSIONS: Myocardial GCH1 activity and intracellular BH4 are a limiting factor for constitutive NOS1 and SERCA2A activity in the healthy myocardium. Our findings suggest that GCH1 may be a valuable target for the treatment of LV diastolic dysfunction.
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Biopterinas/análogos & derivados , GTP Ciclohidrolasa/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Biopterinas/metabolismo , Biopterinas/farmacología , Calcio/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Femenino , GTP Ciclohidrolasa/genética , Corazón/efectos de los fármacos , Corazón/fisiología , Humanos , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Miocardio/citología , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Superóxidos/metabolismoRESUMEN
BACKGROUND: An altered nitric oxide-redox balance has been implicated in the pathogenesis of atrial fibrillation (AF). Statins inhibit NOX2-NADPH oxidases and prevent postoperative AF but are less effective in AF secondary prevention; the mechanisms underlying these findings are poorly understood. METHODS AND RESULTS: By using goat models of pacing-induced AF or of atrial structural remodeling secondary to atrioventricular block and right atrial samples from 130 patients undergoing cardiac surgery, we found that the mechanisms responsible for the NO-redox imbalance differ between atria and with the duration and substrate of AF. Rac1 and NADPH oxidase activity and the protein level of NOX2 and p22phox were significantly increased in the left atrium of goats after 2 weeks of AF and in patients who developed postoperative AF in the absence of differences in leukocytes infiltration. Conversely, in the presence of longstanding AF or atrioventricular block, uncoupled nitric oxide synthase activity (secondary to reduced BH4 content and/or increased arginase activity) and mitochondrial oxidases accounted for the biatrial increase in reactive oxygen species. Atorvastatin caused a mevalonate-reversible inhibition of Rac1 and NOX2-NADPH oxidase activity in right atrial samples from patients who developed postoperative AF, but it did not affect reactive oxygen species, nitric oxide synthase uncoupling, or BH4 in patients with permanent AF. CONCLUSIONS: Upregulation of atrial NADPH oxidases is an early but transient event in the natural history of AF. Changes in the sources of reactive oxygen species with atrial remodeling may explain why statins are effective in the primary prevention of AF but not in its management.
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Antiarrítmicos/uso terapéutico , Fibrilación Atrial/prevención & control , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Arginasa/metabolismo , Bloqueo Atrioventricular/metabolismo , Modelos Animales de Enfermedad , Femenino , Cabras , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/biosíntesis , Persona de Mediana Edad , Mitocondrias/enzimología , NADPH Oxidasa 2 , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/metabolismo , Oxidorreductasas/metabolismo , Proteína de Unión al GTP rac1/metabolismoAsunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Enfermedades de la Aorta/etiología , Oclusión Coronaria/etiología , Policitemia/etiología , Fumar/efectos adversos , Trombosis/etiología , Adulto , Angioplastia Coronaria con Balón , Angiografía Coronaria , Oclusión Coronaria/diagnóstico por imagen , Oclusión Coronaria/terapia , Humanos , Masculino , Infarto del Miocardio/etiología , Choque Cardiogénico/etiología , Trombectomía , Resultado del TratamientoRESUMEN
The exogenous administration of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase (NOS), has been shown to reduce left ventricular hypertrophy, fibrosis, and cardiac dysfunction in mice with pre-established heart disease induced by pressure-overload. In this setting, BH4 re-coupled endothelial NOS (eNOS), with subsequent reduction of NOS-dependent oxidative stress and reversal of maladaptive remodeling. However, recent studies suggest the effective BH4 dosing may be narrower than previously thought, potentially due to its oxidation upon oral consumption. Accordingly, we assessed the dose response of daily oral synthetic sapropterin dihydrochloride (6-R-l-erythro-5,6,7,8-tetrahydrobiopterin, 6R-BH4) on pre-established pressure-overload cardiac disease. Mice (n=64) were administered 0-400mg/kg/d BH4 by ingesting small pre-made pellets (consumed over 15-30 min). In a dose range of 36-200mg/kg/d, 6R-BH4 suppressed cardiac chamber remodeling, hypertrophy, fibrosis, and oxidative stress with pressure-overload. However, at both lower and higher doses, BH4 had less or no ameliorative effects. The effective doses correlated with a higher myocardial BH4/BH2 ratio. However, BH2 rose linearly with dose, and at the 400mg/kg/d, this lowered the BH4/BH2 ratio back toward control. These results expose a potential limitation for the clinical use of BH4, as variability of cellular redox and perhaps heart disease could produce a variable therapeutic window among individuals. This article is part of a special issue entitled ''Key Signaling Molecules in Hypertrophy and Heart Failure.''
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Biopterinas/análogos & derivados , Cardiotónicos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Remodelación Ventricular/efectos de los fármacos , Análisis de Varianza , Animales , Biopterinas/metabolismo , Biopterinas/farmacocinética , Biopterinas/uso terapéutico , Cardiotónicos/farmacocinética , Relación Dosis-Respuesta a Droga , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Humanos , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/fisiopatología , Ligadura , Ratones , Ratones Endogámicos C57BL , Miocardio/patología , Distribución Aleatoria , Superóxidos/metabolismo , Función Ventricular IzquierdaRESUMEN
GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for tetrahydrobiopterin (BH(4)) synthesis. Decreases in GTPCH activity and expression have been shown in late stages of acute cardiac rejection, suggesting a deficit in BH(4). We hypothesized that increasing intracellular levels of BH(4) by cardiac myocyte-targeted overexpression of GTPCH would diminish acute cardiac allograft rejection. Transgenic mice overexpressing GTPCH in the heart were generated and crossed on C57BL6 background. Wild-type and transgenic mouse donor hearts were transplanted into BALB/c recipient mice. Left ventricular (LV) function, histological rejection, BH(4) levels, and inflammatory cytokine gene expression (mRNA) were examined. Expression of human GTPCH was documented by PCR, Western analysis, and function by a significant (P < 0.001) increase in cardiac BH(4) levels. GTPCH transgene decreased histological rejection (46%; P < 0.003) and cardiac myocyte injury (eosin autofluorescence; 56%; P < 0.0001) independent of changes in inflammatory cytokine expression or nitric oxide content. GTPCH transgene decreased IL-2 (88%; P < 0.002), IL-1R2 (42%; P < 0.0001), and programmed cell death-1 (67%; P < 0.0001) expression, whereas it increased fms-like tyrosine kinase 3 (156%; P < 0.0001) and stromal-derived factor-1 (2; 190%; P < 0.0001) expression. There was no difference in ejection fraction or fractional shortening; however, LV mass was significantly increased (P < 0.05) only in wild-type grafts. The decreases in LV mass, cardiac injury, and histological rejection support a protective role of cardiac GTPCH overexpression and increased BH(4) synthesis in cardiac allografts. The mechanism of the decreased rejection appears related to decreased T cell proliferation and modulation of immune function by higher expression of genes involved in hematopoietic/stromal cell development and recruitment.
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Biopterinas/análogos & derivados , GTP Ciclohidrolasa/metabolismo , Rechazo de Injerto/prevención & control , Trasplante de Corazón , Miocitos Cardíacos/enzimología , Enfermedad Aguda , Animales , Biopterinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , GTP Ciclohidrolasa/genética , Genotipo , Rechazo de Injerto/diagnóstico por imagen , Rechazo de Injerto/enzimología , Rechazo de Injerto/inmunología , Rechazo de Injerto/fisiopatología , Trasplante de Corazón/efectos adversos , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/patología , Óxido Nítrico/metabolismo , Fenotipo , ARN Mensajero/metabolismo , Trasplante Homólogo , Ultrasonografía , Función Ventricular IzquierdaRESUMEN
BH4 (tetrahydrobiopterin) supplementation improves endothelial function in models of vascular disease by maintaining eNOS (endothelial nitric oxide synthase) coupling and NO (nitric oxide) bioavailability. However, the cellular mechanisms through which enhanced endothelial function leads to reduced atherosclerosis remain unclear. We have used a pharmaceutical BH4 formulation to investigate the effects of BH4 supplementation on atherosclerosis progression in ApoE-KO (apolipoprotein E-knockout) mice. Single oral dose pharmacokinetic studies revealed rapid BH4 uptake into plasma and organs. Plasma BH4 levels returned to baseline by 8 h after oral dosing, but remained markedly increased in aorta at 24 h. Daily oral BH4 supplementation in ApoE-KO mice from 8 weeks of age, for a period of 8 or 12 weeks, had no effect on plasma lipids or haemodynamic parameters, but significantly reduced aortic root atherosclerosis compared with placebo-treated animals. BH4 supplementation significantly reduced VCAM-1 (vascular cell adhesion molecule 1) mRNA levels in aortic endothelial cells, markedly reduced the infiltration of T-cells, macrophages and monocytes into plaques, and reduced T-cell infiltration in the adjacent adventitia, but importantly had no effect on circulating leucocytes. GCH (GTP cyclohydrolase I)-transgenic mice, with a specific increase in endothelial BH4 levels, exhibited a similar reduction in vascular immune cell infiltration compared with BH4-deficient controls, suggesting that BH4 reduces vascular inflammation via endothelial cell signalling. In conclusion, BH4 supplementation reduces vascular immune cell infiltration in atherosclerosis and may therefore be a rational therapeutic approach to reduce the progression of atherosclerosis.
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Enfermedades de la Aorta/tratamiento farmacológico , Apolipoproteínas E/deficiencia , Aterosclerosis/tratamiento farmacológico , Biopterinas/análogos & derivados , Administración Oral , Animales , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Biopterinas/farmacocinética , Biopterinas/uso terapéutico , Quimiotaxis de Leucocito/efectos de los fármacos , Progresión de la Enfermedad , Esquema de Medicación , Evaluación Preclínica de Medicamentos , Endotelio Vascular/metabolismo , Hemodinámica/efectos de los fármacos , Lípidos/sangre , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/genética , Distribución Tisular , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.
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Biopterinas/análogos & derivados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Antioxidantes/metabolismo , Biopterinas/metabolismo , Línea Celular , Células Cultivadas , Doxiciclina/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/enzimología , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Metotrexato/metabolismo , Ratones , Células 3T3 NIH , Óxido Nítrico Sintasa de Tipo III/genética , Oxidación-Reducción , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Superóxidos/metabolismo , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
BACKGROUND: Atherosclerosis is an inflammatory process that develops in individuals with known risk factors that include hypertension and hyperlipidaemia, influenced by diet. However, the interplay between diet, inflammatory mechanisms and vascular risk factors requires further research. We hypothesised that interleukin-1 (IL-1) signaling in the vessel wall would raise arterial blood pressure and promote atheroma. METHODOLOGY/PRINCIPAL FINDINGS: Apoe(-/-) and Apoe(-/-)/IL-1R1(-/-) mice were fed high fat diets for 8 weeks, and their blood pressure and atherosclerosis development measured. Apoe(-/-)/IL-R1(-/-) mice had a reduced blood pressure and significantly less atheroma than Apoe(-/-) mice. Selective loss of IL-1 signaling in the vessel wall by bone marrow transplantation also reduced plaque burden (p < 0.05). This was associated with an IL-1 mediated loss of endothelium-dependent relaxation and an increase in vessel wall Nox 4. Inhibition of IL-1 restored endothelium-dependent vasodilatation and reduced levels of arterial oxidative stress. CONCLUSIONS/SIGNIFICANCE: The IL-1 cytokine system links atherogenic environmental stimuli with arterial inflammation, oxidative stress, increased blood pressure and atherosclerosis. This is the first demonstration that inhibition of a single cytokine can block the rise in blood pressure in response to an environmental stimulus. IL-1 inhibition may have profound beneficial effects on atherogenesis in man.
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Aterosclerosis/fisiopatología , Grasas de la Dieta/administración & dosificación , Interleucina-1/fisiología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/fisiología , Presión Sanguínea , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/fisiologíaRESUMEN
GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.
Asunto(s)
Biopterinas/análogos & derivados , Proteínas Portadoras/metabolismo , Células Endoteliales/enzimología , GTP Ciclohidrolasa/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Antibacterianos/farmacología , Biopterinas/biosíntesis , Biopterinas/genética , Proteínas Portadoras/genética , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , GTP Ciclohidrolasa/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Células 3T3 NIHRESUMEN
Tetrahydrobiopterin (BH4) is a critical determinant of endothelial nitric-oxide synthase (eNOS) activity. In the absence of BH4, eNOS becomes "uncoupled" and generates superoxide rather than NO. However, the stoichiometry of intracellular BH4/eNOS interactions is not well defined, and it is unclear whether intracellular BH4 deficiency alone is sufficient to induce eNOS uncoupling. To address these questions, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in BH4 synthesis, to selectively induce intracellular BH4 deficiency by incubation with doxycycline. These cells were stably co-transfected to express a human eNOS-green fluorescent protein fusion protein, selecting clones expressing either low (GCH/eNOS-LOW) or high (GCH/eNOS-HIGH) levels. Doxycycline abolished GTPCH mRNA expression and GTPCH protein, leading to markedly diminished total biopterin levels and a decreased ratio of BH4 to oxidized biopterins in cells expressing eNOS. Intracellular BH4 deficiency induced superoxide generation from eNOS, as assessed by N-nitro-L-arginine methyl ester inhibitable 2-hydroxyethidium generation, and attenuated NO production. Quantitative analysis of cellular BH4 versus superoxide production between GCH/eNOS-LOW and GCH/eNOS-HIGH cells revealed a striking linear relationship between eNOS protein and cellular BH4 stoichiometry, with eNOS uncoupling at eNOS:BH4 molar ratio >1. Furthermore, increasing the intracellular BH2 concentration in the presence of a constant eNOS:BH4 ratio was sufficient to induce eNOS-dependent superoxide production. This specific, reductionist approach in a cell-based system reveals that eNOS:BH4 reaction stoichiometry together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling, even in the absence of exogenous oxidative stress.
Asunto(s)
Biopterinas/metabolismo , GTP Ciclohidrolasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Biopterinas/análogos & derivados , Biopterinas/deficiencia , Línea Celular , GTP Ciclohidrolasa/genética , Humanos , Ratones , Óxido Nítrico/biosíntesis , Oxidación-Reducción , Unión Proteica , ARN Interferente Pequeño/genética , Superóxidos/metabolismoRESUMEN
BACKGROUND: Vascular injury results in loss of endothelial nitric oxide (NO), production of reactive oxygen species (ROS), and the initiation of an inflammatory response. Both NO and ROS modulate inflammation through redox-sensitive pathways. Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) that regulates enzymatic synthesis of either nitric oxide or ROS. We hypothesized that endothelial BH4 is an important regulator of inflammation and vascular remodeling. METHODS AND RESULTS: Endothelium-targeted overexpression of GTP cyclohydrolase 1 (GCH), the rate limiting enzyme in BH4 synthesis, increased levels of tetrahydrobiopterin (BH4), reduced endothelial superoxide, improved eNOS coupling, and reduced vein graft atherosclerosis in transgenic GCH/ApoE-KO mice compared to ApoE-KO controls. Immunohistochemistry using anti-MAC-3 and MAC-1 antibody staining revealed a marked reduction in vein graft macrophage content, as did RT-PCR expression of macrophage marker CD68 mRNA levels in GCH/ApoE-KO mice. When we investigated the potential mediators of this reduction, we discovered that mRNA and protein levels of MCP-1 (CCL2) but not RANTES (CCL5) were significantly reduced in GCH/ApoE-KO aortic tissue. Consistent with this finding we found a decrease in CCR2-mediated, but not CCR5-mediated, chemotaxis in vascular tissue and plasma samples from GCH/ApoE-KO animals. CONCLUSIONS: Increased endothelial BH4 reduces vein graft neointimal hyperplasia and atherosclerosis through a reduction in vascular inflammation. These findings highlight the importance of MCP-1/CCR2 signaling in the response to vascular injury and identify novel pathways linking endothelial BH4 to inflammation and vascular remodeling.
Asunto(s)
Aterosclerosis/prevención & control , Biopterinas/análogos & derivados , Vasos Sanguíneos/lesiones , Quimiocina CCL2/metabolismo , Endotelio Vascular/metabolismo , Receptores CCR2/metabolismo , Vasculitis/prevención & control , Animales , Aorta/metabolismo , Apolipoproteínas E/deficiencia , Aterosclerosis/etiología , Biopterinas/metabolismo , Arterias Carótidas/cirugía , Quimiotaxis , Femenino , GTP Ciclohidrolasa/metabolismo , Humanos , Hiperplasia , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Superóxidos/metabolismo , Túnica Íntima/patología , Regulación hacia Arriba , Vasculitis/complicaciones , Venas Cavas/metabolismo , Venas Cavas/patología , Venas Cavas/trasplante , Heridas y Lesiones/complicacionesRESUMEN
OBJECTIVES: This study sought to determine the effects of endogenous tetrahydrobiopterin (BH4) bioavailability on endothelial nitric oxide synthase (eNOS) coupling, nitric oxide (NO) bioavailability, and vascular superoxide production in patients with coronary artery disease (CAD). BACKGROUND: GTP-cyclohydrolase I, encoded by the GCH1 gene, is the rate-limiting enzyme in the biosynthesis of BH4, an eNOS cofactor important for maintaining enzymatic coupling. We examined the associations between haplotypes of the GCH1 gene, GCH1 expression and biopterin levels, and the effects on endothelial function and vascular superoxide production. METHODS: Blood samples and segments of internal mammary arteries and saphenous veins were obtained from patients with CAD undergoing coronary artery bypass grafting (n = 347). The GCH1 haplotypes were defined by 3 polymorphisms: rs8007267G
Asunto(s)
Biopterinas/análogos & derivados
, Enfermedad de la Arteria Coronaria/metabolismo
, Endotelio Vascular/metabolismo
, GTP Ciclohidrolasa/genética
, Óxido Nítrico Sintasa de Tipo III/metabolismo
, Anciano
, Biopterinas/biosíntesis
, Biopterinas/sangre
, Enfermedad de la Arteria Coronaria/genética
, Femenino
, Variación Genética
, Haplotipos
, Humanos
, Masculino
, Arterias Mamarias/metabolismo
, Análisis Multivariante
, Óxido Nítrico/metabolismo
, Vena Safena/metabolismo
, Superóxidos/metabolismo
RESUMEN
BACKGROUND: Sustained pressure overload induces pathological cardiac hypertrophy and dysfunction. Oxidative stress linked to nitric oxide synthase (NOS) uncoupling may play an important role. We tested whether tetrahydrobiopterin (BH4) can recouple NOS and reverse preestablished advanced hypertrophy, fibrosis, and dysfunction. METHODS AND RESULTS: C57/Bl6 mice underwent transverse aortic constriction for 4 weeks, increasing cardiac mass (190%) and diastolic dimension (144%), lowering ejection fraction (-46%), and triggering NOS uncoupling and oxidative stress. Oral BH4 was then administered for 5 more weeks of pressure overload. Without reducing loading, BH4 reversed hypertrophy and fibrosis, recoupled endothelial NOS, lowered oxidant stress, and improved chamber and myocyte function, whereas untreated hearts worsened. If BH4 was started at the onset of pressure overload, it did not suppress hypertrophy over the first week when NOS activity remained preserved even in untreated transverse aortic constriction hearts. However, BH4 stopped subsequent remodeling when NOS activity was otherwise declining. A broad antioxidant, Tempol, also reduced oxidant stress yet did not recouple NOS or reverse worsened hypertrophy/fibrosis from sustained transverse aortic constriction. Microarray analysis revealed very different gene expression profiles for both treatments. BH4 did not enhance net protein kinase G activity. Finally, transgenic mice with enhanced BH4 synthesis confined to endothelial cells were unprotected against pressure overload, indicating that exogenous BH4 targeted myocytes and fibroblasts. CONCLUSIONS: NOS recoupling by exogenous BH4 ameliorates preexisting advanced cardiac hypertrophy/fibrosis and is more effective than a less targeted antioxidant approach (Tempol). These data highlight the importance of myocyte NOS uncoupling in hypertrophic heart disease and support BH4 as a potential new approach to treat this disorder.
Asunto(s)
Biopterinas/análogos & derivados , Cardiomegalia/tratamiento farmacológico , Fibrosis/tratamiento farmacológico , Hipertensión/complicaciones , Miocardio/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Biopterinas/farmacología , Biopterinas/uso terapéutico , Óxidos N-Cíclicos/farmacología , Óxidos N-Cíclicos/uso terapéutico , Modelos Animales de Enfermedad , GTP Ciclohidrolasa/genética , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Marcadores de SpinRESUMEN
Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E-null (ApoE(-/-)) and wild-type mice fed a high cholesterol diet. In ApoE(-/-) mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II(-/-) mice) prevents high-cholesterol diet-dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein-dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.