RESUMEN
Hypercholesterolemia remains one of the leading risk factors for the development of cardiovascular disease. Many large double-blind studies have demonstrated that lowering low-density lipoprotein (LDL) cholesterol using a statin can reduce the risk of having a cardiovascular event by approximately 30%. However, despite the success of statins, some patient populations are unable to lower their LDL cholesterol to meet the targeted lipid levels, due to compliance or potency issues. This is especially true for patients with heterozygous familial hypercholesterolemia who may require additional upregulation of the low-density lipoprotein receptor (LDLR) to reduce LDL cholesterol levels below those achievable with maximal dosing of statins. Here we identify a series of small molecules from a genomic DNA reporter screen that upregulate the LDLR in mouse and human liver cell lines at nanomolar potencies (EC50 = 39 nM). Structure-activity relationship studies carried out on the lead compound, OX03771 [(E)-N,N-dimethyl-3-(4-styrylphenoxy)propan-1-amine], led to the identification of compound OX03050 [(E)-3-(4-styrylphenoxy)propan-1-ol], which had similar potency (EC50 = 26 nM) but a much-improved pharmacokinetic profile and showed in vivo efficacy. Compounds OX03050 and OX03771 were found to inhibit squalene synthase, the first committed step in cholesterol biosynthesis. These squalene synthase inhibitors were shown to act cooperatively with statins to increase LDLR expression in vitro. Overall, we demonstrated here a novel series of small molecules with the potential to be further developed to treat patients either alone or in combination with statins.
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Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Pruebas Genéticas/métodos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Receptores de LDL/biosíntesis , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Regulación hacia Arriba/fisiología , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inhibidores Enzimáticos , Farnesil Difosfato Farnesil Transferasa/metabolismo , Humanos , Masculino , Ratones , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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.
Asunto(s)
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
BACKGROUND: Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. METHODS AND RESULTS: A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. CONCLUSIONS: These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.
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Aneurisma de la Aorta/epidemiología , Disección Aórtica/epidemiología , Susceptibilidad a Enfermedades/epidemiología , Endotelio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Disección Aórtica/etiología , Disección Aórtica/metabolismo , Angiotensina II/efectos adversos , Animales , Aneurisma de la Aorta/etiología , Aneurisma de la Aorta/metabolismo , Ciclofilinas/genética , Ciclofilinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/etiología , Susceptibilidad a Enfermedades/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Transducción de Señal , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Murine models of bone marrow transplantation show that pre-conditioning regimens affect the integrity of the bone marrow endothelium and that the repair of this vascular niche is an essential pre-requisite for successful haematopoietic stem and progenitor cell engraftment. Little is known about the angiogenic pathways that play a role in the repair of the human bone marrow vascular niche. We therefore established an in vitro humanized model, composed of bone marrow stromal and endothelial cells and have identified several pro-angiogenic factors, VEGFA, ANGPT1, CXCL8 and CXCL16, produced by the stromal component of this niche. We demonstrate for the first time that addition of CXCL8 or inhibition of its receptor, CXCR2, modulates blood vessel formation in our bone marrow endothelial niche model. Compared to wild type, Cxcr2(-/-) mice displayed a reduction in bone marrow cellularity and delayed platelet and leucocyte recovery following myeloablation and bone marrow transplantation. The delay in bone marrow recovery correlated with impaired bone marrow vascular repair. Taken together, our data demonstrate that CXCR2 regulates bone marrow blood vessel repair/regeneration and haematopoietic recovery, and clinically may be a therapeutic target for improving bone marrow transplantation.
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Trasplante de Médula Ósea , Médula Ósea/irrigación sanguínea , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Neovascularización Fisiológica , Receptores de Interleucina-8B/metabolismo , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/metabolismo , Animales , Línea Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Noqueados , Receptores de Interleucina-8B/genética , Acondicionamiento PretrasplanteRESUMEN
Endothelial nitric-oxide synthase (eNOS) is a critical regulator of vascular homeostasis by generation of NO that is dependent on the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, eNOS becomes "uncoupled," resulting in superoxide production in place of NO. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, although the functional relationships between BH4 and S-glutathionylation remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we expressed either WT or mutant eNOS rendered resistant to S-glutathionylation in cells with Tet-regulated expression of human GTP cyclohydrolase I to regulate intracellular BH4 availability. We reveal that S-glutathionylation of eNOS, by exposure to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase-specific siRNA, results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:7,8-dihydrobiopterin ratio. In eNOS uncoupling induced by BH4 deficiency, BCNU exposure further exacerbates superoxide production, BH4 oxidation, and eNOS activity. Following mutation of C908S, BCNU-induced eNOS uncoupling and BH4 oxidation are abolished, whereas uncoupling induced by BH4 deficiency was preserved. Furthermore, BH4 deficiency alone is alone sufficient to reduce intracellular GSH:GSSG ratio and cause eNOS S-glutathionylation. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling, although mechanistically distinct, are functionally related. We propose that uncoupling of eNOS by S-glutathionylation- or by BH4-dependent mechanisms exemplifies eNOS as an integrated redox "hub" linking upstream redox-sensitive effects of BH4 and glutathione with redox-dependent targets and pathways that lie downstream of eNOS.
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Biopterinas/análogos & derivados , Regulación Enzimológica de la Expresión Génica , Glutatión/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxidación-Reducción , Animales , Aniones , Biopterinas/química , Carmustina/farmacología , Glutatión Reductasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Modelos Biológicos , Modelos Genéticos , Mutación , Células 3T3 NIH , Oxígeno/química , Interferencia de ARN , Superóxidos/metabolismoRESUMEN
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
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.
Asunto(s)
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
[Figure: see text].
Asunto(s)
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
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
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.
Asunto(s)
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
Stimulation of the beta-adrenergic system is important in the pathological response to sustained cardiac stress, forming the rationale for the use of beta-blockers in heart failure. The beta3-adrenoreceptor (AR) is thought to couple to the inhibitory G-protein, G(i), with downstream signaling through nitric oxide, although its role in the heart remains controversial. In this study, we tested whether lack of beta3-AR influences the myocardial response to pressure-overload. Baseline echocardiography in mice lacking beta3-AR (beta3(-/-)) compared to wild type (WT) showed mild LV hypertrophy at 8 weeks that worsened as they aged. beta3(-/-) mice had much greater mortality after transverse aortic constriction (TAC) than WT controls. By 3 weeks of TAC, systolic function was worse. After 9 weeks of TAC, beta3(-/-) mice also had greater LV dilation, myocyte hypertrophy and enhanced fibrosis. NOS activity declined in beta3(-/-)TAC hearts after 9 weeks, and total and NOS-dependent superoxide rose, indicating heightened oxidative stress and NOS uncoupling. The level of eNOS phosphorylation in beta3(-/-)TAC hearts was diminished, and nNOS and iNOS expression levels were increased. GTP cyclohydrolase-1 expression was reduced, although total BH4 levels were not depleted. 3 weeks of BH4 treatment rescued beta3(-/-) mice from worsened remodeling after TAC, and lowered NOS-dependent superoxide. Thus, lack of beta3-AR signaling exacerbates cardiac pressure-overload induced remodeling and enhances NOS uncoupling and consequent oxidant stress, all of which can be rescued with exogenous BH4. These data suggest a cardioprotective role for the beta3-AR in modulating oxidative stress and adverse remodeling in the failing heart.
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Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/patología , Receptores Adrenérgicos beta 3/fisiología , Remodelación Ventricular/genética , Remodelación Ventricular/fisiología , Factores de Edad , Animales , Biopterinas/análogos & derivados , Biopterinas/uso terapéutico , Western Blotting , Cardiomiopatías/genética , Cardiomiopatías/patología , Ecocardiografía , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Masculino , Ratones , Ratones Mutantes , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Receptores Adrenérgicos beta 3/genética , Superóxidos/metabolismo , Vasoconstricción/fisiología , Remodelación Ventricular/efectos de los fármacosRESUMEN
BACKGROUND AND AIMS: Angiotensin II (Ang II) infusion promotes the development of aortic aneurysms and accelerates atherosclerosis in ApoE-/- mice. In order to elucidate the role of hematopoietic cells in these pathologies, irradiation and bone marrow transplantation (BMT) are commonly utilized. The aim of this study was to investigate the effects of irradiation and BMT on abdominal and thoracic aortic aneurysm formation and acute leukocyte recruitment in the aortic root and descending aorta, in an experimental mouse model of aortic aneurysm formation. METHODS: ApoE-/- mice were either lethally irradiated and reconstituted with ApoE-/- bone marrow or non-irradiated. Following engraftment, mice were treated with Ang II to induce aortic inflammation and accelerate atherosclerosis. RESULTS: Ang II infusion (0.8â¯mg/kg/day) in BMT mice resulted in reduced aortic aneurysms and atherosclerosis with decreased leukocyte infiltration in the aorta compared to non-BMT mice, when receiving the same dose of Ang II. Furthermore, the reduced aortic infiltration in BMT mice was accompanied by increased levels of monocytes in the spleen and bone marrow. A dose of 3â¯mg/kg/day Ang II was required to achieve a similar incidence of aneurysm formation as achieved with 0.8â¯mg/kg/day in non-BMT mice. CONCLUSIONS: This study provides evidence that BMT can alter inflammatory cell recruitment in experimental mouse models of aortic aneurysm formation and atherosclerosis and suggests that irradiation and BMT have a considerably more complex effect on vascular inflammation, which should be evaluated.
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Angiotensina II , Aneurisma de la Aorta Abdominal/prevención & control , Aneurisma de la Aorta Torácica/prevención & control , Aortitis/prevención & control , Aterosclerosis/prevención & control , Trasplante de Médula Ósea , Irradiación Corporal Total , Animales , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Torácica/inducido químicamente , Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/metabolismo , Rotura de la Aorta/inducido químicamente , Rotura de la Aorta/genética , Rotura de la Aorta/metabolismo , Rotura de la Aorta/prevención & control , Aortitis/inducido químicamente , Aortitis/genética , Aortitis/metabolismo , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Aterosclerosis/metabolismo , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Macrófagos/efectos de la radiación , Macrófagos/trasplante , Masculino , Ratones Noqueados para ApoE , Monocitos/metabolismo , Monocitos/efectos de la radiación , Monocitos/trasplante , Placa AteroscleróticaRESUMEN
GTPCH (GTP cyclohydrolase 1, encoded by Gch1) is required for the synthesis of tetrahydrobiopterin; a critical regulator of endothelial NO synthase function. We have previously shown that mice with selective loss of Gch1 in endothelial cells have mild vascular dysfunction, but the consequences of endothelial cell tetrahydrobiopterin deficiency in vascular disease pathogenesis are unknown. We investigated the pathological consequence of Ang (angiotensin) II infusion in endothelial cell Gch1 deficient (Gch1fl/fl Tie2cre) mice. Ang II (0.4 mg/kg per day, delivered by osmotic minipump) caused a significant decrease in circulating tetrahydrobiopterin levels in Gch1fl/fl Tie2cre mice and a significant increase in the Nω-nitro-L-arginine methyl ester inhabitable production of H2O2 in the aorta. Chronic treatment with this subpressor dose of Ang II resulted in a significant increase in blood pressure only in Gch1fl/fl Tie2cre mice. This finding was mirrored with acute administration of Ang II, where increased sensitivity to Ang II was observed at both pressor and subpressor doses. Chronic Ang II infusion in Gch1fl/fl Tie2ce mice resulted in vascular dysfunction in resistance mesenteric arteries with an enhanced constrictor and decreased dilator response and medial hypertrophy. Altered vascular remodeling was also observed in the aorta with an increase in the incidence of abdominal aortic aneurysm formation in Gch1fl/fl Tie2ce mice. These findings indicate a specific requirement for endothelial cell tetrahydrobiopterin in modulating the hemodynamic and structural changes induced by Ang II, through modulation of blood pressure, structural changes in resistance vessels, and aneurysm formation in the aorta.
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Aneurisma de la Aorta Abdominal , Angiotensina II , Animales , Aorta , Biopterinas/análogos & derivados , Presión Sanguínea , Células Endoteliales , Peróxido de Hidrógeno , Ratones , Remodelación VascularRESUMEN
Aims: GTP cyclohydrolase I catalyses the first and rate-limiting reaction in the synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS). Both eNOS and iNOS have been implicated in the progression of atherosclerosis, with opposing effects in eNOS and iNOS knockout mice. However, the pathophysiologic requirement for BH4 in regulating both eNOS and iNOS function, and the effects of loss of BH4 on the progression of atherosclerosis remains unknown. Methods and results: Hyperlipidemic mice deficient in Gch1 in endothelial cells and leucocytes were generated by crossing Gch1fl/flTie2cre mice with ApoE-/- mice. Deficiency of Gch1 and BH4 in endothelial cells and myeloid cells was associated with mildly increased blood pressure. High fat feeding for 6 weeks in Gch1fl/flTie2CreApoE-/- mice resulted in significantly decreased circulating BH4 levels, increased atherosclerosis burden and increased plaque macrophage content. Gch1fl/flTie2CreApoE-/- mice showed hallmarks of endothelial cell dysfunction, with increased aortic VCAM-1 expression and decreased endothelial cell dependent vasodilation. Furthermore, loss of BH4 from pro-inflammatory macrophages resulted in increased foam cell formation and altered cellular redox signalling, with decreased expression of antioxidant genes and increased reactive oxygen species. Bone marrow chimeras revealed that loss of Gch1 in both endothelial cells and leucocytes is required to accelerate atherosclerosis. Conclusion: Both endothelial cell and macrophage BH4 play important roles in the regulation of NOS function and cellular redox signalling in atherosclerosis.
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Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Biopterinas/análogos & derivados , Células Endoteliales/enzimología , GTP Ciclohidrolasa/metabolismo , Macrófagos/enzimología , Animales , Aorta/patología , Aorta/fisiopatología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/fisiopatología , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Biopterinas/metabolismo , Presión Sanguínea , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/patología , Femenino , Células Espumosas/enzimología , Células Espumosas/patología , GTP Ciclohidrolasa/deficiencia , GTP Ciclohidrolasa/genética , Macrófagos/patología , Masculino , Ratones Noqueados para ApoE , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Placa Aterosclerótica , Especies Reactivas de Oxígeno/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Vasoconstricción , VasodilataciónRESUMEN
Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.
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Biopterinas/análogos & derivados , GTP Ciclohidrolasa/fisiología , Óxido Nítrico Sintasa de Tipo II/fisiología , Óxido Nítrico/biosíntesis , Tuberculosis/inmunología , Animales , Biopterinas/genética , Biopterinas/metabolismo , Biopterinas/fisiología , Supervivencia Celular , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The cofactor tetrahydrobiopterin (BH4) is a critical regulator of endothelial NOS (eNOS) function, eNOS-derived NO and ROS signalling in vascular physiology. To determine the physiological requirement for de novo endothelial cell BH4 synthesis for the vasomotor function of resistance arteries, we have generated a mouse model with endothelial cell-specific deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme for BH4 biosynthesis, and evaluated BH4-dependent eNOS regulation, eNOS-derived NO and ROS generation. EXPERIMENTAL APPROACH: The reactivity of mouse second-order mesenteric arteries was assessed by wire myography. High performance liquid chromatography was used to determine BH4, BH2 and biopterin. Western blotting was used for expression analysis. KEY RESULTS: Gch1fl/fl Tie2cre mice demonstrated reduced GTPCH protein and BH4 levels in mesenteric arteries. Deficiency in endothelial cell BH4 leads to eNOS uncoupling, increased ROS production and loss of NO generation in mesenteric arteries of Gch1fl/fl Tie2cre mice. Gch1fl/fl Tie2cre mesenteric arteries had enhanced vasoconstriction to U46619 and phenylephrine, which was abolished by L-NAME. Endothelium-dependent vasodilatations to ACh and SLIGRL were impaired in mesenteric arteries from Gch1fl/fl Tie2cre mice, compared with those from wild-type littermates. Loss of eNOS-derived NO-mediated vasodilatation was associated with increased eNOS-derived H2 O2 and cyclooxygenase-derived vasodilator in Gch1fl/fl Tie2cre mesenteric arteries. CONCLUSIONS AND IMPLICATIONS: Endothelial cell Gch1 and BH4-dependent eNOS regulation play pivotal roles in maintaining vascular homeostasis in resistance arteries. Therefore, targeting vascular Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of microvascular dysfunction in patients with cardiovascular disease.
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Biopterinas/análogos & derivados , Células Endoteliales/metabolismo , Arterias Mesentéricas/citología , Arterias Mesentéricas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Biopterinas/deficiencia , Biopterinas/metabolismo , Células Cultivadas , GTP Ciclohidrolasa/deficiencia , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Familial hypercholesterolemia (FH) is a life-threatening genetic disorder characterized by elevated levels of plasma low-density lipoprotein cholesterol (LDL-cholesterol). Current attempts at gene therapy for FH have been limited by the use of strong heterologous promoters which lack genomic DNA elements essential for regulated expression. Here, we have combined a mini-gene vector expressing the human LDLR cDNA from a 10 kb native human LDLR locus genomic DNA promoter element, with an efficient miRNA targeting 3-hydroxy-3-methylgutaryl-coenzyme A reductase (Hmgcr), to further enhance LDLR expression. We show that the combined vector suppresses endogenous Hmgcr transcripts in vivo, leading to an increase in LDLR transgene expression. In a diet-induced Ldlr-/- mouse model of FH, we show that administration of the combined vector reduces atherogenic plasma lipids by ~32%. Finally, we demonstrate that our episomal nonviral vectors are able to reduce atherosclerosis by ~40% after 12 weeks in vivo. Taken together, the vector system we describe exploits the normal cellular regulation of the LDLR to provide prolonged expression of LDLR through targeted knockdown of Hmgcr. This novel gene therapy system could act alone, or in synergy with current therapies that modulate intracellular cholesterol, such as statins, greatly enhancing its therapeutic application for FH.
RESUMEN
Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1(fl/fl)) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1(fl/fl)Tie2cre). Macrophages from Gch1(fl/fl)Tie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1(fl/fl)Tie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1(fl/fl)Tie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1(fl/fl)Tie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1(fl/fl)Tie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1(fl/fl)Tie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.
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Biopterinas/análogos & derivados , GTP Ciclohidrolasa/genética , Macrófagos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Biopterinas/deficiencia , Biopterinas/metabolismo , Macrófagos/enzimología , Ratones , Óxido Nítrico/metabolismo , Oxidación-ReducciónRESUMEN
Chemokine signalling drives monocyte recruitment in atherosclerosis and aortic aneurysms. The mechanisms that lead to retention and accumulation of macrophages in the vascular wall remain unclear. Regulator of G-Protein Signalling-1 (RGS1) deactivates G-protein signalling, reducing the response to sustained chemokine stimulation. Here we show that Rgs1 is upregulated in atherosclerotic plaque and aortic aneurysms. Rgs1 reduces macrophage chemotaxis and desensitizes chemokine receptor signalling. In early atherosclerotic lesions, Rgs1 regulates macrophage accumulation and is required for the formation and rupture of Angiotensin II-induced aortic aneurysms, through effects on leukocyte retention. Collectively, these data reveal a role for Rgs1 in leukocyte trafficking and vascular inflammation and identify Rgs1, and inhibition of chemokine receptor signalling as potential therapeutic targets in vascular disease.
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Aneurisma de la Aorta/metabolismo , Aterosclerosis/metabolismo , Quimiocinas/metabolismo , Proteínas RGS/metabolismo , Transducción de Señal , Animales , Aorta/metabolismo , Aneurisma de la Aorta/genética , Presión Sanguínea , Trasplante de Médula Ósea , Quimiotaxis , Citometría de Flujo , Humanos , Inflamación , Leucocitos/citología , Leucocitos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Monocitos/citología , Receptores de Quimiocina/metabolismo , Enfermedades Vasculares/metabolismoRESUMEN
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.