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Find video protocols related to scientific articles indexed in Pubmed.
Cell-autonomous role of endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation.
Hypertension
PUBLISHED: 04-28-2014
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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 (•-) 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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Early change in invasive measures of microvascular function can predict myocardial recovery following PCI for ST-elevation myocardial infarction.
Eur. Heart J.
PUBLISHED: 10-17-2013
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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.
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Atrial sources of reactive oxygen species vary with the duration and substrate of atrial fibrillation: implications for the antiarrhythmic effect of statins.
Circulation
PUBLISHED: 08-15-2011
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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.
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Bi-modal dose-dependent cardiac response to tetrahydrobiopterin in pressure-overload induced hypertrophy and heart failure.
J. Mol. Cell. Cardiol.
PUBLISHED: 05-09-2011
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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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Elevated blood pressure in offspring born premature to hypertensive pregnancy: is endothelial dysfunction the underlying vascular mechanism?
Hypertension
PUBLISHED: 05-17-2010
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Offspring born to mothers with hypertensive pregnancy have higher childhood blood pressure. We hypothesized this relates to prenatally programmed differences in the underlying vascular pathophysiology of the offspring and that these would be most apparent in those born preterm because of severe hypertension. We carried out a 20-year follow-up study of 71 subjects born preterm, 19 to a hypertensive pregnancy and 52 to a normotensive pregnancy. Findings were compared with 38 subjects born at term to uncomplicated pregnancies. Peripheral and central blood pressures were measured, and then central arterial stiffness was assessed by carotid-femoral pulse wave velocity using applanation tonometry. Ultrasound was used to assess flow-mediated endothelial-dependent and independent brachial artery responses and common carotid artery intima-media thickness. Offspring born preterm to either hypertensive or normotensive pregnancy had higher peripheral and central blood pressure compared with full-term born offspring (central mean arterial pressure after preterm hypertensive pregnancy: 84.92+/-7.0 mm Hg; preterm normotensive pregnancy: 84.13+/-8.9 mm Hg; full-term pregnancy: 76.24+/-7.96 mm Hg; P=0.0009). However, underlying vascular phenotype differed. Preterm offspring of normotensive pregnancy had greater arterial stiffness than offspring of hypertensive pregnancy (5.92+/-0.84 versus 5.42+/-0.73 m/s; P=0.039), whereas offspring of hypertensive pregnancy had greater carotid intima-media thickness (0.52+/-0.04 versus 0.48+/-0.06 mm; P=0.013) and 30% lower flow-mediated dilatation (4.25+/-4.02% versus 6.79+/-4.38%; P=0.05). Prematurity is associated with elevated blood pressure in later life. However, predominant underlying vascular phenotype depends on maternal pathology. Targeting endothelial function may be particularly important for primary prevention after hypertension in pregnancy.
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Cardiac myocyte-specific overexpression of human GTP cyclohydrolase I protects against acute cardiac allograft rejection.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 04-23-2010
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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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Tetrahydrobiopterin supplementation reduces atherosclerosis and vascular inflammation in apolipoprotein E-knockout mice.
Clin. Sci.
PUBLISHED: 03-27-2010
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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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Critical role for tetrahydrobiopterin recycling by dihydrofolate reductase in regulation of endothelial nitric-oxide synthase coupling: relative importance of the de novo biopterin synthesis versus salvage pathways.
J. Biol. Chem.
PUBLISHED: 08-07-2009
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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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GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.
J. Biol. Chem.
PUBLISHED: 03-13-2009
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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.
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Quantitative regulation of intracellular endothelial nitric-oxide synthase (eNOS) coupling by both tetrahydrobiopterin-eNOS stoichiometry and biopterin redox status: insights from cells with tet-regulated GTP cyclohydrolase I expression.
J. Biol. Chem.
PUBLISHED: 03-07-2009
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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.
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Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding.
PLoS ONE
PUBLISHED: 02-15-2009
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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.
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Unique blood pressure characteristics in mother and offspring after early onset preeclampsia.
Hypertension
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Risk of hypertension in mother and offspring after preeclampsia is greater if preeclampsia develops early in pregnancy. We investigated whether those who develop early onset disease have unique adverse blood pressure characteristics. One hundred forty women were studied 6 to 13 years either after a pregnancy complicated by preeclampsia (45 women with early onset preeclampsia before 34 weeks gestation and 45 women with late-onset preeclampsia) or after a normotensive pregnancy (50 women). Forty-seven offspring from these pregnancies also participated. Data on maternal antenatal and postnatal blood pressures were extracted from maternity records and related to peripheral, central, and ambulatory blood pressure measurements in later life. Compared with late-onset preeclampsia, early onset preeclampsia was associated with higher diastolic blood pressure 6 weeks postnatally (86.25 ± 13.46 versus 75.00 ± 5.00 mm Hg, P<0.05), a greater increase in blood pressure relative to booking blood pressure over the subsequent 6 to 13 years, and higher nocturnal systolic and diastolic blood pressures in later life (111.07 ± 13.18 versus 101.13 ± 11.50 mm Hg, P=0.04, and 67.00 ± 7.25 versus 58.60 ± 5.79 mm Hg, P=0.002). Furthermore, at age 6 to 13 years their offspring had higher systolic blood pressure compared with those born to late-onset preeclampsia (96.27 ± 7.30 versus 88.39 ± 7.57 mm Hg, P=0.005). Mothers who developed early onset preeclampsia, and the offspring of that pregnancy display specific adverse blood pressure characteristics later in life. These are not evident in mothers and offspring after late-onset preeclampsia or normotensive pregnancy.
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Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.
Eur. Heart J.
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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.
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Cardiomyocyte GTP cyclohydrolase 1 and tetrahydrobiopterin increase NOS1 activity and accelerate myocardial relaxation.
Circ. Res.
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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.
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