Regulation of Endothelial Nitric Oxide Synthase by Tetrahydrobiopterin in Vascular Disease

From the Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

Correspondence to Professor K.M. Channon, Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital Oxford OX3 9DU United Kingdom. E-mail keith.channon@cardiov.ox.ac.uk

    Abstract

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a key signaling molecule in vascular homeostasis. Loss of NO bioavailability due to reduced synthesis and increased scavenging by reactive oxygen species is a cardinal feature of endothelial dysfunction in vascular disease states. The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of eNOS activity: when BH4 availability is limiting, eNOS no longer produces NO but instead generates superoxide. In vascular disease states, there is oxidative degradation of BH4 by reactive oxygen species. However, augmentation of BH4 concentrations in vascular disease by pharmacological supplementation, by enhancement of its rate of de novo biosynthesis or by measures to reduce its oxidation, has been shown in experimental studies to enhance NO bioavailability. Thus, BH4 represents a potential therapeutic target in the regulation of eNOS function in vascular disease.

Key Words: tetrahydrobiopterin ? nitric oxide synthase ? superoxide

    Introduction

Nitric oxide (NO), produced by endothelial NO synthase (eNOS), is a key signaling molecule in vascular homeostasis.1 Originally identified as endothelium-derived relaxing factor,2,3 NO is an important regulator of vascular tone and blood pressure. In addition, NO has multiple antiatherogenic roles, including anti-inflammatory, antithrombotic, antiproliferative, and antioxidant effects (reviewed in Ignarro1). Loss of NO bioavailability is a cardinal feature of endothelial dysfunction4 that precedes the development of overt atherosclerosis and is an independent predictor of adverse cardiovascular risk.5,6 Several factors contribute to loss of NO bioavailability in endothelial dysfunction states, including both reduced NO synthesis and NO scavenging by reactive oxygen species.7 The regulation of NO production by eNOS is complex, but the pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of NO synthesis. Indeed, endothelial BH4 availability appears to be a key requirement for maintaining normal endothelial function. In this article, we review the current knowledge on the regulation of eNOS by BH4 and discuss the potential importance of mechanisms linking BH4 availability with endothelial function in vascular disease.

    Tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor for all 3 NO synthase (NOS) isoforms and for the aromatic amino acid hydroxylases (phenylalanine hydroxylase, tyrosine-3-hydroxylase, and tryptophan-5-hydroxylase). BH4 biosynthesis proceeds from GTP via 2 intermediates, 7,8-dyhydroneopterin triphosphate and 6-pyruvoyl-5,6,7,8-tetrahydropterin (reviewed in Thony et al8; Figure 1). In endothelial cells (ECs), the first and rate-limiting step in this pathway is GTP cyclohydrolase I (EC 3.5.4.16; GTPCH). The subsequent steps are catalyzed by the enzymes 6-pyruvoyl tetrahydropterin synthase (EC 4.6.1.10; PTPS) and sepiapterin reductase (EC 1.1.1.153; SR). In some cell types (for example, macrophages), inflammatory cytokines upregulate GTPCH expression,9 but in these conditions, PTPS is not upregulated to the same extent and becomes rate limiting.10 As a result, the intermediate 7,8-dihydroneopterin triphosphate accumulates and is oxidized to the stable metabolite neopterin, which can be detected in plasma and serves as an indirect marker of inflammation, for example, in acute coronary syndromes.11

   Figure 1. Pathways of BH4 biosynthesis. BH4 biosynthesis proceeds from GTP via 7,8-dyhydroneopterin triphosphate and 6-pyruvoyl-5,6,7,8-tetrahydropterin. The first and rate-limiting step in the pathway is GTPCH. Subsequent steps are catalyzed by the enzymes 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase. An alternative pathway for BH4 synthesis has been documented in bacteria12 and in Drosophila,13 whereby 6-pyruvoyl-5,6,7,8-tetrahydropterin is converted to sepiapterin by an enzyme termed "sepiapterin synthase." There is limited evidence for a sepiapterin synthesis pathway in mammals. However, exogenous sepiapterin can be reduced in all cells by sepiapterin reductase to BH2, and further by dihydrofolate reductase to form BH4 (the so-called salvage pathway).

An alternative pathway for BH4 synthesis has been documented in bacteria12 and in Drosophila,13 whereby 6-pyruvoyl-5,6,7,8-tetrahydropterin is converted to sepiapterin by a poorly defined enzyme termed "sepiapterin synthase." The only evidence for a sepiapterin synthesis pathway in mammals comes from a recent study of rare patients with sepiapterin reductase deficiency, in whom sepiapterin levels were elevated in cerebrospinal fluid, which suggests endogenous production of sepiapterin.14 Certainly, exogenous sepiapterin can be reduced in all cells by sepiapterin reductase to 7,8-dihydrobiopterin (BH2) and further by dihydrofolate reductase (EC 1.5.1.3; DHFR) to form BH4, the so-called salvage pathway15 that has been exploited by many investigators as an approach to increase BH4 levels by pharmacological supplementation of sepiapterin (Figure 1).

As a cofactor for the aromatic amino acid hydroxylases, BH4 is oxidized to tetrahydrobiopterin-4a-carbinolamine. BH4 is regenerated by the actions first of pterin-4a-carbinolamine dehydratase (EC 4.2.1.96; PCD), forming the quinonoid dihydrobiopterin intermediate, which is then reduced by dihydropteridine reductase (EC 1.6.99.7; DHPR). These 2 enzymes are expressed in mammalian liver,16 kidney, and brain,17 and mutations in the PCD and DHPR genes are associated with clinical systemic BH4 deficiency and hyperphenylalaninemia. However, in its action as a cofactor for NOS, BH4 is not oxidized to tetrahydrobiopterin-4a-carbinolamine. Instead, it appears that BH4 forms the protonated trihydrobiopterin cation radical (BH3.H+) in the transfer of electrons to the ferrous-dioxygen complex, coupling oxygen reduction to L-arginine oxidation.18 After each electron transfer step, the BH3.H+ radical species returns to the BH4 state. Thus, continuous BH4 regeneration does not appear to be a requirement for eNOS activity in ECs.

Clinical BH4 deficiency is associated with a rare variant of hyperphenylalaninemia that is unresponsive to a low-phenylalanine diet.19 The syndrome is characterized by a deficit of the neurotransmitters dopamine and serotonin and generally presents in childhood with progressive neurological symptoms.20 The variable presentation reflects a range of mutations in genes encoding the enzymes involved in BH4 biosynthesis21 or regeneration.22 A form of BH4 deficiency without hyperphenylalaninemia is also recognized, known as dopa-responsive dystonia,23 and this is associated with mutations in the gene encoding GTPCH, transmitted in an autosomal-dominant fashion.24 Study of these syndromes has helped elucidate the role of BH4 in neurotransmitter synthesis and phenylalanine metabolism, but there are as yet no published data regarding vascular function in these patients.

    GTP Cyclohydrolase I

The human GCH1 gene, which encodes GTPCH, is located on chromosome 14q22.1-q22.2 and consists of 6 exons spanning 30 kb (reviewed in Thony et al8). This gene encodes a subunit protein of 221 amino acid residues (27.9 kDa), and the subunits assemble to form a toroidal homodecamer of 2 pentamers.25 Each active site is located at the interface of 3 subunits, 2 from 1 pentamer and 1 from the other; there are thus 10 equivalent active sites per functional unit.26

As the committing and rate-limiting enzyme for BH4 synthesis, GTPCH is subject to direct regulation at transcriptional and posttranscriptional levels. Cytokines including interferon- and tumor necrosis factor- and inflammatory mediators such as lipopolysaccharide are reported to increase GTPCH transcription,27,28 enzyme activity,29 and BH4 levels in human ECs. Insulin may also upregulate GTPCH expression in ECs via a phosphatidylinositol-3-kinase–dependent pathway.30

GTPCH is also subject to posttranslational modification by phosphorylation. In rat mesangial cells, angiotensin II and platelet-derived growth factor are reported to increase GTPCH activity by phosphorylation via a protein kinase C–dependent mechanism.31 However, the role of GTPCH phosphorylation in ECs has not been determined.

Finally, GTPCH activity is governed by interactions with GTPCH feedback regulatory protein (GFRP), which has been cloned and purified32 and is a homopentamer of 52-kDa subunits.33,34 Recently, the nature of the physical interaction between GFRP and GTPCH has been demonstrated with x-ray crystallography.35,36 Through GFRP, phenylalanine exerts positive regulation of GTPCH activity, and BH4 exhibits negative feedback regulation.37 GFRP expression has been documented in various cell types and is highly expressed in hepatocytes,32 but whether GFRP has a biologically important role in regulating GTPCH activity in ECs remains to be determined.

    Regulation of eNOS Activity in Vascular Disease

In ECs, eNOS is subject to rapid changes in activity by regulation through signaling molecules that are integrated in caveolae, for example, by receptor-mediated agonist stimulation that leads to rapid enzyme activation by the binding of calcium-calmodulin, depalmitoylation, displacement of caveolin-1, and release of eNOS from caveolae.38,39 Changes in eNOS activity are also mediated through phosphorylation of key residues. Akt-dependent phosphorylation at Ser 1177 activates eNOS in response to stimuli such as fluid shear stress, estrogen, and insulin,40 whereas phosphorylation at Thr 459 (eg, by protein kinase C) reduces activation. Dephosphorylation of Thr 459 by protein phosphatase 1 contributes to eNOS activation in response to bradykinin.41

Although eNOS is constitutively expressed in ECs, eNOS gene transcription may be modulated by a variety of factors. Laminar shear stress is an important stimulus in maintaining or upregulating eNOS expression.42 Although eNOS protein levels may be reduced in ECs overlying advanced atherosclerotic plaques,43 eNOS protein may remain normal or even increase, despite marked endothelial dysfunction, in preatherosclerotic states such as diabetes,44 experimental heart failure,45 and hypertension.46 Overexpression of eNOS by gene transfer can increase NO bioactivity in the vessel wall, but constitutive endothelial overexpression of eNOS paradoxically accelerated atherosclerosis in apoE knockout mice, in association with increased oxidative stress.47 Thus, reductions in NO bioactivity in vascular disease are not explained simply by loss of eNOS protein, and indeed, plentiful eNOS protein is no guarantee of adequate NO bioavailability. These findings have led to the concept of "eNOS dysfunction" or "eNOS uncoupling," characterized by a stoichiometric discordance between eNOS protein levels and NO production.

    eNOS Uncoupling: Role of BH4 in eNOS Regulation

Increased reactive oxygen species generation is associated with many vascular disease states. Several oxidase systems contribute to increased oxidative stress, notably the NADPH oxidases.48 However, increasing evidence suggests that eNOS itself can generate superoxide under certain pathophysiological conditions. Electron transfer within the active site becomes "uncoupled" from L-arginine oxidation; instead, molecular oxygen is reduced to form superoxide.49,50 Superoxide generation by eNOS has been implicated in a variety of experimental and clinical vascular disease states, including diabetes,44,51 cigarette smoking,52 hypertension,53 chronic nitrate tolerance,54 and overt atherosclerosis.55 However, demonstration of eNOS-dependent superoxide production has relied on currently available ex vivo methods, all of which have potential limitations and shortcomings.56 The effect of eNOS uncoupling on NO bioavailability in vascular disease may be profound, because NO production is reduced and superoxide production is increased, which leads to further reductions in NO bioactivity by scavenging and increasing oxidative stress within ECs by formation of peroxynitrite.

A number of molecular mechanisms likely contribute to eNOS uncoupling. Limited availability of the substrate L-arginine may reduce NO synthesis, and there is some evidence that superoxide production may be increased.57 The interaction of heat shock protein 90 with eNOS appears to modulate the relative production of NO and superoxide58; this interaction is inhibited in hypercholesterolemia, associated with eNOS uncoupling.59 However, BH4 appears to have a particularly important role in regulating NO and superoxide production by eNOS.

The importance of BH4 in the catalytic process of L-arginine oxidation and NO synthesis by eNOS is well established. There is 1 BH4 binding site in the oxygenase domain of each monomer, so 2 molecules of BH4 are incorporated into each functional eNOS dimer. In vitro experiments with electron paramagnetic resonance spectroscopy have demonstrated that BH4 both stabilizes and donates electrons to the ferrous-dioxygen complex in the oxygenase domain, as the initiating step of L-arginine oxidation.60–62 In this reaction, BH4 forms the protonated trihydrobiopterin cation radical BH3.H+, which is subsequently reduced by electron transfer from eNOS flavins.61 When BH4 is limiting, electron transfer from eNOS flavins becomes uncoupled from L-arginine oxidation, the ferrous-dioxygen complex dissociates, and superoxide is produced from the oxygenase domain.49,63

It is likely that NOS must assemble in its dimeric form for catalytic activity, whether producing NO or superoxide. However, the role of BH4 in stabilizing the dimeric conformation of NOS isoforms is not fully resolved. In vitro enzyme reconstitution experiments suggest that the addition of BH4 has only a minimal effect on eNOS dimerization.64 However, other evidence indicates a possible role and mechanism for BH4 in stabilizing eNOS dimers. A zinc ion is tetrahedrally coordinated to pairs of cysteine residues at the dimer interface, 1 pair from each subunit, close to the BH4 binding site.65 Site-directed mutagenesis studies indicate that the cysteine residues are critical for locating BH4 in its binding site and maintaining NO production by eNOS.66 Recent evidence suggests that disruption of the zinc-thiolate complex by peroxynitrite leads to loss of BH4 from the active site, enzymatic uncoupling, and destabilization of eNOS dimers.67 Results from recombinant enzyme expression experiments68–70 and from endothelial cell culture studies71 also indicate that BH4 indeed promotes eNOS dimerization, protein stability, and NO synthesis. Thus, BH4 may have an important role in the physical stabilization of eNOS in its active dimeric form, in addition to its catalytic functions.

A variety of experimental approaches have been used to investigate BH4 availability as a modulator of eNOS function in vascular disease states, including diabetes, hypertension, and hypercholesterolemia, in both animal models and clinical studies.

Animal Models

Several recent studies have investigated the relationships between BH4 levels and eNOS function in diabetes. ECs isolated from the BB rat (a strain genetically predisposed to diabetes) have reduced BH4 levels compared with cells from control strains, which results in reduced NO production despite normal eNOS protein levels.72 This deficit is restored by supplementation with sepiapterin. Insulin-resistant fructose-fed rats demonstrate increased vascular superoxide production, oxidative degradation of BH4 in the aorta, reduced eNOS activity, and impaired endothelium-dependent vascular relaxations compared with controls.73,74 These abnormalities can be prevented by coadministration of sapropterin (a synthetic precursor of BH4) in the diet75 or by incubation of aortic rings in BH4 solution.73 Similarly, streptozotocin-induced diabetes in rats76 and hyperglycemia in spontaneously diabetic (db/db -/-) mice77 lead to impaired endothelium-dependent relaxations of vascular rings, which can be restored acutely by incubation with high doses of BH4.

Pharmacological BH4 supplementation also improves endothelial dysfunction in spontaneously hypertensive rats.78,79 In a deoxycorticosterone acetate-salt (DOCA-salt) model of hypertension in mice, there is increased aortic superoxide production, BH4 oxidation, and eNOS uncoupling, which can be prevented by oral BH4 supplementation.53 The hyperphenylalaninemic (Hph-1) mouse, generated by N-ethyl-N-nitrosourea mutagenesis of the GCH1 locus, has systemically reduced levels of GTPCH expression and reduced BH4 synthesis. Initial studies of the vascular phenotype in the Hph-1 mouse suggest that BH4 deficiency leads to hypertension, increased vascular oxidative stress, and reduced eNOS activity,80 which demonstrates that reduced BH4 levels appear sufficient to produce eNOS uncoupling in the absence of coexisting vascular disease.

In hypercholesterolemic apoE-knockout mice, endothelium-dependent vascular relaxations are impaired and vascular superoxide production is increased; both can be normalized by in vitro incubation of vessels with sepiapterin.81 Transgenic endothelial overexpression of eNOS in apoE-knockout mice paradoxically increases vascular superoxide production because of enzymatic uncoupling of the increased eNOS protein levels, which leads to accelerated atherosclerosis.47 BH4 levels are reduced in the aortas of these apoE-knockout mice compared with wild-type controls, but dietary BH4 supplementation with sapropterin appears to reduce superoxide production and restore NO synthesis. These results indicate that increased eNOS protein alone is insufficient to maintain NO synthesis in hypercholesterolemia and that adequate BH4 levels are essential to prevent enzymatic uncoupling.

Thus, in these different animal models, multiple risk factors for endothelial dysfunction appear to act via a common pathway that involves reduced bioavailability of BH4, leading to uncoupling of eNOS, reduced NO synthesis, and increased superoxide production.

Clinical Studies

A number of clinical studies have sought to investigate the effects of BH4 on eNOS function by exploring the effects of pharmacological supplementation of BH4 on NO-mediated endothelial function. Using venous occlusion plethysmography as a measure of endothelial function, acute administration of BH4 improves endothelial function of patients with hypercholesterolemia82 or type 2 diabetes.83 Heitzer and colleagues84 showed that the beneficial effect of acute BH4 administration in chronic smokers was likely via a direct effect on eNOS coupling rather than simply an antioxidant effect, because a control infusion of tetrahydroneopterin (NH4) had no effect on endothelial function despite a similar ability to scavenge superoxide anion in vitro. Direct intracoronary infusion of BH4 during cardiac catheterization also improves endothelial function, measured by quantitative coronary angiography and Doppler flow studies, in the coronary arteries of patients with hypercholesterolemia85 or overt coronary atherosclerosis.55

However, many of these experimental and clinical studies are limited by the high doses of sepiapterin or BH4 used (often >100-fold in excess of physiological concentrations), which may increase NO bioactivity via nonspecific antioxidant effects. Conversely, the experimental effects of high-dose BH4 added to vessel rings in organ bath chambers may be confounded by auto-oxidation of BH4, generating superoxide.86 Where sepiapterin has been used to augment BH4 concentrations in a rabbit model of hypercholesterolemia, unexpected uncoupling of eNOS was observed, possibly as a result of competition with BH4 at the active site of the enzyme.87 In addition, all the experimental interventions have been short term, and there were no studies evaluating the effects of chronic low-dose BH4 augmentation on NOS function in vascular disease. Some of these issues have been addressed by the recent generation of a transgenic mouse model of increased vascular BH4 synthesis, in which human GTPCH expression is targeted to vascular endothelium under the control of the mouse Tie2 promoter. These mice demonstrate a persistent modest (3-fold) increase in vascular BH4 levels and have reduced endothelial superoxide production and preserved NO bioavailability compared with wild-type littermates in a streptozotocin model of diabetic vascular disease.88

    Factors Affecting BH4 Bioavailability in Vascular Disease

Reduced BH4 Synthesis

Some evidence suggests that GTPCH expression may be downregulated, and thereby BH4 synthesis reduced, in certain vascular pathological states. This hypothesis has conflicted with the observation that inflammatory cytokines are reported to upregulate GTPCH expression in ECs.27,28 In a glucocorticoid-induced rat model of hypertension, GTPCH mRNA levels were reduced, and impaired endothelium-dependent relaxations could be restored by incubating vessels in sepiapterin, which suggests reduced BH4 bioavailability as a cause of eNOS uncoupling.89 However, it was not determined whether these abnormalities were a direct effect of glucocorticoid treatment or secondary to the experimental hypertension, and the relevance of these findings to human vascular disease remains uncertain. More recent studies in the DOCA-salt hypertensive mouse also found that decreased BH4 levels were related to reduced GTPCH activity; supplementation of BH4 or increased BH4 synthesis by adenoviral gene delivery of GTPCH restored BH4 levels and normalized eNOS function.90 Other mechanisms regulating GTPCH activity, such as phosphorylation and GFRP interactions, have not yet been explored in the endothelium, so the importance of altered BH4 synthesis in determining BH4 bioavailability in vascular disease remains uncertain.

BH4 Oxidation

Many studies have focused on the potential role of BH4 oxidation to BH2 and other oxidized biopterin species in reducing BH4 bioavailability for eNOS. Many preatherosclerotic disease states are associated with increased vascular oxidative stress, particularly superoxide production. Although superoxide can indeed react directly with BH4, the rate constant of this reaction (3.9x105 mol·L-1·s-1)18 is many orders of magnitude lower than that for NO with superoxide (6.7x109 mol · L-1 · s-1),91 which indicates that BH4 is unlikely to be acting merely as an antioxidant at physiological (as opposed to pharmacological) concentrations.

A more likely mechanism for BH4 oxidation is the interaction with peroxynitrite (generated from the interaction between NO and superoxide). Experiments in vitro92 and ex vivo81 indicate that peroxynitrite can oxidize BH4 within minutes, at physiologically relevant concentrations, and lead directly to eNOS uncoupling and endothelial dysfunction. Recent electron paramagnetic resonance experiments have demonstrated that peroxynitrite oxidizes BH4 to the (nonprotonated) BH3 radical, and thence to BH2, with a rate constant estimated to be 6x103 mol · L-1 · s-1, several-fold higher than reactions between peroxynitrite and ascorbate, glutathione, or thiol groups.93 However, the importance of these observations in vivo remains to be confirmed. In mice with streptozotocin-induced diabetes, endothelial dysfunction is associated with reduced BH4 levels but no change in total biopterins, which suggests that BH4 is oxidized to BH2 or biopterin. Increased endothelial BH4 biosynthesis by transgenic GTPCH overexpression partially corrects eNOS uncoupling, but the ratio of reduced BH4 in relation to oxidized biopterins in diabetes remains largely unaffected.88 More direct evidence showing that oxidative loss of BH4 is sufficient to produce eNOS uncoupling comes from studies of DOCA-salt hypertension in mice.53 DOCA-salt hypertension in eNOS-knockout mice did not increase vascular superoxide production to the same extent as in wild-type mice, which clearly implicates eNOS as an important source of vascular superoxide. Furthermore, in DOCA-salt hypertension, p47phox knockout mice were relatively protected from BH4 oxidation and eNOS uncoupling, which suggests that NADPH oxidase–mediated superoxide production is an important contributor to BH4 oxidative loss and eNOS uncoupling. Other interventions that reduce NADPH oxidase activation in vascular disease states, such as angiotensin II receptor blockade94 or inhibition of protein kinase C,95 have also been shown to reduce eNOS uncoupling and enhance NO bioavailability. These observations have contributed to a paradigm whereby superoxide in the endothelium (principally from NADPH oxidase) is thought to generate peroxynitrite (by reacting with NO), which oxidizes BH4, leading to eNOS uncoupling, thus perpetuating a cycle of vascular oxidative stress7 (Figure 2).

   Figure 2. Schematic illustrating the role of BH4 in regulating eNOS activity in vascular disease. A, In healthy vascular endothelium, BH4 availability is not limiting. eNOS production of NO is appropriate for the regulation of multiple antiatherogenic biological effects. Superoxide production by various oxidases acts predominantly in a signaling capacity. Peroxynitrite formed by interaction between NO and superoxide is minimal. B, In vascular disease states such as diabetes, hypertension, or hypercholesterolemia, superoxide production by oxidases is markedly increased. NO production may remain unaffected initially, but NO bioavailability is reduced because of scavenging interactions with superoxide, forming increased peroxynitrite. C, Peroxynitrite and other reactive oxygen species oxidize BH4, via the BH3 radical to BH2 and biopterin, which reduces the bioavailability of BH4 and promotes eNOS uncoupling. eNOS now generates superoxide rather than NO, which contributes to vascular oxidative stress and further reduces NO bioavailability. Ascorbate may protect against oxidative degradation of BH4 by directly reducing the BH3 radical to BH4.

Oxidation not only directly reduces BH4 bioavailability, but the oxidation products themselves (such as BH2), which have no cofactor activity, may compete with BH4 for binding to eNOS.60 However, it is uncertain whether the ratio of BH4 to oxidized biopterins is as important as the absolute BH4 concentration in determining eNOS activity in vivo.

    Tetrahydrobiopterin as a Therapeutic Target in Vascular Disease

What are the prospects for vascular disease therapies that target BH4-dependent eNOS function? High-dose pharmacological supplementation of BH4, given acutely, may not be specific for eNOS cofactor function and is not practical as a therapy for chronic vascular disease states. However, several studies have shown that ascorbate (vitamin C) may be important in maintaining BH4 levels in the setting of vascular oxidative stress. Acute or chronic ascorbate supplementation improves endothelial dysfunction in smokers,96 in subjects with hypercholesterolemia,97 and in patients with diabetes mellitus98,99 or overt coronary artery disease.100,101 Similarly, chronic dietary supplementation with ascorbate increased the BH4:BH2 ratio and NO bioavailability in the aortas of apoE-knockout mice,102 and there is a dose-dependent effect of ascorbate on BH4 levels and NO synthesis in cultured ECs.103,104 The mechanism for this effect is not fully resolved. Initial reports suggested that ascorbate chemically protects BH4 from oxidation by peroxynitrite,105 but recent data indicate that the rate constant for the reaction between peroxynitrite and ascorbate is 10-fold lower than that for peroxynitrite and BH4 (generating the BH3 radical).93 Instead, ascorbate may act by reducing the BH3 radical to regenerate BH4, a reaction with a kinetically favorable rate constant of 1.7x105 mol · L-1 · s-1.106

    Conclusions

Current research indicates that maintenance of adequate BH4 levels in the endothelium is likely to be critical in regulating the balance of NO and superoxide synthesis by eNOS in vascular disease. Strategies to maintain BH4 availability may include measures to increase BH4 biosynthesis or to reduce oxidative degradation. Despite significant recent insights into the molecular interactions between BH4 and eNOS that regulate endothelial biology, a number of important questions remain unanswered. The high concentrations of exogenous BH4 that are required to generate a biological effect in many studies suggest that biopterins do not enter cells merely by passive diffusion. However, the nature of a possible biopterin receptor/transporter is currently unknown. A better understanding of cellular biopterin transport would open new therapeutic approaches for endothelial dysfunction in vascular disease. An additional question concerns the role of the BH4 regeneration pathway (mediated by PCD and DHPR) in maintaining BH4 concentrations; this is a critical pathway in hepatocytes, but its importance in the vascular endothelium is unclear.

It is likely that the development of new mouse models with tissue-specific overexpression or deficiency of enzymes relevant to biopterin metabolism will provide important additional information on the role of BH4 in regulating eNOS function in vascular disease. Similarly, a better understanding of pharmacological approaches to target BH4 synthesis, oxidation, or regeneration in the endothelium may provide new therapeutic opportunities in vascular diseases.

    Acknowledgments

This work was supported by the British Heart Foundation and The Wellcome Trust.

References

Ignarro LJ. Nitric oxide as a unique signaling molecule in the vascular system: a historical overview. J Physiol Pharmacol. 2002; 53: 503–514.

Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980; 288: 373–376.

Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987; 84: 9265–9269.

Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclerotic risk. Arterioscler Thromb Vasc Biol. 2003; 23: 168–175.

Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation. 2001; 104: 2673–2678.

Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000; 101: 1899–1906.

Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.

Thony B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J. 2000; 347 (pt 1): 1–16.

Kaspers B, Gutlich M, Witter K, Losch U, Goldberg M, Ziegler I. Coordinate induction of tetrahydrobiopterin synthesis and nitric oxide synthase activity in chicken macrophages: upregulation of GTP-cyclohydrolase I activity. Comp Biochem Physiol B Biochem Mol Biol. 1997; 117: 209–215.

Werner ER, Werner-Felmayer G, Fuchs D, et al. Biochemistry and function of pteridine synthesis in human and murine macrophages. Pathobiology. 1991; 59: 276–279.

Gupta S, Fredericks S, Schwartzman RA, Holt DW, Kaski JC. Serum neopterin in acute coronary syndromes. Lancet. 1997; 349: 1252–1253.

Woo HJ, Kang JY, Choi YK, Park YS. Production of sepiapterin in Escherichia coli by coexpression of cyanobacterial GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase. Appl Environ Microbiol. 2002; 68: 3138–3140.

Krivi GG, Brown GM. Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from dihydroneopterin triphosphate. Biochem Genet. 1979; 17: 371–390.

Zorzi G, Redweik U, Trippe H, Penzien JM, Thony B, Blau N. Detection of sepiapterin in CSF of patients with sepiapterin reductase deficiency. Mol Genet Metab. 2002; 75: 174–177.

Nichol CA, Lee CL, Edelstein MP, Chao JY, Duch DS. Biosynthesis of tetrahydrobiopterin by de novo and salvage pathways in adrenal medulla extracts, mammalian cell cultures, and rat brain in vivo. Proc Natl Acad Sci U S A. 1983; 80: 1546–1550.

Hauer CR, Rebrin I, Thony B, et al. Phenylalanine hydroxylase-stimulating protein/pterin-4 alpha-carbinolamine dehydratase from rat and human liver: purification, characterization, and complete amino acid sequence. J Biol Chem. 1993; 268: 4828–4831.

Davis MD, Kaufman S, Milstien S. Distribution of 4a-hydroxytetrahydropterin dehydratase in rat tissues: comparison with the aromatic amino acid hydroxylases. FEBS Lett. 1992; 302: 73–76.

Vasquez-Vivar J, Whitsett J, Martasek P, Hogg N, Kalyanaraman B. Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical. Free Radic Biol Med. 2001; 31: 975–985.

Scriver CR, Eisensmith RC, Woo SL, Kaufman S. The hyperphenylalaninemias of man and mouse. Annu Rev Genet. 1994; 28: 141–165.

Smith I, Clayton BE, Wolff OH. New variant of phenylketonuria with progressive neurological illness unresponsive to phenylalanine restriction. Lancet. 1975; 1: 1108–1111.

Thony B, Blau N. Mutations in the GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase genes. Hum Mutat. 1997; 10: 11–20.

Dianzani I, de Sanctis L, Smooker PM, et al. Dihydropteridine reductase deficiency: physical structure of the QDPR gene, identification of two new mutations and genotype-phenotype correlations. Hum Mutat. 1998; 12: 267–273.

Segawa M, Hosaka A, Miyagawa F, Nomura Y, Imai H. Hereditary progressive dystonia with marked diurnal fluctuation. Adv Neurol. 1976; 14: 215–233.

Ichinose H, Suzuki T, Inagaki H, Ohye T, Nagatsu T. Molecular genetics of dopa-responsive dystonia. Biol Chem. 1999; 380: 1355–1364.

Nar H, Huber R, Meining W, Schmid C, Weinkauf S, Bacher A. Atomic structure of GTP cyclohydrolase I. Structure. 1995; 3: 459–466.

Nar H, Huber R, Auerbach G, et al. Active site topology and reaction mechanism of GTP cyclohydrolase I. Proc Natl Acad Sci U S A. 1995; 92: 12120–12125.

Katusic ZS, Stelter A, Milstien S. Cytokines stimulate GTP cyclohydrolase I gene expression in cultured human umbilical vein endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 27–32.

Linscheid P, Schaffner A, Blau N, Schoedon G. Regulation of 6-pyruvoyltetrahydropterin synthase activity and messenger RNA abundance in human vascular endothelial cells. Circulation. 1998; 98: 1703–1706.

Werner-Felmayer G, Werner ER, Fuchs D, et al. Pteridine biosynthesis in human endothelial cells: impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem. 1993; 268: 1842–1846.

Ishii M, Shimizu S, Nagai T, Shiota K, Kiuchi Y, Yamamoto T. Stimulation of tetrahydrobiopterin synthesis induced by insulin: possible involvement of phosphatidylinositol 3-kinase. Int J Biochem Cell Biol. 2001; 33: 65–73.

Lapize C, Pluss C, Werner ER, Huwiler A, Pfeilschifter J. Protein kinase C phosphorylates and activates GTP cyclohydrolase I in rat renal mesangial cells. Biochem Biophys Res Commun. 1998; 251: 802–805.

Milstien S, Jaffe H, Kowlessur D, Bonner TI. Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP. J Biol Chem. 1996; 271: 19743–19751.

Yoneyama T, Brewer JM, Hatakeyama K. GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits: purification, cDNA cloning, and bacterial expression. J Biol Chem. 1997; 272: 9690–9696.

Yoneyama T, Hatakeyama K. Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP. J Biol Chem. 1998; 273: 20102–20108.

Maita N, Okada K, Hirotsu S, Hatakeyama K, Hakoshima T. Preparation and crystallization of the stimulatory and inhibitory complexes of GTP cyclohydrolase I and its feedback regulatory protein GFRP. Schizophr Res. 2001; 57: 1153–1156.

Maita N, Okada K, Hatakeyama K, Hakoshima T. Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP. Proc Natl Acad Sci U S A. 2002; 99: 1212–1217.

Harada T, Kagamiyama H, Hatakeyama K. Feedback regulation mechanisms for the control of GTP cyclohydrolase I activity. Science. 1993; 260: 1507–1510.

Michel JB, Feron O, Sacks D, Michel T. Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J Biol Chem. 1997; 272: 15583–15586.

Prabhakar P, Thatte HS, Goetz RM, Cho MR, Golan DE, Michel T. Receptor-regulated translocation of endothelial nitric-oxide synthase. J Biol Chem. 1998; 273: 27383–27388.

Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605.

Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol. 2003; 284: R1–R12.

Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells can transduce an atheroprotective force. Arterioscler Thromb Vasc Biol. 1998; 18: 677–685.

Wilcox JN, Subramanian RR, Sundell CL, et al. Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 1997; 17: 2479–2488.

Cosentino F, Hishikawa K, Katusic ZS, Luscher TF. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation. 1997; 96: 25–28.

Bauersachs J, Bouloumi A, Fraccarollo D, Hu K, Busse R, Ertl G. Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation. 1999; 100: 292–298.

Bouloumie A, Bauersachs J, Linz W, et al. Endothelial dysfunction coincides with an enhanced nitric oxide synthase expression and superoxide anion production. Hypertension. 1997; 30: 934–941.

Ozaki M, Kawashima S, Yamashita T, et al. Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest. 2002; 110: 331–340.

Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.

Vasquez-Vivar J, Kalyanaraman B, Martasek P, et al. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A. 1998; 95: 9220–9225.

Vasquez-Vivar J, Kalyanaraman B, Martasek P. The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications. Free Radic Res. 2003; 37: 121–127.

Guzik TJ, Mussa S, Gastaldi D, et al. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.

Heitzer T, Yla-Herttuala S, Luoma J, et al. Cigarette smoking potentiates endothelial dysfunction of forearm resistance vessels in patients with hypercholesterolemia: role of oxidized LDL. Circulation. 1996; 93: 1346–1353.

Landmesser U, Dikalov S, Price SR, et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201–1209.

Munzel T, Li H, Mollnau H, et al. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability. Circ Res. 2000; 86: E7–E12.

Maier W, Cosentino F, Lutolf RB, et al. Tetrahydrobiopterin improves endothelial function in patients with coronary artery disease. J Cardiovasc Pharmacol. 2000; 35: 173–178.

Munzel T, Afanas’ev IB, Kleschyov AL, Harrison DG. Detection of superoxide in vascular tissue. Arterioscler Thromb Vasc Biol. 2002; 22: 1761–1768.

Song Y, Cardounel AJ, Zweier JL, Xia Y. Inhibition of superoxide generation from neuronal nitric oxide synthase by heat shock protein 90: implications in NOS regulation. Biochemistry. 2002; 41: 10616–10622.

Pritchard KA Jr, Ackerman AW, Gross ER, et al. Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase. J Biol Chem. 2001; 276: 17621–17624.

Stepp DW, Ou J, Ackerman AW, Welak S, Klick D, Pritchard KA Jr. Native LDL and minimally oxidized LDL differentially regulate superoxide anion in vascular endothelium in situ. Am J Physiol Heart Circ Physiol. 2002; 283: H750–H759.

Vasquez-Vivar J, Martasek P, Whitsett J, Joseph J, Kalyanaraman B. The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study. Biochem J. 2002; 362: 733–739.

Hurshman AR, Krebs C, Edmondson DE, Huynh BH, Marletta MA. Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen. Biochemistry. 1999; 38: 15689–15696.

Schmidt PP, Lange R, Gorren AC, Werner ER, Mayer B, Andersson KK. Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy. J Biol Inorg Chem. 2001; 6: 151–158.

Stroes E, Hijmering M, van Zandvoort M, Wever R, Rabelink TJ, van Faassen EE. Origin of superoxide production by endothelial nitric oxide synthase. FEBS Lett. 1998; 438: 161–164.

Rodríguez-Crespo I, Gerber NC, de Montellano PRO. Endothelial nitric-oxide synthase. J Biol Chem. 1996; 271: 11462–11467.

Raman CS, Li H, Martasek P, Kral V, Masters BS, Poulos TL. Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell. 1998; 95: 939–950.

Chen PF, Tsai AL, Wu KK. Cysteine 99 of endothelial, nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity. Biochem Biophys Res Comm. 1995; 215: 1119–1129.

Zou MH, Shi C, Cohen RA. Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest. 2002; 109: 817–826.

Tzeng E, Billiar TR, Robbins PD, Loftus M, Stuehr DJ. Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active enzyme. Proc Natl Acad Sci U S A. 1995; 92: 11771–11775.

Wever RMF, van Dam T, van Rijn HJ, de Groot F, Rabelink TJ. Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem Biophys Res Commun. 1997; 237: 340–344.

Reif A, Frohlich LG, Kotsonis P, et al. Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase. J Biol Chem. 1999; 274: 24921–24929.

Cai S, Alp NJ, McDonald D, Canevari L, Heales S, Channon KM. GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerization. Cardiovasc Res. 2002; 55: 838–849.

Meininger CJ, Marinos RS, Hatakeyama K, et al. Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency. Biochem J. 2000; 349: 353–356.

Shinozaki K, Kashiwagi A, Nishio Y, et al. Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O2- imbalance in insulin-resistant rat aorta. Diabetes. 1999; 48: 2437–2445.

Shinozaki K, Okamura T, Nishio Y, Kashiwagi A, Kikkawa R, Toda N. Evaluation of endothelial free radical release by vascular tension responses in insulin-resistant rat aorta. Eur J Pharmacol. 2000; 394: 295–299.

Shinozaki K, Nishio Y, Okamura T, et al. Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ Res. 2000; 87: 566–573.

Pieper GM. Acute amelioration of diabetic endothelial dysfunction with a derivative of the nitric oxide synthase cofactor, tetrahydrobiopterin. J Cardiovasc Pharmacol. 1997; 29: 8–15.

Pannirselvam M, Verma S, Anderson TJ, Triggle CR. Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -/-) mice: role of decreased tetrahydrobiopterin bioavailability. Br J Pharmacol. 2002; 136: 255–263.

Hong HJ, Hsiao G, Cheng TH, Yen MH. Supplementation with tetrahydrobiopterin suppresses the development of hypertension in spontaneously hypertensive rats. Hypertension. 2001; 38: 1044–1048.

Cosentino F, Patton S, d’Uscio LV, et al. Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats. J Clin Invest. 1998; 101: 1530–1537.

Cosentino F, Barker JE, Brand MP, et al. Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice. Arterioscler Thromb Vasc Biol. 2001; 21: 496–502.

Laursen JB, Somers M, Kurz S, et al. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001; 103: 1282–1288.

Stroes E, Kastelein J, Cosentino F, et al. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest. 1997; 99: 41–46.

Heitzer T, Krohn K, Albers S, Meinertz T. Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with type II diabetes mellitus. Diabetologia. 2000; 43: 1435–1438.

Heitzer T, Brockhoff C, Mayer B, et al. Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: evidence for a dysfunctional nitric oxide synthase. Circ Res. 2000; 86: E36–E41.

Fukuda Y, Teragawa H, Matsuda K, Yamagata T, Matsuura H, Chayama K. Tetrahydrobiopterin restores endothelial function of coronary arteries in patients with hypercholesterolaemia. Heart. 2002; 87: 264–269.

Tsutsui M, Milstien S, Katusic ZS. Effect of tetrahydrobiopterin on endothelial function in canine middle cerebral arteries. Circ Res. 1996; 79: 336–342.

Vasquez-Vivar J, Duquaine D, Whitsett J, Kalyanaraman B, Rajagopalan S. Altered tetrahydrobiopterin metabolism in atherosclerosis: implications for use of oxidized tetrahydrobiopterin analogues and thiol antioxidants. Arterioscler Thromb Vasc Biol. 2002; 22: 1655–1661.

Alp NJ, Mussa S, Khoo J, et al. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I over-expression. J Clin Invest. 2003; 119: 725–735.

Mitchell BM, Dorrance AM, Webb RC. GTP cyclohydrolase 1 downregulation contributes to glucocorticoid hypertension in rats. Hypertension. 2003; 41: 669–674.

Zheng J-S, Yang X-Q, Lookingland KJ, et al. Gene transfer of human guanosine 5'-triphosphate cyclohydrolase I restores vascular tetrahydrobiopterin level and endothelial function in low renin hypertension. Circulation. 2003; 108: 1238–1245.

Huie RE, Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun. 1993; 18: 195–199.

Milstien S, Katusic Z. Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun. 1999; 263: 681–684.

Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem. 2003; 278: 22546–22554.

Oelze M, Mollnau H, Hoffmann N, et al. Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction. Circ Res. 2000; 87: 999–1005.

Hink U, Li H, Mollnau H, et al. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001; 88: E14–E22.

Heitzer T, Just H, Munzel T. Antioxidant vitamin C improves endothelial dysfunction in chronic smokers. Circulation. 1996; 94: 6–9.

Ting HH, Timimi FK, Haley EA, Roddy MA, Ganz P, Creager MA. Vitamin C improves endothelium-dependent vasodilation in forearm resistance vessels of humans with hypercholesterolemia. Circulation. 1997; 95: 2617–2622.

Ting HH, Timimi FK, Boles KS, Creager SJ, Ganz P, Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with non-insulin-dependent diabetes mellitus. J Clin Invest. 1996; 97: 22–28.

Timimi FK, Ting HH, Haley EA, Roddy MA, Ganz P, Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 1998; 31: 552–557.

Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996; 93: 1107–1113.

Gokce N, Keaney JF Jr, Frei B, et al. Long-term ascorbic acid administration reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1999; 99: 3234–3240.

d’Uscio LV, Milstien S, Richardson D, Smith L, Katusic ZS. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res. 2003; 92: 88–95.

Heller R, Munscher-Paulig F, Grabner R, Till U. L-ascorbic acid potentiates nitric oxide synthesis in endothelial cells. J Biol Chem. 1999; 274: 8254–8260.

Huang A, Vita JA, Venema RC, Keaney J. Ascorbic acid enhances endothelial nitric oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem. 2000; 275: 17399–17406.

Heller R, Unbehaun A, Schellenberg B, Mayer B, Werner-Felmayer G, Werner ER. L-Ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem. 2001; 276: 40–47.

Patel KB, Stratford MR, Wardman P, Everett SA. Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate. Free Radic Biol Med. 2002; 32: 203–211.