Vascular protection bytetrahydrobiopterin: progress andtherapeutic prospectsZvonimir S. Katusic1, Livius V. d’Uscio1 and Karl A. Nath2

1 Departments of Anesthesiology and Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine,

200 First Street SW, Rochester, MN 55905, USA2 Division of Nephrology and Hypertension, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA

Review

Tetrahydrobiopterin (BH4) is an essential cofactorrequired for the activity of endothelial nitric oxide (NO)synthase. Suboptimal concentrations of BH4 in the endo-thelium reduce the biosynthesis of NO, thus contributingto the pathogenesis of vascular endothelial dysfunction.Supplementation with exogenous BH4 or therapeuticapproaches that increase endogenous amounts of BH4can reduce or reverse endothelial dysfunction by restor-ing production of NO. Improvements in formulations ofBH4 for oral delivery have stimulated clinical trials thattest the efficacy of BH4 in the treatment of systemichypertension, peripheral arterial disease, coronary arterydisease, pulmonary arterial hypertension, and sickle celldisease. This review discusses ongoing progress in thetranslation of knowledge, accumulated in preclinical stu-dies, into theclinical application of BH4 in the treatment ofvascular diseases. This review also addresses the emer-ging roles of BH4 in the regulation of endothelial functionand their therapeutic implications.

IntroductionThe role of tetrahydrobiopterin (BH4) in the control ofnitric oxide synthase (NOS) activity was recognized almost20 years ago [1–4]. It is nowwell established that BH4 is anessential cofactor required for enzymatic activity of allthree NOS isoforms, endothelial (eNOS), neuronal (nNOS)and inducible (iNOS). Availability of BH4 is regulated byseveral mechanisms including de novo synthesis, biopterinrecycling, and so-called salvage pathway (Figure 1). In theearly 1990s, biochemical studies demonstrated that theloss of BH4 might cause uncoupling of nNOS therebyincreasing nNOS-derived production of superoxide anionand hydrogen peroxide (Figure 2) [5,6]. In 1995, our groupreported that in coronary arteries, reduced availability ofBH4 in the endothelium causes an increase in eNOS-derived production of reactive oxygen species (ROS) in-cluding superoxide anion and hydrogen peroxide [7]. Giventhe ability of superoxide anion to chemically inactivatenitric oxide (NO), we hypothesized that uncoupled eNOSmight represent an important mechanism underlyingpathogenesis of endothelial dysfunction.

In an attempt to determine the mechanisms responsiblefor the loss of BH4 in diseased arteries, the attention of

Corresponding author: Katusic, Z.S. (katusic.zvonimir@mayo.edu).

48 0165-6147/$ – see front matter � 2008 Elsevier L

many groups, including ours, was initially directedtowards the interaction between BH4 and ROS. First,these studies confirmed the previously reported propensityof BH4 to autooxidize when exposed to oxygen [8]. Secondand more importantly, in 1999 it was discovered that BH4can be oxidized by peroxynitrite, a potent oxidant gener-ated by the reaction between superoxide anion and NO [9].This observation established a chemical antagonism be-tween BH4 and peroxynitrite, thereby suggesting thatconditions associated with elevated production of peroxy-nitrite might induce the uncoupling of eNOS. As a corol-lary, the ability of BH4 to inactivate peroxynitritesuggested that BH4 might be an important molecule pro-tecting endothelium from oxidative and nitrosative stressinduced by peroxynitrite. In this regard, it is important tonote that peroxynitrite might alter protein structure andfunction by reacting with various amino acids in the pep-tide chain. Cysteine oxidation and tyrosine nitration affectmore than 70 different proteins [10]. Theoretically, all ofthese interactions might be modified by BH4 therebyexpanding the influence of BH4 far beyond its role inthe regulation of NO production. However, there is noexperimental evidence to support the notion that oxidationof BH4 by endogenously generated peroxynitrite reducesintracellular concentration of BH4.

With further progress in the understanding of biochem-istry and pharmacology of BH4, it became apparent thatBH4 is a major vasoprotective molecule. In vivo, thisconcept was strongly supported by a series of studiesdemonstrating the beneficial effect of genetic supplement-ation of BH4 on endothelial dysfunction [11–14]. The morerecent development of novel pharmaceutical formulationsof BH4 enabled investigators to administer BH4 orally,leading to ongoing clinical trials designed to determine thetherapeutic efficacy of BH4 in patents with hypertension,peripheral arterial disease, coronary artery disease, andsickle cell disease (Box 1). In this review we will focus ourdiscussion on themost recent developments in the vascularpharmacology of BH4.

BH4 and endothelial dysfunctionHistorically, the discovery of endothelium-derived relaxingfactor (EDFR) and the subsequent identification of thechemical nature of EDRF as NO were among the mostimportant advances in vascular pharmacology in 1980s.

td. All rights reserved. doi:10.1016/j.tips.2008.10.003 Available online 29 November 2008

Figure 1. Biosynthesis of BH4. GTP cyclohydrolase I (GTP-CH) is the rate-limiting

enzyme in the de novo synthetic pathway of BH4. Pyruvoyl tetrahydropterin

synthase (PTPS) and sepiapterin reductase (SR) are two additional enzymes

required for the final production of BH4. BH4 can also be synthesized from

sepiapterin by the so-called salvage pathway. BH4 oxidized to BH2 can be recycled

back to BH4 by activity of dihydrofolate reductase (DHFR). Neopterin is a stable

side-product of BH4 that might serve as an indicator of elevated GTPCH activity.

Box 1. Major steps in the development of BH4 as a

therapeutic agent for cardiovascular diseases

� Identification of enzymatic activity of endothelial nitric oxide

synthase (eNOS) as a source of nitric oxide (NO), a major

vasodilator substance produced and released from vascular

endothelium.

� Recognition of tetrahydrobiopterin (BH4) as an essential cofactor

required for activity of eNOS.

� Discovery of eNOS uncoupling caused by suboptimal intracellular

concentration of BH4 resulting in increased formation of eNOS-

derived superoxide anion and endothelial dysfunction.

� Preclinical studies demonstrating the ability of pharmacological

and genetic supplementation of BH4 to prevent eNOS-derived

production of superoxide anion and endothelial dysfunction in

experimental models of cardiovascular diseases.

� Establishment of the beneficial effect of intravenous BH4 supple-

mentation on endothelial dysfunction in humans.

� Synthesis of orally active dihydrochloride salt of BH4.

� Commencement of randomized placebo-controlled clinical trials

designed to determine therapeutic efficacy of oral BH4 supple-

mentation in the treatment of hypertension, peripheral arterial

disease, coronary artery disease, pulmonary hypertension, and

sickle cell disease.

Review Trends in Pharmacological Sciences Vol.30 No.1

The groundbreaking impact of these discoveries was recog-nized by the Nobel Prize awarded to Drs. Furchgott,Ignarro and Murad in 1998. The recognition of the endo-thelium as a major source of vasodilator substances, in-cluding NO, had a major impact on the understanding ofthe pathogenesis of vascular diseases. The concept of endo-thelial dysfunction evolved as a result of studies on dis-eased arteries both in experimental animals and inpatients with vascular disease [15]. These studies estab-lished the loss of NO as a central mechanism of endothelialdysfunction, thereby stimulating the development of noveltherapeutic approaches that sought to remedy impairedendothelial production of NO. In addition, drugs currentlyemployed in the treatment of cardiovascular diseases werereevaluated so as to determine the extent to whichimproved endothelial biosynthesis of NO accounted fortheir therapeutic effects. Indeed, an increasing focus in

Figure 2. Schematic representation of endothelial nitric oxide synthase (eNOS)

uncoupling. During uncoupling, biosynthesis of nitric oxide (NO) is uncoupled

from consumption of NADPH and electron flow is directed towards formation of

superoxide anion (O2��) and hydrogen peroxide (H2O2). The subsequent reaction

between superoxide anion and NO generates peroxynitrite anion (ONOO�).

Suboptimal concentrations of BH4 (#).

the field of cardiovascular drug development is the searchfor agents that can preserve endothelial function.

Over the past decade, numerous studies demonstratedthat the chemical inactivation of BH4 by oxidative stresscauses uncoupling of eNOS and endothelial dysfunction;the role of BH4 in eNOS uncoupling and the pathogenesisof endothelial dysfunction has been emphasized in recentreviews [16,17]. During the past few years, studies utiliz-ing genetically modifiedmice provided convincing evidencethat eNOS uncoupling alone (in the absence of vasculardisease) was sufficient to initiate and propagate BH4oxidation in vivo [18]. In mice overexpressing eNOS, amismatch between eNOS protein levels and intracellularconcentration of BH4 resulted in uncoupling of overex-pressed eNOS. This, in turn, led to increased formationof eNOS-derived superoxide anion. Indeed, furtherpharmacological and genetic analysis demonstrated thatuncoupled eNOS was a major source of superoxide anion.This study underscored the fact that even in the absence ofvascular disease, upregulation of eNOS might adverselyaffect endothelial function if the concentrations of BH4 donot commensurately increase so as to attain levels requiredfor optimal activity of eNOS. Consistent with this concept,long-term transgenic overexpression of eNOS in apolipo-protein E knockout (ApoE-KO) mouse model of atheroscle-rosis had a detrimental rather than a beneficial effect onplaque formation [19]; most importantly, supplementationwith BH4 abolished the accelerated atherosclerosisdetected in ApoE-KO mice overexpressing eNOS. Theseobservations were explained, and plausibly so, by BH4-induced eNOS recoupling. They also demonstrated thattherapeutic approaches designed to elevate expression ofeNOS protein might not improve production of NO if thelocal environment in the vascular endothelium becomesrelatively deficient in BH4. In fact, activity of uncoupledeNOS might contribute to oxidative stress and exacerbateendothelial dysfunction. In accordance with this concept,numerous in vivo studies in rodent models and in humans

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Table 1. Effect of administration of BH4 in vivo on human vascular function

Condition Effect Refs

Hypercholesterolemia Prevents endothelial dysfunction [60]

Coronary artery disease Prevents endothelial dysfunction [61]

Chronic smokers Prevents endothelial dysfunction [20]

Long-term smokers Prevents endothelial dysfunction [62]

Coronary risk factors Prevents endothelial dysfunction [63]

Normo- or hypertension Augments endothelium-dependent vasodilatation [64]

Vasospastic angina Prevents endothelial dysfunction [65]

Chronic heart failure Prevents endothelial dysfunction [66]

Hypercholesterolemia Prevents endothelial dysfunction [67]

Glucose challenge Prevents endothelial dysfunction [68]

Type 2 diabetes Increases insulin sensitivity but does not improve endothelial function [69]

Endotoxin-induced endothelial dysfunction Prevents endothelial dysfunction [70]

Aging Improves flow-mediated dilatation [71]

Aging Prevents endothelial dysfunction [72]

Erectile dysfunction Prevents erectile dysfunction [73]

Ischemia reperfusion Prevents endothelial dysfunction [21]

Atherosclerosis Does not improve endothelial function [74]

Hypercholesterolemia Prevents endothelial dysfunction and decreases oxidative stress [34]

Hypertension Prevents endothelial dysfunction and decreases arterial blood pressure [35]

Hypercholesterolemia Improves dysfunction of coronary microcirculation [75]

Type 2 diabetes Prevents endothelial dysfunction [33]

Review Trends in Pharmacological Sciences Vol.30 No.1

have consistently demonstrated the beneficial effect ofBH4 on endothelial dysfunction, thereby attesting to thevasoprotective properties of BH4 (Table 1).

It is however also important to keep in mind that BH4 isa potent reducing agent, and that BH4 might act as anantioxidant. Analogues of BH4 that are antioxidant butincapable of supporting eNOS activity have been success-fully employed to dissect the contribution of the antiox-idant capacity of BH4 to its overall beneficial effect onvascular function [20]. Although the existing evidencesuggests that recoupling of eNOS and stimulation of NOproduction are more important protective mechanisms,antioxidant properties of BH4 might also contribute tothe preservation of endothelial function [21].

BH4 and oxidative stressOxidative stress is imposedwhen the generation of oxidantsoutstrips the rate at which they can be metabolized ordegraded, thereby leading to an increase in ambient con-centrations of these oxidizing species. As mentioned pre-viously, BH4 is a potent reducing agent and therefore avulnerable molecular target for oxidation. Oxidative degra-dation of BH4 is complex and depends on several factorsincluding pH [9]. At neutral pH, 7,8-dihydropterin is themost abundant product of BH4 oxidation by peroxynitrite.Thus, a major portion of BH4 is oxidized irreversibly tocofactor inactive molecule (7,8-dihydropterin), thereby dis-abling intracellular regeneration of BH4 [9]. Only about athird of the BH4 that is oxidized to 7,8-dihydrobiopterin(BH2) can be regenerated into BH4 by dihydrofolatereductase. It should be emphasized that BH2 can competeand displace BH4 from eNOS, thereby fostering the uncou-pling of eNOS initially incurred by the oxidation of BH4[22,23]. The potential importance of peroxynitrite as anoxidant responsible for the loss of BH4 is underscored bythe fact that the rate constants for the reactions of perox-ynitritewithmajor cellular antioxidants (specifically, ascor-bate, cysteine and glutathione) are about six to ten timeslower than the rate constant for the reaction of peroxynitritewith BH4 [24]. This makes BH4 highly susceptible to oxi-

50

dative attack by peroxynitrite. However, it is important tonote that direct experimental evidence supporting the abil-ity of endogenous peroxynitrite to oxidize intracellular BH4is still lacking. More recent in vitro findings suggest thatBH4 might be oxidized when exposed to superoxide aniongenerated by the xanthine/xanthine oxidase system [25].Whether such scavenging of superoxide anion by BH4 con-tributes to the beneficial effects of BH4 is unclear andremains to be determined [25].

We also wish to point out that the chemical antagonismbetween BH4 and peroxynitrite raises another importantquestion: namely, does the availability of BH4 reduce therisk for local injury by peroxynitrite? Proinflammatorycytokines, including tumor necrosis factor-a, interferon-g, and interleukin-1b, are powerful stimulators of BH4biosynthesis. In human endothelium, these cytokinesincrease the expression and activity of GTP cyclohydrolaseI, the rate-limiting enzyme in the production of BH4[26,27]. Given that proinflammatory cytokines decreaseexpression of eNOS (but do not increase endothelialexpression of iNOS [26]), the role of high endothelialconcentration of BH4 during inflammation remains poorlyunderstood. One possibility is that the elevation of BH4might enhance antioxidant potential of the endothelium inconditions associated with increased production of perox-ynitrite [28]. Indeed, BH4 (together with ascorbate) pre-vents peroxynitrite-induced tyrosine nitration ofnumerous proteins in cells stimulated with proinflamma-tory cytokines [23]. It is therefore conceivable that underproinflammatory conditions, BH4 might be an importantantioxidant in general, and one that safeguards againstperoxynitrite-induced nitration of tyrosine residues inparticular. Because peroxynitrite might affect the functionof proteins involved in oxidative stress, energy production,apoptosis, and fatty acid metabolism, as well as structuralproteins [29], it is likely that the ambient levels of BH4might thereby influence a wide range of biologic processes.This hypothesis remains to be tested.

Several unpublished observations fromour group furthersupport an important role for BH4 as an antioxidant

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molecule in the endothelium. For example, we haveobtained immunohistochemical evidence that GTP cyclohy-drolase I localizes in the caveolar membrane microdomainalong with caveolin-1 and eNOS. A strategic, cytoprotectivebenefit is likely conferred by such localization because thehigh local concentrations of BH4 within caveolae mightsafeguard the structural and functional integrity of caveolaeunder conditions of oxidative stress [30].Moreover, ourpriorstudies demonstrated that endothelial progenitor cells(EPCs) have a high antioxidant capacity [31], and our recentstudies revealed that EPCs also have an intrinsically highintracellular concentration of BH4 (He and Katusic, unpub-lished observation), despite the fact that expression of eNOSis relatively low [32]. We suggest that these generousamounts of BH4 in EPCs might serve to maintain eNOSin its coupled state in EPCs, to guard against the oxidizingeffect of peroxynitrite on eNOS and other targets, and tosustain, in general, the high antioxidant profile of EPCs. Anenriched antioxidant capability of EPCs likely confers asurvival advantage to EPCs because the regenerative func-tion of EPCs is most needed in injured tissue, the lattercommonly exhibiting a milieu of increased oxidative stress.In this regard, the exact contribution of BH4 to the anti-oxidant capability and regenerative function ofEPCsmeritsfurther investigation.

BH4 and therapy of vascular diseaseExogenous BH4

Over the last decade, numerous studies have examined theeffect of BH4 supplementation on endothelial dysfunctioncaused by a variety of vascular diseases. Initially, thesestudies involved a limited number of patients in whomBH4 was administered acutely or on a short-term basis.Quite remarkably, supplementation with BH4 improvedendothelial dysfunction in various conditions includinghypercholesterolemia, diabetes, hypertension, coronaryartery disease, and heart failure; such administration ofBH4 also improved endothelial function in smokers andaged subjects (Table 1). The beneficial effect of BH4appeared to be endothelium-specific because the effectsof endothelium-independent vasodilators (e.g. nitrogly-cerin) were not affected by BH4 supplementation. Thebeneficial effects of BH4 arose from the activation of eNOSor antioxidant effect of BH4 [21,33].

More recently, the effects of long-term oral adminis-tration of BH4 have been studied in patients with hyperch-olesterolemia and hypertension. Twenty-twohypercholesterolemic patients were randomized to 4weeksof oral BH4 treatment (400 mg twice daily) or placebo; age-matched healthy volunteers served as controls [34]. BH4levels in plasma were significantly increased by oralsupplementation. BH4 normalized the impairment inendothelium-dependent relaxations to acetylcholine inhypercholesterolemic patients but did not affect endo-thelial function in control subjects. Interestingly, theauthors also demonstrated that BH4 significantly reducedplasma levels of 8-F2 isoprostane, a marker of oxidativestress, and that BH4 prevented uncoupling of eNOSinduced by low-density lipoprotein in cultured endothelialcells. Notably, no adverse effects were observed in subjectsadministered BH4, findings that indicate the safety of

BH4,at leastasutilized in this study.Theauthors concludedthat oxidative stress and endothelial dysfunction inducedby hypercholesterolemia can be normalized by chronicadministration of BH4.

To determine whether chronic administration of BH4might have an antihypertensive effect, the effects of 5 or10 mg/kg ofBH4per day (administered orally for 8weeks) or200 mg or 400 mg of BH4 (administered orally for 4 weeks)on arterial blood pressure and endothelial dysfunctionwerestudied in patientswith poorly controlled hypertension [35].These investigators observed a beneficial effect of BH4 onboth arterial blood pressure and endothelial dysfunction inpatients treated with BH4 at doses of 5 or 10 mg/kg for 8weeks, and at doses of 400 mg for 4 weeks. These findingssupport the view that administration of BH4 might providea salutary effect on vascular function. However, as pointedout by the authors, the study lacked a placebo control group,and BH4 was formulated such that vitamin C was presentduring administration of BH4. Thus, it is possible that thebeneficial effects might reflect a synergistic effect of BH4and vitamin C, or a vitamin C-mediated effect.

In contrast to these two studies demonstrating abeneficial effect of BH4, a phase 2 clinical trial sponsoredby the US pharmaceutical company BioMarin failed toobserve an ameliorative effect of oral administration ofBH4 (5 mg/kg twice daily for 2 months) in patients withpoorly controlled hypertension. It is possible that the lackof efficacy observed with such administration of BH4mightreflect aspects of the pharmacokinetic and pharmacody-namic profiles of BH4 that are currently incompletelyappreciated. For example, from a pathophysiologic stand-point, the ultimate intent when BH4 is administered is theelevation of intracellular levels of BH4 in the endothelium.However, only plasma levels of BH4 are measured inpatients treated with BH4. Whether elevation of BH4 inplasma faithfully reflects increased intracellular concen-tration in the endothelium is unknown. In fact, in patientswith coronary artery atherosclerosis, high plasma levels ofBH4 are associated with low BH4 levels in endothelium[36]. This dissociation between plasma and vascular con-tent of BH4 suggests that, in humans, BH4 does notpassively diffuse from circulating blood into the endo-thelium. Indeed, it has been demonstrated that in manycell types, including endothelial cells, BH4 is not freelydiffusible [37]. In order for BH4 to be imported into theendothelium, it has to undergo oxidation to BH2; importedBH2 is then regenerated back to BH4 by dihydrofolatereductase (DHFR) [37]. Intact DHFR activity is thus anessential requirement in enabling an increase in intracellu-lar concentrations of BH4 after oral supplementation withBH4. However, the extent to which DHFR activity is pre-served in the endotheliumof diseased blood vessels is poorlyunderstood, and the limited number of studies that addressthis issue offer divergent findings. For example, underconditions of oxidative stress, as induced by angiotensinII,DHFRactivity is decreased in endothelium, analterationthat favors the elevationofBH2and theuncoupling of eNOS[38]. At variance with this observation are the findings thatoxidativestress inducedbydiabetesmight increasevascularexpression of DHFR, thus raising the possibility thatelevation of DHFR might be an adaptive response to

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oxidative stress [39]. Further investigation of the involve-ment of DHFR in influencing BH4 metabolism and main-taining the ‘coupling’ of eNOS is clearly needed, not only inthe healthy vasculature but also in the diseased bloodvessels. It is also increasingly apparent that the intracellu-lar concentration of BH4 is determined by a complex net-work of mechanisms that influence the anabolism andcatabolism of BH4, the recycling of BH4, and mechanismsresponsible for the transport of BH4 across the endothelialcell membrane. More complete understanding of thesemechanismswill facilitate the realization of the therapeuticpotential of BH4.

As previously discussed, BH4 is easily oxidized, isthermo- and photolabile, and has relatively poor cell mem-brane permeability. Some of these limitations have beenaddressed by development of sapropterin (BioMarin), asynthetic BH4; the US Food and Drug Administration(FDA) has recently approved the oral administration ofsapropterin for the treatment of phenylketonuria. Saprop-terin is a synthetic preparation of the dihydrochloride saltof the biologically active (6R)-5,6,7,8-tetrahydro-L-biop-terin (BH4). Of note, this compound is currently beingtested in clinical trials in patients with peripheral arterialdisease, hypertension, coronary artery disease, pulmonaryarterial hypertension, and sickle cell disease. However, thedevelopment of novel BH4 analogues with oral bioavail-ability, improved access into intracellular space, longerduration of action, and greater potency might facilitatethe therapeutic success of this approach. Now available is anovel BH4 analogue (6-acetyl-7,7-dimethyl-5,6,7,8-tetra-hydropterin; ADDP) [40] that is a stable compound, solublein water and in polar hydrogen-binding organic solvents; itis also capable of crossing cell membranes and it supportsNOS activity. Interestingly, studies on purified nNOSdemonstrated that ADDP requires conversion into5,6,7,8-tetrahydropterin to support enzymatic activity ofnNOS. It was suggested that in vivo, this chemical con-version of ADDP might occur in the presence of DHFR.Based on studies utilizing isolated rat aorta and obser-vations obtained in an experimental model of pulmonaryhypertension, it was suggested that ADDP causes vasodi-latation by stimulating eNOS activity. These studies illus-trate how the discovery of novel BH4 analogues mightsignificantly broaden and diversify the therapeutic optionsavailable for patients with cardiovascular diseases.

The approval of BH4 by the FDA as a drug for thetreatment of patients with phenylketonuria is based onseveral clinical trials that demonstrate the safety of BH4.However, it is important to keep in mind that BH4 is alsocofactor for iNOS and nNOS. Activation of both of theseisoforms might be associated with detrimental effectsresulting from markedly increased, local concentration ofNO. Marked induction of, and attendant cytotoxicityincurred by iNOS might more likely occur in patients withsevere infections, autoimmune disorders, and pathologicalangiogenesis [41]. In addition, in some patients, the stimu-latory effect of BH4 on biosynthesis of catecholamines [42]might adversely affect cardiovascular function. Carefulmonitoring of patients chronically treated with BH4 willprovide important additional information regardingpossible unrecognized adverse effects.

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Endogenous BH4

The deficiency of BH4 in vascular diseases appears toresultmainly from its oxidation by peroxynitrite [9]. There-fore, strategies that protect endogenous BH4 from theoxidative attack by peroxynitrite might result in preser-vation of optimal endothelial concentration of the cofactorrequired for activity of eNOS and biosynthesis of NO. Inhuman studies, administration of folic acid or 5-methylte-trahydrofolate (5-MTHF; the active and circulating form offolic acid) received the most attention as agents with apotentially favorable effect on BH4 metabolism. Althoughthe mechanism of this effect remains incompletely under-stood [43], more recent findings suggest that 5-MTHF is apotent scavenger of peroxynitrite [44]. Moreover, 5-MTHFand folic acid [45] reduce superoxide anion and improveendothelium-dependent relaxations in human arteriesboth ex vivo and in vivo. Most importantly, the adminis-tration of 5-MTHF to patients with atherosclerosis andcoronary artery disease leads to increased tissue content ofBH4 in arteries and veins, the reversal of the uncoupling ofeNOS, and normalization of production of NO [44,46].Thus, the administration of folates provides an effectivestrategy to prevent BH4 oxidation and subsequent uncou-pling of eNOS. Although the beneficial effects of folates aretraditionally explained by their ability to reduce levels ofhomocysteine, these recent findings indicate that preser-vation of vascular content of BH4 and effective coupling ofeNOS contribute to the vasoprotective effects of folates.

In attempt to define the optimal dose of folates requiredfor vascular protection, the effects of folic acid adminis-tered as a low-dose (400 mg/d) or high-dose regimen (5 mg/d) were studied in patients with coronary artery disease[46]. They demonstrated that the low-dose folic acid regi-men (equivalent to dose that is present in fortified grain) issufficient to improve vascular function by increasing levelsof BH4 in the vascular wall, whereas the high-dose folicregimen did not confer any additional benefit. Thus, itappears that folate supplementation is an efficientstrategy in the prevention of BH4 oxidation and eNOSuncoupling in the vasculature. Further studies are neededto determine if genetic variants in methyltetrahydrofolatereductase can detect those patients with impaired syn-thesis of 5-MTHF who therefore might benefit from 5-MTHF supplementation.

In addition to folates, studies on cultured human endo-thelial cells or experimental animals suggest that otherdrugs might increase the availability of BH4: specifically,ascorbic acid [28,47,48], statins [49–52], enalapril [53],captopril [54], telmisartan [39], eplerenone [53], cilostazol[55], insulin [56], and erythropoietin [57] have all beenshown to stimulate biosynthesis of BH4 and/or protect BH4from oxidation. That structurally diverse therapeuticagents employed in cardiovascular diseases all convergeon the common biochemical effect of sustaining levels ofBH4 bespeaks to a fundamentally important role of BH4 inmaintaining the health and function of the vasculature.

SummaryCurrently, the endothelium is recognized as a major thera-peutic target in the prevention and treatment of vasculardisease. From a pathophysiologic standpoint, an important

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focus in the prevention and treatment of vascular disease isthe restoration of normal biosynthesis of NO and thereduction of excessive generation of superoxide anionand ROS. In this regard, supplementation with BH4and/or strategies that augment endogenous levels ofBH4 have been recently identified as novel approachesthat exert salutary effects on endothelial dysfunctioninduced by a variety of vascular diseases. This conceptand its therapeutic implications are the focus of consider-able investigation, from which will likely emerge anincreased spectrum of therapeutic agents available forcardiovascular diseases. Emerging evidence derived fromthe study of genetic polymorphisms in key enzymesinvolved in metabolism of BH4 suggests that constitutivedeficiency of BH4might increase the risk of cardiovasculardisease [58,59]. Studies of gene polymorphisms might thusassist in identifying those patients who are at risk forcardiovascular disease, and who would most likely benefitfrom supplementation with BH4 or its analogues.

AcknowledgementsThis work was supported by the National Institutes of Health (grantsHL-53524, HL-58080, and HL-66958, to Z.S.K.; HL-55552 and DK-70124,to K.A.N.) and by theMayo Foundation. L.V.D. is the recipient of a ScientistDevelopment Grant from the American Heart Association (07–301333N).

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