Tetrahydrobiopterin Improves Endothelial Function in Cardiovascular Disease: A Systematic Review

Background. Tetrahydrobiopterin (BH4) is a cofactor of nitric oxide synthase (NOS). Nitric oxide (NO) bioavailability is reduced during the early stage of vascular diseases, such as coronary artery disease, hypercholesterolemia, hypertension, and diabetic vasculopathy, and even throughout the entire progression of atherosclerosis. Methods. A literature search was performed using electronic databases (up to January 31, 2014), including MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL), using an established strategy. Results. Fourteen articles were selected with a total of 370 patients. Ten of the fourteen studies showed a significant improvement in the endothelial dysfunction of various cardiovascular disease groups with BH4 supplementation compared with the control groups or placebos. Three studies showed no positive outcome, and one study showed that low-dose BH4 had no effect but that high-dose BH4 did have a significantly different result. Conclusions. This review concludes that supplementation with BH4 and/or augmentation of the endogenous levels of BH4 will be a novel approach to improve the endothelial dysfunction observed in various cardiovascular diseases. BH4 might be considered to be a new therapeutic agent to prevent the initiation and progression of cardiovascular disease.


Introduction
Cardiovascular diseases (CVDs), such as coronary artery disease, hypercholesterolemia, diabetes, hypertension, and stroke, remain the largest cause of mortality and morbidity in the world. In 2014, the attributable fractions of adjusted estimated population for the mortality of CVDs are as follows: 40.6% for high blood pressure, 13.7% for smoking, 13.2% for poor diet, 11.9% for insufficient physical activity, and 8.8% for abnormal blood glucose levels [1]. Abnormal endothelial function appeared as an early feature of all CVDs and risk factor syndromes, resulting in the loss of normal homoeostatic pathways that act to inhibit disease processes such as inflammation, thrombosis, and oxidative stress [2,3].
The endothelium is the largest endocrine organ in the human body and can be involved in the control of vascular tone, platelet reactivity, coagulation, and permeability [4]. Thus, healthy endothelium can protect against excessive/abnormal inflammation and coagulation [5], which are the key processes in CVD development and progression. Furthermore, endothelial function was demonstrated to serve as a predictor of cardiovascular events [6,7]. Therefore, the evaluation of endothelial function is vital to generate and determine a more effective or final therapeutic strategy for cardiovascular diseases. From a pathophysiologic standpoint, there is an important focus on the prevention and treatment of vascular diseases via the restoration of the normal biosynthesis of nitric oxide (NO) and the reduction of the excessive generation of superoxide anions and reactive oxygen species (ROS).
Tetrahydrobiopterin (BH 4 ) is an essential cofactor for a set of enzymes that are of pivotal metabolic importance, including four aromatic amino acid hydroxylases (AAAH), three nitric oxide synthases (NOS), and alkylglycerol monooxygenase (AGMO). Phenylalanine hydroxylase (PAH) was the first enzyme recognized to depend on BH 4 [8]. Phenylketonuria is a genetic disorder characterized by a deficiency of PAH; BH 4 may provide good phenylalanine control in the patients who respond to oral administration of BH 4 . NOS is a critical enzyme militated in the production of the messenger molecule NO, which is generated from Larginine. BH 4 is inseparably considered to be a cofactor of NOS enzymes for the progression of NO synthesis [9,10]. When BH 4 is limited, under the conditions of oxidative stress, BH 4 can be readily oxidized to dihydrobiopterin (BH 2 ) and eventually converted into biopterin, especially when NOS cofactor activity is not needed. When NOS is uncoupled, ROS rather than NO is produced. NO is used as a soluble gas continuously synthesized from the amino acid L-arginine in endothelial cells via the constitutive calciumcalmodulin-dependent enzyme NOS. This substance has a wide variety of biological properties that maintain vascular homeostasis, including the modulation of vascular dilator tone, regulation of local cell growth, and protection of the vessel from the injurious consequences of platelets and cells circulating in the blood. In the early period of different CVDs, the bioavailability of NO is reduced. In humans, endothelial function is altered in different subjects with vascular disease status and correlated with risk factor profiles [2,11]. Importantly, several prospective studies have identified that supplementation with BH 4 improves endothelial function in patients with coronary artery disease, hypercholesterolemia, hypertension, and diabetic vasculopathy [12][13][14][15]. Moreover, there are published clinical studies of BH 4 therapy in the pathogenesis of other vascular diseases, such as pulmonary hypertension [16] and smoking [17,18], as well as in aging [19][20][21].
In this regard, supplementation with BH 4 and/or strategies that augment the endogenous levels of BH 4 have been recently identified to be novel approaches that can exert salutary effects on the endothelial dysfunction induced by a variety of vascular diseases. This concept and its therapeutic implications are the focus of considerable investigation, which will likely generate an enlarged spectrum of therapeutic agents available for CVDs.

Materials and Methods
A systematic review of the literature concerning BH 4 to improve vascular endothelial function in adult patients was conducted using the recommended guidelines provided by the Cochrane Handbook for the Systematic Reviews of Interventions.

Search Methods for the Identification of Studies.
To select eligible studies, a search was performed of electronic databases, including PUBMED, MEDLINE, and the Cochrane Library, using a search strategy that depended on combinations of the keywords tetrahydrobiopterin/BH 4 and endothelial function or endothelial dysfunction. The last search was updated to January 31, 2014. We deliberately broadened the search to ensure the inclusion of all relevant articles. All the bibliographies of papers retrieved from the search were also screened for additional articles. Only full publications in peer-reviewed journals were selected for potential inclusion in the review.

Study Selection Criteria.
Two reviewers independently assessed titles, abstracts, and/or the full-text papers of the records retrieved from the electronic database searches for possible inclusion according to the predefined selection criteria: (1) type of study, only RCTs were selected for further assessment; (2) participants, only CVD patients older than 18 years were included, regardless of gender; (3) type of intervention, the intervention used any generation of BH 4 , alone or combined with other substances, irrespective of the administration approach, and the intervention in the control group was a placebo, alone or combined with other substances; and (4) outcomes, trials focused on the effect of supplementation of BH 4 on endothelial function in patients with CVD.

Quality Assessment.
This study is a "qualitative systematic review" without a meta-analysis. The methodological quality of the RCTs was assessed independently by two reviewers (see Table 1) according to the methods recommended in Section Six of the Cochrane Handbook for Systematic Reviews of Interventions, Version 5.1.0.

Description of Selected Studies. Citations and abstracts
were downloaded into Mendeley and Endnote 6 by independent researchers, and any duplicates were deleted. The main search strategy identified 802 publications, and 356 were excluded because of duplication ( Figure 1). A preliminary screening of the titles and abstracts was performed according to the following inclusion criteria: studies related to BH 4 and endothelial function. We excluded reviews, meeting notes, book chapters, animal experiments employing qualitative methods, findings derived from qualitative methods, interviews, and observations or participant observations. Access to the full text of the remaining articles was then sought (see Figure 1).

Tetrahydrobiopterin in the Treatment of Cardiovascular
Disease. In a range of in vivo pharmacological experiments, bioavailability has emerged as a rational therapeutic target in vascular disease states (see Table 2).

Coronary Artery
Disease. BH 4 was administered acutely or on a short-term basis, delivered via intracoronary/intraarterial infusion [13,[22][23][24][25]. In one study, oral BH 4 was used at a low dose (400 mg/d) or high dose (700 mg/d) for 2 to 6 weeks [26]. In another study, BH 4 alone did not influence the vessel area but did prevent vasoconstriction in response to acetylcholine (ACh) (+2 ± 3%, NS, versus baseline) in 15 of the patients with endothelial dysfunction in the trial. Correspondingly, calculated volume flow showed the highest value after coinfusion with Ach and BH 4 [25]. BH 4 significantly improved acetylcholine-induced increases in coronary blood flow (CBF) in patients with diminished flow responses but exerted no effect in those with normal flow responses [22]. However, no difference was observed in the Ach response due to the coinfusion of BH 4 and Ach with respect to the % change in CBF [24]. Settergren et al. found that the endothelium-dependent vasodilatation was significantly less reduced at 15 and 30 min of reperfusion following L-arginine and BH 4 infusion than with saline infusion [25]. BH 4 did not affect the relative changes in the brachial artery diameter from baseline flow-mediated vasodilation (FMD)(%) in type 2 diabetic and coronary heart disease patients [23]. Oral BH 4 treatment for 2 to 6 weeks significantly augmented the BH 4 levels in plasma but had no effect on the vascular redox state or endothelial function [26].

3.4.
Hypercholesterolemia. The method of administration was mainly infusion via the brachial artery [27][28][29] or coronary ostium [30]. 22 hypercholesterolemic patients were randomized into groups receiving 4 weeks of oral BH 4 (400 mg twice daily) or placebo [14]. In all studies, BH 4 restored the vascular function in the patients with hypercholesterolemia. BH 4 also restored the endothelial function of coronary arteries in the patients with hypercholesterolemia [28,30]. BH 4 attenuated the Ach-induced decrease in coronary diameter and restored the Ach-induced increase in coronary blood flow [30]. BH 4 increased exerciseinduced hyperemia in all subjects but had no influence on myocardial blood flow (MBF) at rest or during adenosineinduced hyperemia in all subjects. Flow reserve utilization was increased significantly in hypercholesterolemic subjects but remained unchanged in controls [28]. The vasoconstrictor response to L-monomethyl-arginine (L-NMMA) was significantly increased with BH 4 treatment compared with saline infusion ( < 0.05); additionally, the impaired  [15] Diabetes (type II) Improves endothelium-dependent vasodilatation 6R-BH 4 (Schircks) intra-arterial infusion 500 g/min serotonin-induced vasodilation was restored by this treatment [27]. Localized BH 4 alone or in combination with other substances augmented the NO-dependent vasodilatation in hypercholesterolemic patients but showed no effect in normocholesterolemic subjects [29]. BH 4 restored endotheliumdependent, NO-mediated vasodilatation but had no effect on endothelium-independent vasodilatation due to sodium nitroprusside [14].

3.5.
Hypertension. BH 4 (500 g/min) was infused intraarterially for 5 min. The forearm blood flow (FBF) response to Ach in hypertensive patients increased significantly to the level of normal control subjects [31]. Oral high-dose BH 4 (400 mg/d) produced a significant decrease in systolic ( < 0.03) and mean blood pressure (BP) ( < 0.04). The decrease in diastolic BP did not reach statistical significance ( < 0.08). No significant change in BP was observed in subjects given low-dose BH 4 (200 mg/d). There was a significant improvement in FMD with 400 mg of BH 4 but no significant change with 200 mg of BH 4 [12].

Diabetic Vasculopathy.
In diabetes, cardiovascular disease is a common complication. Endothelial dysfunction occurs as the first step in the pathogenesis of diabetes to promote arteriosclerosis. BH 4 enhanced vascular response to acetylcholine-induced vasodilation, whereas endotheliumindependent vasodilation was not affected in diabetes patients [15,25]. In contrast, Cosentino et al. found that BH 4 improved glucose disposal in individuals with type 2 diabetes but without any discernible changes in vasodilation or macrovascular blood flow [14]. BH 4 restored the endothelium-dependent vasodilation induced by an oral glucose challenge in the forearm of healthy subjects [32].

Discussion
Previous research has indicated that maintaining adequate BH 4 levels in the endothelium is critical in regulating the balance of NO and superoxide synthesis in CVDs. Numerous studies have examined the effect of BH 4 supplementation on endothelial dysfunction in a wide variety of CVDs, including coronary artery disease, hypercholesterolemia, hypertension, and diabetic vasculopathy. The substitution of BH 4 , an essential cofactor of NOS and a scavenger of oxygen-derived free radicals, is able to restore coronary vasomotion in response to Ach [13,22,30]. Supplementation with BH 4 Evidence-Based Complementary and Alternative Medicine 5 augments forearm vessel endothelium-dependent vasodilation by improving endothelial dysfunction [25,27,31]. The flow reserve utilization of the coronary microcirculation in hypercholesterolemic subjects is significantly reduced but is nearly restored after BH 4 infusion [28]. BH 4 augmented NOdependent vasodilatation during local heating by increasing the plateau in skin blood flow in hypercholesterolemic humans [29]. These studies involved a limited number of patients in whom BH 4 was administered acutely or on a short-term basis, and BH 4 was typically delivered via intracoronary/intraarterial infusion, which is not representative of a suitable route of administration for chronic disease management. The breadth of preclinical and acute clinical data implicating BH 4 as a key regulator in endothelial function suggests that oral BH 4 therapy may be able to prevent or treat CVDs. Clinical trials investigating oral BH 4 supplementation have shown varied efficacy in numerous disorders with an apparent lack of efficacy in diseases such as hypertension, hypercholesterolemia, and coronary artery disease. Porkert and coworkers showed that oral BH 4 at a daily dose of 400 mg or higher has a significant and sustained antihypertensive effect in subjects with poorly controlled hypertension but that lower dose (200 mg per day) BH 4 has no effect [12]. Twenty-two hypercholesterolemic patients were randomized to receive 4 weeks of either oral BH 4 (400 mg twice daily) or placebo, and age-matched healthy volunteers served as controls. They found that chronic BH 4 treatment led to an eightfold increase in plasma BH 4 levels and restored the impairment in endothelium-dependent relaxation due to Ach in hypercholesterolemic patients but did not affect control subjects. Importantly, they also demonstrated that BH 4 significantly reduced the plasma levels of 8-F2 isoprostane, a marker of oxidative stress, and that the effect of BH 4 treatment on NO bioavailability is independent of any change in LDL cholesterol [14]. In contrast, oral low-dose (400 mg/d) or high-dose (700 mg/d) BH 4 for 2 to 6 weeks in patients with established coronary artery disease significantly elevated plasma BH 4 levels. However, this elevation in plasma BH 4 was tempered by similar rises in plasma BH 2 and biopterin, so that the ratio of reduced biopterins to oxidized ones (BH 4 /[BH 2 +biopterin]) in plasma remained unchanged after treatment, with neither molecule having an effect on the vascular redox state or endothelial function [26]. In addition, a phase 2 clinical trial sponsored by the US pharmaceutical company BioMarin failed to observe an ameliorative effect of the oral administration of BH 4 in patients with poorly controlled hypertension. There are studies providing preliminary evidence that oral BH 4 could increase artery compliance and decrease arterial stiffness in healthy older men [33] or estrogen-deficient postmenopausal women [34]. 6R-BH 4 was administered starting at a dose of 2.5 mg/kg and increasing to 20 mg/kg over 8 weeks. This treatment produced an improvement in the 6-minute walking distance, with the most significant improvement at a dose of 5 mg/kg, in patients with pulmonary hypertension [16].
The vascular effects following the oral administration of BH 4 appear complex and dose dependent, which may be explained by either the rapid clearance of BH 4 after oral administration and/or an enhanced oxidation to BH 2 , which lacks eNOS cofactor activity. Thus, systemic oxidative stress may play a critical role in determining the degree of oxidation of BH 4 to BH 2 and hence the ratio of BH 4 : BH 2 and efficacy of the treatment [26]. Recent data from cultured endothelial cells [35,36] suggest that the intracellular levels of BH 2 and, more specifically, the ratio between reduced and oxidized biopterins are important in regulating eNOS coupling. Considering that BH 4 is easily oxidized to BH 2 , strategies should increase the supplementation of BH 4 with antioxidants. On one hand, they can reduce the oxidation of BH 4 to BH 2 ; on the other hand, they may synergistically decrease oxidative stress and increase nitric oxide. This hypothesis is supported by observations in which the antioxidant vitamin C stimulates eNOS enzymatic activity by increasing the intracellular concentration of BH 4 [33,37]. Vitamin C likely exerted its beneficial effects in that study through a variety of molecular mechanisms. In its capacity as an antioxidant, it enhances NO bioavailability by quenching O 2− , thus limiting the inactivation of NO that occurs when O 2− and NO combine to produce OONO − [38]. Vitamin C also stabilizes existing BH 4 [39] and increases endothelial BH 4 synthesis [40]. We have demonstrated that plasma biopterin oxidation status is closely linked to the amount of ascorbate in plasma and hence in the diet in vivo [41]. However, studies in larger cohorts of patients would be required to determine whether this dual (BH 4 plus antioxidant) intervention would be efficacious on a chronic basis.
In addition, acute or short-term supplementation with BH 4 via intracoronary/intra-arterial infusion has no beneficial effect on endothelial dysfunction in CVDs [23,24]. These findings suggest that, in humans, BH 4 does not passively diffuse from the circulating blood into the vascular endothelium. Previous work has indicated that biopterin transport is cell type dependent and that both direct uptake (as BH 4 ) and conversion to BH 2 followed by recycling via dihydrofolate reductase (DHFR) are possible mechanisms [42]. In fact, in patients with coronary artery atherosclerosis, high plasma levels of BH 4 are associated with low BH 4 levels in the endothelium [43]. To ensure that BH 4 is imported into the endothelium, it must undergo oxidation to BH 2 ; imported BH 2 is then regenerated back to BH 4 by DHFR. A recent study found that human DHFR has very low affinity for 7,8-BH 2 and that folic acid inhibits 7,8-BH 2 recycling [44]. Thus, we consider that the low activity of endothelial DHFR is an important factor limiting the benefits of BH 4 therapies.

Conclusion
In summary, targeting BH 4 remains a rational therapeutic strategy in CADs. However, we found that oral BH 4 treatment in patients with CADs significantly elevates the BH 4 levels in blood but that this effect is significantly limited by the systemic oxidation of exogenous BH 4 to BH 2 , which lacks eNOS cofactor activity. More studies should be directed toward interventions that can favorably alter the endogenous BH 4 /BH 2 ratio in the human vascular endothelium via a 6 Evidence-Based Complementary and Alternative Medicine selective increase in the absolute BH 4 levels, the prevention of BH 4 oxidation, or an increase in BH 4 recycling. In particular, the effect of antioxidant coadministration to prevent the systemic and vascular oxidation of exogenous BH 4 warrants further attention. Beneficial effects of acute BH 4 supplementation on endothelial function have been reported in many human studies. However, the long-term based clinical trials are deficient. Oral administration can be considered to be representative of a suitable administration route for chronic disease management. Therefore, longterm experiments investigating oral BH 4 supplementation are needed.