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Objective-Increased production of reactive oxygen species and loss of endothelial nitric oxide (NO) bioactivity are key features of vascular disease states such as atherosclerosis. Tetrahydrobiopterin (BH4) is a required cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS); pharmacologic studies suggest that reduced BH4 availability may be an important mediator of endothelial dysfunction in atherosclerosis. We aimed to investigate the importance of endothelial BH4 availability in atherosclerosis using a transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GTPCH). Methods and Results-Transgenic mice were crossed into an ApoE knockout (ApoE-KO) background and fed a high-fat diet for 16 weeks. Compared with ApoE-KO controls, transgenic mice (ApoE-KO/GCH-Tg) had higher aortic BH4 levels, reduced endothelial superoxide production and eNOS uncoupling, increased cGMP levels, and preserved NO-mediated endothelium dependent vasorelaxations. Furthermore, aortic root atherosclerotic plaque was significantly reduced in ApoE-KO/GCH-Tg mice compared with ApoE-KO controls. Conclusions-These findings indicate that BH4 availability is a critical determinant of eNOS regulation in atherosclerosisand is a rational therapeutic target to restore NO-mediated endothelial function and reduce disease progression. Key Words: nitric oxide synthase Ⅲ endothelium Ⅲ atherosclerosis Ⅲ hypercholesterolemia N itric oxide (NO) produced in the endothelium by endothelial nitric oxide synthase (eNOS) is a key mediator of vascular homeostasis. NO bioavailability is reduced early in vascular disease states such as hypercholesterolemia and atherosclerosis because of reduced NO synthesis and increased NO consumption by reactive oxygen species. 1 A critical determinant of eNOS activity is the availability of the NOS cofactor tetrahydrobiopterin (BH4). When BH4 levels are inadequate, the enzymatic reduction of molecular oxygen by eNOS is no longer coupled to L-arginine oxidation, resulting in generation of superoxide rather than NO, thus contributing to vascular oxidative stress and endothelial dysfunction. BH4 bioavailability in the vasculature appears to be regulated at the level of biosynthesis by the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH) and by oxidative degradation of BH4 to dihydrobiopterin (BH2) that is inactive for eNOS cofactor function.Several pharmacologic studies suggest a possible role for BH4 availability in regulating NO-mediated endothelial function. Acute administration of BH4 improves some features of endothelial dysfunction in smokers, 2 and in patients with type See page 397II diabetes, 3 hypercholesterolemia, 4,5 or coronary atherosclerosis. 6 In hypercholesterolemic ApoE-knockout (ApoE-KO) mice, endothelium-dependent vascular relaxations are impaired, NO synthesis is reduced, and vascular superoxide production is increased. 7,8 However, endothelial dysfunction in ApoE-KO mice can be reduced by incubation of vessels in the BH4 p...

Background-Pulmonary hypertension is a fatal disease characterized by vasoconstriction and vascular remodeling. Loss of endothelial nitric oxide bioavailability is implicated in pulmonary hypertension pathogenesis. Recent evidence suggests that the cofactor tetrahydrobiopterin (BH4) is an important regulator of nitric oxide synthase enzymatic function. Methods and Results-In the hph-1 mouse with deficient BH4 biosynthesis, BH4 deficiency caused pulmonary hypertension, even in normoxic conditions, and greatly increased susceptibility to hypoxia-induced pulmonary hypertension. In contrast, augmented BH4 synthesis in the endothelium, by targeted transgenic overexpression of GTP-cyclohydrolase I (GCH), prevented hypoxia-induced pulmonary hypertension. Furthermore, specific augmentation of endothelial BH4 in hph-1 mice by crossing with GCH transgenic mice rescued pulmonary hypertension induced by systemic BH4 deficiency. Lung BH4 availability controlled pulmonary vascular tone, right ventricular hypertrophy, and vascular structural remodeling in a dose-dependent manner in both normoxia and hypoxia. Furthermore, BH4 availability had striking effects on the immediate vasoconstriction response to acute hypoxia. These effects of BH4 were mediated through the regulation of nitric oxide compared with superoxide synthesis by endothelial nitric oxide synthase. Conclusions-Endothelial BH4 availability is essential for maintaining pulmonary vascular homeostasis, is a critical mediator in the pathogenesis of pulmonary hypertension, and is a novel therapeutic target.

This study demonstrates a critical role for BH4 in regulating eNOS function, suggesting that GTPCH is a rational target to augment endothelial BH4 and recover eNOS activity in hyperglycemic endothelial dysfunction states.

Aims/hypothesis: Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice. Methods: Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting. Results: In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged. Conclusions/interpretation: Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.

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MPR was associated with symptom-onset in initially asymptomatic patients with AS, but with moderate accuracy and was not superior to symptom-limited exercise testing. ClinicalTrials.gov (NCT01658345).

Increased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate–cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states