Background-Tetrahydrobiopterin (BH 4 ) is a key regulator of endothelial nitric oxide synthase (eNOS) activity and coupling. However, the extent to which vascular and/or systemic BH 4 levels are altered in human atherosclerosis and the importance of BH 4 bioavailability in determining endothelial function and oxidative stress remain unclear. We sought to define the relationships between plasma and vascular biopterin levels in patients with coronary artery disease and to determine how BH 4 levels affect endothelial function, eNOS coupling, and vascular superoxide production. Methods and Results-Samples of saphenous veins and internal mammary arteries were collected from 219 patients with coronary artery disease undergoing coronary artery bypass grafting. We determined plasma and vascular levels of biopterins, vasomotor responses to acetylcholine, and vascular superoxide production in the presence and absence of the eNOS inhibitor N G -nitro-L-arginine methyl ester. High vascular BH 4 was associated with greater vasorelaxations to acetylcholine (PϽ0.05), whereas high plasma BH 4 was associated with lower vasorelaxations in response to acetylcholine (PϽ0.05). Furthermore, an inverse association was observed between plasma and vascular biopterins (PϽ0.05 for both saphenous veins and internal mammary arteries). High vascular (but not plasma) BH 4 was associated with reduced total and N G -nitro-L-arginine methyl ester-inhibitable superoxide, suggesting improved eNOS coupling. Finally, plasma but not vascular biopterin levels were correlated with plasma C-reactive protein levels (PϽ0.001). Conclusions-An inverse association exists between plasma and vascular biopterins in patients with coronary artery disease. Vascular but not plasma BH 4 is an important determinant of eNOS coupling, endothelium-dependent vasodilation, and superoxide production in human vessels, whereas plasma biopterins are a marker of systemic inflammation.

Background-In experimental animal models, long-term in vivo treatment with nitroglycerin (NTG) induces both endothelial dysfunction and tolerance to nitrates. However, it is still controversial whether nitrate tolerance in humans is associated with both endothelial dysfunction and impaired vascular response to nitrovasodilator-derived NO. Methods and Results-Patients undergoing elective bypass surgery were randomized to receive 48 hours of continuous NTG infusion (NTG group) or no nitrate therapy (control group). Segments of surgically removed arteria mammaria, vena saphena, and arteria radialis not required for the bypass procedure were used to examine (1) the vascular responsiveness to NTG and the endothelium-dependent vasodilator acetylcholine; (2) the expression of the NO target, the soluble guanylyl cyclase; (3) the expression of the soluble guanylyl cyclase/cGMP effector target, the cGMPdependent protein kinase (cGK); and (4) the cGK activity as assessed by the phosphorylation state of its vascular substrate, the vasodilator-stimulated phosphoprotein at serine 239 (P-VASP). NTG treatment caused a marked degree of nitrate tolerance in all 3 vessel types studied and a significant cross-tolerance to the endothelium-dependent vasodilator acetylcholine in A. mammaria and A. radialis. Although soluble guanylyl cyclase, cGK-I, and VASP expression levels were not modified by NTG treatment, a marked decrease of P-VASP, a surrogate parameter for in-vivo cGK-I activity, was observed. Conclusions-We conclude that long-term NTG treatment induces endothelial dysfunction and impaired vascular NO/cGMP signaling in humans, which can be monitored by measuring P-VASP levels.

Abstract-Vascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species (ROS) derived from vascular NADPH oxidases in both vascular smooth muscle cells (VSMCs) and endothelial cells. Recent evidence suggests an important role for VSMC NADPH oxidases in vascular ROS production. However, it is unclear whether increased NADPH oxidase activity in endothelial cells alone is sufficient to alter overall vascular ROS production and hemodynamics. We sought to address these questions using transgenic mice with endothelialtargeted overexpression of the catalytic subunit of NADPH oxidase, Nox2. Aortas of Nox2 transgenic (Nox2-Tg) mice had increased total Nox2 mRNA and protein levels compared with wild-type littermates. Both p22phox mRNA and protein levels were also significantly elevated in Nox2-Tg aortas. Aortic superoxide production was significantly increased in Nox2-Tg mice compared with wild-type, but this difference was abolished by endothelial removal. Superoxide dismutase inhibition increased superoxide release and levels of Mn superoxide dismutase protein were significantly elevated in aortas from Nox2-Tg mice compared with wild type. Increased ROS production from endothelial Nox2 overexpression led to increased endothelial nitric oxide synthase protein and extracellular signalregulated kinase 1/2 phosphorylation in transgenic aortas. Basal blood pressure was similar, however the pressor responses to both acute and chronic angiotensin II administration were significantly increased in Nox2-Tg mice compared with wild type. These results demonstrate that endothelial-targeted Nox2 overexpression is sufficient to increase vascular NADPH oxidase activity, activate downstream signaling pathways, and potentiate the hemodynamic response to angiotensin II, despite compensatory increases in vascular antioxidant enzymes. Endothelial cell Nox2-containing NADPH oxidase plays an important functional role in vascular redox signaling.

Loss of endothelial cells (ECs) in conditions of vascular injury is an important contributor to adverse vascular remodeling that leads to neointimal hyperplasia and accelerated atherosclerosis, for example, in venous bypass grafts, allograft vasculopathy, and after angioplasty or stenting. 1Re-endothelialization has been shown to be a key event in vascular repair. Endothelial nitric oxide synthase (eNOS) is essential for the normal function of ECs in the vascular wall and for the function of circulating endothelial progenitor cells (EPC).2-4 However, the molecular mechanisms relating eNOS regulation to endothelial loss, survival, and regeneration after vascular disease are uncertain. The eNOS cofactor, tetrahydrobiopterin (BH4), is a critical determinant of eNOS enzymatic activity and function. We and others have shown that in vascular disease states BH4 deficiency leads to eNOS uncoupling, whereby reduction of molecular oxygen by eNOS is no longer coupled to L-arginine oxidation and NO synthesis, and instead eNOS generates superoxide rather than NO. 5We hypothesized that BH4-dependent eNOS regulation may have an important role in endothelial loss and regeneration and in the development of neointimal hyperplasia after vascular injury. To investigate the specific role of EC BH4 availability, we compared apolipoprotein E (apoE)-knockout (KO) mice with or without transgenic endothelium-targeted overexpression of GTP cyclohydrolase I (GCH), the ratelimiting enzyme in endothelial BH4 synthesis, 5,6 in a model of venous bypass grafting characterized by acute endothelial loss, neointimal hyperplasia, and endothelial regeneration 2 and investigated how GCH and BH4 regulate primary EC survival and growth.Background-Endothelial cell (EC) survival and regeneration are important determinants of the response to vascular injury that leads to neointimal hyperplasia and accelerated atherosclerosis. Nitric oxide (NO) is a key regulator of EC and endothelial progenitor cell function, but the pathophysiological mechanisms that regulate endothelial NO synthase in endothelial regeneration remain unclear. Methods and Results-Endothelium-targeted overexpression of GTP cyclohydrolase (GCH) I increased levels of the endothelial NO synthase cofactor, tetrahydrobiopterin, in an EC-specific manner and reduced neointimal hyperplasia in experimental vein grafts in GCH/apolipoprotein E-knockout mice. These effects were mediated through enhanced donor-derived survival and recipient-derived repopulation of GCH transgenic ECs, revealed by tracking studies in Tie2-LacZ/GCH-Tg/apolipoprotein E-knockout recipient mice or donor grafts, respectively. Endothelial GCH overexpression increased endothelial NO synthase coupling and enhanced the proliferative capacity of ECs and circulating endothelial progenitor cell numbers after vascular injury. Conclusions-These observations indicate that endothelial tetrahydrobiopterin availability modulates neointimal hyperplasia after vascular injury via accelerated EC repopulation and growth. Targeting tetrahydrobi...