Search

Aims: Endothelin-1 (ET-1) has been shown to increase endothelial superoxide (O(2)(-)) production in experimental animal models. It is unclear whether ET-1 increases O(2)(-) production in humans. We sought to elucidate whether ET-1 increases O(2)(-) production in human vessels and to identify the mechanism behind this effect., Main Methods: Segments of internal mammary artery (IMA) and human saphenous vein (HSV) were harvested from 90 patients undergoing elective coronary artery bypass graft surgery. Paired vessel rings were incubated in the presence and absence of ET-1 (10(-10)M), the ET(A) receptor antagonist BQ123 alone, or in combination with the ET(B) receptor antagonist BQ788 (dual BQ) and known inhibitors of sources of O(2)(-) and further analysed for O(2)(-) production using lucigenin-enhanced chemiluminescence and DHE fluorescence., Key Findings: ET-1 increased O(2)(-) production in both IMA (2.6 ± 1.5 vs. 1.4 ± 0.8 relative light units/s/mg tissue (RLU); n=33; p < 0.0001) and HSV (1.4 ± 0.8 vs. 1.1 ± 0.6 RLU; n=24; p<0.05). The increase in O(2)(-)production induced by ET-1 in IMA was inhibited by co-incubation with dual BQ (p < 0.05; n=15) and BQ123 (p<0.05; n = 17). Of known O(2)(-) inhibitors, only incubation with Tiron and diphenyleneiodonium resulted in a significant reduction in ET-mediated O(2)(-) production., Significance: ET-1 increases O(2)(-) production especially in human arteries and less so in veins from patients with coronary artery disease via a receptor-dependent pathway involving a flavin dependent enzyme which is likely to be NADPH oxidase. Production of O(2)(-) may be an important factor underlying the negative effects of ET-1 on vascular function such as impairment of endothelium-dependent vasodilatation and pro-inflammatory effects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) plays a pivotal role in maintaining endothelial function in experimental vascular disease models and in humans. Augmentation of endogenous BH4 levels by oral BH4 treatment has been proposed as a potential therapeutic strategy in vascular disease states. We sought to determine the mechanisms relating exogenous BH4 to human vascular function and to determine oral BH4 pharmacokinetics in both plasma and vascular tissue in patients with coronary artery disease., Methods and Results: Forty-nine patients with coronary artery disease were randomized to receive low-dose (400 mg/d) or high-dose (700 mg/d) BH4 or placebo for 2 to 6 weeks before coronary artery bypass surgery. Vascular function was quantified by magnetic resonance imaging before and after treatment, along with plasma BH4 levels. Vascular superoxide, endothelial function, and BH4 levels were determined in segments of saphenous vein and internal mammary artery. Oral BH4 treatment significantly augmented BH4 levels in plasma and in saphenous vein (but not internal mammary artery) but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks endothelial nitric oxide synthase cofactor activity. There was no effect of BH4 treatment on vascular function or superoxide production. Supplementation of human vessels and blood with BH4 ex vivo revealed rapid oxidation of BH4 to BH2 with predominant BH2 uptake by vascular tissue., Conclusions: Oral BH4 treatment augments total biopterin levels in patients with established coronary artery disease but has no net effect on vascular redox state or endothelial function owing to systemic and vascular oxidation of BH4. Alternative strategies are required to target BH4-dependent endothelial function in established vascular disease states.

Background: The role of circulating homocysteine as an atherosclerosis risk factor has recently been questioned. However, 5-methyl-tetrahydrofolate (5-MTHF), the circulating metabolite of folic acid participating in homocysteine metabolism, has direct effects on vascular function. We sought to distinguish the effects of plasma versus vascular tissue 5-MTHF and homocysteine on vascular redox and endothelial nitric oxide bioavailability in human vessels., Methods and Results: We used the methyl tetrahydrofolate reductase (MTHFR) gene polymorphism 677C>T as a model of chronic exposure of the vascular wall to varying 5-MTHF levels in 218 patients undergoing coronary artery bypass graft surgery. Vascular superoxide, vascular 5-MTHF, and total homocysteine were determined in saphenous veins and internal mammary arteries obtained during surgery. Nitric oxide bioavailability was evaluated by organ bath studies on saphenous vein rings. MTHFR genotype was a determinant of vascular 5-MTHF (not vascular homocysteine). Both MTHFR genotype and vascular 5-MTHF were associated with vascular nitric oxide bioavailability and superoxide generated by uncoupled endothelial nitric oxide synthase. In contrast, vascular homocysteine was associated only with NADPH-stimulated superoxide., Conclusions: Genetic polymorphism 677 C>T on MTHFR affects vascular 5-MTHF (but not homocysteine) and can be used as a model to distinguish the chronic effects of vascular 5-MTHF from homocysteine on vascular wall. Vascular 5-MTHF, rather than plasma or vascular homocysteine, is a key regulator of endothelial nitric oxide synthase coupling and nitric oxide bioavailability in human vessels, suggesting that plasma homocysteine is an indirect marker of 5-MTHF rather than a primary regulator of endothelial function.

Background: Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), is considered to be a risk factor for atherosclerosis. However, the mechanisms relating ADMA with vascular function have been evaluated in vitro and in animal models, but its effect in human vasculature is unclear., Aims: We examined the impact of serum ADMA on endothelial nitric oxide (NO) bioavailability and vascular superoxide radical (O2-) production in patients with advanced atherosclerosis., Methods and Results: Paired samples of saphenous veins (SVs) and internal mammary arteries (IMAs) were collected from 201 patients undergoing coronary bypass surgery, and serum ADMA was measured pre-operatively. The vasomotor responses of SV segments to acetylcholine (ACh) and bradykinin (Bk) were evaluated ex vivo. Vascular O2- was measured in paired SV and IMA by lucigenin-enhanced chemiluminescence. The l-NAME-inhibitable as well as the NADPH-stimulated vascular O2- generation was also determined by chemiluminescence. High serum ADMA levels were associated with decreased vasorelaxation of SV to ACh (P < 0.05) and Bk (P < 0.05). Similarly, high serum ADMA was associated with higher total O2- production in both SVs and IMAs (P < 0.05) and greater L-NAME-inhibitable vascular O2- (P < 0.05). However, serum ADMA was not associated with NADPH-stimulated vascular O2-. In multivariable linear regression, serum ADMA was independently associated with vascular O2- in both SVs [beta (SE): 0.987 (0.412), P = 0.019] and IMAs [beta (SE): 1.905 (0.541), P = 0.001]. Asymmetrical dimethylarginine was also independently associated with maximum vasorelaxation in response to both ACh [beta (SE): 14.252 (3.976), P = 0.001] and Bk [beta (SE): 9.564 (3.762), P = 0.013]., Conclusion: This is the first study that demonstrates an association between ADMA and important measures of vascular function, such as vascular O2- production and NO bioavailability directly in human vessels. Although serum ADMA has no effect on NADPH-stimulated superoxide in intact vessels, it is associated with greater eNOS uncoupling in the human vascular endothelium of patients with coronary artery disease.

Objectives: This study sought to determine the effects of endogenous tetrahydrobiopterin (BH4) bioavailability on endothelial nitric oxide synthase (eNOS) coupling, nitric oxide (NO) bioavailability, and vascular superoxide production in patients with coronary artery disease (CAD)., Background: GTP-cyclohydrolase I, encoded by the GCH1 gene, is the rate-limiting enzyme in the biosynthesis of BH4, an eNOS cofactor important for maintaining enzymatic coupling. We examined the associations between haplotypes of the GCH1 gene, GCH1 expression and biopterin levels, and the effects on endothelial function and vascular superoxide production., Methods: Blood samples and segments of internal mammary arteries and saphenous veins were obtained from patients with CAD undergoing coronary artery bypass grafting (n = 347). The GCH1 haplotypes were defined by 3 polymorphisms: rs8007267G