Cofactor For Endothelial Nitric Oxide Synthase Research Articles

Tetrahydrobiopterin Restores Microvascular Dysfunction in Young Adult Binge Drinkers.

Repeated binge drinking is associated with reduced microvascular function. However, microvascular responses to pathophysiological stimulus such as high pressure as well as potential mechanisms that underlie binge-induced microvascular dysfunction are unknown. Therefore, using an ex vivo experimental model, we examined microvascular responses following a brief period of high intraluminal pressure in isolated arterioles from young adults who have a history of repeated binge drinking. In addition, we examined whether the application of the endothelial nitric oxide synthase cofactor, tetrahydrobiopterin, would restore microvascular function in response to flow and high intraluminal pressure in young adult binge drinkers. Isolated subcutaneous adipose arterioles were obtained from young adult binge drinkers (BD; n=14), moderate drinkers (MODs; n=10), and alcohol abstainers (ABs; n=12; mean age: 23.7±0.5years; and body mass index: 23.4±0.4kg/m2 ). Arteriolar flow-induced dilation (FID, pressure gradient: ∆10 to 100cm H2 O) was measured before and after acute high intraluminal pressure with and without tetrahydrobiopterin. Before high pressure, FID at Δ60 and Δ100 cm H2 O pressure gradient in BDs was 14% lower and 18% lower, respectively, than ABs (p<0.05), while MODs and ABs had similar FID across all pressure gradients (p≥0.2). After high pressure, FID in BDs was further reduced by 10% (p<0.0005) and this impairment was ameliorated by the treatment of tetrahydrobiopterin (4 to 26% higher, p<0.005). In contrast, FID after high pressure did not change in MODs and ABs (p≥0.5). Microvascular dysfunction in young adult binge drinkers may be exacerbated with acute pathophysiological stimulus. These binge-induced dysfunctions may be reversed by tetrahydrobiopterin, which suggests a role of oxidative stress and/or uncoupled endothelial nitric oxide synthase in binge drinking.

The endothelial nitric oxide synthase cofactor tetrahydrobiopterin shields the remote myocardium from apoptosis after experimental myocardial infarction in vivo.

Following myocardial infarction (MI), apoptosis occurs early in the remote myocardium and contributes to the processes of myocardial remodelling. Increased nitrosative stress is a well-known and potent inductor of myocardial apopto¬sis. Excess activation of endothelial nitric oxide synthase (eNOS) increases its uncoupling potential and results in nitrosative stress via formation of peroxynitrite. However, the pathophysiological role of eNOS signalling in the remote myocardium after MI is as yet undefined. The impact of eNOS activation on pro- and anti-apoptotic signalling in the remote myocardium and the influence of pretreatment with the eNOS cofactor tetrahydrobiopterin (BH4) on eNOS activation, nitrosative stress level, and apoptosis induction and execution were studied in a rat MI model in vivo. Twenty-four hours after anterior MI, eNOS activity in animals treated with left anterior descending coronary artery ligation (LIG) significantly increased in the posterior left ventricular (LV) myocardium as did protein nitrosylation when com¬pared to sham treatment. This was paralleled by induction of apoptosis via the extrinsic and intrinsic pathways. Moreover, anti-apoptotic signalling via protein kinase B/Akt and glycogen synthase-kinase 3 beta was suppressed. Notably, pretreatment with the eNOS cofactor BH4 reduced eNOS activation, prevented excess protein nitrosylation, blunted apoptosis induction, facilitated anti-apoptotic signalling, and eventually prevented apoptosis execution. Here we showed that 24 h after experimental MI in rats in vivo, apoptosis was induced in the posterior non-in¬farcted LV wall. Evidence is presented that pretreatment with the eNOS cofactor BH4 resulted in less nitrosative stress and weakened apoptotic processes, although the stabilisers contained did participate in this phenomenon. Because apoptosis is a crucial component of myocardial remodelling, influencing eNOS signalling might be an interesting pharmacological target for the development of novel anti-remodelling therapies.

Tetrahydrobiopterin Determines Vascular Remodeling Through Enhanced Endothelial Cell Survival and Regeneration

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. 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. These observations indicate that endothelial tetrahydrobiopterin availability modulates neointimal hyperplasia after vascular injury via accelerated EC repopulation and growth. Targeting tetrahydrobiopterin-dependent endothelial NO synthase regulation in the endothelium is a rational therapeutic target to enhance endothelial regeneration and reduce neointimal hyperplasia in vascular injury states.

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Systemic and Vascular Oxidation Limits the Efficacy of Oral Tetrahydrobiopterin Treatment in Patients With Coronary Artery Disease

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. 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. 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.

Induction of Vascular GTP-Cyclohydrolase I and Endogenous Tetrahydrobiopterin Synthesis Protect Against Inflammation-Induced Endothelial Dysfunction in Human Atherosclerosis

The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is essential for maintenance of enzymatic function. We hypothesized that induction of BH4 synthesis might be an endothelial defense mechanism against inflammation in vascular disease states. In Study 1, 20 healthy individuals were randomized to receive Salmonella typhi vaccine (a model of acute inflammation) or placebo in a double-blind study. Vaccination increased circulating BH4 and interleukin 6 and induced endothelial dysfunction (as evaluated by brachial artery flow-mediated dilation) after 8 hours. In Study 2, a functional haplotype (X haplotype) in the GCH1 gene, encoding GTP-cyclohydrolase I, the rate-limiting enzyme in biopterin biosynthesis, was associated with endothelial dysfunction in the presence of high-sensitivity C-reactive protein in 440 coronary artery disease patients. In Study 3, 10 patients with coronary artery disease homozygotes for the GCH1 X haplotype (XX) and 40 without the haplotype (OO) underwent S Typhi vaccination. XX patients were unable to increase plasma BH4 and had a greater reduction of flow-mediated dilation than OO patients. In Study 4, vessel segments from 19 patients undergoing coronary bypass surgery were incubated with or without cytokines (interleukin-6/tumor necrosis factor-α/lipopolysaccharide) for 24 hours. Cytokine stimulation upregulated GCH1 expression, increased vascular BH4, and improved vasorelaxation in response to acetylcholine, which was inhibited by the GTP-cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine. The ability to increase vascular GCH1 expression and BH4 synthesis in response to inflammation preserves endothelial function in inflammatory states. These novel findings identify BH4 as a vascular defense mechanism against inflammation-induced endothelial dysfunction.

Elevated oxidative stress and endothelial dysfunction in right coronary artery of right ventricular hypertrophy

Remodeling of right coronary artery (RCA) occurs during right ventricular hypertrophy (RVH) induced by banding of the pulmonary artery (PA). The effect of RVH on RCA endothelial function and reactive oxygen species (ROS) in vessel wall remains unclear. A swine RVH model (n = 12 pigs) induced by PA banding was used to study RCA endothelial function and ROS level. To obtain longitudinal coronary hemodynamic and geometric data, digital subtraction angiography was used during the progression of RVH. Blood flow in the RCA increased by 82% and lumen diameter of RCA increased by 22% over a 4-wk period of RVH. The increase in blood flow and the commensurate increase in diameter resulted in a constant wall shear stress in RCA throughout the RVH period. ROS was elevated by ∼100% in RCA after 4 wk of PA banding. The expressions of p47(phox), NADPH oxidase (NOX1, NOX2, and NOX4) were upregulated in the range of 20-300% in RCA of RVH. The endothelial function was compromised in RCA of RVH as attributed to insufficient endothelial nitric oxide synthase cofactor tetrahydrobiopterin. In vivo angiographic analysis suggests an increased basal tone in the RCA during RVH. In conclusion, stretch due to outward remodeling of RCA during RVH (at constant wall shear stress), similar to vessel stretch in hypertension, appears to induce ROS elevation, endothelial dysfunction, and an increase in basal tone.

The Role of Tetrahydrobiopterin and Dihydrobiopterin in Ischemia/Reperfusion Injury When Given at Reperfusion

Reduced nitric oxide (NO) bioavailability and increased oxidative stress are major factors mediating ischemia/reperfusion (I/R) injury. Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial NO synthase (eNOS) to produce NO, whereas dihydrobiopterin (BH2) can shift the eNOS product profile from NO to superoxide, which is further converted to hydrogen peroxide (H2O2) and cause I/R injury. The effects of BH4 and BH2 on oxidative stress and postreperfused cardiac functions were examined in ex vivo myocardial and in vivo femoral I (20 min)/R (45 min) models. In femoral I/R, BH4 increased NO and decreased H2O2 releases relative to saline control, and these effects correlated with improved postreperfused cardiac function. By contrast, BH2 decreased NO release relative to the saline control, but increased H2O2 release similar to the saline control, and these effects correlated with compromised postreperfused cardiac function. In conclusion, these results suggest that promoting eNOS coupling to produce NO and decrease H2O2 may be a key mechanism to restore postreperfused organ function during early reperfusion.

Antioxidants and endothelial nitric oxide synthesis

Oxidative stress in the vasculature has been suggested to contribute to the development of endothelial dysfunction via different mechanisms including LDL oxidation, nitric oxide (NO) scavenging, or oxidation of tetrahydrobiopterin, a critical cofactor of endothelial NO synthase (eNOS). Antioxidants may interfere with these processes and protect NO formed in the endothelium. In particular, ascorbic acid at high concentrations seems to be a prerequisite for sufficient NO bioavailability. Moreover, there is accumulating evidence that ascorbic acid improves tetrahydrobiopterin availability in the vasculature most probably via recycling oxidized tetrahydrobiopterin back to the fully reduced pterin. In addition, ascorbic acid may reduce the α-tocopheroxyl radical and may be required for beneficial vascular effects of α-tocopherol. Recent data have shown that apart from indirect protection of NO from inactivation, α-tocopherol exerts a direct stimulatory effect on eNOS activation via serine 1177 phosphorylation. This effect was amplified by ascorbic acid suggesting that both compounds may act synergistically in optimizing endothelial NO synthesis. The data obtained in cell culture and animal studies are promising, but human long-term studies are needed to determine whether the described mechanisms are active in vivo and may provide a rationale for optimizing dietary or supplementary intake of antioxidant vitamins in certain subsets of patients.

The clinical significance of endothelial dysfunction

Endothelial dysfunction is thought to play a pivotal role in the development, progression, and clinical complications of atherosclerosis. Several recent studies have addressed the clinical implications of endothelial dysfunction for cardiovascular events, atherosclerosis, restenosis, and heart failure. Novel findings with respect to endothelial progenitor cells and their alteration by cardiovascular risk factors are characterized and potential therapeutic interventions to improve endothelial and endothelial progenitor cell function are discussed. Over the past 5 years evidence has accumulated from clinical studies for a close association of the degree of endothelial dysfunction and clinical cardiovascular events in patients with cardiovascular risk factors, coronary disease, acute coronary syndrome, or heart failure. Understanding of the mechanisms leading to endothelial dysfunction has improved, including the notion that dysfunctional endothelial nitric oxide synthase, in part due to deficiency of the endothelial nitric oxide synthase cofactor tetrahydrobiopterin, likely plays an important role. Major progress has been made in understanding the role of endothelial progenitor cells, which likely contribute to both ischemia-induced neovascularization and endothelial regeneration after injury. Endothelial progenitor cell function is altered in patients with cardiovascular risk factors. Recent research on endothelial and endothelial progenitor cell dysfunction supports their clinical significance and has led to important insights in the pathophysiology of cardiovascular disease and at the same time provides an important opportunity to develop novel therapeutic approaches. Endothelial function represents a valuable surrogate endpoint to assess the impact of therapeutic interventions.

Augmented BH4 by gene transfer restores nitric oxide synthase function in hyperglycemic human endothelial cells

Endothelial dysfunction in diabetes is characterized by decreased nitric oxide (NO) bioactivity and increased superoxide (SO) production. Reduced levels of tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase (eNOS), appear to be associated with eNOS enzymatic uncoupling. We sought to investigate whether augmented BH4 biosynthesis in hyperglycemic human aortic endothelial cells (HAEC) by adenovirus-mediated gene transfer of GTP cyclohydrolase I (GTPCH, the rate-limiting enzyme for the de novo BH4 synthesis), would be sufficient to rescue eNOS activity and dimerization. HAEC were cultured in media with low glucose (5 mM) or high glucose (30 mM). After 5 days, the cells with/without GTPCH gene transfer (AdeGFP as a control) were prepared for assays of (1) NO with electron paramagnetic resonance (EPR); (2) SO with cytochrome c reduction and dihydroethidine (DHE) fluorescence; (3) BH4 with high-performance liquid chromatography (HPLC); (4) eNOS expression and dimerization with immunoblotting. We found that high glucose decreased HAEC NO and increased SO production, in association with reductions in both total biopterin and BH4 levels. High glucose increased total eNOS protein levels in HAEC 1.5-fold, but this was present principally in the monomeric form. GTPCH gene transfer increased cellular biopterin levels and NO production but decreased SO production. Furthermore, augmenting BH4 increased the eNOS dimer:monomer ratio 2.6-fold. 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.

206. Adenovirus-Mediated GTP Cyclohydrolase I Gene Delivery Augments Tetrahydrobiopterin and Restores Nitric Oxide Synthase Function in Hyperglycaemic Human Aortic Endothelial Cells

Top of pageAbstract Introduction: Endothelial dysfunction is a marker of diabetic vascular disease, characterised by decreased nitric oxide (NO) bioactivity and increased superoxide (SO) production in endothelium. Reduced levels of tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase (eNOS), leads to enzymatic uncoupling of eNOS and instability of eNOS homodimers. We set out to augment BH4 levels in hyperglycaemic human aortic endothelial cells (HAEC) by gene transfer of GTP cyclohydrolase I (GTPCH, the rate-limiting enzyme for the de novo BH4 synthesis), in order to determine whether genetic modulation of BH4 biosynthesis could rescue eNOS activity and dimerisation. Methods: HAEC were cultured in media with low glucose (5.5 mM) or high glucose (30 mM). After 5 days, the cells with/without GTPCH gene transfer (cells alone and AdeGFP as controls) were prepared for assays of (1) NO with EPR; (2) SO with cytochrome C reduction and DHE; (3) BH4 with HPLC; (4) eNOS expression and dimerisation with immunoblotting (densitometric quantification of blots). All experiments were repeated twice (n = 6), and p value < 0.05 is considered to be significant. Results: HAEC in high glucose cultures compared with the low glucose had 30% decrease in NO and two-fold increase in SO, with reduced levels of total biopterin (7.2 vs. 10.8) and BH4 (1.0 vs. 2.9). eNOS expression in the high glucose was increased 1.5-fold, but mainly existed in the monomeric form (dimer:monomer ratio 0.3 vs 0.5). GTPCH gene transfer increased biopterins 30-40 fold as compared with controls. GTPCH gene transfer also increased NO production 1.6-fold, led to 30% SO reduction. Furthermore, augmented BH4 promoted eNOS dimerisation. The dimer:monomer ratio in GTPCH was 2.6-fold higher than in the controls. Conclusion: This study demonstrates a critical role for BH4 in regulating eNOS function in hyperglycaemic human endothelial cells. GTPCH gene transfer is a powerful approach to investigate eNOS regulation and potential therapeutic significance in diabetes.

Endothelial cell dysfunction in type I and II diabetes: The cellular basis for dysfunction

AbstractMicro‐ and macrovascular complications are the leading causes of mortality in Types I and II diabetes. Hyperglycemia results in increased advanced glycosylation end (AGE) products, oxidative stress, increased sorbitol levels, and increased activation of protein kinase C. These effects of hyperglycemia eventually lead to impaired endothelium‐dependent relaxation to vasoactive substances such as acetylcholine and bradykinin. Increased oxidative stress, which will reduce levels of nitric oxide (NO), and/or decreased bioavailability of tetrahydrobiopterin (BH4), a cofactor for endothelial NO synthase (eNOS), may lead to a phenomenon called “uncoupling” of eNOS and this leads to endothelial dysfunction. Uncoupled NOS produces superoxide anions which lead to a further reduction in NO bioavailability. Thus, restoring BH4 levels and antioxidant activity could prove to be novel approaches for the treatment of endothelial dysfunction in Type I and II diabetes. Drug Dev. Res. 58:28–41, 2003. © 2003 Wiley‐Liss, Inc.