Published online before print April 27, 2009, doi: 10.1161/CIRCULATIONAHA.108.808675
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Circulation. 2009;119:2507-2515.)
© 2009 American Heart Association, Inc.
Vascular Medicine |
MTHFR 677 C>T Polymorphism Reveals Functional Importance for 5-Methyltetrahydrofolate, Not Homocysteine, in Regulation of Vascular Redox State and Endothelial Function in Human Atherosclerosis
From the Departments of Cardiovascular Medicine (C.A., C. Shirodaria, P.L., T.V.-A., C.C., R.P., C.R., K.M.C.) and Physiology, Anatomy, and Genetics (H.R.), University of Oxford, Oxford, UK; Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway (O.A.B., H.R.); and Department of Cardiology, Athens University Medical School, Athens, Greece (C.A., D.T., C. Stefanadis).
Correspondence to Professor Keith M. Channon, MD, FRCP, Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK. E-mail keith.channon{at}cardiov.ox.ac.uk
Received July 31, 2008; accepted March 9, 2009.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
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.
Key Words: antioxidants • atherosclerosis • homocysteine • nitric oxide synthase • nitric oxide • superoxide
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Plasma homocysteine is associated with cardiovascular risk1,2 and surrogate markers such as oxidative stress3 and endothelial dysfunction.4 However, the potential causative role of homocysteine in vascular disease pathogenesis remains unclear.5,6 Although homocysteine may have direct effects on important biological processes such as thrombosis and production of reactive oxygen species,1,2,7 interpretation of these effects is confounded by corresponding changes in folate status. Low plasma folate levels are strongly associated with elevated plasma homocysteine,8 and intervention studies to reduce homocysteine typically use folate supplementation.9,10 Recent evidence suggests that plasma folate may play important direct roles in the pathogenesis of atherosclerosis that are independent of homocysteine.11 Indeed, 5-methyl-tetrahydrofolate (5-MTHF), the circulating form of folate, has a number of direct antiatherogenic effects by scavenging peroxynitrite radicals,12 augmenting nitric oxide (NO) bioavailability,13–15 and decreasing vascular superoxide generation in the vascular wall.12 The potential beneficial effects of homocysteine lowering have been tested in recent clinical trials in patients with recent myocardial infarction,9 stable coronary artery disease,10 or stroke.16 Homocysteine lowering was achieved by combined folate and B vitamin supplementation but with no effect on major clinical outcomes,9,10 perhaps except stroke.10,16 Thus, the primary value of homocysteine lowering, as distinct from folate supplementation, remains uncertain as a therapeutic strategy in cardiovascular risk reduction.
Clinical Perspective on p 2515
A widely known functional genetic polymorphism in the methyltetrahydrofolate reductase gene (MTHFR 677 C>T) has been used as a marker of altered folate and homocysteine status to test causality of the folate-homocysteine pathway in relation to other known confounding factors or the effects of reverse causality.17 The MTHFR 677 C>T polymorphism is independently associated with vascular disease surrogates such as endothelial function18 and intima-media thickness.19 However, MTHFR 677 C>T has effects on both folate and homocysteine that are difficult to separate, so it remains uncertain whether the primary importance of MTHFR 677 C>T in cardiovascular disease is as a marker of constitutive reduction in 5-MTHF or as a constitutive increase in homocysteine levels. This uncertainty results in part from incomplete understanding of the mechanistic relationships between levels of 5-MTHF and homocysteine in both plasma and the vascular wall and how each relates to endothelial function and reactive oxygen species production.
We used the MTHFR 677C>T polymorphism as a model of constitutive alteration of 5-MTHF biosynthesis to investigate functional differences between primary alterations in 5-MTHF and secondary alterations in homocysteine in vascular disease pathophysiology. We examined the effects of this polymorphism on 5-MTHF and total homocysteine (tHcy) levels in both plasma and vascular tissue, vascular NO-mediated endothelial function, and vascular superoxide production in patients with coronary artery disease. Our findings indicate that vascular 5-MTHF levels, rather than vascular or plasma tHcy levels, are the most important determinant of endothelial function and vascular oxidative stress. We suggest that plasma tHcy is a secondary marker of vascular folate status rather than a primary therapeutic target. These findings are important for interpreting the results of previous clinical trials and refining future therapeutic strategies.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Study Subjects
We studied 218 patients with coronary artery disease undergoing elective coronary artery bypass grafting surgery at the John Radcliffe Hospital, Oxford, UK. Exclusion criteria were any inflammatory, infective, liver, or renal disease; malignancy; acute coronary event during the last 2 months; or clinically overt heart failure. Patients receiving nonsteroidal antiinflammatory drugs, dietary supplements of folic acid, or antioxidant vitamins also were excluded. Individual characteristics of the patients are presented in Table 1. Importantly, there was no significant difference in the demographic characteristics and medication between the patients in the 3 MTHFR genotype groups or between the overall population and the subgroups of patients in whom samples of saphenous vein (SV) and internal mammary artery (IMA) were available (Tables 1 and II, respectively, of the online Data Supplement). The study was approved by the local Research Ethics Committee, and each patient gave written informed consent.
|
Plasma and Tissue Samples
Blood samples were obtained immediately before surgery after overnight fasting. Samples were centrifuged at 2500 rpm for 10 minutes, and serum or plasma was stored at –80°C until assayed. Samples of SV and IMA were obtained at the time of coronary artery bypass grafting surgery as previously described.12 Paired vessel segments were snap-frozen and stored at –80°C for measurement of tHcy and 5-MTHF content or were transferred to the laboratory for functional studies within 30 minutes in ice-cold Krebs-Henseleit buffer.12
DNA Extraction and Genotyping
Genomic DNA was extracted from 2 mL whole blood using standard methods (QIAamp DNA Blood Midi Kit, Qiagen, Germantown, Md). The MTHFR 677C>T polymorphism (database of single nucleotide polymorphisms, rs1801133) was genotyped using the Amplifluor SNP Genotyping System (Chemicon, Watford, UK) and with the primers and assay conditions previously described.20 The genotype frequencies were in accordance with Hardy-Weinberg equilibrium in both the overall population and the subsets of patients from whom SV and IMA samples were obtained. There was no significant difference in genotype distribution between the overall population and those subjects for whom SV and IMA samples were available (Table III of the online Data Supplement). Furthermore, there were no significant differences in the genotype frequencies between the overall population and the subsets of patients from whom SV and IMA samples were obtained.
Determination of Vascular Superoxide Production
Vascular superoxide production was measured in paired segments of IMA using lucigenin-enhanced chemiluminescence as previously described.12 Vessels were opened longitudinally to expose the endothelial surface and equilibrated for 20 minutes in oxygenated (95% O2/5% CO2) Krebs-HEPES buffer (pH 7.4) at 37°C. Lucigenin-enhanced chemiluminescence was measured with low-concentration lucigenin (5 µmol/L).12 As a measure of endothelial NO synthase (eNOS) coupling, we determined NOS-derived superoxide production, which was estimated as the difference in superoxide production after 20 minutes of incubation with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). The NADPH-stimulated superoxide production was estimated 10 minutes after 100 µmol/L NADPH was added. Because lucigenin-enhanced chemiluminescence favors redox cycling of lucigenin at high concentrations (250 µmol/L) and when NADH is used as a substrate,21 in the present study, we used low lucigenin concentration (5 µmol/L) and NADPH as a substrate.22
Vasomotor Studies
Endothelium-dependent and endothelium-independent dilatations were assessed in SVs obtained at the time of coronary artery bypass grafting surgery using isometric tension studies.12 Four rings from each vessel were precontracted with phenylephrine (3x10–6 mol/L); then, endothelium-dependent relaxations were quantified with acetylcholine (10–9 to 10–5 mol/L). Finally, relaxations to the endothelium-independent NO donor sodium nitroprusside (10–10 to 10–6 mol/L) were evaluated in the presence of L-NAME (100 µmol/L) as previously described.12
Plasma and Vascular 5-MTHF Measurements
Plasma and vascular 5-MTHF levels were determined by high-performance liquid chromatography as previously described.12 Plasma 5-MTHF is expressed in nanomoles per liter and vascular 5-MTHF as nanomoles per gram of tissue.
Plasma and Vascular tHcy and Sulfur Amino Acid Measurements
Plasma tHcy was measured by a fluorescence polarization immunoassay adapted to the IMx analyzer (Abbott Diagnostics, Abbott Park, IL). Liquid chromatography–mass spectometry/mass spectometry was used for analyzing vascular tHcy and vascular/plasma methionine, cystathionine, total cysteine, and total glutathione concentrations using a modification of a method previously described (for details, see the online Data Supplement).23
Determination of Plasma and Vascular Biopterin Levels
Tetrahydrobiopterin (BH4) levels in plasma or vessel tissue lysates were determined by high-performance liquid chromatography, followed by electrochemical detection as previously described.24 BH4 levels were expressed as picomole per gram of tissue for vessels and nanomole per liter for plasma.
Statistical Analysis
All variables were tested for normal distribution with the Kolmogorov-Smirnov test. Normally distributed variables are presented as mean±SEM; nonnormally distributed variables were log transformed for analysis and are presented as median (25th to 75th percentiles) and range. Comparisons of baseline and demographic characteristics between the 3 genotypes were performed with 1-way ANOVA, and the unpaired t test was used to compare variables between 2 groups (for recessive models). Two-way ANOVA for repeated measures was used to compare the dose-response vasorelaxation curves between groups.
Bivariate analysis was performed by calculation of Pearson’s correlation coefficient. Multivariable linear regression analysis was used to examine the effect of vascular 5-MTHF or tHcy levels on L-NAME–induced change in vascular superoxide, NADPH-stimulated superoxide, or maximum relaxations of SVs to acetylcholine. These parameters were used as dependent variables; for independent variables, we used those variables of the following list that showed an association with the dependent variable in bivariate analysis at the level of 10%: 5-MTHF (or 677 C>T genotype), tHcy, age, gender, diabetes mellitus, smoking, dyslipidemia, hypertension, and body mass index. A backward elimination procedure was applied in all models, with P>0.1 as a threshold to remove a variable from the model. All probability values were 2 tailed, and values of P<0.05 were considered statistically significant. All statistical analyses were performed with SPSS 12.0 (SPSS Inc, Chicago, Ill).
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
To establish the relative levels of 5-MTHF and homocysteine-related compounds in the human vascular wall, we first determined both plasma and vascular tissue levels of 5-MTHF, tHcy, total cysteine, total glutathione, cystathione, and methionine in samples from 218 patients with coronary artery disease (Table 2). As expected, plasma tHcy was weakly associated with cystathione (r=0.332, P=0.0001) and total cysteine (r=0.348, P=0.0001). However, the respective associations were much stronger in vascular tissue than in plasma in both IMAs (r=0.744, P=0.0001 for total cysteine; r=0.308, P=0.013 for cystathione; r=0.324, P=0.009 for methionine; and r=0.497, P=0.0001 for total glutathione) and SVs (r=0.363, P=0.001 for cystathione; r=0.734, P=0.0001 for total cysteine; r=0.664, P=0.0001 for total glutathione; and r=0.521, P=0.0001 for methionine). Furthermore, plasma tHcy was only weakly correlated with vascular tHcy (r=0.389, P=0.003 for SVs; r=0.220, P=0.145 for IMAs). Similarly, plasma 5-MTHF was only weakly associated with vascular 5-MTHF (r=0.207, P=0.05 for SVs; r=0.212, P=0.09 in IMAs). Taken together, these novel findings support the notion that folate and homocysteine metabolism in the vascular wall behaves as an independent compartment from plasma.
|
We next evaluated the effect of constitutive alteration of 5-MTHF synthesis, determined by MTHFR 677 C>T genotype, on both plasma 5-MTHF and plasma tHcy levels. As expected, levels of plasma 5-MTHF and plasma tHcy were significantly associated with MTHFR genotype (Figure 1). However, the difference in plasma 5-MTHF between MTHFR 677 CC and TT genotypes was proportionately much greater than the corresponding difference in tHcy. Furthermore, in vascular tissue, MTHFR genotype was significantly associated with 5-MTHF but not with tHcy levels (Figure 2), suggesting that 5-MTHF may be a less important regulator of tHcy in the vascular wall.
|
|
We next used the presence of the MTHFR 677 C>T polymorphism to test the effects of constitutive differences in vascular 5-MTHF levels, without respective variations of vascular tHcy, on endothelial function, as defined by the vasomotor responses of vascular segments. We observed that the presence of the MTHFR 677 T allele was associated with significantly reduced vasorelaxations in response to acetylcholine, indicative of impaired endothelial function (Figure 3). Vascular 5-MTHF levels were significantly associated with vasomotor responses to acetylcholine (Figure 3), whereas plasma 5-MTHF levels showed only a borderline association (P=0.07). In addition, neither the MTHFR 677C>T polymorphism (Figure 3) nor vascular (Figure 3) or plasma (data not shown) levels of 5-MTHF had any impact on the vasorelaxations in response to sodium nitroprusside. In contrast, neither vascular (Figure 3) nor plasma (data not shown) tHcy levels showed any association with vasorelaxations to acetylcholine or sodium nitroprusside. Thus, the effects of the MTHFR 677 C>T polymorphism on endothelial function appear to be mediated through vascular levels of 5-MTHF rather than either vascular or plasma tHcy.
|
To further examine the mechanisms by which 5-MTHF and tHcy affect vascular function, we evaluated their impact on vascular superoxide generation and on the major sources of superoxide in the vascular wall. We observed that the presence of the MTHFR 677 T allele was associated with significantly higher vascular superoxide radical production (Figure 4). To further explore the effect of MTHFR genotype on the individual sources of superoxide radical generation in the vascular wall, we specifically examined the contributions of eNOS uncoupling and NADPH-oxidase activity. We observed that the presence of the T allele was associated with a significantly greater L-NAME–inhibitable superoxide radical generation in IMA segments, suggesting an association between vascular 5-MTHF and eNOS coupling (Figure 4). In contrast, there was no detectable effect of the MTHFR 677 T allele on NADPH-stimulated superoxide radical generation in IMA segments (Figure 4). The presence of the T allele also was associated with significantly lower vascular BH4 in these vessels (Figure 4).
|
To determine whether the relationship between the MTHFR 677 C>T polymorphism and vascular superoxide generation was mediated principally through 5-MTHF or tHcy, we examined the associations between plasma/vascular 5-MTHF or plasma/vascular tHcy and superoxide radical generation. We observed that high plasma 5-MTHF was associated with low vascular superoxide generation (Figure 5) and low L-NAME–inhibitable superoxide generation (Figure 5). However, there was no association between plasma 5-MTHF and NADPH-stimulated vascular superoxide generation (Figure 5). Conversely, plasma tHcy was not associated with vascular superoxide generation or L-NAME–inhibitable or NADPH-stimulated superoxide radical generation. We next examined the corresponding associations with vascular 5-MTHF or tHcy levels. We observed that both vascular 5-MTHF and vascular tHcy were associated with overall vascular superoxide generation (Figure 6). However, high vascular 5-MTHF levels were associated with lower L-NAME–inhibitable superoxide production, indicative of improved eNOS coupling, an effect not observed for vascular tHcy (Figure 6). Conversely, vascular tHcy but not vascular 5-MTHF was significantly associated with NADPH-stimulated superoxide production, suggesting that tHcy is related to NADPH-oxidase activity in human vessels (Figure 6).
|
|
In a multivariable linear regression analysis in which vascular 5-MTHF and vascular tHcy were considered for inclusion in the same multiple regression models, we observed that vascular 5-MTHF was the only predictor of L-NAME–inhibitable superoxide in IMAs (β=1.557; SE=0.733; P=0.042; R2 for the model=0.127), whereas vascular tHcy was the only predictor of NADPH-induced vascular superoxide in IMAs (β=254.774; SE=77.880; P=0.003; R2 for the model=0.263).
When vascular 5-MTHF was replaced in the regression models by the presence of the MTHFR 677 C>T polymorphism, the predictors of total vascular superoxide were MTHFR genotype (β=1.301; SE=0.543; P=0.021), vascular tHcy (β=2.896; SE=1.592; P=0.076), and age (β=0.094; SE=0.042; P=0.030; R2 for the model=0.230). For L-NAME–inhibitable superoxide in IMAs, only the MTHFR 677 C>T polymorphism and age were qualified for inclusion in a regression model, and they were both predictors of this measure (MTHFR 677 C>T: β=–1.506, SE=0.676, P=0.029; age: β=0.095, SE=0.049, P=0.055; R2 for the model=0.093). However, when MTHFR 677 C>T replaced vascular 5-MTHF in vascular NADPH-induced superoxide analyses, vascular tHcy (β=244.419; SE=73.923; P=0.002) and hypertension (β=127.75; SE=37.473; P=0.002) (but not MTHFR 677 C>T) again were the only predictors of NADPH-stimulated superoxide in IMAs (R2 for the model=0.370). These results suggest that vascular 5-MTHF (or MTHFR 677 C>T polymorphism) is principally associated with eNOS uncoupling in human vessels, whereas vascular tHcy is associated with NADPH-induced vascular superoxide generation.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
In the present study, we have demonstrated that the MTHFR 677 C>T polymorphism affects plasma levels of both 5-MTHF and tHcy in patients with coronary artery disease. In vascular tissue, however, where the MTHFR 677 C>T genotype was more strongly associated with 5-MTHF levels than in plasma, there was no association with vascular tHcy levels in the same vessels. Thus, we conclude that the biological effects of the MTHFR 677 C>T polymorphism in vascular pathophysiology are more likely mediated by effects on 5-MTHF availability rather than by secondary effects on tHcy levels. We substantiated this conclusion by a systematic evaluation of the respective associations between MTHFR 677 C>T genotype, plasma and vascular tissue levels of both 5-MTHF and tHcy, and aspects of vascular pathophysiology with known importance in vascular disease: NO-mediated endothelial function, vascular superoxide production, and vascular BH4 levels. Whereas the MTHFR 677 C>T genotype was significantly associated with endothelial function and vascular superoxide production, both univariate and multivariable linear regression analyses revealed associations with vascular 5-MTHF levels but not with vascular tHcy levels. In addition, both 5-MTHF and tHcy are determinants of vascular superoxide radical generation, but 5-MTHF affects mainly eNOS coupling and NO bioavailability, whereas vascular tHcy affects vascular redox state by modifying vascular NADPH-oxidase activity. Taken together, these findings suggest that vascular 5-MTHF and tHcy are independently associated with the vascular redox state by affecting eNOS coupling and NADPH-oxidase activity, respectively.
Elevated plasma tHcy has previously been associated with increased cardiovascular risk.25 Plasma tHcy has been associated with increased oxidative stress and endothelial dysfunction,3 suggesting that tHcy is directly implicated in atherogenesis. On the other hand, it is still unclear whether homocysteine-lowering treatment with folate or B vitamins has the potential to alter cardiovascular risk. Despite the well-described decline in cardiovascular risk and the risk of stroke in North America since the introduction of folate food fortification,26 recent clinical trials failed to demonstrate any effect on cardiovascular risk in secondary prevention in patients with recent myocardial infarction9 or with stable coronary artery disease living in areas with folate fortification.10 Therefore, the existing controversy in the field suggests that further investigation of the mechanisms relating tHcy, folates, and cardiovascular disease is inevitable.
Evidence suggests that eNOS coupling plays a key role in maintaining vascular redox equilibrium.27 We have recently demonstrated that 5-MTHF, the circulating form of folic acid, has the potential to scavenge peroxynitrite radicals inside the vascular wall, thus protecting the oxidative degradation of BH4, an NOS cofactor.12 In line with our observation, 5-MTHF also was demonstrated to improve endothelial function in several clinical conditions such as established atherosclerosis28 or renal failure.13,14 This direct effect of acute 5-MTHF administration12 or chronic folate supplementation29 on vascular BH4 bioavailability appeared to be independent of systemic tHcy lowering.12,29 However, despite the direct effect of folate administration on vascular NO bioavailability in patients with coronary artery disease,12,29,30 there are no mechanistic data supporting that endogenous 5-MTHF may affect eNOS coupling and the vascular redox state in humans. Indeed, it is likely that 5-MTHF administration induces a rapid decrease in intracellular tHcy, before any systemic change in tHcy is witnessed. Therefore, it is crucial to define the exact contribution of 5-MTHF and tHcy on vascular function in human atherosclerosis.
To explore the role of 5-MTHF on vascular function, we used the well-known functional 677 C>T polymorphism in the MTHFR gene as a model system to study the impact of chronic exposure of human vessels to differential levels of 5-MTHF. Evidence suggests that the presence of the T allele is associated with significantly lower plasma 5-MTHF levels and higher plasma tHcy31 compared with other genotypes, but it is unclear whether this also applies for intravascular folate and homocysteine concentrations. Although MTHFR is responsible for the endogenous synthesis of 5-MTHF from tetrahydrofolate and 5-MTHF is necessary for the conversion of homocysteine back to methionine (by homocysteine-methyltransferase), it is likely that the expression of MTHFR may vary in different cell types in the human body.32 Indeed, liver is the main contributor of tHcy in the circulation, and the mechanisms controlling intracellular 5-MTHF and therefore affecting its ability to saturate the enzymatic conversion of homocysteine to methionine may be different in liver and vascular endothelium.
In the present study, we demonstrate for the first time that despite the significant effect of the MTHFR 677 C>T polymorphism on plasma 5-MTHF and tHcy, this polymorphism does not affect vascular tHcy, despite its strong effect on vascular 5-MTHF levels in both human arteries and veins. This is compatible with our previous observation that vascular endothelium behaves as a different compartment than systemic circulation regarding the regulation of 5-MTHF levels.29 It is likely that endothelial cells express sufficient amounts of MTHFR to maintain the critical intracellular 5-MTHF levels necessary for the normal conversion of tHcy to methionine, even in the presence of the T allele. This hypothesis explains the observation that the presence of the T allele does not affect vascular tHcy levels in human arteries and veins.
We have previously demonstrated that high-dose intravenous administration of 5-MTHF affects eNOS coupling, resulting into an improvement in NO bioavailability and a reduction in superoxide radicals generation in human arteries and veins.12 We suggested that this effect was due partly to the ability of 5-MTHF to scavenge peroxynitrite radicals in human vessels, thus protecting the oxidation of the eNOS cofactor BH4.12 However, it was unclear whether physiological variations of endogenous 5-MTHF levels have the potential to define endothelial function in humans. In addition, it was unclear whether 5-MTHF exerts its effects independently of tHcy levels in systemic circulation or in plasma. In the present study, we have demonstrated that resting vascular 5-MTHF levels are strongly associated with total vascular superoxide production and with eNOS coupling in human arteries, whereas they have no effect on NADPH-oxidase activity in these vessels. However, vascular but not plasma tHcy had an effect on vascular superoxide production by these vessels, mainly by modifying NADPH-oxidase activity. Most important, the effect of 5-MTHF on eNOS coupling and the effect of vascular tHcy on NADPH-oxidase activity in human arteries were independent of each other, suggesting that 5-MTHF and tHcy exert their effect on the vascular redox state by affecting different enzymatic pathways of superoxide generation.
The finding that vascular tHcy affects superoxide generation in human arteries by modulating NADPH-oxidase activity is consistent with previous observations in cell culture systems, which have demonstrated that incubation of monocytes with homocysteine increases NADPH-oxidase activity by affecting the phosphorylation of p47phox and p67phox subunits of this enzyme, enhancing their translocation to cellular membranes.33,34 However, the effect of vascular tHcy on NADPH-oxidase activity does not necessarily lead also to impaired NO bioavailability and endothelial dysfunction, as we have shown in this study.
Because previous reports have suggested that treatment with 5-MTHF12 or folates29 improves NO bioavailability in human vessels, we sought to examine whether the same effect is observed for the variations of plasma and vascular 5-MTHF within the normal range. We observed that high plasma or vascular 5-MTHF was associated with better vasorelaxations of SVs in response to acetylcholine compared with low 5-MTHF levels. Most important, neither plasma nor vascular tHcy had any effect on the vasorelaxations in response to acetylcholine, suggesting that variations of tHcy within the normal range have a minor effect on endothelial function in patients with coronary artery disease.
The experiments in the present study were done in SVs and IMAs, both of which do not develop atherosclerosis, although these vessels seem to be reliable model systems to study the mechanisms involved in atherogenesis in humans. Because the present study was done entirely in human vessels, the limited amount of tissue obtained from each patient did not allow every experiment to be done on every vessel, which is a limitation of the study. Furthermore, the vasorelaxation experiments were done in SV samples, but as previously shown,35 IMAs exhibit much better endothelial function than SVs, and this had been suggested as a mechanism making these vessels resistant to atherosclerosis.35 Therefore, the absence of further replication of our vasorelaxation findings in IMA points out that extrapolation of these observations to arteries should be made with caution. Another limitation of the study comes from the use of lucigenin-enhanced chemiluminescence to evaluate superoxide generation. Lucigenin-enhanced chemiluminescence favors redox cycling of lucigenin at high concentrations (250 µmol/L),21 a phenomenon that is minimally observed when a low lucigenin concentration (5 µmol/L) is used.21 In addition, lucigenin redox recycling is detectable even at lucigenin concentrations as low as 5 µmol/L when NADH is used as a substrate, a phenomenon that is further reduced when NADPH is used as a substrate.22 Therefore, by using low lucigenin concentrations and NADPH as a substrate in our studies, we reduce these artifacts to the minimum.
|
Conclusions |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
Vascular endothelium circulation and systemic circulation behave as independent compartments regarding the metabolism of homocysteine. Despite the strong impact of the MTHFR 677 C>T polymorphism on vascular and plasma 5-MTHF and on plasma tHcy, it appears to have a minor role in the regulation of vascular tHcy levels in humans. In addition, plasma or circulating 5-MTHF and the MTHFR 677 C>T polymorphism exert a direct effect on vascular BH4 levels, NO bioavailability, and eNOS coupling in human vessels in vivo, whereas vascular but not plasma tHcy is a regulator of vascular NADPH-oxidase activity. Therefore, 5-MTHF and tHcy have independent effects on the vascular redox state by affecting different enzymatic sources of superoxide such as uncoupled eNOS or NADPH-oxidase.
|
Acknowledgments |
|---|
We would like to thank Donald Warden for performing the MTHFR genotype analyses, Carole Johnston for carrying out the homocysteine analyses, and Elfrid Blomdal for help with laboratory management.
Sources of Funding
This study was supported by a fellowship from the European Society of Cardiology (to Dr Antoniades), by the National Institute for Health Research, Oxford Biomedical Research Centre Programme, and by grants from the British Heart Foundation (RG/02/006 to Dr Channon, FS/03/105/16340 to Dr Shirodaria). This project has also been supported by the Advanced Research Program Norway, Research Council of Norway, and the Johan Throne Holst Foundation for Nutrition Research.
Disclosures
None.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
|---|
1. Danesh J, Lewington S. Plasma homocysteine and coronary heart disease: systematic review of published epidemiological studies. J Cardiovasc Risk. 1998; 5: 229–232.[Medline] [Order article via Infotrieve]
2. Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med. 1998; 49: 31–62.[CrossRef][Medline] [Order article via Infotrieve]
3. Weiss N, Heydrick S, Zhang YY, Bierl C, Cap A, Loscalzo J. Cellular redox state and endothelial dysfunction in mildly hyperhomocysteinemic cystathionine beta-synthase-deficient mice. Arterioscler Thromb Vasc Biol. 2002; 22: 34–41.
4. McDowell IF, Lang D. Homocysteine and endothelial dysfunction: a link with cardiovascular disease. J Nutr. 2000; 130 (suppl): 369S–372S.[Medline] [Order article via Infotrieve]
5. Tomlinson DR, Lang D, Lewis MJ. Homocysteine, B vitamins, and cardiovascular disease. N Engl J Med. 2006; 355: 209.[Medline] [Order article via Infotrieve]
6. Moat SJ, Lang D, McDowell IF, Clarke ZL, Madhavan AK, Lewis MJ, Goodfellow J. Folate, homocysteine, endothelial function and cardiovascular disease. J Nutr Biochem. 2004; 15: 64–79.[CrossRef][Medline] [Order article via Infotrieve]
7. Weiss N, Keller C, Hoffmann U, Loscalzo J. Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia. Vasc Med. 2002; 7: 227–239.
8. El-Khairy L, Ueland PM, Refsum H, Graham IM, Vollset SE. Plasma total cysteine as a risk factor for vascular disease: the European Concerted Action Project. Circulation. 2001; 103: 2544–2549.
9. Bonaa KH, Njolstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T, Wang H, Nordrehaug JE, Arnesen E, Rasmussen K. Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med. 2006; 354: 1578–1588.
10. Heart Outcomes Prevention Evaluation(HOPE) 2 Investigators. Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med. 2006; 354: 1567–1577.
11. Das UN. Folic acid says NO to vascular diseases. Nutrition. 2003; 19: 686–692.[CrossRef][Medline] [Order article via Infotrieve]
12. Antoniades C, Shirodaria C, Warrick N, Shijie C, DeBono J, Lee J, Leeson P, Neubauer S, Ratnatunga C, Pillai R, Refsum H, Channon K. 5-Methyltetrahydrofolate rapidly improves endothelial function and decreases superoxide production in human vessels: effects on vascular tetrahydrobiopterin availability and eNOS coupling. Circulation. 2006; 114: 1193–1201.
13. Buccianti G, Raselli S, Baragetti I, Bamonti F, Corghi E, Novembrino C, Patrosso C, Maggi FM, Catapano AL. 5-Methyltetrahydrofolate restores endothelial function in uraemic patients on convective haemodialysis. Nephrol Dial Transplant. 2002; 17: 857–864.
14. Baragetti I, Raselli S, Stucchi A, Terraneo V, Furiani S, Buzzi L, Garlaschelli K, Alberghini E, Catapano AL, Buccianti G. Improvement of endothelial function in uraemic patients on peritoneal dialysis: a possible role for 5-MTHF administration. Nephrol Dial Transplant. 2007; 22: 3292–3297.
15. Verhaar MC, Wever RM, Kastelein JJ, van Dam T, Koomans HA, Rabelink TJ. 5-Methyltetrahydrofolate, the active form of folic acid, restores endothelial function in familial hypercholesterolemia. Circulation. 1998; 97: 237–241.
16. Spence JD. Homocysteine-lowering therapy: a role in stroke prevention? Lancet Neurol. 2007; 6: 830–838.[CrossRef][Medline] [Order article via Infotrieve]
17. Casas JP, Bautista LE, Smeeth L, Sharma P, Hingorani AD. Homocysteine and stroke: evidence on a causal link from mendelian randomisation. Lancet. 2005; 365: 224–232.[Medline] [Order article via Infotrieve]
18. Imamura A, Okumura K, Matsui H, Mizuno T, Ogawa Y, Imai H, Numaguchi Y, Sakai K, Murohara T. Endothelial nitric oxide synthase and methylenetetrahydrofolate reductase gene polymorphisms are associated with endothelial dysfunction in young, healthy men. Can J Cardiol. 2004; 20: 1229–1234.[Medline] [Order article via Infotrieve]
19. Passaro A, Vanini A, Calzoni F, Alberti L, Zamboni PF, Fellin R, Solini A. Plasma homocysteine, methylenetetrahydrofolate reductase mutation and carotid damage in elderly healthy women. Atherosclerosis. 2001; 157: 175–180.[CrossRef][Medline] [Order article via Infotrieve]
20. Warden DR, Refsum H. Detection of single-nucleotide polymorphisms by PCR with universal energy transfer-labeled primers: application to folate- and cobalamin-related genes. Clin Chem. 2005; 51: 1713–1716.
21. Skatchkov MP, Sperling D, Hink U, Mulsch A, Harrison DG, Sindermann I, Meinertz T, Munzel T. Validation of lucigenin as a chemiluminescent probe to monitor vascular superoxide as well as basal vascular nitric oxide production. Biochem Biophys Res Commun. 1999; 254: 319–324.[CrossRef][Medline] [Order article via Infotrieve]
22. Janiszewski M, Souza HP, Liu X, Pedro MA, Zweier JL, Laurindo FR. Overestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts. Free Radic Biol Med. 2002; 32: 446–453.[CrossRef][Medline] [Order article via Infotrieve]
23. Refsum H, Grindflek AW, Ueland PM, Fredriksen A, Meyer K, Ulvik A, Guttormsen AB, Iversen OE, Schneede J, Kase BF. Screening for serum total homocysteine in newborn children. Clin Chem. 2004; 50: 1769–1784.
24. Antoniades C, Shirodaria C, Crabtree M, Rinze R, Alp N, Cunnington C, Diesch J, Tousoulis D, Stefanadis C, Leeson P, Ratnatunga C, Pillai R, Channon KM. Altered plasma versus vascular biopterins in human atherosclerosis reveal relationships between endothelial nitric oxide synthase coupling, endothelial function, and inflammation. Circulation. 2007; 116: 2851–2859.
25. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med. 1997; 337: 230–236.
26. Yang Q, Botto LD, Erickson JD, Berry RJ, Sambell C, Johansen H, Friedman JM. Improvement in stroke mortality in Canada and the United States, 1990 to 2002. Circulation. 2006; 113: 1335–1343.
27. Verhaar MC, Westerweel PE, van Zonneveld AJ, Rabelink TJ. Free radical production by dysfunctional eNOS. Heart. 2004; 90: 494–495.
28. Doshi SN, McDowell IF, Moat SJ, Lang D, Newcombe RG, Kredan MB, Lewis MJ, Goodfellow J. Folate improves endothelial function in coronary artery disease: an effect mediated by reduction of intracellular superoxide? Arterioscler Thromb Vasc Biol. 2001; 21: 1196–1202.
29. Shirodaria C, Antoniades C, Lee J, Jackson CE, Robson MD, Francis JM, Moat SJ, Ratnatunga C, Pillai R, Refsum H, Neubauer S, Channon KM. Global improvement of vascular function and redox state with low-dose folic acid: implications for folate therapy in patients with coronary artery disease. Circulation. 2007; 115: 2262–2270.
30. Doshi SN, McDowell IF, Moat SJ, Payne N, Durrant HJ, Lewis MJ, Goodfellow J. Folic acid improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering. Circulation. 2002; 105: 22–26.
31. Ueland PM, Hustad S, Schneede J, Refsum H, Vollset SE. Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci. 2001; 22: 195–201.[CrossRef][Medline] [Order article via Infotrieve]
32. Ueland PM, Refsum H, Beresford SA, Vollset SE. The controversy over homocysteine and cardiovascular risk. Am J Clin Nutr. 2000; 72: 324–332.
33. Siow YL, Au-Yeung KK, Woo CW, OK. O K. Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation. Biochem J. 2006; 398: 73–82.[CrossRef][Medline] [Order article via Infotrieve]
34. Alvarez-Maqueda M, El Bekay R, Monteseirin J, Alba G, Chacon P, Vega A, Santa Maria C, Tejedo JR, Martin-Nieto J, Bedoya FJ, Pintado E, Sobrino F. Homocysteine enhances superoxide anion release and NADPH oxidase assembly by human neutrophils: effects on MAPK activation and neutrophil migration. Atherosclerosis. 2004; 172: 229–238.[CrossRef][Medline] [Order article via Infotrieve]
35. Luscher TF, Diederich D, Siebenmann R, Lehmann K, Stulz P, von Segesser L, Yang ZH, Turina M, Gradel E, Weber E. Difference between endothelium-dependent relaxation in arterial and in venous coronary bypass grafts. N Engl J Med. 1988; 319: 462–467.[Abstract]
|
Footnotes |
|---|
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.808675/DC1.
Related Article:
Circulation 2009 119: 2417-2419.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||