Jean-Claude Guilland

Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: Possible therapeutic targets?

Nitric oxide (NO) is synthetized enzymatically from l-arginine (l-Arg) by three NO synthase isoforms, iNOS, eNOS and nNOS. The synthesis of NO is selectively inhibited by guanidino-substituted analogs of l-Arg or methylarginines such as asymmetric dimethylarginine (ADMA), which results from protein degradation in cells. Many disease states, including cardiovascular diseases and diabetes, are associated with increased plasma levels of ADMA. The N-terminal catalytic domain of these NOS isoforms binds the heme prosthetic group as well as the redox cofactor, tetrahydrobiopterin (BH(4)) associated with a regulatory protein, calmodulin (CaM). The enzymatic activity of NOS depends on substrate and cofactor availability. The importance of BH(4) as a critical regulator of eNOS function suggests that BH(4) may be a rational therapeutic target in vascular disease states. BH(4) oxidation appears to be a major contributor to vascular dysfunction associated with hypertension, ischemia/reperfusion injury, diabetes and other cardiovascular diseases as it leads to the increased formation of oxygen-derived radicals due to NOS uncoupling rather than NO. Accordingly, abnormalities in vascular NO production and transport result in endothelial dysfunction leading to various cardiovascular disorders. However, some disorders including a wide range of functions in the neuronal, immune and cardiovascular system were associated with the over-production of NO. Inhibition of the enzyme should be a useful approach to treat these pathologies. Therefore, it appears that both a lack and excess of NO production in diseases can have various important pathological implications. In this context, NOS modulators (exogenous and endogenous) and their therapeutic effects are discussed.

Impact of Asymmetric Dimethylarginine on Mortality After Acute Myocardial Infarction

Objective—Asymmetrical dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthases. From a prospective cohort of patients with acute myocardial infarction (MI), we aimed to analyze the predictive value of circulating ADMA concentrations on prognosis. Methods and Results—Blood samples from 249 consecutive patients hospitalized for acute MI <24 hours were taken on admission. Serum levels of ADMA and its stereoisomer, symmetrical dimethylarginine (SDMA), were determined using high-performance liquid chromatography. The independent predictors of ADMA were glomerular filtration rate, female sex, and SDMA (R2=0. 25). Baseline ADMA levels were higher in patients who had died than in patients who were alive at 1 year follow-up (1.23 [0.98 to 1.56] versus 0.95 [0.77 to 1.20] &mgr;mol/L, P<0.001). By Cox multivariate analysis, the higher tertile of ADMA (median [interquartile range]: 1.45 [1.24 to 1.70] &mgr;mol/L) was a predictor for mortality (Hazard Ratio [95% CI], 4.83 [1.59 to 14.71]), when compared to lower tertiles, even when adjusted for potential confounders, such as acute therapy, biological, and clinical factors. Conclusion—Our study suggests that the baseline ADMA level has a strong prognostic value for mortality after MI, beyond traditional risk factors and biomarkers.

5,10-methylenetetrahydrofolate reductase common mutations, folate status and plasma homocysteine in healthy French adults of the Supplementation en Vitamines et Mineraux Antioxydants (SU.VI.MAX) cohort

The 677cytosine mutation identified in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene has been frequently associated with an elevated plasma homocysteine concentration. The aim of the present study was to determine the impact of this MTHFR common mutation on plasma and erythrocyte folate (RCF) and plasma total homocysteine (tHcy) concentrations in healthy French adults. A cohort of 291 subjects living in the Paris area and participating in the Supplementation en Vitamines et Mineraux Antioxydants (SU.VI.MAX) study were analysed to assess the impact of MTHFR polymorphism 677C-->T on folate status and plasma tHcy concentration. The frequency of the mutant homozygote for 677C-->T polymorphism (677TT genotype) in the present cohort was 16.8%. There were significant differences in plasma tHcy between 677CC, 677CT and 677TT genotype groups. The RCF concentrations were significantly different between each genotype, the lowest levels being associated with the 677TT genotype. When segregated by gender, no differences in tHcy between homozygous 677TT, heterozygous 677CT and wild-type 677CC genotype groups in women were observed. The fasting tHcy in women was unrelated to the 677C-->T mutation. However, tHcy was significantly increased in men with the homozygous 677TT genotype. We also analysed the possible implication of a second new MTHFR polymorphism (1298A-->C) in subjects with mild hyperhomocysteinaemia (4th quartile of homocysteinaemia; tHcy >11.1 micromol/l). The polymorphism 1298A-->C did not have a notable effect on tHcy or on the RCF levels. Our observations confirm a relatively high frequency of the 677TT genotype in the French population. Women with this genotype did not show the same increase in tHcy observed in men. In the present study dietary folate intake was not measured. Thus, the interaction of dietary folate with the MTHFR genotype in the French population needs further study.

Vitamin E-coated dialyzer reduces oxidative stress in hemodialysis patients.

The high incidence of cardiovascular disease in hemodialyzed (HD) patients is well established and oxidative stress has been involved in this phenomenon. The aim of our study was to evaluate if a vitamin E-coated dialyzer could offer protection to HD patients against oxidative stress. Sixteen HD patients were successively assessed for one month (i) on a high biocompatible synthetic dialyzer (AN) and (ii) on a vitamin E-coated dialyzer (VE). Blood samples were taken before and after the dialysis session at the end of each treatment period. HD session conducted with the AN dialyzer was responsible for acute oxidative stress, significantly assessed after HD by a decreased plasma vitamin C level and an increased ascorbyl free radical (AFR)/vitamin C ratio used as an index of oxidative stress. Plasma elastase activity, reflecting neutrophil activation, was also increased; soluble P-selectin, reflecting platelet activation, did not show any variation. The use of the VE dialyzer was associated with a less extended oxidative stress compared with the AN membrane: basal vitamin C level was higher, and after the HD session AFR/vitamin C ratio and elastase activity were not significantly increased. Plasma vitamin E levels were not affected. Our study demonstrates that HD is associated with oxidative stress, which can be partially prevented by the use of a vitamin E-coated dialyzer. Our data suggest that this dialyzer may exert a site-specific scavenging effect on free radical species in synergy with a reduced activation of neutrophils.

Effects of exhaustive exercise and vitamin b6 deficiency on free radical oxidative process in male trained rats

There is growing evidence that oxygen free radical production and subsequent lipid peroxidation are normal sequelae to the rise in oxygen consumption concomitant with exercise. In addition, increased lipid peroxidation has also been shown in vitamin B6-deficient rat plasma, liver, and kidney. To investigate the potential for a role of vitamin B6 in exercise-induced oxidative stress, 36 male Sprague-Dawley rats received 0 (n = 12), 2 (n = 12), and 8 mg pyridoxine (PN)-HCl/kg diet (n = 12) and were trained by a 9-week swimming program. After 9-weeks of training, six rats (exhausted: E rats) of each vitamin group were exercised to exhaustion by swimming while the other six rats rested (nonexhausted: NE rats). Ascorbate, ascorbate free radical and antilipoperoxidant capability (AC) were evaluated in plasma. These parameters were higher in E rats than in NE rats. Free radical-mediated lipid peroxidation was measured in tissue and plasma by evaluation of thiobarbituric acid reactive substances (TBARS) content. This index of peroxidation was significantly increased in liver of E rats but not in plasma, heart, and gastrocnemius muscle. Concentration of TBARS in liver was the highest in vitamin B6-deficient rats (consuming 0 mg PN-HCl/kg diet) and the lowest in vitamin B6-sufficient rats (consuming 8 mg PN-HCl/kg diet). Vitamin E (alpha-tocopherol) levels in liver and heart were negatively related to vitamin B6 levels in the diet. Independently of vitamin B6, liver and muscle alpha-tocopherol levels were significantly higher in E animals than in NE animals. There is good evidence according to our results that exercise induced an oxidative stress, as indicated by a significant increase of ascorbyl radical levels in the plasma. The effects of vitamin B6 deficiency on the free radical metabolism are low in trained rats. On the contrary, exhaustive exercise induced modifications in the metabolism pathways of vitamin C and E objectivated by variations of levels of vitamin C in the plasma and vitamin E in liver.

Distribution of tyrosine hydroxylase, dopamine, and serotonin in the central nervous system of amphioxus (Branchiostoma lanceolatum): Implications for the evolution of catecholamine systems in vertebrates

To investigate the evolutionary transition that has shaped the catecholaminergic systems of vertebrates, the organization of catecholamine‐synthesizing neurons and the nature of the catecholamines were examined in the central nervous system of adult amphioxus (Branchiostoma lanceolatum), a cephalochordate. We isolated a gene transcript encoding tyrosine hydroxylase (TH), the limiting enzyme of catecholamine biosynthesis, and studied its distribution together with that of dopamine and serotonin. Dopamine and TH are found in the same neurons of which they are three separate populations. Two are located in the anterior brain, the first being dorsal and lying in a row and the second being more posterior and lateral. A third population comprising a few dorsal commissural neurons was found in the posterior brain. The anterior dopaminergic cells innervate the ventral commissure of the cephalic vesicle, the hindbrain, and the spinal cord. A serotonin‐containing cell group is located in the same plane as the second dopaminergic cell population but is more caudal, marking the probable transition between anterior brain and hindbrain, as deduced from gene expression patterns. The overall distribution of dopaminergic and serotoninergic systems is similar in amphioxus and vertebrate central nervous system and could be an ancestral character of chordates. As assayed by high‐performance liquid chromatrography and electrochemical detection, significant amounts of dopamine and octopamine, but not of noradrenaline, are present in amphioxus head. This finding is consistent with data obtained from most prostomian species. We conclude that the noradrenergic system is probably an innovation of vertebrates that appeared along with the neural crest and specific hindbrain nuclei. J. Comp. Neurol. 468:135–150, 2004.

L’hyperhomocystéinémie : facteur de risque cardiovasculaire ou simple marqueur ? ☆: 1. Données fondamentales

: Recent epidemiological studies have suggested that hyperhomocysteinemia is associated with increased risk of vascular disease. Homocysteine is a sulphur-containing amino acid whose metabolism stands at the intersection of two pathways: remethylation to methionine, which requires folate and vitamin B12 (or betaine in an alternative reaction); and transsulfuration to cystathionine which requires vitamin B6. The two pathways are coordinated by S-adenosylmethionine which acts as an allosteric inhibitor of the methylenetetrahydrofolate reductase (MTHFR) and as an activator of cystathionine beta-synthase (CBS). Hyperhomocysteinemia arises from disrupted homocysteine metabolism. Severe hyperhomocysteinemia is due to rare genetic defects resulting in deficiencies in CBS, MTHFR, or in enzymes involved in methyl cobalamine synthesis and homocysteine methylation. Mild hyperhomocysteinemia seen in fasting condition is due to mild impairment in the methylation pathway (i.e. folate or B12 deficiencies or MTHFR thermolability). Post-methionine-load hyperhomocysteinaemia may be due to heterozygous cystathionine-beta-synthase defect or B6 deficiency. Patients with homocystinuria and severe hyperhomocysteinemia develop arterial thrombotic events, venous thromboembolism, and more seldom premature arteriosclerosis. Experimental evidence suggests that an increased concentration of homocysteine may result in vascular changes through several mechanisms. High levels of homocysteine induce sustained injury of arterial endothelial cells, proliferation of arterial smooth muscle cells and enhance expression/activity of key participants in vascular inflammation, atherogenesis, and vulnerability of the established atherosclerotic plaque. These effects are supposed to be mediated through its oxidation and the concomitant production of reactive oxygen species. Other effects of homocysteine include: impaired generation and decreased bioavailability of endothelium-derived relaxing factor/nitric oxide; interference with many transcription factors and signal transduction; oxidation of low-density lipoproteins; lowering of endothelium-dependent vasodilatation. In fact, the effect of elevated homocysteine appears multifactorial affecting both the vascular wall structure and the blood coagulation system.Resume L’homocysteine est un acide amine soufre qui est soit methyle en methionine soit transforme en cystathionine et cysteine par la voie de la transsulfuration dependant de la vitamine B 6 . La premiere voie a pour cofacteur la vitamine B 12 et utilise comme donneur de methyle soit un derive de l’acide folique, le N5-methyltetrahydrofolate, soit la betaine. La remethylation et la transsulfuration de l’homocysteine sont regulees de facon coordonnee par la S-adenosylmethionine (SAM) qui inhibe l’activite methylenetetrahydrofolate reductase (MTHFR), l’enzyme catalysant la synthese du N5-CH 3 THF a partir du methylenetetrahydrofolate et stimule l’activite cystathionine beta-synthase. Toute perturbation metabolique de l’une de ces deux voies entraine une hyperhomocysteinemie. Une hyperhomocysteinemie severe est generalement engendree par un deficit congenital en cystathionine beta-synthase (CBS). Une hyperhomocysteinemie moderee est observee a jeun soit du fait de la mutation du gene codant pour la MTHFR soit a cause d’une alteration du statut vitaminique B 9 ou B 12 . L’hyperhomocysteinemie moderee observee apres une surcharge en methionine traduit plutot un defaut de la voie de transsulfuration (mutation du gene codant pour la CBS, deficience en vitamine B 6 ). Les sujets atteints d’homocystinurie ont des valeurs d’homocysteinemie tres elevees et presentent des l’enfance des accidents vasculaires, thromboemboliques ou plus rarement lies a l’atherosclerose. Des concentrations elevees d’homocysteine provoquent des lesions anatomiques de l’endothelium vasculaire, stimulent la proliferation des cellules musculaires lisses arterielles et la production des mediateurs de l’inflammation. Ces effets sont supposes medies par l’oxydation de l’homocysteine et la production d’especes radicalaires derivees de l’oxygene et aussi par interference avec la production du NO et de nombreux facteurs de transcription, la transduction de signaux cellulaires, l’oxydation des LDL et la diminution de la vasodilatation dependant de l’endothelium.

Plasma antioxidant status in septic critically ill patients: a decrease over time.

Severe septic states in humans are responsible for intense intravascular oxidative stress, which induces numerous adaptive mechanisms. We determined time sequence changes in total plasma antioxidant capacity (TAC) and major plasma antioxidant concentrations, which have not been fully explained in septic conditions. A cohort of 56 consecutive septic patients (septic shock n = 37, severe sepsis n = 19) and six healthy volunteers. We compared TAC and antioxidant levels in patients with one of two degrees of septic states, at the onset of illness, to those of healthy volunteers. Thereafter, over a 10‐day follow‐up, we observed daily the time sequence changes of the two septic populations in terms of TAC and antioxidants. At the onset, there was no difference between the three groups in terms of TAC values (healthy subjects 2.18 ± 0.04; severe sepsis 2.03 ± 0.07; septic shock 2.09 ± 0.09), then an equivalent time decline was observed in the two septic populations whatever the severity. TAC was statistically linked to uric acid, proteins in particular albumin and bilirubin (multivariate analysis), but no correlation was found with any vitamin (A, C and E). A sharp and persistent decrease in vitamin C concentrations was underlined. TAC, unaffected at first, deteriorated over time whatever the severity of the infection in these critically ill patients. TAC, unable to distinguish severe sepsis and septic shock, is unlikely to be a particularly useful outcome measure.

Anti-hypertensive effects of Rosuvastatin are associated with decreased inflammation and oxidative stress markers in hypertensive rats.

Among their pleiotropic effects, statins exert antioxidant and anti-inflammatory properties. The aim of this study was to evaluate in normotensive (WKY) and in spontaneously hypertensive rats (SHR) the effect of rosuvastatin (ROSU) treatment on (1) plasma inflammation markers and endogenous NO synthase inhibitor (ADMA) levels, (2) reactive oxygen species (ROS) generated by circulating leukocytes and (3) vascular oxidative stress and tissue inflammation markers. Plasma cytokines were higher in SHR than in WKY, except for IL-4, which was lower in SHR than in WKY. SHR monocytes exhibited higher production of ROS than did WKY monocytes. In the experimental conditions, ROSU did not modify plasma cholesterol levels in SHR but attenuated the increase in systolic blood pressure. In SHR only, ROSU lessened pro-inflammatory cytokines and ADMA levels, increased IL-4 and reduced ROS production in circulating monocytes. These results demonstrate the beneficial effects of ROSU in SHR, independently of any lowering of cholesterol levels.

Folate status worsens in recently institutionalized elderly people without evidence of functional deterioration

Objective: To follow folate status, hematological and cognitive changes during the first year of institutionalization among elderly subjects. Design: Prospective study. Setting: Long-stay unit of the Dijon University Geriatric Hospital. Subjects: Twenty women and four men older than 65 years admitted consecutively. Main outcome measures: Folate and vitamin B-6 dietary intake was evaluated by a five-day record on admission (day 1 or d 1), at day 45 (d 45), day 90 (d 90), day 135 (d 135), day 180 (d 180), day 360 (d 360). Circulating levels of folate, vitamin B-6, total homocysteine (tHcy), blood counts and cognitive performance were determined in parallel. Results: From d 1 to d 360, mean folate and vitamin B-6 intakes remained below the French RDA and mean folate intakes decreased significantly (Δ = −10.2%, p <0.05). Mean plasma or erythrocyte folate decreased significantly (Δ = −33.7%, p <0.05 and Δ = −30.2%, p <0.001, respectively) from d 1 to d 360; no significant change was observed for the other blood parameters. The incidence of folate deficiency increased (8% vs. 37% for plasma folate <6.8 nmol/L and 8% vs. 17% for erythrocyte folate <340 nmol/L) from d 1 to d 360. Mean plasma pyridoxal 5′-phosphate (PLP) remained <20 nmol/L during the one-year follow-up. There was no difference between genders for plasma tHcy. Although mean plasma tHcy was <14 μmol/L, plasma tHcy was >14 μmol/L in about one-third of the subjects. At each period, 50% or more subjects were anemic (Hct <35% in women and Hct <40% in men), but the anemia was normocytic (MCV <100 fL). Subjects had a moderate dementia at admission, and no change was observed during the study. Conclusions: Subjects were already vitamin B-6 deficient at admission. Folate status was impaired during the study. Low vitamin intakes were the main cause of vitamin B-6 deficiency and folate status deterioration. Hematology and mental status capacity were not aggravated by folate status deterioration. Plasma tHcy didn’t appear to be an earlier predictor of folate deficiency.