P13
Cytochrome P450 2c19 polymorphism and cardiovascular recurrences in patients under clopidogrel treatment: a meta-analysis
F Sofi, B Giusti, AM Gori, F Cesari, R Marcucci, R Abbate, GF Gensini
University of Florence, Florence, Italy
Topic: Genetic-environmental interactions
Introduction: Nonresponsiveness to clopidogrel has been reported to be an independent predictor of clinical recurrences in patients with coronary artery disease under clopidogrel treatment. Recently, several polymorphisms in gene encoding platelet components or cytochrome P450 (CYP) enzymes have been proposed as possible mechanisms for nonresponsiveness to clopidogrel. Among them, a great deal of attention has been posed on a loss-of-function CYP2C19∗2 (or 681 G>A) polymorphism. To evaluate the role of such polymorphism in recurrent atherothrombotic events in patients with CAD under clopidogrel treatment, we considered all available studies in a meta-analysis.
Material and Methods: Cohort prospective studies evaluating the association between CYP2C19∗2 polymorphism and adverse clinical outcome were searched in MEDLINE, EMBASE, Web of Science, the Cochrane Systematic Review Database and bibliographies of retrieved articles up to January, 2009. the principal prior hypothesis was that the presence of the ∗2 variant allele of the polymorphism would be associated with an increased risk of clinical recurrence.
Results: Data were available for a total of 7,950 patients from 6 cohort prospective studies who were followed for a time ranging from 6 months to 8.4 years. the summary risk ratios for included cohort prospective studies showed a significant association between the CYP2C19∗2 polymorphism and an increased risk of major adverse cardiac events in the follow-up [RR: 1.80 (1.02–3.19); p=0.04]. When studies evaluating stent thrombosis (n=4) for a total of 4,975 patients were considered, the presence of the variant allele was associated with an increased risk of stent thrombosis [RR: 2.82 (1.43–5.56); p=0.0001].
Conclusion: the present meta-analysis performed on nearly 8,000 patients with CAD under clopidogrel treatment shows that the CYP2C19∗2 polymorphism is associated with an increased risk of major adverse cardiac events and stent thrombosis. these results need to be confirmed by further studies.
P152
Nitric oxide (NO) induces AT-2 receptor expression in a cGMP-dependent manner
S Agouri, VT Dao, O Kocgirli, M Oppermann, T Suvorava, G Kojda
Heinrich-Heine-University, Dusseldorf, Germany
Topic: Hypertension
Purpose: the aim of this study is to investigate whether endothelial nitric oxide (NO) has any influence on the expression on angiotensin (AT) type 1 (AT-1) and (AT-2) receptors.
Methods: Primary porcine aortic endothelial cells (PAEC) were routinely cultured in growth medium which was supplemented with 10% fetal serum, and 1% penicillin-streptomycin in an atmosphere of 10% CO2. Cells were grown until passage 2 in 75-cm flasks to 80% confluency. Cells were incubated for 1, 3, 6 hours with the NO- donor SNAP (S-nitroso-N-acetyl-D-L-pencilliamin) 10mM stock SNAP was dissolved in DMSO (dimetheyl sulfoxide) 5%. Cells incubated with DMSO served as controls, cells were incubated for 3 asd 6 hours with 10cM SNAP, in the presence of 10cM of the soluble guanylate cyclase inhibitor ODQ (oxidiazolol quinoxalin) or with 10cM 8- Br-cGMP (Bromoguanosine-3,5 cyclicmonophosphate) which is an activator of cGMP dependent kinase. AT1 - AT2 protein level was measured by western blot using polyclonal antibodies.
Results: SNAP incubation in PAEC results in significant higher expression levels of AT2 receptors (166±23.90, p=0.33, n=7) after 3 hours compared with the control. this effect disappeared after 6 hours of incubation indicating the short halflife of SNAP. In striking contrast the expression on the AT1 receptors did not change (p>0.05, n=4). ODQ inhibit the effect of SNAP on AT2 receptor expression (p>0.05), while Incubation with 8-Br cGMP results in upregulation of AT2 receptor protein levels (186.6±59.3, p=0.2183, n=5).
Conclusion: these results suggest that NO can upregulate vascular AT-2 receptor in PAEC. this newly discovered regulation might contribute to the vasoprotective effects of NO. Further research is necessary to understand the molecular signaling pathway and possible in vivo outcomes of increased vascular AT2 receptors density.
P155
Effects of NO on Endothelial Vascular Bradykinin Type-2 Receptor
O Kocgirli, S Valcaccia, VT Dao, T Suvorava, S Kumpf, M Floeren, M Oppermann, G Kojda
Institute of Pharmacology, Dusseldorf, Germany
Topic: Stable angina pectoris
Purpose: the activation of vascular bradykinin type-2 receptor (BKR-2) produces vasodilatory effects mediated by prostacyclin and NO. In eNOS knockout mice, coronary flow responses to bradykinin are largly increased. these data suggest that NO might act as a negative feed-back regulator on vascular BKR-2 expression and/or activity.
Methods: We used a human head and neck squamous carcinoma cell line expressing BKR-2 (UDSCC-2) and another one without BKR-2 expression (UDSCC-3) which were established in our labaratory to confirm the detection of BKR-2. For in vivo experiments we used eNOS++ mice which has more than 2 times higher endothelial NOS, eNOSn (transnegative litter mates) for negative control, eNOS knockout and C57Bl/6 mice. For in vitro experiments we used primary cultured porcine aortic endothelial cells which were incubated with 10 çM S-nitroso-N-acetylpenicillamine (SNAP) (NO-Donor) or Ca-Ionophor together with NG-nitro-L-Arginine-Methyl-Ester (L-NAME) (NOS inhibitor). C57Bl/6 mice were fed with NO-donor pentaerythrityl-tetranitrate (PETN, 0mg, 6mg or 60mg kg/KG/d).
Results: the expression of BKR-2 in eNOS++ compared to eNOSn mice lung (124.6±28.4%) and heart (114.5±18.1%) have not shown any significant difference. the knockout mice have shown an increase of BKR-2 expression (lung 130.7±6.5%, p>0.005, heart 172±26%, p>0.05). the C57Bl/6 mice which were treated with PETN and the cells which were incubated with SNAP or L-NAME showed no significant difference in BKR-2 expession.
Conclusion: these data suggest that an increase of BKR-2 expression in knockout eNOS mice could cause the largely increase of coronary flow mediated by bradykinin. NO does not seem to have any effect on vascular BKR-2 expression, in vitro and in vivo.
P156
Inherited human GP 91PHOX deficiency is associated with impaired isoprostane formation and reduced platelet recruitmen
P Pignatelli1, R Carnevale1, S Di Santo1, V Sanguigni1, M Proietti1, A Plebani2, F Violi2
1
Sapienza University of Rome, Rome, Italy, 2University of Brescia, Brescia, Italy
Topic: Biomarkers
Platelet isoprostane PGF2a-III, a pro-aggregating molecule, is believed to derive from non enzymatic oxidation of arachidonic acid. We studied PGF2a-III in platelets from 8 male patients with hereditary deficiency of gp91phox, the catalytic subunit of NADPH oxidase, and 8 male controls. Upon stimulation platelets from healthy subjects produced PGF2a-III, that was inhibited 8% by aspirin and 43% by an inhibitor of gp91phox. Also, in platelets incubated with a gp91phox inhibitor or with SQ29548, a thromboxane A2 receptors inhibitor, platelet recruitment, an in vitro model of thrombus growth, was reduced by 52% and 84% respectively; a lower effect (–13%) was seen with aspirin. Incubation of normal platelets with PGF2a-III dose-dependently (1–100 pM) increased platelet recruitment by mobilizing platelet Ca2+ and activating gpIIb/IIIa. Platelets from patients with gp91phox hereditary deficiency had normal thromboxane A2 formation and 75% PGF2a-III reduction compared to controls. In gp91phox-deficient patients agonist-induced platelet aggregation was within the normal range while platelet recruitment was reduced compared to controls. Incubation of gp91phox-deficient platelets with PGF2a-III (1–100 pM) partially restored platelet recruitment. this study provides the first evidence that platelet PGF2a-III maximally derives from gp91phox activation and contributes to platelet recruitment via activation of gpIIb/IIIa.
P157
No interaction between alcohol consumption and selected HDL-related genes on HDL levels
P Marques-Vidal1, M Bochud1, F Paccaud1, D Waterworth2, S Bergmann3, M Preisig4, G Waeber4, P Vollenweider4
1
University Institute of Social and Preventive Medicine Lausanne (IUMSP), Lausanne, Switzerland, 2Genetics Division, GlaxoSmithKline, Philadelpha, United States of America, 3University of Lausanne, Faculty of Biology and of Medicine, Department of Medical Genetics, Lausanne, Switzerland, 4University Hospital Center Vaudois, Lausanne, Switzerland
Topic: Genetic-environmental interactions
Objective: To assess the interactions between several polymorphisms related to HDL levels and alcohol consumption.
Methods: cross-sectional population-based study including 2,976 women and 2,658 men aged 3575 years (CoLaus study). Alcohol intake was assessed by the reported alcohol consumption of the last seven days. Nine candidate genes known to influence HDL levels: ABCA1, ATP-binding cassette, sub-family A (ABC1), member 1; APOA2, apolipoprotein A-II; APOC1, apolipoprotein C-I; CETP, cholesteryl ester transfer protein; GALNT2, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2; LIPC, hepatic lipase; LPL, lipoprotein lipase; MVK, mevalonate kinase; MMAB, methylmalonic aciduria (cobalamin deficiency) cblB type were studied.
Results: on bivariate analysis, four SNPs (two from ABCA1, one from APOC1 and one from LIPC) showed a significant (p>10–4) effect on HDL cholesterol levels. Multivariate adjustment for gender, age, body mass index, smoking, hypolipidaemic drug treatment, physical activity and alcohol consumption showed a SNP from LIPC and another from GALNT2 to be significantly related with HDL, ABCA1 and APOC1 remained borderline significant (p>0.001, see table). Smoothing of p-values showed a region in LIPC (positions 56,511,998 to 56,523,115) and in ABCA1 (106,693,247 to 106,721,246) to be significantly related to HDL cholesterol. Conversely, no geneæ alcohol intake interaction was found for all SNPs studied.
Conclusion: our data confirm the effect of selected polymorphisms of LIPC, ABCA1, APOC1 and GALNT2 on HDL cholesterol levels. those effects do not appear to be influenced by alcohol consumption.
Results are expressed as adjusted mean +/– standard error. a non-adjusted; b adjusting for gender, age (continuous), BMI (continuous), smoking (never, former, current), hypolipidaemic drug treatment (yes/no), physical activity (yes/no) and alcohol consumption (continuous); c after multivariate adjustment, using alcohol consumption in quintiles.
(W) P158: Impaired intracellular calcium handling in atrial cardiomyocytes from rats selected for low aerobic capacity and metabolic syndrome
A B Johnsen, M Alvez, G Kaurstad, N Rolim, U Wisloff, MA Hoydal
Norwegian University of Science and Technology, Department of Circulation and Medical Imaging, Trondheim, Norway
Topic: Heart disease
Purpose: Atrial fibrillation (AF) is the most common arrhythmia in humans and is often associated with altered Ca2+ handling. Evidence points to low aerobic capacity as predictor of cardiovascular disease and AF. the hypothesis of the present study was that rats, genetically selected and breed on the basis of low running capacity has impaired Ca2+ handling compared to rats selected for high running capacity.
Methods: Low capacity runners (LCR) (n=5) develop characteristics of the metabolic syndrome, and high capacity runners (n=5) develop a healthy and athletic phenotype. the exercise capacity was measured by VO2max. Ca2+ handling was measured in isolated Fura-2/AM loaded atrial cardiomyocytes.
Results: VO2max was 30% lower in LCR than HCR. Cardiomyocyte function was significantly depressed in LCR as compared to HCR; fractional shortening was 52% lower and time to 50% relenghtening was 42% prolonged. We observed no differences in peak systolic and diastolic Ca2+, as well as no significant difference in amplitude of Ca2+ transient between the two groups. this indicate reduced Ca2+sensitivity in cardiomyocytes from LCR. SR Ca2+ load, assessed by caffeine-induced Ca2+ release, was not different between groups. Time to 50% Ca2+ transient decay was prolonged by 58% in LCR. We found no difference in Ca2+ decay between the two groups during caffeine induced Ca2+ transient, reflecting unaltered NCX function; neither did we find any changes in the plasma membrane Ca2+ exchanger (PMCA), assessed by caffeine induced transients in a 0Na+/0Ca2+ solution. the impaired diastolic Ca2+ removal is therefore mainly attributed to reduced SERCA function in LCR (39%). Diastolic SR Ca2+ leak was significantly increased in LCR compared to HCR; this observation was quantitative assessed by measuring diastolic Ca2+ in quiescent myocytes with and without tetracaine.
Conclusion: Our data demonstrate differences in atrial Ca2+ handling between rats selected for high and low aerobic capacity and we suggest that impaired Ca2+ handling in LCR may be an important factor in initiation of AF.
