Trine Baur Opstad

Influence of highly concentrated n-3 fatty acids on serum lipids and hemostatic variables in survivors of myocardial infarction receiving either oral anticoagulants or matching placebo

Forty patients with previous myocardial infarction were given 4 capsules with 1 g concentrated fish oil preparation daily for 4 weeks. No special diet was applied. The supplementation was equivalent to 3.4 grams of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) daily. Twenty-two of the 40 subjects received concomitant treatment with long-term oral anticoagulants (OAC). The fatty acid composition of serum after the supplementation period showed a significant increase in the proportion of EPA and DHA, while arachidonic acid (AA) remained essentially constant. This resulted in a rise of the EPA/AA ratio from 0.59 to 1.49 (p less than 0.001), confirming satisfying absorption of the concentrate. Blood lipids showed an overall decrease of triglycerides (TG) by 25% (p = 0.02), while total cholesterol rose by 5% (p = 0.03) and HDL-cholesterol was unaffected. Blood glucose and the TG associated factors plasminogen activator inhibitor and factor VII-phospholipid complex revealed trends towards reduction. Ivy bleeding time showed a significant prolongation, the median increasing from 240 to 270 seconds. A significant increase of fibrinogen was seen, as was a decrease of clotting time in the combined prothrombin test in patients receiving concomitant OAC. Thus, given for 4 weeks, the investigated concentrate of n-3 fatty acids exerts not merely beneficial effects as far as the risk profile for atherosclerotic disease is concerned. The results also point towards interactions with OAC that may be of clinical relevance.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating levels of IL-18 are significantly influenced by the IL-18 +183 A/G polymorphism in coronary artery disease patients with diabetes type 2 and the metabolic syndrome: an Observational Study

BackgroundIncreased IL-18 serum levels have been associated with diabetes type 2, metabolic syndrome and the severity of atherosclerosis. The present study investigated the presence and influence of IL-18 genetic variants on gene- and protein expression in stable coronary artery disease (CAD) patients.MethodsThe +183 A/G (rs 5744292), -137 G/C (rs 187238) and -607 C/A (rs 1946518) polymorphisms were determined in 1001 patients with angiographically verified stable CAD, and in 204 healthy controls. IL-18 gene-expression was measured in circulating leukocytes in 240 randomly selected patients. Circulating IL-18 and IL-18 binding protein levels were measured immunologically in all patients.ResultsThe +183 G-allele associated significantly with lower serum levels of IL-18 (p = 0.002, adjusted for age, glucose, body mass index and gender) and a 1.13- fold higher IL-18 gene-expression (p = 0.010). No influence was observed for the -137 G/C and -607 C/A polymorphisms. The IL-18 binding protein levels were not influenced by IL-18 genotypes. IL-18 levels were significantly higher in men as compared to women, and in patients with diabetes type 2 and metabolic syndrome compared to those without (p ≤ 0.001, all). The reduction in IL-18 levels according to the +183 G-allele was 3-4 fold more pronounced in diabetes and metabolic syndrome as compared to unaffected patients.Finally, the +183 AA genotype was more frequent in patients with hypertension (p = 0.042, adjusted for age, body mass index and gender).ConclusionThe reduction in serum IL-18 levels across increasing numbers of +183G-alleles was especially apparent in patient with diabetes type 2 and metabolic syndrome, suggesting a beneficial GG genotype in relation to cardiovascular outcome in these patients.Clinical Trial Registration NumberClinicalTrials.gov: NCT00222261

Genetic variation, gene-expression and circulating levels of matrix metalloproteinase-9 in patients with stable coronary artery disease.

BACKGROUND Mediators involved in atherosclerosis and plaque rupture may have importance as risk markers for coronary artery disease (CAD). We have investigated the influence of matrix metalloproteinase (MMP)-9 genetic variations on gene- and protein expression in stable CAD patients. METHODS The promoter -1562C/T and exon 6 R279Q A/G polymorphisms were determined in 1001 patients with angiographically verified stable CAD and in 204 healthy controls. Genotype and gene-expression were determined by real-time PCR. Serum levels of MMP-9 and its inhibitor TIMP-1were measured immunologically and by zymography (MMP-9 activity). RESULTS None of the polymorphisms associated with the presence of CAD, myocardial infarction or type 2 diabetes, whereas the variant allele of the R279Q polymorphism associated with hypertension (adjusted p=0.015). The T- and G alleles associated with lower and higher mRNA levels, respectively (p<0.005 both), also shown in an experimental ex-vivo LPS stimulated model. T-allele carriers had higher concentrations of MMP-9 (adjusted p=0.032) and the GG genotype induced lower MMP-9 gelatinolytic activity (p=0.01). Higher MMP-9 gene-expression and TIMP-1 levels were observed in patients with previous myocardial infarction, the latter also was elevated in diabetics (<0.05, all). CONCLUSION The investigated MMP-9 polymorphisms influenced gene- and protein expression differently and the R279Q polymorphism associated significantly with hypertension.

The Co-Existence of the IL-18+183 A/G and MMP-9 -1562 C/T Polymorphisms Is Associated with Clinical Events in Coronary Artery Disease Patients

Objective Interleukin (IL)-18 has been associated with severity of atherosclerosis and discussed to predict cardiovascular (CV) events. We have previously shown that the IL-18+183 G-allele significantly reduces IL-18 levels. This study was aimed to investigate the prognostic significance of the IL-18+183 A/G polymorphism (rs5744292), single and in coexistence with the matrix metalloproteinase (MMP)-9 -1562 C/T (rs3918242) polymorphism, in patients with stable coronary artery disease (CAD). Serum levels of IL-18, MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were additionally assessed. Methods 1001 patients with angiographically verified CAD were genotyped and the biomarkers were measured accordingly. After two years follow-up, 10.6% experienced new clinical events; acute myocardial infarction (AMI), stroke, unstable angina pectoris and death. Results The IL-18+183 G-allele associated with 35% risk reduction in composite endpoints after adjusting for potential covariates (p = 0.044). The IL-18+183 AA/MMP-9 -1562 CT/TT combined genotypes associated with a significant increase in risk of composite endpoints (OR = 1.87; 95% CI = 1.13–3.11, p = 0.015, adjusted). Patients with clinical events presented with significantly higher IL-18 levels as compared to patients without (p = 0.011, adjusted). The upper tertile of IL-18 levels associated with an increase in risk of AMI as compared to lower tertiles (OR = 2.36; 95% CI = 1.20–4.64, p = 0.013, adjusted). Conclusion The IL-18+183 A/G polymorphism, single and in combination with MMP-9 genotypes, may influence the risk of clinical events in stable CAD patients.

The influence of CYP 2C19*2 polymorphism on platelet function testing during single antiplatelet treatment with clopidogrel

BackgroundDifferent platelet function tests can be used to evaluate the degree of achieved platelet inhibition in patients treated with clopidogrel. The presence of CYP 2C19*2 polymorphism can reduce the formation of the active metabolite of clopidogrel, resulting in less platelet inhibition.Patients and MethodsPatients with symptomatic coronary artery disease, all on chronic single aspirin treatment were randomized to continue on aspirin or change to clopidogrel. In 219 randomly selected clopidogrel treated patients, platelet reactivity was evaluated by VASP-PRI determination and by use of VerifyNow P2Y12-PRU. The CYP 2C19*2 G/A polymorphism was further determined.ResultsThe total frequency of clopidogrel resistance was 29.0% by VASP-PRI and 31.6% by VerifyNow-PRU. The number of patients being hetero- and homozygous combined for the CYP 2C19*2 polymorphism (GA/AA) was 64 (29%). Platelet reactivity was significantly higher in patients with the polymorphism compared to wild-type patients (GG). VASP-PRI was 50.9% (SD19) in patients having the polymorphism compared to 38.3% (SD21) in patients with the GG genotype (p = 0.001). Correspondingly, the mean PRU was 165 (SD67) compared to 124 (SD69) (p < 0.001). The frequency of clopidogrel resistance in patients with the polymorphism was 32% compared to 16% in wild-type patients when defined by VASP-PRI (p = 0.006). When defined by PRU (VerifyNow), the corresponding frequencies were 53% and 22% (p < 0.001).ConclusionsClopidogrel treated patients with the CYP 2C19*2 polymorphism have significantly increased platelet reactivity compared to patients with the wild-type, evaluated with the VASP determination, and even more pronounced with the VerifyNow P2Y12 method.Trial RegistrationClinicalTrials.gov: NCT00222261

The MMP-9 -1562 C/T Polymorphism in the Presence of Metabolic Syndrome Increases the Risk of Clinical Events in Patients with Coronary Artery Disease

Background and Objectives Elevated levels of matrix metalloproteinase (MMP)-9 have been associated with the metabolic syndrome (MetS) and cardiovascular events. The MMP-9 −1562 C/T polymorphism has furthermore been shown as a risk factor for coronary artery disease (CAD). The non-favourable cardiometabolic state in MetS may increase the risk. We aimed to investigate the influence of MMP-9 −1562 C/T polymorphism in subjects with CAD and MetS. Methods Patients (n = 1000) with verified CAD stratified in Mets +/− (n = 244/756), were analyzed for the MMP-9 −1562 C/T polymorphism and related to clinical events after 2 years follow-up. Serum levels of total MMP-9 and tissue inhibitor of matrix metalloproteinases (TIMP)-1were analyzed in all, whereas MMP-9 activity, extracellular matrix metalloproteinase inducer (EMMPRIN), and expression of the two genes were analyzed in a subset of 240 randomly selected patients. Results Totally, 106 clinical endpoints were recorded. In MetS; the T-allele associated with 5.5 fold increase in event rate (p<0.0001), increased with number of MetS components, a 117% increase in total MMP-9 levels (TT homozygous, p = 0.05), significantly higher total- and endogenous active MMP-9 and TIMP-1 levels (p<0.01 all), and EMMPRIN was inversely correlated with pro- and endogenous active MMP-9 (p<0.05, both). In non-MetS; the T-allele was not associated with new events, nor higher MMP-9 levels. EMMPRIN was significantly correlated with total MMP-9 and TIMP-1 (p<0.01, both) and the two genes were inter-correlated (p<0.001). Conclusion In CAD patients with MetS, the MMP-9 T-allele increased the risk of clinical events, probably mediated through elevated MMP-9 levels and altered MMP-9 regulation.

Markers of hypercoagulability in CAD patients. Effects of single aspirin and clopidogrel treatment

BackgroundCardiovascular disease with disturbances in the haemostatic system, might lead to thrombotic complications with clinical manifestations like acute myocardial infarction (AMI) and stroke. Activation of the coagulation cascade with subsequent increased thrombin generation, characterizes a prothrombotic phenotype. In the present study we investigated whether prothrombotic markers were associated with risk factors and clinical subgroups in a cohort of patients with angiographically verified coronary artery disease (CAD). The patients were randomized to long-term treatment with the antiplatelet drugs aspirin or clopidogrel, and we further investigated the effect on hypercoagulability of such treatment for 1 year, of which limited data exists.MethodsVenous blood samples were collected in fasting condition between 08:00 and 10:30 am, at baseline when all patients were on aspirin therapy (n = 1001) and in 276 patients after 1 year follow-up on aspirin or clopidogrel. In vivo thrombin generation was assessed by prothrombin fragment 1 + 2 (F1+2) and D-dimer, and the endogenous thrombin potentiale (ETP) in the calibrated automated thrombogram (CAT) assay, representing ex vivo thrombin generation. In addition soluble tissue factor (sTF) and free- and total tissue factor pathway inhibitor (TFPI) were measured.ResultsWe found age to be significantly associated with F1+2 and D-dimer (β = 0.229 and β =0.417 respectively, p <0.001, both). Otherwise, only weak associations were found. F1+2 and D-dimer were higher in women compared to men (p <0.001 and p = 0.033, respectively). Smokers had elevated levels of ETP compared to non-smokers (p = 0.014). Additionally, patients on renin-angiotensin system (RAS) inhibition showed significantly higher levels of F1+2, compared to non-users (p = 0.013). Both aspirin and clopidogrel reduced levels of ETP after 12 months intervention (p = 0.003 and p <0.001, respectively) and the levels of F1+2 were significantly more reduced on aspirin compared to clopidogrel (p = 0.023).ConclusionsIn the present population of stable CAD, we could demonstrate a more hypercoagulable profile among women, smokers and patients on RAS medication, assessed by the prothrombotic markers F1+2, D-dimer and ETP. Long-term antiplatelet treatment with aspirin alone seems to attenuate thrombin generation to a greater extent than with clopidogrel alone. The study is registered at http://www.clinicaltrials.gov: NCT00222261.

The Time Profile of Pentraxin 3 in Patients with Acute ST-Elevation Myocardial Infarction and Stable Angina Pectoris Undergoing Percutaneous Coronary Intervention

Background. High levels of Pentraxin 3 (PTX3) are reported in acute myocardial infarction (AMI). Aim. To investigate circulating levels and gene expression of PTX3 in patients with AMI and stable angina pectoris (AP) undergoing PCI. Methods. Ten patients with AP and 20 patients with AMI were included. Blood samples were drawn before PCI in the AP group and after 3 and 12 hours and days 1, 3, 5, 7, and 14 in both groups. Results. Circulating PTX3 levels were higher in AMI compared to AP at 3 and 12 hours (P < 0.001 and P = 0.003). Within the AMI group, reduction from 3 hours to all later time points was observed (all P ≤ 0.001). Within the AP group, increase from baseline to 3 hours (P = 0.022), followed by reductions thereafter (all P < 0.05), was observed. PTX3 mRNA increased in the AMI group from 3 hours to days 7 and 14 in a relative manner of 62% and 73%, while a relative reduction from baseline to 3 and 12 hours of 29% and 37% was seen in the AP group. Conclusion. High circulating PTX3 levels shortly after PCI in AMI indicate that AMI itself influences PTX3 levels. PTX3 mRNA might be in response to fluctuations in circulating levels.

Fractalkine and its receptor (CX3CR1) in patients with stable coronary artery disease and diabetes mellitus.

BACKGROUND Fractalkine and its receptor CX3CR1 are associated with atherosclerosis. In vitro studies have shown increased expression of fractalkine in endothelial and vascular smooth muscle cells when stimulated with a high concentration of glucose. Increased serum levels of fractalkine have been shown in patients with type 2 diabetes mellitus (T2DM) and also in unstable coronary artery disease (CAD) patients. We investigated whether CAD patients with T2DM or metabolic syndrome have increased circulating and gene expression levels of fractalkine compared to CAD patients without these conditions. METHODS Serum levels of fractalkine were analyzed by the enzyme-linked immunosorbent assay (ELISA) method in 1001 patients with angiographically verified CAD, of which 200 had T2DM and 244 had metabolic syndrome. All patients were taking aspirin as an antithrombotic treatment. Gene expression of fractalkine and CX3CR1 in circulating leukocytes was explored in a subset of patients (n=168). RESULTS We found no significant difference in circulating levels of fractalkine in patients with T2DM [653 (556, 775) pg/mL] compared to patients without T2DM [646 (553, 761) pg/mL], p=0.50. There was also no difference between patients with and without metabolic syndrome (p=0.60). Fractalkine was not expressed in circulating leukocytes, and CX3CR1 was not expressed differently between any of the groups (p=0.13 and p=0.32, respectively). Smokers had lower fractalkine levels (p<0.001), and patients on angiotensin II receptor blockers had higher levels (p=0.047) compared to nonaffected patients. CONCLUSIONS In the present CAD population, no differences in circulating levels of fractalkine or expression levels of CX3CR1 were observed between patients with and without T2DM, or with and without metabolic syndrome, which may be related to their underlying disease.

Tissue factor pathway inhibitor polymorphisms in women with and without a history of venous thrombosis and the effects of postmenopausal hormone therapy

Postmenopausal hormone therapy is associated with marked reduction in tissue factor pathway inhibitor (TFPI) levels, and low TFPI levels have been associated with increased risk of venous thrombosis. Polymorphisms in the TFPI gene may affect the expression of TFPI. We aimed to investigate the influences of such polymorphisms on plasma TFPI levels and to investigate the effect of hormone therapy. Four single nucleotide polymorphisms in the TFPI gene (the −287T/C and the −399C/T polymorphisms in the 5′ upstream region, and the intron 7 −33T/C and the exon 9 874G/A polymorphisms) were studied with regard to frequency, phenotype, and their influence on hormone therapy in postmenopausal women with a history of venous thrombosis (n = 138), in healthy postmenopausal women (n = 202), and in normal controls (n = 212). The frequencies of the −287C and the −33C variants were nonsignificantly lower in cases than in controls, and the polymorphisms were associated with slightly higher levels of free TFPI antigen (−287C; P = 0.076) and higher TFPI activity (−33C; P < 0.001). The −399T variant showed equal distribution in cases and controls, but was associated with lower levels of TFPI activity (P = 0.036). Conventional-dose hormone therapy induced significant reductions in TFPI levels irrespective of genotypes. In healthy women treated with low-dose hormone therapy, the reduction in TFPI levels was less pronounced with the −287C variant (P = 0.054). Our study indicates that polymorphisms in the TFPI gene may be of importance for plasma TFPI levels and for the effects of hormone therapy.