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Objectives: Matrix metalloproteinase-3 (MMP-3) is implicated in the formation of atherosclerotic plaques, and the MMP-3 -1612 5A/6A polymorphism is associated with myocardial infarction (MI) and stable coronary artery disease (CAD). The present study examined whether the -1612 5A/6A polymorphism in the promoter region of the MMP-3 gene influences serum concentrations of MMP-3 and whether serum concentrations of MMP-3 are related to extent of coronary atherosclerosis and risk of MI. Design and Subjects: This case-control study was conducted in three hospitals in the northern part of Stockholm. A total of 755 MI patients aged below 60 were screened, 433 entered and 387 completed the study. Three hundred and eighty-seven sex- and age-matched control subjects were recruited from the general population of the same county. Methods: The MMP-3 genotype was determined by Pyrosequencing(TM) and the serum MMP-3 concentration was quantified with an immunoassay. Severity and extension of CAD was assessed by quantitative coronary angiography in a subgroup of patients (n=243). Results: Patients had lower serum MMP-3 concentration than controls. There was a strong association between MMP-3 -1612 5A/6A genotype and serum concentrations of MMP-3. The presence of one or two copies of the 6A-allele was associated with a graded increase in serum MMP-3. In female patients there was an inverse correlation (r=-0.39, P<0.05) between serum MMP-3 concentration and plaque area. Conclusion. In conclusion, the serum concentration of MMP-3 is influenced by MMP-3 -1612 5A/6A genotype and associated with MI. [ABSTRACT FROM AUTHOR]

Aims Tumour necrosis factor-α (TNF-α) is a proinflammatory cytokine, which is implicated in some metabolic disorders and may play a role in the development of cardiovascular disease. We examined whether plasma TNF-α is related to established cardiovascular risk indicators, plasma levels of soluble cellular adhesion molecules and carotid artery intima–media thickness determined by ultrasound examination in a population-based cohort of 96 healthy 50-year-old men.Methods and Results TNF-α and cellular adhesion molecules were measured with enzyme-linked immunosorbent assays. Plasma TNF-α concentration was associated with systolic and diastolic blood pressure, degrees of alimentary lipaemia, plasma very low density lipoprotein triglyceride, low density lipoprotein (LDL) cholesterol concentrations and peak LDL particle size. Two indices of insulin resistance as well as all soluble cellular adhesion molecules correlated positively with TNF-α. The plasma TNF-α concentration was associated with common carotid intima–media thickness in univariate analysis. In contrast, soluble E-selectin and postprandial triglycerides, but not TNF-α, were independent determinants of common carotid intima–media thickness.Conclusion The plasma TNF-α concentration is associated with degrees of early atherosclerosis and correlates with metabolic and cellular perturbations that are considered important for the vascular process. [ABSTRACT FROM PUBLISHER]

Background: Low levels of free activated coagulation factor VII (VIIa) are normally present in plasma to prime the coagulation of blood in normal hemostasis and during thrombus formation. VIIa also circulates in inactive form, in complex with antithrombin (VIIaAT) formed when VIIa is bound to tissue factor (TF). This study evaluated VIIaAT in relation to cardiovascular disease (CVD)., Methods: We determined the plasma VIIaAT concentration in samples from the Stockholm Coronary Atherosclerosis Risk Factor (SCARF) study, a population-based case-control study of myocardial infarction (MI) and in samples from the Stockholm study of 60-years-old individuals, a prospective study of CVD. VIIaAT was measured with a sandwich ELISA that captures the complex between a monoclonal antibody to VIIa and a polyclonal antibody to AT., Results: In the SCARF study (200 post-MI cases, 340 controls), VIIaAT was statistically significantly associated with patient status [odds ratio (95% confidence interval (CI)] 1.51 (1.09-2.08), p=0.0126). The case-control differences were however small, with VIIaAT values that largely overlap between the two groups. When a nested case-control design (211 incident CVD cases and 633 matched controls) was applied on 5- to 7-year follow-up results of the Stockholm prospective study of 60-year-olds, plasma VIIaAT concentration was not associated with incident CVD (odds ratio (95% CI) 1.001 (0.997-1.005), p=0.5447)., Conclusions: Plasma VIIaAT concentration had no predictive value for future CVD in our study population. Slightly increased plasma VIIaAT concentrations observed after MI may reflect processes that occur in connection with the acute event when TF and VIIa availability is increased., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The TNFSF4/TNFRSF4 system, along with several other receptor-ligand pairs, is involved in the recruitment and activation of T-cells and is therefore tentatively implicated in atherosclerosis and acute coronary syndromes. We have previously shown that genetic variants in TNFSF4 are associated with myocardial infarction (MI) in women. This prompted functional studies of TNFSF4 expression., Methods and Results: Based on a screening of the TNFSF4 genomic region, a promoter polymorphism (rs45454293) and a haplotype were identified, conceivably involved in gene regulation. The rs45454293T-allele, in agreement with the linked rs3850641G-allele, proved to be associated with increased risk of MI in women. Haplotype-specific chromatin immunoprecipitation of activated polymerase II, as a measure of transcriptional activity in vivo, suggested that the haplotype including the rs45454293 and rs3850641 polymorphisms is functionally important, the rs45454293T- and rs3850641G-alleles being associated with lower transcriptional activity in cells heterozygous for both polymorphisms. The functional role of rs45454293 on transcriptional levels of TNFSF4 was clarified by luciferase reporter assays, where the rs45454293T-allele decreased gene expression when compared with the rs45454293C-allele, while the rs3850641 SNP did not have any effect on TNFSF4 promoter activity. Electromobility shift assay showed that the rs45454293 polymorphism, but not rs3850641, affects the binding of nuclear factors, thus suggesting that the lower transcriptional activity is attributed to binding of one or more transcriptional repressor(s) to the T-allele., Conclusions: Our data indicate that the TNFSF4 rs45454293T-allele is associated with lower TNFSF4 expression and increased risk of MI.

Background and Aim: Enhanced and prolonged postprandial lipaemia is associated with coronary heart disease (CHD). However, the mechanisms linking postprandial lipaemia to the increased risk of atherosclerosis and CHD remain to be determined. The aim of the present study was to examine the effects of a high-fat meal on plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) and cellular adhesion molecules in CHD patients and control subjects., Methods and Results: Forty-one middle-aged men with premature CHD and 26 healthy male controls were investigated. The plasma triglyceride response to the high-fat meal was significantly greater among cases than controls. The oral fat load induced a twofold increase in plasma concentrations of IL-6, an increase that was similar in CHD patients and control subjects. No changes could be detected in plasma concentrations of cellular adhesion molecules in response to postprandial lipaemia in either CHD patients or control subjects., Conclusion: The results of the present study suggest that a high-fat meal affects mechanisms that induce increased inflammatory activity, which is recognised as a key modulator in the development of atherosclerosis and CHD. However, the increased levels of plasma IL-6 appear not to be determined by the magnitude of the postprandial triglyceridaemia.

We recently showed that genetic variants in OX40L are associated with myocardial infarction (MI) and severity of coronary artery disease in human. A number of studies also suggest a possible role for OX40 (the OX40L receptor) as a factor contributing to atherosclerosis. In the present study, the OX40 gene was screened for variants associated with precocious MI, using individuals with MI before the age of 60 and controls. Despite the fact that the OX40 gene is highly conserved between species and that relatively few common genetic variants were encountered, an association with MI was seen for a polymorphism in intron 5 (rs2298212). In silico investigation suggested that genetic variation (rs2298211), linked to this intronic variant, is possibly affecting spliceosome function. Our results provide evidence that variants in human OX40 might influence susceptibility to MI. The relevance of these findings is supported by the vital functions fulfilled by OX40 in mammals as reflected by the high level of evolutionary conservation.

Objectives: In this study, we examined the apolipoprotein (apo) CI content of triglyceride-rich lipoproteins (TRLs) in relation to established coronary heart disease (CHD) risk factors and early atherosclerosis., Background: In Western society, the postprandial state constitutes a nearly constant stress on the vasculature and the metabolism of lipoproteins. Delayed clearance of postprandial TRL remnants has repeatedly been associated with premature CHD and may include the enrichment of these remnants with apoCI., Methods: We examined 72 healthy 50-year-old men with an apoE3/E3 genotype who had undergone an oral fat load test and B-mode ultrasound examination of the intima-media thickness (IMT) of the common carotid artery., Results: In the fasting state, plasma, very-low-density lipoprotein (VLDL), and low-density lipoprotein cholesterol, proinsulin, and apoB100-containing intermediate density lipoprotein levels were related to IMT (p < 0.05). In the postprandial state, IMT was related to triglycerides at 2 h (p < 0.01), large VLDL concentration at 3 h (p < 0.05), the apoCI plasma and TRL concentrations at 6 h (p < 0.05, p < 0.05), and the apoCI content of TRLs at 6 h (p < 0.002). Multivariate analysis revealed that the apoCI content of TRLs at 6 h (p < 0.0001), plasma triglyceride concentrations at 2 h (p < 0.006), and fasting plasma cholesterol concentration (p < 0.05) independently predicted IMT. In addition, the apoCI content of postprandial TRLs correlated strongly with the cholesterol content (r = 0.64, p < 0.0001)., Conclusions: Our results indicate that the apoCI content of postprandial TRLs is a novel independent risk factor for early atherosclerosis in normolipidemic healthy middle-aged men with possible implication for the enrichment of TRL remnant lipoproteins with cholesterol.

Fibrinogen has consistently been recognized as an independent predictor of myocardial infarction (MI). Multiple mechanisms link fibrinogen to MI; therefore disentangling the factors underlying variation in plasma fibrinogen concentration is essential. Candidate regions in the fibrinogen gamma (FGG), alpha (FGA) and beta (FGB) genes were screened for single nucleotide polymorphisms (SNPs). Several novel SNPs were detected in the FGG and FGA genes in addition to the previously known SNPs in the fibrinogen genes. Tight linkage disequilibrium extending over various physical distances was observed between most SNPs. Consequently, eight SNPs were chosen and determined in 377 postinfarction patients and 387 healthy individuals. None of the SNPs were associated with plasma fibrinogen concentration or MI. Haplotype analyses revealed a consistent pattern of haplotypes associated with variation in risk of MI. Of the four haplotypes inferred using the FGA -58G>A and FGG 1299 +79T>C SNPs, the most frequent haplotype, FGG-FGA*1 (prevalence 46.6%), was associated with increased risk of MI (OR 1.51; 95%CI 1.18, 1.93), whereas the least frequent haplotype, FGG-FGA*4 (11.8%), was associated with lower risk of MI (OR 0.79 95%CI 0.64, 0.98). In conclusion, fibrinogen haplotypes, but not SNPs in isolation, are associated with variation in risk of MI.

The plasma CRP concentration has consistently been shown to be associated with the risk of future coronary heart disease (CHD) and recent studies have suggested that CRP has a pathogenic role in CHD. Family studies and genotype-phenotype association studies of known polymorphisms in the intron, second exon and 3'-untranslated region (UTR) have suggested that plasma CRP concentrations are under genetic control. However, no functional polymorphisms have so far been reported in the promoter region of the CRP gene. Screening of 1600 base pair (bp) of the promoter region of the CRP gene, using denaturing high performance liquid chromatography, revealed two novel common single nucleotide polymorphisms (SNPs). One of them, a three allelic SNP located at position -286 from the transcription start, was strongly associated with the plasma CRP concentration, predominantly in patients with CHD. No difference in allele frequency was seen between middle-aged post-infarction patients and population-based controls. The prognostic role and therapeutic implications in CHD and the functionality of this polymorphism remain to be determined.

Objective: Overexpression of elastolytic cysteine and aspartic proteases, known as cathepsins, is implicated in atherogenesis. The potential significance of imbalance in expression between cathepsins and their inhibitor cystatin C in cardiovascular disease has been highlighted by the demonstration of cystatin C deficiency in human atherosclerosis and abdominal aortic aneurysms., Methods and Results: We identified and characterized physiologically relevant polymorphisms in the promoter region of the cystatin C gene that influence cystatin C production and used these polymorphisms as a tool to examine the significance of cystatin C in coronary atherosclerosis in vivo in humans. Seven polymorphisms, all in strong-linkage disequilibrium, were identified in the cystatin C gene, of which 2 promoter polymorphisms (-82G/C and -78T/G) were functional in vitro in electromobility shift and transient transfection assays. Genotyping of 1105 individuals (237 survivors of a first myocardial infarction before age 60 and 2 independent groups comprising a total of 868 healthy individuals) revealed that the plasma cystatin C concentration was significantly lower in carriers of the mutant haplotype. Furthermore, the mutant haplotype was associated with a higher average number of stenoses per coronary artery segment in unselected postinfarction patients (N=237) undergoing routine coronary angiography., Conclusions: These results provide human evidence for an important role of cystatin C in coronary artery disease.

Objectives: Recently, we reported that exaggerated postprandial triglyceridemia in normolipidemic patients with coronary artery disease is associated with enrichment of remnant lipoproteins with apolipoprotein C-I (apoC-I). In this study, the number and composition of chylomicron remnants and very low density lipoproteins (VLDLs) were examined in 30 asymptomatic normolipidemic 50-year-old men with and without early carotid atherosclerotic lesions., Methods and Results: Intima-media thickness of the far wall of the common carotid artery was determined by B-mode ultrasound. Triglyceride-rich lipoproteins were subfractionated by density gradient ultracentrifugation and separated into VLDL and chylomicron remnant fractions by immunoaffinity chromatography. The postprandial triglyceridemia and increase in triglyceride-rich lipoprotein particle number (ie, apolipoprotein B concentrations) were not exaggerated in men with early atherosclerosis. In contrast, their large (Svedberg flotation rate 60 to 400) and small (Svedberg flotation rate 20 to 60) chylomicron remnants and VLDL were greatly enriched with apoC-I, and their small chylomicron remnants and VLDL particles were relatively enriched with cholesterol. Moreover, the number of apoC-I molecules on small chylomicron remnants was strongly associated with the degree of atherosclerosis., Conclusions: Early asymptomatic atherosclerosis in normolipidemic men without exaggerated postprandial triglyceridemia is associated with the enrichment of postprandial chylomicron and VLDL particles with apoC-I. Therefore, it is conceivable that the apoC-I content of lipoprotein remnants may serve as an early marker of coronary artery disease risk.

Enhanced and prolonged postprandial lipaemia is implicated in coronary and carotid artery disease. This study assessed the effects of atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, on postprandial plasma concentrations of triglyceride-rich lipoproteins (TRLs). Sixteen middle-aged men with combined hyperlipidaemia (baseline low density lipoprotein (LDL) cholesterol and plasma triglyceride concentrations (median (interquartile range) of 4.54 (4.17-5.26)) and 2.66 (2.04-3.20) mmol/l, respectively) and previous myocardial infarction were randomised to atorvastatin 40 mg or placebo once daily for 8 weeks in a double-blind, cross-over design. The apolipoprotein (apo) B-48 and B-100 contents were determined in subfractions of TRLs as a measure of chylomicron remnant and very low density lipoprotein (VLDL) particle concentrations (expressed as mg apo B-48 or apo B-100 per litre of plasma), in the fasting state and after intake of a mixed meal. Atorvastatin treatment reduced significantly the fasting plasma concentrations of VLDL cholesterol, LDL cholesterol and VLDL triglycerides (median% change) by 29, 44 and 27%, respectively, and increased high density lipoprotein (HDL) cholesterol by 19%, compared with baseline. The postprandial plasma concentrations of large (Svedberg flotation rate (Sf) 60-400) and small (Sf 20-60) VLDLs and chylomicron remnants were almost halved compared with baseline (mean 0-6 h plasma concentrations were reduced by 48% for Sf 60-400 apo B-100, by 46% for Sf 60-400 apo B-48, by 46% for Sf 20-60 apo B-100 and by 27% for Sf 20-60 apo B-48), and the postprandial triglyceridaemia was reduced by 23% during active treatment. In conclusion, atorvastatin 40 mg once daily causes profound reductions of postprandial plasma concentrations of all TRLs in combined hyperlipidaemic patients with premature coronary artery disease.

Aims: Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine, which is implicated in some metabolic disorders and may play a role in the development of cardiovascular disease. We examined whether plasma TNF-alpha is related to established cardiovascular risk indicators, plasma levels of soluble cellular adhesion molecules and carotid artery intima-media thickness determined by ultrasound examination in a population-based cohort of 96 healthy 50-year-old men., Methods and Results: TNF-alpha and cellular adhesion molecules were measured with enzyme-linked immunosorbent assays. Plasma TNF-alpha concentration was associated with systolic and diastolic blood pressure, degrees of alimentary lipaemia, plasma very low density lipoprotein triglyceride, low density lipoprotein (LDL) cholesterol concentrations and peak LDL particle size. Two indices of insulin resistance as well as all soluble cellular adhesion molecules correlated positively with TNF-alpha. The plasma TNF-alpha concentration was associated with common carotid intima-media thickness in univariate analysis. In contrast, soluble E-selectin and postprandial triglycerides, but not TNF-alpha, were independent determinants of common carotid intima--media thickness., Conclusion: The plasma TNF-alpha concentration is associated with degrees of early atherosclerosis and correlates with metabolic and cellular perturbations that are considered important for the vascular process., (Copyright 2001 The European Society of Cardiology.)

Background: Alimentary lipemia has been associated with coronary heart disease and common carotid artery intima-media thickness (IMT). This study was designed to investigate the relations of subclasses of postprandial triglyceride-rich lipoproteins (TRLs) with IMT., Methods and Results: Ninety-six healthy 50-year-old men with an apolipoprotein (apo) E3/E3 genotype underwent an oral fat tolerance test and B-mode carotid ultrasound examination. The apo B-48 and apo B-100 contents of each fraction of TRLs were determined as a measure of chylomicron remnant and VLDL particle concentrations. In the fasting state, LDL cholesterol (P<0.05) and basal proinsulin (P<0. 05) were significantly related to IMT, whereas HDL cholesterol, plasma triglycerides, and insulin were not. In the postprandial state, plasma triglycerides at 1 to 4 hours (P<0.01 at 2 hours), total triglyceride area under the curve (AUC) (P<0.05), incremental triglyceride AUC (P<0.01), and the large VLDL (Sf 60 to 400 apo B-100) concentration at 3 hours (P<0.05) were significantly related to IMT. Multivariate analyses showed that plasma triglycerides at 2 hours, LDL cholesterol, and basal proinsulin were consistently and independently related to IMT when cumulative tobacco consumption, alcohol intake, waist-to-hip circumference ratio, and systolic blood pressure were included as confounders., Conclusions: These results provide further evidence for postprandial triglyceridemia as an independent risk factor for early atherosclerosis and also suggest that the postprandial triglyceridemia is a better predictor of IMT than particle concentrations of individual TRLs.

Tumour necrosis factor-alpha (TNF-alpha) plays a key role in orchestrating the complex events involved in inflammation and immunity. Accordingly, TNF-alpha has been implicated in a wide range of autoimmune and infectious diseases, but also in conditions such as obesity and insulin resistance. The regulation of TNF-alpha expression in man is indicated to be partly genetically determined. We therefore screened a 1263 bp section of the proximal promoter of the TNF-alpha gene for common genetic variants affecting the transcriptional activity of the gene. Here we report the characterization of a common functional polymorphism in the promoter region of the TNF-alpha gene, a C-->A substitution at position -863. Electromobility shift assays provided evidence for a distinct difference in the binding of monocytic and hepatic nuclear factors to the -863C and -863A alleles. The rare -863A allele was associated with 31% lower transcriptional activity ( P < 0.001) in chloramphenicol acetyltransferase (CAT) reporter gene studies in human hepatoblastoma (HepG2) cells, indicating that the-863C/A polymorphism influences the basal rate of transcription of the TNF-alpha gene in vitro. Allele frequencies were 0.83/0.17 amongst 254 apparently healthy men of Swedish origin, aged 35-50 years. In 156 men, the -863C/A polymorphism was associated with the serum TNF-alpha concentration, carriers of the rare A allele having a significantly lower TNF-alpha level ( P < 0.05). It is concluded that the common-863C/A polymorphism in the promoter region of the TNF-alpha gene is functional in vitro in monocytic and hepatic cells and influences the serum TNF-alpha concentration in vivo in healthy middle-aged men.

Beta-adrenergic receptor-blocking agents are commonly used for treatment of hypertension, angina pectoris and arrhythmias and as secondary prevention after myocardial infarction. The modest protection against myocardial infarction conferred by these compounds in primary-preventive studies has suggested that beneficial effects of beta-blockade are counteracted by known adverse influences on lipid and glucose metabolism. As most beta-blockers increase plasma triglycerides and decrease the high density lipoprotein (HDL) cholesterol concentration, a randomized, double-blind, cross-over study was conducted to evaluate whether a 12-week treatment with metoprolol (100 mg o.d.) or placebo affected the metabolism of postprandial triglyceride-rich lipoproteins in 15 middle-aged men with a modestly increased cardiovascular risk. Metoprolol treatment significantly increased the postprandial responses of very low density lipoprotein (VLDL) and VLDL remnants to a mixed meal-type of oral fat tolerance test. The effect was particularly prominent for larger (Svedberg flotation rate (Sf) > 400 and Sf 60-400) particle species (P < 0.001 in repeated measures ANOVA), whereas the smaller (Sf 20-60) particles were less affected (P < 0.05). The changes in the postprandial responses of the different VLDL species were mainly related to an effect on the fasting plasma concentrations, with limited or no influences on VLDL catabolism during the postprandial state. In contrast, metoprolol treatment did not significantly influence the postprandial responses of chylomicrons and chylomicron remnants. Notably, the enhanced fasting and postprandial triglyceridaemia during metoprolol treatment was neither accompanied by a rise in fasting or postprandial free fatty acid concentrations, nor by alterations of the glucose and insulin responses to a standard oral glucose challenge. The ensuing shift in the LDL particle size distribution towards smaller particles was limited (fraction small LDL: metoprolol 22.8 +/- 15.7% versus placebo 19.3 +/- 15.0%, P < 0.05). In conclusion, metoprolol treatment primarily enhances fasting and postprandial triglyceridaemia in middle-aged men by increasing the basal hepatic production of VLDL.