Ann Samnegård

Positional identification of TNFSF4, encoding OX40 ligand, as a gene that influences atherosclerosis susceptibility

Ath1 is a quantitative trait locus on mouse chromosome 1 that renders C57BL/6 mice susceptible and C3H/He mice resistant to diet-induced atherosclerosis. The quantitative trait locus region encompasses 11 known genes, including Tnfsf4 (also called Ox40l or Cd134l), which encodes OX40 ligand. Here we report that mice with targeted mutations of Tnfsf4 had significantly (P ≤ 0.05) smaller atherosclerotic lesions than did control mice. In addition, mice overexpressing Tnfsf4 had significantly (P ≤ 0.05) larger atherosclerotic lesions than did control mice. In two independent human populations, the less common allele of SNP rs3850641 in TNFSF4 was significantly more frequent (P ≤ 0.05) in individuals with myocardial infarction than in controls. We therefore conclude that Tnfsf4 underlies Ath1 in mice and that polymorphisms in its human homolog TNFSF4 increase the risk of myocardial infarction in humans.

Serum matrix metalloproteinase-3 concentration is influenced by MMP-3 )1612 5A/6A promoter genotype and associated with myocardial infarction

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.

Human Evidence That the Cystatin C Gene Is Implicated in Focal Progression of Coronary Artery Disease

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.

Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: the Stockholm Atherosclerosis Gene Expression (STAGE) study.

Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10−27and−30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2–deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research.

Elevated plasma fibrinogen γ′ concentration is associated with myocardial infarction: Effects of variation in fibrinogen genes and environmental factors

Summary.  Background:  Fibrinogen γ′, a fibrinogen γ‐chain variant generated via alternative mRNA processing, has been associated with susceptibility to thrombotic disease.

Haplotype Effect of the Matrix Metalloproteinase-1 Gene on Risk of Myocardial Infarction

Myocardial infarction (MI) is commonly caused by atherosclerotic plaque rupture following excessive degradation of collagen fibers in the atherosclerotic lesion. We investigated whether interindividual variability in risk of MI was related to polymorphisms in the gene encoding matrix metalloproteinase (MMP)-1, a key fibrillar collagen–degrading enzyme. Several single nucleotide polymorphisms in the MMP1 gene promoter were identified following sequencing DNA samples from 30 individuals. An analysis of the polymorphisms in a cohort of British whites with coronary atherosclerosis, including 639 patients with MI and 538 non-MI subjects, revealed a haplotype effect of the −519A>G and −340T>C polymorphisms on risk of MI, with the A−519-C−340 and G−519-T−340 haplotypes being protective (odds ratio=0.70 [0.57 to 0.86]; P=0.0007), whereas the G−519-C−340 haplotype increased MI risk (odds ratio=1.94 [1.15 to 3.28]; P=0.013). This finding was replicated in a subsequent analysis of 387 Swedish MI patients and 387 healthy controls (odds ratio=0.70 [0.55 to 0.89], P=0.003, for A−519-C−340 and G−519-T−340; odds ratio=1.54 [0.97 to 2.46], P=0.07, for G−519-C−340). In vitro assays showed that compared with the A−519-T−340 haplotype, the A−519-C−340 and G−519-T−340 haplotypes had lower promoter activity, whereas the G−519-C−340 haplotype had greater promoter strength, in driving gene expression in human macrophages. Haplotype-specific differences in MMP1 mRNA level in atherosclerotic tissues were also detected. The data indicate that MMP1 gene variation is a genetic factor contributing to interindividual differences in MI risk.

High plasma concentrations of autoantibodies against native peptide 210 of apoB-100 are related to less coronary atherosclerosis and lower risk of myocardial infarction

AIMS We examined whether antibodies against peptides 45 and 210 of apoB-100 are related to myocardial infarction (MI) and severity of coronary atherosclerosis. METHODS AND RESULTS Three hundred and eighty-seven survivors of a first MI (aged <60 years) and 387 sex- and age-matched controls were characterized in detail. IgG and IgM autoantibodies against native and malondialdehyde (MDA)-modified peptides 45 and 210 of apoB-100 (amino acids 661-680 and 3136-3155) were quantified in plasma and quantitative coronary angiography was performed in 243 patients. Post-infarction patients had significantly lower IgG against the native peptide 210 (IgG-p210(nat)) and higher IgM against the MDA-modified peptide 210 (IgM-p210(MDA)) compared with controls, whereas no differences were found for other antibodies. Plasma concentrations of IgG-p210(nat), but not IgM-p210(MDA), were independently and inversely related to the degree of coronary atherosclerosis in patients. In multiple logistic regression analysis (including established risk indicators), MI risk was 0.55 (95%CI: 0.37-0.81) for individuals in the IgG-p210(nat) upper quartile compared with the remaining individuals. CONCLUSION Circulating IgG antibodies against the native peptide 210 of apoB-100 are inversely related to the severity of coronary atherosclerosis and associated with lower risk of MI. Epitope 210 of apoB-100 emerges as a target for immunization against atherosclerosis in humans.

Accumulation of Apolipoprotein C-I–Rich and Cholesterol-Rich VLDL Remnants During Exaggerated Postprandial Triglyceridemia in Normolipidemic Patients With Coronary Artery Disease

BACKGROUND Exaggerated postprandial triglyceridemia is common in normolipidemic patients with coronary artery disease (CAD). Alterations in the composition of triglyceride-rich lipoproteins (TRLs) are likely to underlie this metabolic disturbance. However, the composition of very-low-density lipoproteins (VLDLs), which are the most abundant postprandial TRLs, has never been defined in CAD patients. METHODS AND RESULTS We examined postprandial changes in the number and composition of VLDLs in middle-aged, normolipidemic CAD patients and control subjects. TRLs from 14 patients and 14 control subjects aged 45 to 55 years were subfractionated by density gradient ultracentrifugation into Svedberg flotation rate (Sf) fractions >400, 60 to 400, and 20 to 60. The VLDLs were separated from chylomicron remnants by immunoaffinity chromatography. In CAD patients, the postprandial concentrations of triglycerides and large (Sf 60 to 400) VLDL particles were elevated. In addition, their postprandial large VLDLs were enriched in apolipoprotein (apo) C-I and their postprandial small (Sf 20 to 60) VLDL remnants were enriched with apo C-I and cholesterol. CONCLUSIONS Perturbed handling of postprandial triglycerides in normolipidemic CAD patients involves the accumulation of apo C-I-rich large VLDL particles and the generation of small, apo C-I- and cholesterol-rich VLDL remnants.

Contribution of haplotypes across the fibrinogen gene cluster to variation in risk of myocardial infarction

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.

Severity of coronary artery stenosis is associated with a polymorphism in the CXCL16/SR‐PSOX gene

Objective.  Enhanced expression of CXCL16 has been demonstrated in atherosclerotic plaques and several properties have been attributed to CXCL16 that could influence the atherosclerotic process. CXCL16 exists in transmembrane and soluble forms. The transmembrane form acts as a scavenger receptor for oxidised LDL whereas the soluble form acts a chemoattractant for mainly CD8+ T cells. In addition, the soluble form of CXCL16 influences human aortic smooth muscle cell proliferation in vitro. In the present work, a human molecular genetic approach employing a common polymorphism within exon 4 of CXCL16 (181 Ala>Val) was used to investigate whether CXCL16 may be involved in the development of coronary artery disease. The polymorphism is located within the spacer region between the chemokine and transmembrane region and potentially influences an Ala/Val cleavage site, a site commonly used for the release of chemokines by tumour necrosis factor‐α converting enzyme.