Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide, particularly in individuals with diabetes. The current study objective was to determine the circulating metabolite profiles associated with the risk of future cardiovascular events, with emphasis on diabetes status. Nontargeted metabolomics analysis was performed by LC-HRMS in combination with targeted quantification of eicosanoids and endocannabinoids. Plasma from 375 individuals from the IMPROVE pan-European cohort was included in a case-control study design. Following data processing, the three metabolite data sets were concatenated to produce a single data set of 267 identified metabolites. Factor analysis identified six factors that described 26.6% of the variability in the given set of predictors. An association with cardiovascular events was only observed for one factor following adjustment (p = 0.026). From this factor, we identified a free fatty acid signature (n = 10 lipids, including saturated, monounsaturated, and polyunsaturated fatty acids) that was associated with lower risk of future cardiovascular events in nondiabetics only (OR = 0.65, 0.27-0.80 95% CI, p = 0.030), whereas no association was observed among diabetic individuals. These observations support the hypothesis that increased levels of circulating omega-6 and omega-3 fatty acids are associated with protective effects against future cardiovascular events. However, these effects were only observed in the nondiabetic population, further highlighting the need for patient stratification in clinical investigations.

To characterize type 2 diabetes (T2D)-associated variation across the allele frequency spectrum, we conducted a meta-analysis of genome-wide association data from 26,676 T2D case and 132,532 control subjects of European ancestry after imputation using the 1000 Genomes multiethnic reference panel. Promising association signals were followed up in additional data sets (of 14,545 or 7,397 T2D case and 38,994 or 71,604 control subjects). We identified 13 novel T2D-associated loci (P < 5 × 10-8), including variants near the GLP2R, GIP, and HLA-DQA1 genes. Our analysis brought the total number of independent T2D associations to 128 distinct signals at 113 loci. Despite substantially increased sample size and more complete coverage of low-frequency variation, all novel associations were driven by common single nucleotide variants. Credible sets of potentially causal variants were generally larger than those based on imputation with earlier reference panels, consistent with resolution of causal signals to common risk haplotypes. Stratification of T2D-associated loci based on T2D-related quantitative trait associations revealed tissue-specific enrichment of regulatory annotations in pancreatic islet enhancers for loci influencing insulin secretion and in adipocytes, monocytes, and hepatocytes for insulin action-associated loci. These findings highlight the predominant role played by common variants of modest effect and the diversity of biological mechanisms influencing T2D pathophysiology.

Type 2 diabetes and cardiovascular disease are complex disorders involving metabolic and inflammatory mechanisms. Here we investigated whether sCD93, a group XIV c-type lectin of the endosialin family, plays a role in metabolic dysregulation or carotid intima-media thickness (IMT). Although no association was observed between sCD93 and IMT, sCD93 levels were significantly lower in subjects with type 2 diabetes (n = 901, mean ± SD 156.6 ± 40.0 ng/mL) compared with subjects without diabetes (n = 2,470, 164.1 ± 44.8 ng/mL, P < 0.0001). Genetic variants associated with diabetes risk (DIAGRAM Consortium) did not influence sCD93 levels (individually or combined in a single nucleotide polymorphism score). In a prospective cohort, lower sCD93 levels preceded the development of diabetes. Consistent with this, a cd93-deficient mouse model (in addition to apoe deficiency) demonstrated no difference in atherosclerotic lesion development compared with apoe−/− cd93-sufficient littermates. However, cd93-deficient mice showed impaired glucose clearance and insulin sensitivity (compared with littermate controls) after eating a high-fat diet. The expression of cd93 was observed in pancreatic islets, and leaky vessels were apparent in cd93-deficient pancreases. We further demonstrated that stress-induced release of sCD93 is impaired by hyperglycemia. Therefore, we propose CD93 as an important component in glucometabolic regulation.

IL-5 is a Th2 cytokine which activates eosinophils and is suggested to have an atheroprotective role. Genetic variants in the IL5 locus have been associated with increased risk of CAD and ischemic stroke. In this study we aimed to identify genetic variants associated with IL-5 concentrations and apply a Mendelian randomisation approach to assess IL-5 levels for causal effect on intima-media thickness in a European population at high risk of coronary artery disease. We analysed SNPs within robustly associated candidate loci for immune, inflammatory, metabolic and cardiovascular traits. We identified 2 genetic loci for IL-5 levels (chromosome 5, rs56183820, BETA=0.11, P=6.73E(-5) and chromosome 14, rs4902762, BETA=0.12, P=5.76E(-6)) and one for eosinophil count (rs72797327, BETA=-0.10, P=1.41E(-6)). Both chromosome 5 loci were in the vicinity of the IL5 gene, however the association with IL-5 levels failed to replicate in a meta-analysis of 2 independent cohorts (rs56183820, BETA=0.04, P=0.2763, I(2)=24, I(2)-P=0.2516). No significant associations were observed between SNPs associated with IL-5 levels or eosinophil count and IMT measures. Expression quantitative trait analyses indicate effects of the IL-5 and eosinophil-associated SNPs on RAD50 mRNA expression levels (rs12652920 (r2=0.93 with rs56183820) BETA=-0.10, P=8.64E(-6) and rs11739623 (r2=0.96 with rs72797327) BETA=-0.23, P=1.74E(-29), respectively). Our data do not support a role for IL-5 levels and eosinophil count in intima-media thickness, however SNPs associated with IL-5 and eosinophils might influence stability of the atherosclerotic plaque via modulation of RAD50 levels.

An analysis of 16 health-related quantitative traits in approximately 350,000 individuals reveals statistically significant associations between genome-wide homozygosity and four complex traits (height, lung function, cognitive ability and educational attainment); in each case increased homozygosity associates with a decreased trait value, but no evidence was seen of an influence on blood pressure, cholesterol, or ten other cardio-metabolic traits. This consortium meta-analysis of 102 cohorts and more than 350,000 individuals investigates the effects of homozygosity on traits of public health importance by observing contiguous homozygous segments (runs of homozygosity, ROH), which are inferred to be homozygous along their complete length. Focusing on 16 health-related quantitative traits, the authors find statistically significant associations between summed runs of homozygosity and four complex traits: height, forced expiratory lung volume in a second, general cognitive ability and educational attainment. In each case increased homozygosity associates with decreased trait value. No evidence was seen of an influence of genome-wide homozygosity on blood pressure and low density lipoprotein cholesterol, or ten other cardio-metabolic traits. Homozygosity has long been associated with rare, often devastating, Mendelian disorders1, and Darwin was one of the first to recognize that inbreeding reduces evolutionary fitness2. However, the effect of the more distant parental relatedness that is common in modern human populations is less well understood. Genomic data now allow us to investigate the effects of homozygosity on traits of public health importance by observing contiguous homozygous segments (runs of homozygosity), which are inferred to be homozygous along their complete length. Given the low levels of genome-wide homozygosity prevalent in most human populations, information is required on very large numbers of people to provide sufficient power3,4. Here we use runs of homozygosity to study 16 health-related quantitative traits in 354,224 individuals from 102 cohorts, and find statistically significant associations between summed runs of homozygosity and four complex traits: height, forced expiratory lung volume in one second, general cognitive ability and educational attainment (P < 1 × 10−300, 2.1 × 10−6, 2.5 × 10−10 and 1.8 × 10−10, respectively). In each case, increased homozygosity was associated with decreased trait value, equivalent to the offspring of first cousins being 1.2 cm shorter and having 10 months' less education. Similar effect sizes were found across four continental groups and populations with different degrees of genome-wide homozygosity, providing evidence that homozygosity, rather than confounding, directly contributes to phenotypic variance. Contrary to earlier reports in substantially smaller samples5,6, no evidence was seen of an influence of genome-wide homozygosity on blood pressure and low density lipoprotein cholesterol, or ten other cardio-metabolic traits. Since directional dominance is predicted for traits under directional evolutionary selection7, this study provides evidence that increased stature and cognitive function have been positively selected in human evolution, whereas many important risk factors for late-onset complex diseases may not have been.

Fasting glucose and insulin are intermediate traits for type 2 diabetes. Here we explore the role of coding variation on these traits by analysis of variants on the HumanExome BeadChip in 60,564 non-diabetic individuals and in 16,491 T2D cases and 81,877 controls. We identify a novel association of a low-frequency nonsynonymous SNV in GLP1R (A316T; rs10305492; MAF=1.4%) with lower FG ( β =−0.09±0.01 mmol l −1 , P =3.4 × 10 −12 ), T2D risk (OR[95%CI]=0.86[0.76–0.96], P =0.010), early insulin secretion (β =−0.07±0.035 pmol insulin mmol glucose −1 , P =0.048), but higher 2-h glucose (β =0.16±0.05 mmol l −1 , P =4.3 × 10 −4 ). We identify a gene-based association with FG at G6PC2 ( p SKAT =6.8 × 10 −6 ) driven by four rare protein-coding SNVs (H177Y, Y207S, R283X and S324P). We identify rs651007 (MAF=20%) in the first intron of ABO at the putative promoter of an antisense lncRNA, associating with higher FG (β =0.02±0.004 mmol l −1 , P =1.3 × 10 −8 ). Our approach identifies novel coding variant associations and extends the allelic spectrum of variation underlying diabetes-related quantitative traits and T2D susceptibility.

BackgroundCollagen type IV is the major constituent of basement membranes underlying endothelial cells and is important for endothelial cell attachment and function. Autoantibodies against native collagen type IV have been found in various autoimmune diseases. Oxidation of LDL in the vascular wall results in the formation of reactive aldehydes, which could modify surrounding matrix proteins. Like oxidized LDL, these modified matrix proteins are likely to induce immune responses. We examined whether autoantibodies against native or aldehyde-modified collagen type IV are associated with myocardial infarction.MethodsIgM and IgG against native and aldehyde-modified collagen type IV were measured by ELISA in serum from 387 survivors of a first myocardial infarction and 387 age- and sex-matched controls.ResultsPost-infarction patients had significantly increased levels of IgM against native collagen type IV, and IgG against native collagen type IV was present at detectable level in 17% of patients as opposed to 7% of controls (p<0.001). Controlling for major cardiovascular risk factors demonstrated that the presence of IgG against native collagen type IV was associated with myocardial infarction (OR 2.9 (1.6–5.4), p=0.001). Similarly, subjects in the highest quartile of IgM against native collagen type IV had increased risk of having suffered myocardial infarction (OR 3.11 (1.8–5.4), p<0.001) after adjusting for cardiovascular risk factors. In contrast, IgG against aldehyde-modified collagen type IV was decreased in myocardial infarction patients, but this association was not independent of established cardiovascular risk factors.ConclusionAutoantibodies against collagen type IV are associated with myocardial infarction independently of traditional cardiovascular risk factors.

Objective: Genetic variants robustly associated with coronary artery disease were reported in the vicinity of the interleukin (IL)-5 locus, and animal studies suggested a protective role for IL-5 in atherosclerosis. Therefore, we set this work to explore IL-5 as a plasma biomarker for early subclinical atherosclerosis, as determined by measures of baseline severity and change over time of carotid intima-media thickness (cIMT).Methods: We used biobank and databases of IMPROVE, a large European prospective cohort study of high-risk individuals (n = 3534) free of clinically overt cardiovascular disease at enrollment, in whom composite and segment-specific measures of cIMT were recorded at baseline and after 15 and 30 months. IL-5 was measured with an immunoassay in plasma samples taken at baseline.Results: IL-5 levels were lower in women than in men, lower in the South than in North of Europe, and showed positive correlations with most established risk factors. IL-5 showed significant inverse relationships with cIMT change over time in the common carotid segment in women, but no significant relationships to baseline cIMT in either men or women.Conclusions: Our results suggest that IL-5 may be part of protective mechanisms operating in early atherosclerosis, at least in women. However, the relationships are weak and whereas IL-5 has been proposed as a potential molecular target to treat allergies, it is difficult to envisage such a scenario in coronary artery disease. (C) 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NCND license.

Background Plasma adiponectin levels have previously been inversely associated with carotid intima‐media thickness ( IMT ), a marker of subclinical atherosclerosis. In this study, we used a sex‐stratified Mendelian randomization approach to investigate whether adiponectin has a causal protective influence on IMT . Methods and Results Baseline plasma adiponectin concentration was tested for association with baseline IMT , IMT progression over 30 months, and occurrence of cardiovascular events within 3 years in 3430 participants (women, n=1777; men, n=1653) with high cardiovascular risk but no prevalent disease. Plasma adiponectin levels were inversely associated with baseline mean bifurcation IMT after adjustment for established risk factors (β=−0.018, P <0.001) in men but not in women (β=−0.006, P =0.185; P for interaction=0.061). Adiponectin levels were inversely associated with progression of mean common carotid IMT in men (β=−0.0022, P =0.047), whereas no association was seen in women (0.0007, P =0.475; P for interaction=0.018). Moreover, we observed that adiponectin levels were inversely associated with coronary events in women (hazard ratio 0.57, 95% CI 0.37 to 0.87) but not in men (hazard ratio 0.82, 95% CI 0.54 to 1.25). A gene score of adiponectin‐raising alleles in 6 loci, reported recently in a large multi‐ethnic meta‐analysis, was inversely associated with baseline mean bifurcation IMT in men (β=−0.0008, P =0.004) but not in women (β=−0.0003, P =0.522; P for interaction=0.007). Conclusions This report provides some evidence for adiponectin protecting against atherosclerosis, with effects being confined to men; however, compared with established cardiovascular risk factors, the effect of plasma adiponectin was modest. Further investigation involving mechanistic studies is warranted.

Objective: Experimental studies have suggested that autoimmunity is involved in atherosclerosis and provided evidence that both protective and pro-atherogenic immune responses exist. This concept has received support from small clinical studies implicating autoantibodies directed against apolipoprotein B-100 (apoB-100) in human atherosclerosis. We examined circulating autoantibodies directed against native and malondialdehyde (MDA)-modified epitope p210 of apoB-100 (IgG-p210(nat) and IgM-p210(MDA)) in relation to early atherosclerosis in a large, European longitudinal cohort study of healthy high-risk individuals.Approach and results: IgG-p210(nat) and IgM-p210(MDA) were quantified in baseline plasma samples of 3430 participants in the IMPROVE study and related to composite and segment-specific measures of severity and rate of progression of carotid intima-media thickness (cIMT) determined at baseline and after 30 months. IgM-p210(MDA) autoantibody levels were independently related to several cIMT measures both in the common carotid artery and in the carotid bulb, including measures of cIMT progression, higher levels being associated with lower cIMT or slower cIMT progression. Consistent inverse relationships were also found between plasma levels of IgG-p210(nat) and baseline composite measures of cIMT. These associations disappeared when adjusting for established and emerging risk factors, and there were no associations with rate of cIMT progression besides in certain secondary stratified analyses.Conclusions: The present study provides further evidence of involvement of autoantibodies against native and MDA-modified apoB-100 peptide 210 in cardiovascular disease in humans and demonstrates that these associations are present already at a subclinical stage of the disease. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Objective: Low levels of IgM anti-phosphorylcholine (anti-PC) increase the risk of cardiovascular events (CVE). Here we investigate the association of low anti-PC with the progression of carotid intima media thickness (C-IMT) and incidence of CVE in a large cohort of individuals at high risk of CVE, the IMPROVE, a prospective multicenter European study.Methods: 3711 subjects (54-79 years) with at least three established cardiovascular risk factors were enrolled. Baseline serum levels of IgM anti-PC were measured by ELISA. Carotid ultrasound investigations were performed at baseline and after 15 and 30 months of follow-up. The risk of C-IMT progression and ischemic CVE associated with low anti-PC levels was tested by logistic regression and Cox regression analysis, respectively. Risk estimates were adjusted by center and conventional cardiovascular risk factors.Results: 3670 study participants were included in the present analysis and 213 CVE were recorded during a 3 year follow up. Anti-PC levels (U/ml) were classified into quartiles [Q1 <= 40, Q2 >40-<= 64, Q3 >64- <= 102, Q4 > 102]. In men, low levels of anti-PC (Q1) were associated with the highest (> 90th) percentile of the fastest C-IMT progression, i.e. the segment showing the fastest progression over 30 months in the whole carotid tree, with an OR of 1.41 (95%CI, 1.02-1.9) and with an increased risk of CVE with a multivariable adjusted HR of 1.85 (95%CI, 1.1-3.1). No significant associations were found in women.Conclusions: Low anti-PC levels increase the risk of CVE in men. This effect may be partly mediated by a fast C-IMT progression. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

AIMS/HYPOTHESIS:The findings of studies investigating whether or not low serum 25-hydroxyvitamin D [25(OH)D] concentration promotes development of atherosclerosis have been contradictory. The present study employed a Mendelian randomisation approach and carotid artery intima-media thickness (cIMT), a surrogate marker of coronary artery disease, to address this question. METHODS:The multicentre, longitudinal Carotid Intima-Media Thickness and IMT-Progression as Predictors of Vascular Events in a High-Risk European Population (IMPROVE) cohort study, which enrolled individuals with at least three cardiovascular risk factors and no history or symptoms of cardiovascular disease, was used for the present investigation. Participants underwent carotid ultrasound examination at baseline and at months 15 and 30. Six single nucleotide polymorphisms (SNPs) associated with serum 25(OH)D concentration in genome-wide association studies were identified and genotyped in 3,418 individuals, of whom 929 had type 2 diabetes. RESULTS:SNPs in the genes encoding vitamin D binding protein (GC; rs2282679 and rs7041) and 7-dehydrocholesterol reductase/NAD synthetase-1 (DHCR7; rs12785878 and rs3829251) were negatively associated with 25(OH)D levels. Effect sizes and significance of associations between SNPs and 25(OH)D levels differed between individuals with and without type 2 diabetes, although no significant interactions were observed. A SNP in DHCR7 interacted with type 2 diabetes to significantly influence progression of cIMT measures independent of 25(OH)D levels and established risk factors. Expression analysis demonstrated that this SNP modulates DHCR7 mRNA levels in aortic adventitia. CONCLUSIONS/INTERPRETATION:SNPs in GC and DHCR7 were associated with serum levels of 25(OH)D, but only rs3829251 (DHCR7) influenced progression of subclinical atherosclerosis, as measured by cIMT, in a manner dependent on type 2 diabetes status but independent of 25(OH)D levels.