Alternative Splicing of FOXP3 Controls Regulatory T Cell Effector Functions and Is Associated With Human Atherosclerotic Plaque Stability.

RATIONALE: Regulatory T (Treg) cells suppress immune responses and have been shown to attenuate atherosclerosis. The Treg cell lineage-specification factor FOXP3 (forkhead box P3) is essential for Treg cells' ability to uphold immunologic tolerance. In humans, FOXP3 exists in several different isoforms, however, their specific role is poorly understood. OBJECTIVE: To define the regulation and functions of the 2 major FOXP3 isoforms, FOXP3fl and FOXP3Δ2, as well as to establish whether their expression is associated with the ischemic atherosclerotic disease. METHODS AND RESULTS: Human primary T cells were transduced with lentiviruses encoding distinct FOXP3 isoforms. The phenotype and function of these cells were analyzed by flow cytometry, in vitro suppression assays and RNA-sequencing. We also assessed the effect of activation on Treg cells isolated from healthy volunteers. Treg cell activation resulted in increased FOXP3 expression that predominantly was made up of FOXP3Δ2. FOXP3Δ2 induced specific transcription of GARP (glycoprotein A repetitions predominant), which functions by tethering the immunosuppressive cytokine TGF (transforming growth factor)-ß to the cell membrane of activated Treg cells. Real-time polymerase chain reaction was used to determine the impact of alternative splicing of FOXP3 in relation with atherosclerotic plaque stability in a cohort of >150 patients that underwent carotid endarterectomy. Plaque instability was associated with a lower FOXP3Δ2 transcript usage, when comparing plaques from patients without symptoms and patients with the occurrence of recent (<1 month) vascular symptoms including minor stroke, transient ischemic attack, or amaurosis fugax. No difference was detected in total levels of FOXP3 mRNA between these 2 groups. CONCLUSIONS: These results suggest that activated Treg cells suppress the atherosclerotic disease process and that FOXP3Δ2 controls a transcriptional program that acts protectively in human atherosclerotic plaques.

Increased Levels of Circulating Fatty Acids Are Associated with Protective Effects against Future Cardiovascular Events in Nondiabetics.

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.

Discovering Genetic Interactions in Large-Scale Association Studies by Stage-wise Likelihood Ratio Tests.

Despite the success of genome-wide association studies in medical genetics, the underlying genetics of many complex diseases remains enigmatic. One plausible reason for this could be the failure to account for the presence of genetic interactions in current analyses. Exhaustive investigations of interactions are typically infeasible because the vast number of possible interactions impose hard statistical and computational challenges. There is, therefore, a need for computationally efficient methods that build on models appropriately capturing interaction. We introduce a new methodology where we augment the interaction hypothesis with a set of simpler hypotheses that are tested, in order of their complexity, against a saturated alternative hypothesis representing interaction. This sequential testing provides an efficient way to reduce the number of non-interacting variant pairs before the final interaction test. We devise two different methods, one that relies on a priori estimated numbers of marginally associated variants to correct for multiple tests, and a second that does this adaptively. We show that our methodology in general has an improved statistical power in comparison to seven other methods, and, using the idea of closed testing, that it controls the family-wise error rate. We apply our methodology to genetic data from the PROCARDIS coronary artery disease case/control cohort and discover three distinct interactions. While analyses on simulated data suggest that the statistical power may suffice for an exhaustive search of all variant pairs in ideal cases, we explore strategies for a priori selecting subsets of variant pairs to test. Our new methodology facilitates identification of new disease-relevant interactions from existing and future genome-wide association data, which may involve genes with previously unknown association to the disease. Moreover, it enables construction of interaction networks that provide a systems biology view of complex diseases, serving as a basis for more comprehensive understanding of disease pathophysiology and its clinical consequences.

Genetic loci on chromosome 5 are associated with circulating levels of interleukin-5 and eosinophil count in a European population with high risk for cardiovascular disease.

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.

ACE-Inhibition Benefit on Lung Function in Heart Failure is Modulated by ACE Insertion/Deletion Polymorphism.

PURPOSE: The benefit of angiotensin converting enzyme (ACE) inhibition in chronic heart failure (HF) is partially due to its effects on pulmonary function and particularly on lung diffusion, the latter being counteracted by acetylsalicylic acid (ASA). Tissue ACE activity is largely determined by an insertion/deletion (I/D) polymorphism resulting in three possible genotypes (DD, ID and II). It is not clear if ACE inhibitor therapy could exert different effects in these genotypes. The aim of the study was to understand whether I/D polymorphism interferes with ACE inhibitor's protection of the lungs in HF during acute fluid overload. METHODS: 100 HF patients (left ventricular ejection fraction ≤40 %) in stable clinical conditions, treated with enalapril but without ASA performed pulmonary function tests including lung diffusion (DLco) and its subcomponents, membrane diffusion (Dm) and capillary volume (Vcap), and a cardiopulmonary exercise test before and immediately after rapid infusion of 500 cc saline. RESULTS: ACE I/D genotype prevalence was: DD = 28, ID =55 and II = 17 cases. No significant differences in major pulmonary function and exercise parameters were observed before saline infusion among ACE genotypes. After fluid challenge, DD patients presented a higher DLco and Dm reduction than ID and II (DLco -2.3 ± 1.3 vs. -0.8 ± 1.9 and -0.6 ± 1 mL/mmHg/min, p < 0.0001 and p < 0.01; Dm -7 ± 5 vs. -3.2 ± 7.4 and -1.3 ± 5 mL/mmHg/min, p < 0.05, respectively) and a higher increase in VE/VCO2 slope than II (1.8 ± 1.9 vs. -0.8 ± 2.3, p = 0.01). CONCLUSIONS: ACE DD genotype is associated with higher vulnerability of the alveolar-capillary membrane to acute fluid overload in HF patients treated with ACE inhibitors.

A serum 25-hydroxyvitamin D concentration-associated genetic variant in DHCR7 interacts with type 2 diabetes status to influence subclinical atherosclerosis (measured by carotid intima-media thickness).

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.

Human genetic evidence for involvement of CD137 in atherosclerosis.

Atherosclerosis is an inflammatory disease and the main cause of cardiovascular disease. Inflammation promotes plaque instability and clinical disease, such as myocardial infarction, stroke and peripheral vascular disease. Subclinical atherosclerosis begins with thickening of the arterial intimal layer, and increased intima-media thickness (IMT) in the carotid artery is a widely used measurement of subclinical atherosclerosis. Activation of CD137 (tumor necrosis factor receptor super family 9) promotes inflammation and disease development in murine atherosclerosis. CD137 is expressed in human atherosclerosis, but its role is largely unknown. This study uses a genetic approach to investigate CD137 in human atherosclerotic disease. In publicly available data on genotype and gene expression from the HapMap project, the minor T allele of rs2453021, a single nucleotide polymorphism in CD137, was significantly associated with CD137 gene expression. In the PROCARDIS and Wellcome Trust Case Control Consortium (WTCCC) cohorts of 13,029 cases and controls, no significant association was detected between the minor T allele of rs2453021 and risk for coronary artery disease or myocardial infarction. However, in the IMPROVE multicenter study of 3,418 individuals, the minor T allele of rs2453021 was associated with increased IMT of the common carotid artery (CCA), as measured by ultrasonography, with presence of plaque in CCA and with increased incidence of adverse noncardiac vascular events. Taken together, this study shows that the minor T allele of rs2453021 is associated with increased IMT in the CCA and increased risk of incident noncardiac vascular events, thus providing the first human genetic evidence for involvement of CD137 in atherosclerosis.

Serum 25-hydroxyvitamin D concentration in subclinical carotid atherosclerosis.

OBJECTIVE: Vitamin D deficiency has been implicated in cardiovascular disease and is associated with multiple cardiovascular risk factors. We investigated the serum 25-hydroxyvitamin D (25(OH)D) concentration in relation to latitude, baseline carotid intima-media thickness (IMT), and IMT progression, the carotid IMT measures being surrogate markers of subclinical atherosclerosis and cardiovascular disease risk. APPROACH AND RESULTS: Serum 25(OH)D concentration was related to high-resolution carotid IMT measures in 3430 middle-aged and elderly subjects with high cardiovascular risk but no prevalent disease, who were recruited at 7 centers in Finland, Sweden, The Netherlands, France, and Italy. Participants underwent carotid ultrasound examination at baseline and at months 15 and 30 after entry into the study, whereas blood samples, clinical data, and information about lifestyle were collected at baseline. Serum 25(OH)D levels were positively associated with latitude (Jonckheere-Terpstra χ=166.643; P<0.001) and, as previously reported, associated with a range of cardiovascular risk factors. There were no independent relationships between 25(OH)D and segment-specific or composite IMT measures in the entire cohort. In analyses stratified by sex, diabetes mellitus, and statin treatment, weak associations with some baseline and progression measures of carotid IMT were observed in males, diabetics, and nonstatin-treated individuals. CONCLUSIONS: Levels of 25(OH)D differed across Europe, were highest in the North, showed multiple associations with established and emerging cardiovascular risk factors but were not consistently, independently related to measures of carotid IMT. This argues against a protective role of vitamin D against subclinical atherosclerosis in high-risk individuals.

Healthcare resource utilisation and direct medical cost for individuals with 5q spinal muscular atrophy in Sweden.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare, progressive, neuromuscular disorder. Recent advances in treatment require an updated assessment of burden to inform reimbursement decisions. OBJECTIVES: To quantify healthcare resource utilisation (HCRU) and cost of care for patients with SMA. METHODS: Cohort study of patients with SMA identified in the Swedish National Patient Registry (2007-2018), matched to a reference cohort grouped into four SMA types (1, 2, 3, unspecified adult onset [UAO]). HCRU included inpatient admissions, outpatient visits, procedures, and dispensed medications. Direct medical costs were estimated by multiplying HCRU by respective unit costs. Average annual HCRU and medical costs were modelled for SMA versus reference cohorts to estimate differences attributable to the disease (i.e., average treatment effect estimand). The trajectory of direct costs over time were assessed using synthetic cohorts. RESULTS: We identified 290 SMA patients. Annualised HCRU was higher in SMA patients compared with reference cohorts. Highest risk ratios were observed for inpatient overnight stays for type 1 (risk ratio [RR]: 29.2; 95% confidence interval [CI]: 16.0, 53.5) and type 2 (RR: 23.3; 95% CI: 16.4,33.1). Mean annual direct medical costs per patient for each year since first diagnosis were greatest for type 1 (€114,185 and SMA-attributable: €113,380), type 2 (€61,876 and SMA-attributable: €61,237), type 3 (€45,518 and SMA-attributable: €44,556), and UAO (€4046 and SMA-attributable: €2098). Costs were greatest in the 2-3 years after the first diagnosis for all types. DISCUSSION AND CONCLUSION: The economic burden attributable to SMA is significant. Further research is needed to understand the burden in other European countries and the impact of new treatments.

Fatty acid binding protein 4 in circulating leucocytes reflects atherosclerotic lesion progression in Apoe(-/-) mice.

Discovery of novel biomarkers for atherosclerosis is important to aid in early diagnosis of pre-symptomatic patients at high risk of cardiovascular events. The aim of the present study was therefore to identify potential biomarkers in circulating cells reflecting atherosclerotic lesion progression in the vessel wall. We performed gene arrays on circulating leucocytes from atherosclerosis prone Apoe(-/-) mice with increasing ages, using C57BL/6 mice as healthy controls. We identified fatty acid binding protein 4 (FABP4) mRNA to be augmented in mice with established disease compared with young Apoe(-/-) or controls. Interestingly, the transcript FABP4 correlated significantly with lesion size, further supporting a disease associated increase. In addition, validation of our finding on protein level showed augmented FABP4 in circulating leucocytes whereas, importantly, no change could be observed in plasma. Immunofluorescence analysis demonstrated FABP4 to be present mainly in circulating neutrophils and to some extent in monocytes. Moreover, FABP4-positive neutrophils and macrophages could be identified in the subintimal space in the plaque. Using human circulating leucocytes, we confirmed the presence of FABP4 protein in neutrophils and monocytes. In conclusion, we have showed that cellular levels of FABP4 in circulating leucocytes associate with lesion development in the experimental Apoe(-/-) model. The increased expression is primarily localized to neutrophils, but also in monocytes. We have identified FABP4 in leucocytes as a potential and easy accessible biomarker of atherosclerosis which could be of future clinical relevance.

Tissue factor in patients with acute coronary syndromes: expression in platelets, leukocytes, and platelet-leukocyte aggregates.

OBJECTIVE: Activated platelets and circulating platelet-leukocyte aggregates (PLA) are significantly higher in patients with unstable angina than in those with stable angina (SA). Platelets from healthy subjects express TF on activation. The aim of this study was to investigate the expression of TF in PLA, in platelets, and in monocytes of acute coronary syndrome (ACS) patients compared to SA patients and healthy subjects (Controls). METHODS AND RESULTS: We enrolled 26 consecutive patients with ACS, 29 patients with SA, and 25 Controls. A significantly greater number of total and TF positive platelet-monocyte aggregates was found by flow cytometry in blood of ACS patients than in either SA patients (3-fold) or Controls (5-fold). ACS patients also had a significantly higher amount of TF-positive platelets than SA or Controls (>3-fold) and significantly higher thrombin generation capacity. TF mRNA expression in platelets was significantly higher in ACS patients than in SA or Controls. CONCLUSIONS: In ACS patients the greater expression of TF in platelets and PLA strengthens the link between platelet activation, blood coagulation, and thrombus formation and may further contribute to the hypercoagulability associated with the disease.

Identification of a novel proinsulin-associated SNP and demonstration that proinsulin is unlikely to be a causal factor in subclinical vascular remodelling using Mendelian randomisation.

BACKGROUND AND AIMS: Increased proinsulin relative to insulin levels have been associated with subclinical atherosclerosis (measured by carotid intima-media thickness (cIMT)) and are predictive of future cardiovascular disease (CVD), independently of established risk factors. The mechanisms linking proinsulin to atherosclerosis and CVD are unclear. A genome-wide meta-analysis has identified nine loci associated with circulating proinsulin levels. Using proinsulin-associated SNPs, we set out to use a Mendelian randomisation approach to test the hypothesis that proinsulin plays a causal role in subclinical vascular remodelling. METHODS: We studied the high CVD-risk IMPROVE cohort (n = 3345), which has detailed biochemical phenotyping and repeated, state-of-the-art, high-resolution carotid ultrasound examinations. Genotyping was performed using Illumina Cardio-Metabo and Immuno arrays, which include reported proinsulin-associated loci. Participants with type 2 diabetes (n = 904) were omitted from the analysis. Linear regression was used to identify proinsulin-associated genetic variants. RESULTS: We identified a proinsulin locus on chromosome 15 (rs8029765) and replicated it in data from 20,003 additional individuals. An 11-SNP score, including the previously identified and the chromosome 15 proinsulin-associated loci, was significantly and negatively associated with baseline IMTmean and IMTmax (the primary cIMT phenotypes) but not with progression measures. However, MR-Eggers refuted any significant effect of the proinsulin-associated 11-SNP score, and a non-pleiotropic SNP score of three variants (including rs8029765) demonstrated no effect on baseline or progression cIMT measures. CONCLUSIONS: We identified a novel proinsulin-associated locus and demonstrated that whilst proinsulin levels are associated with cIMT measures, proinsulin per se is unlikely to have a causative effect on cIMT.

Functional Analysis of a Carotid Intima-Media Thickness Locus Implicates BCAR1 and Suggests a Causal Variant.

BACKGROUND: Carotid intima-media thickness (IMT) is a marker of subclinical atherosclerosis that can predict cardiovascular disease events over traditional risk factors. This study examined the BCAR1-CFDP1-TMEM170A locus on chromosome 16, associated with carotid IMT and coronary artery disease in the IMT and IMT-Progression as Predictors of Vascular Events (IMPROVE) cohort, to identify the functional variant. METHODS AND RESULTS: In analysis of the locus lead single nucleotide polymorphism (SNP; rs4888378, intronic in CFDP1) in Progressione della Lesione Intimale Carotidea (PLIC), the protective AA genotype was associated with slower IMT progression in women (P=0.04) but not in men. Meta-analysis of 5 cohort studies also supported a protective effect of the A allele on common carotid IMT in women only (women: ß=-0.0047, P=1.63 × 10(-4); men: ß=-0.0029, P=0.0678). Two hundred fourteen noncoding variants in strong linkage disequilibrium (r(2) ≥ 0.8) with rs4888378 were identified from 1000 Genome Project. ENCODE regulatory chromatin marks were used to create a shortlist of 6 possible regulatory variants. Electrophoretic mobility shift assays on the shortlist detected allele-specific protein binding to the lead SNP rs4888378; multiplexed competitor electrophoretic mobility shift assays implicated FOXA as the protein. Luciferase reporter assays on rs4888378 showed a significant 35% to 92% (P=0.0057; P=4.0 × 10(-22)) decrease in gene expression with the A allele. Expression quantitative trait loci analysis confirmed previously reported associations of rs4888378 with BCAR1 in vascular tissues. CONCLUSIONS: Molecular studies suggest the lead SNP as a potentially causal SNP at the BCAR1-CFDP1-TMEM170A locus, and expression quantitative trait loci studies implicate BCAR1 as the causal gene. This variant showed stronger effects on common carotid IMT in women, raising questions about the mechanism of the causal SNP on atherosclerosis.

Plasma IL-5 concentration and subclinical carotid atherosclerosis.

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.

Sex-Specific Effects of Adiponectin on Carotid Intima-Media Thickness and Incident Cardiovascular Disease.

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 (ß=-0.018, P<0.001) in men but not in women (ß=-0.006, P=0.185; P for interaction=0.061). Adiponectin levels were inversely associated with progression of mean common carotid IMT in men (ß=-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 (ß=-0.0008, P=0.004) but not in women (ß=-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.

Expression of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma co-activator 1, IL-6 and CD45 in subcutaneous adipose tissue in lipodystrophy associated with highly active antiretroviral therapy.

OBJECTIVE: To determine the expressions of multiple genes in the subcutaneous adipose tissue of HIV-positive, highly active antiretroviral therapy (HAART)-treated patients with and without lipodystrophy. DESIGN AND METHODS: Real-time polymerase chain reaction was used to measure gene expressions in this cross-sectional study. RESULTS: The messenger RNA concentrations of adipose transcription factors (peroxisome proliferator-activated receptor (PPAR) gamma and delta and sterol regulatory element binding protein 1c) were all significantly lower in the lipodystrophic than the non-lipodystrophic group. The mRNA concentration of PPAR-gamma co-activator 1 (PGC-1), which regulates mitochondrial biogenesis, was lower in the lipodystrophic than the non-lipodystrophic group. The mRNA expression of lipoprotein lipase, acyl coenzyme A synthase and glucose transport protein 4 were significantly lower in the lipodystrophic than the non-lipodystrophic group, but the mRNA concentrations of fatty acid transport and binding proteins were similar in both groups. The mRNA concentrations of IL-6 and CD45 (a common leukocyte marker) were significantly higher in the lipodystrophic than the non-lipodystrophic group. CONCLUSION: Multiple alterations characterize gene expression in the subcutaneous adipose tissue of patients with HAART-associated lipodystrophy compared with HIV-positive, HAART-treated patients without lipodystrophy. The low expression of transcription factors inhibits adipocyte differentiation. The low expression of PGC-1 may contribute to mitochondrial defects. In addition, IL-6 and CD45 expressions are increased, the latter implying an excessive number of cells of leukocyte origin in lipodystrophic adipose tissue. Mitochondrial injury and an excess of proinflammatory cytokines may lead to increased apoptosis. All these changes may contribute to the loss of subcutaneous fat in HAART-associated lipodystrophy.

Influence of coronary artery disease-associated genetic variants on risk of venous thromboembolism.

INTRODUCTION: We investigated whether genetic variations robustly associated with coronary artery disease are also associated with risk of venous thromboembolism in a well-defined, female case-control study (n=2753) from Sweden. MATERIALS AND METHODS: 39 single nucleotide polymorphisms in 32 loci associated with coronary artery disease in genome-wide association studies were identified in a literature search and genotyped in the ThromboEmbolism Hormone Study (TEHS). Association with venous thromboembolism was assessed by logistic regression. RESULTS: Only rs579459 in the ABO locus demonstrated a significant association with VTE. A tentative association between ANRIL and VTE in the discovery analysis failed to replicate in a meta-analysis of 4 independent cohorts (total n=7181). CONCLUSIONS: It appears that only the ABO locus is a shared risk factor for coronary artery disease and VTE.

Low levels of IgM antibodies against phosphorylcholine are associated with fast carotid intima media thickness progression and cardiovascular risk in men.

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.

Plasma autoantibodies against apolipoprotein B-100 peptide 210 in subclinical atherosclerosis.

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-p210nat and IgM-p210MDA) in relation to early atherosclerosis in a large, European longitudinal cohort study of healthy high-risk individuals. APPROACH AND RESULTS: IgG-p210nat and IgM-p210MDA 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-p210MDA 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-p210nat 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.

Causal relevance of blood lipid fractions in the development of carotid atherosclerosis: Mendelian randomization analysis.

BACKGROUND: Carotid intima-media thickness (CIMT), a subclinical measure of atherosclerosis, is associated with risk of coronary heart disease events. Statins reduce progression of CIMT and coronary heart disease risk in proportion to the reduction in low-density lipoprotein cholesterol. However, interventions targeting triglycerides (TGs) or high-density lipoprotein cholesterol (HDL-C) have produced inconsistent effects on CIMT and coronary heart disease risk, making it uncertain whether such agents are ineffective for coronary heart disease prevention or whether CIMT is an inadequate marker of HDL-C or TG-mediated effects. We aimed to determine the causal association among the 3 major blood lipid fractions and common CIMT using mendelian randomization analysis. METHODS AND RESULTS: Genetic scores specific for low-density lipoprotein cholesterol, HDL-C, and TGs were derived based on single nucleotide polymorphisms from a gene-centric array in ≈5000 individuals (Cardiochip scores) and from a genome-wide association meta-analysis in >100 000 individuals (Global Lipids Genetic Consortium scores). These were used as instruments in a mendelian randomization analysis in 2 prospective cohort studies. A genetically predicted 1 mmol/L higher low-density lipoprotein cholesterol concentration was associated with a higher common CIMT by 0.03 mm (95% confidence interval, 0.01-0.04) and 0.04 mm (95% confidence interval, 0.02-0.06) based on the Cardiochip and Global Lipids Genetic Consortium scores, respectively. HDL-C and TGs were not causally associated with CIMT. CONCLUSIONS: Our findings confirm a causal relationship between low-density lipoprotein cholesterol and CIMT but not with HDL-C and TGs. At present, the suitability of CIMT as a surrogate marker in trials of cardiovascular therapies targeting HDL-C and TGs is questionable and requires further study.