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Background: Atherosclerosis is a chronic inflammatory disease in part caused by lipid uptake in the vascular wall, but the exact underlying mechanisms leading to acute myocardial infarction and stroke remain poorly understood. Large consortia identified genetic susceptibility loci that associate with large artery ischemic stroke and coronary artery disease. However, deciphering their underlying mechanisms are challenging. Histological studies identified destabilizing characteristics in human atherosclerotic plaques that associate with clinical outcome. To what extent established susceptibility loci for large artery ischemic stroke and coronary artery disease relate to plaque characteristics is thus far unknown but may point to novel mechanisms., Methods: We studied the associations of 61 established cardiovascular risk loci with 7 histological plaque characteristics assessed in 1443 carotid plaque specimens from the Athero-Express Biobank Study. We also assessed if the genotyped cardiovascular risk loci impact the tissue-specific gene expression in 2 independent biobanks, Biobank of Karolinska Endarterectomy and Stockholm Atherosclerosis Gene Expression., Results: A total of 21 established risk variants (out of 61) nominally associated to a plaque characteristic. One variant (rs12539895, risk allele A) at 7q22 associated to a reduction of intraplaque fat, P=5.09×10 -6 after correction for multiple testing. We further characterized this 7q22 Locus and show tissue-specific effects of rs12539895 on HBP1 expression in plaques and COG5 expression in whole blood and provide data from public resources showing an association with decreased LDL (low-density lipoprotein) and increase HDL (high-density lipoprotein) in the blood., Conclusions: Our study supports the view that cardiovascular susceptibility loci may exert their effect by influencing the atherosclerotic plaque characteristics.

Background: Tobacco smoking is a major risk factor for atherosclerotic disease and has been associated with DNA methylation (DNAm) changes in blood cells. However, whether smoking influences DNAm in the diseased vascular wall is unknown but may prove crucial in understanding the pathophysiology of atherosclerosis. In this study, we associated current tobacco smoking to epigenome-wide DNAm in atherosclerotic plaques from patients undergoing carotid endarterectomy., Methods: DNAm at commonly methylated sites (cytosine-guanine nucleotide pairs separated by a phospho-group [CpGs]) was assessed in atherosclerotic plaque samples and peripheral blood samples from 485 carotid endarterectomy patients. We tested the association of current tobacco smoking with DNAm corrected for age and sex. To control for bias and inflation because of cellular heterogeneity, we applied a Bayesian method to estimate an empirical null distribution as implemented by the R package bacon. Replication of the smoking-associated methylated CpGs in atherosclerotic plaques was executed in the second sample of 190 carotid endarterectomy patients, and results were meta-analyzed using a fixed-effects model., Results: Tobacco smoking was significantly associated to differential DNAm in atherosclerotic lesions of 4 CpGs (false discovery rate <0.05) mapped to 2 different genes ( AHRR, ITPK1) and 17 CpGs mapped to 8 genes and RNAs in blood. The strongest associations were found for CpGs mapped to the gene AHRR, a repressor of the aryl hydrocarbon receptor transcription factor involved in xenobiotic detoxification. One of these methylated CpGs were found to be regulated by local genetic variation., Conclusions: The risk factor tobacco smoking associates with DNAm at multiple loci in carotid atherosclerotic lesions. These observations support further investigation of the relationship between risk factors and epigenetic regulation in atherosclerotic disease.

Background and Aims: Mitochondrial damage and augmented production of reactive oxygen species (ROS) may represent an intermediate step by which hypercholesterolemia exacerbates atherosclerotic lesion formation., Methods: To test this hypothesis, in mice with severe but genetically reversible hypercholesterolemia (i.e. the so called Reversa mouse model), we performed time-resolved analyses of mitochondrial transcriptome in the aortic arch employing a systems-level network approach., Results: During hypercholesterolemia, we observed a massive down-regulation (>28%) of mitochondrial genes, specifically at the time of rapid atherosclerotic lesion expansion and foam cell formation, i.e. between 30 and 40 weeks of age. Both phenomena - down-regulation of mitochondrial genes and lesion expansion - were largely reversible by genetically lowering plasma cholesterol (by >80%, from 427 to 54 ± 31 mg/L) at 30 weeks. Co-expression network analysis revealed that both mitochondrial signature genes were highly connected in two modules, negatively correlating with lesion size and supported as causal for coronary artery disease (CAD) in humans, as expression-associated single nucleotide polymorphisms (eSNPs) representing their genes overlapped markedly with established disease risk loci. Within these modules, we identified the transcription factor estrogen related receptor (ERR)-α and its co-factors PGC1-α and -β, i.e. two members of the peroxisome proliferator-activated receptor γ co-activator 1 family of transcription regulators, as key regulatory genes. Together, these factors are known as major orchestrators of mitochondrial biogenesis and antioxidant responses., Conclusions: Using a network approach, we demonstrate how hypercholesterolemia could hamper mitochondrial activity during atherosclerosis progression and pinpoint potential therapeutic targets to counteract these processes., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: As genome-wide association efforts, such as CARDIoGRAM and METASTROKE, are ongoing to reveal susceptibility loci for their underlying disease-atherosclerotic disease-identification of candidate genes explaining the associations of these loci has proven the main challenge. Many disease susceptibility loci colocalize with DNA regulatory elements, which influence gene expression through chromatin interactions. Therefore, the target genes of these regulatory elements can be considered candidate genes. Applying these biological principles, we used an alternative approach to annotate susceptibility loci and identify candidate genes for human atherosclerotic disease based on circular chromosome conformation capture followed by sequencing., Methods and Results: In human monocytes and coronary endothelial cells, we generated 63 chromatin interaction data sets for 37 active DNA regulatory elements that colocalize with known susceptibility loci for coronary artery disease (CARDIoGRAMplusC4D) and large artery stroke (METASTROKE). By circular chromosome conformation capture followed by sequencing, we identified a physical 3-dimensional interaction with 326 candidate genes expressed in at least 1 of these cell types, of which 294 have not been reported before. We highlight 16 genes based on expression quantitative trait loci., Conclusions: Our findings provide additional candidate-gene annotation for 37 disease susceptibility loci for human atherosclerotic disease that are of potential interest to better understand the complex pathophysiology of cardiovascular diseases., (© 2017 American Heart Association, Inc.)

Objective: Recently, poliovirus receptor-related 2 ( Pvrl2 ) emerged as a top gene in a global gene expression study aiming to detect plasma cholesterol-responsive genes causally related to atherosclerosis regression in hypercholesterolemic mice. PVRL2 is an adherens junction protein implied to play a role in transendothelial migration of leukocytes, a key feature in atherosclerosis development. In this study, we investigated the effect of Pvrl2 deficiency on atherosclerosis development and transendothelial migration of leukocytes activity. APPROACH AND RESULTS: Pvrl2 -deficient mice bred onto an atherosclerosis-prone background ( Pvrl2 -/- Ldlr -/- during atherosclerosis progression coincided with an increased gene expression of migrating leukocytes into the vessel wall. Both in human and mice, gene and protein expression of PVRL2 was predominantly observed in the vascular endothelium according to the immunohistochemical and gene expression data. In addition, the cholesterol responsiveness of Apob 100/100 ) had less atherosclerotic lesions and more stable plaques compared with littermate controls ( Pvrl2 +/+ Ldlr -/- Apob 100/100 ). Pvrl2 -/- Ldlr -/- Apob 100/100 mice also showed a 49% decrease in transendothelial migration of leukocytes activity observed using the in vivo air pouch model. In accordance, augmented arterial wall expression of Pvrl2 during atherosclerosis progression coincided with an increased gene expression of migrating leukocytes into the vessel wall. Both in human and mice, gene and protein expression of PVRL2 was predominantly observed in the vascular endothelium according to the immunohistochemical and gene expression data. In addition, the cholesterol responsiveness of PVRL2 was also observed in humans., Conclusions: PVRL2 is a plasma cholesterol-responsive gene acting at endothelial sites of vascular inflammation that could potentially be a new therapeutic target for atherosclerosis prevention through its suggested transendothelial migration of leukocytes modulating activity., (© 2017 American Heart Association, Inc.)

Background: The eicosanoid genes ALOX5, ALOX5AP and LTA4H have been implicated in atherosclerosis. We assessed the impact of common variants in these genes on gene expression, circulating protein levels, and atherosclerotic plaque phenotypes., Methods: We included patients from the Stockholm Atherosclerosis Gene Expression study (STAGE, N = 109), and the Athero-Express Biobank Study (AE, N = 1443). We tested 1453 single-nucleotide variants (SNVs) in ALOX5, ALOX5AP and LTA4H for association with gene expression in STAGE. We also tested these SNVs for association with seven histologically defined plaque phenotypes in the AE (which included calcification, collagen, cellular content, atheroma size, and intraplaque vessel density and hemorrhage)., Results: We replicate a known cis-eQTL (rs6538697, p = 1.96 × 10(-6)) for LTA4H expression in whole blood of patients from STAGE. We found no significant association for any of the SNVs tested with serum levels of ALOX5 or ALOX5AP (p > 5.79 × 10(-4)). For atherosclerotic plaque phenotypes the strongest associations were found for intraplaque vessel density and smooth muscle cells in the ALOX5AP locus (p > 1.67 × 10(-4))., Conclusions: We replicate a known eQTL for LTA4H expression in whole blood using STAGE data. We found no associations of variants in and around ALOX5, ALOX5AP and LTA4H with serum ALOX5 or ALOX5AP levels, or plaque phenotypes. On the supposition that these genes play a causal role in atherosclerosis, these results suggest that common variants in these loci play a limited role (if any) in influencing advanced atherosclerotic plaque morphology to the extent that it impacts atherosclerotic disease., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr(-/-)Apob (100/100) Mttp (flox/flox)Mx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions.

ObjectiveGenome-wide association studies have to date identified 159 significant and suggestive loci for coronary artery disease (CAD). We now report comprehensive bioinformatics analyses of sequence variation in these loci to predict candidate causal genes.Approach and resultsAll annotated genes in the loci were evaluated with respect to protein-coding single-nucleotide polymorphism and gene expression parameters. The latter included expression quantitative trait loci, tissue specificity, and miRNA binding. High priority candidate genes were further identified based on literature searches and our experimental data. We conclude that the great majority of causal variations affecting CAD risk occur in noncoding regions, with 41% affecting gene expression robustly versus 6% leading to amino acid changes. Many of these genes differed from the traditionally annotated genes, which was usually based on proximity to the lead single-nucleotide polymorphism. Indeed, we obtained evidence that genetic variants at CAD loci affect 98 genes which had not been linked to CAD previously.ConclusionsOur results substantially revise the list of likely candidates for CAD and suggest that genome-wide association studies efforts in other diseases may benefit from similar bioinformatics analyses.

Supplemental Digital Content is available in the text. Background: Tobacco smoking is a major risk factor for atherosclerotic disease and has been associated with DNA methylation (DNAm) changes in blood cells. However, whether smoking influences DNAm in the diseased vascular wall is unknown but may prove crucial in understanding the pathophysiology of atherosclerosis. In this study, we associated current tobacco smoking to epigenome-wide DNAm in atherosclerotic plaques from patients undergoing carotid endarterectomy. Methods: DNAm at commonly methylated sites (cytosine-guanine nucleotide pairs separated by a phospho-group [CpGs]) was assessed in atherosclerotic plaque samples and peripheral blood samples from 485 carotid endarterectomy patients. We tested the association of current tobacco smoking with DNAm corrected for age and sex. To control for bias and inflation because of cellular heterogeneity, we applied a Bayesian method to estimate an empirical null distribution as implemented by the R package bacon. Replication of the smoking-associated methylated CpGs in atherosclerotic plaques was executed in the second sample of 190 carotid endarterectomy patients, and results were meta-analyzed using a fixed-effects model. Results: Tobacco smoking was significantly associated to differential DNAm in atherosclerotic lesions of 4 CpGs (false discovery rate <0.05) mapped to 2 different genes (AHRR , ITPK1) and 17 CpGs mapped to 8 genes and RNAs in blood. The strongest associations were found for CpGs mapped to the gene AHRR , a repressor of the aryl hydrocarbon receptor transcription factor involved in xenobiotic detoxification. One of these methylated CpGs were found to be regulated by local genetic variation. Conclusions: The risk factor tobacco smoking associates with DNAm at multiple loci in carotid atherosclerotic lesions. These observations support further investigation of the relationship between risk factors and epigenetic regulation in atherosclerotic disease. [ABSTRACT FROM AUTHOR]