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Aims Physical activity (PA) is a mainstay of cardiovascular prevention. This study aimed to identify metabolic mediators of PA that protect against the development of atherosclerosis. Methods and results A total of 2160 participants in the LIFE heart study were analysed with data on PA and vascular phenotyping. In a targeted metabolomic approach, 61 metabolites (amino acids and acylcarnitines) were measured using liquid chromatography–tandem mass spectrometry. We investigated the interactions between PA, metabolites and markers of atherosclerosis in order to uncover possible mediation effects. Intended sports activity, but no daily PA, was associated with a lower degree of atherosclerosis, odds ratio (OR) for total atherosclerotic burden of 0.76 (95% confidence interval 0.62–0.94), carotid artery plaque OR 0.79 (0.66–0.96), and peripheral artery disease OR 0.74 (0.56–0.98). Twelve amino acids, free carnitine, five acylcarnitines were associated with sports activity. Of these, eight metabolites were also associated with the degree of atherosclerosis. In the mediation analyses, a cluster of amino acids (arginine, glutamine, pipecolic acid, taurine) were considered as possible mediators of atheroprotection. In contrast, a group of members of the carnitine metabolism (free carnitine, acetyl carnitine, octadecenoyl carnitine) were associated with inactivity and higher atherosclerotic burden. Conclusion Our metabolomic approach, which is integrated into a mediation model, provides transformative insights into the complex metabolic processes involved in atheroprotection. Metabolites with antioxidant and endothelial active properties are believed to be possible mediators of atheroprotection. The metabolomic mediation approach can support the understanding of complex diseases in order to identify targets for prevention and therapy.

Background While numerous interventions effectively interfered with abdominal aortic aneurysm (AAA) formation/progression in preclinical models, none of the successes translated into clinical success. Hence, a systematic exploration of parallel and divergent processes in clinical AAA disease and its 2 primary models (the porcine pancreatic elastase and angiotensin‐II infusion [AngII] murine model) was performed to identify mechanisms relevant for aneurysm disease. Methods and Results This study combines Movat staining and pathway analysis for histological and genomic comparisons between clinical disease and its models. The impact of a notable genomic signal for metabolic reprogramming was tested in a rescue trial (AngII model) evaluating the impact of 1‐(4‐pyridinyl)‐3‐(2‐quinolinyl)‐2‐propen‐1‐one (PFK15)‐mediated interference with main glycolytic switch PFKFB3. Histological evaluation characterized the AngII model as a dissection model that is accompanied by adventitial fibrosis. The porcine pancreatic elastase model showed a transient inflammatory response and aortic dilatation, followed by stabilization and fibrosis. Normalization of the genomic responses at day 14 confirmed the self‐limiting nature of the porcine pancreatic elastase model. Clear parallel genomic responses with activated adaptive immune responses, and particularly strong signals for metabolic switching were observed in human AAA and the AngII model. Rescue intervention with the glycolysis inhibitor PFK15 in the AngII model showed that interference with the glycolytic switching quenches aneurysm formation. Conclusions Despite clear morphological contrasts, remarkable genomic parallels exist for clinical AAA disease and the AngII model. The metabolic response appears causatively involved in AAA progression and provides a novel therapeutic target. The clear transient genomic response classifies the porcine pancreatic elastase model as a disease initiation model.

Background: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. Methods: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreER T2 -driven)–specific or smooth muscle cell (SMC, SmmhcCreER T2 - or TaglnCre-driven)–specific deficiency of CXCR4 in an apolipoprotein E–deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/β-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. Results: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12–15) or SMCs (n=13–24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/β-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. Conclusions: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Objective— ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase releasing different types of membrane-bound proteins, including adhesion molecules, cytokines, and their receptors as well as inflammatory mediators. Because these substrates modulate important mechanisms of atherosclerosis, we hypothesized that ADAM17 might be involved in the pathogenesis of this frequent disease. Approach and Results— Because Adam17 -knockout mice are not viable, we studied the effect of Adam17 deficiency on atherosclerosis in Adam17 hypomorphic mice ( Adam17 ex/ex ), which have low residual Adam17 expression. To induce atherosclerosis, mice were crossed onto the low-density lipoprotein receptor ( Ldlr )-deficient background. We found that Adam17 ex/ex .Ldlr −/− mice developed ≈1.5-fold larger atherosclerotic lesions, which contained more macrophages and vascular smooth muscle cells than wild-type littermate controls ( Adam17 wt/wt .Ldlr −/− ). Reduced Adam17 -mediated shedding led to significantly increased protein levels of membrane-resident TNFα (tumor necrosis factor) and TNFR2 (tumor necrosis factor receptor 2), resulting in a constitutive activation of TNFR2 signaling. At the same time, Adam17 deficiency promoted proatherosclerotic cellular functions, such as increased proliferation and reduced apoptosis in cultured macrophages and vascular smooth muscle cells and increased adhesion of macrophages to vascular endothelial cells. Because siRNA (small interfering RNA)-mediated knockdown of Tnfr2 rescued from aberrant proliferation and from misregulation of apoptosis in Adam17 -depleted cells, our data indicate that TNFR2 is an important effector of ADAM17 in our mouse model. Conclusions— Our results provide evidence for an atheroprotective role of ADAM17, which might be mediated by cleaving membrane-bound TNFα and TNFR2, thereby preventing overactivation of endogenous TNFR2 signaling in cells of the vasculature.

Objective— Cholesterol homeostasis is fundamental to human health and is, thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. Approach and Results— Here, we identify oxysterol-binding protein-like 6 ( OSBPL6 ) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by liver X receptor and in response to cholesterol loading and in mice and nonhuman primates by Western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains dual membrane- and endoplasmic reticulum–targeting motifs. Subcellular localization studies showed that ORP6 is associated with the endolysosomal network and endoplasmic reticulum, suggesting a role for ORP6 in cholesterol trafficking between these compartments. Accordingly, knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures, resulting in reduced cholesterol esterification at the endoplasmic reticulum. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of high-density lipoprotein cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. Conclusions— These studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

Background Despite the widespread use of noninvasive testing prior to invasive coronary diagnostic the diagnostic yield of elective coronary angiography has been reported low in subjects with suspected obstructive CAD. Objective To determine the predictive value of noncoronary atherosclerosis (NCA) in subjects with suspected stable coronary artery disease (CAD) intended to invasive coronary angiography. Methods Ultrasound-based assessment of carotid artery plaque (CAP), carotid intima-media thickness (CIMT) and ankle-brachial index (ABI) was performed in 2216 subjects with suspected CAD prior to coronary angiography. Logistic regression and c-statistics were used to analyze the diagnostic value of NCA for the presence of obstructive CAD and the intention to revascularization. Results Percentage of positive results of elective coronary angiography was low but comparable to other studies (41 % obstructive CAD). We identified 1323 subjects (60 %) with NCA, most of them were characterized by CAP (93 %). CAP independently predicted obstructive CAD in addition to traditional risk factors and clinical factors while CIMT and ABI failed to improve the prediction. The presence of NCA and typical angina were the strongest predictors for obstructive CAD (OR 4.0 and 2.4, respectively). A large subgroup of patients (n = 703, 32 %) with atypical clinical presentation and lack of NCA revealed a low indication for revascularization

Background Clinical decision making in abdominal aortic aneurysms (AAA) relies completely on diameter. At this point, improved decision tools remain an unmet medical need. Our goal was to identify changes at the molecular level specifically leading up to AAA rupture. Methods and Results Aortic wall tissue specimens were collected during open elective (e AAA ; n=31) or emergency repair of ruptured AAA (r AAA ; n=17), and gene expression was investigated using microarrays. Identified candidate genes were validated with quantitative real‐time polymerase chain reaction in an independent sample set (e AAA : n=46; r AAA : n=18). Two gene sets were identified, 1 set containing 5 genes linked to terminal progression, that is, positively associated with progression of larger AAA , and with rupture ( HILPDA , ANGPTL 4 , LOX , SRPX 2 , FCGBP ), and a second set containing 5 genes exclusively upregulated in rAAA ( ADAMTS 9 , STC 1 , GFPT 2 , GAL 3 ST 4 , CCL 4L1 ). Genes in both sets essentially associated with processes related to impaired tissue remodeling, such as angiogenesis and adipogenesis. In gene expression experiments we were able to show that upregulated gene expression for identified candidate genes is unique for AAA . Functionally, the selected upregulated factors converge at processes coordinated by the canonical HIF ‐1α signaling pathway and are highly expressed in fibroblasts but not inflammatory cells of the aneurysmatic wall. Histological quantification of angiogenesis and exploration of the HIF ‐1α network in r AAA versus e AAA shows enhanced microvessel density but also clear activation of the HIF ‐1α network in r AAA . Conclusions Our study shows a specific molecular fingerprint for terminal AAA disease. These changes appear to converge at activation of HIF ‐1α signaling in mesenchymal cells. Aspects of this cascade might represent targets for rupture risk assessment.

Coronary artery disease (CAD) has a complex etiology involving numerous environmental and genetic factors of disease risk. To date, the genetic 9p21 locus represents the most robust genetic finding for prevalent and incident CAD. However, limited information is available on the genetic background of the severity and distribution of CAD. CAD manifests itself as stable CAD or acute coronary syndrome. The Gensini score quantifies the extent CAD but requires coronary angiography. Here, we aimed to identify novel genetic variants associated with Gensini score severity and distribution of CAD. A two-stage approach including a discovery and a replication stage was used to assess genetic variants. In the discovery phase, a meta-analysis of genome-wide association data of 4,930 CAD-subjects assessed by the Gensini score was performed. Selected single nucleotide polymorphisms (SNPs) were replicated in 2,283 CAD-subjects by de novo genotyping. We identified genetic loci located on chromosome 2 and 9 to be associated with Gensini score severity and distribution of CAD in the discovery stage. Although the loci on chromosome 2 could not be replicated in the second stage, the known CAD-locus on chromosome 9p21, represented by rs133349, was identified and, thus, was confirmed as risk locus for CAD severity.

Background and aims In a previous work, a female-specific atherosclerosis risk locus on chromosome (Chr) 3 was identified in an intercross of atherosclerosis-resistant FVB and atherosclerosis-susceptible C57BL/6 (B6) mice on the LDL-receptor deficient (Ldlr−/−) background. It was the aim of the current study to identify causative genes at this locus. Methods We established a congenic mouse model, where FVB.Chr3B6/B6 mice carried an 80 Mb interval of distal Chr3 on an otherwise FVB.Ldlr−/− background, to validate the Chr3 locus. Candidate genes were identified using genome-wide expression analyses. Differentially expressed genes were validated using quantitative PCRs in F0 and F2 mice and their functions were investigated in pathophysiologically relevant cells. Results Fine-mapping of the Chr3 locus revealed two overlapping, yet independent subloci for female atherosclerosis susceptibility: when transmitted by grandfathers to granddaughters, the B6 risk allele increased atherosclerosis and downregulated the expression of the secreted phospholipase Pla2g12a (2.6 and 2.2 fold, respectively); when inherited by grandmothers, the B6 risk allele induced vascular cell adhesion molecule 1 (Vcam1). Down-regulation of Pla2g12a and up-regulation of Vcam1 were validated in female FVB.Chr3B6/B6 congenic mice, which developed 2.5 greater atherosclerotic lesions compared to littermate controls (p=0.039). Pla2g12a was highly expressed in aortic endothelial cells in vivo, and knocking-down Pla2g12a expression by RNAi in cultured vascular endothelial cells or macrophages increased their adhesion to ECs in vitro. Conclusions Our data establish Pla2g12a as an atheroprotective candidate gene in mice, where high expression levels in ECs and macrophages may limit the recruitment and accumulation of these cells in nascent atherosclerotic lesions.

Rationale : Quantitative trait locus mapping of an intercross between C57. Apoe −/− and FVB. Apoe −/− mice revealed an atherosclerosis locus controlling aortic root lesion area on proximal chromosome 10, Ath11 . In a previous work, subcongenic analysis showed Ath11 to be complex with proximal (10a) and distal (10b) regions. Objective : To identify the causative genetic variation underlying the atherosclerosis modifier locus Ath11 10b. Methods and Results: We now report subcongenic J, which narrows the 10b region to 5 genes, Myb , Hbs1L , Aldh8a1 , Sgk1 , and Raet1e. Sequence analysis of these genes revealed no amino acid coding differences between the parental strains. However, comparing aortic expression of these genes between F1. Apoe −/− Chr10SubJ (B/F) and F1. Apoe −/− Chr10SubJ (F/F) uncovered a consistent difference only for Raet1e , with decreased, virtually background, expression associated with increased atherosclerosis in the latter. The key role of Raet1e was confirmed by showing that transgene-induced aortic overexpression of Raet1e in F1. Apoe −/− Chr10SubJ (F/F) mice decreased atherosclerosis. Promoter reporter constructs comparing C57 and FVB sequences identified an FVB mutation in the core of the major aortic transcription start site abrogating activity. Conclusions : This nonbiased approach has revealed Raet1e , a major histocompatibility complex class 1–like molecule expressed in lesional aortic endothelial cells and macrophage-rich regions, as a novel atherosclerosis gene and represents one of the few successes of the quantitative trait locus strategy in complex diseases.

Purpose of review Since 2007, genome-wide association studies (GWAS) have led to the identification of numerous loci of atherosclerotic cardiovascular disease. The majority of these loci harbor genes previously not known to be involved in atherogenesis. In this review, we summarize the recent progress in understanding the pathophysiology of genetic variants in atherosclerosis. Recent findings Fifty-eight loci with P

Rationale: Currently, there are no blood-based biomarkers with clinical utility for acute ischemic stroke (IS). MicroRNAs show promise as disease markers because of their cell type–specific expression patterns and stability in peripheral blood. Objective: To identify circulating microRNAs associated with acute IS, determine their temporal course up to 90 days post-stroke, and explore their utility as an early diagnostic marker. Methods and Results: We used RNA sequencing to study expression changes of circulating microRNAs in a discovery sample of 20 patients with IS and 20 matched healthy control subjects. We further applied quantitative real-time polymerase chain reaction in independent samples for validation (40 patients with IS and 40 matched controls), replication (200 patients with IS, 100 healthy control subjects), and in 72 patients with transient ischemic attacks. Sampling of patient plasma was done immediately upon hospital arrival. We identified, validated, and replicated 3 differentially expressed microRNAs, which were upregulated in patients with IS compared with both healthy control subjects (miR-125a-5p [1.8-fold; P =1.5×10 −6 ], miR-125b-5p [2.5-fold; P =5.6×10 −6 ], and miR-143-3p [4.8-fold; P =7.8×10 −9 ]) and patients with transient ischemic attack (miR-125a-5p: P =0.003; miR-125b-5p: P =0.003; miR-143-3p: P =0.005). Longitudinal analysis of expression levels up to 90 days after stroke revealed a normalization to control levels for miR-125b-5p and miR-143-3p starting at day 2 while miR-125a-5p remained elevated. Levels of all 3 microRNAs depended on platelet numbers in a platelet spike-in experiment but were unaffected by chemical hypoxia in Neuro2a cells and in experimental stroke models. In a random forest classification, miR-125a-5p, miR-125b-5p, and miR-143-3p differentiated between healthy control subjects and patients with IS with an area under the curve of 0.90 (sensitivity: 85.6%; specificity: 76.3%), which was superior to multimodal cranial computed tomography obtained for routine diagnostics (sensitivity: 72.5%) and previously reported biomarkers of acute IS (neuron-specific enolase: area under the curve=0.69; interleukin 6: area under the curve=0.82). Conclusions: A set of circulating microRNAs (miR-125a-5p, miR-125b-5p, and miR-143-3p) associates with acute IS and might have clinical utility as an early diagnostic marker.

Background and aims Carotid artery plaque is an established marker of subclinical atherosclerosis and common patho-mechanisms with coronary artery disease (CAD) are hypothesized. We aimed to identify genetic variants associated with carotid plaque and to examine the potential shared genetic basis with CAD. Methods After investigating the reliability of plaque detection, we performed a genome-wide meta-association study in two independent cohorts (LIFE-Adult, n = 4037 and LIFE-Heart, n = 3152) for carotid plaque score (PS), defined as the sum of the plaque load of common carotid artery and carotid bulb. Further, we analyzed whether previously reported CAD and stroke loci were also associated with PS. Results We identified two loci with genome-wide significance for PS. One locus is the known CAD-locus at chromosome 9p21 (lead SNP rs9644862, p = 8.73 × 10 −12 ). We also describe a novel locus on chromosome 10q24 within the SFXN2 gene as the most probable candidate (lead SNP rs2902548, p = 1.97 × 10 −8 ). In addition, 17 out of 58 known CAD loci and six of 17 known stroke loci were associated with PS at a nominal level of significance. Conclusions We showed that PS is a reliable trait to analyze genetics of atherosclerosis. Two new loci of genome-wide significant association with PS were found. The observed non-random overlap of CAD and PS associations strengthens the hypothesis of a shared genetic basis for these atherosclerotic manifestations.

The discovery of microRNAs (miRNA) targeting gene pathways involved in HDL and LDL metabolism illuminated the potent role of non-coding RNAs in the regulation of cholesterol homeostasis. Long non-coding RNAs (lncRNA) have also been identified as crucial regulators of gene expression; however, few have been fully characterized. Here we report a novel human lncRNA, CHROME (Cholesterol Homeostasis Regulator Of MicroRNA Expression), that functions as a competing endogenous RNA to regulate cellular cholesterol homeostasis. We show that CHROME has 7 broadly expressed variants that are transcriptionally regulated by the cholesterol-sensing liver X receptors. Computational analyses revealed that CHROME harbors binding sites for multiple (11) miRNAs involved in cholesterol homeostasis, including miR-27b and miR-33a/b, which function as hubs controlling the expression of genes involved in cholesterol efflux and HDL metabolism. Using CHROME knock-down and overexpression, we demonstrate that CHROME acts as a ‘miRNA sponge’ that sequesters these miRNAs, limiting their ability to repress target genes, including ABCA1, OSBPL6 and ANGPTL3. Consistent with this, we show that overexpression of CHROME increases cholesterol efflux, whereas its silencing reduces cholesterol efflux from primary human hepatocytes and macrophages. As hepatic cholesterol efflux via ABCA1 plays a central role in HDL biogenesis, we investigated the relationship of CHROME to its miRNA targets and plasma levels of HDL cholesterol in liver samples from a cohort of 200 healthy individuals. This analysis showed that CHROME is inversely correlated with miR-27b and miR-33a/b levels, and positively correlated with levels of their target genes and plasma HDL cholesterol. Collectively, these findings identify CHROME as a key regulatory component of the non-coding RNA circuitry that controls cellular cholesterol efflux and plasma HDL levels in humans.

Background— Muscle wasting occurs in both chronic heart failure (CHF) and normal aging and contributes to exercise intolerance and increased morbidity/mortality. However, the molecular mechanisms of muscle atrophy in CHF and their interaction with aging are still largely unknown. We therefore measured the activation of the ubiquitin-proteasome system and the lysosomal pathway of intracellular proteolysis in muscle biopsies of CHF patients and healthy controls in two age strata and assessed the age-dependent effects of a 4-week endurance training program on the catabolic-anabolic balance. Methods and Results— Sixty CHF patients (30 patients aged ≤55 years, mean age 46±5 years; 30 patients aged ≥65 years, mean age 72±5 years) and 60 healthy controls (30 subjects aged ≤55 years, mean age 50±5 years; 30 subjects aged ≥65 years, mean age 72±4 years) were randomized to 4 weeks of supervised endurance training or to a control group. Before and after the intervention, vastus lateralis muscle biopsies were obtained. The expressions of cathepsin-L and the muscle-specific E3 ligases MuRF-1 and MAFbx were measured by real-time polymerase chain reaction and confirmed by Western blot. At baseline, MuRF-1 expression was significantly higher in CHF patients versus healthy controls (mRNA: 624±59 versus 401±25 relative units; P =0.007). After 4 weeks of exercise training, MuRF-1 mRNA expression was reduced by −32.8% ( P =0.02) in CHF patients aged ≤55 years and by −37.0% ( P Conclusions— MuRF-1, a component of the ubiquitin-proteasome system involved in muscle proteolysis, is increased in the skeletal muscle of patients with heart failure. Exercise training results in reduced MuRF-1 levels, suggesting that it blocks ubiquitin-proteasome system activation and does so in both younger and older CHF patients. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00176319.

Aims Diastolic dysfunction (DD) was identified as a predictor of adverse prognosis in heart failure with reduced ejection fraction (HFREF). It is, however, unknown if DD is improved by exercise training, which is known to induce reverse remodelling, and if the training effect is attenuated in elderly HFREF patients. We therefore assessed DD in a cohort of referent controls (RCs) and HFREF patients and studied the response of DD to endurance exercise in two age groups (≤55 years and ≥65 years). Methods and results Sixty RC (30 ≤ 55 years, mean age 50 ± 5 years; 30 ≥ 65 years, 72 ± 4 years) and 60 HFREF patients (30 ≤ 55 years, 46 ± 5 years; 30 ≥ 65 years, 72 ± 5 years, EF 28 ± 5%) were randomized to 4 weeks of supervised endurance training or to a control group. Exercise training was effective in reducing LV isovolumetric relaxation time by 29% in young and by 26% in old HFREF patients ( P < 0.05 for both). As assessed by tissue Doppler, septal E′ increased by 37% in young and by 39% among old HFREF patients ( P < 0.005 for both) resulting in a significant decrease in the E/E′ ratio from 13 ± 1 to 10 ± 1 in young and 14 ± 1 to 11 ± 1 in old HFREF patients ( P < 0.05 for both). Serum levels of N-terminal pro brain natriuretic peptide were significantly reduced after endurance training in HFREF patients of all ages. Conclusion In HFREF, diastolic function is significantly impaired in all age groups. Endurance training is highly effective in improving left ventricular diastolic function in HFREF patients regardless of age. This study is registered at ClinicalTrials.gov (number: [NCT00176319][1]). [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00176319&atom=%2Fehj%2F33%2F14%2F1758.atom

Objective The pathophysiology underlying the chromosome (Chr) 9p21 locus of atherosclerosis susceptibility is presently unknown. Here, we sought to determine whether protein coding genes in the Chr9p21 region, i.e. cyclin-dependent kinase inhibitors CDKN2B (p15 INK4b ), CDKN2A (p16 INK4a , p14 ARF ) and methylthioadenosine phosphorylase (MTAP) were expressed in human atherosclerotic lesions and whether expression was correlated with lesion composition. Methods and results Protein expression of p15 INK4b , p16 INK4a , p14 ARF and MTAP was demonstrated by immunostaining in normal and atherosclerotic coronary arteries and co-localized with CD68 and smooth muscle alpha-actin positive cells. Quantitative RT-PCR in human endarteryectomy specimens ( n =57) revealed increased p16 INK4a and decreased MTAP expression in macrophage-rich lesions ( P P =0.007, respectively). Functional studies suggest that decreased MTAP expression in macrophage-rich lesions might be mediated through down-regulation by TNF-alpha. No clear association of p15 INK4b , p16 INK4a , p14 ARF , and MTAP expression in plaque tissue with Chr9p21 haplotypes was found. The latter finding was corroborated by the lack of correlation of RNA expression of 9p21-regulated transcripts EU741058 and NR_003529 of antisense non-coding RNA in the INK4 locus ( ANRIL ) with mRNA expression of these genes. In contrast, ANRIL DQ485454 which is not genetically determined by the 9p21 genotype was significantly correlated with MTAP expression ( P =0.01). Conclusion CDKN2B (p15 INK4b ), CDKN2A (p16 INK4a , p14 ARF ), and MTAP are abundantly expressed in atherosclerotic lesions. While expression levels showed no clear association with Chr9p21 genotype, association of high p16 INK4a and low MTAP expression with a less stable plaque phenotype suggests a more general role of these proteins in atherogenesis.

Objective— Ath11 , an atherosclerosis susceptibility locus on proximal chromosome 10 (0 to 21 cM) revealed in a cross between apolipoprotein E deficient C57BL/6 (B6) and FVB mice, was recently confirmed in congenic mice. The objectives of this study were to assess how Ath11 affects lesion development and morphology, to determine aortic gene expression in congenics, and to narrow the congenic interval. Methods and Results— Assessing lesion area over time in congenic mice showed that homozygosity for the FVB allele increased lesion area at 6 weeks persisting through to 24 weeks of age. Staining of aortic root sections at 16 weeks did not reveal obvious differences between congenics. Aortic expression-array analysis at 6 weeks revealed 97 genes that were >2-fold regulated, including 1 gene in the quantitative trait locus interval, Aldh8a1 , and 2 gene clusters regulated by Hnf4α and Esr1 . Analysis of lesion area in 11 subcongenic strains revealed 2 narrowed regions, 10a (21 genes), acting in females, and 10b (7 genes), acting in both genders. Conclusion— Ath11 appears to act early in lesion formation, with significant effects on aortic gene expression. This quantitative trait locus is genetically complex, containing a female-specific region 10a from 0 to 7.3 megabases (21 genes) and a gender-independent region 10b from 20.1 to 21.9 megabases (7 genes).

Background— Phytosterols are plant-derived sterols that are taken up from food and can serve as biomarkers of cholesterol uptake. Serum levels are under tight genetic control. We used a genomic approach to study the molecular regulation of serum phytosterol levels and potential links to coronary artery disease (CAD). Methods and Results— A genome-wide association study for serum phytosterols (campesterol, sitosterol, brassicasterol) was conducted in a population-based sample from KORA (Cooperative Research in the Region of Augsburg) (n=1495) with subsequent replication in 2 additional samples (n=1157 and n=1760). Replicated single-nucleotide polymorphisms (SNPs) were tested for association with premature CAD in a metaanalysis of 11 different samples comprising 13 764 CAD cases and 13 630 healthy controls. Genetic variants in the ATP-binding hemitransporter ABCG8 and at the blood group ABO locus were significantly associated with serum phytosterols. Effects in ABCG8 were independently related to SNPs rs4245791 and rs41360247 (combined P =1.6×10 −50 and 6.2×10 −25 , respectively; n=4412). Serum campesterol was elevated 12% for each rs4245791 T-allele. The same allele was associated with 40% decreased hepatic ABCG8 mRNA expression ( P =0.009). Effects at the ABO locus were related to SNP rs657152 (combined P =9.4×10 −13 ). Alleles of ABCG8 and ABO associated with elevated phytosterol levels displayed significant associations with increased CAD risk (rs4245791 odds ratio, 1.10; 95% CI, 1.06 to 1.14; P =2.2×10 −6 ; rs657152 odds ratio, 1.13; 95% CI, 1.07 to 1.19; P =9.4×10 −6 ), whereas alleles at ABCG8 associated with reduced phytosterol levels were associated with reduced CAD risk (rs41360247 odds ratio, 0.84; 95% CI, 0.78 to 0.91; P =1.3×10 −5 ). Conclusion— Common variants in ABCG8 and ABO are strongly associated with serum phytosterol levels and show concordant and previously unknown associations with CAD.

Objective— We tested the hypothesis that expression of transcripts adjacent to the chromosome 9p21 (Chr9p21) locus of coronary artery disease was affected by the genotype at this locus and associated with atherosclerosis risk. Methods and Results— We replicated the locus for coronary artery disease ( P =0.007; OR=1.28) and other manifestations of atherosclerosis such as carotid plaque ( P =0.003; OR=1.31) in the Leipzig Heart Study, a cohort of 1134 patients with varying degree of angiographically assessed coronary artery disease. Expression analysis in peripheral blood mononuclear cells (n=1098) revealed that transcripts EU741058 and NR_003529 of antisense noncoding RNA in the INK4 locus ( ANRIL ) were significantly increased in carriers of the risk haplotype ( P =2.1×10 −12 and P =1.6×10 −5 , respectively). In contrast, transcript DQ485454 remained unaffected, suggesting differential expression of ANRIL transcripts at Chr9p21. Results were replicated in whole blood (n=769) and atherosclerotic plaque tissue (n=41). Moreover, expression of ANRIL transcripts was directly correlated with severity of atherosclerosis ( EU741058 and NR_003529 ; P =0.02 and P =0.001, respectively). No consistent association of Chr9p21 or atherosclerosis was found with expression of other genes such as CDKN2A , CDKN2B , C9orf53 , and MTAP . Conclusion— Our data provide robust evidence for an association of ANRIL but not CDKN2A, CDKN2B, C9orf53 , and MTAP , with atherosclerosis and Chr9p21 genotype in a large cohort.

Objective— Susceptibility to atherosclerosis is genetically complex, and modifier genes that do not operate via traditional risk factors are largely unknown. A mouse genetics approach can simplify the genetic analysis and provide tools for mechanistic studies. Methods and Results— We previously identified atherosclerosis susceptibility QTL ( Athsq1 ) on chromosome 4 acting independently of systemic risk factors. We now report confirmation of this locus in congenic strains carrying the MOLF-derived susceptibility allele in the C57BL/6J- Ldlr −/− genetic background. Homozygous congenic mice exhibited up to 4.5-fold greater lesion area compared to noncongenic littermates ( P Athsq1 congenic interval contains the mouse region homologous to a widely-replicated CHD locus on human chromosome 9p21. Conclusion— These studies confirm the proatherogenic activity of a novel gene(s) in the MOLF-derived Athsq1 locus and provide in vivo evidence for a causative role of versican in lesion development.

Background— We have previously identified an atherosclerosis quantitative trait locus (QTL) on mouse chromosome (Chr) 12 in an F2-intercross of atherosclerosis-resistant FVB and atherosclerosis-susceptible C57BL/6 (B6) mice on the LDL-receptor deficient (LDL −/− ) background. The aim of the present study was to identify potentially causative genes at this locus. Methods and Results— Expression QTL (eQTL) analysis of candidate genes in livers of F2-mice revealed that a disintegrin and metalloproteinase 17 (ADAM17 ) mRNA expression mapped to the physical position of ADAM17 on proximal Chr12 (21.6 Mb, LOD 3.3) and colocalized with the atherosclerosis QTL. The FVB allele was associated with significantly higher ADAM17 mRNA expression (39%) than the B6 allele. Likewise, ADAM17 mRNA levels in the parental strains were significantly elevated in FVB.LDLR −/− compared to B6.LDLR −/− mice in liver, macrophages, and aorta (68%, 58%, and 32%, respectively). Reporter gene assays revealed a genetic variant that might explain these expression differences. Moreover, FVB.LDLR −/− macrophages showed 5-fold increased PMA-induced shedding of tumor necrosis factor (TNF)-α and 32% increased release of TNF-receptor I compared to B6.LDLR −/− . The atherosclerosis locus and expression differences were confirmed in Chr12 interval-specific congenic mice. Conclusion— Our data provide functional evidence for ADAM17 as a candidate gene of atherosclerosis susceptibility at the murine Chr12 QTL.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression in diverse biological contexts. Although several thousand lncRNAs have been identified in humans, only a small number have been fully characterized. Here we identify a novel human lncRNA, linc-OSBPL6, that functions as a competing endogenous RNA to regulate cellular cholesterol homeostasis. Linc-OSBPL6 has 7 variants that are broadly expressed in various cell types and tissues, and are controlled at the transcriptional level by the cholesterol-sensing nuclear receptor liver X receptor. We noted that each of the linc-OSBPL6 variants harbors binding sites for miR-27b and miR-33a/b, microRNAs recently identified as hubs controlling the expression of genes involved in cellular cholesterol efflux and HDL metabolism. Using linc-OSBPL6 knockdown and overexpression, combined with RNA profiling, we demonstrate that linc-OSBPL6 acts as a ‘microRNA sponge’ to limit availability of miR-27b and miR33a/b and inversely regulate expression of their target genes (ABCA1, OSBPL6, ANGPTL3, CROT, ABCB11). Consistent with the role of miR-27b and miR-33a/b in repressing cellular cholesterol export, we show that overexpression of linc-OSBPL6 increases cholesterol efflux, while shRNA-silencing of linc-OSBPL6 reduces cholesterol efflux from hepatocytes and macrophages. As cholesterol efflux in the liver plays a central role in HDL biogenesis, we investigated the relationship of hepatic linc-OSBPL6 to its microRNA-targets and plasma levels of HDL cholesterol in a cohort of 200 healthy individuals. We found that expression of linc-OSBPL6 is negatively correlated with levels of miR-27b and miR-33a/b in the liver, and positively correlated with levels of their target genes and plasma HDL cholesterol. These findings identify linc-OSBPL6 as a key regulatory component of the non-coding RNA circuitry that controls cellular cholesterol efflux and plasma HDL levels in humans.

A reduction in number and function of endothelial progenitor cells (EPCs) occurs in both physiologic aging and chronic heart failure (CHF). We assessed whether disease and aging have additive effects on EPCs or whether beneficial effects of exercise training are diminished in old age.We randomized 60 patients with stable CHF and 60 referent controls to a training or a control group. To detect possible aging effects we included subjects below 55 (young) and above 65 years (older). Subjects in the training group exercised four times daily at 60% to 70% of VO2max for four weeks under supervision. At baseline and after the intervention the number and function of EPCs were assessed.As compared with young referent controls, older referent controls showed at baseline a reduced EPC number (young: 190 ± 37 CD34/KDR positive cells/ml blood; older: 131 ± 26 CD34/KDR positive cells/ml blood; p 0.05) and function (young: 230 ± 41 migrated cells/1000 plated cells; older: 185 ± 28 cells/1000 plated cells; p 0.05). In young and older CHF patients EPC-number (young: 85 ± 21 CD34/KDR positive cells/ml blood; older: 78 ± 20 CD34/KDR positive cells/ml blood) and EPC-function (young: 113 ± 26 cells/1000 plated cells; older: 120 ± 27 cells/1000 plated cells) were impaired. As a result of exercise training, EPC function improved by 24% in older referent controls (p 0.05), while it remained unchanged in young training referent controls and controls respectively. In young and older patients with CHF four weeks of exercise training resulted in a significant improvement in EPC numbers and EPC function (young: number +66% function +43%; p 0.05; older: number +69% function +36%; p 0.05). These results were accompanied by a significant increase in flow mediated dilatation in the training groups of young/older CHF patients and in older referent controls.Four weeks of exercise training are effective in improving EPC number and EPC function in CHF patients. These training effects were not impaired among older patients, emphasizing the potentials of rehabilitation interventions in a patient group where CHF has a high prevalence.

Objective—Our laboratory has previously created 2 strains of rabbits with genetically determined high-atherosclerotic response (HAR) and low-atherosclerotic response (LAR). The aim of the present study was to identify new genes of atherosclerosis susceptibility in macrophages from the 2 strains.Methods and Results—Suppression subtractive hybridization was used to screen for genes with higher expression in macrophages from LAR rabbits. We identified a cDNA fragment with high homology to human arginase I (AI; 91%) and subsequently cloned the full-length cDNA of the rabbit homologue. Quantitative RT-PCR revealed a significantly higher macrophage AI mRNA expression in LAR rabbits than in HAR rabbits (77428±10941 versus 34344±4538;P=0.002; copies/106copies β-actin), which also correlated with a significantly higher arginase enzyme activity. Northern blot analysis led to the identification of a size polymorphism of AI mRNA. This was because of a 413 bp C-repeat insertion in the 3′ untranslated region. The shorter transcript variant was predominantly expressed in LAR rabbits and associated with significantly higher AI mRNA expression levels. Transfection experiments indicated decreased mRNA stability of the long AI variant.Conclusions—High expression of arginase I in macrophages may contribute to atherosclerosis resistance of LAR rabbits, possibly by conferring antiinflammatory effects in the vessel wall.

Quantitative trait locus (QTL) mapping in an F2 intercross (n=452) of atherosclerosis-susceptible C57BL/6 (B6) and atherosclerosis-resistant FVB mice on the LDL-receptor deficient background revealed a novel atherosclerosis susceptibility locus on mouse chromosome (Chr) 3. In previous work the susceptible genetic region on Chr3 was narrowed to 80 - 160 MB and validated by congenic FVB.Chr3 B6/B6 mice. We hypothesized that underlying genetic variation in this region leads to differential expression of causal genes, thereby affecting atherosclerosis susceptibility. We performed transcriptome-wide expression analyses in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=4/4) using Illumina Ref-8 arrays followed by validation in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=8/9) as well as in livers of B6 and FVB mice (n=5/5) by quantitative real-time PCR (qRT-PCR). C is -regulation was investigated in F2 livers (n=47) by correlating the expression to the genotype. Tissue-specific expression of genes was examined by qRT-PCR in parental B6 and FVB mice. Western blot analysis and immunohistochemical staining (IHC) were performed. Mechanisms of atherogenesis were investigated by RNAi. Pla2g12a and Elovl6 were identified as candidate genes co-segregating with the atherosclerosis QTL at marker rs13464244. Pla2g12a mRNA expression was inversely correlated (r 2 =0.2, p=0.002) with atherosclerotic lesion size in F2 mice while Elovl6 expression was positively correlated (r 2 =0.18, p=0.002). qRT-PCR revealed a strong expression of Pla2g12a in muscle and fat tissues whereas Elovl6 was highly expressed in liver and fat tissues. Western blot analysis revealed significantly decreased protein expression of Pla2g12a in livers of B6 compared to FVB and an increased expression of Elovl6 in B6 mice. IHC staining of Pla2g12a and Elovl6 in aortic roots indicated high expression in macrophages and predominantly in endothelial cells. siRNA knockdown of Elovl6 was associated with reduced adhesion and increased apoptosis. In conclusion, we identified Elovl6 and Pla2g12a as promising candidate genes of atherosclerosis susceptibility on mouse Chr3. Further work is necessary to better understand the influence of these two genes on atherosclerosis development.

Previous work in an F2 intercross between atherosclerosis-susceptible C57BL/6 and atherosclerosis-resistant BALB/cByJ (BALB) mice on the LDL-receptor deficient background revealed a quantitative trait locus (QTL) for atherosclerotic lesion size at rs33142586 on chromosome (Chr) 2 in female mice (n=183) (Burkhardt et al., Arterioscler Thromb Vasc Biol; 31:775-84, 2011). The aim of the present study was to identify genetic variants responsible for differences in atherosclerosis-susceptibility between the two strains. We hypothesized that the causal genes could be identified by transcriptome-wide expression analyses followed by functional validation. 25,697 transcripts were analyzed in aortas (n=165) and livers (n=176) of F2 mice using Illumina Ref8 expression arrays. Expression levels were correlated with single nucleotide polymorphism (n=31) on Chr2 by expression QTL (eQTL) mapping. Genes co-segregating with the atherosclerosis QTL were identified as potential candidate genes. Results were validated by quantitative real-time PCR (qRT-PCR) in livers (n=176) and bone marrow-derived macrophages (BMDM, n=184) of F2 mice. Expression patterns of candidate genes were examined in different tissues of F0 mice, followed by functional studies. eQTL mapping identified Surf4 as the only cis -regulated gene co-segregating with the Chr2 QTL in aortas (LOD array =17.6), livers (LOD array =11.5, LOD qRT-PCR =23.2) and BMDM (LOD qRT-PCR =15.7). Surf4 mRNA was significantly increased in F2 mice carrying the BALB allele at rs33142586 and BALB F0 mice. Increased Surf4 expression was associated with reduced atherosclerosis lesion size in F2 mice. Immunohistochemical staining revealed expression of Surf4 in vascular endothelial cells of the aorta. RNAi of Surf4 lead to increased staurosporine- and cycloheximide-induced apoptosis and decreased proliferation compared to control siRNA in RAW 264.7 cells, which are two key mechanisms of atherosclerosis. In conclusion, we identified Surf4 as a novel candidate gene of atherosclerosis susceptibility co-segregating a QTL for atherosclerosis on mouse Chr2. In current work, its functional role in atherogenesis is being further investigated in in-vitro experiments.

Background— Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction. Methods and Results— Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1 , MIR33B/SREBF1 , and TNIP1 were identified. CpG cg06500161, located in ABCG1 , was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=−0.049; P =8.26E-17) and triglyceride levels (β=0.070; P =1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06–1.25). Conclusions— Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases.

Background —Endothelin-converting enzymes (ECEs) are the key enzymes in endothelin-1 (ET-1) generation. However, their pathophysiological role in patients with cardiovascular disease remains elusive. Methods and Results —Vascular reactivity to big endothelin-1 (bigET-1; 10 − 9 to 10 − 7 mol/L) and ET-1 (10 − 9 to 10 − 7 mol/L) were examined in the internal mammary artery (IMA, n=33) and saphenous vein (SV, n=27) of patients with coronary artery disease with identified cardiovascular risk factors. Vascular ECE activity was determined by conversion of exogenously added bigET-1 to ET-1. Tissue contents of bigET-1 and ET-1 were measured by radioimmunoassay. In addition, the effects of LDL and oxidized LDL on ECE-1 protein levels were determined by Western blot analysis in human IMA endothelial cells. In the IMA, vascular ECE activity showed an inverse correlation with serum LDL levels ( r =−0.76; P r =0.58; P P P Conclusions —These data demonstrate, for the first time, that vascular ECE activity is (1) inversely correlated with serum LDL levels and blood pressure and (2) positively associated with fibrinogen in human vascular tissue. Hence, ECE-1 activity may modulate cardiovascular risk in patients with coronary artery disease.

Objectives:\ud This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.\ud \ud Background:\ud Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is causal. A recent phase III clinical trial of an sPLA2 inhibitor (varespladib) was stopped prematurely for lack of efficacy.\ud \ud Methods:\ud We conducted a Mendelian randomization meta-analysis of 19 general population studies (8,021 incident, 7,513 prevalent major vascular events [MVE] in 74,683 individuals) and 10 acute coronary syndrome (ACS) cohorts (2,520 recurrent MVE in 18,355 individuals) using rs11573156, a variant in PLA2G2A encoding the sPLA2-IIA isoenzyme, as an instrumental variable.\ud \ud Results:\ud PLA2G2A rs11573156 C allele associated with lower circulating sPLA2-IIA mass (38% to 44%) and sPLA2 enzyme activity (3% to 23%) per C allele. The odds ratio (OR) for MVE per rs11573156 C allele was 1.02 (95% confidence interval [CI]: 0.98 to 1.06) in general populations and 0.96 (95% CI: 0.90 to 1.03) in ACS cohorts. In the general population studies, the OR derived from the genetic instrumental variable analysis for MVE for a 1-log unit lower sPLA2-IIA mass was 1.04 (95% CI: 0.96 to 1.13), and differed from the non-genetic observational estimate (OR: 0.69; 95% CI: 0.61 to 0.79). In the ACS cohorts, both the genetic instrumental variable and observational ORs showed a null association with MVE. Instrumental variable analysis failed to show associations between sPLA2 enzyme activity and MVE.\ud \ud Conclusions:\ud Reducing sPLA2-IIA mass is unlikely to be a useful therapeutic goal for preventing cardiovascular events.

The objective of this study was to investigate the effect of a specific endothelin-A receptor antagonist on mRNA expression of genes encoding vasoactive mediators and proinflammatory cytokines following complete vascular exclusion of the porcine liver. Fourteen adult German Landrace pigs were subjected to 120 minutes of warm hepatic ischemia by total vascular exclusion. The animals were divided into two groups: the control group received saline solution and the therapy group was given the selective endothelin-A receptor antagonist BSF 208075. Liver tissue samples were collected 1 hour after reperfusion and mRNA expression for preproendothelin-1, prointerleukin-1beta, prointerleukin- 6, pro-tumor necrosis factor-alpha and endothelial nitric oxide synthase was analyzed quantitatively using the TaqMan system. Additionally, immunohistochemical analysis using a semiquantitative score for endothelin-1 and endothelin-A receptor was performed. One hour after reperfusion, quantitative reverse transcriptase-polymerase chain reaction revealed significantly lower expression of preproendothelin-1, pro-tumor necrosis factor-alpha, and prointerleukin-6 in the therapy group compared to controls. Immunohistochemical analysis demonstrated significantly reduced endothelin-1 immunostaining after therapy. Treatment with the selective endothelin-A receptor antagonist exerts a protective effect on the microcirculation after liver ischemia and reperfusion. We were able to show that the endothelin-A receptor antagonist not only has effects on the expression of vasoactive genes, it also decreases gene expression of proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6.

The chromosome 9p21 (Chr9p21) locus was discovered in 2007 by independent genome-wide association studies for coronary artery disease. Since then, the locus has been replicated numerous times and can be considered the most robust genetic marker of coronary artery disease today. Subsequent work has shown associations of Chr9p21 with a number of additional cardiovascular disease traits, such as carotid artery plaque, stroke, aneurysms, peripheral artery disease, heart failure, and cardiovascular mortality, suggesting a more general role in vascular pathology. Importantly, Chr9p21 lacks associations with common cardiovascular risk factors, such as lipids and hypertension, indicating that the locus exerts its effect through a completely novel mechanism. One of the challenges is that the core haplotype block at Chr9p21 resides in a region of the genome devoid of protein-coding genes. Recent progress has been made by functional studies focusing on differential expression of antisense noncoding RNA in the INK4 locus ( ANRIL ), which is transcribed from the Chr9p21 locus, as well as neighboring protein-coding genes at the INK4 / ARF locus. The emerging concept suggests that ANRIL might constitute a regulator of epigenetic modification and thus modulate cardiovascular risk. Here, we review the current clinical, mechanistic, and diagnostic implications of the Chr9p21 locus in cardiovascular disease.

In the current issue, Hu et al1 provide evidence for the transcription factor nuclear factor 1A ( NFIA ) as a novel atherosclerosis candidate gene, which is regulated through expression of the long noncoding RNA (lncRNA) RP5-833A20.1 , embedded in intron 2 of NFIA , and microRNA (miRNA) hsa-miR-382-5p. In addition to providing compelling evidence for a role of NFIA in atherogenesis, the article is important for strengthening the concept of lncRNAs as master-regulators of this frequent disease. See accompanying article on page 87 lncRNAs are a class of RNAs gaining increasing attention in biomedical research. They lack an open reading frame and are distinguished from other classes of noncoding RNAs, such as miRNAs, by an arbitrary size limit of >200 bp.2 Until now, >10 000 mammalian lncRNAs have been discovered across the genome and it has only been recognized recently that lncRNAs constitute an important layer of transcriptional regulation.3 In a hypothesis-free approach, the authors used expression arrays in a THP-1 foam cell model and identified several differentially expressed mRNAs and lncRNAs. They subsequently focused on NFIA , which overlapped with the genomic position of a yet uncharacterized lncRNA, designated RP5-833A20.1 …

Proliferation signal inhibitors/mTOR-inhibitors have been shown to reduce de novo development of hypercholesterolemic atherosclerosis in animal models. However, their effect on pre-existing atherosclerosis has not yet been studied.Feeding LDL-R-KO mice a high cholesterol diet for 12 weeks resulted in formation of moderate fibroatheroma (induction phase). Sixty mice received either everolimus (1 or 5 mg/kg) or no everolimus for further 12 weeks (treatment phase). Everolimus significantly enhanced hypercholesterolemia (plasma cholesterol +45%, p0.001). Atherosclerosis progressed obstructively in treated and non-treated mice. Everolimus (5 mg/kg) tended to reduced progression in aortic root lesions (0.28±0.02 vs. 0.33±0.03 mm(2), p=ns) and brachiocephalic lesions (0.044±0.006 vs. 0.066±0.012 mm(2), p=ns) but without significance. Everolimus (5mg/kg) resulted in an arrest of CD68 positive plaque area (p=0.03) and nearly halved CD68 fraction (p=0.05) in aortic root lesions but not in brachiocephalic lesions. Taken together, despite a trend to reduced progression and inflammatory cell content there was less conclusive net effect of everolimus treatment than expected.A higher potential of everolimus in the treatment of atherosclerosis might be obscured by its concomitant hypercholesterolemia. Considering stronger effects in previous studies we suggest that everolimus might exert more potent anti-atherogenic properties in earlier stages of atherogenesis than in advanced atherosclerosis.

Objective— Adiponectin is an adipocyte-derived, secreted protein that is implicated in protection against a cluster of related metabolic disorders. Mice lacking adiponectin display impaired hepatic insulin sensitivity and respond only partially to peroxisome proliferator-activated receptor γ agonists. Adiponectin has been associated with antiinflammatory and antiatherogenic properties; however, the direct involvement of adiponectin on the atherogenic process has not been studied. Methods and Results— We crossed adiponectin knockout mice ( Adn −/− ) or mice with chronically elevated adiponectin levels ( Adn Tg ) into the low-density lipoprotein receptor–null ( Ldlr −/− ) and the apoliprotein E–null ( Apoe −/− ) mouse models. Adiponectin levels did not correlate with a suppression of the atherogenic process. Plaque volume in the aortic root, cholesterol accumulation in the aorta, and plaque morphology under various dietary conditions were not affected by circulating adiponectin levels. In light of the strong associations reported for adiponectin with cardiovascular disease in humans, the lack of a phenotype in gain- and loss-of-function studies in mice suggests a lack of causation for adiponectin in inhibiting the buildup of atherosclerotic lesions. Conclusion— These data indicate that the actions of adiponectin on the cardiovascular system are complex and multifaceted, with a minimal direct impact on atherosclerotic plaque formation in preclinical rodent models.

Despite the advances in interventional techniques, the management of stable atherosclerosis remains the domain of optimal guideline-oriented therapy. Recent studies on the effects of aggressive lipid lowering on atheroma volume changes using intravascular ultrasound indicate that it is possible to achieve atherosclerosis regression by reaching low-density lipoprotein (LDL) levels less than 75 mg/dl. The pleiotropic anti-inflammatory effects of statins contribute to the reduction of cardiovascular (CV) event observed with aggressive lipid lowering. As a second important strategy to prevent disease progression, lifestyle changes with regular physical exercise are capable of halting the atherosclerotic process and reducing angina symptoms and CV events. Optimal medical therapy, a healthy lifestyle with regular physical exercise, and coronary interventions are not mutually exclusive treatment strategies. Over the last few decades, both have proved to be effective in significantly reducing the CV mortality in the Western world. However, risk factor modification contributed to at least half the effect in the reduction of CV mortality. This figure provides an estimate of what could be achieved if we were to take risk factor modification more seriously – especially in the acute care setting. The knowledge is there: today we have a better understanding on how to stop progression and even induce regression of atherosclerosis. Much research still needs to be done and will be done. In the meantime, however, our primary focus should lie in implementing what is already known. In addition, it is essential not just to treat CV risk factors, but also to treat them to achieve the target values as set by the guidelines of European Society of Cardiology.

Objective— We have previously identified a quantitative trait locus (QTL) for atherosclerosis susceptibility on proximal chromosome 10 (Chr10) ( Ath11 ) in independent crosses of FVB and C57BL/6 (B6) mice on the apolipoprotein E (ApoE −/− ) and LDL receptor (LDLR −/− ) deficient backgrounds. The aims of the current study were to (1) test a novel strategy for validating QTLs using interval-specific congenic strains that were heterozygous (F1) across the genome, (2) validate the Chr10 QTL, and (3) to assess whether the phenotype is transferable by bone marrow transplantation. Methods and Results— We generated Chr10 (0 to 21 cM) interval-specific mice on the F1.ApoE −/− background by crossing congenic FVB.ApoE −/− Chr10 B6/FVB with B6.ApoE −/− , and B6.ApoE −/− Chr10 B6/FVB with FVB.ApoE −/− mice. Lesion size was significantly larger in the resultant F1.ApoE −/− Chr10 FVB/FVB mice compared to F1.ApoE −/− Chr10 B6/FVB and F1.ApoE −/− Chr10 B6/B6 mice, validating the Chr10 QTL. The effect of the congenic interval was more robust on the F1.ApoE −/− than on the FVB.ApoE −/− and B6.ApoE −/− backgrounds. Bone marrow transplantation in congenic mice showed that the effect of the proximal Chr10 interval was not transferable by bone marrow–derived cells. Conclusions— A novel strategy of congenic strains on an F1 background proved useful to validate an atherosclerosis susceptibility QTL on mouse proximal Chr10.

Background— The nuclear liver X receptor-α (LXR-α) has been implicated in the regulation of intracellular cholesterol homeostasis, inflammatory response, and atherosclerosis susceptibility. The aim of the present study was to test whether transgenic expression of LXR-α might affect these mechanisms and result in a reduction of atherosclerosis. Methods and Results— We generated mice with macrophage overexpression of mouse LXR -α, evidenced by significantly elevated expression levels of LXR-target genes ( ABCA1 , ABCG1 ) in these cells. For atherosclerosis studies, mice were crossed onto the LDL-receptor deficient background. Plasma lipids and lipoproteins as well as liver triglycerides were not significantly different between transgenic animals and nontransgenic controls. However, lesion area at the brachiocephalic artery (BCA) was significantly reduced (−83%, P =0.02) in male LXR -α transgenic mice. This was associated with a significantly increased cholesterol efflux to acceptor-free media (+24%, P =0.002) and ApoA1 containing media (+20%, P Conclusion— Our data show for the first time that transgenic overexpression of LXR -α in macrophages has significant antiatherogenic properties. We conclude that overexpression of LXR -α in macrophages might be useful as a therapeutic principle for the prevention of atherosclerosis.

Background: In previous work we have identified arginase I (ARG1) as a novel candidate gene of atherosclerosis resistance in macrophages (Teupser et al ATVB 2006). In macrophages, L-arginine can be metabolized by NO synthase and arginase to form NO and urea, respectively. Thus, high expression of ARG1 in macrophages (as observed in atherosclerosis resistant our rabbits) may be an effective mechanism of reducing macrophage NO-production and thus vascular inflammation. Methods and Results: To test the hypothesis that ARG1 exerts anti-atherogenic properties, we generated ARG1-transgenic mice, expressing human ARG1 under the control of a CMV promoter. In these animals, ARG1 was over-expressed in various tissues including bone-marrow-derived macrophages. Under basal conditions, expression of human ARG1 in macrophages of transgenic mice was significantly higher compared to endogenous murine ARG1 (27062±13652 vs. 176±81 copies ARG1/10 6 copies cyclophilin, respectively). To test the functionality of the human transgene, macrophages were incubated with lipopolysaccharide (1 μg/mL). After 6 hours of incubation, NO-release was significantly diminished in macrophages of transgenic mice, compared to non-transgenic controls (94±9 vs. 140±16 μmol/ng cell protein, respectively p = 0.03), indicating that hARG1 transgene expression influences NO synthesis by competing with iNOS for the common substrate L-arginine. To test the effect of the transgene on atherosclerosis susceptibility, bone marrow from ARG1-transgenic and non-transgenic animals were transplanted onto LDL-receptor deficient C57BL/6 recipients at 8 weeks of age. Animals were sacrified at 20 weeks of age and no significant differences in lipid and lipoprotein levels were noted. However, atherosclerosis of animals transplanted with transgenic bone marrow (n=16) was significantly reduced compared to controls (n=15) (75160±9914 vs. 121500±18410 μm 2 , p=0.03). Conclusion: Our data show for the first time that over-expression of human ARG1 in macrophages has direct anti-atherogenic properties by reducing NO-release in macrophages. We conclude that ARG1 might constitute an interesting novel target for the prevention of atherosclerosis.

Everolimus inhibits the mammalian target of rapamycin (mTOR) in proliferating cells. It is widely used in transplant patients and has also been exploited by drug-eluting stents for the treatment of cardiovascular disease. However, there is only limited data on the pathophysiological effects of mTOR-inhibitors on the vascular wall. We aimed to unravel the effects of everolimus on cholesterol-induced atherosclerosis and on circulating cell mediators in LDL-receptor-deficient (LDLR(-/-)) mice. Male hypercholesterolemic LDLR(-/-) mice received either solvent (group A; n=28) or everolimus at 0.05 mg/kg (group B, n=22) and 1.5 mg/kg (group C, n=29) per body weight per day by subcutaneously implanted osmotic minipumps for the study period of 12 weeks. Group B showed 44% reduction of atherosclerotic lesions at the brachiocephalic artery (BCA). In group C atherosclerotic lesions were reduced by 85% in the BCA and by 60% at the aortic root. This was associated with a significantly lower complexity of lesions in both treated groups (p

Bioresorbable vascular scaffolds (BVS) promise to address pending issues of current-generation stents. While promising data of this novel platform is accumulating, signals of scaffold thrombosis (ST) were noted in recent registry data. Our study aimed to determine whether such signals could be

Objective— A semisynthetic diet with varying amounts of cholesterol was used to achieve hypercholesterolemia and atherosclerosis in LDL receptor–deficient (LDLR−/−) mice. Atherosclerotic lesions were measured as cross-sectional area at the aortic root and brachiocephalic artery and by en face analysis of aortic lesion area in 209 male and female animals on the C57BL/6J (B6.LDLR−/−) and FVB/NJ (FVB.LDLR−/−) backgrounds. Methods and Results— The semisynthetic diet containing 4.3% fat and 0.00% or 0.02% cholesterol was sufficient to induce hypercholesterolemia (12.6±2.4 mmol/L) and atherosclerosis in B6.LDLR−/− mice at the aortic root (98 980±37 727 μm 2 ) and brachiocephalic artery (12 039±12 750 μm 2 ) but did not produce significant lesions in the aorta measurable by the en face method. Raising dietary cholesterol to 0.15%, 0.30%, or 0.50% more than doubled plasma cholesterol levels (35.9±8.5 mmol/L) and resulted in significant en face lesions. It also led to a significant increase in atherosclerotic lesion area at the aortic root (547 753±182 151 μm 2 ) and brachiocephalic arteries (125 666±59 339 μm 2 ). Although FVB.LDLR−/− mice developed comparable cholesterol levels, they were relatively atherosclerosis resistant and had many-fold smaller lesions. Conclusions— These results should aid investigations of atherosclerosis in LDLR−/− mice by informing the selection of diet to be used and the location of lesions to be scored.

The aim of this study was to investigate the effect of alpha-tocopherol on scavenger receptor (SR) activity, SR class A (SR-A) mRNA expression and transcriptional regulation in macrophages. Scavenger receptor activity was determined quantitatively by uptake of DiI-acLDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein) in rabbit peritoneal macrophages and human monocytes/macrophages in the presence and absence of different tocopherol homologues. SR-A mRNA expression was determined by Northern blotting, the activity of the transcription factor activator protein-1 (AP-1) by electrophoretic mobility shift assay. We could demonstrate that alpha-tocopherol down-regulates scavenger receptor activity in macrophages in a dose-dependent manner. Scavenger receptor activity was reduced by 13, 16, 18 and 24% in the presence of 1, 5, 10 and 50 microM alpha-tocopherol, respectively. This effect was associated with a reduced SR-A mRNA expression and activity of AP-1 binding transcription factors in the presence of alpha-tocopherol. The activity of scavenger receptors in human monocyte derived macrophages incubated with 100 microM alpha-tocopherol for 15 days was reduced up to 60%. Interestingly, gamma-tocopherol, which is a homologue of alpha-tocopherol with a comparable antioxidative capacity, showed only a weak suppression of SR activity, SR-A expression and AP-1 activity. Our observations point to the conclusion that the reduction of SR-A expression and activity in presence of alpha-tocopherol is possibly related to its direct action on cell signaling.