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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)-β 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 (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.

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.

Background Hyperlipidemia is a major risk factor for cardiovascular disease and atherosclerosis is the underlying cause of both myocardial infarction and stroke. We have previously shown that the Pro251 variant of perilipin-2 reduces plasma triglycerides and may therefore be beneficial for atherosclerosis development. Purpose We sought to delineate putative beneficial effects of the Pro251 variant of perlipin-2 on subclinical atherosclerosis and the mechanism by which it acts. Methods A pan-European cohort of high-risk individuals where carotid intima-media thickness has been assessed was adopted. Human primary monocyte-derived macrophages were prepared from whole blood from individuals recruited by perilipin-2 genotype, or from buffy coats from the our University hospital blood central. Results The Pro251 variant of perilipin-2 is associated with decreased intima-media thickness at baseline and 30 months follow-up. Using human primary monocyte-derived macrophages from carriers of the beneficial Pro251 variant we show that this variant increases autophagy activity, cholesterol efflux, and a controlled inflammatory response. Through extensive mechanistic studies we demonstrate that increase in autophagy activity is accompanied with an increase in LXR activity and that LXR and autophagy reciprocally activate each other in a feed-forward loop, regulated by CYP27A1 and 27OH-cholesterol. Conclusions For the first time, we show that perilipin-2 affects susceptibility to human atherosclerosis through activation of autophagy and stimulation of cholesterol efflux. We demonstrate that perilipin-2 modulates levels of the LXR ligand 27OH-cholesterol and initiates a feed-forward loop where LXR and autophagy reciprocally activate each other; the mechanism by which perilipin-2 exerts its beneficial effects on subclinical atherosclerosis. Acknowledgement/Funding The Swedish Research Council, Swedish Heart-Lung Foundation, Marianne and Marcus Wallenberg's Foundation, Swedish Medical Society

Background Hyperlipidaemia is a major risk factor for cardiovascular disease, and atherosclerosis is the underlying cause of both myocardial infarction and stroke. We have previously shown that the Pro251 variant of perilipin‐2 reduces plasma triglycerides and may therefore be beneficial to reduce atherosclerosis development. Objective We sought to delineate putative beneficial effects of the Pro251 variant of perlipin‐2 on subclinical atherosclerosis and the mechanism by which it acts. Methods A pan‐European cohort of high‐risk individuals where carotid intima‐media thickness has been assessed was adopted. Human primary monocyte‐derived macrophages were prepared from whole blood from individuals recruited by perilipin‐2 genotype or from buffy coats from the Karolinska University hospital blood central. Results The Pro251 variant of perilipin‐2 is associated with decreased intima‐media thickness at baseline and over 30 months of follow‐up. Using human primary monocyte‐derived macrophages from carriers of the beneficial Pro251 variant, we show that this variant increases autophagy activity, cholesterol efflux and a controlled inflammatory response. Through extensive mechanistic studies, we demonstrate that increase in autophagy activity is accompanied with an increase in liver‐X‐receptor (LXR) activity and that LXR and autophagy reciprocally activate each other in a feed‐forward loop, regulated by CYP27A1 and 27OH‐cholesterol. Conclusions For the first time, we show that perilipin‐2 affects susceptibility to human atherosclerosis through activation of autophagy and stimulation of cholesterol efflux. We demonstrate that perilipin‐2 modulates levels of the LXR ligand 27OH‐cholesterol and initiates a feed‐forward loop where LXR and autophagy reciprocally activate each other; the mechanism by which perilipin‐2 exerts its beneficial effects on subclinical atherosclerosis.

Publisher's version (útgefin grein), Carotid artery intima media thickness (cIMT) and carotid plaque are measures of subclinical atherosclerosis associated with ischemic stroke and coronary heart disease (CHD). Here, we undertake meta-analyses of genome-wide association studies (GWAS) in 71,128 individuals for cIMT, and 48,434 individuals for carotid plaque traits. We identify eight novel susceptibility loci for cIMT, one independent association at the previously-identified PINX1 locus, and one novel locus for carotid plaque. Colocalization analysis with nearby vascular expression quantitative loci (cis-eQTLs) derived from arterial wall and metabolic tissues obtained from patients with CHD identifies candidate genes at two potentially additional loci, ADAMTS9 and LOXL4. LD score regression reveals significant genetic correlations between cIMT and plaque traits, and both cIMT and plaque with CHD, any stroke subtype and ischemic stroke. Our study provides insights into genes and tissue-specific regulatory mechanisms linking atherosclerosis both to its functional genomic origins and its clinical consequences in humans., The work was supported by the following grants: National Institute of Health grants: R21HL123677, R21-HL140385, DK104806-01A1, R01-MD012765-01A1 (NF), National Institutes of Health awards R01HG009120, R01HG006399, U01CA194393, T32NS048004 (CG), the American Heart Association Grant #17POST33350042 (PV), the British Heart Foundation (RG/13/5/30112) and the National Institute for Health Research University College London Hospitals Biomedical Research Centre (RCL and RPH), the British Heart Foundation FS/14/55/30806 (JCH), the German Federal Ministry of Education and Research (BMBF) in the context of the e:Med program (e:AtheroSysMed), the DFG as part of the CRC 1123 (B3), and the FP7/2007-2103 European Union project CVgenes@target (grant agreement number Health-F2-2013-601456). We thank Li-Ming Gan for assistance with the STARNET study and Jon White for assistance with UCLEB analyses. Additional acknowledgements are included in Supplementary Note 2.

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.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.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)-β to the cell membrane of activated Treg cells. Real-time polymerase chain reaction was used to determine the impact of alternative splicing ofThese 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.

Background Ischemic stroke and coronary heart disease are important contributors to the global disease burden and share atherosclerosis as the main underlying cause. Recent evidence from a genome-wide association study (GWAS) suggested that single nucleotide polymorphisms (SNP) near the MMP12 gene at chromosome 11q22.3 were associated with large-vessel ischemic stroke. Here we evaluated and extended these results by examining the relationship between MMP12 and atherosclerosis in clinical and experimental studies. Methods and Results Plasma concentrations of MMP-12 were measured at baseline in 3 394 subjects with high-risk for cardiovascular disease (CVD) using the Olink ProSeek CVD I array. The plasma MMP-12 concentration showed association with incident cardiovascular and cerebrovascular events (130 and 67 events respectively over 36 months) and carotid intima-media thickness progression (p=3.6x10-5). A GWAS of plasma MMP-12 concentrations revealed that SNPs rs499459, rs613084 and rs1892971 at chr11q22.3 were independently associated with plasma MMP-12 (p

Arterial wall remodeling is a central multifactorial process in the development and progression of cardiovascular diseases. We employed an approach aimed at observing genetic variants associated with the progression of carotid intima-media thickness (cIMT) in order to identify novel pathways effecting vessel remodeling. This was achieved by conducting gene-centric analysis of 400,000 variants in 3,042 subjects with repeated cIMT measurements. Rs16997464 on chr22 intergenic between neutrophil cytosolic factor-4 ( NCF4) and colony stimulating factor 2 receptor beta ( CSF2RB ) was associated with cIMT progression at array-wide significance (p -7 ). The potential causative genes within this locus were investigated using a human vascular and non-vascular tissue biobank. Expression of 9 genes near rs16997464, were analyzed with the most significant association being with NCF4 in aortic adventitia. The effect of the variant on the function of the NCF4 gene product was further analyzed by comparing the oxidative burst capacity of neutrophils from subjects with different rs16997464 genotypes. We observed that neutrophils homozygous for the minor T allele, associated with slower cIMT progression, produced more extracellular ROS than neutrophils homozygous for the G allele, indicating a functional effect of rs16997464 on the NCF4 gene product p40 phox , a component of the NADPH oxidase 2 complex (NOX2). In parallel, we investigated if the chr22 locus also influenced the cellular composition of the atherosclerotic plaque, by utilizing data from the Athero-Express Biobank. Here we found that the minor T allele associated with a higher smooth muscle cell (SMC) content in the plaque. Finally, using a partial ligation model in mice where ncf4 is mutated, resulting in a reduced but not absent NOX2-associated ROS formation, we observed a reduced neointima formation in the ncf4 -mutated strain compared with wild-type littermates. Thus, this study identified rs16997464 in the NCF4-CSF2RB locus as a novel genetic determinant of cIMT progression, and provides evidence suggesting that NCF4 is involved in SMC proliferation and alteration of vessel wall pathophysiology.

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

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution., A full list of acknowledgments appears in the Supplementary Note 4. Co-author A.J.M.d.C. recently passed away while this work was in process. This work was performed under the auspices of the Genetic Investigation of ANthropometric Traits (GIANT) consortium. We acknowledge the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium for encouraging CHARGE studies to participate in this effort and for the contributions of CHARGE members to the analyses conducted for this research. Funding for this study was provided by the Aase and Ejner Danielsens Foundation; Academy of Finland (41071, 77299, 102318, 110413, 117787, 121584, 123885, 124243, 124282, 126925, 129378, 134309, 286284); Accare Center for Child and Adolescent Psychiatry; Action on Hearing Loss (G51); Agence Nationale de la 359 Recherche; Agency for Health Care Policy Research (HS06516); ALF/LUA research grant in Gothenburg; ALFEDIAM; ALK-Abelló A/S; Althingi; American Heart Association (13POST16500011); Amgen; Andrea and Charles Bronfman Philanthropies; Ardix Medical; Arthritis Research UK; Association Diabète Risque Vasculaire; Australian National Health and Medical Research Council (241944, 339462, 389875, 389891, 389892, 389927, 389938, 442915, 442981, 496739, 552485, 552498); Avera Institute; Bayer Diagnostics; Becton Dickinson; BHF (RG/14/5/30893); Boston Obesity Nutrition Research Center (DK46200), Bristol-Myers Squibb; British Heart Foundation (RG/10/12/28456, RG2008/08, RG2008/014, SP/04/002); Medical Research Council of Canada; Canadian Institutes for Health Research (FRCN-CCT-83028); Cancer Research UK; Cardionics; Cavadis B.V., Center for Medical Systems Biology; Center of Excellence in Genomics; CFI; CIHR; City of Kuopio; CNAMTS; Cohortes Santé TGIR; Contrat de Projets État-Région; Croatian Science Foundation (8875); Danish Agency for Science, Technology and Innovation; Danish Council for Independent Research (DFF-1333-00124, DFF-1331-00730B); County Council of Dalarna; Dalarna University; Danish Council for Strategic Research; Danish Diabetes Academy; Danish Medical Research Council; Department of Health, UK; Development Fund from the University of Tartu (SP1GVARENG); Diabetes Hilfs- und Forschungsfonds Deutschland; Diabetes UK; Diabetes Research and Wellness Foundation Fellowship; Donald W. Reynolds Foundation; Dr Robert Pfleger-Stiftung; Dutch Brain Foundation; Dutch Diabetes Research Foundation; Dutch Inter University Cardiology Institute; Dutch Kidney Foundation (E033); Dutch Ministry of Justice; the DynaHEALTH action No. 633595, Economic Structure Enhancing Fund of the Dutch Government; Else Kröner-Fresenius-Stiftung (2012_A147, P48/08//A11/08); Emil Aaltonen Foundation; Erasmus University Medical Center Rotterdam; Erasmus MC and Erasmus University Rotterdam; the Municipality of Rotterdam; Estonian Government (IUT20-60, IUT24-6); Estonian Research Roadmap through the Estonian Ministry of Education and Research (3.2.0304.11-0312); European Research Council (ERC Starting Grant and 323195:SZ-245 50371-GLUCOSEGENES-FP7-IDEAS-ERC); European Regional Development Fund; European Science Foundation (EU/QLRT-2001-01254); European Commission (018947, 018996, 201668, 223004, 230374, 279143, 284167, 305739, BBMRI-LPC-313010, HEALTH-2011.2.4.2-2-EU-MASCARA, HEALTH-2011-278913, HEALTH-2011-294713-EPLORE, HEALTH-F2-2008-201865-GEFOS, HEALTH-F2-2013-601456, HEALTH-F4-2007-201413, HEALTH-F4-2007-201550-HYPERGENES, HEALTH-F7-305507 HOMAGE, IMI/115006, LSHG-CT-2006-018947, LSHG-CT-2006-01947, LSHM-CT-2004-005272, LSHM-CT-2006-037697, LSHM-CT-2007-037273, QLG1-CT-2002-00896, QLG2-CT-2002-01254); Faculty of Biology and Medicine of Lausanne; Federal Ministry of Education and Research (01ZZ0103, 01ZZ0403, 01ZZ9603, 03IS2061A, 03ZIK012); Federal State of Mecklenburg-West Pomerania; Fédération Française de Cardiologie; Finnish Cultural Foundation; Finnish Diabetes Association; Finnish Foundation of Cardiovascular Research; Finnish Heart Association; Fondation Leducq; Food Standards Agency; Foundation for Strategic Research; French Ministry of Research; FRSQ; Genetic Association Information Network (GAIN) of the Foundation for the NIH; German Federal Ministry of Education and Research (BMBF, 01ER1206, 01ER1507); GlaxoSmithKline; Greek General Secretary of Research and Technology; Göteborg Medical Society; Health and Safety Executive; Healthcare NHS Trust; Healthway; Western Australia; Heart Foundation of Northern Sweden; Helmholtz Zentrum München—German Research Center for Environmental Health; Hjartavernd; Ingrid Thurings Foundation; INSERM; InterOmics (PB05 MIUR-CNR); INTERREG IV Oberrhein Program (A28); Interuniversity Cardiology Institute of the Netherlands (ICIN, 09.001); Italian Ministry of Health (ICS110.1/RF97.71); Italian Ministry of Economy and Finance (FaReBio di Qualità); Marianne and Marcus Wallenberg Foundation; the Ministry of Health, Welfare and Sports, the Netherlands; J.D.E. and Catherine T, MacArthur Foundation Research Networks on Successful Midlife Development and Socioeconomic Status and Health; Juho Vainio Foundation; Juvenile Diabetes Research Foundation International; KfH Stiftung Präventivmedizin e.V.; King's College London; Knut and Alice Wallenberg Foundation; Kuopio University Hospital; Kuopio, Tampere and Turku University Hospital Medical Funds (X51001); La Fondation de France; Leenaards Foundation; Lilly; LMUinnovativ; Lundberg Foundation; Magnus Bergvall Foundation; MDEIE; Medical Research Council UK (G0000934, G0601966, G0700931, MC_U106179471, MC_UU_12019/1); MEKOS Laboratories; Merck Santé; Ministry for Health, Welfare and Sports, The Netherlands; Ministry of Cultural Affairs of Mecklenburg-West Pomerania; Ministry of Economic Affairs, The Netherlands; Ministry of Education and Culture of Finland (627;2004-2011); Ministry of Education, Culture and Science, The Netherlands; Ministry of Science, Education and Sport in the Republic of Croatia (108-1080315-0302); MRC centre for Causal Analyses in Translational Epidemiology; MRC Human Genetics Unit; MRC-GlaxoSmithKline pilot programme (G0701863); MSD Stipend Diabetes; National Institute for Health Research; Netherlands Brain Foundation (F2013(1)-28); Netherlands CardioVascular Research Initiative (CVON2011-19); Netherlands Genomics Initiative (050-060-810); Netherlands Heart Foundation (2001 D 032, NHS2010B280); Netherlands Organization for Scientific Research (NWO) and Netherlands Organisation for Health Research and Development (ZonMW) (56-464-14192, 60-60600-97-118, 100-001-004, 261-98-710, 400-05-717, 480-04-004, 480-05-003, 481-08-013, 904-61-090, 904-61-193, 911-11-025, 985-10-002, Addiction-31160008, BBMRI–NL 184.021.007, GB-MaGW 452-04-314, GB-MaGW 452-06-004, GB-MaGW 480-01-006, GB-MaGW 480-07-001, GB-MW 940-38-011, Middelgroot-911-09-032, NBIC/BioAssist/RK 2008.024, Spinozapremie 175.010.2003.005, 175.010.2007.006); Neuroscience Campus Amsterdam; NHS Foundation Trust; National Institutes of Health (1RC2MH089951, 1Z01HG000024, 24152, 263MD9164, 263MD821336, 2R01LM010098, 32100-2, 32122, 32108, 5K99HL130580-02, AA07535, AA10248, AA11998, AA13320, AA13321, AA13326, AA14041, AA17688, AG13196, CA047988, DA12854, DK56350, DK063491, DK078150, DK091718, DK100383, DK078616, ES10126, HG004790, HHSN268200625226C, HHSN268200800007C, HHSN268201200036C, HHSN268201500001I, HHSN268201100046C, HHSN268201100001C, HHSN268201100002C, HHSN268201100003C, HHSN268201100004C, HHSN271201100004C, HL043851, HL45670, HL080467, HL085144, HL087660, HL054457, HL119443, HL118305, HL071981, HL034594, HL126024, HL130114, KL2TR001109, MH66206, MH081802, N01AG12100, N01HC55015, N01HC55016, N01C55018, N01HC55019, N01HC55020, N01HC55021, N01HC55022, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086, N01HC95159, N01HC95160, N01HC95161, N01HC95162, N01HC95163, N01HC95164, N01HC95165, N01HC95166, N01HC95167, N01HC95168, N01HC95169, N01HG65403, N01WH22110, N02HL6‐4278, N01-HC-25195, P01CA33619, R01HD057194, R01HD057194, R01AG023629, R01CA63, R01D004215701A, R01DK075787, R01DK062370, R01DK072193, R01DK075787, R01DK089256, R01HL53353, R01HL59367, R01HL086694, R01HL087641, R01HL087652, R01HL103612, R01HL105756, R01HL117078, R01HL120393, R03 AG046389, R37CA54281, RC2AG036495, RC4AG039029, RPPG040710371, RR20649, TW008288, TW05596, U01AG009740, U01CA98758, U01CA136792, U01DK062418, U01HG004402, U01HG004802, U01HG007376, U01HL080295, UL1RR025005, UL1TR000040, UL1TR000124, UL1TR001079, 2T32HL007055-36, T32GM074905, HG002651, HL084729, N01-HC-25195, UM1CA182913); NIH, National Institute on Aging (Intramural funding, NO1-AG-1-2109); Northern Netherlands Collaboration of Provinces; Novartis Pharma; Novo Nordisk; Novo Nordisk Foundation; Nutricia Research Foundation (2016-T1); ONIVINS; Parnassia Bavo group; Pierre Fabre; Province of Groningen; Päivikki and Sakari Sohlberg Foundation; Påhlssons Foundation; Paavo Nurmi Foundation; Radboud Medical Center Nijmegen; Research Centre for Prevention and Health, the Capital Region of Denmark; the Research Institute for Diseases in the Elderly; Research into Ageing; Robert Dawson Evans Endowment of the Department of Medicine at Boston University School of Medicine and Boston Medical Center; Roche; Royal Society; Russian Foundation for Basic Research (NWO-RFBR 047.017.043); Rutgers University Cell and DNA Repository (NIMH U24 MH068457-06); Sanofi-Aventis; Scottish Government Health Directorates, Chief Scientist Office (CZD/16/6); Siemens Healthcare; Social Insurance Institution of Finland (4/26/2010); Social Ministry of the Federal State of Mecklenburg-West Pomerania; Société Francophone du 358 Diabète; State of Bavaria; Stiftelsen för Gamla Tjänarinnor; Stockholm County Council (560183, 592229); Strategic Cardiovascular and Diabetes Programmes of Karolinska Institutet and Stockholm County Council; Stroke Association; Swedish Diabetes Association; Swedish Diabetes Foundation (2013-024); Swedish Foundation for Strategic Research; Swedish Heart-Lung Foundation (20120197, 20150711); Swedish Research Council (0593, 8691, 2012-1397, 2012-1727, and 2012-2215); Swedish Society for Medical Research; Swiss Institute of Bioinformatics; Swiss National Science Foundation (3100AO-116323/1, 31003A-143914, 33CSCO-122661, 33CS30-139468, 33CS30-148401, 51RTP0_151019); Tampere Tuberculosis Foundation; Technology Foundation STW (11679); The Fonds voor Wetenschappelijk Onderzoek Vlaanderen, Ministry of the Flemish Community (G.0880.13, G.0881.13); The Great Wine Estates of the Margaret River Region of Western Australia; Timber Merchant Vilhelm Bangs Foundation; Topcon; Tore Nilsson Foundation; Torsten and Ragnar Söderberg's Foundation; United States – Israel Binational Science Foundation (Grant 2011036), Umeå University; University Hospital of Regensburg; University of Groningen; University Medical Center Groningen; University of Michigan; University of Utrecht; Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) (b2011036); Velux Foundation; VU University’s Institute for Health and Care Research; Västra Götaland Foundation; Wellcome Trust (068545, 076113, 079895, 084723, 088869, WT064890, WT086596, WT098017, WT090532, WT098051, 098381); Wissenschaftsoffensive TMO; Yrjö Jahnsson Foundation; and Åke Wiberg Foundation. The views expressed in this manuscript are those of the authors and do not necessarily represent the views of the National Heart, Lung, and Blood Institute (NHLBI); the National Institutes of Health (NIH); or the U.S. Department of Health and Human Services.

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 40-64- 102]. In men, low levels of anti-PC (Q1) were associated with the highest (> 90th) percentile of the fastest C-IMT progression, i.e. the segment showing the fastest progression over 30 months in the whole carotid tree, with an OR of 1.41 (95%CI, 1.02-1.9) and with an increased risk of CVE with a multivariable adjusted HR of 1.85 (95%CI, 1.1-3.1). No significant associations were found in women.Conclusions: Low anti-PC levels increase the risk of CVE in men. This effect may be partly mediated by a fast C-IMT progression. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

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.

To access publisher's full text version of this article click on the hyperlink at the bottom of the page To further understanding of the genetic basis of type 2 diabetes (T2D) susceptibility, we aggregated published meta-analyses of genome-wide association studies (GWAS), including 26,488 cases and 83,964 controls of European, east Asian, south Asian and Mexican and Mexican American ancestry. We observed a significant excess in the directional consistency of T2D risk alleles across ancestry groups, even at SNPs demonstrating only weak evidence of association. By following up the strongest signals of association from the trans-ethnic meta-analysis in an additional 21,491 cases and 55,647 controls of European ancestry, we identified seven new T2D susceptibility loci. Furthermore, we observed considerable improvements in the fine-mapping resolution of common variant association signals at several T2D susceptibility loci. These observations highlight the benefits of trans-ethnic GWAS for the discovery and characterization of complex trait loci and emphasize an exciting opportunity to extend insight into the genetic architecture and pathogenesis of human diseases across populations of diverse ancestry. Canadian Institutes of Health Research Medical Research Council UK G0601261 Mexico Convocatoria SSA/IMMS/ISSSTE-CONACYT 2012-2 clave 150352 IMSS R-2011-785-018 CONACYT Salud-2007-C01-71068 US National Institutes of Health DK062370 HG000376 DK085584 DK085545 DK073541 DK085501 Wellcome Trust WT098017 WT090532 WT090367 WT098381 WT081682 WT085475 info:eu-repo/grantAgreement/EC/FP7/201413

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.

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.

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.

Recent evidence suggests that single nucleotide polymorphisms (SNPs) in the matrix metalloproteinase (MMP) gene cluster located at chromosome 11q22.3 are associated with large-vessel stroke. In the present study, we evaluated and extended the reported association by examining the relationship between MMPs and vascular disease in both clinical and experimental samples. Plasma concentrations of MMP-1, MMP-3, MMP-7, MMP-10 and MMP-12 were measured in 3 394 subjects with high-risk for cardiovascular disease (CVD) using the Olink ProSeek CVD array. Plasma MMP-12 concentration showed association with incident cardiovascular events (199 events over 36 months) and intima-media thickness progression over time (p=3.6x10 -5 ). The SNP variant rs1892971 was strongly associated with plasma MMP-12 concentration (p=8x10 -29 ) and weakly with susceptibility to coronary heart disease in the CardiogramplusC4D consortium study (p=8.8x10 -5 ). The same SNP was also significantly associated with MMP-12 gene expression in peripheral blood mononuclear cells using microarrays from patients with carotid atherosclerosis (n=96; p=1.8x10 -4 ). Expression of MMP-12 was strongly increased in carotid plaques (n=127) compared with undiseased arteries (n=10; pMMP-12 using siRNA in differentiated THP-1 cells indicated that MMP-12 has a role in macrophage migration. In conclusion, our study suggests that MMP-12 is a causal factor in CVD that is highly upregulated in human atherosclerotic plaques where it interacts with elastin and appears to enhance macrophage invasion.

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. 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. 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

To dissect the genetic architecture of blood pressure (BP) and assess how its elevation promotes downstream cardiovascular diseases, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry. Genotypes from an additional 140,886 individuals of European ancestry were used as validation for loci reaching genome-wide significance but without prior support in the literature. We identified 66 BP loci, of which 17 were novel and 15 harbored multiple distinct association signals, and which together explain up to 3.5% of BP variation. The 66 index SNPs were enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in BP control through modulating blood vessel tone and fluid filtration across multiple tissues, not solely the kidney. Importantly, the 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent (South-Asian, East-Asian and African), confirming that these are ancestral physiological effects that arose prior to human migration out of Africa. The 66-SNP BP risk score was significantly associated with target-organ damage in multiple tissues, with minor effects in the kidney. Our data expand current knowledge of BP pathways, and also, highlight that BP regulation and its effects may occur in multiple organs and tissues beyond the classic renal system.

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation.

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.

Fatty acid desaturases introduce double bonds into growing fatty acid chains. The key desaturases in humans are Delta5-desaturase (D5D), Delta6-desaturase (D6D) and stearoyl-CoA desaturase (SCD). Animal and human data implicate hepatic desaturase activities in insulin resistance, obesity and dyslipidaemia. However, the role of desaturase activity in adipose tissue is uncertain. We therefore evaluated relationships between adipose mRNA expression, estimated desaturase activities (fatty acid ratios) in adipose tissue and insulin resistance.Subcutaneous adipose tissue mRNA expression of D5D (also known as FADS1), D6D (also known as FADS2) and SCD was determined in 75 individuals representative of the study population of 294 healthy 63-year-old men. Desaturation indexes (product/substrate fatty acid ratios) were generated from adipose tissue fatty acid composition in all individuals. Insulin resistance was defined as the upper quartile of the updated homeostasis model assessment (HOMA-2) index.The relevant desaturation indexes (16:1/16:0, 18:1/18:0, 20:4/20:3 and 18:3/18:2) reflected expression of SCD, but not of D5D or D6D in adipose tissue. Insulin-resistant individuals had a higher adipose tissue 18:1/18:0, but not 16:1/16:0 ratio than insulin-sensitive individuals. Individuals with a high adipose tissue 18:1/18:0 ratio were 4.4-fold (95% CI 1.8-11.8) more likely to be insulin resistant [threefold (95% CI 1.1-8.6) after adjustment for waist circumference and plasma triacylglycerol]. In a multiple regression model predicting HOMA-2, the independent effect of the 18:1/18:0 ratio was borderline (p=0.086).Adipose tissue desaturation indexes of SCD reflect the expression of the gene encoding the enzyme in this tissue. Elevated SCD activity within adipose tissue is closely coupled to the development of insulin resistance.

To access publisher's full text version of this article click on the hyperlink at the bottom of the page We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease. cademy of Finland 77299 102318 10493 118065 123885 124243 129293 129680 136895 139635 211119 213506 251217 263836 Agence National de la Recherche Association de Langue Francaise pour l'Etude du Diabete et des Maladies Metaboliques Association Diabeete Risque Vasculaire Association Francaise des Diabetiques Association of Danish Pharmacies Augustinus Foundation Becket Foundation British Diabetes Association (BDA) Research British Heart Foundation Central Norway Health Authority Central Finland Hospital District Center for Inherited Disease Research (CIDR) City of Kuopio City of Leutkirch Copenhagen County Danish Centre for Evaluation and Health Technology Assessment Danish Council for Independent Research Danish Heart Foundation Danish Research Councils Deutsche Forschungsgemeinschaft ER 155/6-2 Diabetes Research Foundation Diabetes UK Doris Duke Charitable Foundation Erasmus Medical Center Erasmus University Estonian government SF0180142s08 European Commission ENGAGE HEALTH-F4-2007-201413 FP7-201413 FP7-245536 EXGENESIS LSHM-CT-2004-005272 FP6 LSHM_CT_2006_037197 LSHM-CT-2007-037273 C-Public Health 2004310 European Regional Development Fund Federal Ministry of Education and Research, Germany FKZ 01GI1128 FKZ 01EO1001 Federal Ministry of Health, Germany Finnish Diabetes Association Finnish Diabetes Research Foundation Finnish Foundation for Cardiovascular Research Finnish Medical Society Folkhalsan Research Foundation Foundation for Life and Health in Finland Foundation for Old Servants Fredrick och Ingrid Thuring Foundation French region of Nord-Pas-de-Calais (Contrat de Projets Etat-Region) German Center for Diabetes Research German Research Council GRK1041 German National Genome Research Network Groupe d'Etude des Maladies Metaboliques et Systemiques Health Care Centers in Vasa, Narpes and Korsholm, Finland Health Foundation Heinz Nixdorf Foundation Helmholtz Zentrum Munchen Helsinki University Central Hospital Research Foundation Hospital District of Southwest Finland Ib Henriksens Foundation IngaBritt and Arne Lundberg's Research Foundation 359 Karolinska Institutet Knut and Alice Wallenberg Foundation KAW 2009.0243 Kuopio University Hospital Lundbeck Foundation Magnus Bergvall Foundation Medical Faculty of University Duisburg-Essen Medical Research Council, UK G0000649 G0601261 Ministry for Health, Welfare and Sports, the Netherlands Ministry of Education and Culture, Finland 722 627 Ministry of Education, Culture and Science, the Netherlands Ministry of Health and Prevention, Denmark Ministry of Social Affairs and Health, Finland Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia, Germany Munich Center of Health Sciences Municipal Health Care Center and Hospital in Jakobstad, Finland municipality of Rotterdam, the Netherlands Narpes Health Care Foundation National Health Screening Service of Norway National Heart, Lung, and Blood Institute, USA HHSN268201100005C HHSN268201100006C HHSN268201100007C HHSN268201100008C HHSN268201100009C HHSN268201100010C HHSN268201100011C HHSN268201100012C N01HC25195 N02HL64278 R01HL087641 R01HL59367 R01HL086694 National Human Genome Research Institute, USA U01HG004402 N01HG65403 National Institute for Diabetes and Digestive and Kidney Diseases, USA R01DK078616 U01DK085526 K24DK080140 R01DK073490 National Institute for Health and Welfare, Finland National Institutes of Health, USA HHSN268200625226C UL1RR025005 R01DK062370 R01DK072193 1Z01HG000024 AG028555 AG08724 AG04563 AG10175 AG08861 U01HG004399 DK58845 CA055075 DK085545 DK098032 Netherlands Genomics Initiative Netherlands Organisation for Health Research and Development Netherlands Organisation of Scientific Research NOW Investments 175.010.2005.011 911-03-012 050-060-810 Nord-Trondelag County Council Nordic Center of Excellence in Disease Genetics Norwegian Institute of Public Health Norwegian Research Council Novo Nordisk Foundation Ollquist Foundation Oxford National Institute for Health Research (NIHR) Biomedical Research Centre Paavo Nurmi Foundation Paivikki and Sakari Sohlberg Foundation Perklen Foundation Pirkanmaa Hospital District, Finland Programme Hospitalier de Recherche Clinique Programme National de Recherche sur la Diabete Research Institute for Diseases in the Elderly 014-93-015 Robert Dawson Evans Endowment, Department of Medicine, Boston University School of Medicine and Boston Medical Center Royal Swedish Academy of Sciences Sarstedt, Germany Signe and Ane Gyllenberg Foundation Sigrid Juselius Foundation Slottery Machine Association, Finland Social Insurance Institution of Finland South OstroBothnia Hospital District state of Baden-Wurttemberg, Germany Stockholm County Council 560183 Swedish Cultural Foundation, Finland Swedish Diabetes Foundation Swedish e-science Research Center Swedish Foundation for Strategic Research Swedish Heart-Lung Foundation Swedish Research Council SFO EXODIAB 2009-1039 521-2010-3490 521-2007-4037 521-2008-2974 ANDIS 825-2010-5983 LUDC 349-2008-6589 8691 Swedish Society of Medicine Tore Nilsson Foundation Torsten and Ragnar Soderbergs Stiftelser MT33/09 University Hospital Essen University of Tromso University College London NIHR Biomedical Research Centre UK NIHR Cambridge Biomedical Research Centre Uppsala University Uppsala University Hospital Vaasa Hospital District Velux Foundation Wellcome Trust GR072960 076113 083948 090367 090532 083270 086596 098017 095101 098051 098381

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: β=−0.0047, P =1.63×10 –4 ; men: β=−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.

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 concentrationwas 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 (beta=-0.018, PFunding: IMPROVE was supported by the European Commission (Contract number: QLG1-CT-2002-00896), the Swedish Heart-Lung Foundation, the Swedish Research Council (projects 8691 and 0593), the Knut and Alice Wallenberg Foundation, the Foundation for Strategic Research, the Stockholm County Council (project 592229), the Strategic Cardiovascular and Diabetes Programmes of Karolinska Institutet and Stockholm County Council, the European Union Framework Programme 7 (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement no. IMI/115006 (the SUMMIT consortium), the Academy of Finland (Grant #110413), the British Heart Foundation (RG2008/08, RG2008/014) and the Italian Ministry of Health (Ricerca Corrente). The SNP Technology Platform is supported by Uppsala University, Uppsala University Hospital and the Swedish Research Council for Infrastructures. Persson is supported by the Stockholm County Council (clinical postdoctorial appointment). Strawbridge is supported by Swedish Heart-Lung Foundation (20120600), the Tore Nilsson, Gamla Tjanarinnor and Thurings foundations. Gertow acknowledges support from the Swedish Heart-Lung Foundation and Stiftelsen for Gamla Tjanarinnor. Sabater-Lleal is supported by the Swedish Heart-Lung Foundation (20130399), and acknowledges funding from Ake Wiberg and Tore Nilssons foundations. Sennblad acknowledges funding from the Magnus Bergvall Foundation and the Foundation for Old Servants. Rauramaa acknowledges the Ministry of Education and Culture in Finland. S.So. is supported by the Vasterbotten County Council (ALF) and the Swedish Heart and Lung Foundation. AGT is supported by TAMOP 4.2.4.A/1-11-1-2012-0001 National Excellence Program - research fellowship co-financed by the European Union and the European Social Fund. M.K. is supported by the UK Medical Research Council (K013351), the Economic and Social Research Council and the Academy of Finland. The University College London Genetics Institute supported S.Sh.

The fatty acid transport proteins (FATPs) have been implicated in facilitated cellular uptake of non-esterified fatty acids (NEFAs), thus having the potential to regulate local and systemic NEFA concentrations and metabolism. Hypothesising that genetic variation within the FATP genes may affect lipid metabolism, we investigated a G/A substitution at position 48 in intron 8 of the fatty acid transport-1 (FATP1) gene with respect to associations with fasting and post-prandial plasma lipid and lipoprotein variables in 628 healthy 50-year-old Swedish men and 426 Swedish women, aged 37-65 years. A subset of 105 men with the apoE3/E3 genotype underwent an oral fat tolerance test. Although fasting plasma TG concentrations were not different, male A/A individuals had significantly higher post-prandial TG concentrations and VLDL(1) (S(f) 60-400 apoB100)-to-VLDL(2) (S(f) 20-60 apoB100) ratio compared to male G/A and G/G individuals. A/A individuals apparently failed to suppress plasma NEFA concentrations during the oral fat tolerance test. Furthermore, fasting plasma concentrations of the largest, most buoyant LDL subfraction (LDL-I) were significantly lower in carriers of the A allele in the male cohort. Electromobility shift assays and reporter gene studies indicated that binding of nuclear factors and effect on transcriptional activity differ between the intron 8 alleles. These findings suggest that through regulation of NEFA trafficking, FATP1 might play a role in post-prandial lipid metabolism and development of cardiovascular disease.

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

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.

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 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.

Objective To determine whether genetic variation robustly associated with coronary artery disease (CAD) also influences risk of venous thromboembolism (VTE) in a well-defined, female case-control study (n=2753) from Sweden (the ThromboEmbolism Hormone Study (TEHS)). Approach and Results Single nucleotide polymorphisms associated with CAD in genome-wide association studies were genotyped in TEHS. Association with VTE was assessed by logistic regression (Bonferroni-corrected p-values). A strong association was found between rs579459 in the ABO locus and VTE risk (p=3.8×10 13 ). Adjustment for known VTE-associated variants within the ABO , F5 , F2 , F11 and FGG loci, along with age and body-mass index, identified a further suggestive association for rs1333049 in the ANRIL locus (p=0.003). However, replication analysis in 3 French cohorts (total n=5503) failed to confirm the ANRIL association with VTE, but indicated that the effect direction is consistent across cohorts. Furthermore, marked variation was found in minor allele and minor allele frequency of the ANRIL variant in a survey of studies conducted across Europe. Conclusions CAD-associated genetic variants in the ABO and ANRIL loci appear to influence risk of VTE. Differences in cohort characteristics, including allele frequencies, may account for the failure to replicate the association with rs1333049 in the French cohorts.

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.

Background— Carotid intima-media thickness (cIMT) is a widely accepted marker of subclinical atherosclerosis. To date, large-scale investigations of genetic determinants of cIMT are sparse. Methods and Results— To identify cIMT-associated genes and genetic variants, a discovery analysis using the Illumina 200K CardioMetabochip was conducted in 3430 subjects with detailed ultrasonographic determinations of cIMT from the IMPROVE (Carotid Intima Media Thickness [IMT] and IMT-Progression as Predictors of Vascular Events in a High Risk European Population) study. Segment-specific IMT measurements of common carotid, bifurcation, and internal carotid arteries, and composite IMT variables considering the whole carotid tree (IMT mean , IMT max , and IMT mean-max ), were analyzed. A replication stage investigating 42 single-nucleotide polymorphisms for association with common carotid IMT was undertaken in 5 independent European cohorts (total n=11 590). A locus on chromosome 16 (lead single-nucleotide polymorphism rs4888378, intronic in CFDP1 ) was associated with cIMT at significance levels passing multiple testing correction at both stages (array-wide significant discovery P =6.75×10 −7 for IMT max ; replication P =7.24×10 −6 for common cIMT; adjustments for sex, age, and population substructure where applicable; minor allele frequency 0.43 and 0.41, respectively). The protective minor allele was associated with lower carotid plaque score in a replication cohort ( P =0.04, n=2120) and lower coronary artery disease risk in 2 case-control studies of subjects with European ancestry (odds ratio [95% confidence interval] 0.83 [0.77–0.90], P =6.53×10 −6 , n=13 591; and 0.95 [0.92–0.98], P =1.83×10 −4 , n=82 297, respectively). Queries of human biobank data sets revealed associations of rs4888378 with nearby gene expression in vascular tissues (n=126–138). Conclusions— This study identified rs4888378 in the BCAR1-CFDP1-TMEM170A locus as a novel genetic determinant of cIMT and coronary artery disease risk in individuals of European descent.

To extend understanding of the genetic architecture and molecular basis of type 2 diabetes (T2D), we conducted a meta-analysis of genetic variants on the Metabochip, including 34,840 cases and 114,981 controls, overwhelmingly of European descent. We identified ten previously unreported T2D susceptibility loci, including two showing sex-differentiated association. Genome-wide analyses of these data are consistent with a long tail of additional common variant loci explaining much of the variation in susceptibility to T2D. Exploration of the enlarged set of susceptibility loci implicates several processes, including CREBBP-related transcription, adipocytokine signaling and cell cycle regulation, in diabetes pathogenesis. © 2012 Nature America, Inc. All rights reserved.

Objectives The purpose of this study is investigate the effects of variants in the apolipoprotein(a) gene (LPA) on vascular diseases with different atherosclerotic and thrombotic components.Background It is unclear whether the LPA variants rs10455872 and rs3798220, which correlate with lipoprotein(a) levels and coronary artery disease (CAD), confer susceptibility predominantly via atherosclerosis or thrombosis.Methods The 2 LPA variants were combined and examined as LPA scores for the association with ischemic stroke (and TOAST [Trial of Org 10172 in Acute Stroke Treatment] subtypes) (effective sample size [n(e)] = 9,396); peripheral arterial disease (n(e) = 5,215); abdominal aortic aneurysm (ne = 4,572); venous thromboembolism (ne = 4,607); intracranial aneurysm (ne = 1,328); CAD (n(e) = 12,716), carotid intima-media thickness (n = 3,714), and angiographic CAD severity (n = 5,588). Results LPA score was associated with ischemic stroke subtype large artery atherosclerosis (odds ratio [OR]: 1.27; p = 6.7 X 10(-4)), peripheral artery disease (OR: 1.47; p = 2.9 x 10(-14)), and abdominal aortic aneurysm (OR: 1.23; p = 6.0 x 10(-5)), but not with the ischemic stroke subtypes cardioembolism (OR: 1.03; p = 0.69) or small vessel disease (OR: 1.06; p = 0.52). Although the LPA variants were not associated with carotid intima-media thickness, they were associated with the number of obstructed coronary vessels (p = 4.8 x 10(-12)). Furthermore, CAD cases carrying LPA risk variants had increased susceptibility to atherosclerotic manifestations outside of the coronary tree (OR: 1.26; p = 0.0010) and had earlier onset of CAD (-1.58 years/allele; p = 8.2 x 10(-8)) than CAD cases not carrying the risk variants. There was no association of LPA score with venous thromboembolism (OR: 0.97; p = 0.63) or intracranial aneurysm (OR: 0.85; p = 0.15).Conclusions LPA sequence variants were associated with atherosclerotic burden, but not with primarily thrombotic phenotypes. (J Am Coll Cardiol 2012; 60: 722-9) (C) 2012 by the American College of Cardiology Foundation

Introduction Platelets play a key role in coronary artery disease. They have the capacity of protein synthesis through translation of megakaryocyte-derived mRNAs, which may influence pathophysiological functions. The present study aimed to prove the concept that platelets from patients with non-ST elevation acute coronary syndrome (NSTE-ACS) have differential mRNA expression profiles, in the hypothesis that this may influence their thrombogenicity. Materials and Methods Gene expression profiles were determined in RNA pools from resting platelets of patients with stable angina (SA, n = 14) or NSTE-ACS (n = 15) using a glass microarray platform. Validation was done by real-time PCR and immunoblot analyses in independent sets of individual samples (26 SA and 17 NSTE-ACS patients, in total). Parallel comparison with healthy subjects was performed to relate the relative abundance of validated genes in CAD patients to a control expression level. Results Microarray analysis identified 45 transcripts with a significant ≥ ± 2.0-fold difference in expression between NSTE-ACS and SA platelet pools. Thus, gene expression profiles at least partially discriminate unstable from stable CAD. Validation confirmed a significant over-expression of 3 genes in NSTE-ACS at both mRNA and protein level. In particular, the glycoprotein Ib β-polypeptide (GP1BB) was increased in NSTE-ACS also in comparison with healthy subjects. Conclusion This study provides evidence that NSTE-ACS platelets are potentially preconditioned to a higher degree of reactivity on the transcriptional level. Our data suggest that a different composition of the mRNA pool might mediate an increased platelet prothrombotic potential in NSTE-ACS patients.

It is important that RNA molecules representing members of gene families are distinguished in expression analyses, and even greater resolving power may be required to identify allelic variants of transcripts in order to investigate imprinting or to study the distribution of mutant genes in tissues. Ligase-mediated gene detection allows precise distinction of DNA sequence variants, but it is not known if ligases can also be used to distinguish variants of RNA sequences. Here we present conditions for efficient ligation of pairs of DNA oligonucleotides hybridizing next to one another on RNA strands, permitting discrimination of any single nucleotide probe-target mismatch by a factor of between 20- and 200-fold. The mechanism allows padlock probes to be used to distinguish single-nucleotide variants in RNA. Ligase-mediated gene detection could therefore provide highly sensitive and accurate ligase-mediated detection and distinction of RNA sequence variants in solution, on DNA microarrays, and in situ.

Objective Tissue factor (TF), key initiator of coagulation, has been ascribed roles not only in thrombosis but also in atherosclerosis. TF gene promoter haplotypes modulate TF expression, thereby potentially affecting atherosclerosis. The objective of this study was to evaluate functionally relevant TF promoter haplotypes as determinants of carotid intima-media thickness (C-IMT), a marker of atherosclerosis. Methods The haplotype-tagging TF A-603G polymorphism and C-IMT were determined in 611 subjects referred to cardiovascular risk prevention. Subjects were aged 59.7 (58.1–61.1) years (mean (95% C.I.)), 79% were male, and 74% in secondary prevention with a history of coronary, cerebrovascular, or peripheral atherosclerotic disease. TF plasma levels were measured in 120 subjects. Results Significant dose-dependent associations were found between A-603G genotype and both IMT max and IMT mean-max after adjusting for potential confounders. IMT values were highest in A/A ( n =173), intermediate in A/G ( n =312) and lowest in G/G ( n =126). IMT max values (average and 95% C.I., in mm) for A/A, A/G and G/G, were 2.22 (2.11–2.34), 2.09 (2.01–2.18) and 2.02 (1.90–2.15), respectively ( p trend =0.019), and IMT mean-max values were 1.28 (1.23–1.32), 1.25 (1.22–1.28) and 1.21 (1.16–1.26), respectively ( p trend =0.041). A significant interaction between A-603G genotype and prevention status was found ( p =0.046 and p =0.042 for IMT max and IMT mean-max , respectively). TF plasma levels did not differ between genotypes and were not determinants of C-IMT. Conclusion The haplotype-tagging TF A-603G polymorphism is associated with C-IMT, independently of TF levels in plasma. These findings provide clinical evidence of an involvement of TF in atherosclerosis, in addition to its well-known roles in hemostasis and thrombosis.

Proteins involved in cellular fatty acid (FA) uptake and metabolism may be of relevance in the context of disturbed FA metabolism associated with insulin resistance. Therefore this study investigated relationships between FA handling protein mRNA expression in adipose tissue, FA composition of adipose tissue and serum, and markers of insulin resistance.75 subjects with a range of insulin sensitivities recruited from a cohort of 294 healthy 63-year-old Swedish men.Anthropometric and biochemical variables (e.g. waist-hip-ratio (WHR) and homeostasis model assessment (HOMA) index of insulin sensitivity), FA composition of the subcutaneous (s.c.) gluteal adipose tissue, serum nonesterified FA (NEFA) and serum phospholipid compartments (by gas-liquid chromatography; n = 294), and mRNA levels of FA handling proteins (adipocyte and keratinocyte lipid binding proteins, fatty acid transport protein (FATP) -1 and -4, CD36/fatty acid translocase, plasma membrane fatty acid binding protein, and acyl-CoA synthase-1 (ACS1)) in s.c. gluteal adipose tissue (by quantitative real-time polymerase chain reaction; n = 75).ACS1 expression was negatively correlated with measures of insulin resistance and central obesity (ACS1 versus HOMA: r = -0.28, P0.05; ACS1 versus WHR: r = -0.23, P0.05), with an opposite trend for FATP4. Further analysis of ACS1 expression levels revealed correlations with adipose tissue 16:0 (r = -0.27, P0.05) and NEFA 16:1 (r = 0.29, P0.05), FA composition variables which in turn correlated with HOMA index (r = 0.39, P0.001 and r = -0.23, P0.05, respectively, n = 75). Moreover, NEFA 16:1 predicted ACS1 expression independently of HOMA, WHR and adipose tissue 16:0 in multiple regression analysis (standardized coefficient = 0.27, P0.05).Significant associations were found between measures of insulin sensitivity, adipose tissue FA handling protein expression, and specific FA composition variables. Although causal relationships could not be identified these findings suggest a role of FA handling proteins in relation to insulin sensitivity, via their involvement in FA trafficking and metabolism. In particular they indicate links between ACS1 activity, the distribution of 16:0 and 16:1, and insulin sensitivity, which may be of physiological relevance.

PURPOSE OF REVIEW Disturbed fatty acid metabolism and homeostasis is associated with insulin resistance. The aim of this review, therefore, is to summarize recent developments relating to the relevance and importance of the fatty acid transport proteins (FATPs) in the aetiology of insulin resistance. In particular, the potential differences between the six members of the FATP family will be considered. RECENT FINDINGS FATP1 knockout mice failed to develop insulin resistance associated with lipid infusion or a high-fat diet, as wild-type mice did. FATP1-mediated fatty acid uptake may cause intramuscular lipid accumulation leading to insulin resistance in muscle if the fatty acids are not oxidized. While mouse models demonstrated an absolute requirement for FATP4 for survival, they provided no direct evidence for a role of FATP4 in insulin resistance. However, expression of FATP4 in human adipose tissue was increased in obesity (independent of genetic factors). While other members of the FATP family have important roles in fatty acid metabolism, they have not been clearly linked to insulin resistance. FATP-mediated fatty acid uptake may be driven by intrinsic acyl-CoA synthase activity. SUMMARY Any role in the development of insulin resistance is likely to be different for each member of the FATP family. So far, both FATP1 and FATP4 have been associated with parameters related to insulin resistance. Whether increased FATP-mediated fatty acid uptake is beneficial or detrimental may be dependent on the tissue in question and on the subsequent fate of the fatty acids. These issues remain to be resolved.

Protein-mediated trans-membrane and intracellular fatty acid trafficking are becoming increasingly recognised as biochemically and physiologically important concepts. Obesity and insulin resistance are polygenic disorders, heavily influenced by environmental and life-style factors, and are virtually always associated with disturbed fatty acid metabolism in adipose and other tissues. The aim of this study was to investigate mRNA expression levels of fatty-acid-handling proteins in adipose tissue in relation to markers of genetic and acquired obesity and insulin resistance.We quantified mRNA expression of subcutaneous adipose tissue fatty-acid-handling proteins (ALBP, KLBP, FATP1, FATP4, CD36, ACS1) in 17 monozygotic twin-pairs with a range of intra-pair differences (Delta) in BMI and detailed measures of obesity and insulin resistance, allowing influences of genetic and non-genetic factors to be distinguished.In acquired obesity FATP4 expression was up-regulated independently of genetic background (DeltaFATP4 versus DeltaBMI; r=0.50, p=0.04; DeltaFATP4 versus Deltabody fat; r=0.59, p=0.01). Similarly, CD36 and FATP1 expression correlated with acquired differences in HDL cholesterol and non-esterified fatty acid concentrations respectively. Moreover, FATP4 and CD36 expression levels correlated with measures of obesity and insulin resistance that are influenced by both genetic and non-genetic factors (FATP4 versus BMI: r=0.53, p=0.0001; FATP4 versus body fat: r=0.51, p=0.002; FATP4 versus homeostasis model assessment [HOMA]: r=0.49, p=0.001; CD36 versus BMI: r=0.50, p=0.02; CD36 versus body fat: r=0.63, p=0.001; CD36 versus HOMA: r=0.34, p=0.06).These findings indicate that expression of specific adipose tissue fatty-acid-handling proteins is related to obesity and insulin resistance, and that, in particular, FATP4 plays a role in acquired obesity. Our results suggest that facilitated fatty acid trafficking is a physiologically and pathologically relevant phenomenon in man.

Disturbances in fatty acid metabolism are involved in the etiology of insulin resistance and the related dyslipidemia, hypertension, and procoagulant state. The fatty acid transport proteins (FATPs) are implicated in facilitated cellular uptake of nonesterified fatty acids (NEFAs), thus potentially regulating NEFA concentrations and metabolism. The aim of this study was to investigate polymorphic loci in the FATP4 gene with respect to associations with fasting and postprandial lipid and lipoprotein variables and markers of insulin resistance in 608 healthy, middle-aged Swedish men and to evaluate possible mechanisms behind any associations observed. Heterozygotes for a Gly209Ser polymorphism (Ser allele frequency 0.05) had significantly lower body mass index and, correcting for body mass index, significantly lower triglyceride concentrations, systolic blood pressure, insulin concentrations, and homeostasis model assessment index compared with common homozygotes. A three-dimensional model of the FATP4 protein based on structural and functional similarity with adenylate-forming enzymes revealed that the variable residue 209 is exposed in a region potentially involved in protein-protein interactions. Furthermore, the model indicated functional regions with respect to NEFA transport and acyl-coenzyme A synthase activity and membrane association. These findings propose FATP4 as a candidate gene for the insulin resistance syndrome and provide a structural basis for understanding FATP function in NEFA transport and metabolism.

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 1 c) were all significantly lower in the lipodystrophic than the non-lipodystrophic group. The mRNA concentration of PPAR-y 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.

The interaction between HIV-1 protease and inhibitors has been studied with optical biosensor technology. Optimized experimental procedures and mathematical analysis permitted determination of association and dissociation rate constants. A sensor surface with native enzyme was unstable and exhibited a drift that was influenced by the binding of inhibitor. This was hypothesized to be due to a specific mechanism involving autoproteolysis and/or dimer dissociation. The use of a mutant predicted to be less susceptible to autoproteolysis (Q7K) than wild-type enzyme resulted in a minor effect on surface stability, while a completely stable surface was obtained by treatment of the immobilized enzyme with N-ethyl-N'-(dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide; the most stable surface was achieved by chemically modifying the Q7K enzyme. The stabilized surface was enzymatically active and the interaction with inhibitors was similar to that for native enzyme. Several of the inhibitors had very high association rates, and estimation of kinetic constants was therefore performed with a binding equation accounting for limited mass transport. Of the clinical inhibitors studied, saquinavir had the highest affinity for the enzyme, a result of the lowest dissociation rate. Although the dissociation rate for ritonavir was sixfold faster, the affinity was only twofold lower than that for saquinavir since the association rate was threefold faster. Nelfinavir and indinavir exhibited lower affinities relative to the other inhibitors, a consequence of a slower association for nelfinavir and a relatively fast dissociation for indinavir. These results show that biosensor-based interaction studies can resolve affinity into association and dissociation rates, and that these are characteristic parameters for the interaction between enzymes and inhibitors.