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21 COMPREHENSIVE METABOLIC PROFILING IMPROVES PREDICTION OF SUBCLINICAL ATHEROSCLEROSIS IN YOUNG ADULTS
79th EAS Congress
Atherosclerosis Supplements 12, no. 1 (2011) 1–11
remained unchanged in course of KRP-203 treatment. However, KRP-203 induced marked peripheral blood lymphopenia, reduced total (CD4+ , CD8+ ) and activated (CD69+ /CD8+ , CD69+ /CD4+ ) T-cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased splenocyte proliferation and IL-2 and IFN-gamma production in response concanavalin A or phytohaemagglutinin as well as reduced RANTES levels in plasma. Plasma concentrations of macrophage-derived cytokines TNF-alpha and IL-6 were reduced by KRP-206 administration. Moreover, peritoneal macrophages from KRP-206 treated mice showed reduced surface expression of activation markers MCH-II and CD86 as well as LPS-elicited production of TNF-alpha and IL-6. In vitro experiments demonstrated reduced production of TNF-alpha and IL-6 in LPS-stimulated and IP-10 in INF-gamma stimulated bone marrow macrophages. Conclusions: Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P(1) at least partly mediates anti-atherogenic effects of S1P. 20 LCAT , HDL CHOLESTEROL AND MYOCARDIAL INFARCTION − A MENDELIAN RANDOMIZATION STUDY OF HDL CHOLESTEROL IN 54,500 INDIVIDUALS C.L. Haase1 , A. Tybjærg-Hansen1,2,3 , A.A. Qayyum1 , J. Schou1 , B.G. Nordestgaard2,3,4 , R. Frikke-Schmidt1,3 . 1 Dept. Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Copenhagen, 2 The Copenhagen City Heart Study, Bispebjerg Hospital, Copenhagen, 3 The Copenhagen General Population Study, Herlev Hospital, Herlev, 4 Dept. Clinical Biochemistry, Herlev Hospital, Herlev, Denmark Background: Epidemiologically, high-density lipoprotein cholesterol (HDL-C) levels relate inversely to risk of ischemic cardiovascular disease. We tested whether genetic variation in lecithin:cholesterol acyltransferase (LCAT), a key enzyme in HDL metabolism, contributes to HDL-C levels and risk of myocardial infarction (MI) in the general population and whether HDL-C levels associate causally with risk of MI. Methods: We resequenced the regulatory and coding regions of LCAT in 380 individuals from the Copenhagen City Heart Study (CCHS) with the lowest 2% and highest 2% HDL-C levels. All six identified variants were genotyped in the CCHS (n = 10,281), and a common genetic variant (S208T, rs4986970), which affected HDL-C levels in the CCHS, was further genotyped in the Copenhagen General Population Study (CGPS, n = 50,523). Instrumental variable analysis with MI as endpoint and S208T as the genetic instrument was performed in the combined study (n = 54,500). Results: S208T associated with decreased HDL-C levels of up to 0.2 mmol/L resulting in a predicted increase in risk of 18% for MI. However, S208T did not associate with increased risk of MI. The estimated causal increase in risk of MI by instrumental variable analysis for a 50% reduction in genetically reduced HDL-C levels was −51% (−89–116%) compared to an observational increase of 111% (70–162%) for a similar reduction in HDL-C on Cox regression (P-comparison = 0.03). Conclusion: Common genetic variation in LCAT associated with decreased HDL-C levels did not associate with increased risk of MI. Low HDL-C levels robustly associated with increased risk of MI, however genetically decreased HDL-C did not. This suggests that HDL-C levels are not causally related to risk of MI. 21 COMPREHENSIVE METABOLIC PROFILING IMPROVES PREDICTION OF SUBCLINICAL ATHEROSCLEROSIS IN YOUNG ADULTS P. Wurtz ¨ 1,2,3 , J. Raiko2 , C.G. Magnussen2,4,5 , P. Soininen1,6 , A.J. Kangas1 , T. Tynkkynen1,6 , R. Thomson4 , R. Laatikainen6 , M.J. Savolainen1 , A. Jula7 , 9 J.S. Viikari8 , M. Kah ¨ onen ¨ , T. Lehtimaki ¨ 9 , M. Juonala2,8 , M. Ala-Korpela1,6 , O.T. Raitakari2 . 1 Computational Medicine, University of Oulu, Oulu, 2 Cardiovascular Research Centre, University of Turku, Turku, 3 Institute of Molecular Medicine Finland, University of Helsinki, Helsinki, Finland, 4 Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS, 5 Murdoch Childrens Research Institute, Melbourne, VIC, Australia, 6 NMR Metabonomics Laboratory, University of Eastern Finland, Kuopio, 7 National Institute for Health and Welfare, Helsinki, 8 Department of Medicine, Turku University Hospital, Turku, 9 Tampere University Hospital and University of Tampere, Tampere, Finland Background: Metabolic profiling holds potential in cardiovascular risk assessment. We evaluated whether high-throughput metabolic profiling improves prediction of subclinical atherosclerosis as compared with conventional lipid testing. Methods: Serum lipids, lipoprotein subclasses, and small molecule metabolites were analyzed by nuclear magnetic resonance (NMR) for 1570 individuals aged 24−39 years in the population-based Cardiovascular Risk in Young Finns Study. Carotid intima-media thickness, a marker of subclinical atherosclerosis, was measured in 2001 and 2007. Baseline conventional risk factors and circulating metabolites were used to predict 6-year incidence of high intimamedia thickness (90th percentile) or plaque.
5
Results: The best prediction of high intima-media thickness was achieved when NMR-based lipoprotein lipids, and two novel biomarkers replaced conventional LDL-C and HDL-C in models including conventional risk factors. This model improved risk stratification beyond established risk factors alone; area under the ROC curve 0.77 vs. 0.74, P = 0.03; net reclassification improvement 15%, P = 0.003. The novel biomarkers had odds ratios for incident high intima-media thickness of similar magnitude as cholesterol measures but were independent of these. Conclusions: Metabolic profiling, with new systemic biomarkers, improved risk stratification for subclinical atherosclerosis over and above conventional lipids, and would be useful for early cardiovascular risk assessment.
22 BIOLOGICAL ROLES OF THE SECRETED SOLUBLE FORM OF LR11/SORLA W. Schneider1 , H. Bujo2 , M. Jiang2 , K. Ohwaki2 , Y. Saito2 . 1 Medical Biochemistry, Medical University Vienna, Vienna, Austria, 2 Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba, Japan Objectives: The receptor LR11 (also termed SorLA) exists as a membranebound full-length protein as well as a soluble form shed from the cell surface, sLR11, which is present in the circulation. The aim of our studies is to fully understand the roles of sLR11 in macrophage-foam cell formation and in smooth muscle cell (SMC) migration. Results: LR11−/− mice were protected from macrophage infiltration into cuffinjured arteries, while incubation with sLR11 increased by twofold the migration activity of THP-1 monocyte-macrophages and their adhesion to extracellular matrices. Serum concentrations of sLR11 were positively correlated with carotid intima-media thickness (IMT). Compared to wildtype mice, in LR11 −/− mice i. angiotensin-II (AngII)-stimulated migration and attachment of SMCs were largely abolished, ii. overproduction of sLR11 decreased the sensitivity of AngII-induced activation pathways to inhibition by an AngII-type1 receptor-blocker, and iii. femoral artery intimal thickness after cuff-placement decreased. In isolated wt murine SMCs, exposure to sLR11 caused membrane ruffle formation via activation of focal adhesion kinase/ERK/Rac1 accompanied by complex formation between uPAR and integrin avb3, a process accelerated by AngII. The effects of sLR11 were also inhibited by neutralizing anti-urokinasetype plasminogen activator receptor (uPAR) antibody. Conclusions: SLR11, originating from intimal SMCs, modulates adhesion, AngII-stimulated migration, and foam cell formation through activation of uPAR. Since SLR11 is also required for AngII-induced SMC migration, we propose that sLR11 may serve a novel role as a bio-marker of IMT.
23 GENETIC POLYMORPHISMS OF THE MAIN TRANSCRIPTION FACTORS FOR ADIPONECTIN GENE PROMOTER IN REGULATION OF PLASMA ADIPONECTIN LEVEL L. Kedenko1 , C. Lamina2 , I. Kedenko1 , F. Kronenberg2 , B. Paulweber1 . 1 University Clinic for Internal Medicine I, Paracelsus Medical University, Salzburg, 2 Division of Genetic Epidemiology, Innsbruck Medical University, Innsbruck, Austria Adiponectin is one of the most promising biomarkers in cardiovascular epidemiology. It is estimated that 30−70% of the variability in plasma adiponectin levels is accounted for by genetic factors and variability at adiponectin gene locus is thought to account for only 2−8% of serum adiponectin variance. We hypothesized that genetic polymorphisms in the main transcription factor genes for adiponectin gene might influence serum adiponectin levels. We selected 26 SNPs based on their potential ability to modify serum adiponectin concentration and the haplotype block structure. Genotyping was performed using TaqMan method. The association analyses on the relation of selected SNPs at FOXO1, SREBP1c, SIRT1, PPARgamma, PPARdelta, PGC1alpha and TFAP-2beta genes on adiponectin levels in SAPHIR population (1738 healthy unrelated subjects, Caucasian origin) was performed using linear regression models adjusted for age, gender and BMI assuming an additive as well as recessive genetic model. Assuming an additive genetic model we found associations between adiponectin levels and three correlated SNPs in the SREBP1c gene with the strongest effect for rs1889018, (b = −0.054, SE = 0.016, p = 0.001). Another SNP (rs2267668) at the PPARdelta gene (b = −0.062, SE = 0.021, p = 0.003) was borderline significant, if correction for multiple testing was applied. Haplotype analysis of the FOXO1, SREBP1c, SIRT1, TFAP-2beta genes, stratified anaylsis for gender and analysis using a recessive model did not give additional information for the association of plasma adiponectin levels. The role of genetic variation at SREBP1c and PPARdelta genes in regulation of adiponectin level has to be further investigated and should be confirmed in other populations.
Atherosclerosis Supplements 12, no. 1 (2011) 1–11
remained unchanged in course of KRP-203 treatment. However, KRP-203 induced marked peripheral blood lymphopenia, reduced total (CD4+ , CD8+ ) and activated (CD69+ /CD8+ , CD69+ /CD4+ ) T-cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased splenocyte proliferation and IL-2 and IFN-gamma production in response concanavalin A or phytohaemagglutinin as well as reduced RANTES levels in plasma. Plasma concentrations of macrophage-derived cytokines TNF-alpha and IL-6 were reduced by KRP-206 administration. Moreover, peritoneal macrophages from KRP-206 treated mice showed reduced surface expression of activation markers MCH-II and CD86 as well as LPS-elicited production of TNF-alpha and IL-6. In vitro experiments demonstrated reduced production of TNF-alpha and IL-6 in LPS-stimulated and IP-10 in INF-gamma stimulated bone marrow macrophages. Conclusions: Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P(1) at least partly mediates anti-atherogenic effects of S1P. 20 LCAT , HDL CHOLESTEROL AND MYOCARDIAL INFARCTION − A MENDELIAN RANDOMIZATION STUDY OF HDL CHOLESTEROL IN 54,500 INDIVIDUALS C.L. Haase1 , A. Tybjærg-Hansen1,2,3 , A.A. Qayyum1 , J. Schou1 , B.G. Nordestgaard2,3,4 , R. Frikke-Schmidt1,3 . 1 Dept. Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Copenhagen, 2 The Copenhagen City Heart Study, Bispebjerg Hospital, Copenhagen, 3 The Copenhagen General Population Study, Herlev Hospital, Herlev, 4 Dept. Clinical Biochemistry, Herlev Hospital, Herlev, Denmark Background: Epidemiologically, high-density lipoprotein cholesterol (HDL-C) levels relate inversely to risk of ischemic cardiovascular disease. We tested whether genetic variation in lecithin:cholesterol acyltransferase (LCAT), a key enzyme in HDL metabolism, contributes to HDL-C levels and risk of myocardial infarction (MI) in the general population and whether HDL-C levels associate causally with risk of MI. Methods: We resequenced the regulatory and coding regions of LCAT in 380 individuals from the Copenhagen City Heart Study (CCHS) with the lowest 2% and highest 2% HDL-C levels. All six identified variants were genotyped in the CCHS (n = 10,281), and a common genetic variant (S208T, rs4986970), which affected HDL-C levels in the CCHS, was further genotyped in the Copenhagen General Population Study (CGPS, n = 50,523). Instrumental variable analysis with MI as endpoint and S208T as the genetic instrument was performed in the combined study (n = 54,500). Results: S208T associated with decreased HDL-C levels of up to 0.2 mmol/L resulting in a predicted increase in risk of 18% for MI. However, S208T did not associate with increased risk of MI. The estimated causal increase in risk of MI by instrumental variable analysis for a 50% reduction in genetically reduced HDL-C levels was −51% (−89–116%) compared to an observational increase of 111% (70–162%) for a similar reduction in HDL-C on Cox regression (P-comparison = 0.03). Conclusion: Common genetic variation in LCAT associated with decreased HDL-C levels did not associate with increased risk of MI. Low HDL-C levels robustly associated with increased risk of MI, however genetically decreased HDL-C did not. This suggests that HDL-C levels are not causally related to risk of MI. 21 COMPREHENSIVE METABOLIC PROFILING IMPROVES PREDICTION OF SUBCLINICAL ATHEROSCLEROSIS IN YOUNG ADULTS P. Wurtz ¨ 1,2,3 , J. Raiko2 , C.G. Magnussen2,4,5 , P. Soininen1,6 , A.J. Kangas1 , T. Tynkkynen1,6 , R. Thomson4 , R. Laatikainen6 , M.J. Savolainen1 , A. Jula7 , 9 J.S. Viikari8 , M. Kah ¨ onen ¨ , T. Lehtimaki ¨ 9 , M. Juonala2,8 , M. Ala-Korpela1,6 , O.T. Raitakari2 . 1 Computational Medicine, University of Oulu, Oulu, 2 Cardiovascular Research Centre, University of Turku, Turku, 3 Institute of Molecular Medicine Finland, University of Helsinki, Helsinki, Finland, 4 Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS, 5 Murdoch Childrens Research Institute, Melbourne, VIC, Australia, 6 NMR Metabonomics Laboratory, University of Eastern Finland, Kuopio, 7 National Institute for Health and Welfare, Helsinki, 8 Department of Medicine, Turku University Hospital, Turku, 9 Tampere University Hospital and University of Tampere, Tampere, Finland Background: Metabolic profiling holds potential in cardiovascular risk assessment. We evaluated whether high-throughput metabolic profiling improves prediction of subclinical atherosclerosis as compared with conventional lipid testing. Methods: Serum lipids, lipoprotein subclasses, and small molecule metabolites were analyzed by nuclear magnetic resonance (NMR) for 1570 individuals aged 24−39 years in the population-based Cardiovascular Risk in Young Finns Study. Carotid intima-media thickness, a marker of subclinical atherosclerosis, was measured in 2001 and 2007. Baseline conventional risk factors and circulating metabolites were used to predict 6-year incidence of high intimamedia thickness (90th percentile) or plaque.
5
Results: The best prediction of high intima-media thickness was achieved when NMR-based lipoprotein lipids, and two novel biomarkers replaced conventional LDL-C and HDL-C in models including conventional risk factors. This model improved risk stratification beyond established risk factors alone; area under the ROC curve 0.77 vs. 0.74, P = 0.03; net reclassification improvement 15%, P = 0.003. The novel biomarkers had odds ratios for incident high intima-media thickness of similar magnitude as cholesterol measures but were independent of these. Conclusions: Metabolic profiling, with new systemic biomarkers, improved risk stratification for subclinical atherosclerosis over and above conventional lipids, and would be useful for early cardiovascular risk assessment.
22 BIOLOGICAL ROLES OF THE SECRETED SOLUBLE FORM OF LR11/SORLA W. Schneider1 , H. Bujo2 , M. Jiang2 , K. Ohwaki2 , Y. Saito2 . 1 Medical Biochemistry, Medical University Vienna, Vienna, Austria, 2 Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba, Japan Objectives: The receptor LR11 (also termed SorLA) exists as a membranebound full-length protein as well as a soluble form shed from the cell surface, sLR11, which is present in the circulation. The aim of our studies is to fully understand the roles of sLR11 in macrophage-foam cell formation and in smooth muscle cell (SMC) migration. Results: LR11−/− mice were protected from macrophage infiltration into cuffinjured arteries, while incubation with sLR11 increased by twofold the migration activity of THP-1 monocyte-macrophages and their adhesion to extracellular matrices. Serum concentrations of sLR11 were positively correlated with carotid intima-media thickness (IMT). Compared to wildtype mice, in LR11 −/− mice i. angiotensin-II (AngII)-stimulated migration and attachment of SMCs were largely abolished, ii. overproduction of sLR11 decreased the sensitivity of AngII-induced activation pathways to inhibition by an AngII-type1 receptor-blocker, and iii. femoral artery intimal thickness after cuff-placement decreased. In isolated wt murine SMCs, exposure to sLR11 caused membrane ruffle formation via activation of focal adhesion kinase/ERK/Rac1 accompanied by complex formation between uPAR and integrin avb3, a process accelerated by AngII. The effects of sLR11 were also inhibited by neutralizing anti-urokinasetype plasminogen activator receptor (uPAR) antibody. Conclusions: SLR11, originating from intimal SMCs, modulates adhesion, AngII-stimulated migration, and foam cell formation through activation of uPAR. Since SLR11 is also required for AngII-induced SMC migration, we propose that sLR11 may serve a novel role as a bio-marker of IMT.
23 GENETIC POLYMORPHISMS OF THE MAIN TRANSCRIPTION FACTORS FOR ADIPONECTIN GENE PROMOTER IN REGULATION OF PLASMA ADIPONECTIN LEVEL L. Kedenko1 , C. Lamina2 , I. Kedenko1 , F. Kronenberg2 , B. Paulweber1 . 1 University Clinic for Internal Medicine I, Paracelsus Medical University, Salzburg, 2 Division of Genetic Epidemiology, Innsbruck Medical University, Innsbruck, Austria Adiponectin is one of the most promising biomarkers in cardiovascular epidemiology. It is estimated that 30−70% of the variability in plasma adiponectin levels is accounted for by genetic factors and variability at adiponectin gene locus is thought to account for only 2−8% of serum adiponectin variance. We hypothesized that genetic polymorphisms in the main transcription factor genes for adiponectin gene might influence serum adiponectin levels. We selected 26 SNPs based on their potential ability to modify serum adiponectin concentration and the haplotype block structure. Genotyping was performed using TaqMan method. The association analyses on the relation of selected SNPs at FOXO1, SREBP1c, SIRT1, PPARgamma, PPARdelta, PGC1alpha and TFAP-2beta genes on adiponectin levels in SAPHIR population (1738 healthy unrelated subjects, Caucasian origin) was performed using linear regression models adjusted for age, gender and BMI assuming an additive as well as recessive genetic model. Assuming an additive genetic model we found associations between adiponectin levels and three correlated SNPs in the SREBP1c gene with the strongest effect for rs1889018, (b = −0.054, SE = 0.016, p = 0.001). Another SNP (rs2267668) at the PPARdelta gene (b = −0.062, SE = 0.021, p = 0.003) was borderline significant, if correction for multiple testing was applied. Haplotype analysis of the FOXO1, SREBP1c, SIRT1, TFAP-2beta genes, stratified anaylsis for gender and analysis using a recessive model did not give additional information for the association of plasma adiponectin levels. The role of genetic variation at SREBP1c and PPARdelta genes in regulation of adiponectin level has to be further investigated and should be confirmed in other populations.