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P218 TWO NOVEL GENETIC LOCI FOR HIGH AND LOW PLASMA HDL-C LEVELS IN 2 LARGE FAMILIES FROM DUTCH ANCESTRY
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Atherosclerosis Supplements 11, no. 2 (2010) 17–108
which includes data from multiple models. The most strongly co-varying genes are mitochondrial (p-value < 10−121 ); with sub-networks corresponding to oxidative phosphorylation, lipogenesis and b-oxidation (p-value < 10−55 ). Distinct inflammatory sub-networks were also discovered (p-values < 10−5 ). Genes associated with metabolic phenotypes in GWASs were identified within the metabolic sub-networks, and included Mlxipl, Pank1, Mtch2, Ide and Jazf1, respectively associated with hypertriglyceridaemia, insulin levels, obesity and T2D. We evaluated gene expression data from human WAT, and also found suppression of oxidative phosphorylation and b-oxidation (P value < 10−12 ) and activation of inflammation (P value < 10−4 ). We tested human SNPs in genes corresponding to those in the mouse networks for genome-wide association with metabolic phenotypes and these are undergoing replication. We conclude that mitochondrial dysfunction in response to high-fat feeding and in obesity favours obesity and inflammation in WAT. P218 TWO NOVEL GENETIC LOCI FOR HIGH AND LOW PLASMA HDL-C LEVELS IN 2 LARGE FAMILIES FROM DUTCH ANCESTRY Mo. Mahdi Motazacker1 , I. Tietjen2 , M. Hayden2 , J.P.P. Kastelein1 , E.S.G. Stroes1 , G.K. Hovingh1 , J.A. Kuivenhoven1 . 1 Academic Medical Center Amsterdam, Amsterdam, The Netherlands, 2 Xenon Pharmaceuticals Inc., Burnaby, BC, Canada Plasma high-density lipoprotein-cholesterol (HDL-c) levels are inversely correlated with cardiovascular disease (CVD). In this light, therapeutic targeting of HDL is regarded as an interesting strategy to reduce atherosclerosis. Plasma HDL-c level is a complex trait and its genetic component has recently been studied by many genome-wide association studies (GWAS). Interestingly, only 7 loci have thus far been associated with this trait which can explain a mere 5.2% of the heritability. This may indicate that rare variants with large or intermediate effect on the phenotype may account for a large portion of this unexplained heritability. In search for rare variants determining plasma HDL-c levels, we have performed linkage analysis studies in 2 large families with an autosomal dominant pattern of high HDL-c (>95th %) or low HDL-c (<5th %) phenotypes for age and gender (mutations in major candidate genes were excluded). We identified a novel locus for high plasma HDL-c levels (4.5 MB interval with multipoint parametric LOD score 3.4) and a novel locus for low plasma HDL-c levels (8 MB interval with multipoint parametric LOD score 4). To identify underlying genetic defects in these linkage intervals, we have designed capture arrays for targeted enrichment of the coding sequence and regulatory elements of the genes within each interval which will be processed by massive parallel sequencing. Our Pilot data indicates that many more ‘HDL genes’ are waiting to be discovered and that family studies are likely to remain an important tool for progress in this field. P219 CARRIERS OF LCAT GENE MUTATIONS HAVE HIGH DENSITY LIPOPROTEIN WITH DECREASED ANTI-OXIDATIVE CAPACITY A.G. Holleboom1 , G. Daniel2 , G.K. Hovingh1 , A.W. Schimmel1 , J.N. van Miert1 , J.J.P. Kastelein1 , E.S.G. Stroes1 , J.A. Kuivenhoven1 , A. Chroni2 . 1 Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands, 2 Institute of Biology, National Center for Scientific Research ‘Demokritos’, Athens, Greece Background: In vitro and animal studies have shown that lecithin:cholesterol acyltransferase (LCAT), an enzyme involved in high density lipoprotein (HDL) metabolism, has anti-oxidant properties. It is unclear, however, whether HDL of carriers of LCAT gene mutations has decreased anti-oxidative capacity. Methods: HDL of 69 heterozygous carriers and 62 unaffected family members was isolated from plasma and incubated in equal concentrations with human oxidized LDL. The capacity of HDL to inactivate the oxidized phospholipids of LDL was measured in the presence of 2,7 dichlorofluorescin (DCFH) that reacts with lipid peroxidation products. Results: Statistically significant differences in lipid levels of heterozygous carriers and controls were: HDL-c 0.8 (SD 0.3) versus 1.4 (SD 0.55) mmol/l (p < 0.0001), apolipoprotein A-I 159 (SD 33) versus 127 (SD 24) mg/dl (p < 0.0001) and triglycerides [median (interquartile range)] 1.6 (0.9−2.3) versus 1.0 (0.69–1.21) mmol/l (p after log-transformation < 0.05). The anti-oxidative capacity of HDL was significantly decreased in the carriers compared to controls: fluorescence intensity in carriers was 3.4×104 arbritrary units (AU) versus 2.3×104 AU in controls (p < 0.0001). This association was independent of age and gender. When 4 patients homozygous for a functional LCAT mutation were included in the analysis, the decrease in anti-oxidative capacity was found to be gene dose-dependent (p for ANOVA < 0.0001). Conclusion: This first report of the anti-oxidative properties of LCAT in humans suggests that LCAT has a physiologically relevant anti-oxidative function in human lipoprotein metabolism, which is abolished in LCAT deficiency.
Poster Presentations
P220 THE LEPTIN RECEPTOR GLN223ARG POLYMORPHISM MEDIATES THE POSTPRANDIAL LIPAEMIC RESPONSE IN ADULT MALES K. Jackson1 , E. Olano-Martin1 , E. Abraham1 , R. Gill2 , A. Valdes3 , F. Tang1 , J. Lovegrove1 , C. Williams1 , A. Minihane1 . 1 University of Reading, Reading, UK , 2 Ipsogen Inc, Stamford, CT, USA, 3 King’s College London, London, UK Background and Aims: An exaggerated postprandial triacylglycerol (TAG) response is an important determinant of cardiovascular disease risk. The main genetic modulators of TAG metabolism include variants of the apolipoprotein (apo)E, apoCIII, apoAV and lipoprotein lipase genes. Our aim was to examine the impact of the leptin receptor (LEPR) Gln223Arg polymorphism on postprandial lipaemia in healthy individuals. Methods: Males (n = 122) and females (n = 109) underwent a sequential meal postprandial investigation, in which blood samples were taken at regular intervals after a test breakfast and lunch given at 0 and 330 min respectively. Results: Fasting total- and low density lipoprotein cholesterol were 9% lower in the ArgArg compared with the GlnArg genotype (P < 0.04), whereas fasting TAG was 27% lower in the ArgArg compared with the GlnGln group (P < 0.02). The magnitude of the postprandial TAG response was also significantly lower in the ArgArg compared with the GlnArg and GlnGln genotypes, with a 26% (P = 0.010) and 31% (P = 0.023) lower area under the curve (AUC) and incremental AUC (IAUC) in the ArgArg individuals. Gender × genotype interactions were evident for the fasting TAG, TAG AUC and IAUC (P < 0.05), with the genotype effect only evident in males. Regression analysis indicated that the LEPR genotype and genotype × gender interactions were independent predictors of the TAG AUC, accounting for 6.3% of the variance. Conclusion: We report for the first time that the common LEPR Gln223Arg genotype is an important predictor of the postprandial TAG response in males. The mechanistic basis of these associations remains to be evaluated. P221 LIPID DROPLETS IN HUMAN MACROPHAGES RECRUIT CYTOSOLIC PHOSPHOLIPASE A2 AND PROMOTE SECRETION OF INFLAMMATORY MEDIATORS C. De Pascale, P. Bostrom, Y. Wickstrom, ¨ C. Mogensen, L. Mattsson Hulten, ´ R. Perkins, M. Stahlman, ˚ O. Wiklund, S.-O. Olofsson, J. Boren. ´ Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, ¨ University of Gothenburg, Sahlgrenska University Hospital, Goteborg, Sweden Foam cells, inflammation and hypoxia characterize early atherosclerotic lesions. Previous studies have shown that exposure of human macrophages to hypoxia and oleic acid results in the accumulation of neutral lipids (triglycerides and cholesteryl esters) in cytosolic droplets. Foam cell formation is a lipid disorder but it is also associated with inflammatory events. In this study, we investigated the relationship between lipid droplet formation and the induction of inflammation in human macrophages. Incubation of macrophages with oleic acid and in hypoxic conditions resulted in increased secretion of the eicosanoids leukotriene B4 (LTB4 ) and prostacyclin (PGI2 ) and the chemokines RANTES and interferon-inducible protein 10 (IP-10). Both treatments also promoted the translocation of cytosolic phospholipase A2 (cPLA2 ), an enzyme involved in the eicosanoid production, to the lipid droplet fraction. Transfection of hypoxic macrophages with SNAP23 siRNA, which inhibits lipid droplet fusion and thus increases the lipid droplet surface area, induced substantial increases in cPLA2 levels on lipid droplets and in the secretion of inflammatory mediators. Chemokine secretion was inhibited by cPLA2 inhibitors and LTB4 receptor antagonists, suggesting that this is mediated by increased production of eicosanoids. In conclusion, our data show that cPLA2 may be a direct link between cellular metabolism and inflammatory responses by mediating the formation of eicosanoids and the secretion of chemokines. These inflammatory events may be critical in pathologies such as type 2 diabetes and atherosclerosis where high levels of fatty acids are observed. P222 COMPOSITE DEFICIENCY OF THE LIPOLYTIC COMPLEX IN PREGNANCY-INDUCED MAJOR HYPERTRIGLYCERIDEMIA P. Benlian1 , B. Donadille2 , C. Bouche´ 3 , C. Vincent Dejean4 , R. Valero5 , F. Dufernez6 , V. Sapin7 , F. Paye8 , P. Bouchard2 . 1 Biochemistry and ˆ Molecular Biology, Universite´ Pierre et Marie Curie, 2 Endocrinology, Hopital ˆ Saint Louis APHP, Paris, Saint Antoine, APHP, 3 Endocrinology, Hopital 4 ˆ ˆ Endocrinology, Hopital Pasteur, Chartres, 5 Endocrinology, Hopital Timone, ˆ Saint APHM, Marseille, 6 Biochemistry and Molecular Biology, Hopital Antoine, APHP, 7 Biochemistry and Molecular Biology, CHU Clermont Ferrand, 8 ˆ Digestive Surgery, Hopital Saint Antoine, APHP, Paris, France Background: Moderate hypertriglyceridemia (HTG) is common at the last trimester of normal pregnancy, as the result of high oestrogen secretion. Although exceptional, major HTG may have life-threatening consequences for the mother and child, through a risk of acute pancreatitis before delivery or in post partum. Chylomicronemia may occur as the result of genetic variations of the endothelial lipolytic complex, in metabolic conditions challenging lipoprotein lipase (LPL) activity, however no extensive analysis is usually reported beyond one disease-locus.
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
which includes data from multiple models. The most strongly co-varying genes are mitochondrial (p-value < 10−121 ); with sub-networks corresponding to oxidative phosphorylation, lipogenesis and b-oxidation (p-value < 10−55 ). Distinct inflammatory sub-networks were also discovered (p-values < 10−5 ). Genes associated with metabolic phenotypes in GWASs were identified within the metabolic sub-networks, and included Mlxipl, Pank1, Mtch2, Ide and Jazf1, respectively associated with hypertriglyceridaemia, insulin levels, obesity and T2D. We evaluated gene expression data from human WAT, and also found suppression of oxidative phosphorylation and b-oxidation (P value < 10−12 ) and activation of inflammation (P value < 10−4 ). We tested human SNPs in genes corresponding to those in the mouse networks for genome-wide association with metabolic phenotypes and these are undergoing replication. We conclude that mitochondrial dysfunction in response to high-fat feeding and in obesity favours obesity and inflammation in WAT. P218 TWO NOVEL GENETIC LOCI FOR HIGH AND LOW PLASMA HDL-C LEVELS IN 2 LARGE FAMILIES FROM DUTCH ANCESTRY Mo. Mahdi Motazacker1 , I. Tietjen2 , M. Hayden2 , J.P.P. Kastelein1 , E.S.G. Stroes1 , G.K. Hovingh1 , J.A. Kuivenhoven1 . 1 Academic Medical Center Amsterdam, Amsterdam, The Netherlands, 2 Xenon Pharmaceuticals Inc., Burnaby, BC, Canada Plasma high-density lipoprotein-cholesterol (HDL-c) levels are inversely correlated with cardiovascular disease (CVD). In this light, therapeutic targeting of HDL is regarded as an interesting strategy to reduce atherosclerosis. Plasma HDL-c level is a complex trait and its genetic component has recently been studied by many genome-wide association studies (GWAS). Interestingly, only 7 loci have thus far been associated with this trait which can explain a mere 5.2% of the heritability. This may indicate that rare variants with large or intermediate effect on the phenotype may account for a large portion of this unexplained heritability. In search for rare variants determining plasma HDL-c levels, we have performed linkage analysis studies in 2 large families with an autosomal dominant pattern of high HDL-c (>95th %) or low HDL-c (<5th %) phenotypes for age and gender (mutations in major candidate genes were excluded). We identified a novel locus for high plasma HDL-c levels (4.5 MB interval with multipoint parametric LOD score 3.4) and a novel locus for low plasma HDL-c levels (8 MB interval with multipoint parametric LOD score 4). To identify underlying genetic defects in these linkage intervals, we have designed capture arrays for targeted enrichment of the coding sequence and regulatory elements of the genes within each interval which will be processed by massive parallel sequencing. Our Pilot data indicates that many more ‘HDL genes’ are waiting to be discovered and that family studies are likely to remain an important tool for progress in this field. P219 CARRIERS OF LCAT GENE MUTATIONS HAVE HIGH DENSITY LIPOPROTEIN WITH DECREASED ANTI-OXIDATIVE CAPACITY A.G. Holleboom1 , G. Daniel2 , G.K. Hovingh1 , A.W. Schimmel1 , J.N. van Miert1 , J.J.P. Kastelein1 , E.S.G. Stroes1 , J.A. Kuivenhoven1 , A. Chroni2 . 1 Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands, 2 Institute of Biology, National Center for Scientific Research ‘Demokritos’, Athens, Greece Background: In vitro and animal studies have shown that lecithin:cholesterol acyltransferase (LCAT), an enzyme involved in high density lipoprotein (HDL) metabolism, has anti-oxidant properties. It is unclear, however, whether HDL of carriers of LCAT gene mutations has decreased anti-oxidative capacity. Methods: HDL of 69 heterozygous carriers and 62 unaffected family members was isolated from plasma and incubated in equal concentrations with human oxidized LDL. The capacity of HDL to inactivate the oxidized phospholipids of LDL was measured in the presence of 2,7 dichlorofluorescin (DCFH) that reacts with lipid peroxidation products. Results: Statistically significant differences in lipid levels of heterozygous carriers and controls were: HDL-c 0.8 (SD 0.3) versus 1.4 (SD 0.55) mmol/l (p < 0.0001), apolipoprotein A-I 159 (SD 33) versus 127 (SD 24) mg/dl (p < 0.0001) and triglycerides [median (interquartile range)] 1.6 (0.9−2.3) versus 1.0 (0.69–1.21) mmol/l (p after log-transformation < 0.05). The anti-oxidative capacity of HDL was significantly decreased in the carriers compared to controls: fluorescence intensity in carriers was 3.4×104 arbritrary units (AU) versus 2.3×104 AU in controls (p < 0.0001). This association was independent of age and gender. When 4 patients homozygous for a functional LCAT mutation were included in the analysis, the decrease in anti-oxidative capacity was found to be gene dose-dependent (p for ANOVA < 0.0001). Conclusion: This first report of the anti-oxidative properties of LCAT in humans suggests that LCAT has a physiologically relevant anti-oxidative function in human lipoprotein metabolism, which is abolished in LCAT deficiency.
Poster Presentations
P220 THE LEPTIN RECEPTOR GLN223ARG POLYMORPHISM MEDIATES THE POSTPRANDIAL LIPAEMIC RESPONSE IN ADULT MALES K. Jackson1 , E. Olano-Martin1 , E. Abraham1 , R. Gill2 , A. Valdes3 , F. Tang1 , J. Lovegrove1 , C. Williams1 , A. Minihane1 . 1 University of Reading, Reading, UK , 2 Ipsogen Inc, Stamford, CT, USA, 3 King’s College London, London, UK Background and Aims: An exaggerated postprandial triacylglycerol (TAG) response is an important determinant of cardiovascular disease risk. The main genetic modulators of TAG metabolism include variants of the apolipoprotein (apo)E, apoCIII, apoAV and lipoprotein lipase genes. Our aim was to examine the impact of the leptin receptor (LEPR) Gln223Arg polymorphism on postprandial lipaemia in healthy individuals. Methods: Males (n = 122) and females (n = 109) underwent a sequential meal postprandial investigation, in which blood samples were taken at regular intervals after a test breakfast and lunch given at 0 and 330 min respectively. Results: Fasting total- and low density lipoprotein cholesterol were 9% lower in the ArgArg compared with the GlnArg genotype (P < 0.04), whereas fasting TAG was 27% lower in the ArgArg compared with the GlnGln group (P < 0.02). The magnitude of the postprandial TAG response was also significantly lower in the ArgArg compared with the GlnArg and GlnGln genotypes, with a 26% (P = 0.010) and 31% (P = 0.023) lower area under the curve (AUC) and incremental AUC (IAUC) in the ArgArg individuals. Gender × genotype interactions were evident for the fasting TAG, TAG AUC and IAUC (P < 0.05), with the genotype effect only evident in males. Regression analysis indicated that the LEPR genotype and genotype × gender interactions were independent predictors of the TAG AUC, accounting for 6.3% of the variance. Conclusion: We report for the first time that the common LEPR Gln223Arg genotype is an important predictor of the postprandial TAG response in males. The mechanistic basis of these associations remains to be evaluated. P221 LIPID DROPLETS IN HUMAN MACROPHAGES RECRUIT CYTOSOLIC PHOSPHOLIPASE A2 AND PROMOTE SECRETION OF INFLAMMATORY MEDIATORS C. De Pascale, P. Bostrom, Y. Wickstrom, ¨ C. Mogensen, L. Mattsson Hulten, ´ R. Perkins, M. Stahlman, ˚ O. Wiklund, S.-O. Olofsson, J. Boren. ´ Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, ¨ University of Gothenburg, Sahlgrenska University Hospital, Goteborg, Sweden Foam cells, inflammation and hypoxia characterize early atherosclerotic lesions. Previous studies have shown that exposure of human macrophages to hypoxia and oleic acid results in the accumulation of neutral lipids (triglycerides and cholesteryl esters) in cytosolic droplets. Foam cell formation is a lipid disorder but it is also associated with inflammatory events. In this study, we investigated the relationship between lipid droplet formation and the induction of inflammation in human macrophages. Incubation of macrophages with oleic acid and in hypoxic conditions resulted in increased secretion of the eicosanoids leukotriene B4 (LTB4 ) and prostacyclin (PGI2 ) and the chemokines RANTES and interferon-inducible protein 10 (IP-10). Both treatments also promoted the translocation of cytosolic phospholipase A2 (cPLA2 ), an enzyme involved in the eicosanoid production, to the lipid droplet fraction. Transfection of hypoxic macrophages with SNAP23 siRNA, which inhibits lipid droplet fusion and thus increases the lipid droplet surface area, induced substantial increases in cPLA2 levels on lipid droplets and in the secretion of inflammatory mediators. Chemokine secretion was inhibited by cPLA2 inhibitors and LTB4 receptor antagonists, suggesting that this is mediated by increased production of eicosanoids. In conclusion, our data show that cPLA2 may be a direct link between cellular metabolism and inflammatory responses by mediating the formation of eicosanoids and the secretion of chemokines. These inflammatory events may be critical in pathologies such as type 2 diabetes and atherosclerosis where high levels of fatty acids are observed. P222 COMPOSITE DEFICIENCY OF THE LIPOLYTIC COMPLEX IN PREGNANCY-INDUCED MAJOR HYPERTRIGLYCERIDEMIA P. Benlian1 , B. Donadille2 , C. Bouche´ 3 , C. Vincent Dejean4 , R. Valero5 , F. Dufernez6 , V. Sapin7 , F. Paye8 , P. Bouchard2 . 1 Biochemistry and ˆ Molecular Biology, Universite´ Pierre et Marie Curie, 2 Endocrinology, Hopital ˆ Saint Louis APHP, Paris, Saint Antoine, APHP, 3 Endocrinology, Hopital 4 ˆ ˆ Endocrinology, Hopital Pasteur, Chartres, 5 Endocrinology, Hopital Timone, ˆ Saint APHM, Marseille, 6 Biochemistry and Molecular Biology, Hopital Antoine, APHP, 7 Biochemistry and Molecular Biology, CHU Clermont Ferrand, 8 ˆ Digestive Surgery, Hopital Saint Antoine, APHP, Paris, France Background: Moderate hypertriglyceridemia (HTG) is common at the last trimester of normal pregnancy, as the result of high oestrogen secretion. Although exceptional, major HTG may have life-threatening consequences for the mother and child, through a risk of acute pancreatitis before delivery or in post partum. Chylomicronemia may occur as the result of genetic variations of the endothelial lipolytic complex, in metabolic conditions challenging lipoprotein lipase (LPL) activity, however no extensive analysis is usually reported beyond one disease-locus.