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P230 ABC TRANSPORTERS-MEDIATED CHOLESTEROL EFFLUX FROM HUMAN MACROPHAGE FOAM CELLS IS IMPAIRED IN AN ACIDIC EXTRACELLULAR ENVIRONMENT
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Atherosclerosis Supplements 11, no. 2 (2010) 17–108
was significantly lower in FCH (46±58×102 mmol/mmol) than in PH or controls (79±81 and 82±59). Sitosterol was significantly higher in PH than in controls (130±81 vs 95±37×102 mmol/mmol, p < 0.05). Lathosterol showed a direct relationship with BMI, waist circumference and HOMA (r 0.297, 0.322, 0.196, respectively, p < 0.001) Campesterol and sitosterol had direct relationships with HDL-cholesterol (r = 0.39 and r = 0.41, respectively). Multivariate regression analysis showed female gender and sitosterol as independent predictors of HDL-C (b 0.230 and 0.246, p < 0.05). Conclusions: FCH is characterized by increased cholesterol biosynthesis and reduction in absorption; higher cholesterol absorption was found in polygenic hypercholesterolemia. Dietary cholesterol influences HDL-C levels. Investigating cholesterol synthesis and absorption by dosing non-cholesterol sterols is a promising approach to a better insight into the pathogenesis of hyperlipemias. P228 POTENTIAL ATHEROGENICITY OF POSTPRANDIAL LIPEMIA IN MIXED DYSLIPIDEMIA: THERAPEUTIC TARGET FOR CETP INHIBITION Z. Julia1 , E. Duchene1,2 , N. Fournier3,4 , N. Bellanger1 , M.J. Chapman1 , W. Le Goff1 , M. Guerin1 . 1 UMRS939, INSERM-UPMC, 2 Department of Endocrinology, AP-HP Pitie-Salpetriere, Paris, 3 UMR INRA 1154, University Paris-Sud, Chatenay-Malabry, 4 Service de Biochimie, AP-HP Hopital Europeen Georges Pompidou, Paris, France Hypothesis: Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodelling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. Methods and Results: We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular FC efflux, CETP-mediated CE transfer and selective hepatic CE uptake during the postprandial phase in subjects displaying mixed dyslipidemia (n = 16). Results: Postprandial large HDL2 displayed an enhanced capacity to mediate FC efflux via both SR-BI- (+12%;p < 0.02) and ABCG1- (+31%;p < 0.008) dependent pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; p < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB-lipoproteins, and with attenuated capacity (−17%; p < 0.02) of total HDL to deliver CE to hepatic cells (HepG2) in vitro. Conclusion: Postprandially, cellular cholesterol efflux was preferentially enhanced through both SR-BI and ABCG1 pathways in mixed dyslipidemic subjects. Equally, CETP-mediated transfer of CE to TRL particles was significantly elevated. Moreover hepatic uptake of HDL-CE via SR-BI was diminished. Considered together, these findings strongly suggest that postprandial changes in HDL particle metabolism and function in mixed dyslipidemia may constitute a therapeutic target to attenuate the proatherogenic features of postprandial lipemia in dyslipidemic subjects at high cardiovascular risk. Clearly then, CETP inhibition may represent an efficacious strategy in this metabolic context. P229 EFFECTS OF HIGH DOSE STATIN ON THE HUMAN HEPATIC EXPRESSION OF GENES INVOLVED IN CARBOHYDRATE AND TRIGLYCERIDE METABOLISM C. Pramfalk1 , P. Parini1 , U. Gustafsson2 , S. Sahlin2 , M. Eriksson3 . 1 Department of Laboratory Medicine, 2 Department of Surgery, 3 Department of Medicine, Karolinska Institute, Stockholm, Sweden Background: Atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, lowers plasma cholesterol and triglyceride (TG) levels in a dose-dependent manner. Here we aimed to investigate the molecular mechanism(s) that results in lower TG in patients treated with 80 mg/d atorvastatin for four weeks. Methods: Lipoprotein separation and plasma analysis of lipids, glucose, and insulin were performed. Liver TG mass was determined in pooled samples. Hepatic gene expressions of several genes involved in carbohydrate and TG metabolism were analysed. Results: Atorvastatin lowered plasma levels of very low density lipoprotein (VLDL) TG (>40%, p < 0.05), low density lipoprotein (LDL) TG (N.S.) and liver TG mass compared to placebo. Except for cholesterol changes, no other significant differences in plasma lipids, glucose, and insulin occurred. However, atorvastatin reduced mRNA expressions of sterol regulatory element binding protein 1c (SREBP1c) (>50%, p < 0.05), glucokinase (GK) (>75%, p < 0.05), angiopoietin-like protein 3 (ANGPTL3) (~30%, p < 0.01), and induced mRNA expressions of acetyl-coenzyme A carboxylase 1 (~45%, p < 0.05), and glucose-6-phosphatase (G6Pase) (~70%, p < 0.05) compared to placebo. Conclusions: Reduced ANGPTL3 mRNA expression, following treatment with atorvastatin, may contribute to reduced plasma levels of VLDL TG. The reduced liver TG mass together with decreased hepatic SREBP1c and GK expressions and increased G6Pase expression suggests that treatment with atorvastatin do
Poster Presentations
not have negative effects on the insulin-sensitivity in humans. The reduced liver TG mass by high dosage of atorvastatin may be important as a treatment option for fatty liver in humans. P230 ABC TRANSPORTERS-MEDIATED CHOLESTEROL EFFLUX FROM HUMAN MACROPHAGE FOAM CELLS IS IMPAIRED IN AN ACIDIC EXTRACELLULAR ENVIRONMENT M. Lee-Rueckert1 , J. Lappalainen1 , H. Leinonen1 , M. Jauhiainen2 , P. Kovanen1 . 1 Wihuri Research Institute, 2 National Public Health Institute and Finnish Institute for Molecular Medicine, Biomedicum, Helsinki, Finland In the deep microenvironments of advanced human atherosclerotic lesions, the intimal fluid becomes acidic. We hypothesized that an acidic extracellular pH may disturb macrophage-lipoprotein interactions, so compromising cholesterol efflux from these cells. 3 H-cholesterol efflux from human monocyte-derived macrophage foam cells to various cholesterol acceptors was evaluated in culture media of pH 5.5, 6.5, and 7.5. It appeared that the lower the pH, the more was cholesterol efflux reduced, the reduction being strongest to lipidfree apoA-I and less so to HDL2 or to plasma. Of note, cholesterol efflux to apoA-I and HDL2 at neutral pH was also compromised from foam cells that had been generated at acidic pH. Compatible with this observation, the gene expression of the membrane proteins ABCA1 and ABCG1, which is typically up-regulated by incubation of macrophages with acetyl-LDL in neutral medium, was progressively blocked at acidic pH. ApoE secretion from the foam cells increased at pH 6.5 but was blocked at pH 5.5, so further reducing the cholesterol efflux potential of these cells. In overall, the results revealed that cholesterol efflux is pH-dependent and, moreover, demonstrated multiple mechanisms involved in the inhibitory effect induced by a low pH. The present study discloses a novel effect of the extracellular pH which, by regulating cholesterol efflux mediated by the ABC transporters from macrophage foam cells, may play a critical role in acceleration of atherosclerosis in local areas where the intimal fluid becomes acidic. P231 IDENTIFICATION OF COMPOUND HETEROZYGOUS DEFICIENCY WITH A NOVEL LARGE DELETION AND Y61X OF THE LIPOPROTEIN LIPASE GENE IN JAPANESE SUBJECT A. Takagi1 , M. Nagano2 , T. Iwanaga1 , N. Ito2 , H. Hattori2 , T. Egashira2 , Y. Ikeda1 . 1 National Cardiovascular Center, Suita, 2 BML, Inc., Kawagoe, Japan Objectives: The early diagnosis of the LPL gene mutations is important for preventing the development of hypertriglyceridemia (HTG) that is one of the defined components of metabolic syndrome. In the Japanese, 24 LPL mutations resulting in non-functional LPL molecule have been reported, in which 20 mutations were accumulated by us. Accumulation of these LPL mutations makes it possible to establish an early diagnostic system of LPL gene mutations. Here we aimed to furthermore identify novel LPL mutations from a subject with HTG. Methods: A Japanese hypertriglyceridemic subject, #521 (one month, male; TG, 779 mg/dl, TC, 280 mg/dl) was examined. LPL activity and immunoreactive mass in postheparin plasma (PHP) were measured with a selective immunoinactivation assay and MARKIT-M LPL ELISA kit (Dainippon Sumitomo Pharma Co., Japan), respectively. LPL mutations were identified with PCRSSCP, Invader, Southern blot and DNA sequencing. Results: Patient #521 showed extremely low levels of LPL mass (1.3 ng/ml) and activity (0.37 unit/h/ml) in PHP. The patient was compound heterozygous deficiency with a novel large deletion (a 54 kb containing 5 upper region and a part of intron 1 in the LPL gene; 53,918 bp from 7,594,420 to 7,648,337 in NT-030737.9) and a known nonsense mutation (Y61X) in exon 3 of the LPL gene. We established a simple PCR method to detect this deletion mutation. Conclusion: Establishment of a simple genetic analysis for the large deletion can promote efficiency for the detection of its LPL gene mutation and may thereby contribute to treatment and prevention of HTG in the Japanese. P232 LDL RECEPTOR RELATED PROTEIN 1 (LRP1) IS NECESSARY FOR APOLIPOPROTEIN A5 (APOA5) MEDIATED TRIGLYCERIDE REDUCTION 1 K. Brugelmann ¨ , D. Teupser2 , A. Bartelt1 , P. Gordts3 , J. Heeren1 , M. Merkel4 . 1 Institute for Molecular Cell Biology, University Hospital Hamburg-Eppendorf, Hamburg, 2 Institute of Laboratory Medicine, University Hospital Leipzig, Leipzig, Germany, 3 Department of Human Genetics, Developmental and Molecular Genetics Section, University of Leuven, Leuven, Belgium, 4 Department of Internal Medicine, Asklepios Clinic St. George, Hamburg, Germany Objective: ApoA5 is an important regulator of plasma triglyceride levels (TG) in mice and humans. It was proposed that apoA5 activates lipoprotein lipase and thereby accelerates plasma TG hydrolysis. Recent in vitro studies suggest that apoA5 may also mediate lipoprotein uptake by members of the LDL receptor family. This study investigates apoA5 interactions with lipoprotein receptors in vivo.
Atherosclerosis Supplements 11, no. 2 (2010) 17–108
was significantly lower in FCH (46±58×102 mmol/mmol) than in PH or controls (79±81 and 82±59). Sitosterol was significantly higher in PH than in controls (130±81 vs 95±37×102 mmol/mmol, p < 0.05). Lathosterol showed a direct relationship with BMI, waist circumference and HOMA (r 0.297, 0.322, 0.196, respectively, p < 0.001) Campesterol and sitosterol had direct relationships with HDL-cholesterol (r = 0.39 and r = 0.41, respectively). Multivariate regression analysis showed female gender and sitosterol as independent predictors of HDL-C (b 0.230 and 0.246, p < 0.05). Conclusions: FCH is characterized by increased cholesterol biosynthesis and reduction in absorption; higher cholesterol absorption was found in polygenic hypercholesterolemia. Dietary cholesterol influences HDL-C levels. Investigating cholesterol synthesis and absorption by dosing non-cholesterol sterols is a promising approach to a better insight into the pathogenesis of hyperlipemias. P228 POTENTIAL ATHEROGENICITY OF POSTPRANDIAL LIPEMIA IN MIXED DYSLIPIDEMIA: THERAPEUTIC TARGET FOR CETP INHIBITION Z. Julia1 , E. Duchene1,2 , N. Fournier3,4 , N. Bellanger1 , M.J. Chapman1 , W. Le Goff1 , M. Guerin1 . 1 UMRS939, INSERM-UPMC, 2 Department of Endocrinology, AP-HP Pitie-Salpetriere, Paris, 3 UMR INRA 1154, University Paris-Sud, Chatenay-Malabry, 4 Service de Biochimie, AP-HP Hopital Europeen Georges Pompidou, Paris, France Hypothesis: Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodelling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. Methods and Results: We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular FC efflux, CETP-mediated CE transfer and selective hepatic CE uptake during the postprandial phase in subjects displaying mixed dyslipidemia (n = 16). Results: Postprandial large HDL2 displayed an enhanced capacity to mediate FC efflux via both SR-BI- (+12%;p < 0.02) and ABCG1- (+31%;p < 0.008) dependent pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; p < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB-lipoproteins, and with attenuated capacity (−17%; p < 0.02) of total HDL to deliver CE to hepatic cells (HepG2) in vitro. Conclusion: Postprandially, cellular cholesterol efflux was preferentially enhanced through both SR-BI and ABCG1 pathways in mixed dyslipidemic subjects. Equally, CETP-mediated transfer of CE to TRL particles was significantly elevated. Moreover hepatic uptake of HDL-CE via SR-BI was diminished. Considered together, these findings strongly suggest that postprandial changes in HDL particle metabolism and function in mixed dyslipidemia may constitute a therapeutic target to attenuate the proatherogenic features of postprandial lipemia in dyslipidemic subjects at high cardiovascular risk. Clearly then, CETP inhibition may represent an efficacious strategy in this metabolic context. P229 EFFECTS OF HIGH DOSE STATIN ON THE HUMAN HEPATIC EXPRESSION OF GENES INVOLVED IN CARBOHYDRATE AND TRIGLYCERIDE METABOLISM C. Pramfalk1 , P. Parini1 , U. Gustafsson2 , S. Sahlin2 , M. Eriksson3 . 1 Department of Laboratory Medicine, 2 Department of Surgery, 3 Department of Medicine, Karolinska Institute, Stockholm, Sweden Background: Atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, lowers plasma cholesterol and triglyceride (TG) levels in a dose-dependent manner. Here we aimed to investigate the molecular mechanism(s) that results in lower TG in patients treated with 80 mg/d atorvastatin for four weeks. Methods: Lipoprotein separation and plasma analysis of lipids, glucose, and insulin were performed. Liver TG mass was determined in pooled samples. Hepatic gene expressions of several genes involved in carbohydrate and TG metabolism were analysed. Results: Atorvastatin lowered plasma levels of very low density lipoprotein (VLDL) TG (>40%, p < 0.05), low density lipoprotein (LDL) TG (N.S.) and liver TG mass compared to placebo. Except for cholesterol changes, no other significant differences in plasma lipids, glucose, and insulin occurred. However, atorvastatin reduced mRNA expressions of sterol regulatory element binding protein 1c (SREBP1c) (>50%, p < 0.05), glucokinase (GK) (>75%, p < 0.05), angiopoietin-like protein 3 (ANGPTL3) (~30%, p < 0.01), and induced mRNA expressions of acetyl-coenzyme A carboxylase 1 (~45%, p < 0.05), and glucose-6-phosphatase (G6Pase) (~70%, p < 0.05) compared to placebo. Conclusions: Reduced ANGPTL3 mRNA expression, following treatment with atorvastatin, may contribute to reduced plasma levels of VLDL TG. The reduced liver TG mass together with decreased hepatic SREBP1c and GK expressions and increased G6Pase expression suggests that treatment with atorvastatin do
Poster Presentations
not have negative effects on the insulin-sensitivity in humans. The reduced liver TG mass by high dosage of atorvastatin may be important as a treatment option for fatty liver in humans. P230 ABC TRANSPORTERS-MEDIATED CHOLESTEROL EFFLUX FROM HUMAN MACROPHAGE FOAM CELLS IS IMPAIRED IN AN ACIDIC EXTRACELLULAR ENVIRONMENT M. Lee-Rueckert1 , J. Lappalainen1 , H. Leinonen1 , M. Jauhiainen2 , P. Kovanen1 . 1 Wihuri Research Institute, 2 National Public Health Institute and Finnish Institute for Molecular Medicine, Biomedicum, Helsinki, Finland In the deep microenvironments of advanced human atherosclerotic lesions, the intimal fluid becomes acidic. We hypothesized that an acidic extracellular pH may disturb macrophage-lipoprotein interactions, so compromising cholesterol efflux from these cells. 3 H-cholesterol efflux from human monocyte-derived macrophage foam cells to various cholesterol acceptors was evaluated in culture media of pH 5.5, 6.5, and 7.5. It appeared that the lower the pH, the more was cholesterol efflux reduced, the reduction being strongest to lipidfree apoA-I and less so to HDL2 or to plasma. Of note, cholesterol efflux to apoA-I and HDL2 at neutral pH was also compromised from foam cells that had been generated at acidic pH. Compatible with this observation, the gene expression of the membrane proteins ABCA1 and ABCG1, which is typically up-regulated by incubation of macrophages with acetyl-LDL in neutral medium, was progressively blocked at acidic pH. ApoE secretion from the foam cells increased at pH 6.5 but was blocked at pH 5.5, so further reducing the cholesterol efflux potential of these cells. In overall, the results revealed that cholesterol efflux is pH-dependent and, moreover, demonstrated multiple mechanisms involved in the inhibitory effect induced by a low pH. The present study discloses a novel effect of the extracellular pH which, by regulating cholesterol efflux mediated by the ABC transporters from macrophage foam cells, may play a critical role in acceleration of atherosclerosis in local areas where the intimal fluid becomes acidic. P231 IDENTIFICATION OF COMPOUND HETEROZYGOUS DEFICIENCY WITH A NOVEL LARGE DELETION AND Y61X OF THE LIPOPROTEIN LIPASE GENE IN JAPANESE SUBJECT A. Takagi1 , M. Nagano2 , T. Iwanaga1 , N. Ito2 , H. Hattori2 , T. Egashira2 , Y. Ikeda1 . 1 National Cardiovascular Center, Suita, 2 BML, Inc., Kawagoe, Japan Objectives: The early diagnosis of the LPL gene mutations is important for preventing the development of hypertriglyceridemia (HTG) that is one of the defined components of metabolic syndrome. In the Japanese, 24 LPL mutations resulting in non-functional LPL molecule have been reported, in which 20 mutations were accumulated by us. Accumulation of these LPL mutations makes it possible to establish an early diagnostic system of LPL gene mutations. Here we aimed to furthermore identify novel LPL mutations from a subject with HTG. Methods: A Japanese hypertriglyceridemic subject, #521 (one month, male; TG, 779 mg/dl, TC, 280 mg/dl) was examined. LPL activity and immunoreactive mass in postheparin plasma (PHP) were measured with a selective immunoinactivation assay and MARKIT-M LPL ELISA kit (Dainippon Sumitomo Pharma Co., Japan), respectively. LPL mutations were identified with PCRSSCP, Invader, Southern blot and DNA sequencing. Results: Patient #521 showed extremely low levels of LPL mass (1.3 ng/ml) and activity (0.37 unit/h/ml) in PHP. The patient was compound heterozygous deficiency with a novel large deletion (a 54 kb containing 5 upper region and a part of intron 1 in the LPL gene; 53,918 bp from 7,594,420 to 7,648,337 in NT-030737.9) and a known nonsense mutation (Y61X) in exon 3 of the LPL gene. We established a simple PCR method to detect this deletion mutation. Conclusion: Establishment of a simple genetic analysis for the large deletion can promote efficiency for the detection of its LPL gene mutation and may thereby contribute to treatment and prevention of HTG in the Japanese. P232 LDL RECEPTOR RELATED PROTEIN 1 (LRP1) IS NECESSARY FOR APOLIPOPROTEIN A5 (APOA5) MEDIATED TRIGLYCERIDE REDUCTION 1 K. Brugelmann ¨ , D. Teupser2 , A. Bartelt1 , P. Gordts3 , J. Heeren1 , M. Merkel4 . 1 Institute for Molecular Cell Biology, University Hospital Hamburg-Eppendorf, Hamburg, 2 Institute of Laboratory Medicine, University Hospital Leipzig, Leipzig, Germany, 3 Department of Human Genetics, Developmental and Molecular Genetics Section, University of Leuven, Leuven, Belgium, 4 Department of Internal Medicine, Asklepios Clinic St. George, Hamburg, Germany Objective: ApoA5 is an important regulator of plasma triglyceride levels (TG) in mice and humans. It was proposed that apoA5 activates lipoprotein lipase and thereby accelerates plasma TG hydrolysis. Recent in vitro studies suggest that apoA5 may also mediate lipoprotein uptake by members of the LDL receptor family. This study investigates apoA5 interactions with lipoprotein receptors in vivo.