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Th-P15:98 Macrophage scavenger receptor 1 is associated with atherosclerosis susceptibility in humans
P15
514
Thurs&ty, June 22, 2006: Poster Session Pathophysiology of lipids attd lipoproteins
I
Th- P 15:961 A P O L I P O P R O T E I N A - V I N T E R A C T I O N W I T H i M E M B E R S OF T H E L O W D E N S I T Y L I P O P R O T E I N R E C E P T O R G E N E FAMILY
IT h - P 1 5 : 9 8 1
I i
M A C R O P H A G E S C A V E N G E R R E C E P T O R 1 IS ASSOCIATED WITH ATHEROSCLEROSIS S U S C E P T I B I L I T Y IN H U M A N S
S.K. Nilsson I , A. Lookene "~, J.B. Beckstead 3 , J. Gliemann 4 , R.O. Ryan 3, G. Olivecrona I . 1Department of Medical Biosciences/Physiological
R. Durst I , Y. Neumark 2, A. Polack I , M. M u s s e d I , R. Beeri I , H. Dannenberg 1 , Y. Friedlander 2 , E. Leitersdorf 3 , V. Meiner 4 , H. Lotan 1 .
Chemistry, Ume& Sweden: 2Department of Gene Technology, Tallinn Universi~ of Technology, Tallinn, Estonia." 3Lipid Biology In Health arm Disease Researeh Group, Childrens Hospital OaklatM Researeh Institute, Oakland, USA." 4Department of Medical Biochemistt T, Universi~ of Aarhus, Aarhus, Denmark
1Cardiology Department, Jerusalem, Israel: 2Braun School of Public Health & Communi~ Medicine, Jerusalem, Israel." 31ntetTtal Medicine B, Jerusalem, Israel." 4Genetic Departnwnt, Hadassah Hebrew Universi~ Medical School, Jerusalem, Israel
Objective: Apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels. To investigate the molecular basis for this phenomenon we explored the ability of apoA-V to interact with two members of the low-density lipoprotein receptor family (LDLR-family), the low density lipoprotein receptor-related protein (LRP) and the mosaic type-1 receptor, SorLA. A double site mutant was also evaluated regarding heparin binding and receptor binding. Methods: Investigation of recombinant apolipoprotein A-V, as free protein and in complex with dimyristoyl-phosphatidylcholine (DMPC), by surface plasmon resonance (SPR) and HPLC. Results: Experiments using surface plasmon resonance showed specific binding of apoA-V to both receptors. ApoA-V binding was calcium dependent and was inhibited by the Receptor Associated Protein (RAP), a known ligand for members of the LDLR-family. Pre-incubation with hepaxin decreased the receptor binding ability of apoA-V-DMPC, indicating that overlap exists between the recognition sites for these receptors and for heparin. A double site mutant showed decreased heparin affinity, but retained receptor binding ability. Association of apoA-V with LRP or SorLA enhanced the ability of the receptors to bind human chylomicrons. Conclusions: ApoA-V may influence plasma lipid homeostasis by enhancing receptor-mediated endocytosis of TG-rich lipoproteins. F u n d i n g : This work was supported by the Swedish Research Council, the King Gusta£ V Research Foundation, The Kempe Research Foundation, Tallinn University of Technology and the National Institutes of Health. I
Th-P 15:97 Ii I D E N T I F I C A T I O N OF A P O L I P O P R O T E I N A - I A S A PLASMA LIGAND FOR MACROPHAGE SCAVENGER RECEPTOR A C. Neyen;, A. Pltiddemann I , B. Thomas 1, A. Akoulitchev 1 , L. Cal.~-, D. Van Der Westhuyzen 2, R. Sire;, S. Gordon;. 1Universi~ ofOaford, Oaford,
United Kingdom: 2 Universi~ of Kentucky, Lexington, USA Objectives: Macrophages adhere to serum-coated tissue culture plastic in an integrin-independent, scavenger receptor A-mediated way. Studies in SR-A-/mice have shown a potentially atherogenic role for SR-A, but the mechanism remains undeax. Our aim was to identify plasma SR-A ligands that mediate adhesion and/or endocytosis, and find possible hypotheses for SR-A atherogenicity. Methods: Human plasma was fractionated by ion exchange and size exclusion chromatography and SR-A binding ability traced in ELISA, on SDS-PAGE and in Western blots. Ligand-containing bands were analysed by mass spectrometry and candidate ligands tested for binding, with the above methods or by flow cytometry and fluorescence microscopy. Results: One of the identified plasma SR-A ligands is apolipoprotein A-I, the major apoprotein of high density lipoprotein. In ELISA, a p t A-I and HDL binding to SR-A can be inhibited by known SR-A ligands. Uptake assays by CHO cells expressing human SR-A show binding and endocytosis of fluorescently labelled a p t A-I and HDL. Conclusions: This work presents a novel ligand-receptor pair with possible relevance to atherosclerosis, namely scavenger receptor A and apolipoprotein A-I. A p t A-I is found in amyloid deposits of atherosclerotic lesions and may promote retention of inflammatory macrophages via SR-A, thereby enhancing disease progression. Alternatively, SR-A might deplete atheroprotective HDL molecules from plasma by an undefined endocytosis mechanism. Further work will need to define SR-A's contribution to HDL metabolism among the large multiligand lipoprotein receptor family. F u n d i n g : Wellcome Trust
B a c k g r o u n d : The macrophage scavenger receptor 1 (MSR1) mediates uptake of modified LDL- c and may thus modulate genetic susceptibility to atherosclerosis through variations in its genomic structure. Several MSR1 gene single nudeotide polymorphisms (SNPs) have recently been described. The present study investigated the association between genotypic variation of the MSR1 gene and atherosclerosis. Methods: DNA samples from 136 non-diabetic males under age 65 undergoing routine coronary angiography, were genotyped for MSR1 SNPs. Data on classical risk factors were collected from patients" files. The number of coronary segments with m a x i m u m stenosis > 2 0 % ( C A G E > 2 0 % ) and the total number of diseased vessels, were used as measures for coronaxy-axtery disease. Associations between MSR1 SNPs and atherosclerotic burden were examined using linear regression modeling accounting for covaxiates. Results: We tested 6 polymorphic sites. Of them 3 were highly polymorphic: a 3-bp insertion deletion of "TTA" in intron 7 (Indel 7), an intron 5 SNP (IVS5-59) and a missense change in exon 6 (P275A). All SNP's were within Haxdy-Weinberg equilibrium. A significant association was noted between the IVS5-59 and number of diseased vessels (p=0.009) and C A G E > 2 0 % (0.017). These relationships remained significant upon controlling for age, cholesterol levels, hypertension, smoking and the effects of the INDEL7 and P275A. Gene-environment interaction effects on C A G E > 2 0 % were noted, including INDEL7 and smoking (p=0.022), P275A and smoking (p=0.034), INDEL7 and age (p=0.024), and IVS5-59 and hypertension (p=0.092). C o n d n s i o n : We demonstrate an association between MSR1 polymorphisms and atherosclerosis in the studied population. Our data suggest that the risk associated with classical risk factors might be modified by MSR1 polymorphisms. These findings point to a significant role of MSR1 in human atherosclerosis development. HEROL INTERFERES WITH THE ITh-P15:991 AR LE PGHUAL-ATTOICOONPOF CD36 A N D A B C A 1 IN M A C R O P H A G E S , K E Y R E C E P T O R S OF P L A Q U E R E G R E S S I O N IN T H E V E S S E L W A L L
S. Rode, R. Lorenz. Kreisla~nstitut, Klinikum Universitaet Muenchen,
Munich, Gerntany Based on the oxidation hypothesis of atherogenesis high doses of alphatocopherol have been advocated for the prevention of vascular events. Unexpectedly several clinical trials suggested proatherogenic effects of high dose tocopherol. The regulation of key effectors of macrophage cholesterol handling in the vessel wall involves the activation of the transcription factors PPARg and L X R a by oxidized fatty acids and oxysterols. We therefore tested, whether the antioxidant alpha-tocopherol impairs the adequate cellular expression of CD36 and ABCA1 by interfering with intracellular signalling via oxidized lipids. Human THP cells served as macrophage model and were preincubated with alpha-tocopherol (20 and 40 Itg/ml) or carrier before exposure to oxLDL (100 Itg/ml), delipidated HDL (20 Itg/ml) or control buffer. CelluDx CD36, ABCA1 and L D L - R specific mRNA was measured by real-time RT-PCR, celluDx cholesterol handling by uptake-efflux assays and LXRalpha-activation by a LXRE-luc construct transfected into the cells. Preincubation with alpha-tocopherol significantly reduced baseline expression of CD36 and ABCA1 and attenuated the increase of CD36 and ABCA1 by LXRalpha activation upon stimulation with oxLDL. Tocopherol also attenuated the ABCA1 down-regulation in macrophages depleted of cholesterol by dHDL exposure. Thereby, tocopherol reduced baseline cellular cholesterol content, cellular cholesterol uptake from oxLDL and cellular cholesterol efflux to dHDL, initializing reverse cholesterol transport. Exposure to pharmacologic concentrations of alpha-tocopherol interferes with the transcriptional response of macrophages to oxLDL. The effect of tocopherol on CD36 and ABCA1 expression of macrophages will retard the scavenging of extracellular cholesterol deposits in the vessel wall and their channeling into reverse cholesterol transport expDhning the proatherogenic effects of high dose alpha-tocopherol observed in several clinical trials.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
514
Thurs&ty, June 22, 2006: Poster Session Pathophysiology of lipids attd lipoproteins
I
Th- P 15:961 A P O L I P O P R O T E I N A - V I N T E R A C T I O N W I T H i M E M B E R S OF T H E L O W D E N S I T Y L I P O P R O T E I N R E C E P T O R G E N E FAMILY
IT h - P 1 5 : 9 8 1
I i
M A C R O P H A G E S C A V E N G E R R E C E P T O R 1 IS ASSOCIATED WITH ATHEROSCLEROSIS S U S C E P T I B I L I T Y IN H U M A N S
S.K. Nilsson I , A. Lookene "~, J.B. Beckstead 3 , J. Gliemann 4 , R.O. Ryan 3, G. Olivecrona I . 1Department of Medical Biosciences/Physiological
R. Durst I , Y. Neumark 2, A. Polack I , M. M u s s e d I , R. Beeri I , H. Dannenberg 1 , Y. Friedlander 2 , E. Leitersdorf 3 , V. Meiner 4 , H. Lotan 1 .
Chemistry, Ume& Sweden: 2Department of Gene Technology, Tallinn Universi~ of Technology, Tallinn, Estonia." 3Lipid Biology In Health arm Disease Researeh Group, Childrens Hospital OaklatM Researeh Institute, Oakland, USA." 4Department of Medical Biochemistt T, Universi~ of Aarhus, Aarhus, Denmark
1Cardiology Department, Jerusalem, Israel: 2Braun School of Public Health & Communi~ Medicine, Jerusalem, Israel." 31ntetTtal Medicine B, Jerusalem, Israel." 4Genetic Departnwnt, Hadassah Hebrew Universi~ Medical School, Jerusalem, Israel
Objective: Apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels. To investigate the molecular basis for this phenomenon we explored the ability of apoA-V to interact with two members of the low-density lipoprotein receptor family (LDLR-family), the low density lipoprotein receptor-related protein (LRP) and the mosaic type-1 receptor, SorLA. A double site mutant was also evaluated regarding heparin binding and receptor binding. Methods: Investigation of recombinant apolipoprotein A-V, as free protein and in complex with dimyristoyl-phosphatidylcholine (DMPC), by surface plasmon resonance (SPR) and HPLC. Results: Experiments using surface plasmon resonance showed specific binding of apoA-V to both receptors. ApoA-V binding was calcium dependent and was inhibited by the Receptor Associated Protein (RAP), a known ligand for members of the LDLR-family. Pre-incubation with hepaxin decreased the receptor binding ability of apoA-V-DMPC, indicating that overlap exists between the recognition sites for these receptors and for heparin. A double site mutant showed decreased heparin affinity, but retained receptor binding ability. Association of apoA-V with LRP or SorLA enhanced the ability of the receptors to bind human chylomicrons. Conclusions: ApoA-V may influence plasma lipid homeostasis by enhancing receptor-mediated endocytosis of TG-rich lipoproteins. F u n d i n g : This work was supported by the Swedish Research Council, the King Gusta£ V Research Foundation, The Kempe Research Foundation, Tallinn University of Technology and the National Institutes of Health. I
Th-P 15:97 Ii I D E N T I F I C A T I O N OF A P O L I P O P R O T E I N A - I A S A PLASMA LIGAND FOR MACROPHAGE SCAVENGER RECEPTOR A C. Neyen;, A. Pltiddemann I , B. Thomas 1, A. Akoulitchev 1 , L. Cal.~-, D. Van Der Westhuyzen 2, R. Sire;, S. Gordon;. 1Universi~ ofOaford, Oaford,
United Kingdom: 2 Universi~ of Kentucky, Lexington, USA Objectives: Macrophages adhere to serum-coated tissue culture plastic in an integrin-independent, scavenger receptor A-mediated way. Studies in SR-A-/mice have shown a potentially atherogenic role for SR-A, but the mechanism remains undeax. Our aim was to identify plasma SR-A ligands that mediate adhesion and/or endocytosis, and find possible hypotheses for SR-A atherogenicity. Methods: Human plasma was fractionated by ion exchange and size exclusion chromatography and SR-A binding ability traced in ELISA, on SDS-PAGE and in Western blots. Ligand-containing bands were analysed by mass spectrometry and candidate ligands tested for binding, with the above methods or by flow cytometry and fluorescence microscopy. Results: One of the identified plasma SR-A ligands is apolipoprotein A-I, the major apoprotein of high density lipoprotein. In ELISA, a p t A-I and HDL binding to SR-A can be inhibited by known SR-A ligands. Uptake assays by CHO cells expressing human SR-A show binding and endocytosis of fluorescently labelled a p t A-I and HDL. Conclusions: This work presents a novel ligand-receptor pair with possible relevance to atherosclerosis, namely scavenger receptor A and apolipoprotein A-I. A p t A-I is found in amyloid deposits of atherosclerotic lesions and may promote retention of inflammatory macrophages via SR-A, thereby enhancing disease progression. Alternatively, SR-A might deplete atheroprotective HDL molecules from plasma by an undefined endocytosis mechanism. Further work will need to define SR-A's contribution to HDL metabolism among the large multiligand lipoprotein receptor family. F u n d i n g : Wellcome Trust
B a c k g r o u n d : The macrophage scavenger receptor 1 (MSR1) mediates uptake of modified LDL- c and may thus modulate genetic susceptibility to atherosclerosis through variations in its genomic structure. Several MSR1 gene single nudeotide polymorphisms (SNPs) have recently been described. The present study investigated the association between genotypic variation of the MSR1 gene and atherosclerosis. Methods: DNA samples from 136 non-diabetic males under age 65 undergoing routine coronary angiography, were genotyped for MSR1 SNPs. Data on classical risk factors were collected from patients" files. The number of coronary segments with m a x i m u m stenosis > 2 0 % ( C A G E > 2 0 % ) and the total number of diseased vessels, were used as measures for coronaxy-axtery disease. Associations between MSR1 SNPs and atherosclerotic burden were examined using linear regression modeling accounting for covaxiates. Results: We tested 6 polymorphic sites. Of them 3 were highly polymorphic: a 3-bp insertion deletion of "TTA" in intron 7 (Indel 7), an intron 5 SNP (IVS5-59) and a missense change in exon 6 (P275A). All SNP's were within Haxdy-Weinberg equilibrium. A significant association was noted between the IVS5-59 and number of diseased vessels (p=0.009) and C A G E > 2 0 % (0.017). These relationships remained significant upon controlling for age, cholesterol levels, hypertension, smoking and the effects of the INDEL7 and P275A. Gene-environment interaction effects on C A G E > 2 0 % were noted, including INDEL7 and smoking (p=0.022), P275A and smoking (p=0.034), INDEL7 and age (p=0.024), and IVS5-59 and hypertension (p=0.092). C o n d n s i o n : We demonstrate an association between MSR1 polymorphisms and atherosclerosis in the studied population. Our data suggest that the risk associated with classical risk factors might be modified by MSR1 polymorphisms. These findings point to a significant role of MSR1 in human atherosclerosis development. HEROL INTERFERES WITH THE ITh-P15:991 AR LE PGHUAL-ATTOICOONPOF CD36 A N D A B C A 1 IN M A C R O P H A G E S , K E Y R E C E P T O R S OF P L A Q U E R E G R E S S I O N IN T H E V E S S E L W A L L
S. Rode, R. Lorenz. Kreisla~nstitut, Klinikum Universitaet Muenchen,
Munich, Gerntany Based on the oxidation hypothesis of atherogenesis high doses of alphatocopherol have been advocated for the prevention of vascular events. Unexpectedly several clinical trials suggested proatherogenic effects of high dose tocopherol. The regulation of key effectors of macrophage cholesterol handling in the vessel wall involves the activation of the transcription factors PPARg and L X R a by oxidized fatty acids and oxysterols. We therefore tested, whether the antioxidant alpha-tocopherol impairs the adequate cellular expression of CD36 and ABCA1 by interfering with intracellular signalling via oxidized lipids. Human THP cells served as macrophage model and were preincubated with alpha-tocopherol (20 and 40 Itg/ml) or carrier before exposure to oxLDL (100 Itg/ml), delipidated HDL (20 Itg/ml) or control buffer. CelluDx CD36, ABCA1 and L D L - R specific mRNA was measured by real-time RT-PCR, celluDx cholesterol handling by uptake-efflux assays and LXRalpha-activation by a LXRE-luc construct transfected into the cells. Preincubation with alpha-tocopherol significantly reduced baseline expression of CD36 and ABCA1 and attenuated the increase of CD36 and ABCA1 by LXRalpha activation upon stimulation with oxLDL. Tocopherol also attenuated the ABCA1 down-regulation in macrophages depleted of cholesterol by dHDL exposure. Thereby, tocopherol reduced baseline cellular cholesterol content, cellular cholesterol uptake from oxLDL and cellular cholesterol efflux to dHDL, initializing reverse cholesterol transport. Exposure to pharmacologic concentrations of alpha-tocopherol interferes with the transcriptional response of macrophages to oxLDL. The effect of tocopherol on CD36 and ABCA1 expression of macrophages will retard the scavenging of extracellular cholesterol deposits in the vessel wall and their channeling into reverse cholesterol transport expDhning the proatherogenic effects of high dose alpha-tocopherol observed in several clinical trials.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006