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4.W26.1 Common carotid artery intima-media thickness and the risk of coronary heart disease
294 4 .W25.3
W26
Thursday 9 October 1997: Workshops Non-invasive assessment of atherosclerosis
Interaction of the cholesteryl ester transfer protein with high density lipoproteins from human and mouse plasmas
Laurent Lagrost l , David Masson', Florence Emmanuel2 , Nicolas Duverger2 . I INSERM CJF 93-10, CHRU-Dijon; 2 Rhone-Poulenc Rorer, France In human plasma, the cholesteryl ester transfer protein (CETP) promotes the exchange of cholesteryl esters and triglycerides between various lipoprotein fractions . Previous studies conducted with transgenic mice expressing human high density lipoprotein (HDL) apolipoproteins and CETP provided new insights into the role of apolipoproteins in modulating plasma cholesteryl ester transfer activity in vivo. In order to further elucidate the mechanism by which the expression of human apolipoproteins can affect CETP activity in plasma from transgenic mice, plasma HDL from humans, from transgenic mice to human apolipoprotein A-I (HuAITg mice), from transgenic mice to human apolipoproteins A-I and A-II (HuAIAIITg mice), and from C57BL/6 control mice were isolated, and their ability to interact with CETP was studied . In vitro studies revealed that human and control mouse HDL contain a specific, heat-labile lipid transfer inhibitor activity that is absent from HDL of HuAI and HuAIAII transgenic mice . In complementary experiments, lipid transfer inhibitor activity was shown to be absent from the lipoprotein-deficient plasma fraction from HuAITg mice . Although affinity binding experiments indicated that the interaction of CETP with HDL from humans, control mice or transgenic mice is driven by the electronegativity of HDL, alterations in CETP-lipoprotein binding did not account for differential lipid transfer inhibitor activities of plasma samples from various sources .
4 .W25 .4
Role of the central domain of apolipoprotein A-I in reverse cholesterol transport
Y.L . Marcel, P.G . Frank, V. Franklin, P. Denefle, E. Rassart . Lipoprotein & Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Canada Three human apolipoprotein (apo) A-I mutants, each with a deletion of two consecutive a-helices [A(100-143), A(122-165), 0(144-186)], have been expressed in E. coli. They have been previously shown to exhibit a reduced phospholipid (PL) binding capacity as well as reduced kinetics of association with PL for the last 2 mutants . The deletion 100-143, and within it the deletion 100 and 121, decreases the stability of the protein, possibly through the loss of helix interactions . To determine if these properties affect diffusional efflux of cellular cholesterol, discoidal complexes containing recombinant wild-type apoA-I (Rec .-apoA-I) or the mutant proteins with a constant PL/apoA-I molar ratio have been prepared and incubated with 3 H-cholesterol-labeled human fibroblasts . All mutants and Rec .-apoA-I have similar abilities to promote diffusional cholesterol efflux with comparable kinetics and saturating concentrations . Since apoA-I can also promote cholesterol as well as PL efflux from cholesterol-loaded fibroblasts, we compared cholesterol efflux from cholesterol-loaded fibroblasts to lipid-free Rec .-apoA-I and mutants . Again very little differences are observed between the four proteins . These results suggest that deletions of central a-helices do not affect the ability of the resulting mutants to associate and to retain cellular cholesterol, and that the remaining a-helices are sufficient to promote cholesterol efflux from human fibroblasts. We also investigated the effect of deletions on the capacity of lipid-free apoA-I to act as a shuttle protein for the transfer of cholesterol and phospholipids between cells and lipoproteins . Finally we analyzed the effect of the deletions on the esterification of cell-derived cholesterol by lecithin : cholesterol acyltransferase . The results show that the cholesterol esterification reaction is more dependent on the conformation of apoA-I central domain than the cholesterol efflux process .
with the apo A-II WT, the LCAT 56-68 WT and 0° peptides . None of the other mutant peptides had any fusogenic activity . Vesicle fusion was further accompanied by an increase of the size of the pyrene-labeled DMPC vesicles, after mixing with the apo A-II WT and the LCAT 56-68 WT peptides, whereas the mutants had no effect. The membrane destabilization properties of the apo A-II and the LCAT peptides were monitored by measuring the release of calcein, entrapped inside PC/PE/chol vesicles, upon addition of the apo A-II and LCAT peptides. Calcein leakage amounted to 55% for apo A-II 53-70, 20% for LCAT 56-6 and 90% for the LCAT 56--68, 0° peptide, whereas it was negligible for the mutant peptides . The effect observed for the LCAT 56-68, 0° peptide could be explained by the formation of small phospholipid-peptide complexes . We further investigated the role of the apo A-II 53-70 peptide and its mutants in the displacement of apo A-I from HDL3, by incubating HDL3 with increasing amounts of the apo A-II peptides . Gel filtration on a Superose 12 PG column, demonstrated that the amount of apo A-I released from HDL3 increased up to 55% with increasing apo A-II WT peptidelapo A-I ratios . The corresponding 0° and 90° mutants were unable to displace apo A-I from HDL3 . These data suggest that the angle of insertion of the C-terminal apo A-Il helical peptide into a lipid bilayer, is critical for both its fusogenic activity and its ability to displace apo A-I from HDL3 . Furthermore, the results obtained for the LCAT 56-68 peptide suggest that the 56-68 domain might be involved in destabilizing the lipid core of lipoproteins in order to enable diffusion of a phospholipid monomer into the active site of the enzyme .
W26
NON-INVASIVE ASSESSMENT OF ATHEROSCLEROSIS
I 4.W26 .1 Common carotid artery intima-media thickness and the risk of coronary heart disease
J.R . Crouse Ill. Bowman Gray School of Medicine, Winston-Salem, NC 27157, USA The association between disease of the extracranial carotid arteries and coronary artery disease has been accepted for several years . Chambers and Norris first noted that patients with extracranial carotid stenosis are likely to develop clinical coronary events . Ultrasound studies utilizing B-mode alone or combined with Doppler have identified increased risk of clinical coronary and cerebrovascular events associated with either increased intima-media thickening (IMT) or stenosis of the extracranial carotid arteries . The greatest risk appears to be associated with internal carotid stenosis. One of the advantages of use of the extracranial carotid atherosclerosis as a marker for risk of coronary events relates to its ability to reflect the integrated exposure of the arterial bed to risk factors over a lifetime (as opposed to risk factor levels obtained at a single point in time) . On the other hand, whereas risk of incident coronary events may change rapidly with introduction of pharmacologic agents or life style changes (e .g . cholesterol lowering, estrogen treatment, weigh loss) the changes in IMT occur more slowly . Thus, while IMT is a good marker for incident clinical events in epidemilogic studies or in controlled clinical trials, for individuals exposed to various interventions other measures of vascular disease (e .g . measures of vascular dimensions or function) may be of considerable supportive value.
4 .W26 .2 J MR evaluation of atherosclerotic disease 4 .W25 .5
Contribution apo A-II and LCAT oblique peptides to HDL metabolism
B .Vanloot, 0 . Pdrez-MEndezr , G . Lambert2 , R . Brasseur3 , M . Rosseneut . 'Dept. Biochemistry, University Gent; 3Centre de Biophysique Moleculaire Numerique, Fac. Sci. Agmn . Gembloux, Belgium; 2 CJF INSERM 9508, Institut des Cordeliers, Paris, France Computer modelling of the apo A-II 53-70 and the LCAT 56-68 segments suggests that these amphipathic helices are oriented at an angle of 30° at a lipid/water interface, due to the C-N hydrophobicity gradient along the helix . Mutant peptides were designed by computer modelling to be either parallel (0°) or perpendicular (90°) to a lipid bilayer, and the capacity of the WT and variant peptides to induce fusion of pyrene-labeled phospholipid vesicles was measured . The excimer/monomer fluorescence ratio decreased down to 35 and 50% of its initial value for dimyristoylphosphatidylcholine (DMPC) vesicles
R .R. Edelman. Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts, USA Flowing blood has an appearance in magnetic resonance (MR) images that is distinct from that of stationary tissue . This unique appearance permits the creation of MR "angiograms" that mimic conventional angiograms without the need for potentially nephrotoxic contrast agents or r adiation . MR angiography has numerous clinical applications, but most experience has been accumulated in imaging of the extracranial carotid bifurcations . Several authors have evaluated the combined utility of MRA and duplex ultrasonography, and recommend carotid angiography only for discrepant results(l)(2) ; . An assessment of preoperative imaging strategies for symptomatic patients using a decision-analytic model to minimize morbidity, mortality, and cost was performed by our group(3) . We concluded that the combination of MRA and duplex sonography, with contrast arteriography for discrepant results in 21% of patients, maximizes the quality adjusted life-expectancy with the lowest
11th International Symposium on Atherosclerosis, Paris, October 1997
W26
Thursday 9 October 1997: Workshops Non-invasive assessment of atherosclerosis
Interaction of the cholesteryl ester transfer protein with high density lipoproteins from human and mouse plasmas
Laurent Lagrost l , David Masson', Florence Emmanuel2 , Nicolas Duverger2 . I INSERM CJF 93-10, CHRU-Dijon; 2 Rhone-Poulenc Rorer, France In human plasma, the cholesteryl ester transfer protein (CETP) promotes the exchange of cholesteryl esters and triglycerides between various lipoprotein fractions . Previous studies conducted with transgenic mice expressing human high density lipoprotein (HDL) apolipoproteins and CETP provided new insights into the role of apolipoproteins in modulating plasma cholesteryl ester transfer activity in vivo. In order to further elucidate the mechanism by which the expression of human apolipoproteins can affect CETP activity in plasma from transgenic mice, plasma HDL from humans, from transgenic mice to human apolipoprotein A-I (HuAITg mice), from transgenic mice to human apolipoproteins A-I and A-II (HuAIAIITg mice), and from C57BL/6 control mice were isolated, and their ability to interact with CETP was studied . In vitro studies revealed that human and control mouse HDL contain a specific, heat-labile lipid transfer inhibitor activity that is absent from HDL of HuAI and HuAIAII transgenic mice . In complementary experiments, lipid transfer inhibitor activity was shown to be absent from the lipoprotein-deficient plasma fraction from HuAITg mice . Although affinity binding experiments indicated that the interaction of CETP with HDL from humans, control mice or transgenic mice is driven by the electronegativity of HDL, alterations in CETP-lipoprotein binding did not account for differential lipid transfer inhibitor activities of plasma samples from various sources .
4 .W25 .4
Role of the central domain of apolipoprotein A-I in reverse cholesterol transport
Y.L . Marcel, P.G . Frank, V. Franklin, P. Denefle, E. Rassart . Lipoprotein & Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Canada Three human apolipoprotein (apo) A-I mutants, each with a deletion of two consecutive a-helices [A(100-143), A(122-165), 0(144-186)], have been expressed in E. coli. They have been previously shown to exhibit a reduced phospholipid (PL) binding capacity as well as reduced kinetics of association with PL for the last 2 mutants . The deletion 100-143, and within it the deletion 100 and 121, decreases the stability of the protein, possibly through the loss of helix interactions . To determine if these properties affect diffusional efflux of cellular cholesterol, discoidal complexes containing recombinant wild-type apoA-I (Rec .-apoA-I) or the mutant proteins with a constant PL/apoA-I molar ratio have been prepared and incubated with 3 H-cholesterol-labeled human fibroblasts . All mutants and Rec .-apoA-I have similar abilities to promote diffusional cholesterol efflux with comparable kinetics and saturating concentrations . Since apoA-I can also promote cholesterol as well as PL efflux from cholesterol-loaded fibroblasts, we compared cholesterol efflux from cholesterol-loaded fibroblasts to lipid-free Rec .-apoA-I and mutants . Again very little differences are observed between the four proteins . These results suggest that deletions of central a-helices do not affect the ability of the resulting mutants to associate and to retain cellular cholesterol, and that the remaining a-helices are sufficient to promote cholesterol efflux from human fibroblasts. We also investigated the effect of deletions on the capacity of lipid-free apoA-I to act as a shuttle protein for the transfer of cholesterol and phospholipids between cells and lipoproteins . Finally we analyzed the effect of the deletions on the esterification of cell-derived cholesterol by lecithin : cholesterol acyltransferase . The results show that the cholesterol esterification reaction is more dependent on the conformation of apoA-I central domain than the cholesterol efflux process .
with the apo A-II WT, the LCAT 56-68 WT and 0° peptides . None of the other mutant peptides had any fusogenic activity . Vesicle fusion was further accompanied by an increase of the size of the pyrene-labeled DMPC vesicles, after mixing with the apo A-II WT and the LCAT 56-68 WT peptides, whereas the mutants had no effect. The membrane destabilization properties of the apo A-II and the LCAT peptides were monitored by measuring the release of calcein, entrapped inside PC/PE/chol vesicles, upon addition of the apo A-II and LCAT peptides. Calcein leakage amounted to 55% for apo A-II 53-70, 20% for LCAT 56-6 and 90% for the LCAT 56--68, 0° peptide, whereas it was negligible for the mutant peptides . The effect observed for the LCAT 56-68, 0° peptide could be explained by the formation of small phospholipid-peptide complexes . We further investigated the role of the apo A-II 53-70 peptide and its mutants in the displacement of apo A-I from HDL3, by incubating HDL3 with increasing amounts of the apo A-II peptides . Gel filtration on a Superose 12 PG column, demonstrated that the amount of apo A-I released from HDL3 increased up to 55% with increasing apo A-II WT peptidelapo A-I ratios . The corresponding 0° and 90° mutants were unable to displace apo A-I from HDL3 . These data suggest that the angle of insertion of the C-terminal apo A-Il helical peptide into a lipid bilayer, is critical for both its fusogenic activity and its ability to displace apo A-I from HDL3 . Furthermore, the results obtained for the LCAT 56-68 peptide suggest that the 56-68 domain might be involved in destabilizing the lipid core of lipoproteins in order to enable diffusion of a phospholipid monomer into the active site of the enzyme .
W26
NON-INVASIVE ASSESSMENT OF ATHEROSCLEROSIS
I 4.W26 .1 Common carotid artery intima-media thickness and the risk of coronary heart disease
J.R . Crouse Ill. Bowman Gray School of Medicine, Winston-Salem, NC 27157, USA The association between disease of the extracranial carotid arteries and coronary artery disease has been accepted for several years . Chambers and Norris first noted that patients with extracranial carotid stenosis are likely to develop clinical coronary events . Ultrasound studies utilizing B-mode alone or combined with Doppler have identified increased risk of clinical coronary and cerebrovascular events associated with either increased intima-media thickening (IMT) or stenosis of the extracranial carotid arteries . The greatest risk appears to be associated with internal carotid stenosis. One of the advantages of use of the extracranial carotid atherosclerosis as a marker for risk of coronary events relates to its ability to reflect the integrated exposure of the arterial bed to risk factors over a lifetime (as opposed to risk factor levels obtained at a single point in time) . On the other hand, whereas risk of incident coronary events may change rapidly with introduction of pharmacologic agents or life style changes (e .g . cholesterol lowering, estrogen treatment, weigh loss) the changes in IMT occur more slowly . Thus, while IMT is a good marker for incident clinical events in epidemilogic studies or in controlled clinical trials, for individuals exposed to various interventions other measures of vascular disease (e .g . measures of vascular dimensions or function) may be of considerable supportive value.
4 .W26 .2 J MR evaluation of atherosclerotic disease 4 .W25 .5
Contribution apo A-II and LCAT oblique peptides to HDL metabolism
B .Vanloot, 0 . Pdrez-MEndezr , G . Lambert2 , R . Brasseur3 , M . Rosseneut . 'Dept. Biochemistry, University Gent; 3Centre de Biophysique Moleculaire Numerique, Fac. Sci. Agmn . Gembloux, Belgium; 2 CJF INSERM 9508, Institut des Cordeliers, Paris, France Computer modelling of the apo A-II 53-70 and the LCAT 56-68 segments suggests that these amphipathic helices are oriented at an angle of 30° at a lipid/water interface, due to the C-N hydrophobicity gradient along the helix . Mutant peptides were designed by computer modelling to be either parallel (0°) or perpendicular (90°) to a lipid bilayer, and the capacity of the WT and variant peptides to induce fusion of pyrene-labeled phospholipid vesicles was measured . The excimer/monomer fluorescence ratio decreased down to 35 and 50% of its initial value for dimyristoylphosphatidylcholine (DMPC) vesicles
R .R. Edelman. Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts, USA Flowing blood has an appearance in magnetic resonance (MR) images that is distinct from that of stationary tissue . This unique appearance permits the creation of MR "angiograms" that mimic conventional angiograms without the need for potentially nephrotoxic contrast agents or r adiation . MR angiography has numerous clinical applications, but most experience has been accumulated in imaging of the extracranial carotid bifurcations . Several authors have evaluated the combined utility of MRA and duplex ultrasonography, and recommend carotid angiography only for discrepant results(l)(2) ; . An assessment of preoperative imaging strategies for symptomatic patients using a decision-analytic model to minimize morbidity, mortality, and cost was performed by our group(3) . We concluded that the combination of MRA and duplex sonography, with contrast arteriography for discrepant results in 21% of patients, maximizes the quality adjusted life-expectancy with the lowest
11th International Symposium on Atherosclerosis, Paris, October 1997