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A novel approach to cholesterol lowering: Lipoprotein charge control
Stein
demonstrated the presence of immunoreactive FAT/CD36 in pBCEC lysates. In summary, our findings indicate that lipoprotein-associated PL are taken up via SR-BI mediated selective uptake and are partially hydrolyzed in the sn-2 position, in line with the localization of PUFAs in PL. The fatty acid pool that is generated by EL-dependent hydrolysis could then be incorporated by the action of FAT/CD36. This SR-BI/EL/FAT-dependent pathway could represent a feasible uptake route for PUFAs from the peripheral circulation into the brain.
I670 A NOVEL
APPROACH TO CHOLESTEROL LIPOPROTEIN CHARGE CONTROL
LOWERING:
D.L. Sparks. Uniuersify of Ottawa Heart Institute and Liponex Inc., Ottawa, ON, Canada Studies show that lipoprotein charge can regulate interfacial interactions with vascular proteins and cell surface receptors. A novel therapeutic approach has therefore been designed to selectively manipulate lipoprotein charge and therein affect the metabolism of cholesterol. CRD5 was developed to increase the negative charge of all major lipoprotein classes in viva. These alterations to lipoprotein charge turn on an excretory pathway that lowers plasma cholesterol levels by liberating cholesterol from LDL and tissues and promoting its transport to the liver for clearance. CRD5 lowers LDL cholesterol levels by inhibiting LCAT-dependent cholesterol esterification and by promoting a CETP mediated transfer of cholesteryl ester from LDL to HDL. CRD5 also promotes a 30-fold increase in the rate of clearance of [3H]cholesterol (FC) from the plasma. Enhanced clearance of cholesterol from the blood stream is associated with an increased hepatic uptake and a 20-fold increase in biliary cholesterol output. In addition, a single bolus dose of CRD5 promotes an almost 3-fold increase in the fecal excretion of cholesterol from a rabbit. Studies in cholesterol-fed hypercholesterolemic rabbits show that CRD5 has value as a cholesterol lowering therapeutic. LDL and VLDL cholesterol levels are reduced to near normal values after 1 week of treatment with CRDS. In addition, pre-clinical toxicology studies show that CRD5 is well tolerated; no adverse clinical signs or organ damage was observed following escalatory doses of CRD5 up to 120 mg/kg over a 14d study. These data show that the transport and clearance of cholesterol is affected by lipoprotein charge and can be stimulated by the administration of CRDS. The work shows that reverse cholesterol transport can now be selectively manipulated in viva.
I671 NEW
DEVELOPMENTS IN FAMILIAL HYPERLIPIDEMIA (FCH)
A.F.H. Stalenhoef, M.J. Veerkamp, Nijmegen, The Netherlands
.I. DeGraaf.
COMBINED
Uniuersify Medical Centre
FCH is characterized by a variable expression of dyslipidemia. The molecular mechanisms are still unclear. Although several gene mapping studies have surfaced candidate regions, no causative genes have been identified so far. A mutant mouse strain has been described, which shares features with FCH, in which a nonsense mutation in the gene encoding a cytoplasmatic protein, thioredoxin interacting protein, was found, possibly responsible for the hyperlipidemia (Bodnar et al, Nat Genet 2002;30:110). FCH is until now diagnosed by a total cholesterol (TC) and/or triglyceride (TG) concentration above the 90th percentile adjusted for age and gender. We have shown that the diagnosis FCH based on these criteria was inconsistent in 26% of the subjects over a five years period. These results emphasised the need for re-evaluation of the diagnostic criteria. Further analyses demonstrated that apoB and small dense LDL (density gradient ultracentrifugation), were less variable in time compared to TC and TG levels. The optimal cut-off point for apoB was >1200 mg/l and for K-value (reflecting small dense LDL) i-0.10. Because measuring small dense LDL is not routinely available for clinical laboratories and small dense LDL increases substantially with TG>1.5 mmol/l, we also used TG>1.5 mmol/l as cut-off point. Now, different definitions of FCH are defined and we are comparing these in our FCH cohort with a 5 years follow-up. We aim to formulate a new valid proposal to re-define FCH, which will be necessary to elucidate the genetic background and molecular mechanisms causing this disease.
I672 CURRENT
RESEARCH.
PERSPECTIVES IN CLINICAL BACK TO THE LAB?
211
HOMOCYSTEINE
0. Stanger. Department of Cardiac Surgery, Clinical Atherosclerosis Research Group, Karl-Franzens University Graz, Austria
Homocysteine research has emerged among the most dynamic fields in expanding insight of atherosclerosis progression. However, a number of issues require clarification to overome limitations in clinical use. Under conditions of hyperhomocysteinemia (HHcy), high-risk groups need to be clearly defined, as interactions and treatment may not be identically effective in all. Most criteria for causality have been fulfilled. New associations, e. g. with infectious agents like Chlamydia, and underestimation of the risk due to regression dilution bias require critical handling of available data. HHcy is frequently found after acute and chronic tissue damage, reflecting damaged cell integrity with accelerated methylation demand. HHcy may efficiently activate the inflammatory process in the vessel wall, promote oxidative modifications of lipids and proteins and may dysregulate cholesterol and triglyceride metabolism through activation of sterol regulatory transcription factors (PPARs). Knock-out models will help investigating these mechanisms. Timing of blood sampling after acute events must be established and will increase the value of homocysteine measurements for clinical purposes as a monitoring and prognostic parameter. Importantly, pathomechanisms may differ fundamentally under acute and chronic conditions, and in the healthy and diseased tissue thus explaining in part conflicting results. Adaptive mechanisms to chonic HHcy exposure and hypomethylation require further investigation. Especially the study of ischemia-reperfusion damages appears being of particular clinical significance. Interventional studies designed to distinguish the intrinsic actions of folate and homocysteine will help clarify whether HHcy is rather cause or effect, most likely the most important answer sought in the near future. I673 ULTRASENSITIVE
MICROSCOPY OF THE UPTAKE OF INDIVIDUAL HDL PARTICLES VIA SCAVENGER RECEPTOR CLASS B TYPE I IN LIVING CELLS
J. Hesse’, M. Sonnleitne?, A. Breue?, G.J. Schuetz’, M. Doringer’, H. Stangl' ‘Institute for Medical Chemistry, Uniuersify of Vienna, ‘Institute for Biophysics, Uniuersify of Linz, Austria
The scavenger receptor class B type I (SR-BI) plays an important role in mediating selective uptake of HDL-derived cholesterol and cholesteryl ester in liver and steroidogenic tissues. However the molecular mechanism by which this receptor mediates selective uptake remains still enigmatic. In hepatocytes SRBI was shown to mediate the transcytosis of cholesterol into the bile and appears to be expressed on both basolateral and apical membranes. For visualizing the intracellular transport routes of SR-BI derived lipids and/or the whole HDL particle uptake, we applied ultrasensitive 3 dimensional fluorescence microscopy. SR-BI mediated uptake of individual HDL particles was directly observed in a Chinese Hamster Ovarian cell line expressing high levels of SR-BI. Simultaneous excitation and observation of 2 different colors allowed to discriminate different transport routes for SR-BI derived lipids, labeled via DiI, and for the apolipoprotein, covalently labeled via Cy5, of the HDL particle. I674 COADMINISTRATION
OF EZETIMIBE
PLUS
ATORVASTATIN
E. Stein’ S. Stende?, P. Mata3, D. Ponsonnet4, L. Melani4, L. Lipka4, _> R. Suresh4, E. Veltri4. ‘Metabolic and Atherosclerosis Research Center, Cincinnati, OH, USA: ‘Gentofte University Hospital, Hellerup, Denmark, ‘Fundacion Jiminez Diaz, Madrid, Spain: 4Schering-Plough Research Institute, Kenilworth, NJ, USA Objectives: This study evaluated the efficacy and safety of ezetimibe coadministered with atorvastatin in patients with heterozygous familial hypercholesterolemia, coronary heart disease, or multiple cardiovascular risk factors. Methods: After dietary stabilization, 6- to lo-week washout, and atorvastatin run-in period (open-label atorvastatin 10 mg/day), 621 patients with baseline LDL-C 33.4 mmol/L and TG < 4.0mmol/L while receiving atorvastatin 10 mg were randomized to ezetimibe 10 mg or additional double-blind atorvastatin (10 mg) administered daily for 4 weeks. The atorvastatin dose was doubled if LDL-C was >2.6 mmol/L after 4 and/or 9 weeks of treatment (maximum 80 mg/day with atorvastatin alone;
73rd EAS Congress
demonstrated the presence of immunoreactive FAT/CD36 in pBCEC lysates. In summary, our findings indicate that lipoprotein-associated PL are taken up via SR-BI mediated selective uptake and are partially hydrolyzed in the sn-2 position, in line with the localization of PUFAs in PL. The fatty acid pool that is generated by EL-dependent hydrolysis could then be incorporated by the action of FAT/CD36. This SR-BI/EL/FAT-dependent pathway could represent a feasible uptake route for PUFAs from the peripheral circulation into the brain.
I670 A NOVEL
APPROACH TO CHOLESTEROL LIPOPROTEIN CHARGE CONTROL
LOWERING:
D.L. Sparks. Uniuersify of Ottawa Heart Institute and Liponex Inc., Ottawa, ON, Canada Studies show that lipoprotein charge can regulate interfacial interactions with vascular proteins and cell surface receptors. A novel therapeutic approach has therefore been designed to selectively manipulate lipoprotein charge and therein affect the metabolism of cholesterol. CRD5 was developed to increase the negative charge of all major lipoprotein classes in viva. These alterations to lipoprotein charge turn on an excretory pathway that lowers plasma cholesterol levels by liberating cholesterol from LDL and tissues and promoting its transport to the liver for clearance. CRD5 lowers LDL cholesterol levels by inhibiting LCAT-dependent cholesterol esterification and by promoting a CETP mediated transfer of cholesteryl ester from LDL to HDL. CRD5 also promotes a 30-fold increase in the rate of clearance of [3H]cholesterol (FC) from the plasma. Enhanced clearance of cholesterol from the blood stream is associated with an increased hepatic uptake and a 20-fold increase in biliary cholesterol output. In addition, a single bolus dose of CRD5 promotes an almost 3-fold increase in the fecal excretion of cholesterol from a rabbit. Studies in cholesterol-fed hypercholesterolemic rabbits show that CRD5 has value as a cholesterol lowering therapeutic. LDL and VLDL cholesterol levels are reduced to near normal values after 1 week of treatment with CRDS. In addition, pre-clinical toxicology studies show that CRD5 is well tolerated; no adverse clinical signs or organ damage was observed following escalatory doses of CRD5 up to 120 mg/kg over a 14d study. These data show that the transport and clearance of cholesterol is affected by lipoprotein charge and can be stimulated by the administration of CRDS. The work shows that reverse cholesterol transport can now be selectively manipulated in viva.
I671 NEW
DEVELOPMENTS IN FAMILIAL HYPERLIPIDEMIA (FCH)
A.F.H. Stalenhoef, M.J. Veerkamp, Nijmegen, The Netherlands
.I. DeGraaf.
COMBINED
Uniuersify Medical Centre
FCH is characterized by a variable expression of dyslipidemia. The molecular mechanisms are still unclear. Although several gene mapping studies have surfaced candidate regions, no causative genes have been identified so far. A mutant mouse strain has been described, which shares features with FCH, in which a nonsense mutation in the gene encoding a cytoplasmatic protein, thioredoxin interacting protein, was found, possibly responsible for the hyperlipidemia (Bodnar et al, Nat Genet 2002;30:110). FCH is until now diagnosed by a total cholesterol (TC) and/or triglyceride (TG) concentration above the 90th percentile adjusted for age and gender. We have shown that the diagnosis FCH based on these criteria was inconsistent in 26% of the subjects over a five years period. These results emphasised the need for re-evaluation of the diagnostic criteria. Further analyses demonstrated that apoB and small dense LDL (density gradient ultracentrifugation), were less variable in time compared to TC and TG levels. The optimal cut-off point for apoB was >1200 mg/l and for K-value (reflecting small dense LDL) i-0.10. Because measuring small dense LDL is not routinely available for clinical laboratories and small dense LDL increases substantially with TG>1.5 mmol/l, we also used TG>1.5 mmol/l as cut-off point. Now, different definitions of FCH are defined and we are comparing these in our FCH cohort with a 5 years follow-up. We aim to formulate a new valid proposal to re-define FCH, which will be necessary to elucidate the genetic background and molecular mechanisms causing this disease.
I672 CURRENT
RESEARCH.
PERSPECTIVES IN CLINICAL BACK TO THE LAB?
211
HOMOCYSTEINE
0. Stanger. Department of Cardiac Surgery, Clinical Atherosclerosis Research Group, Karl-Franzens University Graz, Austria
Homocysteine research has emerged among the most dynamic fields in expanding insight of atherosclerosis progression. However, a number of issues require clarification to overome limitations in clinical use. Under conditions of hyperhomocysteinemia (HHcy), high-risk groups need to be clearly defined, as interactions and treatment may not be identically effective in all. Most criteria for causality have been fulfilled. New associations, e. g. with infectious agents like Chlamydia, and underestimation of the risk due to regression dilution bias require critical handling of available data. HHcy is frequently found after acute and chronic tissue damage, reflecting damaged cell integrity with accelerated methylation demand. HHcy may efficiently activate the inflammatory process in the vessel wall, promote oxidative modifications of lipids and proteins and may dysregulate cholesterol and triglyceride metabolism through activation of sterol regulatory transcription factors (PPARs). Knock-out models will help investigating these mechanisms. Timing of blood sampling after acute events must be established and will increase the value of homocysteine measurements for clinical purposes as a monitoring and prognostic parameter. Importantly, pathomechanisms may differ fundamentally under acute and chronic conditions, and in the healthy and diseased tissue thus explaining in part conflicting results. Adaptive mechanisms to chonic HHcy exposure and hypomethylation require further investigation. Especially the study of ischemia-reperfusion damages appears being of particular clinical significance. Interventional studies designed to distinguish the intrinsic actions of folate and homocysteine will help clarify whether HHcy is rather cause or effect, most likely the most important answer sought in the near future. I673 ULTRASENSITIVE
MICROSCOPY OF THE UPTAKE OF INDIVIDUAL HDL PARTICLES VIA SCAVENGER RECEPTOR CLASS B TYPE I IN LIVING CELLS
J. Hesse’, M. Sonnleitne?, A. Breue?, G.J. Schuetz’, M. Doringer’, H. Stangl' ‘Institute for Medical Chemistry, Uniuersify of Vienna, ‘Institute for Biophysics, Uniuersify of Linz, Austria
The scavenger receptor class B type I (SR-BI) plays an important role in mediating selective uptake of HDL-derived cholesterol and cholesteryl ester in liver and steroidogenic tissues. However the molecular mechanism by which this receptor mediates selective uptake remains still enigmatic. In hepatocytes SRBI was shown to mediate the transcytosis of cholesterol into the bile and appears to be expressed on both basolateral and apical membranes. For visualizing the intracellular transport routes of SR-BI derived lipids and/or the whole HDL particle uptake, we applied ultrasensitive 3 dimensional fluorescence microscopy. SR-BI mediated uptake of individual HDL particles was directly observed in a Chinese Hamster Ovarian cell line expressing high levels of SR-BI. Simultaneous excitation and observation of 2 different colors allowed to discriminate different transport routes for SR-BI derived lipids, labeled via DiI, and for the apolipoprotein, covalently labeled via Cy5, of the HDL particle. I674 COADMINISTRATION
OF EZETIMIBE
PLUS
ATORVASTATIN
E. Stein’ S. Stende?, P. Mata3, D. Ponsonnet4, L. Melani4, L. Lipka4, _> R. Suresh4, E. Veltri4. ‘Metabolic and Atherosclerosis Research Center, Cincinnati, OH, USA: ‘Gentofte University Hospital, Hellerup, Denmark, ‘Fundacion Jiminez Diaz, Madrid, Spain: 4Schering-Plough Research Institute, Kenilworth, NJ, USA Objectives: This study evaluated the efficacy and safety of ezetimibe coadministered with atorvastatin in patients with heterozygous familial hypercholesterolemia, coronary heart disease, or multiple cardiovascular risk factors. Methods: After dietary stabilization, 6- to lo-week washout, and atorvastatin run-in period (open-label atorvastatin 10 mg/day), 621 patients with baseline LDL-C 33.4 mmol/L and TG < 4.0mmol/L while receiving atorvastatin 10 mg were randomized to ezetimibe 10 mg or additional double-blind atorvastatin (10 mg) administered daily for 4 weeks. The atorvastatin dose was doubled if LDL-C was >2.6 mmol/L after 4 and/or 9 weeks of treatment (maximum 80 mg/day with atorvastatin alone;
73rd EAS Congress