- Email: [email protected]
Kinetic study of HDL selective cholesteryl ester uptake in dogs using stable isotopes
72
Bailhache
factors (such as free radicals, homocysteine, estrogenic imbalance, etc.) induce endothelial dysfunction. Because of its position covering the luminal aspect of the vessel wall, the endothelium is additionally under the regulation of flow forces and blood derived mitogenes that can cooperate to induce endothelial dysfunction. We have recently shown by RT-DD-PCR that high concentrations of LDL are able to produce downregulation of CYP-51 in endothelial cells. The effect is time and concentration dependent and follows a pattern similar to that of the endothelial cell SRBP-2 gene. New light into endothelial gene expression regulation and function may help to uncover new mechanism to explain the initial modification of endothelial cells that contributes to atherosclerotic lesion formation. 0~]
KINETIC STUDY OF HDL SELECTIVE CHOLESTERYL ESTER UPTAKE IN DOGS USING STABLE ISOTOPES
E. Bailhache 1'2, K. Ouguerram1, P. Nguyen2, P. Maugere 1, M. Krempf1, T. Magot 1. JlNSERM U539, Groupe Metabolisme," 2Ecole Nationale
Veterinaire, Nantes, France In humans, the transport of HDL cholesteryl ester (CE) from plasma to the liver (reverse cholesterol transport) involves 3 pathways: two direct way mediated either by apoA-I or by hepatic scavenger receptor B-I responsible of selective HDL-CE uptake and another indirect way where HDL CE is transferred into VLDL and LDL by CETR The aim of our study was to evaluate the contribution of HDL CE selective uptake in dogs, devoid in CETP activity and with HDL as main carrier of cholesterol. 13C-acetate and D3-1eucine, as labeled precursors of cholesterol and apoA-I, were infused over 8 h intravenously to eight healthy dogs. Blood samples were obtained during and after the end of the perfusion. HDL were isolated by ultracentrifugation. After isolation of unesterified cholesterol, CE and apoA-I, isotopic enrichments were measured by GC-C-IRMS for cholesterol and GC-MS for apoA-I. Compartmental modeling (SAAM II) was used for the analysis of tracer data. Kinetic analysis allowed to measure the activity of cholesterol esterification (0.18±0.03 h-t), rate of apoA-I catabolism (0.008±0.003 h-t), HDL CE turnover (0.078±0.013 h -t) and CE selective uptake (0.070±0.013 h-t). Our results suggested that reverse cholesterol transport in dogs is characterized by a high rate of esterification and selective HDL CE uptake (89% of HDL CE turnover). This could explain the low atherosclerosis risk observed in dogs. These results suggest that dog could be a useful animal model to study modulation of CE uptake by pathological, nutritional and pharmacological factors. ~THE SCAVENGER RECEPTOR CLASS B, TYPE I COLOCALIZES W I T H CAVEOLAE AND PROMOTES PARTITIONING OF HDL ASSOCIATED ALPHA-TOCOPHEROL INTO CAVEOLAE IN PORCINE BRAIN CAPILLARY ENDOTHELIAL C E L L S Z. Balazs, U. PanzenbSck, A. Sovic, W. Sattler. Karl-Franzens-University, Institute of Medical Biochemistry and Medical Molecular Biology, Graz, Austria Efficient supply of the brain with alpha-tocopherol (alphaTocH) the member of the vitamin E family with the highest biological activity for proper neurological functioning is clearly established. We could recently demonstrate that scavenger receptor class B, type I (SR-BI) plays a pivotal role in the supply of brain capillary endothelial cells (pBCECs) that constitute the blood-brain barrier with alphaTocH. During the present study we have investigated whether selective uptake (and subsequent transcytosis) of alphaTocH could be facilitated by caveolae in a similar manner as described for HI)L-associated cholesteryl esters. Therefore, we tested whether uptake of HDL-associated alphaTocH takes place in caveolae. Our findings indicate that >80% of radiolabeled alphaTocH is present in sucrose gradient fractions containing caveolae. Furthermore we have shown by immunoblotting that SR-BI colocalizes with caveolin-1 in the caveolar membrane fraction of pBCECs. As the polarized distribution of proteins is a fundamental property of endothelial cells, the appearance of SR-BI on apical and basolateral membrane surfaces was investigated by selective biotin labeling of pBCECs grown on TranswellT M filters and subsequent immunoprecipitations. These experiments demonstrated that the bulk of SR-BI is sorted to the apical side of the plasma membrane. The results of the present study suggest that uptake and transcytosis of HDL-associated alphaTocH by pBCECs is facilitated by a two-step mechanism: the first step of uptake involves SR-BI dependent selective uptake in caveolae, while the actual transcytosis step occurs in conjunction with 'remnant' HDL particles that are remarkably depleted of cholesteryl esters and alphaTocH.
•3-•
EFFICACY AND SAFETY OF SIMVASTATIN VERSUS ATORVASTATIN: RESULTS OF THE COMPARATIVE H D L - C EFFICACY AND SAFETY STUDY (CHESS)
C.M. Ballantyne 1, C.M. Hustad2, Z. Yuan2, P. DeLucca 2, J. Palmisano 2.
JBaylor College of Medicine, Houston, TX," 2Merck & Co., Inc., West Point, PA, USA Objectives: To assess the comparative efficacy of simvastatin 80mg (S) versus atorvastatin 80mg (A) on HDL-C (primary) and Apolipoprotein (Apo) A-I values and to evaluate the effects of S and A on ALT/AST and drugrelated clinical GI adverse events (AEs). Methods: In a multicenter, randomized, double-blind, parallel-dose study, 917 patients with hypercholesterolemia were treated with S or A for 24 weeks. Prespecified assessment of efficacy was the mean of weeks 6 and 12 (primary) and weeks 18 and 24. Results: S increased HDL-C and Apo A-I values significantly more than A at weeks 6/12 (HDL-C: 8.9±0.6% vs. 3.6±0.6%; Apo A-l: 4.9±0.7% vs. -0.9±0.7%, p<0.001) and 18/24 (HDL-C: 8.3±0.7% vs. 4.2±0.7%; Apo A-l: 3.7±0.7% vs. -1.4±0.7%, p <0.001) overall and across all HDL-C subgroups (<40 mg/dL, ~>40 mg/dL). Consecutive elevations in ALT/AST greater than 3X upper limit of normal occurred in fewer patients treated with S than with A [2/453 (0.4%) versus 13/464 (2.8%), p-0.007], with more observed in women [S 1/199 (0.5%); A 11/209 (5.3%)]. There were no significant differences in clinical GI AEs. At the 6/12 week timepoint, A and S lowered LDL-C by 53.5±0.6% and 45.4±0.6% and triglycerides by 34.1±1.4% and 25.8±1.4%, respectively (p<0.001). Conclusions: Simvastatin 80mg increased HDL-C and Apo A-1 significantly more than atorvastatin 80mg at weeks 6/12 and 18/24 of treatment. Significantly fewer elevations in ALT/AST occurred in patients treated with simvastatin. ~]
EFFECT OF ATORVASTATIN ON THE KINETICS OF APOLIPOPROTEIN AIV IN NON INSULIN DEPENDENT DIABETICS
E Ferrer 1, H. Nazih 1'2, K. Ouguerram 1'3, M. Krempf1, H. Laouenan4, J.-M. Bard 1'2. JlNSERM U539 et CHU," 2UFR de Pharmacie," 3UFR de
Sciences, Nantes," 4Laboratoires Pfizer, Paris, France The present study was undertaken to determine, in diabetic patients, the relationship of triglyceride rich lipoproteins (TGRL) with the kinetic parameters of apo AIV and the effect of atorvastatin on apo AIV concentration and kinetics. Apo AIV kinetics were determined by GC-MS, in 10 diabetic patients with moderate hypertriglyceridemia. Analyses were carried out following a 14h IV infusion of deuterated leucine, before and after 8 weeks of therapy with atorvastatin 40 mg/d. This therapy resulted in a significant decrease in cholesterol (CH), triglycerides (TG) and apo B (B) (CH: 262 vs 174 mg/dl, p<0.001; TG: 279 vs 201 mg/dl, p<0.05; B: 133 vs 178, p<0.001; before vs after therapy). In contrast, an increase in apo AIV was observed (16 vs 19 mg/dl, p<0.05, before vs after therapy). This was mainly due to an increase in the absolute production rate (APR), while the fractional catabolic rate (FCR) did not change (APR: 2.34 vs 2.73 mg/kg/h, p-0.07; FCR: 0.362 vs 0.382 /d, NS). Before therapy, FCR was positively correlated to TGRL concentration (r-0.79, p<0.01; r-0.82, p<0.001; r-0.71, p<0.05, for TG, VLDL-C and VLDL-TG respectively), while APR was positively correlated only with HDL-TG (r-0.74, p<0.01). After therapy, these correlations were lost except for APR which was still positively correlated with HDL-TG (r-0.79, p<0.01). The kinetics of apo AIV in hypertriglyceridemic diabetics are related to TGRL concentration. Atorvastatin increases apo AIV concentration, most probably by increasing its production rate. This could represent an additional beneficial effect of atorvastatin therapy. ~THE 5A/6A STROMELYSIN POLYMORPHISM AND ECHOGENIC PLAQUES: DEWSBURY AND MAIDSTONE PART OF THE BRITISH REGIONAL HEART STUDY E. Bashiardes 1, M. Cariolou 1, M. Giffin 2, A. Nicolaidis 1, A. Karagrigoriou3, S. Ebrahim4, S.E. Humphries5. JThe Cyprus
Institute of Neurology and Genetics, Nicosia, Cyprus," 2Imperial College, London, UK," 3 University of Cyprus, Nicosia, Cyprus," 4Bristol University," SRoyal Free and University College London, UK Ultrasonic images of atherosclerotic plaques of the carotid bifurcation can be characterized as echogenic/stable with grey scale median (GSM>32) or
73rd EAS Congress
Bailhache
factors (such as free radicals, homocysteine, estrogenic imbalance, etc.) induce endothelial dysfunction. Because of its position covering the luminal aspect of the vessel wall, the endothelium is additionally under the regulation of flow forces and blood derived mitogenes that can cooperate to induce endothelial dysfunction. We have recently shown by RT-DD-PCR that high concentrations of LDL are able to produce downregulation of CYP-51 in endothelial cells. The effect is time and concentration dependent and follows a pattern similar to that of the endothelial cell SRBP-2 gene. New light into endothelial gene expression regulation and function may help to uncover new mechanism to explain the initial modification of endothelial cells that contributes to atherosclerotic lesion formation. 0~]
KINETIC STUDY OF HDL SELECTIVE CHOLESTERYL ESTER UPTAKE IN DOGS USING STABLE ISOTOPES
E. Bailhache 1'2, K. Ouguerram1, P. Nguyen2, P. Maugere 1, M. Krempf1, T. Magot 1. JlNSERM U539, Groupe Metabolisme," 2Ecole Nationale
Veterinaire, Nantes, France In humans, the transport of HDL cholesteryl ester (CE) from plasma to the liver (reverse cholesterol transport) involves 3 pathways: two direct way mediated either by apoA-I or by hepatic scavenger receptor B-I responsible of selective HDL-CE uptake and another indirect way where HDL CE is transferred into VLDL and LDL by CETR The aim of our study was to evaluate the contribution of HDL CE selective uptake in dogs, devoid in CETP activity and with HDL as main carrier of cholesterol. 13C-acetate and D3-1eucine, as labeled precursors of cholesterol and apoA-I, were infused over 8 h intravenously to eight healthy dogs. Blood samples were obtained during and after the end of the perfusion. HDL were isolated by ultracentrifugation. After isolation of unesterified cholesterol, CE and apoA-I, isotopic enrichments were measured by GC-C-IRMS for cholesterol and GC-MS for apoA-I. Compartmental modeling (SAAM II) was used for the analysis of tracer data. Kinetic analysis allowed to measure the activity of cholesterol esterification (0.18±0.03 h-t), rate of apoA-I catabolism (0.008±0.003 h-t), HDL CE turnover (0.078±0.013 h -t) and CE selective uptake (0.070±0.013 h-t). Our results suggested that reverse cholesterol transport in dogs is characterized by a high rate of esterification and selective HDL CE uptake (89% of HDL CE turnover). This could explain the low atherosclerosis risk observed in dogs. These results suggest that dog could be a useful animal model to study modulation of CE uptake by pathological, nutritional and pharmacological factors. ~THE SCAVENGER RECEPTOR CLASS B, TYPE I COLOCALIZES W I T H CAVEOLAE AND PROMOTES PARTITIONING OF HDL ASSOCIATED ALPHA-TOCOPHEROL INTO CAVEOLAE IN PORCINE BRAIN CAPILLARY ENDOTHELIAL C E L L S Z. Balazs, U. PanzenbSck, A. Sovic, W. Sattler. Karl-Franzens-University, Institute of Medical Biochemistry and Medical Molecular Biology, Graz, Austria Efficient supply of the brain with alpha-tocopherol (alphaTocH) the member of the vitamin E family with the highest biological activity for proper neurological functioning is clearly established. We could recently demonstrate that scavenger receptor class B, type I (SR-BI) plays a pivotal role in the supply of brain capillary endothelial cells (pBCECs) that constitute the blood-brain barrier with alphaTocH. During the present study we have investigated whether selective uptake (and subsequent transcytosis) of alphaTocH could be facilitated by caveolae in a similar manner as described for HI)L-associated cholesteryl esters. Therefore, we tested whether uptake of HDL-associated alphaTocH takes place in caveolae. Our findings indicate that >80% of radiolabeled alphaTocH is present in sucrose gradient fractions containing caveolae. Furthermore we have shown by immunoblotting that SR-BI colocalizes with caveolin-1 in the caveolar membrane fraction of pBCECs. As the polarized distribution of proteins is a fundamental property of endothelial cells, the appearance of SR-BI on apical and basolateral membrane surfaces was investigated by selective biotin labeling of pBCECs grown on TranswellT M filters and subsequent immunoprecipitations. These experiments demonstrated that the bulk of SR-BI is sorted to the apical side of the plasma membrane. The results of the present study suggest that uptake and transcytosis of HDL-associated alphaTocH by pBCECs is facilitated by a two-step mechanism: the first step of uptake involves SR-BI dependent selective uptake in caveolae, while the actual transcytosis step occurs in conjunction with 'remnant' HDL particles that are remarkably depleted of cholesteryl esters and alphaTocH.
•3-•
EFFICACY AND SAFETY OF SIMVASTATIN VERSUS ATORVASTATIN: RESULTS OF THE COMPARATIVE H D L - C EFFICACY AND SAFETY STUDY (CHESS)
C.M. Ballantyne 1, C.M. Hustad2, Z. Yuan2, P. DeLucca 2, J. Palmisano 2.
JBaylor College of Medicine, Houston, TX," 2Merck & Co., Inc., West Point, PA, USA Objectives: To assess the comparative efficacy of simvastatin 80mg (S) versus atorvastatin 80mg (A) on HDL-C (primary) and Apolipoprotein (Apo) A-I values and to evaluate the effects of S and A on ALT/AST and drugrelated clinical GI adverse events (AEs). Methods: In a multicenter, randomized, double-blind, parallel-dose study, 917 patients with hypercholesterolemia were treated with S or A for 24 weeks. Prespecified assessment of efficacy was the mean of weeks 6 and 12 (primary) and weeks 18 and 24. Results: S increased HDL-C and Apo A-I values significantly more than A at weeks 6/12 (HDL-C: 8.9±0.6% vs. 3.6±0.6%; Apo A-l: 4.9±0.7% vs. -0.9±0.7%, p<0.001) and 18/24 (HDL-C: 8.3±0.7% vs. 4.2±0.7%; Apo A-l: 3.7±0.7% vs. -1.4±0.7%, p <0.001) overall and across all HDL-C subgroups (<40 mg/dL, ~>40 mg/dL). Consecutive elevations in ALT/AST greater than 3X upper limit of normal occurred in fewer patients treated with S than with A [2/453 (0.4%) versus 13/464 (2.8%), p-0.007], with more observed in women [S 1/199 (0.5%); A 11/209 (5.3%)]. There were no significant differences in clinical GI AEs. At the 6/12 week timepoint, A and S lowered LDL-C by 53.5±0.6% and 45.4±0.6% and triglycerides by 34.1±1.4% and 25.8±1.4%, respectively (p<0.001). Conclusions: Simvastatin 80mg increased HDL-C and Apo A-1 significantly more than atorvastatin 80mg at weeks 6/12 and 18/24 of treatment. Significantly fewer elevations in ALT/AST occurred in patients treated with simvastatin. ~]
EFFECT OF ATORVASTATIN ON THE KINETICS OF APOLIPOPROTEIN AIV IN NON INSULIN DEPENDENT DIABETICS
E Ferrer 1, H. Nazih 1'2, K. Ouguerram 1'3, M. Krempf1, H. Laouenan4, J.-M. Bard 1'2. JlNSERM U539 et CHU," 2UFR de Pharmacie," 3UFR de
Sciences, Nantes," 4Laboratoires Pfizer, Paris, France The present study was undertaken to determine, in diabetic patients, the relationship of triglyceride rich lipoproteins (TGRL) with the kinetic parameters of apo AIV and the effect of atorvastatin on apo AIV concentration and kinetics. Apo AIV kinetics were determined by GC-MS, in 10 diabetic patients with moderate hypertriglyceridemia. Analyses were carried out following a 14h IV infusion of deuterated leucine, before and after 8 weeks of therapy with atorvastatin 40 mg/d. This therapy resulted in a significant decrease in cholesterol (CH), triglycerides (TG) and apo B (B) (CH: 262 vs 174 mg/dl, p<0.001; TG: 279 vs 201 mg/dl, p<0.05; B: 133 vs 178, p<0.001; before vs after therapy). In contrast, an increase in apo AIV was observed (16 vs 19 mg/dl, p<0.05, before vs after therapy). This was mainly due to an increase in the absolute production rate (APR), while the fractional catabolic rate (FCR) did not change (APR: 2.34 vs 2.73 mg/kg/h, p-0.07; FCR: 0.362 vs 0.382 /d, NS). Before therapy, FCR was positively correlated to TGRL concentration (r-0.79, p<0.01; r-0.82, p<0.001; r-0.71, p<0.05, for TG, VLDL-C and VLDL-TG respectively), while APR was positively correlated only with HDL-TG (r-0.74, p<0.01). After therapy, these correlations were lost except for APR which was still positively correlated with HDL-TG (r-0.79, p<0.01). The kinetics of apo AIV in hypertriglyceridemic diabetics are related to TGRL concentration. Atorvastatin increases apo AIV concentration, most probably by increasing its production rate. This could represent an additional beneficial effect of atorvastatin therapy. ~THE 5A/6A STROMELYSIN POLYMORPHISM AND ECHOGENIC PLAQUES: DEWSBURY AND MAIDSTONE PART OF THE BRITISH REGIONAL HEART STUDY E. Bashiardes 1, M. Cariolou 1, M. Giffin 2, A. Nicolaidis 1, A. Karagrigoriou3, S. Ebrahim4, S.E. Humphries5. JThe Cyprus
Institute of Neurology and Genetics, Nicosia, Cyprus," 2Imperial College, London, UK," 3 University of Cyprus, Nicosia, Cyprus," 4Bristol University," SRoyal Free and University College London, UK Ultrasonic images of atherosclerotic plaques of the carotid bifurcation can be characterized as echogenic/stable with grey scale median (GSM>32) or
73rd EAS Congress