- Email: [email protected]
Clinical and molecular characteristics of homozygous CETP deficiency
Wednesday June 28.2000: Poster Abstracts P: W22 Reverse Cholesterol Transport
1WeP25:W22
1 Release of cholesteryl esters fipm macrophage foam cells in culture
T. Mikkola, M. Leigh& R. St. Clair. Wake Forest University School ofMed., Winston-Salem, NC, USA
Thioglycollate-elicited peritoneal macrophages from cholesterol-fed, atherosclerosis-susceptible White Carneau pigeons contain high concentrations of free (FC) and ester&d (EC) cholesterol (200- z 1000 &mg cell protein), similar to what has been reported for macrophage foam cells from atherosclerotic plaques. when incubated in culture medium containing apo HDL/phosphtidylcholine (APC) vesicles for 48 hrs. there was a stimulation of cholesterol efflux with up to 50% of the cellular cholesterol appearing in the culture medium. Of the cholesterol in the medium, 2MO% was EC. Both the FC and EC in the medium appeared to be associated with the APC vesicles based on their ability to pass through a 0.45 urn filter. The appearance of EC in the medium could not be explained by gross cell death, based on changes in cell protein, or by esterification of FC in the culture medium via LCAT secreted by the macrophages. These findings were unexpected since most studies of cholesterol efflux from cells show that EC must first be hydrolyzed to FC prior to efflux. The majority of the published studies, however, have been done with cells either not loaded with EC or loaded to levels much less than seen in these macrophage foam cells. Although the mechanism by which macrophages release EC is unclear, it may represent a unique property of macrophage foam cells that could play an important role in the pathogenesis of atherosclerosis.
1WeP26:W22
1 Cyclodextrins acting as sinks and shuttles diierentially mobilise free and e&r&d foam ceil macrophages
cholesterol from human
S. Liu’ , R.T. Dean*, W. Jessup*, L. Kritharides *a3.‘~3Clinical Research; ‘Cell Biology Groups The Heart Research Institute; 3Dept. Cardiology _. Concord H&pital, Sydney, Australia Objective:
To investigate whether cyclodextrins (CD) with high cholesterol affinity acting as sinks, or low cholesterol affinity acting as shuttles to phospholipid vesicles (PV), deplete esterified cholesterol (EC) and free cholesterol (C) from primary human foam cell macrophages (HMFC) with equivalent efficacy. Methods: HMFC were generated by incubating monocyte derived macrophages with acetylated low density lipoprotein radiolabelled with [3H]-C. HMFC were incubated for up to 24 hrs in medium containing PV (200 &ml), CD (1 .Omg/ml) with differing affinity for C (low affinity hydroxypropyl-CD, hp-CD, or high affinity trimethyl-CD, tm-CD), and hp-CD or tm-CD with PV. Cells and media were analysed for C and EC by HPLC, TLC and radiometric detection. Results: Within 2 hrs, tm-CD depleted C to 42.3 f 10% of HMFC in control medium RPMI, but did not deplete CE even after 24 brs (95.0 f 6.1% of control). hp-CD caused minimal C efflux on its own, but with PV it depleted C to 36.7 & 5.0% and CE to 35.2 f 4.9% of control at 24 hrs. tm-CD with PV was also ineffective at depleting EC (102.5 f 5.0% of control) precluding acceptor saturation as the mechanism of failed CE clearance by tm-CD. Initial efflux at 10 minutes to tm-CD (1.3 f 0.2% of total cholesterol), hp-CD + PV (2.2 f 0.3%) and tm-CD + PV (3.0 f 0.5%) did not correlate with relative efficacy of CE depletion. Concluskms: In HMFC, synergistic efflux with CD and PV achieves superior depletion of CE than does efflux to high affinity CD alone.
IWeP27.W22
Stimulation of apo E secretion from human foam cell macrophages by apotipoproteh A-I mutants
T. Sloane’, D. Sviridov’, L. Pyle2, R.T. Dean’, W. Jessup’, L. Kritharide~‘,~. mart Research Institute Sydney; 2Baker Medical Research Institute Melbourne; ‘Dept. of Cardiology Concord Hospital Sydney, Australia Objective:
We have recently identified that apolipoprotein A-I (apo A-I) induces secretion of apo E from foam cell macrophages (J. Biol. Chem. 1999, 274,27925), and that proapo A-I mutants achieve differential cholesterol (C) and phospholipid efflux (J. Biol. Chem. 1996, 271, 33277). This study has investigated whether specific sequences of apo A-I mediate apo E secretion from human foam cell macrophages. Methods: Primary human monocyte-derived macrophages were incubated with acetylated low density lipoprotein radiolabelled with [3H]-C to generate HMFC, and HMFC then incubated in RPM1 and delipidated proapo A-I mutants (25 wg/ml). Apo A-I mutants were expressed as fusion proteins in an XlIth hmmational
185
E. Coli/pGEX vector system with whole pro apo A-I (-6-243) and truncated forms (-6-222) and (-6-150) used herein. Lipid analyses in cells and media were performed by HPLC, TLC and radiometric detection of [3H]-C, and apo E quantified by Western blotting. Results: Mutants -6-150, -6-243, -6-222 respectively achieved 3.5-fold, 2.8-fold and 2.0-fold the C efflux achieved in controls at 8 hrs. All 3 mutants stimulated time-dependent apo E secretion, respectively 5.0-fold, 3.0-fold and 3.0-fold the apo E Secretion of controls at 8 hrs. Interestingly, -6-243 stimulated the initial secretion of a lower MW form of apo E which did not accumulate further after 2 hours. Conclusions: In HMFC, apo A-I mutants induce C efflux and overall apo E secretion approximately in parallel. Residues 222-243 may specifically stimulate the secretion of lower MW, presumably less glycosylated, apo E.
IWeP28
W22
Mechanisms of 7.ketocholesteml-mediated inhibition of cholesterol efllux from human foam cell macrophages
K Gaus C.R. Dass, I.C. Gelissen, L. Kritharides, A.J. Brown, R.T. Dean, i) W. Jessup. Heart Research Institute, Sydney, Australia Macrophage foam cells are early and characteristic components of atherosclerotic lesions. They contain large cytoplasmic deposits of esterified cholesterol, which presumably results from a disparity between the rates of uptake, synthesis and export of cholesterol. Generally cells are able to closely control their cholesterol content through regulation of lipoprotein uptake and of cholesterol synthesis. In addition, most cells efficiently export excess free cholesterol to appropriate extracellular acceptors such as high-density lipoproteins (HDL) or its major protein component, apolipopmtein A-l (apoA-1). Addition of such acceptors to foam cells in vitro leads to a progressive release of free cholesterol and concomitant hydrolysis and depletion of intracellular cholesteryl esters Human foam cells contain a range of oxidised cholesterols (oxysterols), of which 7-ke.tocholesteml (7KC) is a major component. The effect of 7KC on sterol efflux to apolipoprotein A-I (apoA-I) was examined in human monocyte-derived macrophages (HMDM) loaded with cholesterol only or with a combination of cholesterol and 7KC. The amount of 7KC loaded was within the range found in human lesion foam cells. Efflux of cholesterol from HMDMs was stimulated by apoA-I, but was inhibited to basal levels when 7KC was present. Only 7% of cell phospholipid was exported to apoA-I from cells loaded with 7KC compared with 25% from AcLDL- loaded cells. ApoA-I-did not stimulate efflux of 7KC. Similar but less profound effects were also found in comparable murine foam cells. These data suggest that 7KC may contribute to atherogenesis by its ability to inhibit reverse cholesterol transport in human macrophage foam cells. Likely mechanisms under investigation include inhibition of plasma membrane cholesterol and phospholipid desorption to apoA-1 and inhibition of phospholipid synthesis by 7KC.
IWeP29
W22
Chid and molecular characteristics CETP deficiency
of homozygous
A. Inazu, Y. Noji, Y. Toudo, A. Nohara, J. Koizumi, H. Mabuchi. The Second Dept of Internal Medicine and Dept of Clinical Laboratory Science, Kanazawa University, Kanazawa, Japan Objective:
Relationship between CETP deficiency and atherosclerosis remains controversial and appears dependent on serum HDL-C levels. Methods: By PCR genotyping and/or plasma CETP measurement by ELISA in hyperalphalipoproteinemic subjects (n = 1000, HDL-C >= 80 mg/dl), 44 cases (18 men and 26 women) with homozygous CETP deficiency from 35 unrelated families (age 22-89 yrs, 61 yr f 13 [SD]) were identified, and 11 cases of them were followed-up. Results: Subjects with homozygous CETP deficiency had BMI 21.4 kg/m2 f 3.1, CHOL 267 mg/dl f 40, TG 99 f 63, HDL-C 147 f 42, apo A-I 228 f 53, A-II 44 f 15, B 74 f 53, C-II 7.5 rt 5.5, C-III 20.9 f 11.5, E 10.7 f 5.2, and Lp(a) 11.9 f 9.1. There were 30 complete CETP deficiency caused by 27 true homozygotes of intron 14 G (+l)-to-A (intronl4A) mutation (mean HDL-C 167 mg/dl), 1 compound heterozygote of intron 14A and intron 14 T (+3) insertion (HDL-C 106), 1 compound heterozygote of intron 14A and unknown mutation (HDL-C 223). and 1 unknown mutation (HDL-C 130). There were 14 partial CETP deficiency caused by 7 compound heterozygotes of intron 14A and D442G (HDL-C 124), and 7 true homozygotes of D442G (HDL-C 93). Plasma CETP levels in intron 14A/D442G and D442G homozygotes were 0.7 &ml and 0.9, respectively. Hypertension (SBP >= 160 and/or DBP >= 95 mmHg or drug treatment) was found in 8 cases. Cerebral infarction was found in 2 cases (ages of 50 and 66), polycystic kidney disease
Symposium on Atherosclerosis,
Stockholm, Sweden, June 25-29, 2@_XI
Wednesday June 28,200O: Poster Abstracts P: W22 Reverse Cholesterol Transport
186
(PKD) in 2 cases of 1 family; (probable) angina pectoris in 2 cases (ages of 63 and 77); SAH in 1 case of PKD family; liver cirrhosis in 1 case; COPD in 1 case; FH in 1 case; and DM in 1 case. Conclusions: Prevalence of premature atherosclerotic diseases was not increased in homozygous CETP deficiency despite hypercholesterolemia.
IWeP30
W22
Inhibition of lecithin: Cholesterol acyltransferase cholesterol oxides
by
EC. Pincinato’, A. Sevaniru?, D.S.P. Abdalla’. ‘Depto. of Clin and Toxicol. Analyses, FCF USP Scio Paulo, SP Brazil; Inst. Toxicol., University of Southern California, IA, CA, USA Objective: To investigate the effect of cholesterol oxides on esteritication of cholesterol by LCAT. Methods: HDL particles enriched with increasing concentration of cholesterol oxides were incubated with fresh plasma as source of LCAT. The esterification of cholesterol and cholesterol oxides was followed by measuring the consumption of the respective free sterol and oxysterols. Measurements of cholesterol and cholesterol oxides were done by gas chromatography with flame ionization detection. Results: All the studied cholesterol oxides were esterified by LCAT after incorporation into HDL particle, competing with cholesterol by LCAT-mediated esterification. Cholesterol esterification by LCAT was inversely related to the cholesterol oxide concentration in HDL particles. Kinetic studies with cholestan- 38, 5cr, 6B-trio1 and 5-cholesten- 3/I-25diol (25-hidroxycholesterol) showed a non-competitive inhibition with a Kiapparent of 103 and 15.02 ng/pL, respectively. This study shows markedly differences for inhibition parameters and apparent Ki among the different cholesterol oxides. Conclusions: Data suggest that cholesterol esterification by LCAT is inhibited in the cholesterol oxide-enriched HDL particles which could disturb the reverse cholesterol transport. In contrast, the esterification of cholesterol oxides by LCAT may have an anti-atherogenic effect considering that this step may increase the catabolism of these atherogenic compounds by enhancing their liver uptake either by direct HDL removal or by the previous transfer of the cholesteryl oxide esters to apo, B-containing lipoproteins followed by their hepatic catabolism. Supported by FAPESP
Conclusions: The hyperalphalipoproteinemia was explained, part, by the lower plasma levels of HL in this population.
I WeP32 W22
1 Lipases and transfer proteins in patients with hyperalphaiipoproteinemia
S. Alarcon’,V. Castanho’, D. Kaplan’, H. Oliveira*, V Nunes3, P. Cazita3, E. Quintio3, E. de Faria’ I Dept. of Clinical Pathology/NMCE/Il;NICAMP; 2Dept. of Physiology/UNICAM; 3Lipid Lab., FM-LISP Sr70 Paulo, Brazil Objective: To determine in Brazilian hyperalphalipoproteinemic (HALP) patients the activities of factors that modulate the metabolism of HDL: lipoprotein lipase (LPL), hepatic lipase (HL), cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP). Methods: Ninety-five volunteers were defined by their HDL-chol as controls (CTL-n = 35, below 68 mg!dL) and HALP (n = 60, equal and above 68 mg/dL), the 90th percentile for a local population. HL, (post-heparin plasma), CETP and PLTP were measured by exogenous radiometric methods using respectively triolein emulsion, “C-CE-HDL and phospholipid lipossomes as substrates. Results: HL was significantly lower (p = 0.04) in HALP, but no differences were found for LPL, CETP and PLTP (Mann-Whitney). The scatterplots are shown below:
HALP
i
3
CT1
LCAT-dependent cholesterol esterhkation rate is slower in in vi&o giycated HDL but is not altered in plasma HDL drawn from NIDDM patients
M. Passarelli, E.R. Nakandakare, S. Catanozi, J.C. Rocha, S.A. Lottenberg, E.C.R. Ouinfio. Lipids Lab. (LIM IO), Univ. S. Paul0 Med. School, S. Paulo, SP Brazil Objective: Alterations in the reverse cholesterol transport has been related to glycation of plasma lipoproteins. Present study investigates the influence of HDL glycation on the in vitro activity of the enzyme lecithin cholesterol acyl transferase (LCAT). Methods: HDL obtained from normal controls (N) and poorly controlled NIDDM (D) patients was separated by ultracentrifugation, and plasma d > 1.21 g/mL from N was utilized as the source of LCAT. Reconstituted HDL (ret HDL) from N were prepared by sonication of delipidated HDL previously submitted to selective glycation of the lipid and protein components separately, or both together, by exposure to a concentrated glucose solution. RecHDL as well as intact HDL from N and D were then labeled with “C-unesterified cholesterol (14C-C) and incubated with LCAT at 37°C along time. 14C-C ester and 14C-C were isolated after TLC and the percent of esterification was calculated. Results: The in vitro glycation of the intact HDL N particle, as well as of its protein, but not of its lipid component, impaired the activity of plasma LCAT. However, plasma LCAT esterified equally well 14C-C HDL that had been drawn from N (n = 11) and from D patients (n = 12). In spite of the elevated level of HBA’, in D, there was no difference in the level of HDL glycation between N and D subjects. Conclusion: In vitro HDL particle glycation rate impairs the LCAT-dependent 14C-C esterification rate. However, this rate is not modified in HDL D as compared to HDL N plasma, possibly because the faster removal rate of HDL D brings on a low plasma glycated HDL level.
I WeP33 W22 1WeP31 :W22
at least in
The stimuiation of cholesterol efilux by cpt-CAMP and free apo A-I from various cell lines
A.E. Bortnick, G.R. Rothblat, G. Stoudt, K.L. Hoppe’, L. Royer’, 0. Francone’. MCP Hahnemann University, Dept. of Biochem., 2900 Queen Lane, Phila., PA 19129; ‘PJizer Inc., Dept. of Cardiovascular and Metabolic Diseases, Central Research Division, Groton, CT, 06340, USA Studies have shown that unassociated, lipid-free apo A-I stimulates the release of cholesterol and phospholipid from cells. ATP-Binding Cassette 1 (ABCl) is implicated in this release, providing evidence that it is critical in the formation of HDL. Specific binding of apo A-I is upregulated by CAMP or enrichment with cholesterol. In this study, we determined the kinetics of cholesterol efflux from 5774 mouse macrophages with and without I exposure to cpt-CAMP. Upregulation is correlated to increased cholesterol efflux in a dose- and time-dependent manner with efflux first detectable 2-4 hours after treatment with cpt-CAMP. Efflux is upregulated by cholesterol enrichment of the same cell line. Stimulated efflux exhibits specificity for apo A-I, HDL, apo E and apo C as acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin, demonstrating that a protein-specific interaction is required. We have extended this study to 13 other cell lines under a standardized protocol, including fibroblasts (normal, transformed, and sitosterolemic), human and murine macrophages, hepatocytes, kidney cells, ovarian cells, and human enterocytes. Only J774 land elicited mouse macrophages show a significant increase in efflux with treatment. Apo A-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment. Apo A-I-stimulated efflux varied greatly among cell types, both in the percent and calculated mass of sterol released. Other data are consistent with upregulation of ABC1 by cpt-CAMP and cholesterol enrichment.
HALP
I
WeP34.W22
Cholesteryl ester transfer protein activity and hyperalphalipoproteinaemia in Chinese
K.C.B. Tan, S.W.M. Shiu, E.D. Janus’. Department of Medicine, University of Hong Kong, Hong kong,China;Melboume Lipid Clinic, Melbourne, Australia 4
CTL
HALP
Objective: Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and plays a significant role in HDL metaboKHth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 20130
1WeP25:W22
1 Release of cholesteryl esters fipm macrophage foam cells in culture
T. Mikkola, M. Leigh& R. St. Clair. Wake Forest University School ofMed., Winston-Salem, NC, USA
Thioglycollate-elicited peritoneal macrophages from cholesterol-fed, atherosclerosis-susceptible White Carneau pigeons contain high concentrations of free (FC) and ester&d (EC) cholesterol (200- z 1000 &mg cell protein), similar to what has been reported for macrophage foam cells from atherosclerotic plaques. when incubated in culture medium containing apo HDL/phosphtidylcholine (APC) vesicles for 48 hrs. there was a stimulation of cholesterol efflux with up to 50% of the cellular cholesterol appearing in the culture medium. Of the cholesterol in the medium, 2MO% was EC. Both the FC and EC in the medium appeared to be associated with the APC vesicles based on their ability to pass through a 0.45 urn filter. The appearance of EC in the medium could not be explained by gross cell death, based on changes in cell protein, or by esterification of FC in the culture medium via LCAT secreted by the macrophages. These findings were unexpected since most studies of cholesterol efflux from cells show that EC must first be hydrolyzed to FC prior to efflux. The majority of the published studies, however, have been done with cells either not loaded with EC or loaded to levels much less than seen in these macrophage foam cells. Although the mechanism by which macrophages release EC is unclear, it may represent a unique property of macrophage foam cells that could play an important role in the pathogenesis of atherosclerosis.
1WeP26:W22
1 Cyclodextrins acting as sinks and shuttles diierentially mobilise free and e&r&d foam ceil macrophages
cholesterol from human
S. Liu’ , R.T. Dean*, W. Jessup*, L. Kritharides *a3.‘~3Clinical Research; ‘Cell Biology Groups The Heart Research Institute; 3Dept. Cardiology _. Concord H&pital, Sydney, Australia Objective:
To investigate whether cyclodextrins (CD) with high cholesterol affinity acting as sinks, or low cholesterol affinity acting as shuttles to phospholipid vesicles (PV), deplete esterified cholesterol (EC) and free cholesterol (C) from primary human foam cell macrophages (HMFC) with equivalent efficacy. Methods: HMFC were generated by incubating monocyte derived macrophages with acetylated low density lipoprotein radiolabelled with [3H]-C. HMFC were incubated for up to 24 hrs in medium containing PV (200 &ml), CD (1 .Omg/ml) with differing affinity for C (low affinity hydroxypropyl-CD, hp-CD, or high affinity trimethyl-CD, tm-CD), and hp-CD or tm-CD with PV. Cells and media were analysed for C and EC by HPLC, TLC and radiometric detection. Results: Within 2 hrs, tm-CD depleted C to 42.3 f 10% of HMFC in control medium RPMI, but did not deplete CE even after 24 brs (95.0 f 6.1% of control). hp-CD caused minimal C efflux on its own, but with PV it depleted C to 36.7 & 5.0% and CE to 35.2 f 4.9% of control at 24 hrs. tm-CD with PV was also ineffective at depleting EC (102.5 f 5.0% of control) precluding acceptor saturation as the mechanism of failed CE clearance by tm-CD. Initial efflux at 10 minutes to tm-CD (1.3 f 0.2% of total cholesterol), hp-CD + PV (2.2 f 0.3%) and tm-CD + PV (3.0 f 0.5%) did not correlate with relative efficacy of CE depletion. Concluskms: In HMFC, synergistic efflux with CD and PV achieves superior depletion of CE than does efflux to high affinity CD alone.
IWeP27.W22
Stimulation of apo E secretion from human foam cell macrophages by apotipoproteh A-I mutants
T. Sloane’, D. Sviridov’, L. Pyle2, R.T. Dean’, W. Jessup’, L. Kritharide~‘,~. mart Research Institute Sydney; 2Baker Medical Research Institute Melbourne; ‘Dept. of Cardiology Concord Hospital Sydney, Australia Objective:
We have recently identified that apolipoprotein A-I (apo A-I) induces secretion of apo E from foam cell macrophages (J. Biol. Chem. 1999, 274,27925), and that proapo A-I mutants achieve differential cholesterol (C) and phospholipid efflux (J. Biol. Chem. 1996, 271, 33277). This study has investigated whether specific sequences of apo A-I mediate apo E secretion from human foam cell macrophages. Methods: Primary human monocyte-derived macrophages were incubated with acetylated low density lipoprotein radiolabelled with [3H]-C to generate HMFC, and HMFC then incubated in RPM1 and delipidated proapo A-I mutants (25 wg/ml). Apo A-I mutants were expressed as fusion proteins in an XlIth hmmational
185
E. Coli/pGEX vector system with whole pro apo A-I (-6-243) and truncated forms (-6-222) and (-6-150) used herein. Lipid analyses in cells and media were performed by HPLC, TLC and radiometric detection of [3H]-C, and apo E quantified by Western blotting. Results: Mutants -6-150, -6-243, -6-222 respectively achieved 3.5-fold, 2.8-fold and 2.0-fold the C efflux achieved in controls at 8 hrs. All 3 mutants stimulated time-dependent apo E secretion, respectively 5.0-fold, 3.0-fold and 3.0-fold the apo E Secretion of controls at 8 hrs. Interestingly, -6-243 stimulated the initial secretion of a lower MW form of apo E which did not accumulate further after 2 hours. Conclusions: In HMFC, apo A-I mutants induce C efflux and overall apo E secretion approximately in parallel. Residues 222-243 may specifically stimulate the secretion of lower MW, presumably less glycosylated, apo E.
IWeP28
W22
Mechanisms of 7.ketocholesteml-mediated inhibition of cholesterol efllux from human foam cell macrophages
K Gaus C.R. Dass, I.C. Gelissen, L. Kritharides, A.J. Brown, R.T. Dean, i) W. Jessup. Heart Research Institute, Sydney, Australia Macrophage foam cells are early and characteristic components of atherosclerotic lesions. They contain large cytoplasmic deposits of esterified cholesterol, which presumably results from a disparity between the rates of uptake, synthesis and export of cholesterol. Generally cells are able to closely control their cholesterol content through regulation of lipoprotein uptake and of cholesterol synthesis. In addition, most cells efficiently export excess free cholesterol to appropriate extracellular acceptors such as high-density lipoproteins (HDL) or its major protein component, apolipopmtein A-l (apoA-1). Addition of such acceptors to foam cells in vitro leads to a progressive release of free cholesterol and concomitant hydrolysis and depletion of intracellular cholesteryl esters Human foam cells contain a range of oxidised cholesterols (oxysterols), of which 7-ke.tocholesteml (7KC) is a major component. The effect of 7KC on sterol efflux to apolipoprotein A-I (apoA-I) was examined in human monocyte-derived macrophages (HMDM) loaded with cholesterol only or with a combination of cholesterol and 7KC. The amount of 7KC loaded was within the range found in human lesion foam cells. Efflux of cholesterol from HMDMs was stimulated by apoA-I, but was inhibited to basal levels when 7KC was present. Only 7% of cell phospholipid was exported to apoA-I from cells loaded with 7KC compared with 25% from AcLDL- loaded cells. ApoA-I-did not stimulate efflux of 7KC. Similar but less profound effects were also found in comparable murine foam cells. These data suggest that 7KC may contribute to atherogenesis by its ability to inhibit reverse cholesterol transport in human macrophage foam cells. Likely mechanisms under investigation include inhibition of plasma membrane cholesterol and phospholipid desorption to apoA-1 and inhibition of phospholipid synthesis by 7KC.
IWeP29
W22
Chid and molecular characteristics CETP deficiency
of homozygous
A. Inazu, Y. Noji, Y. Toudo, A. Nohara, J. Koizumi, H. Mabuchi. The Second Dept of Internal Medicine and Dept of Clinical Laboratory Science, Kanazawa University, Kanazawa, Japan Objective:
Relationship between CETP deficiency and atherosclerosis remains controversial and appears dependent on serum HDL-C levels. Methods: By PCR genotyping and/or plasma CETP measurement by ELISA in hyperalphalipoproteinemic subjects (n = 1000, HDL-C >= 80 mg/dl), 44 cases (18 men and 26 women) with homozygous CETP deficiency from 35 unrelated families (age 22-89 yrs, 61 yr f 13 [SD]) were identified, and 11 cases of them were followed-up. Results: Subjects with homozygous CETP deficiency had BMI 21.4 kg/m2 f 3.1, CHOL 267 mg/dl f 40, TG 99 f 63, HDL-C 147 f 42, apo A-I 228 f 53, A-II 44 f 15, B 74 f 53, C-II 7.5 rt 5.5, C-III 20.9 f 11.5, E 10.7 f 5.2, and Lp(a) 11.9 f 9.1. There were 30 complete CETP deficiency caused by 27 true homozygotes of intron 14 G (+l)-to-A (intronl4A) mutation (mean HDL-C 167 mg/dl), 1 compound heterozygote of intron 14A and intron 14 T (+3) insertion (HDL-C 106), 1 compound heterozygote of intron 14A and unknown mutation (HDL-C 223). and 1 unknown mutation (HDL-C 130). There were 14 partial CETP deficiency caused by 7 compound heterozygotes of intron 14A and D442G (HDL-C 124), and 7 true homozygotes of D442G (HDL-C 93). Plasma CETP levels in intron 14A/D442G and D442G homozygotes were 0.7 &ml and 0.9, respectively. Hypertension (SBP >= 160 and/or DBP >= 95 mmHg or drug treatment) was found in 8 cases. Cerebral infarction was found in 2 cases (ages of 50 and 66), polycystic kidney disease
Symposium on Atherosclerosis,
Stockholm, Sweden, June 25-29, 2@_XI
Wednesday June 28,200O: Poster Abstracts P: W22 Reverse Cholesterol Transport
186
(PKD) in 2 cases of 1 family; (probable) angina pectoris in 2 cases (ages of 63 and 77); SAH in 1 case of PKD family; liver cirrhosis in 1 case; COPD in 1 case; FH in 1 case; and DM in 1 case. Conclusions: Prevalence of premature atherosclerotic diseases was not increased in homozygous CETP deficiency despite hypercholesterolemia.
IWeP30
W22
Inhibition of lecithin: Cholesterol acyltransferase cholesterol oxides
by
EC. Pincinato’, A. Sevaniru?, D.S.P. Abdalla’. ‘Depto. of Clin and Toxicol. Analyses, FCF USP Scio Paulo, SP Brazil; Inst. Toxicol., University of Southern California, IA, CA, USA Objective: To investigate the effect of cholesterol oxides on esteritication of cholesterol by LCAT. Methods: HDL particles enriched with increasing concentration of cholesterol oxides were incubated with fresh plasma as source of LCAT. The esterification of cholesterol and cholesterol oxides was followed by measuring the consumption of the respective free sterol and oxysterols. Measurements of cholesterol and cholesterol oxides were done by gas chromatography with flame ionization detection. Results: All the studied cholesterol oxides were esterified by LCAT after incorporation into HDL particle, competing with cholesterol by LCAT-mediated esterification. Cholesterol esterification by LCAT was inversely related to the cholesterol oxide concentration in HDL particles. Kinetic studies with cholestan- 38, 5cr, 6B-trio1 and 5-cholesten- 3/I-25diol (25-hidroxycholesterol) showed a non-competitive inhibition with a Kiapparent of 103 and 15.02 ng/pL, respectively. This study shows markedly differences for inhibition parameters and apparent Ki among the different cholesterol oxides. Conclusions: Data suggest that cholesterol esterification by LCAT is inhibited in the cholesterol oxide-enriched HDL particles which could disturb the reverse cholesterol transport. In contrast, the esterification of cholesterol oxides by LCAT may have an anti-atherogenic effect considering that this step may increase the catabolism of these atherogenic compounds by enhancing their liver uptake either by direct HDL removal or by the previous transfer of the cholesteryl oxide esters to apo, B-containing lipoproteins followed by their hepatic catabolism. Supported by FAPESP
Conclusions: The hyperalphalipoproteinemia was explained, part, by the lower plasma levels of HL in this population.
I WeP32 W22
1 Lipases and transfer proteins in patients with hyperalphaiipoproteinemia
S. Alarcon’,V. Castanho’, D. Kaplan’, H. Oliveira*, V Nunes3, P. Cazita3, E. Quintio3, E. de Faria’ I Dept. of Clinical Pathology/NMCE/Il;NICAMP; 2Dept. of Physiology/UNICAM; 3Lipid Lab., FM-LISP Sr70 Paulo, Brazil Objective: To determine in Brazilian hyperalphalipoproteinemic (HALP) patients the activities of factors that modulate the metabolism of HDL: lipoprotein lipase (LPL), hepatic lipase (HL), cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP). Methods: Ninety-five volunteers were defined by their HDL-chol as controls (CTL-n = 35, below 68 mg!dL) and HALP (n = 60, equal and above 68 mg/dL), the 90th percentile for a local population. HL, (post-heparin plasma), CETP and PLTP were measured by exogenous radiometric methods using respectively triolein emulsion, “C-CE-HDL and phospholipid lipossomes as substrates. Results: HL was significantly lower (p = 0.04) in HALP, but no differences were found for LPL, CETP and PLTP (Mann-Whitney). The scatterplots are shown below:
HALP
i
3
CT1
LCAT-dependent cholesterol esterhkation rate is slower in in vi&o giycated HDL but is not altered in plasma HDL drawn from NIDDM patients
M. Passarelli, E.R. Nakandakare, S. Catanozi, J.C. Rocha, S.A. Lottenberg, E.C.R. Ouinfio. Lipids Lab. (LIM IO), Univ. S. Paul0 Med. School, S. Paulo, SP Brazil Objective: Alterations in the reverse cholesterol transport has been related to glycation of plasma lipoproteins. Present study investigates the influence of HDL glycation on the in vitro activity of the enzyme lecithin cholesterol acyl transferase (LCAT). Methods: HDL obtained from normal controls (N) and poorly controlled NIDDM (D) patients was separated by ultracentrifugation, and plasma d > 1.21 g/mL from N was utilized as the source of LCAT. Reconstituted HDL (ret HDL) from N were prepared by sonication of delipidated HDL previously submitted to selective glycation of the lipid and protein components separately, or both together, by exposure to a concentrated glucose solution. RecHDL as well as intact HDL from N and D were then labeled with “C-unesterified cholesterol (14C-C) and incubated with LCAT at 37°C along time. 14C-C ester and 14C-C were isolated after TLC and the percent of esterification was calculated. Results: The in vitro glycation of the intact HDL N particle, as well as of its protein, but not of its lipid component, impaired the activity of plasma LCAT. However, plasma LCAT esterified equally well 14C-C HDL that had been drawn from N (n = 11) and from D patients (n = 12). In spite of the elevated level of HBA’, in D, there was no difference in the level of HDL glycation between N and D subjects. Conclusion: In vitro HDL particle glycation rate impairs the LCAT-dependent 14C-C esterification rate. However, this rate is not modified in HDL D as compared to HDL N plasma, possibly because the faster removal rate of HDL D brings on a low plasma glycated HDL level.
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The stimuiation of cholesterol efilux by cpt-CAMP and free apo A-I from various cell lines
A.E. Bortnick, G.R. Rothblat, G. Stoudt, K.L. Hoppe’, L. Royer’, 0. Francone’. MCP Hahnemann University, Dept. of Biochem., 2900 Queen Lane, Phila., PA 19129; ‘PJizer Inc., Dept. of Cardiovascular and Metabolic Diseases, Central Research Division, Groton, CT, 06340, USA Studies have shown that unassociated, lipid-free apo A-I stimulates the release of cholesterol and phospholipid from cells. ATP-Binding Cassette 1 (ABCl) is implicated in this release, providing evidence that it is critical in the formation of HDL. Specific binding of apo A-I is upregulated by CAMP or enrichment with cholesterol. In this study, we determined the kinetics of cholesterol efflux from 5774 mouse macrophages with and without I exposure to cpt-CAMP. Upregulation is correlated to increased cholesterol efflux in a dose- and time-dependent manner with efflux first detectable 2-4 hours after treatment with cpt-CAMP. Efflux is upregulated by cholesterol enrichment of the same cell line. Stimulated efflux exhibits specificity for apo A-I, HDL, apo E and apo C as acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin, demonstrating that a protein-specific interaction is required. We have extended this study to 13 other cell lines under a standardized protocol, including fibroblasts (normal, transformed, and sitosterolemic), human and murine macrophages, hepatocytes, kidney cells, ovarian cells, and human enterocytes. Only J774 land elicited mouse macrophages show a significant increase in efflux with treatment. Apo A-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment. Apo A-I-stimulated efflux varied greatly among cell types, both in the percent and calculated mass of sterol released. Other data are consistent with upregulation of ABC1 by cpt-CAMP and cholesterol enrichment.
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Cholesteryl ester transfer protein activity and hyperalphalipoproteinaemia in Chinese
K.C.B. Tan, S.W.M. Shiu, E.D. Janus’. Department of Medicine, University of Hong Kong, Hong kong,China;Melboume Lipid Clinic, Melbourne, Australia 4
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Objective: Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and plays a significant role in HDL metaboKHth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 20130