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Apolipoprotein A-I removes lipids from oxidized-LDL-derived foam cell macrophages inefficiently
224
Wednesday 12 October 1994: Poster Abstracts HDL and reverse cholesterol transport
tamed from cells labeled with [14C]cholesterol showed no differences in cholesterol efflux. Additionally, no differences in the esterification of membrane [t4C]cholesterol released into serum, or of its distribution among lipoprotein subfractions (LDL, pm& HDLs and a-HDL) was observed. We conclude that the availability of cellular cholesterol for efflux from HepG2 cells is influenced more by its subcellular location than by properties of the membranes themselves. )1671
.
Apolipoprotein A-I removes lipids from oxidixed-
. LDL-derived foam cell macrophages inefticiently
Jessup W, Dean RT, Cell Biol. Group, The Heart Res. Inst., 145 Missenden Rd., Camperdown, Sydney, NSW, 2050, Australia
B
Although oxidized LDL (OxLDL) can accumulate in macrophages in vitro, thus generating ‘foam cells’, little attention has been paid to the capacity of macrophages loaded with OxLDL to release cholesterol or oxidized sterol moieties. In vitro foam-cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated (AcLDL) or copper-OxLDL for 24 h. These foam-cells were then incubated with medium containing apolipoprotein A-I and albumin or albumin alone for up to 24 h. The content of oxidized and unoxidized cholesterol and cholesteryl esters in both the cells and the medium was determined using HPLC. Whereas cells loaded with AcLDL had accumulated cholesterol and large quantities of cholesteryl esters, OxLDL-derived foam-cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a very small quantity of cholestetyl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esteritied) to apo A-I-containing medium over 24 h in the form of unesterified cholesterol, while OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux into apo A-I-free medium. The proportions of cholesterol and 7-ketocholesterol released were not changed significantly by inhibiting cellular acyl-CoA: cholesterol acyl transferase (ACAT) using Sandoz 58-035. We conclude that OxLDL-derived foam-cells release cholesterol less efficiently than do cells derived from AcLDL. In addition, OxLDL-derived foam-cells release 7-ketocholesterol much less efficiently than cholesterol. It is likely that this impairment of efflux from OxLDL-derived foam-cells contributes to the generation and persistence of the foam-cell phenotype in vivo, and may therefore contribute to the adherogenicity of OxLDL. Influence of cholesteryl ester transfer protein on the composition, size and structure of spherical, reconstituted high density lipoproteins Rye K& Hime NJ, Barter PJ, Lipid Res. Lab., Level 1, Hanson
(
Centre, IMVS, Adelaide, South Australia, Australia, 5000
Spherical reconstituted HDL (rHDL) containing apo A-I were incubated with choksteryl ester transfer protein (CETP) and Intralipid for up to 24 h. During this time CETP promoted transfers of cholestetyl esters (CE) and triglyceride (TG) between rHDL and Intralipid, generating rHDL which were enriched in TG and depleted of CE. However, as the loss of CE from the rHDL was greater than the gain of TG, the concentration of core lipids in the rHDL decreased. The decrease in the concentration of rHDL core lipids, which was evident throughout the incubation, was accompanied by a reduction in rHDL diameter from 9.2 nm to 8.0 nm, the dissociation of apo A-I from rHDL and a decrease in the number of apo A-I molecules, from three/particle in the 9.2 nm rHDL, to two/particle in the 8.0 nm rHDL. The 8.0 nm rHDL had a more polar lipid-water interface and less ordered phospholipid packing
than the 9.2 nm rHDL. Fluorescence quenching studies revealed that the number of apo A-I Trp residues exposed to quencher in the 9.2 nm and 8.0 nm rHDL was two and three respectively. The 9.2 nm and 8.0 mn rHDL had identical apo A-I a-helical contents and surface charges. Denaturation studies with guanidine hydrochloride established that apo A-I was mote stable in the 8.0 nm rHDL than in the 9.2 nm rHDL. It is concluded that CETP converts rHDL to small TGenriched, apo A-I-depleted particles which have increased surface hydration and less ordered phospholipid packing. These changes are associated with an altered conformation and increased stability of the apo A-I remaining with the rHDL. Incorporation of lipid-free apoiipoprotein A-I into high density lipoproteins Rye K-A, Barter PJ, Depf. of Med., Royal Adelaide U&&Q,
pJ
Hospital, Adelaide, South Australia 5000
We have shown previously that the reduction in particle size of HDL which follows incubation with the cholesteryl ester transfer protein (CETP) plus VLDL or LDL is accompanied by the dissociation of lipid-free apo A-I from HDL. In the present study we demonstrate that it is possible to reincorporate lipid-free apo A-I into HDL by increasing the HDL particle size during incubation with 1ecithin:cholesterol acyltmnsferase &CAT). The lipoprotein fraction (d < 1.21 g/ml) of human plasma was mixed with CETP and incubated under conditions which resulted in a reduction in HDL particle sire and the dissociation of about 30% of the apo A-I from HDL. Following this incubation, the d < 1.21 g/ml fraction was re-isolated, supplemented with purified apo A-I and m-incubated in the presence and absence of LCAT. In the absence of LCAT, HDL size did not increase and there was no incorporation of the added apo A-I into the HDL density range. In contrast, when LCAT was present the cholesteryl ester content and the particle size of HDL increased. This coincided with an incorporation of lipid-free apo A-I into HDL in an amount sufficient to return the apo A-I content of HDL to that observed in the original, unmodified particles. This incorporation of lipid-free apo A-I into the HDL density range was dependent on both the presence of preexisting HDL and an increase in their size. Thus, just as a reduction in HDL size is accompanied by the dissociation of lipid-free apo A I, we have now shown that a subsequent increase in HDL size is accompanied by the reincorporation of lipid-free apo A-I into the particle. Production of small I-IDL particles after intravenous heparin in hypertriglyceridemic (ERG) diabetic and non-diabetic individuals. Studies before and after gemtibrozil therapy w, Cabana VG, Uffelman K, Steiner G, Polonsky KS, Getz GS, Div. of Endocrinol., Dept. of Med., Univ. of Toronto,
(170)
Toronto Hosp., 200 Elizabeth Str, EN 11-229, Toronto, Canada. MSG2C4; Depts. of Pathol. and Med., Univ. of Chicago, Chicago, USA
We have previously shown that light but small HDL particles are generated in vitro by incubating HDL with FFA in a dosedependent fashion (Circulation 1993; 88(no. 4, pt 2): 1269). Seven HTG Type 2 diabetic (D) patients (TG 3.2 f 0.7 mmoV1, HDL-C 0.72 i 0.05 mmobl, I-IbAtc 8.6 f 0.4%) and 5 HTG nondiabetic controls (C) (TG 3.7 f 0.3 mmobl, HDL-chol 0.74 f 0.07 mmobl) ingested a mixed meal containing 60 g fat/m’. Plasma TG increased postprandially by 5 h in D to 7.5 f 1.3 mmol/l and C to 7.4 f 0.9 mmobl and HDL became mom TGenriched in both groups. Heparin (6Op/kg i.v.) at 5 h postprandially generated lighter and smaller HDL particles in D (preheparin HDL peak d = 1.105 g/ml with 5.7 i 1.9% of the total
Atherosclerosis X, Montreal, October I994
Wednesday 12 October 1994: Poster Abstracts HDL and reverse cholesterol transport
tamed from cells labeled with [14C]cholesterol showed no differences in cholesterol efflux. Additionally, no differences in the esterification of membrane [t4C]cholesterol released into serum, or of its distribution among lipoprotein subfractions (LDL, pm& HDLs and a-HDL) was observed. We conclude that the availability of cellular cholesterol for efflux from HepG2 cells is influenced more by its subcellular location than by properties of the membranes themselves. )1671
.
Apolipoprotein A-I removes lipids from oxidixed-
. LDL-derived foam cell macrophages inefticiently
Jessup W, Dean RT, Cell Biol. Group, The Heart Res. Inst., 145 Missenden Rd., Camperdown, Sydney, NSW, 2050, Australia
B
Although oxidized LDL (OxLDL) can accumulate in macrophages in vitro, thus generating ‘foam cells’, little attention has been paid to the capacity of macrophages loaded with OxLDL to release cholesterol or oxidized sterol moieties. In vitro foam-cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated (AcLDL) or copper-OxLDL for 24 h. These foam-cells were then incubated with medium containing apolipoprotein A-I and albumin or albumin alone for up to 24 h. The content of oxidized and unoxidized cholesterol and cholesteryl esters in both the cells and the medium was determined using HPLC. Whereas cells loaded with AcLDL had accumulated cholesterol and large quantities of cholesteryl esters, OxLDL-derived foam-cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a very small quantity of cholestetyl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esteritied) to apo A-I-containing medium over 24 h in the form of unesterified cholesterol, while OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux into apo A-I-free medium. The proportions of cholesterol and 7-ketocholesterol released were not changed significantly by inhibiting cellular acyl-CoA: cholesterol acyl transferase (ACAT) using Sandoz 58-035. We conclude that OxLDL-derived foam-cells release cholesterol less efficiently than do cells derived from AcLDL. In addition, OxLDL-derived foam-cells release 7-ketocholesterol much less efficiently than cholesterol. It is likely that this impairment of efflux from OxLDL-derived foam-cells contributes to the generation and persistence of the foam-cell phenotype in vivo, and may therefore contribute to the adherogenicity of OxLDL. Influence of cholesteryl ester transfer protein on the composition, size and structure of spherical, reconstituted high density lipoproteins Rye K& Hime NJ, Barter PJ, Lipid Res. Lab., Level 1, Hanson
(
Centre, IMVS, Adelaide, South Australia, Australia, 5000
Spherical reconstituted HDL (rHDL) containing apo A-I were incubated with choksteryl ester transfer protein (CETP) and Intralipid for up to 24 h. During this time CETP promoted transfers of cholestetyl esters (CE) and triglyceride (TG) between rHDL and Intralipid, generating rHDL which were enriched in TG and depleted of CE. However, as the loss of CE from the rHDL was greater than the gain of TG, the concentration of core lipids in the rHDL decreased. The decrease in the concentration of rHDL core lipids, which was evident throughout the incubation, was accompanied by a reduction in rHDL diameter from 9.2 nm to 8.0 nm, the dissociation of apo A-I from rHDL and a decrease in the number of apo A-I molecules, from three/particle in the 9.2 nm rHDL, to two/particle in the 8.0 nm rHDL. The 8.0 nm rHDL had a more polar lipid-water interface and less ordered phospholipid packing
than the 9.2 nm rHDL. Fluorescence quenching studies revealed that the number of apo A-I Trp residues exposed to quencher in the 9.2 nm and 8.0 nm rHDL was two and three respectively. The 9.2 nm and 8.0 mn rHDL had identical apo A-I a-helical contents and surface charges. Denaturation studies with guanidine hydrochloride established that apo A-I was mote stable in the 8.0 nm rHDL than in the 9.2 nm rHDL. It is concluded that CETP converts rHDL to small TGenriched, apo A-I-depleted particles which have increased surface hydration and less ordered phospholipid packing. These changes are associated with an altered conformation and increased stability of the apo A-I remaining with the rHDL. Incorporation of lipid-free apoiipoprotein A-I into high density lipoproteins Rye K-A, Barter PJ, Depf. of Med., Royal Adelaide U&&Q,
pJ
Hospital, Adelaide, South Australia 5000
We have shown previously that the reduction in particle size of HDL which follows incubation with the cholesteryl ester transfer protein (CETP) plus VLDL or LDL is accompanied by the dissociation of lipid-free apo A-I from HDL. In the present study we demonstrate that it is possible to reincorporate lipid-free apo A-I into HDL by increasing the HDL particle size during incubation with 1ecithin:cholesterol acyltmnsferase &CAT). The lipoprotein fraction (d < 1.21 g/ml) of human plasma was mixed with CETP and incubated under conditions which resulted in a reduction in HDL particle sire and the dissociation of about 30% of the apo A-I from HDL. Following this incubation, the d < 1.21 g/ml fraction was re-isolated, supplemented with purified apo A-I and m-incubated in the presence and absence of LCAT. In the absence of LCAT, HDL size did not increase and there was no incorporation of the added apo A-I into the HDL density range. In contrast, when LCAT was present the cholesteryl ester content and the particle size of HDL increased. This coincided with an incorporation of lipid-free apo A-I into HDL in an amount sufficient to return the apo A-I content of HDL to that observed in the original, unmodified particles. This incorporation of lipid-free apo A-I into the HDL density range was dependent on both the presence of preexisting HDL and an increase in their size. Thus, just as a reduction in HDL size is accompanied by the dissociation of lipid-free apo A I, we have now shown that a subsequent increase in HDL size is accompanied by the reincorporation of lipid-free apo A-I into the particle. Production of small I-IDL particles after intravenous heparin in hypertriglyceridemic (ERG) diabetic and non-diabetic individuals. Studies before and after gemtibrozil therapy w, Cabana VG, Uffelman K, Steiner G, Polonsky KS, Getz GS, Div. of Endocrinol., Dept. of Med., Univ. of Toronto,
(170)
Toronto Hosp., 200 Elizabeth Str, EN 11-229, Toronto, Canada. MSG2C4; Depts. of Pathol. and Med., Univ. of Chicago, Chicago, USA
We have previously shown that light but small HDL particles are generated in vitro by incubating HDL with FFA in a dosedependent fashion (Circulation 1993; 88(no. 4, pt 2): 1269). Seven HTG Type 2 diabetic (D) patients (TG 3.2 f 0.7 mmoV1, HDL-C 0.72 i 0.05 mmobl, I-IbAtc 8.6 f 0.4%) and 5 HTG nondiabetic controls (C) (TG 3.7 f 0.3 mmobl, HDL-chol 0.74 f 0.07 mmobl) ingested a mixed meal containing 60 g fat/m’. Plasma TG increased postprandially by 5 h in D to 7.5 f 1.3 mmol/l and C to 7.4 f 0.9 mmobl and HDL became mom TGenriched in both groups. Heparin (6Op/kg i.v.) at 5 h postprandially generated lighter and smaller HDL particles in D (preheparin HDL peak d = 1.105 g/ml with 5.7 i 1.9% of the total
Atherosclerosis X, Montreal, October I994