Gideon Halperin

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.

Lipoprotein lipase mediated uptake of non-degradable ether analogues of phosphatidylcholine and cholesteryl ester by cultured cells.

Lipoprotein lipase mediated transfer of cholesteryl ester and its ether analog, cholesteryl linoleyl ether, from unilamellar liposomes, prepared from a nonhydrolyzable ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleyl ether-sn-glycero-3-phosphocholine (DOEPC), was studied in various cells in culture. It was found that lipoprotein lipase enhanced the uptake of cholesteryl linoleyl ether and of DOEPC. These findings provided a definitive proof that hydrolysis of liposomal PC is not needed for the lipoprotein lipase catalyzed transfer of cholesteryl linoleyl ether and cholesteryl ester to cells. The lipids transferred by lipoprotein lipase to cells were localized in three compartments, trypsin-releasable, resistant and metabolic; the latter was a chloroquine-sensitive pool as evidenced by inhibition of cholesteryl ester hydrolysis. Labeled PC and, to a lesser extent DOEPC, in the trypsin-releasable pool was able to return to the medium, while cholesteryl linoleyl ether and cholesteryl ester required cholesteryl ester transfer protein for release. The transfer of cholesteryl linoleyl ether and cholesteryl ester into a trypsin-resistant compartment did not require metabolic energy and occurred also in formaldehyde-fixed cells. Metabolic energy was needed for the translocation of cholesteryl linoleyl ether and cholesteryl ester into the lysosomal compartment, presumably by a process of endocytosis. The physiological relevance of the present findings is that as intravascular hydrolysis of triacylglycerol-rich lipoproteins is mediated by lipoprotein lipase attached to endothelial cells, the latter can provide a very extensive surface for removal and metabolism of phospholipids and cholesteryl ester by a mechanism mediated by lipoprotein lipase.

Comparison of cholesterol egress from cultured cells enriched with cholesterol ester after exposure to cationized LDL or to LDL and chloroquine.

Abstract Human skin fibroblasts in culture were incubated with low density lipoprotein (LDL) and 50 μM chloroquine (Method 1) or with cationized LDL (Method 2) and a 20–50-fold increase in cholesterol ester was achieved. Incubation with cationized LDL resulted in an increase in cellular lipid droplets and in membrane bound vacuoles, containing the cationized 125I-labeled LDL as shown by radioautography. Large amounts of cationized 125I-labeled LDL were seen to adhere to cell surfaces both in cells fixed in the petri dish and in cells subjected to trypsinization and washing. Adhesion of cationized LDL to cells amounted to 30% of the material added during trypsinization as compared to less than 1% with native LDL. When the cells had been enriched with cholesterol ester by Method 1 and postincubated with high density apolipoprotein-sphingomyelin liposomes for 24–48 h, a 50–80% reduction in cellular cholesterol ester was found. Little or no reduction in cholesterol ester occurred when Method 2 was used to induce cholesterol accumulation. The slow egress of cholesterol from cells incubated with cationized LDL could be due to (a) over-loading of the lysosomal system, (b) overestimation of cellular cholesterol ester content owing to tenacious adherence of the cationized LDL to cell surfaces, and (c) reesterification of the free cholesterol liberated after intralysosomal hydrolysis, to cytoplasmic cholesterol ester. The present findings indicate that Method 2 for enrichment of cells with cholesterol ester is more complicated than the chloroquine LDL model, especially when the primary aim is quantitative determination of cholesterol egress from cells enriched in cholesterol ester.

Reverse cholesterol transport in mice expressing simian cholesteryl ester transfer protein

The role of cholesteryl ester transfer protein (CETP) in atherogenesis remains ambiguous, as both pro and antiatherogenic effects have been described. Expression of CETP increases HDL-cholesteryl ester turnover, but there is no direct evidence whether CETP mobilizes cholesterol in vivo. The rate of cholesterol removal injected into a leg muscle as cationized low density lipoprotein (cat-LDL) was compared in CETP transgenic and control mice. Four days after injection the exogenous cholesterol mass retained in muscle was 65% in CETP transgenic and 70% of injected dose in controls; it decreased to 52-54% by day 8 and negligible amounts remained on day 28. The cat-LDL was labeled with either 3H-cholesterol oleate (3H-CE) or 3H-cholesteryl oleoyl ether (3H-COE), a nonhydrolyzable analog of 3H-CE. After injection of 3H-CE cat-LDL, clearance of 3H-cholesterol had a t(1/2) of 4 days between day 4 and 8 but there was little loss of 3H-COE between day 4 and 51. Liver radioactivity on day 4 was 1.7% in controls and 3.4% in CETP transgenics; it was 2.8 and 4.6%, respectively, on day 8. 3H-COE in liver accounted for 60% of label in CETP transgenics. In conclusion, high levels of plasma CETP in mice do not enhance reverse cholesterol transport in vivo but may act on extracellularly located cholesteryl ester.

Relative resistance of the hamster to aortic atherosclerosis in spite of prolonged vitamin E deficiency and dietary hypercholesterolemia. Putative effect of increased HDL

UNLABELLED Male golden hamsters were rendered hypercholesterolemic by feeding diets enriched with cholesterol and fat. In the first series of experiments, 5% butter and 1% cholesterol were added to a chow diet and plasma cholesterol levels were maintained at 350-390 mg/dl over the entire experimental period. Groups of hamsters and their age controls consuming the chow diet, were killed after 7, 15 and 20 months when the aorta was examined for atherosclerosis by determination of cholesterol mass. In the controls, aortic total cholesterol (TC) increased with age by 28% and esterified cholesterol increased to 11% of TC. In the hypercholesterolemic animals aortic TC was only 28% higher than in the controls and cholesteryl ester was also 11.5% of TC. In the second series, one group of hamsters were fed a semi-purified diet deficient in vitamin E, containing 1% cholesterol and 10% lard; a second group received the same diet, but supplemented with vitamin E. Controls consumed local chow. After 7 months on the vitamin E deficient diet plasma alpha-tocopherol was 0.05 mg/l, in those supplemented with vitamin E it was 20 mg/l, while in the controls it was 3.3 mg/l. Plasma thiobarbituric acid reactive substances (TBARS) were higher in the vitamin E deficient group and there was a greater propensity of lipoproteins (d < 1.063 g/ml) to peroxidation in vitro than in the vitamin E supplemented group. Plasma cholesterol was 366 mg/dl in the vitamin E deficient, 336 mg/dl in the vitamin E supplemented group, and 64 mg/dl in controls. Aortic cholesterol was 79.1 in vitamin E supplemented and 84.4 micrograms/10 mg dry weight in vitamin E deficient hamsters. In both series of experiments, HDL amounted to 36-41% of plasma TC in the hypercholesterolemic animals and 59-62% in the controls. IN CONCLUSION the hamster appears to be quite resistant to atherosclerosis in face of sustained hypercholesterolemia, even in the presence of increased peroxidative stress caused by vitamin E deficiency. This relative resistance could be related to commensurate increase in plasma HDL which was observed in both series of experiments. Since vitamin E deficiency did not enhance aortic cholesteryl ester deposition, the protective effect of HDL seems to be related to its role in reverse cholesterol transport, rather than in prevention of peroxidation.

Can lipoprotein lipase be the culprit in cholesteryl ester accretion in smooth muscle cells in atheroma

Bovine aortic smooth muscle cells and human skin fibroblasts were incubated with beta-very low density lipoprotein (beta VLDL) isolated from cholesterol-fed rabbits and labeled with [3H]cholesteryl oleate. Addition of lipoprotein lipase resulted in a 3.2-4.8-fold increase in cell associated radioactivity of which 45-61% was in free cholesterol, i.e., derived after intracellular hydrolysis. After exposure of smooth muscle cells to beta VLDL for up to 9 days and 60 min sodium heparin wash at 4 degrees C to remove extracellular surface bound lipoprotein, cellular cholesterol increase was 2 micrograms in controls and in the presence of lipoprotein lipase (LPL) it was tenfold higher. Addition of [3H]cholesteryl ester labeled beta VLDL during the last 48 h of incubation showed that 30-40% of total cellular label was in free cholesterol. This value represents the minimal cellular uptake of the added lipoprotein cholesteryl ester. Addition of recombinant apolipoprotein (apo) E to smooth muscle cells incubated with beta VLDL and [3H]oleate induced no further increase in [3H]cholesteryl oleate. We propose that following LPL-mediated binding of beta VLDL to heparan sulphate, this complex either undergoes endocytosis, or translocation of cholesteryl ester into the smooth muscle cells (SMC) occurs without endocytosis of the entire particle. The present results indicate that in the aortic wall macrophage-derived lipoprotein lipase could play a role in cholesteryl ester accretion in smooth muscle cells during atherogenesis.

Dexamethasone impairs cholesterol egress from a localized lipoprotein depot in vivo

Plasma high density lipoproteins play a central role in the prevention and regression of atherosclerosis, as they are known to promote egress of cholesterol from cells. Glucocorticoids increase plasma HDL, but enhance esterification of cholesterol in macrophages in vitro. A novel model to measure cholesterol egress from a well defined depot in vivo was used currently to study the effect of dexamethasone on reverse cholesterol transport. Cationized LDL (cat LDL) (200 microg cholesterol) was injected into the rectus femoris muscle of mice and the egress of cholesterol was studied as a function of time. Daily subcutaneous injection of dexamethasone (1.25 microg) raised plasma HDL levels by 40-80%. In mice injected with cat LDL labeled with 3H-cholesterol, daily treatment with dexamethasone slowed the loss of labeled cholesterol from the depot. With dexamethasone, there was no removal of the mass of lipoprotein cholesterol up to 14 days after injection of cat LDL, while in the controls 75% of the exogenous cholesterol mass had been cleared from the depot. When the cat LDL had been labeled with 3H-cholesteryl ester (3H-CE), apparent hydrolysis of 3H-CE amounted to 46, 75 and 97% in controls, but only to 20, 48 and 65% in dexamethasone treated mice on days 4, 8 and 14, respectively. In addition, dexamethasone stimulated cholesterol re-esterification as evidenced by recovery of 80% of the retained cholesterol mass as CE. In experiments with cultured macrophages exposed to modified LDL, dexamethasone increased the amount of labeled cholesteryl ester by 50-75% as compared to controls. Histological examination of the rectus femoris muscle after injection of cat LDL showed that in dexamethasone treated mice cellular infiltration was sparser on day 4, but not on day 8, and persisted longer than in controls. In conclusion, dexamethasone treatment impeded cholesterol egress from a lipoprotein depot by: a) reduction of early inflow of mononuclear cells; b) partial inhibition of cholesteryl ester hydrolysis, and c) enhancement of cholesterol esterification. The latter effect did not permit cholesterol egress from the injected site even in the presence of high plasma HDL in dexamethasone treated mice.

Preferential uptake of cholesteryl ester-HDL by cultured macrophages

The interaction between HDL and macrophages in culture was studied using HDL labeled with 125I and with [3H]cholesteryl linoleyl ether. Mouse peritoneal macrophages and the macrophage-like cell lines J-774 and CT2, of mouse origin, took up and metabolized rat HDL and human HDL3. In all 3 cell types using both rat and human HDL, the uptake of the cholesteryl ester moiety as measured with the nondegradable cholesteryl ether analog, was 2-5-fold higher when compared to the protein moiety. Modulation of the cholesterol content of the cultured macrophages affected the uptake of both protein and lipid moieties of HDL to the same extent. When the macrophages had interacted with the labeled HDL for 5 h and were post-incubated for 20 h, the amount of [125I]HDL which reappeared in the post incubation medium was twice that of [3H]cholesteryl linoleyl ether-HDL. The site from which the HDL may have returned to the culture medium was tentatively localized to the trypsin-releasable, cell surface-related compartment. The present results indicate that interaction between macrophages and HDL may result in some loss of cholesteryl ester and possibly render the particle more receptive for cellular cholesterol removal.

Cholesterol removal by peritoneal lavage with phospholipid-HDL apoprotein mixtures in hypercholesterolemic hamsters

Syrian hamsters were rendered hypercholesterolemic by supplementation of their diet with 1% cholesterol and 15% butter. The hamsters were injected intraperitoneally (i.p.) with about 20 mg of phospholipid liposomes containing trace amounts of [3H]cholesteryl linoleyl ether ([ 3H]CLE) alone or combined with 10 mg delipidated high-density lipoprotein (apoHDL). After 2 h the peritoneal cavity was washed repeatedly with up to 15 ml phosphate-buffered saline. 60%-70% of [3H]CLE were retained after i.p. injection without apoHDL, 30-50% in the presence of apoHDL. The amount of free cholesterol recovered in the peritoneal lavage was significantly higher when apoHDL was combined with 18:2 sphingomyelin or dilinoleyl phosphatidylcholine liposomes, when compared to either liposomes or apoHDL alone. It is suggested that supplementation of dialysate with HDL apolipoproteins and phospholipids in patients undergoing continuous peritoneal dialysis could be of use in a cholesterol depletion regimen.