Removal Of Cellular Cholesterol Research Articles

Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface.

Cultured cells have on their cell surface a specific high-affinity binding site (receptor) for high density lipoproteins (HDL) which appears to promote cholesterol efflux. In this study we characterized the cellular mechanisms involved in HDL receptor-mediated transport of cholesterol from cultured human fibroblasts and bovine aortic endothelial cells. HDL3, chemically modified by tetranitromethane (TNM-HDL3), is not recognized by this receptor and was used as a control for efflux not mediated by HDL receptor binding. HDL3 and TNM-HDL3 were found to be equally effective in causing efflux of plasma membrane cholesterol radiolabeled with [3H]cholesterol. However, HDL3 was much more effective than TNM-HDL3 in causing efflux of [3H]cholesterol associated with intracellular membranes. By measuring movement of endogenously synthesized [3H]cholesterol to the plasma membrane, and into the medium, we found that HDL3 induced a rapid movement of [3H]cholesterol from a preplasma membrane compartment to the plasma membrane that preceded [3H]cholesterol efflux. This effect was not observed with TNM-HDL3. Thus, receptor binding of HDL3 appears to facilitate removal of cellular cholesterol from specific intracellular pools by initiation of translocation of intracellular cholesterol to the plasma membrane.

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 CT 2, of mouse origin, took up and metabolized rat HDL and human HDL 3. 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.

Interaction of Tangier lipoproteins with cholesteryl ester-laden mouse peritoneal macrophages.

Cholesterol efflux was studied from cholesteryl esterladen mouse peritoneal macrophages in the presence of Tangier lipoproteins derived from fasting and postprandial sera of three patients homozygous for Tangier disease (analphalipoproteinemia). The d greater than 1.063 g/ml fractions isolated from fasting patients and 3 hr and 18 hr after an oral fat load were all effective in cellular cholesterol removal. By contrast, the d greater than 1.063 g/ml fractions isolated 6 hr and 12 hr after fat ingestion did not affect net removal of cellular cholesterol. The d greater than 1.21 g/ml protein fractions derived from fasting as well as postprandial sera were all effective in removing cholesterol. D 1.063-1.21 g/ml fractions from fasting Tangier patients contained HDLT. In the corresponding postprandial fractions, in addition to HDLT, apoB-100- and apoB-48-containing lipoproteins were present. Furthermore, the 6 hr and 12 hr postprandial Tangier HDL fractions contained apoB-immunoreactive proteins of lower molecular weight. The abnormal activity of the elastase/alpha 1-antitrypsin proteolytic system and the abnormal fibronectin concentration we found in Tangier plasma suggests a possible relationship to the in vivo degradation of apoB. The peculiar type of membrane-bound lipid droplets in Tangier splenic macrophages points to a lipoprotein source of lipid accumulation which possibly originates from the uptake of chylomicrons or chylomicron-derived particles. It is concluded that cholesteryl ester storage in Tangier macrophages results from an imbalance of cholesterol influx and efflux. In the absence of HDL, the net increase of cholesterol caused by abnormal lipoproteins in certain postprandial states cannot be fully compensated by effective efflux and ultimately leads to macrophage cholesteryl ester accumulation.

Phospholipase A 2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferae reactivity

Human HDL 3 ( d 1.125−1.21 g/ml) were treated by an exogenous phospholipase A 2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. 1. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5–10% haematocrit and HDL 3 (0.6 mM total cholesterol) from 0 to 12–15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 μM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. 2. (2) The lecithin: cholesterol acyltransferase reactivity of HDL 3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A 2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21–1.25 g /ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15–30% lipolysis, the lecithin:cholesterol acyltransferase reactivity of HDL 3 was reduced about 30–40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. 3. (3) The addition of the lecithin:cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL 3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin:cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin:cholesterol acyltransferase-induced removal of cellular cholesterol.

Effects of diet and high density lipoprotein subfractions on the removal of cellular cholesterol.

AbstractThe effects of isocaloric substitutions of dietary polyunsaturated and saturated fat on the composition and function of plasma high density lipoproteins (HDLs) were studied in 3 normal subjects who were fed saturate‐rich and polyunsaturate‐rich diet programs. Compared to the saturated diets (P/S=0.4), polyunsaturated fat diets (P/S=4 or 2) reduced both plasma cholesterol and triglyceride levels. In 2 of the subjects, HDL cholesterol concentrations increased with polyunsaturated fat caused a reduction in HDL fatty acyl content of oleate and an increase in linoleate. To determine whether the altered composition affected the removal of cell membrane cholesterol, HDL and their subfractions, HDL2 and HDL3, which were isolated from each of the diets, were incubated with Ehrlich ascites cells in vitro. The cells were prelabeled with [3H] cholesterol, and the release of labeled cholesterol from the cells into the medium containing the various HDL fractions was determined. HDL, irrespective of the type of dietary fat, caused a release of [3H] cholesterol from the cells into the medium. The amount of [3H] cholesterol recovered in the medium was dependent on the absolute concentration of HDL cholesterol added to the cells and was independent of the type of diet. These results indicate that HDL facilitates the removal of cholesterol from cells, but that the amount and rate of removal are independent of the changes in HDL composition that can be obtained by dietary perturbations.

Removal of cholesterol from fibroblasts and smooth muscle cells in culture in the presence and absence of cholesterol esterification in the medium

Removal of cellular cholesterol from human skin fibroblasts and rat aortic smooth muscle cells was studied in tissue culture. The cells were prelabeled with [ 3H] cholesterol and incubated with medium containing very low density-and low density lipoprotein-free serum at concentrations of 5–20%. Labeled cholesterol was recovered in the medium containing the very low density-and low density lipoprotein-free serum and up to 40% of the label was found in the form of cholesterol ester in the presence of unheated serum. Heating and pretreatment of the serum with N-ethylmaleimide resulted in an almost complete inhibition of formation of labeled cholesterol ester in the medium. However, the amount of labeled cholesterol transferred from the cells into the medium as well as the loss of cholesterol mass from the cells, was similar irrespective of whether lecithin:cholesterol acyltransferase was active and cholesterol esterification occurred in the medium. When the cells had been enriched in cholesterol ester by pretreatment with chloroquine and low density lipoprotein, net loss of cellular cholesterol was neither dependent on nor increased in the presence of cholesterol esterification in the medium. The present results indicate that lecithin:cholesterol acyltransferase activity is not indispensable for cholesterol removal from cells in culture under conditions which were designed to mimic interactions between extracellular fluid and peripheral cells in vivo.

Cellular control of cholesterol metabolism

Cholesterol is a structural component of cell membranes in mammalian tissues. The cholesterol content of cells fluctuates within narrow limits and reflects synthesis within the cells and the rates of influx and efflux which are related to the metabolism of circulating lipoproteins. These events have been studied in cultured arterial smooth muscle cells and fibroblasts; accumulation of cholesterol within these cells is the hallmark of atherosclerosis. The cholesterol content of cells is more influenced by plasma lipoproteins than by cellular synthesis. Uptake of cholesterol into cells occurs through the binding of low density lipoprotein (LDL) to surface receptors, the internalization of the whole lipoprotein, the degradation of the lipoprotein and the release of its cholesterol. This in turn suppresses synthesis within the cell. Other lipoproteins are also taken up into smooth muscle cells, notably in cholesterol-fed animals. This uptake is enhanced by rendering the cells hypoxic. By contrast, uptake of LDL which transport the bulk of circulating cholesterol in man, is reduced in cells from subjects with the deficiency of the specific surface receptors; this leads to the accumulation of cholesterol in LDL in plasma and to an over-production of cholesterol within the cell. The removal of cellular cholesterol appears to be controlled by another lipoprotein, the high density lipoprotein (HDL). The proteins of HDL have a strong affinity for cholesterol and stimulate efflux from the cell. Furthermore, HDL interferes with the binding of LDL to cells. These interactions between HDL and LDL at the cellular level determine cholesterol homeostasis within cells. This can be translated to documented events leading to atherosclerosis in man: athe mass of the body's exchangeable cholesterol is inversely related to the concentration of plasma HDL bthe cholesterol content of some tissues varies directly with the plasma LDL and inversely with the plasma HDL concentrations cplasma LDL is raised and the HDL level reduced in severe hypercholesterolaemia dthe epidemiological probability of developing premature coronary artery disease is a direct function of the plasma LDL level and an inverse function of the plasma HDL level. Cholesterol is a structural component of cell membranes in mammalian tissues. The cholesterol content of cells fluctuates within narrow limits and reflects synthesis within the cells and the rates of influx and efflux which are related to the metabolism of circulating lipoproteins. These events have been studied in cultured arterial smooth muscle cells and fibroblasts; accumulation of cholesterol within these cells is the hallmark of atherosclerosis. The cholesterol content of cells is more influenced by plasma lipoproteins than by cellular synthesis. Uptake of cholesterol into cells occurs through the binding of low density lipoprotein (LDL) to surface receptors, the internalization of the whole lipoprotein, the degradation of the lipoprotein and the release of its cholesterol. This in turn suppresses synthesis within the cell. Other lipoproteins are also taken up into smooth muscle cells, notably in cholesterol-fed animals. This uptake is enhanced by rendering the cells hypoxic. By contrast, uptake of LDL which transport the bulk of circulating cholesterol in man, is reduced in cells from subjects with the deficiency of the specific surface receptors; this leads to the accumulation of cholesterol in LDL in plasma and to an over-production of cholesterol within the cell. The removal of cellular cholesterol appears to be controlled by another lipoprotein, the high density lipoprotein (HDL). The proteins of HDL have a strong affinity for cholesterol and stimulate efflux from the cell. Furthermore, HDL interferes with the binding of LDL to cells. These interactions between HDL and LDL at the cellular level determine cholesterol homeostasis within cells. This can be translated to documented events leading to atherosclerosis in man: athe mass of the body's exchangeable cholesterol is inversely related to the concentration of plasma HDL bthe cholesterol content of some tissues varies directly with the plasma LDL and inversely with the plasma HDL concentrations cplasma LDL is raised and the HDL level reduced in severe hypercholesterolaemia dthe epidemiological probability of developing premature coronary artery disease is a direct function of the plasma LDL level and an inverse function of the plasma HDL level.

The removal of cholesterol from aortic smooth muscle cells in culture and landschutz ascites cells by fractions of human high-density apolipoprotein

Ascites cells were labeled by intraperitoneal injection of [ 3H] cholesterol and aortic smooth muscle cells by addition of [ 3H] cholesterol to the serum component of the culture medium. The release of cholesterol from cells into a serum-free medium supplemented with the various “acceptors” was studied using ascites cells in suspension and aortic smooth muscle cells in a multilayer culture. Unfractionated human high-density apolipoprotein was somewhat more effective in the removal of labeled cellular free cholesterol, in both cell types, than apolipoprotein derived from rat high-density lipoprotein. Following separation of human high-density apolipoprotein into four fractions by Sephadex chromatography, the effect of each fraction on the removal of cellular cholesterol from ascites cells was studied. The individual fractions had a lower capacity for cholesterol removal than the original unfractionated high-density apolipoprotein and the lowest activity was detected in Fraction II which comprised 75% of the total apolipoprotein. The effectiveness to remove cholesterol could be restored to all the fractions, as well as enhanced, by addition of sonicated suspensions of lecithin or sphingomyelin, which by themselves promoted a more limited removal of cellular cholesterol. Negatively stained preparations of mixtures of the four fractions and sonicated dispersion of lecithin were shown to consist of vesicles and discs of various sizes. Addition of the apolipoprotein fractions (especially Fractions II and IV) to sonicated dispersion of sphingomyelin resulted in a pronounced formation of discs which showed a high tendency towards stack formation. Mixtures of Fraction II and lecithin or sphingomyelin were effective in the release of cellular cholesterol from multilayers of aortic smooth muscle cells in culture. These results show the feasibility of net removal of cholesterol from cells which grow in a form resembling a tissue and thus provide a model to study the role of apolipoprotein-phospholipid mixtures in cholesterol removal from cells and tissues in vivo.

The removal of cholesterol from landschütz ascites cells by high-density apolipoprotein

Albino mice inoculated with Landschütz ascites cells were injected with [ 3H]-cholesterol and 2–4 days later the cells were used for the study of cholesterol release in vitro. The [ 3H]cholesterol in the cells was 75 % in the free and 25 % in esterified form. Following incubation in the presence of human low- or high-density lipoproteins, 10–30 % of the labeled cellular cholesterol was present in the incubation medium. In order to study net removal of cellular cholesterol use was made of delipidated, water-soluble apolipoprotein of human high-density lipoprotein. The apolipoprotein was 4 times more effective in removal of cellular free cholesterol than serum albumin and the reaction was time and temperature dependent. The affinity of the high-density apolipoprotein for cellular cholesterol was enhanced when the latter had been sonified with either rat liver lecithin and/or sphingomyelin and the rate of release was related to the concentration of the phospholipid. Labeled cellular lecithin was also removed by high-density apolipoprotein but not by serum albumin. The release was lower in the presence of sonicated non-labeled lecithin or sphingomyelin in the medium, while the release of cellular free cholesterol was enhanced. These findings suggest that an apoprotein-phospholipid complex is probably operative in the removal of membrane cholesterol and that in the absence of exogenous phospholipid, cellular lecithin may be utilized to form such a complex.