Cholesterol Accumulation In Cells Research Articles

Free radical modification of lipoproteins and cholesterol accumulation in cells upon atherosclerosis

An electron spin probe study was made of the effect of lipid peroxidation (LPO) on the structure of surface proteolipid layer of human serum low-density lipoproteins (LDL). The results obtained with a positively charged spin label and stearic acid spin probes with doxyl labels at positions 5, 12, and 16 revelaed that LPO caused a decreased in phospholipid molecule mobility both in the region of polar heads and in the region of acyl chains till the depth of a least 1.7 mm from water-lipid interface. Under relatively high levels of oxidation (more than 6 μmol MDA/g LDL phospholipid) the polarity of lipid phase increased. The decrease in efficiency of tryptophan fluorescence quenching by nitroxide fragments incoporated in hydrophobic regions at the depth of ∼2 nm from water-lipid interface indicated that lipid-protein interaction was disturbed as a result of oxidation of LDL lipids. In addition, the LPO-induced modification of apo-B, the main protein of LDL, was examined with maleimide spin label. LPO led to increase in mobility of strongly immobilized maleimide labels and in the number of weakly immobilized ones. Oxidized LDL revealed decreased ability to incorporate spin-labeled steroid (androstane) as compared to antive ones. LPO-induced structural changes of LDL surface are supposed to be a reason of enhanced accumulation of cholesterol in human monocytes during their incubation with oxidized LDL. The cholesterol content in red cells was shown to be directly correlated to MDA content in apo-B containing lipoproteins but not in whole serum. Our findings suggest that free radical modification of serum lipoproteins but not solely an increased level of LPO products in blood is one important cause for cholesterol accumulation in cells and, apparently, for their transformation into foam cells during atherosclerosis.

Correlation between cholesterol content in circulating immune complexes and atherogenic properties of CHD patients' serum manifested in cell culture

Blood serum of most patients with coronary heart disease (CHD) caused a 2–5-fold increase in the total cholesterol content of smooth muscle cells cultured from unaffected human aortic intima, i.e. possessed an atherogenic potential manifested in culture. Removal of immunoglobulins G and M from an atherogenic serum brought about a fall in its atherogenic potential. The serum deficient in immunoglobulins A retained its ability to induce the cholesterol accumulation in cells. Treatment of the CHD patients' serum with 2.5% polyethylene glycol 6000 removed the circulating immune complexes. The serum subjected to this treatment lost its atherogenicity, i.e. failed to increase the cholesterol content in cultured cells. Incubation of smooth muscle cells derived from human aortic intima with circulating immune complexes isolated from an atherogenic patients' serum caused a 1.5-3-fold rise in the intracellular cholesterol. Blood sera of most (89%) CHD patients was characterized by a high cholesterol content in circulating immune complexes. More than 75% of healthy subjects and patients without stenosis of coronary arteries had low level of cholesterol in immune complexes. Blood sera atherogenicity manifested in culture directly correlated with the cholesterol level of circulating immune complexes (r = 0.90). These findings suggest that the atherogenicity of CHD patients blood serum is due to cholesterol-containing immune complexes.

Effects of hypoxia on sterol synthesis, acyl-CoA:cholesterol acyltransferase activity, and efflux of cholesterol in cultured rabbit skin fibroblasts.

To elucidate the mechanisms of cholesterol accumulation in cells under hypoxic conditions, we tested the effects of hypoxia on sterol synthesis, on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), and on the efflux of cholesterol in cultured rabbit skin fibroblasts. Sterol synthesis was measured by the incorporation of 14C-acetate into sterol, and ACAT activity, by the incorporation of 14C-oleate into cholesteryl ester. Hypoxia suppressed both sterol synthesis and the efflux of cholesterol but increased ACAT activity. These results suggest that hypoxia disturbs the balance of cholesterol metabolism in cells and induces intracellular lipid accumulation.

Free radical modification of lipoproteins and cholesterol accumulation in cells upon atherosclerosis

Free radical modification of lipoproteins and cholesterol accumulation in cells upon atherosclerosis

Reversible inhibitory effects of apo-B-carrying lipoproteins on acid lipase activity of mononuclear leukocytes from cholesterol-fed rabbits.

Acid lipase (AL) activity of mixed mononuclear leukocytes from cholesterol-fed rabbits exhibited reduced values when compared with that of normolipidemic rabbits. The observed suppression of the AL activity was reversed commensurate with a fall in the plasma concentrations of cholesterol and lipid peroxides (LPO) after the rabbits were returned to a basal diet. The AL activity was negatively correlated with both plasma cholesterol and LPO levels in rabbits. This reversible inhibition was caused by apo-B-carrying lipoproteins from the hypercholesterolemic rabbits, and protective effects were produced by preincubation of lipoproteins with anti apo-B serum. The increase in plasma LPO in the hypercholesterolemic rabbits suggests that the observed reversible inhibition of the AL activity might be due to an increase in free radicals produced in the process of lipid peroxidation in the hypercholesterolemic state. The current findings also suggest that, through the combined effects of suppression of lysosomal hydrolysis and stimulation of microsomal synthesis of esterified cholesterol, β-migrating very low density lipoprotein in cholesterol-fed rabbits accelerates hypercholesterolemia and causes accumulation of esterified cholesterol in cells.

Correction of sphingomyelinase deficiency in Niemann‐Pick type C fibroblasts by removal of lipoprotein fraction from culture media

The average sphingomyelinase activity of fibroblasts obtained from 12 Niemann-Pick type C patients was 37.9% of that of normal fibroblasts (27.2 versus 72 nmol (mg protein)-1 h-1) when the cells were cultured in minimum essential medium containing 13% fetal bovine serum. Following replacement of the above medium with medium in which the lipoprotein fraction had been removed from the fetal bovine serum, the sphingomyelinase activity rose over a 7-day period from about 1/3 of normal to normal or above. Upon reintroduction of medium containing 10% fetal bovine serum which had not been extracted, the sphingomyelinase activity of the Niemann-Pick type C cells again fell within 48 h to 30% of the normal controls. In contrast, cell lines from patients with either Niemann-Pick Type A or B were not influenced by the presence or the absence of lipoprotein, i.e. lacked sphingomyelinase activity under all culture conditions examined. Histochemical staining with filipin showed an inverse relationship between the sphingomyelinase activity and intracellular, free, unesterified, cholesterol level. Moreover, immunochemical staining with an antibody against a lysosomal membrane protein provided direct evidence that the accumulation of unesterified cholesterol in cells cultured in regular (non-extracted) medium occurred within lysosomes and/or related organelles.

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells.

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.

Induction of corticosteroid biosynthetic pathway by ACTH and high-density lipoprotein in newborn rat adrenocortical cells cultured in serum-free medium

In order to investigate the role of rat high-density lipoprotein (HDL) on adrenal cholesterol accumulation and steroidogenic pathways (corticosteroid, i.e., 21-hydroxysteroid biosynthesis and reductive metabolism of progesterone), newborn rat adrenal cells cultured in serum-free medium were used. Incubation of [4- 14C]cholesterol-HDL (i) in serum-free medium compared to those in medium with lipoprotein-deficient serum, (ii) in serum-free medium with ACTH compared to those without ACTH, both showed an increase of labelled cholesterol in cells and of labelled 21-hydroxysteroids excreted in medium. Substitution of serum-supplemented medium by serum-free and cholesterol-free medium led to a deep decrease of ACTH-induced steroid biosynthesis with a predominance of 20α-reduced steroids; addition of HDL restored the corticosteroid biosynthesis and decreased the reductive metabolism. Addition of increased concentrations of HDL (7–150 μg cholesterol/ml) enhanced, in a saturable fashion, the total cholesterol uptake and the corticosteroid biosynthesis. The total cholesterol accumulation in cells exceeded by 4-fold the steroid production at saturation. The ratio between the two steroidogenic pathways increased up to 40 at saturation in favor of corticosteroids. These results suggest that HDL is at least partly internalized and that probably its constituents contribute greatly to the control of the two different steroidogenic pathways.

細胞内コレステロール処理機構に対する低酸素の影響

To elucidate the mechanism of cholesterol accumulation in cultured cells under hypoxic conditions which we previously reported, the effect of hypoxia on the efflux of cholesterol from cultured rabbit skin fibroblasts was tested.Cells were incubated with hyperlipemic rabbit serum (HRS) for 48 hours (load phase), then washed three times with phosphate buffered saline and incubated with normolipemic rabbit serum (NRS) for 24 hours (efflux phase). In the efflux phase cells were divided into two groups, one group was incubated under 95% N2 and 5% CO2 (hypoxic group), another was 95% room air and 5% CO2 (control group).At the end of load phase cellular content of esterified cholesterol was 39.0±1.5nmol/mg protein and after efflux phase it was remarkably decreased in control group (22.4±2.5nmol/mg protein) while it was almost unchanged in hypoxic group (38.2±1.7nmol/mg protein).When cells were incubated with lipoprotein deficient serum instead of NRS to exclude the uptake of extracellular cholesterol in efflux phase, the cellular content of esterified cholesterol was also significantly higher in hypoxic group than control group (53.1±1.5, 36.2±3.2nmol/mg protein respectively).These results suggested that the affected efflux may play a role in accumulation of esterified cholesterol in cells under hypoxic conditions.

Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein

The mass efflux of free and esterified cholesterol was studied in skin fibroblasts loaded with cholesterol by incubation with low density lipoproteins (LDL) isolated from normal or hypercholesterolemic cynomolgus monkeys. Cells incubated with hypercholesterolemic LDL accumulated 2-3 times more cholesteryl ester than did cells incubated with the same amount of normal LDL. Cholesteryl oleate was the principal cholesteryl ester species to accumulate in cells incubated with both normal and hypercholesterolemic LDL. Efflux of this accumulated cholesterol was absolutely dependent on the presence of a cholesterol acceptor in the culture medium. Lipoprotein-deficient serum (LPDS) was the most potent promoter of cholesterol efflux tested, with maximum efflux occurring at LPDS concentrations greater than 1.5 mg protein/ml. Upon addition of efflux medium containing LPDS, there was a reduction in both the free and esterified cholesterol concentration of the cells. Greater than 90% of the cholesteryl esters that were lost from the cells appeared in the culture medium as free cholesterol, indicating that hydrolysis of cholesteryl esters preceded efflux. Efflux was not inhibited by chloroquine, however, suggesting a mechanism independent of lysosomes. Loss of cellular free cholesterol was maximum by 6 hr and changed very little thereafter up to 72 hr. Cholesteryl ester loss from cells decreased in a log linear fashion for efflux periods of 6-72 hr, with an average half-life for cholesteryl ester efflux of 30 hr, but with a range of 20-50 hr, depending upon the specific cell line. The rate of efflux of cellular cholesteryl esters was similar for cells loaded with normal or hypercholesterolemic LDL. In cells loaded with cholesteryl esters, cholesterol synthesis was suppressed and cholesterol esterification and fatty acid synthesis were enhanced. During efflux, cholesterol synthesis remained maximally suppressed while cholesterol esterification decreased for the first 24 hr of efflux, then plateaued at a level approximately 5-fold higher than control levels, while fatty acid synthesis was slightly stimulated. There was little difference in the rate of efflux of individual cholesteryl ester species. There was, however, the suggestion that reesterification of cholesterol principally to palmitic acid occurred during efflux. Since the rate of cellular cholesteryl ester efflux was similar regardless of whether the cells had been loaded with cholesterol by incubation with normal LDL or hypercholesterolemic LDL, the greater accumulation of cholesterol in cells incubated with hypercholesterolemic LDL cannot be explained by differences in rates of efflux.-St. Clair, R. W., and M. A. Leight. Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein.

Enhanced cholesterol delivery to cells in culture by low density lipoproteins from hypercholesterolemic monkeys.

Previous studies have shown that the large molecular weight low density lipoprotein (LDL) of abnormal composition isolated from hypercholesterolemic nonhuman primates stimulates greater cholesterol accumulation in cells in culture than does the same amount of normal LDL. The purpose of the present study was to determine if a correlation existed over a range of LDL molecular weights with cholesterol accumulation in cells in culture, if the differential in cholesterol accumulation was the result of increased delivery of cholesterol to the cells and to evaluate the extent to which this differential was dependent on a functional LDL receptor pathway. Monkey and human skin fibroblasts were incubated for 24 hours with LDL isolated from individual normal or hypercholesterolemic cynomolgus monkeys having LDL molecular weights ranging from 2.58-6.39 x 10(6), and the cellular free and esterified cholesterol contents were determined. There was no correlation of LDL molecular weight with accumulation of cellular free or esterified cholesterol with LDL from normal animals having molecular weight of 2.58 to 3.08 X 10(6) or from hypercholesterolemic animals with LDL molecular weights greater than 4.5 x 10(6). There was a positive and significant correlation of LDL molecular weight with the accumulation of cellular free and esterified cholesterol with LDL molecular weights of 3.0 x 4.5 x 10(6). These differences were present when the LDL were added at equivalent protein or cholesterol concentrations and cannot be entirely explained by the increased amounts of cholesterol in LDL particles of larger molecular weight. The enhanced cellular cholesterol accumulation with hypercholesterolemic LDL seems to be the result of increased delivery of LDL cholesterol to the cells as shown by the increased rate of suppression of cellular sterol synthesis and LDL receptor activity, the increased stimulation of cholesterol esterification, and the increased accumulation of cellular 3H-cholesterol from LDL labeled with 3H-cholesteryl oleate. This difference in cellular cholesterol accumulation requires that the LDL must be both bound and internalized by a functional LDL receptor pathway, since cells that lack LDL receptors or are unable to internalize their LDL receptors do not show increased accumulation of cholesterol when incubated with hypercholesterolemic LDL.

MEtabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia

Diet-induced hypercholesterolemia in non-human primates results in the production of a low-density lipoprotein (LDL) of abnormal size and composition. This LDL from hypercholesterolemic monkeys has been shown to be more atherogenic than the same amount of LDL from normocholesterolemic animals. Previous studies have demonstrated that hypercholesterolemic LDL is approximately twice as effective as normal LDL in stimulating cholesterol accumulation and esterification in arterial smooth muscle cells in culture. The purpose of the present study was to determine whether this effect was secondary to differences in metabolism of the normal and hypercholesterolemic LDL. For this, the metabolism of 125I-labeled normal and hypercholesterolemic LDL from rhesus and cynomolgus monkeys was compared in several lines of skin fibroblasts and smooth muscle cells. Both normal and hypercholesterolemic LDL bound with high affinity to the same cell surface receptor. However, the affinity for binding of hypercholesterolemic LDL was about twice that of normal LDL (apparent dissociation constant for binding, K d , was 2.63 μg protein/ ml and 4.35 μg protein/ml, respectively). Conversely, only about 50% as many particles of hypercholesterolemic were able to bind to the receptor, compared with normal LDL. Those cells with the greatest capacity to metabolize LDL generally accumulated the most cholesterol with either hypercholesterolemic or normal LDL. In all cell lines, nearly twice as much cholesterol accumulated in cells incubated with hypercholesterolemic LDL compared with normal LDL, and this differential could not be explained by differences in metabolism of the two lipoproteins, suggesting that some cholesterol entered the cells independent of the uptake of the intact LDL molecule. LDL receptors appear necessary for this to occur, since no difference in cholesterol accumulation was observed in cells genetically deficient in LDL receptors.

Differential effects of isolated lipoproteins from normal and hypercholesterolemic rhesus monkeys on cholesterol esterification and accumulation in arterial smooth muscle cells in culture

Whole serum obtained from hypercholesterolemic rhesus monkeys was found to stimulate cholesterol esterification and cholesteryl ester accumulation in rhesus monkey arterial smooth muscle cells in culture to a significantly greater extent than normocholesterolemic serum. This was true even when the cholesterol concentration of the culture medium was equalized. Isolation and characterization of the low density lipoproteins (LDL) from rhesus monkeys indicated that the LDL from hypercholesterolemic animals was 33% larger than LDL from normocholesterolemic animals due principally to an increase in the amount of cholesteryl ester per molecule. As a result, LDL from hypercholesterolemic animals transported over 50% more cholesterol per molecule than did normal LDL. The LDL of altered composition from hypercholesterolemic animals, when added to smooth muscle cells in culture, was nearly twice as effective in stimulating cholesterol esterification and cholesteryl ester accumulation than was LDL of normal composition. Results suggest that at least part of the exaggerated ability of whole hypercholesterolemic serum to stimulate the esterification and accumulation of cholesterol in cells in culture is due to the presence of LDL of altered composition.