Influx Of Free Cholesterol Research Articles

HDL to macrophage free cholesterol influx and transfer to LDL - new assays for dysfunctional HDL: The Houston heart study

HDL to macrophage free cholesterol influx and transfer to LDL - new assays for dysfunctional HDL: The Houston heart study

Abstract MP30: Excess HDL Free Cholesterol Bioavailability Drives Free Cholesterol Accretion Into Macrophages And Erythrocytes In Scarb1 -/- Mice

Aim: In humans, very high plasma HDL-cholesterol concentrations are associated with increased all cause- and atherosclerotic cardiovascular disease (ASCVD)-mortality. The HDL receptor-deficient mouse (Scarb1 -/- ), a robust model of this phenotype, is characterized by high free cholesterol (FC) bioavailability due to too many HDL particles that are FC-rich. Clinically, plasma LDL and HDL are quantified according to total cholesterol content, the sum of FC and esterified cholesterol, which likely contribute to ASCVD pathophysiology differently. A Western diet induces ASCVD in Scarb1 -/- mice, despite an attendant increase in HDL. We tested the hypothesis that high HDL-FC bioavailability contributes to ASCVD in Scarb1 -/- mice by increasing FC flux into macrophage cells, erythrocytes and other major tissues. Methods: Influx of HDL-FC and efflux of macrophage FC were determined between WT and Scarb1 -/- HDL and J774 macrophage cells. HDL of both genotypes were radiolabelled with [ 3 H]FC, injected into autologous mice, and the rates of plasma clearance and erythrocyte uptake were determined. Results: The magnitude of FC transfer from Scarb1 -/- HDL to LDL is greater than that from WT HDL; APOB-containing lipoproteins from Scarb1 -/- vs. WT mice are FC-enriched due likely to greater HDL-FC transfer. While macrophage efflux to HDL of Scarb1 -/- vs. WT HDL was not different, FC influx from Scarb1 -/- vs. WT HDL to macrophages was three-fold greater, a net effect that increased the FC burden of macrophages. In vivo studies showed that compared to WT mice, in Scarb1 -/- mice, autologous HDL-FC cleared more slowly and more FC transferred to erythrocytes. We compared the FC, CE, PL, and TG contents of all major tissues and determined that FC accretion by some tissues is higher among Scarb1 -/- vs. WT mice whereas in other tissues FC homeostasis is maintained. Lastly, we determined that the tissue compositions and plasma FC clearance kinetics varied according to sex, particularly among Scarb1 -/- mice. Conclusions: These findings are relevant to pathologies specific to Scarb1 -/- mice and to the evolving model of the role of HDL-FC in RCT. They provide a rationale for human studies to determine the utility of HDL-FC bioavailability as a risk factor for ASCVD and other pathologies.

Ginkgo biloba extract (Egb 761) inhibits β-amyloid production by lowering free cholesterol levels

Ginkgo biloba extract (EGb 761) can improve cognitive function in patients with Alzheimer's disease, but the molecular mechanisms underlying this effect remain undefined. Because free cholesterol may be involved in the production of β-amyloid precursor protein and amyloid β-peptide, key events in the development of Alzheimer's disease, we examined EGb 761 in relation to cholesterol and amyloidogenesis. In aging rats, EGb 761 treatment lowered circulating free cholesterol and inhibited the production of brain β-amyloid precursor protein and amyloid β-peptide. Exposure of PC12 cells to EGb 761 decreased the processing of β-amyloid precursor protein and abolished cholesterol-induced overproduction of this protein. Exposure of human NT2 cells to EGb 761 decreased free cholesterol influx and increased free cholesterol efflux. Our findings indicate that free circulating and intracellular cholesterol levels affect the processing of β-amyloid precursor protein and amyloidogenesis. Our findings also provide the first demonstration that EGb 761 can influence these mechanisms.

The influence of cellular and lipoprotein cholesterol contents on the flux of cholesterol between fibroblasts and high density lipoprotein.

Previous studies indicate that free cholesterol moves passively between high density lipoprotein (HDL) and cell plasma membranes by uncatalyzed diffusion of cholesterol molecules in the extracellular aqueous phase. By this mechanism, the rate constants for free cholesterol influx (Cli) and efflux (ke) should not be very sensitive to the free cholesterol content of cells or HDL. Thus, at a given HDL concentration, the unidirectional influx and efflux of cholesterol mass (Fi, Fe) should be proportional to the cholesterol content of HDL and cells, respectively, and net efflux of cholesterol mass (Fe-Fi greater than 0) should occur when either cells are enriched with cholesterol or HDL is depleted of cholesterol. We have examined the influence of cell and HDL free cholesterol contents on the bidirectional flux of free cholesterol between HDL and human fibroblasts and also attempted to detect some dependence of flux on the binding of HDL to the cells. In the range of HDL concentrations from 1 to 1000 micrograms of protein/ml, ke for cell free cholesterol approximately doubled for every 10-fold increase in HDL concentration, reaching 0.04 h-1 at 1000 micrograms of HDL/ml. ke and Cli were not influenced by the doubling of fibroblast free cholesterol content (from 31 +/- 5 to 62 +/- 13 micrograms of cholesterol/mg of protein). There was an approximate exchange of cholesterol between HDL and the unenriched fibroblasts (e.g. at [HDL] = 100 micrograms/ml, Fe and Fi = 3.2 and 3.0 micrograms of cholesterol/[4 h.mg of cell protein], respectively). In contrast, there was substantial net efflux from the enriched cells (at [HDL] = 100 micrograms/ml, Fe and Fi = 5.5 and 3.1 micrograms of cholesterol/[4 h.mg of cell protein], respectively). The rate constants for cholesterol flux were not influenced by changing the free cholesterol content of HDL, so that there was net efflux of cell cholesterol in the presence of cholesterol-depleted HDL and net influx from cholesterol-rich HDL. The Kd of HDL binding to fibroblasts was reduced from 1.7 to 0.9 micrograms/ml by the enrichment of the cells with free cholesterol; this increase in affinity for HDL was not reflected in enhanced rate constants for cholesterol flux. The inhibition of specific HDL binding by treatment of the lipoprotein with dimethyl suberimidate did not affect cholesterol flux using either control or cholesterol-rich cells at any HDL concentration in the range 1-1000 micrograms/ml. The above results are consistent with the concept that net movement of free cholesterol between cells and HDL occurs by passive, mass-action effects.(ABSTRACT TRUNCATED AT 400 WORDS)

Accumulation and mobilization of cholesteryl esters in cultured human fibroblasts exposed to free cholesterol-rich phospholipid vesicles

The uptake of free cholesterol (FC) into cells by surface transfer and its esterification to cholesteryl esters (CE) has been studied with a system of FC-egg phosphatidylcholine (EPC) vesicles and human lung fibroblasts in serum-free growth medium. The influx of FC was dependent on the molar ratio of FC to EPC in the donor vesicles. The FC incorporated by surface transfer was available for esterification in the cells. Incubations with FC/EPC vesicles with a FC/EPC molar ratio of 0.5:1 gave a small net decrease in cellular CE, while 1:1 vesicles gave a mild increase. When the cells were incubated with 2:1 FC/EPC vesicles an extensive accumulation of CE was demonstrated, which was accentuated if albumin was present in the medium. The CE accumulated in the form of lipid droplets within the cells. The largest of these droplets exhibited positive birefringence with formée crosses, that is typical for liquid crystals of cholesteryl esters. If cells loaded with CE were incubated with vesicles with low FC/EPC ratios a net efflux of CE was noted. The present study demonstrates that the uptake of FC from lipid vesicles by surface transfer can reproduce typical features of foam cells in early atherosclerosis.

In vivo influx of free and esterified plasma cholesterol into human aortic tissue without atherosclerotic lesions.

In order to determine the in vivo influx of plasma cholesterol into human aortic intimamedia tissue, specimens of the ascending aortic wall without visible atherosclerosis were obtained from patients undergoing aortic valve replacement. Before the operation the patients were intravenously injected with autologous plasma in which the lipoproteins were labeled with radioactive cholesterol. The influence of the duration of the exposure time (0.3-114 h) and of the distribution of radioactivity between free and esterified cholesterol in plasma on the amount of radioactivity found in the arterial wall was studied by the simultaneous use of 3H- and 14C-cholesterol. It was shown that the influx of free and esterified cholesterol into the intima-media layer of the tissue could be calculated from a set of linear equations that relate the labeled sterols in the tissue to the average specific activities in plasma. In nine patients between 50 and 70 yr of age with 4.2-5.9 mM total cholesterol in plasma, the influx of free cholesterol and of esterified cholesterol was 1.2-8.8 and 1.0-12.5 nmol X cm-2 X d-1, respectively. Both hydrolysis and esterification of the sterol fractions in the aortic tissue and exchange of free cholesterol between the plasma lipoproteins and the tissue were demonstrated. The cholesterol content of the intima-media layer was 0.6-2.3 mumol X cm-2. This corresponds to the influx of esterified cholesterol during a period of only 0.1-3.5 yr, which is short compared with the lifespan of the patient. Our data thus suggest that removal of esterified cholesterol from aortic tissue without visible atherosclerosis represents a major importance for the cholesterol concentration in the tissue.

No effect of nifedipine on atherogenesis in cholesterol-fed rabbits.

It was recently reported that the calcium antagonist nifedipine suppresses aortic cholesterol accumulation in cholesterol-fed rabbits without reducing hypercholesterolemia. We extended this study on plasma lipoprotein levels and aortic influx of cholesteryl ester. We gave 40 mg per day of nifedipine orally to 17 rabbits fed a 2% cholesterol diet for 8 weeks. For the same period of time 15 control rabbits received placebo capsules and the same diet. During the study, total cholesterol, HDL, LDL, and VLDL cholesterol concentrations in plasma were not significantly different in the experimental and control animals. At the end of the study we found no difference in the two groups in accumulation of cholesterol in the intima media of the proximal thoracic aorta, the distal thoracic aorta, and the corresponding media layers. Furthermore, aortic influx of free cholesterol, cholesteryl ester, and albumin from plasma measured by radioactive tracers was not significantly affected by nifedipine.

The in vivo transfer of free and esterified cholesterol from plasma into the arterial wall of hypercholesterolemic rabbits

With a new technique based on the simultaneous use of 3H and 14C-cholesterol, the fluxes of free and esterified cholesterol into the thoracic aorta of the hypercholesterolemic rabbits have been measured. By additional use of 3H-leucine as tracer for apoproteins, it was shown that esterified cholesterol in plasma enters the arterial tissue mainly as part of a lipoprotein influx. In contrast, only between 20 and 90% of the influx of free cholesterol could be accounted for by an influx of plasma lipoproteins. The excess probably represents an exchange of free cholesterol between the plasma lipoproteins and the intimal surface of the artery. Some 5-40% of the esterified cholesterol which had entered the artery during a 3-6 hours period was hydrolyzed in the artery during that period, whereas only negligible amounts of newly-entered free cholesterol was esterified. The arterial influx of very low, low and high density lipoproteins or albumin was determined in the same animal. The influx of each fraction was expressed as an intimal clearance, i.e., the influx divided by its plasma concentration. The intimal clearances of these macromolecules decreased linearly with the logarithm of their diameters. This suggests that the arterial influx of plasma lipoproteins and of albumin proceed by the same mechanism, which involves a steric hindrance for these macromolecules in the influx pathway.

Interaction of free cholesterol and apoproteins of low and high density lipoproteins with isolated rabbit hepatocytes.

Primary cultures of rabbit hepatocytes were incubated with rabbit high density (HDL) and low density (LDL) lipoproteins in order to compare the surface transfer of free cholesterol with the uptake of apoproteins. Hepatocytes were maintained for various intervals with either LDL or HDL which contained both 125I-labeled protein and free [4-14C]cholesterol. After a 3-hr incubation with an LDL concentration equivalent to 25% of the normal rabbit serum level, the percentage of media 14C in hepatocytes was 2.3 times greater than the percentage of 125I; cells that had been incubated with HDL showed an eight-fold selectivity for 14C. Although the influx of free cholesterol from HDL was greater than that from LDL, there was no difference between the uptake of LDL protein and of HDL protein. The degradation of lipoproteins labeled with [3H]-leucine or 125I was compared. Hepatocytes incubated with lipoproteins labeled with [4-14C]cholesterol showed a greater influx of cholesterol from HDL2 than from LDL. The efflux of labeled cellular cholesterol was also greater to HDL2 than to LDL, whether the cellular cholesterol was labeled by prior exchange with labeled HDL2 or by endogenous synthesis of cholesterol from [2-3H]mevalonic acid lactone.

Arterial influx of esterified cholesterol from two plasma lipoprotein fractions and its hydrolysis in vtvo in hype rcholesterolemic rabbits

Arterial influx of esterified cholesterol from 2 plasma lipoprotein fractions, d < 1.019 and d > 1.019, and influx of plasma free cholesterol were determined in each of 15 hypercholesterolemic rabbits with approximately the same plasma cholesterol concentrations but with different extents of arterial lesions. The procedure consisted of injecting intravenously into recipient rabbits [ 14C]- or [ 3H]cholesterol-labeled lipoproteins prepared from donor rabbits. The esterified cholesterol of one lipoprotein fraction was labeled primarily with one isotope and that of the other lipoprotein fraction was labeled with the other isotope. Thoracic aortas were removed 4–6 h after lipoprotein injections. The arterial influx of esterified cholesterol was up to 50 times higher in rabbits with maximal lesions than in those with minimal cholesterol deposits. The arterial influx of cholesteryl ester derived from d < 1.019 lipoproteins was about equal to that derived from the d > 1.019 fraction. The amount of cholesteryl ester in plasma d < 1.019 was approximately 3 times that in d > 1.019. Thus, per unit of cholesteryl ester concentration the d < 1.019 lipoproteins delivered about 1 3 as much cholesteryl ester to the artery as the lipoproteins in the higher density fractions. Some 5–40% of plasma esterified cholesterol which had entered the artery was hydrolyzed in the artery during the experimental period. The influx of free cholesterol that could not be accounted for by the influx of intact plasma lipoproteins was 5–80% of the free cholesterol influx. This excess probably represents free cholesterol influx by an exchange between the plasma lipoproteins and the intimal surface of the artery.

In vivo influx, tissue esterification and hydrolysis of free and esterified plasma cholesterol in the cholesterol-fed rabbit

The influx of free and esterified cholesterol into various tissues of cholesterol-fed rabbits is calculated from the tissue [ 3H] cholesterol and [ 14C]cholesterol content—corrected for radioactivity in contaminating plasma—after a 3–6 h exposure to in vivo-labeled plasma. The plasma free cholesterol was labeled primarily with 3H and the esterified cholesterol with 14C or vice versa. The influx calculation is based on total 3H and 14C in tissues and two linear equations that take into account esterification and hydrolysis of sterol fractions by the tissues. The influx of esterified cholesterol into tissue samples from aorta, heart, small intestine and lung was 10–80 nmol · g −1 · h −1, whereas the influx into adrenal, spleen and liver was from 400–2500 nmol · g −1 · h −1. The influx of free cholesterol was considerably higher than expected if free and esterified cholesterol had entered the tissues together as part of plasma lipoproteins. This excess of free cholesterol influx can be ascribed to cholesterol exchange between plasma lipoproteins and tissues, which in several tissues amounted to more than 80% of the total free cholesterol influx. From tissue free and esterified cholesterol radioactivity, one can calculate that 20–70% of the newly entered esterified cholesterol was hydrolyzed by various tissues and that most tissues esterified less than 10% of newly entered cholesterol during the experimental period. However, esterification of plasma cholesterol by adrenals averaged 50% of that taken up during a 3–6 h period.

The bi-directional transfer of cholesterol in normal aorta, fatty streaks, and atheromatous plaques.

SummaryIsotopic cholesterol was administered per os for 30 days to cholesterol-fed White Carneau pigeons. At weekly intervals and for 100 days after cessation of isotope administration, subgroups were killed and their aortas were divided into normal tissue, fatty streaks, and atherosclerotic plaques. Cryostat sections from each plaque were examined for morphologic features, and from the different areas of the aorta cholesterol concentration and radioactivity were determined. From the values obtained, rates of influx and efflux of plasma cholesterol were calculated. The results indicated that in normal aorta the influx of free cholesterol greatly exceeds that of cholesteryl esters. There appeared to be no net positive cholesterol balance. In fatty streaks, the influx of both free and esterified cholesterol was greater than for normal tissue, and influx rates exceeded those for efflux. In plaques, still greater rates of influx and efflux were seen, and the influx of cholesteryl esters frequently exceeded th...

Effect of estradiol benzoate on lipid metabolism in the rat.

The effect of estradiol benzoate on the concentration of lipids in the plasma, liver and heart and on the incorporation of acetate 1-14C into lipids in these tissues has been studied in adult male rats. Following the daily injection of 1.7 mg of estradiol benzoate for 5 days, estrogen- treated and control animals were injected with 14 μc of sodium acetate 1-14C and killed 2, 4, 12, 24, 48 and 60 hr after the injection. There was a marked decrease in plasma free and esterified cholesterol, plasma phospholipids and the plasma cholesterol/phospholipid (C/P) ratio. The accompanying increase in liver cholesterol ester suggests that estradiol benzoate causes the transfer of cholesterol ester from the plasma to the liver. The increased turnover rate of plasma free cholesterol but lack of increase in liver free cholesterol may be due to an influx of free cholesterol from the plasma to the liver, the effect of which is balanced by a decreased rate of hepatic free cholesterol synthesis. There was a decreased incorporation of acetate 1-14C into liver cholesterolcholesterol ester and free cholesterol. In the plasma cholesterol ester fractions of the estrogen-treated animals there was an increase in the percentage of cholesterol oleate. The decrease in plasma phospholipids was not reflected by significant changes in corresponding liver or myocardial tissue fractions. Estradiol benzoate had no apparent effect on the concentration of tri- and diglycerides in the tissues examined but decreased the plasma monoglyceride and the liver free fatty acid (FFA) concentrations. The incorporation of acetate 1-14C into the plasma FFA was decreased in the estrogen-treated animals. There was also a decreased incorporation of the acetate 1-14C into the plasma triglycerides and the FFA liver tri-, di- and monoglycerides and phospholipids. The specific activity values for myocardial tissue were too low to determine whether estradiol benzoate might have any effect on the lipids in this tissue. There was no apparent effect on myocardial lipid concentrations. (Endocrinology80: 263, 1967)