Véronique Atger

A cell culture system for screening human serum for ability to promote cellular cholesterol efflux. Relations between serum components and efflux, esterification, and transfer.

A cell culture system was employed to test a large number of samples of human serum for the ability to stimulate the efflux of cell cholesterol. The extent of efflux obtained with each specimen was correlated with the serum concentrations of cholesterol, triglycerides, apoprotein (apo) B, apo A-I, apo A-II, and lipoprotein subfractions (ie, high-density lipoprotein2 [HDL2], HDL3, lipoprotein [Lp] A-I, and LpA-I:A-II). In addition, the subsequent esterification of the released cholesterol and the distribution of the synthesized exogenous cholesteryl esters between HDL and low-density lipoprotein/very-low-density lipoprotein provided estimates of the lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities of each serum. The values for these activities were analyzed for correlations with cell efflux and the various serum parameters. Cell cholesterol efflux best correlated with serum total HDL cholesterol values. HDL2 and HDL3 correlated about equally well with efflux, whereas LpA-I demonstrated a much greater association with efflux than did LpA-I:A-II. Analysis of the data by partial correlation analysis indicated that HDL3 and LpA-I were the HDL subfractions most closely associated with efflux. Esterification of the released radiolabeled cholesterol was strongly and positively correlated with serum triglyceride concentrations and negatively related to the serum concentrations of HDL2. There was no relation between esterification values, which reflect LCAT activity, and efflux. The transfer of the labeled cholesteryl esters between HDL and apoB-containing lipoproteins was used as a measure of CETP activity and demonstrated a pattern in which all apoB-related parameters were positively correlated to transfer of esterified cholesterol, and all HDL associated parameters, particularly HDL3, were negatively related to transfer. No relations were observed between efflux, esterification, and transfer.

HDL Phospholipid Content and Composition as a Major Factor Determining Cholesterol Efflux Capacity From Fu5AH Cells to Human Serum

The relationships of cell cholesterol efflux to HDL phospholipid (PL) content and composition in human serum were analyzed in two groups of subjects selected on the basis of their HDL cholesterol (HDL-C) levels: a norm-HDL group (1.10 mmol/L < HDL-C < 1.50 mmol/L) and a high-HDL group (HDL-C > 1.75 mmol/L). In the high-HDL group, the relative fractional efflux was significantly higher than in the norm-HDL group, and in both groups, fractional efflux was correlated with a number of lipoprotein parameters, the best correlation and the only one that remained significant after multivariate analysis being with HDL phospholipid (HDL-PL). Analysis of the HDL-PL subclasses revealed that HDL in the high-HDL sera was enriched with phosphatidylethanolamine (HDL-PE) and relatively deficient in sphingomyelin (HDL-SM) compared with norm-HDL sera. Moreover, the fractional efflux values in the high-HDL group were negatively correlated with the proportion of HDL-PE (r = -.64, P < .0001) and positively correlated with the proportion of HDL-SM (r = .43, P < .01). Thus, this study provides evidence that HDL-PL concentration can be used to predict the capacity of serum to accept cellular cholesterol. Among the differences described between norm-HDL and high-HDL sera, the variability in PE to SM ratio might reflect changes in serum cholesterol acceptors that modulate the first step of reverse cholesterol transport.

Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells.

Low concentrations of cyclodextrins (< 1.0 mM) added to serum act catalytically, accelerating the exchange of cholesterol between cells and lipoproteins. J774 macrophages incubated with serum and 2-hydroxypropyl-beta-cyclodextrin (< or = 1 mM) released fivefold more labeled cholesterol than with serum alone. Increased efflux was not accompanied by a change in cell cholesterol mass; thus, cyclodextrin functioned as a cholesterol shuttle, enhancing cholesterol bidirectional flux without changing the equilibrium cholesterol distribution between cells and medium. The addition of phospholipid vesicles to serum and cyclodextrin shifted the equilibrium distribution to favor the medium, producing rapid and extensive depletion of cell cholesterol mass. The combination of serum, phospholipid vesicles, and cyclodextrin also stimulated the rapid clearance of both free and esterified cholesterol from mouse peritoneal macrophages loaded with free and esterified cholesterol. This study: (a) demonstrates that a compound can function as a catalyst to enhance the movement of cholesterol between cells and serum, (b) illustrates the difference between cholesterol exchange and net transport in a cell/serum system, (c) demonstrates how net movement of cholesterol is linked to concentration gradients established by phospholipids, (d) provides a basis for the development of the shuttle/sink model for the first steps in reverse cholesterol transport, (e) validates the model using artificial shuttles (cyclodextrins) and sinks (large unilamellar vesicles), and (f) suggests that cyclodextrin-like cholesterol shuttles might be of pharmacological significance in treating unstable atherosclerotic plaques.

Serum Lp(a) as a discriminant marker of early atherosclerotic plaque at three extracoronary sites in hypercholesterolemic men. The PCVMETRA Group.

To investigate the role of lipoprotein (a) (Lp[a]) as an atherogenic condition related to hypercholesterolemia, we studied the serum concentration of Lp(a) as measured by immunonephelometry in relation to the presence of asymptomatic echographic plaques in the peripheral arteries of 103 untreated hypercholesterolemic, normotensive, middle-aged men. Plaque was found at carotid, aortic, and femoral sites in 36%, 51%, and 53% of subjects, respectively. The Lp(a) level was higher in the group with carotid plaques than in the group without (0.29 +/- 0.20 versus 0.17 +/- 0.14 g/l, p < 0.01), not significantly higher in the group with aortics plaque than in the group without (0.24 +/- 0.19 versus 0.19 +/- 0.16 g/l), and not different between groups with and without femoral plaques (0.21 +/- 0.18 versus 0.22 +/- 0.17 g/l). A logistic regression analysis confirmed that Lp(a) was associated with carotid plaques (p = 0.004), independent of other risk factors. However, in patients with low density lipoprotein cholesterol values above the group median value (4.7 mmol/l), Lp(a) was associated not only with carotid plaques (p < 0.01) but also with aortic plaques (p < 0.05), as well as with the number of diseased sites (p = 0.02). In contrast, in patients with low density lipoprotein cholesterol levels below or equal to 4.7 mmol/l, Lp(a) only remained associated with carotid plaques (p < 0.05). Thus, in symptom-free, hypercholesterolemic men, early atherosclerosis was influenced by serum Lp(a), particularly in the carotid arteries, as well as by the presence of a higher level of low density lipoprotein cholesterol.

Human ApoA-IV Overexpression in Transgenic Mice Induces cAMP-Stimulated Cholesterol Efflux From J774 Macrophages to Whole Serum

The role of apolipoprotein A-IV (apoA-IV) in lipoprotein metabolism has not been established. The aim of the present study was to investigate the role of apoA-IV in reverse cholesterol transport by comparing cellular cholesterol efflux to serum or serum fractions from control mice and from mice transgenic for human apoA-IV (HuA-IVTg mice). When Fu5AH hepatoma cells were used, the cholesterol efflux to serum from either control or transgenic mice was similar. When control J774 macrophage cells were used, a comparison of efflux to serum or lipoprotein-deficient serum (LPDS) failed to demonstrate any differences between control and transgenic mice. In contrast, when the J774 cells were pretreated with cAMP, there was a stimulation of efflux to whole serum or LPDS from HuA-IVTg mice. cAMP treatment had no effect on efflux to serum or LPDS from control mice. Pretreatment of the cells with cAMP did not enhance the efflux response to high density lipoprotein isolated from HuA-IVTg mouse serum. Our results suggest that apoA-IV, unassociated with high density lipoprotein particles, is responsible for enhanced cholesterol efflux. This study illustrates the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux with use of a biological fluid and is potentially of physiological relevance, especially in apolipoprotein-rich extravascular fluids.

Analysis of the relationship between triglyceridemia and HDL-phospholipid concentrations: consequences on the efflux capacity of serum in the Fu5AH system.

The high triglyceride/low HDL-cholesterol trait is a common finding in the general population. The aim of the present study was to analyze and interpret the relationships between triglycerides (TG), HDL-related parameters and serum cholesterol efflux potential in an asymptomatic population including both normo- and hyperlipidemic individuals. In a large sample (n = 1143) of this population, there was a negative correlation between TG and HDL-cholesterol (HDL-C) (r = -0.49, P<0.0001) whereas the negative correlation between TG and HDL-phospholipid (HDL-PL) (r = -0.29, P<0.0001) was weaker, leading to a strong positive correlation between TG and HDL-PL/C ratio (r = 0.58, P<0.0001). Thus, increased TG concentrations were associated with an enrichment of HDL with PL. Since we have demonstrated previously that HDL-PL is the major determinant for cholesterol efflux potential from Fu5AH rat hepatoma cells, we determined the effect of the variations in HDL lipid composition on the cholesterol efflux capacity in a subsample of 198 subjects. Compared with normolipidemic subjects (NLP) (TG< or = 1.7 mmol/l; LDL-C< or = 4.1 mmol/l, n=58), hypertriglyceridemic subjects (HTG) (TG>1.7 mmol/l, n=63) exhibited lower HDL-C levels (1.08+/-0.21 vs. 1.25+/-0.32, P=0.0003) whereas they showed similar HDL-PL concentrations (1.25+/-0.21 vs. 1.25+/-2.7) and, thus, higher HDL-PL/C ratio (1.17+/-0.15 vs. 1.02+/-0.14, P=0.0001). The relative efflux capacity of serum measured in the Fu5AH system (5% serum, 4 h incubation at 37 degrees C) was on average identical in the HTG and NLP groups. Thus, this study provides evidence that despite decreased HDL concentrations, as determined routinely by the HDL-C assay, some HTG subjects maintained serum cholesterol efflux capacity thanks to the enrichment of HDL with PL.

Structural and Functional Comparison of HDL From Homologous Human Plasma and Follicular Fluid: A Model for Extravascular Fluid

In the preovulatory period, follicular fluid contains only HDL. Biochemical characterization of such lipoproteins showed that follicular fluid HDLs were cholesterol-poor particles compared with serum HDLs, whereas the amount of phospholipids, expressed as percent weight, was significantly higher in follicular fluid HDLs (28.5%) than in serum HDLs (25.0%, P < .05). The amount of apolipoprotein (apo) A-IV per apo A-I was significantly higher in follicular fluid than in serum (0.77 versus 0.58 mg/g apo A-I, P < .02). To explore the role of HDLs as cholesterol acceptors in physiological media, we compared the ability of either whole human follicular fluids or homologous sera to promote cellular cholesterol efflux using Fu5AH rat hepatoma cells. At equivalent concentrations of HDL cholesterol in follicular fluid and in serum, t1/2 values for cholesterol efflux were in the same range. In addition, estimated maximal efflux values were not significantly different in follicular fluid and serum (45.9% and 49.6%, respectively), as were K(m) values (0.064 and 0.071 mmol/L HDL cholesterol respectively). In addition, isolated HDLs displayed the same capacity to promote cellular cholesterol efflux in both media. Thus, the kinetics and dose-response data between these two physiological media showed that HDLs play the major role in cellular cholesterol efflux. The rate of cholesterol esterification, as measured in the presence of cells, was significantly higher in follicular fluid than in serum at constant HDL cholesterol concentrations, whereas the rate of esterified cholesterol transfer toward added LDL was lower. In contrast, in a cell-free system, lecithin:cholesterol acyltransferase activity represented only 26% of that in serum HDL, whereas cholesterol ester transfer protein activities were comparable. In summary, in this particular model, we confirmed the essential role of HDLs as physiological acceptors in the removal of cellular cholesterol.

Cholesterol efflux potential of sera from mice expressing human cholesteryl ester transfer protein and/or human apolipoprotein AI.

The ability of whole serum to promote cell cholesterol efflux and the relationships between apoprotein and lipoprotein components of human serum efflux have been investigated previously (de la Llera Moya, M., V. Atger, J.L. Paul, N. Fournier, N. Moatti, P. Giral, K.E. Friday, and G.H. Rothblat. 1994. Arterioscler. Thromb. 14:1056-1065). We have now used this experimental system to study the selective effects of two human lipoprotein-related proteins, apoprotein AI (apo AI) and cholesteryl ester transfer protein (CETP) on cell cholesterol efflux, when these proteins are expressed in transgenic mice. The percent efflux values for cholesterol released in 4 h from Fu5AH donor cells to 5% sera from the different groups of mice were in the order: background = human apo AI transgenic (HuAITg) > human CETP transgenic (HuCETPTg) > human apo AI and CETP transgenic (HuAICETPTg) >> apo AI knockout mice. In each group of mice a strong, positive correlation (r2 ranging from 0.64 to 0.76) was found between efflux and HDL cholesterol concentrations. The slopes of these regression lines differed between groups of mice, indicating that the cholesterol acceptor efficiencies of the sera differed among groups. These differences in relative efficiencies can explain why cholesterol efflux was not proportional to the different HDL levels in the various groups of mice. We can conclude that: (a) HDL particles from HuAITg mice are less efficient as cholesterol acceptors than HDL from the background mice; (b) despite a lower average efflux due to lower HDL cholesterol concentrations, HDL particles are more efficient in the HuCETPTg mice than in the background mice; and (c) the coexpression of both human apo AI and CETP improves the efficiency of HDL particles in the HuAICETPTg mice when compared with the HuAITg mice. We also demonstrated that the esterification of the free cholesterol released from the cells by lecithin cholesterol acyltransferase in the serum was reduced in the HuAITg and AI knockout mice, whereas it was not different from background values in the two groups of mice expressing human CETP.

Erythrocyte antioxidant status in asymptomatic hypercholesterolemic men.

An imbalance between antioxidant and oxidant-generating systems leading to an oxidative stress has already been proposed in the pathogenesis of atherosclerosis. In the present study we investigated the antioxidant status in 60 asymptomatic hypercholesterolemic (HC) men compared with 48 normocholesterolemic (NC) men. Hypercholesterolemic subjects had a significantly lower red blood cell vitamin E (vit E-RBC) content in spite of their normal total plasma and HDL vitamin E concentrations. Activities of erythrocyte superoxide dismutase and glutathione peroxidase were not significantly different between groups. We also determined the resistance of RBCs to an oxidative stress by determining the extent of hemolysis induced by a water-soluble azo-compound. This resistance was significantly decreased in HC men compared with NC subjects. These results demonstrate an altered antioxidant status of RBC in asymptomatic HC men associated with an increased erythrocyte susceptibility to an oxidative stress. The measure of the vitamin E content in RBC might be the most sensitive parameter for evidencing early oxidative stress which does not need an adaptation of enzymatic protective systems.

Influence of HDL subfractions on erythrocyte aggregation in hypercholesterolemic men. PCVMETRA Group.

Recent studies have suggested that rheological mechanisms may be involved in the pathogenesis of ischemic syndromes in hyperlipidemias. We investigated the association between erythrocyte aggregation and components of lipoproteins in the blood of 60 normotensive, hypercholesterolemic men aged 45 +/- 8 years. The rheological parameters assessed were aggregation index (AI) and disaggregation shear rate threshold (gamma t) as determined by laser reflectometry, plasma fibrinogen, total serum protein, and hematocrit. The lipoprotein variables included total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol and its subfractions HDL2 cholesterol and HDL3 cholesterol, apolipoprotein (apo) B, apoA-I, HDL particles containing apoA-I without apoA-II (LpA-I), and HDL particles containing both apoA-I and apoA-II (LpA-I/A-II). Covariables considered for possible confounding effects were age, body mass index, and smoking behavior. Fibrinogen, total serum protein, and both aggregation parameters (AI and gamma t) were elevated in this hypercholesterolemic population. Univariate analysis showed that both AI and gamma t correlated positively with fibrinogen (P < .001) and total serum protein (P < .01) and negatively with HDL2 cholesterol (P < .01) and LpA-I (P < .01); gamma t also provided a positive correlation with LpA-I/A-II (P < .05). A multivariate model analysis demonstrated that HDL2 cholesterol, LpA-I, and LpA-I/A-II also emerged as significant factors influencing erythrocyte aggregation; 60% to 68% of the variance of AI and 47% to 64% of the variance of gamma t could be explained by these factors.(ABSTRACT TRUNCATED AT 250 WORDS)