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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1283-1292

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1283.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

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

Natalie Fournier; Véronique Atger; Jean-Louis Paul; Marie Sturm; Nicolas Duverger; George H. Rothblat; Nicole Moatti

From the Laboratoire de Biochimie Appliquée (N.F., J.-L.P., M.S., N.M.), Faculté des Sciences Pharmaceutiques et Biologiques, Châtenay-Malabry, France; the Laboratoire de Biochimie (N.F., V.A., J.-L.P., N.M.), Hôpital Broussais, Assistance Publique-Hôpitaux de Paris, Paris, France; the Laboratoire Rhône-Poulenc-Rorer (N.D.), Division Gencell, Département d’Athérosclérose, Vitry sur Seine, France; and the MCP Hahnemann School of Medicine (G.H.R.), Biochemistry Department, Philadelphia, Pa.

Correspondence to Dr N. Fournier, Laboratoire de Biochimie, Hôpital Broussais, 96 rue Didot, F-75674 Paris Cedex 14, France. E-mail natalie.fournier{at}brs.ap-hop-paris.fr


*    Abstract
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Abstract—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.


Key Words: free apolipoprotein A-IV • J774 mouse macrophages • cAMP • cholesterol efflux • transgenic mice


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Human apoA-IV is 46-kDa protein that is synthesized primarily in the intestine1 2 and is present in plasma,3 interstitial fluid,4 5 and peripheral lymph.6 The distribution of apoA-IV in plasma has been difficult to characterize because of its relatively low concentration and because of its weak lipid-binding affinity.7 After plasma ultracentrifugation, apoA-IV is mainly found in the lipoprotein-free fraction, whereas when plasma is fractionated by nondenaturing gel electrophoresis or by gel filtration, apoA-IV is primarily associated with the HDL fraction.8 Moreover, the analysis of human serum by affinity chromatography or by 2D gel electrophoresis has shown that only a small fraction of apoA-IV is associated with apoA-I and that most of the protein forms heterogeneous particles designated LpA-IV.5 9 From these observations, it can be deduced that apoA-IV is easily displaced from lipoprotein particles on analytical or metabolic conditions, and it has been proposed that unassociated apoA-IV has the ability to pass into the extracellular space to exert its metabolic function. The physiological role of apoA-IV is not completely understood. There is growing evidence that apoA-IV plays a role in fat and fat-soluble vitamin absorption10 and could be involved in the short-term and long-term regulations of food intake and in upper gastrointestinal function modulation.11 In addition, 1 of the main metabolic functions that has been assigned to apoA-IV is related to reverse cholesterol transport (RCT). ApoA-IV is a potent activator of lecithin-cholesterol acyltransferase12 and can promote cellular cholesterol efflux. It has been demonstrated that either apoA-IV recombined with phosphatidylcholine in a liposome or serum LpA-IV is an efficient cholesterol acceptor from fibroblasts9 13 or from mouse adipose cells.5 14 With regard to these functions, apoA-IV has been proposed as a "surrogate" for apoA-I–containing particles in promoting RCT in conditions characterized by a low plasma apoA-I level, such as Tangier disease or lecithin-cholesterol acyltransferase deficiency.15 The contribution of apoA-IV to RCT in these conditions could be part of the explanation for the reduced prevalence of atherosclerosis in these patients.

The involvement of apoA-IV in preventing atherosclerosis has been recently emphasized by studies using human apoA-IV transgenic mice (HuA-IVTg mice), which (compared with control mice) exhibited a significant reduction of arterial lesions under atherogenic conditions.16 Because it has been suggested that this effect could have been the result of an increase in RCT, the aim of the present study was to evaluate the capacity of serum from the previously described HuA-IVTg mice to stimulate the efflux of cholesterol from cells, taking into account the fact that apoA-IV is partly associated with HDL but is mainly recovered in a form in which it is unassociated with lipoproteins. The efflux capacity of serum was tested first in the Fu5AH rat hepatoma cell system, in which the concentration of HDL phospholipids was demonstrated to be the main determinant for efflux (for a review see Rothblat et al17 ).18 19 20 The efflux capacity of serum was also measured with the use of J774 mouse macrophages under conditions in which preincubation of the cells with cAMP enhances the release of cholesterol mediated by lipid-free apolipoproteins.21 The stimulating effect of cAMP on cholesterol efflux has been previously demonstrated with the use of purified lipid-free apoA-I and apoE.21 22 Thus, in the present study, we addressed (1) the issue of the response of apoA-IV in a cAMP-stimulated macrophages system and (2) the issue of the response of lipid-free apolipoprotein in a biologically complex environment with the use of whole serum.


*    Methods
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Materials
Tissue culture medium RPMI was obtained from GIBCO-BRL (Life Sciences). Tissue culture medium MEM, L-glutamine, and penicillin-streptomycin were purchased from BioWhittaker. Heat-inactivated FBS and newborn calf serum (CS) were from Boehringer-Mannheim. Tissue culture flasks and plates were from Falcon (Polylabo). The radiolabeled [1,2-3H]cholesterol was provided by Amersham. BSA (essentially fatty acid free), 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), EDTA, and guanidine hydrochloride were purchased from Sigma. The inhibitor of acyl coenzyme A:cholesterol acyl transferase (ACAT), GW 447C88, was a gift from Wellcome Pharmaceuticals (Les Ulis, France). Enzymatic kits for total cholesterol (TC) and triglyceride (TG) determinations were from Merck. The phospholipid enzymatic colorimetric procedure was provided by Biomérieux.

Control and HuA-IVTg Mice
We used HuA-IVTg mice, the heterozygous transgenic mice expressing human apoA-IV originally described by Duverger et al.16 The transgenic animals were 13- to 16-week-old males or females expressing human apoA-IV in the liver. C57/BL6 wild-type mice of similar age were considered as controls. Purina laboratory mouse chow diet contained <0.03% (wt/wt) cholesterol, 4.5% (wt/wt) animal fat, and no casein or sodium cholate. Analysis were performed after 6 weeks of the regular diet. Blood was collected from the retro-orbital plexus of animals that had been deprived of food for 4 hours. Sera were then collected by following the standardized procedures that have been described for human serum23 and were immediately frozen at -70°C until use.

Serum Lipoprotein Characterization
TC, TG, and phospholipid levels were determined by using commercial enzymatic kits. HDL cholesterol and phospholipid concentrations were measured by using the kits mentioned above after precipitation of apoB-containing lipoproteins by a dextran sulfate/MgCl2 reagent in pooled sera as described elsewhere.24 Non-HDL cholesterol was calculated by subtraction of HDL cholesterol from TC concentrations. Human apoA-IV was determined by Laurell electroimmunoassay performed in hydrated agarose gels containing rabbit polyclonal antibodies.16

Human Lipid-Free ApoA-I, Human Lipid-Free ApoA-IV, Isolated HDL, and Lipoprotein-Deficient Serum From Mouse Sera
Pure human apoA-I was isolated as previously described,25 solubilized in a 6 mol/L guanidine hydrochloride solution at a concentration of {approx}3 mg/mL, and then extensively dialyzed against 10 mmol/L Tris/HCl-buffered saline (0.15 mol/L NaCl) supplemented with 0.25 mmol/L EDTA. Pure human apoA-IV was a gift from P. Tso (Department of Pathology, University of Cincinnati, Cincinnati, Ohio); it was isolated as previously described26 and handled in the same manner as apoA-I. The apolipoproteins were added to the culture medium at the indicated concentrations.

HDL and lipoprotein-deficient serum (LPDS) from mouse sera were obtained by sequential ultracentrifugation. HDL was isolated at a density (d) between 1.063 and 1.210 g/mL, and LPDS corresponded to the d>1.210 g/mL fraction.

Fu5AH Rat Hepatoma Cell Cholesterol Efflux
The capacity of serum to promote cholesterol efflux from Fu5AH cells in culture was assayed as described previously.23 Briefly, stock cultures of Fu5AH cells were grown in Eagle’s MEM supplemented with 5% CS, buffered with bicarbonate, 1% L-glutamine, and 0.75% penicillin-streptomycin. For experiments, 80 000 cells were seeded in 22-mm wells in 12-well multiwell plates and grown in MEM/5% CS for 3 days. Radiolabeling was then achieved by the addition of 2 µCi per well of [3H]cholesterol in ethanol to medium containing 5% CS and incubation of the cells for 36 hours to obtain confluent monolayers. After an additional 18-hour equilibration period with 0.5% BSA, serum samples were incubated with the labeled cells for 4 hours at 37°C. At the end of the 4-hour incubation, the efflux media were collected and centrifuged at 1000 rpm to remove floating cells. The labeled cell cholesterol released was measured in an aliquot of the medium by liquid scintillation counting. Fractional efflux values were calculated by dividing the amount of cholesterol released to the medium by the total amount of labeled cholesterol in the cells at time zero.

J774 Cell Culture, Cholesterol Labeling, and Efflux Determinations
Cholesterol efflux determinations that used J774 cells as cholesterol donors were performed by use of a modification of the general technique described by Sakr et al.22 Stock cultures of the J774 mouse macrophage cell line were grown in RPMI 1640 supplemented with 10% FBS, buffered with bicarbonate, and containing 1% L-glutamine and 0.75% penicillin-streptomycin. For experiments, cells in RPMI containing 10% FBS were plated in 22-mm multiwell plates (300 000 cells per well). Three days after plating, cellular cholesterol was labeled by exposing the cells for 20 hours to 2 µCi per well of [3H]cholesterol added in ethanol to the culture media, which contained 2.5% FBS. The final ethanol concentration was 0.1%. To allow equilibration of the label among the various pools of cell cholesterol, the monolayers were rinsed 2 times with RPMI after the labeling phase and incubated for an additional 12 hours in FBS-free culture medium containing 0.2% BSA. To ensure that all of the radiolabeled cholesterol was present as free cholesterol, the ACAT inhibitor 447C88 compound (2 µg per well, Wellcome) was added to the medium during the labeling period and all subsequent stages of the experiment. After this equilibration period, cells were washed 2 times with RPMI and then incubated for 10 to 12 hours in FBS-free culture medium with or without 0.2 mmol/L CPT-cAMP. CPT-cAMP was dissolved in distilled water at 30 mmol/L and stored at -20°C. At the end of the pretreatment period with cAMP, cells were washed 2 times with RPMI and then incubated for 2 hours with 1 mL of medium containing the test samples. At the end of this experimental period, aliquots of medium were centrifuged at 1000 rpm to remove floating cells and counted in a liquid scintillation counter (Packard). Parallel monolayers of control or treated cells were also incubated with cholesterol acceptor–free medium to measure background (non–acceptor-mediated) cholesterol efflux. Before the start of the cholesterol efflux assay, triplicate wells of control or treated cells were washed 2 times with RPMI, cellular lipids were extracted with isopropanol, and cell radioactivity at time zero was measured by liquid scintillation counting. The percent cholesterol efflux was calculated by dividing the amount of radioactivity released into the medium by the total radioactivity in the cells at time zero. The background cholesterol efflux obtained in the absence of any acceptor was subtracted from the efflux values obtained with the test samples. The percent stimulation of efflux produced by cAMP pretreatment of the J774 cells was calculated as follows: [(fractional cholesterol efflux from cAMP pretreated J774 cells-fractional cholesterol efflux from control J774 cells)/fractional cholesterol efflux from control J774 cells]x100.

Data Analysis
All data are presented as mean±SD. The statistical analysis was performed on an Apple Macintosh computer with the use of Statview software (Abacus Concepts). The nonparametric Mann-Whitney U test was used to determine statistical differences at the P<0.05 level of significance.


*    Results
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Characterization of Control and ApoA-IV Transgenic Mouse Sera
The lipoprotein profiles of the individual serum specimens from the mice used for the efflux experiments exhibited a high degree of homogeneity among the samples within the control and the HuA-IVTg groups. Therefore, efflux experiments have been performed with the use of different pools of serum from either transgenic or control animals. Five individual specimens were used in each pool.

As shown on Table 1Down, the concentration of human apoA-IV in the transgenic mice was, on average, 90 times higher than in the human serum.8 This overexpression of human apoA-IV had no significant impact on the serum lipid profiles of the transgenic animals because TG, TC, HDL cholesterol, HDL phospholipid, and non-HDL cholesterol were similar in the 2 groups of mice.


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  		Table 1. Serum Lipis, Lipoproteins, and Human ApoA-IV Concentrations in Control and HuAIVTg Mice Sera

Efflux of Cholesterol From Fu5AH Rat Hepatoma Cells to Mouse Serum
Using the Fu5AH rat hepatoma cells as cholesterol donors and the experimental protocols previously used to assess the efflux capacity of human and mouse sera,23 27 we assayed the pools of serum from either transgenic or control animals for their ability to promote the efflux of radiolabeled cell cholesterol. The mean 4-hour percent efflux values were not significantly different between the control and the HuA-IVTg groups (30.0±1.1% versus 27.3±1.7%, respectively).

Effect of cAMP on Efflux of Cholesterol From J774 Cells to Mouse Serum
Cholesterol efflux from J774 cells mediated by the serum from control and transgenic mice was first examined in a dose-response experiment with concentrations of serum ranging from 0.1% to 5%. Figure 1ADown shows that when serum was incubated with J774 cells that had not been pretreated with cAMP, the dose-response curves obtained with the control and the transgenic sera were similar. Preincubation of the cells with cAMP did not change the efflux response to the serum of control mice. However, we observed that cAMP treatment of the J774 cells produced an increase in the efflux to the serum from HuA-IVTg mice. Figure 1BDown illustrates the percent stimulation of cholesterol efflux observed with the HuA-IVTg serum, compared with control serum, on treatment of cells with cAMP. The apparent stimulation of efflux produced by cAMP treatment was inversely proportional to the concentration of HuA-IVTg serum added to the culture medium. Thus, the maximum cAMP-mediated stimulation of efflux with use of the serum from the HuA-IVTg mice, {approx}80%, was obtained with the most dilute serum, which in this experiment was 0.1% serum.



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  		Figure 1. Effect of cAMP pretreatment of J774 mouse macrophages on the dose-response relation of cholesterol efflux to control and transgenic mouse serum. A, Dose-response relation of cholesterol fractional efflux from cells incubated with (solid and stippled symbols with broken lines) or without (open symbols with solid lines) 0.2 mmol/L cAMP to serum of control (Ct, squares) or HuA-IVTg (AIV, circles) mice. B, Dose-response curve of percent cAMP stimulation of efflux to serum of Ct (square) or AIV (circle) mice. Increasing doses were determined on the basis of serum dilutions from 0.1% up to 5%. J774 cells were labeled with 2 µCi per well [3H]cholesterol in the presence of 2 µg per well ACAT inhibitor, equilibrated with BSA, and then incubated for 10 to 12 hours in FBS-free culture medium with or without 0.2 mmol/L cAMP as indicated in Methods. At the end of the pretreatment period, cells were washed and then incubated for 2 hours with 1 mL of medium containing the test samples. Percent cholesterol efflux was calculated by dividing amount of radioactivity released into medium by total radioactivity in cells at time zero. Background cholesterol efflux obtained in the absence of any acceptor was subtracted from the efflux values obtained with the test samples. Each fractional cholesterol efflux value is the mean of triplicate wells. Results are expressed as mean±SD from 6 experiments. Error bars not shown are within the symbols. *P<0.05 and **P<0.01 with respect to control group (nonparametric Mann-Whitney U test).

Effect of cAMP on Efflux of Cholesterol From J774 Cells to Mouse LPDS
ApoA-IV in serum is known to be distributed among different lipoproteins, with HDL containing the largest fraction.8 In addition, because of its relatively low affinity for lipids, apoA-IV is also present in serum unassociated with any lipoprotein and can be recovered in the d>1.21 g/mL fraction of serum (LPDS).28 Because the very high overexpression of apoA-IV in the transgenic mice did not change the lipid concentrations in the serum, it was likely that a large fraction of apoA-IV in these sera was not associated with any lipoprotein. Thus, the efflux capacity of the LPDS from control and transgenic animals was tested with or without pretreatment of the J774 cells with cAMP. The dilutions of LPDS chosen for this experiment were selected according to the previous results, which showed that the maximum stimulation by cAMP was obtained at the lower concentrations of serum. Thus, the LPDS was tested at concentrations equivalent to 0.05% up to 0.4% serum.

Figure 2ADown shows that the dose-response curves for the LPDS obtained from control and transgenic sera gave similar efflux values when the donor J774 cells were not exposed to cAMP. In contrast, when the cells had been pretreated with cAMP, the LPDS obtained from the apoA-IV transgenic serum produced a marked increase in efflux compared with the LPDS obtained from control mouse sera. As illustrated in Figure 2BDown, the maximum stimulation of {approx}200% above the control LPDS was obtained at a concentration of LPDS equivalent to 0.2% serum. Interestingly, the stimulation of efflux produced by cAMP preincubation was greater with LPDS than with whole serum. This observation was further documented in a subsequent experiment in which whole serum and LPDS were obtained from the same pool of HuA-IVTg mouse serum. Serum- and LPDS-mediated efflux values were compared with and without pretreatment of the cells with cAMP on the basis of increasing concentrations of human apoA-IV. The apoA-IV concentrations chosen, ranging from 5 to 35 µg/mL, were selected according to the previous results and corresponded to serum concentrations ranging from 0.05% to 0.40%. Figure 3ADown shows that without preincubation of the cells with cAMP, the efflux induced by the serum was higher than the efflux produced by LPDS. Efflux response mediated by serum and LPDS were increased when the cells were pretreated with cAMP. However, in the presence of cAMP, the efflux to LPDS was greater than the cholesterol efflux obtained with the total serum. Thus, as illustrated in Figure 3BDown, the stimulation of efflux by cAMP was almost 4 times greater with the LPDS than with the whole serum.



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  		Figure 2. Effect of the cAMP pretreatment of J774 mouse macrophages on dose-response relation of cholesterol efflux to control and transgenic mouse LPDS. A, Dose-response relation of cholesterol fractional efflux from cells incubated with (solid and stippled symbols with broken lines) or without (open symbols with solid lines) 0.2 mmol/L cAMP to the LPDS of Ct (triangles) or AIV (diamonds) mice. B, Dose-response curve of percent cAMP stimulation of efflux to the LPDS of Ct (triangle) or AIV (diamond) mice. Increasing doses were determined on the basis of LPDS dilutions corresponding to serum concentrations from 0.05% up to 0.4%. Cholesterol efflux was determined as described in legend to Figure 1Up. Results are expressed as mean±SD from triplicate wells. Error bars not shown are within the symbols. **P<0.01 with respect to control group (nonparametric Mann-Whitney U test).



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  		Figure 3. Effect of cAMP pretreatment of J774 mouse macrophages on dose-response relation of cholesterol efflux to HuA-IVTg (human apo AIV) mouse serum or LPDS. A, Dose-response relation of cholesterol fractional efflux from cells incubated with (solid and stippled symbols with broken lines) or without (open symbols with solid lines) 0.2 mmol/L cAMP to the serum (circles) or LPDS (diamonds) of human apo AIV transgenic mice. B, Dose-response curve of percent cAMP stimulation of efflux to the serum (circle) or the LPDS (diamond) of human apo AIV transgenic mice. Increasing doses were determined on the basis of human apo AIV concentrations in the serum or LPDS ranging from 5 µg/mL up to 35 µg/mL, corresponding to equivalent serum concentrations ranging from 0.05% to 0.4%. Cholesterol efflux was determined as described in legend to Figure 1Up. Results are expressed as mean±SD from triplicate wells. Error bars not shown are within the symbols.

Effect of cAMP on Efflux of Cholesterol From J774 Cells to Isolated Mouse HDL
HDLs were isolated from a pool of 5 transgenic mice sera to determine the effect of cAMP stimulation on cholesterol efflux from J774 mediated by human apoA-IV. Human apoA-IV recovered in the HDL fraction represented 2.3% of the total concentration of apoA-IV in serum and accounted for 28% of the HDL protein composition.

When J774 cells were incubated with isolated HDLs from HuA-IVTg mice, the efflux response was proportional to the amount of HDL protein added (Figure 4Down). However, no stimulation of efflux occurred by preincubating the cells with cAMP. Thus, we can conclude that cAMP stimulation of the efflux observed with the serum of HuA-IVTg was not due to the contribution of HDL to efflux but rather to human apoA-IV that was not associated with lipoproteins.



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  		Figure 4. Effect of the cAMP pretreatment of J774 mouse macrophages on cholesterol efflux mediated by HDL isolated from the serum of HuA-IVTg mice. The cholesterol fractional efflux from cells incubated with (hatched bars) or without (open bars) 0.2 mmol/L cAMP to the isolated HDL was determined as described in legend to Figure 1Up. Prot indicates protein. Results are expressed as mean±SD from triplicate wells.

Comparison of Efflux-Stimulating Capacity of Human ApoA-I and ApoA-IV
The stimulatory effect of cAMP on efflux from macrophage cells, mediated by unassociated apolipoproteins, has been demonstrated by use of purified human apoA-I.21 22 Thus, we compared the efflux-stimulating capacity of LPDS from HuA-IVTg with purified human apoA-I in its lipid-free form or added to either the total serum or LPDS of control mice. In addition, we tested within the same experiment the effect of purified human apoA-IV in its lipid-free form or added to control mouse serum fractions. The efflux experiment with J774 cells was performed with the use of a 15 µg/mL concentration of human apoA-I or apoA-IV. Efflux results in each condition are illustrated in Table 2Down. The greatest stimulation was observed when purified apoA-IV or apoA-I was incubated alone with the cells. The lowest stimulation was observed when apoA-IV or apoA-I was added to the total serum of control mice. Intermediate levels of stimulation were obtained either by adding back apoA-IV or apoA-I to the LPDS from control mice or by using the LPDS from apoA-IV transgenic mice.


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  		Table 2. Effect of cAMP Pretreatment of J774 Mouse Macrophages on Cholesterol Efflux Mediated by LPDS From HuA-IVTg Mouse Serum Compared with Human Lipid-Free ApoA-IV or ApoA-I

When the absolute values for efflux without cAMP pretreatment were examined, we observed that the highest efflux values were obtained with total serum from control mice supplemented with free apoA-IV or apoA-I and that the lowest fractional efflux values were obtained with purified apoA-IV or apoA-I alone. Thus, the HDL in total serum promotes a significant efflux from J774 cells that is not stimulated by cAMP and that will "mask" the contribution of the stimulation of efflux produced by unassociated apoA-IV.

The "masking" effect of HDL on the cAMP-stimulated efflux mediated by unassociated apolipoprotein was confirmed in the next series of experiments, in which increasing concentrations of isolated human HDL were added back to the LPDS from apoA-IV transgenic mice. Table 3Down shows that increasing concentrations of HDL progressively reduced the stimulation of efflux induced by cAMP.


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  		Table 3. Effect of Increasing Concentrations of Isolated HDL Added to LPDS Fraction of HuA-IVTg Mice on cAMP Stimulation of Cholesterol Efflux From J774 Mouse Macrophages

Comparative Response of cAMP-Stimulated J774 Cells to Varying Concentrations of Mouse HDL and LPDS
At least 2 different mechanisms have been proposed to be operating in the efflux of cholesterol from J774 cells to serum: an HDL-driven mechanism and a second mechanism linked to the release of cell cholesterol and phospholipid to unassociated apolipoproteins (for a review, see Rothblat et al17 ).

Thus, the aim of the present experiment was to quantify the relative contribution of HDL and of unassociated apoA-IV on cholesterol efflux from cAMP-pretreated cells. To accomplish this, J774 cells preincubated with cAMP were exposed to either increasing concentrations of LPDS obtained from HuA-IVTg mouse serum or isolated HDL obtained from the same pool of serum. The HDL was added at increasing concentrations with respect to its equivalent concentrations in whole serum.

The dose-response curves for cAMP-stimulated efflux mediated by the HuA-IVTg LPDS showed a linear increase up to a concentration equivalent to 1% serum and then tended to reach a plateau, demonstrating a saturation of the capacity of the lipid-free apolipoproteins of LPDS to promote cholesterol efflux (Figure 5Down). In contrast, the dose-response curve for efflux mediated by isolated HDL remained approximately linear over the whole range of concentrations tested. However, at the lower concentrations, the cAMP-stimulated efflux mediated by LPDS was greater than the efflux mediated by HDL, whereas above the 2% serum concentration, the efflux mediated by HDL was predominant.



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  		Figure 5. Comparison of the dose-response relations for isolated HDL (circle, broken line) and LPDS (diamond, solid line) from HuA-IVTg (HuAIVTg) mice in mediating cholesterol efflux from cAMP-treated J774 mouse macrophages. Increasing concentrations of HDL and LPDS were determined on the basis of equivalent concentrations in whole serum. Cholesterol efflux from cAMP-pretreated cells was determined as described in legend to Figure 1Up. Each fractional cholesterol efflux value is the mean of triplicate wells. Results are expressed as mean±SD from 2 experiments. Error bars not shown are within the symbols.

These studies, which dissociated the effects of HuA-IVTg LPDS and isolated HDL on cholesterol efflux from J774 cells pretreated with cAMP, provide an explanation for the results obtained in the initial experiment with the use of whole serum (Figure 1Up). It is apparent that the cAMP stimulation observed with the addition of up to 2% serum was due to the effect of unassociated apoA-IV in the serum of transgenic mice, whereas the lack of stimulation with >2% serum was predominantly due to the HDL-mediated efflux, which is independent of any effect produced by cAMP pretreatment.


*    Discussion
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A number of previous studies29 30 have demonstrated that lipid-free or lipid-poor apolipoproteins can stimulate the release of cellular cholesterol (for reviews, see Oram and Yokoyama31 and von Eckardstein32 ) and have established that some macrophages are among the most responsive cells to apolipoproteins.31 33 34 35 Moreover, it has been shown that exposure of macrophages to cAMP stimulates the release of lipid to either apoA-I or apoE, and evidence has been obtained indicating that in some macrophage systems, the apolipoprotein-mediated release of cholesterol involves the interaction of apolipoproteins with cellular surface proteins,36 which in some systems are upregulated by the exposure of the cells to cAMP.21 22 To date, most of the studies demonstrating the ability of unassociated or lipid-free apolipoproteins to mediate cellular cholesterol efflux have been conducted with the use of experimental systems in which the apolipoproteins have been provided to the cells in their purified isolated form and were present in the culture medium in the absence of alternative cholesterol acceptors.

In the present series of experiments, we have attempted to determine the extent to which unassociated apolipoproteins contribute to cell cholesterol efflux when cells are exposed to serum or serum fractions. For this purpose, we have studied sera from control mice and HuA-IVTg mice. In the transgenic apoA-IV mouse model, which has previously been described by Duverger at al,16 human apoA-IV was expressed in the liver, and its plasma concentration reached high levels without producing a major change in serum lipid concentration. Among all of the exchangeable apolipoproteins, apoA-IV is most easily dissociated from intact lipoprotein particles; thus, the high overexpression of human apoA-IV in our mouse model ensured the presence of the unassociated form of the apoA-IV in the serum specimens and allowed us to investigate, for the first time, the contribution of unassociated apolipoprotein to cellular cholesterol efflux when it was present in serum. Although the efflux capacity of lipoprotein particles containing apoA-IV has been demonstrated,5 9 14 32 few studies have used delipidated apoA-IV as a cholesterol acceptor. However, early studies have shown that lipid-free apoA-IV can mediate cholesterol efflux from human fibroblasts with an efficiency similar to that of lipid-free apoA-I.29

Our present results provide data demonstrating that in a biological fluid, apoA-IV, dissociated from whole lipoprotein particles, is able to stimulate the release of cholesterol from macrophages and that this efflux is enhanced if the cells are pretreated with cAMP to upregulate a putative receptor. Measurements of cholesterol efflux to dilutions of whole serum have been performed with the use of either Fu5AH rat hepatoma cells or J774 mouse macrophages under experimental conditions that allow the quantification of cell cholesterol efflux mediated by either HDL or by lipid-free apoA-IV. For this purpose, we first showed that cholesterol efflux to serum from either control or transgenic mice was similar when the cholesterol donor was Fu5AH hepatoma cells. This result was expected because cholesterol efflux from these cells is largely a reflection of the presence of high levels of scavenger receptor BI,37 and the main determinant for cholesterol efflux in the Fu5AH system is the concentration of HDL cholesterol and HDL phospholipids.18 19 23 38 Neither of these parameters was different in the sera from the 2 types of mice. In addition, cholesterol efflux from Fu5AH cells is relatively unresponsive to lipid-free apolipoproteins and to cAMP stimulation.22 When control J774 cells were used as cholesterol donors, a comparison of efflux to serum or 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, compared with that obtained with control untreated cells, when whole serum or LPDS from HuA-IVTg mice was used as a cholesterol acceptor, whereas no cAMP stimulation was observed when the control mice serum or LPDS was used. However, pretreatment of the cells with cAMP did not enhance the efflux response to HDL isolated from HuA-IVTg mouse serum.

It has been shown that cAMP can stimulate cholesterol efflux from cholesteryl ester–loaded J774 cells to isolated HDLs, primarily as a result of increased cholesteryl esters hydrolysis.39 40 However, this effect is distinct from the one observed in the present study because our cells were grown in the presence of an ACAT inhibitor and contained no esterified cholesterol. Thus, cholesterol efflux occurred in the absence of cholesteryl ester hydrolysis. The cAMP stimulation of cholesterol efflux from J774 cells exposed to serum or LPDS from HuA-IVTg mice can be attributed to the upregulation of an apolipoprotein-mediated response that is associated with the expression of a cell surface protein.22 Although HDL contributed a substantial part to the efflux of cholesterol from J774 cells to the mouse sera, the efflux to HDL did not contribute to the efflux that was stimulated by cAMP treatment. Actually, in our model, which used various dilutions of serum, the presence of increasing concentrations of HDL appeared to mask the cAMP-stimulated efflux mediated by unassociated apolipoproteins. A number of observations illustrate this relation between unassociated apolipoproteins and intact HDL. First, there is an inverse relation between the concentration of HuA-IVTg serum exposed to the cells and the percent of cAMP stimulation of efflux (Figure 1BUp). Second, the magnitude of cAMP stimulation of efflux was much more pronounced with the LPDS fraction from HuA-IVTg mice than with whole serum (Figure 3Up). Third, the degree of cAMP stimulation of efflux obtained with purified human apoA-IV or apoA-I was as follows: free apolipoproteins incubated alone with the cells>free apolipoproteins added to the LPDS from control mice>free apolipoproteins added to the serum from control mice (Table 2Up). Fourth, by adding increasing amounts of HDL to a constant concentration of LPDS, we observed a dose-dependent decrease in the apparent fractional stimulation of efflux by cAMP (Table 3Up). Finally, whereas HDL isolated from the serum of HuA-IVTg mice produced a near-linear dose-response curve for efflux, the cAMP-stimulated efflux mediated by LPDS tended to be saturable (Figure 5Up). The relations between HDL-mediated efflux and efflux produced by the unassociated apoA-IV in LPDS indicate that when cAMP-treated J774 cells are used as cholesterol donors, the efflux to unassociated apolipoproteins makes the predominant contribution at serum concentrations <2%, whereas at serum concentrations >2%, HDL-mediated efflux becomes increasingly more important. The observation that serum HDL and unassociated apolipoproteins are contributing by different mechanisms to the efflux of cholesterol from cells has been proposed in a number of previous models reported in a recent review.17 A process of cooperation between the 2 types of acceptors has been demonstrated in an artificial shuttle and sink model, with the use of cyclodextrins as cholesterol shuttles and phospholipid vesicles as cholesterol sinks.41 From our results, it is apparent that lipid-free apolipoproteins can be expected to have a significant impact on cellular cholesterol release under conditions in which HDL concentrations are low and cholesterol donor cells are equipped with mechanisms that promote cholesterol efflux to unassociated apolipoproteins.

Biological fluids other than plasma, such as interstitial fluid,4 5 peripheral lymph,42 or follicular fluid,43 are the best candidate media to be enriched, at least temporarily, with lipid-free apolipoproteins. Although the relations between unassociated apolipoproteins and pre-ß HDLs have not been established, it is probable that at least some of the unassociated apolipoproteins, either apoA-I or apoA-IV, would have the characteristics attributed to pre-ß HDL. The presence of an increased proportion of pre-ß–migrating HDL particles has been demonstrated in the extravascular fluids,44 45 46 47 and it is not clear which part of these pre-ß–migrating particles might reflect lipid-poor or lipid-free apolipoproteins. A general model that has been proposed for the role of unassociated/lipid-free apolipoproteins is that apoA-I and apoA-IV, which dissociate from HDL during the remodeling of plasma lipoproteins, are available for filtration through the vascular bed and for interaction with peripheral cells.48 49 These unassociated apolipoproteins interact directly with the plasma membrane, resulting in the release of phospholipids and free cholesterol from the cell surface,33 34 50 with the subsequent generation of pre-ß HDL particles.51 This process would contribute to cellular cholesterol homeostasis in peripheral tissues and to the generation of nascent HDL particles.51 52 Within the arterial wall, lipid-poor apolipoproteins could participate in the removal of excess cholesterol from macrophage-derived foam cells. Consistent with this concept is the recent observation that treatment of serum with mast cell chymase produced a proteolysis of lipid-poor particles, such as pre-ß LpA-I and LpA-IV, which consequently reduced the ability of serum to remove cholesterol from cholesterol-loaded mouse peritoneal macrophages.53

In the present study, we have used cAMP treatment of J774 cells as a tool to demonstrate the ability of unassociated apolipoproteins to stimulate cell cholesterol efflux. The mechanism underlying the action of cAMP involves the induction of apolipoprotein binding sites on the cell surface.21 22 Whether this apolipoprotein-binding protein is constitutively expressed on macrophage-derived foam cells within the atherosclerotic lesion or whether the protein is induced under specific metabolic conditions can only be resolved after this apolipoprotein-binding protein has been isolated and characterized. The cell culture models that have been shown to respond to cAMP are limited to RAW, J774, elicited mouse peritoneal macrophages,22 and, more recently, human skin fibroblasts.54 At the present time, there is growing speculation55 56 and some preliminary data indicating that the cAMP-inducible protein in J774 macrophages (A.E. Bortnick, O. Francone, G.H. Rothblat, unpublished data, 1999) is the ATP-binding cassette transporter 1, which has recently been shown to be defective in patients with Tangier disease57 58 59 60 and which appears to be responsible for the lipidation of apoA-I.61 In the present study, we have shown that apoA-I and apoA-IV can stimulate cell cholesterol efflux, and a previous study by Smith et al21 has demonstrated the effectiveness of apoE. Thus, there does not seem to be a high degree of specificity associated with the lipidation of unassociated apolipoproteins, but this process is linked to the presence of amphipathic helix and the extent of lipid affinity of the acceptor protein or peptide.30 34 50 There are a number of metabolic events that can be expected to produce an increase in the intracellular cAMP content in macrophages, including physiologically relevant compounds such as prostaglandins,62 63 64 vasoactive intestinal peptide, and pituitary adenylate cyclase–activating polypeptide,65 all of which have links to the inflammatory reaction that is becoming an increasingly important component in the atherosclerotic process.66 67

In conclusion, the present study demonstrates that serum and LPDS from HuA-IVTg mice produce a cAMP-sensitive efflux of cholesterol from J774 macrophages and strongly suggests that apoA-IV unassociated with lipoprotein particles is responsible for this process. Overall, the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux is emphasized for the first time in a biological fluid. The cAMP-stimulated J774 macrophage cell system might be proposed as a new tool for investigating those physiopathological aspects of reverse cholesterol transport that are linked to lipid-free or lipid-poor apolipoproteins.


*    Acknowledgments
 
This study was supported in part by grant HL-22633 from the National Institutes of Health (Dr Rothblat). We thank P. Tso (Department of Pathology, University of Cincinnati, Ohio) for providing purified human apoA-IV.

Received July 7, 1999; accepted December 20, 1999.


*    References
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