This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 24/12/2345    most recent 01.ATV.0000148706.15947.8av1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Favari, E. Articles by Rothblat, G. H. Search for Related Content PubMed PubMed Citation Articles by Favari, E. Articles by Rothblat, G. H. Related Collections Cardiovascular Pharmacology Cell biology/structural biology Lipid and lipoprotein metabolism Other Vascular biology

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:2345.)
© 2004 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Probucol Inhibits ABCA1-Mediated Cellular Lipid Efflux

Elda Favari; Ilaria Zanotti; Francesca Zimetti; Nicoletta Ronda; Franco Bernini; George H. Rothblat

From the Departments of Pharmacological and Biological Sciences and Applied Chemistry (E.F., I.Z., F.Z., F.B.) and Clinical Medicine, Nephrology, and Health Sciences (N.R.), University of Parma, Italy; and Gastroenterology and Nutrition (G.H.R.), Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, Pa.

Correspondence to Franco Bernini, Departments of Pharmacological and Biological Sciences and Applied Chemistry, University of Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy. E-mail fbernini{at}unipr.it

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of lipids from cells to lipid-poor apolipoproteins. In this article, we characterize the effect of probucol on cellular ABCA1-mediated lipid efflux.

Methods and Results— Probucol inhibited cholesterol efflux up to 80% in J774 macrophages expressing ABCA1. In Fu5AH hepatoma cells that contain scavenger receptor class B, type I, but not functional ABCA1, we observed no effect of probucol on cholesterol efflux. Probucol inhibited cholesterol efflux from normal human skin fibroblasts but not from fibroblasts from a Tangier patient. Fluorescent confocal microscopy and biotinylation assay demonstrated that in J774 cells probucol impaired the translocation of ABCA1 from intracellular compartments to the plasma membrane. Probucol also inhibited the formation of an ABCA1-linked cholesterol oxidase sensitive plasma membrane domain. Consistent with the inhibitory effect on ABCA1 translocation to the plasma membrane, probucol reduced cell surface–specific [¹²⁵I]-labeled apolipoprotein-AI binding.

Conclusions— We conclude that probucol is an effective inhibitor of ABCA1-mediated cholesterol efflux without influencing scavenger receptor class B type I–mediated efflux. The inhibition of ABCA1 translocation to the plasma membrane may in part explain the reported in vivo high-density lipoprotein–lowering action of probucol.

This study characterized probucol effect on ABCA1-mediated lipid efflux. Probucol inhibits ABCA1-mediated efflux but not SR-BI–mediated efflux by affecting the ABCA1 translocation to the plasma membrane, the membrane cholesterol pool, and the ¹²⁵I-apo-AI binding.

Key Words: probucol • lipid efflux • ABCA1 • macrophages • fibroblasts

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Probucol is a lipid-lowering drug that has been extensively investigated since its introduction in the early 1970s.¹ Among its most dramatic effects is the ability to promote the regression of cutaneous and tendinous xanthoma, and this effect appears to be independent of its cholesterol-lowering effect.^1,2 However, probucol also significantly reduces plasma high-density lipoproteins (HDLs). These facts have made this drug very controversial. It has been reported that probucol treatment induced a more rapid progression of atherosclerotic lesions³ in apolipoprotein E (apoE) knockout mice. In contrast, a recent study demonstrated that probucol treatment of scavenger receptor class B type I (SR-BI)/apoE double knockout mice prevents the dramatic early coronary heart disease and death that is a characteristic of these animals.⁴

Just as the data on probucol effects in vivo are contradictory, so too are the results from a number of experiments that have been conducted to study the action of probucol on cholesterol metabolism in vitro. Yamamoto et al⁵ measured the effect of probucol on the change in cholesterol mass on incubation of THP-1 macrophages with HDLs and observed a significant reduction consistent with a probucol-mediated increase in the net efflux of cholesterol. A similar result was obtained by Goldberg and Mendez⁶ using human skin fibroblasts. In contrast, a subsequent investigation failed to observe any probucol stimulation in the efflux of cholesterol from a number of cell types.⁷ It has been demonstrated that specific cell surface proteins play an important role in mediating the flux of cholesterol between cells and extracellular acceptors (for a review see Yancey et al⁸). SR-BI facilitates the selective uptake of HDL cholesteryl ester (CE) and enhances the bidirectional flux of free cholesterol (FC) between cells and HDLs.⁹ ATP-binding cassette AI (ABCA1) has been shown to bind lipid-free or lipid-poor apoproteins and to transfer both cholesterol and phospholipid from the cell membrane to the apoprotein.^10,11

Even before the identification of ABCA1 as a mediator of cell lipid efflux to apoproteins, it was demonstrated that probucol treatment of cells produced a marked inhibition of apo-AI–mediated lipid efflux from macrophages.^12,13 It has now been shown that this inhibition is accompanied by complete or partial inhibition of binding of the apoprotein to the probucol-treated cells.^14–16

Thus, there is no question that treatment of cells with probucol can impact cellular lipid efflux. However, the effect of the drug and the mechanisms by which it produces changes in efflux remain obscure and contradictory. Because of a renewed interest in probucol, and because of our growing knowledge of the roles of SR-BI and ABCA1 on cell cholesterol flux, we have now conducted studies to obtain additional information on the effect of probucol on lipid flux.

For this investigation, we have focused on the impact of probucol treatment on the expression, intracellular distribution, and cholesterol efflux–mediating ability of ABCA1.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Acetylated low-density lipoproteins (LDLs) were prepared as previously described.¹⁷ Apo-AI was purified from human blood plasma, as previously described,¹⁸ and it was provided by L. Calabresi at University of Milan (Italy). [¹²⁵I]-labeled apo-AI (¹²⁵I apo-AI) was provided by M.A. Connelly at State University of New York (SUNY, Stony Brook, NY). For details, please see the online Methods, available at http://atvb.ahajournals.org.

Cells
J774 mouse macrophages were cultured in RPMI medium 1640 supplemented with 10% FCS. Fu5AH rat hepatoma cells were grown in DMEM with 5% FCS. Human fibroblasts were grown in DMEM supplemented with glutamine, nonessential amino acids, sodium pyruvate, and FCS. All the culture media were supplemented with 50 µg/mL gentamicin.

Assay of Cellular Cholesterol and Phospholipid Efflux
Cells were seeded in 24-well plates until 80% confluent. Cells were then labeled with 2 µCi/mL [1,2-³H] cholesterol for 24 hours and subsequently incubated overnight in medium containing 0.2% BSA with or without either 5 µg/mL 22-hydroxycholesterol (22-OH)/10 µmol/L 9-cis retinoic acid (9cRA) or 0.3 mmol/L of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (cpt-cAMP).¹⁹ Efflux was promoted to various acceptors after the incubation of cells with or without 10 µmol/L probucol. Time zero values were obtained from cell walls harvested before lipid acceptor addition. The percent release of lipid was calculated as: (cpm in medium per cpm time zero) x100. For phospholipid efflux assay, cells were treated as previously described and phospholipids were extracted by the Bligh and Dyer method.²⁰

Assay of Cholesterol Oxidase
Cholesterol oxidase treatment was essentially as previously described.²¹ Briefly, cells were labeled with 3 µCi/mL[³H] cholesterol. Cholesterol oxidase (1 U/mL) was added, and cells were incubated for 4 hours. Lipid was extracted with isopropanol, and radioactive cholesterol and cholestenone were separated using thin-layer chromatography and quantified.

Western Blots
Cell monolayers were lysed with a 1% Triton X-100, 0.5% Nonidet P-40, 10 mmol/L tris buffer and homogenized through a 27-gauge needle. Equal amounts of protein (24 µg) were separated on 3% to 8% tris-acetate gels and transferred to polyvinylidene fluoride membrane. Blot was performed with an anti-ABCA1 rabbit polyclonal antibody. Chemiluminescence was used to visualize the proteins according to the manufacturer’s instructions.

Confocal Microscopy
J774 macrophages were grown on 8-well multichamber slides and incubated with 0.3 mmol/L cpt-cAMP for 18 hours to induce the expression of ABCA1. In each slide, half of the wells were then treated with 10 µmol/L probucol in serum-free medium for 2 hours. Cells were fixed with methanol and incubated with an anti-ABCA1 rabbit polyclonal antibody for 40 minutes at room temperature, then incubated with an anti-rabbit Ig coupled with fluorescein isothiocyanate. Slides were mounted with a cover slide and observed by confocal microscopy (Molecular Dynamics Multiprobe 2001). For further details on the experimental conditions please see online Methods.

J774 Cell Surface Binding of ¹²⁵I-Lipid–Free Apo-AI
J774 were treated according to the following protocol: during the efflux period, cells were treated with ¹²⁵I apo-AI in presence or absence of a 20-fold excess of unlabeled apo-AI for 4 hours. After the incubation, cells were washed several times with PBS and lysed with 0.1 mol/L NaOH. Radioactivity in cell lysate was measured by -counting, after which aliquots were taken for protein determination. Specific binding was calculated by subtraction of nonspecific from total binding.

Cell Surface Biotinylation Assay
Cell monolayers were grown in 60-mm Petri dishes and incubated with 0.3 mmol/L cpt-cAMP for 18 hours to induce the expression of ABCA1. Half of the dishes were then treated with 10 µmol/L probucol in serum-free medium for 2 hours. Cells were washed with PBS and treated with Sulfo-NHS-LC-LC-Biotin (Pierce) in PBS/calcium-magnesium buffer for 30 minutes at room temperature. Monolayers were washed with PBS supplemented by 100 mmol/L glycine and were lysed with a 1% Triton X-100, 0.5% Nonidet P-40, 10 mmol/L tris buffer. Biotinylated proteins were separated from nonbiotinylated proteins by using Monomeric Avidin Column (ImmunoPure Immobilized Monomeric Avidin Kit, Pierce). Biotinylated proteins (14.2 µg) were separated on 3% to 8% tris-acetate gels and transferred to polyvinylidene fluoride membrane. Western blot analysis was performed as described.

Statistical Analysis
Results were presented as means and standard deviations of triplicate determinations. Significant differences were established by t test using Graph Pad Prism (GraphPad Software Inc).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Efflux of cholesterol to apo-AI from J774 macrophages was stimulated up to 4-fold by the exposure to cpt-cAMP. Probucol treatment for 2 hours inhibited this efflux by 80%±0.06 (Figure 1). Stimulation with cpt-cAMP upregulated ABCA1 expression,¹³ and probucol interfered with the ABCA1 efflux to lipid-free apolipoproteins without affecting the protein expression (Figure 1, inset). We tested the probucol effect on Fu5AH cells, a cell line that expresses high levels of SR-BI protein, but not functional ABCA1.²² Probucol did not influence cholesterol efflux from Fu5AH to HDL (% efflux to HDL was 9.98±1.28 and 11.24±0.69 after probucol pretreatment), showing that the probucol effect was specific for ABCA1-mediated cholesterol efflux. In J774 cells upregulated for ABCA1 using the liver X receptor and retinoic X receptor (LXR-RXR) agonists 22-OH and 9cRA and exposed to probucol for 2 hours, 1 µmol/L of probucol inhibited efflux by 50% and reached the maximum inhibition at 5 µmol/L (Figure IA, available online at http://atvb.ahajournals.org). We performed a time course experiment using 10 µmol/L of probucol. The results show a 60% inhibition of apo-AI–mediated cholesterol efflux from J774 cells treated with LXR-RXR agonists after only 15 minutes exposure to probucol, and by 2 hours cholesterol efflux was maximally inhibited (Figure IB). We next evaluated phospholipid efflux to further test the probucol effect on ABCA1 activity. The 3-fold increase of phospholipid efflux to apo-AI stimulated by LXR-RXR agonists was abolished in the presence of probucol (% efflux to apo-AI was 4.42±0.001 and 1.20±0.12 after probucol pretreatment).

View larger version (20K): [in this window] [in a new window]   Figure 1. Probucol activity on FC efflux from J774 cells. Monolayers were labeled with 2 µCi/mL [3H]cholesterol for 24 hours in RPMI medium 1640 with 1% FCS in the presence of 2 µg/mL acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. Cells were then incubated for 18 hours with 0.2% BSA in the presence (solid bars) or absence (hatched bars) of 0.3 mmol/L cpt-cAMP followed by incubation with or without 10 µmol/L probucol as described in Methods. After 2 hours of probucol treatment, cells were washed and then incubated with RPMI medium 1640 containing 25 µg/mL lipid-free apo-AI for 4 hours. Data are from a representative experiment with triplicate wells (n=3). Values are expressed as mean±SD. Inset, Effect of probucol on ABCA1 protein expression in cpt-cAMP–stimulated J774 macrophages. Cells were treated as described above, and after probucol incubation they were washed with PBS and solubilized for Western blot analysis as described in Methods. Letter a shows cpt-cAMP–treated cells; b, control cells; and c, cpt-cAMP–treated cells plus probucol.

As shown in Figure II (available online at http://atvb.ahajournals.org), using normal and Tangier fibroblasts upregulated for ABCA1 by incubation with 9-cRA and 22-OH, apo-AI–mediated efflux was inhibited by probucol in normal fibroblasts but probucol treatment had no effect on Tangier cells that are unable to express ABCA1.

The turnover of ABCA1 protein can be rapid,^11,23 thus probucol inhibition of lipid efflux could be linked to accelerated ABCA1 turnover and degradation. However, this does not appear to be the case, because the probucol inhibition is exceptionally rapid (15 minutes; Figure IB) and the level of ABCA1 protein is not significantly reduced after a 2-hour exposure to probucol (Figure 1, inset). Because ABCA1 localization is in the plasma membrane and late endosome compartments,²⁴ we used fluorescent confocal microscopy to determine whether probucol could interfere with the protein distribution in cells. Consistent with previous studies,²⁴ we observed ABCA1 protein both at the cell periphery and intracellular locations in cells stimulated by cpt-cAMP (Figure 2A). After a 2-hour exposure to probucol, ABCA1 protein was almost exclusively present in an intracellular location (Figure 2B). Similar results were obtained using J774-loaded cells by 50 µg/mL of acetylated LDLs in the same experimental conditions (Figure III, available online at http://atvb.ahajournals.org).

View larger version (76K): [in this window] [in a new window]   Figure 2. Fluorescent confocal microscopy analysis of probucol effect on ABCA1 localization in J774 mouse macrophages. Cells were plated in RPMI medium 1640 with 10% FCS in a 8-well chamber slides. After 24 hours, cells were incubated in medium with 1% FCS in the presence of 2 µg/mL ACAT inhibitor. Cells were then equilibrated with 0.2% BSA and incubated with 0.3 mmol/L cpt-cAMP for 18 hours before the probucol exposure. After the cpt-cAMP treatment, cells were incubated with (B) or without (A) 10 µM probucol as described in Methods. After 2 hours, cells were chilled on ice and then fixed by exposure to methanol for 2 minutes. After fixation, cells were washed and then incubated 1 hour with the primary rabbit polyclonal antibody to ABCA1 diluted 1:50 in DPBS. Cells were then extensively washed and incubated with a fluorescent secondary antibody for 1 hour.

Vaughan and Oram²⁵ demonstrated that the expression of ABCA1 in cells produces an increase in the size of membrane cholesterol pool. Thus, we tested whether probucol interfered with the movement of cholesterol to plasma membrane domains. To this end, we evaluated cholestenone formation after exposure of cells to cholesterol oxidase under conditions leading to oxidation of FC present in the plasma membrane.²¹ As expected,²⁵ in J774 macrophages the upregulation of ABCA1 by cpt-cAMP increased by almost 3-fold the cholesterol in the plasma membrane sensitive to oxidation by cholesterol oxidase. This process was absent in cells exposed to probucol (Figure 3A). The experiment was also performed using only 2-hour incubation with the enzyme, and we obtained similar results (% cholestenone moves from 4.50±0.17 in control cells to 9.90±0.16 in cAMP treated cells, and 2-hour incubation with probucol reduces this value back to 5.90±0.60).

View larger version (20K): [in this window] [in a new window]   Figure 3. Probucol effect on FC oxidation by cholesterol oxidase in J774 mouse macrophages (A) and in normal and Tangier human fibroblasts (B). A, Monolayers were labeled with 4 µCi/mL [3H]cholesterol for 24 hours in RPMI medium 1640 with 1% FCS in the presence of 2 µg/mL ACAT inhibitor. Cells were then incubated for 18 hours with 0.2% BSA in the presence or absence of 0.3 mmol/L cpt-cAMP followed by incubation with or without 10 µmol/L probucol. After 2 hours of probucol treatment, cells were washed and incubated with (solid bars) or without (open bars) 0.5 U/mL cholesterol oxidase enzyme in DPBS for 4 hours at 37°C. B, Cells were treated as described above, but the incubation with cAMP was done with 10 µmol/L 9cRA and 5 µg/mL 22-OH. Data are from a representative experiment with triplicate wells (n=3). Values are expressed as mean±SD.

We performed a similar experiment using normal and Tangier human skin fibroblasts incubated with the LXR-RXR agonists to upregulate ABCA1. Only in normal cells LXR-RXR stimulation induced an increase in the cholesterol oxidase sensitive pool. Probucol completely inhibited this effect. In Tangier cells, the cholestenone formation was unaffected by either LXR-RXR stimulation or probucol treatment (Figure 3B).

To test the effect of probucol on apo-AI cell surface binding, J774 macrophages, cpt-cAMP–stimulated, were treated with probucol for 2 hours and ¹²⁵I-apo-AI binding was measured at 4°C. As shown in Figure 4, binding was increased by cAMP treatment, and exposure to probucol reduced the specific binding of ¹²⁵I-apo-AI. Probucol had no effect on the nonspecific binding. To further provide biochemical support for the concept that probucol impaired the ABCA1 trafficking from the intracellular compartments to the plasma membrane, we measured the ABCA1 localization on the cell surface by biotinylation assay. The results showed that the expression of ABCA1 protein on the cell surface is fully inhibited by probucol (Figure 4, inset).

View larger version (27K): [in this window] [in a new window]   Figure 4. Effect of probucol pretreatment of cAMP-stimulated J774 macrophages on apo-AI–specific binding. Cells were incubated in medium with 1% FCS in the presence of 2 µg/mL ACAT inhibitor. Monolayers were then equilibrated with 0.2% BSA and incubated with or without 0.3 mmol/L cpt-cAMP for 18 hours before the probucol exposure. After the cpt-cAMP treatment, cells were incubated with or without 10 µmol/L probucol. After 2 hours, cells were chilled on ice and then exposed to 10 µg/mL [125I]apo-AI, with or without a 40-fold excess of unlabeled apo-AI. After 1 hour on ice, cells were washed and aliquots of cell lysate were taken for -counting and protein mass assay. [125I]apo-AI total, nonspecific, and specific binding were calculated as described in Methods. Results are presented as mean±SD from 3 determinations from 3 experiments. Inset, Effect of probucol on biotinylated ABCA1 protein expression in cpt-cAMP–stimulated J774 macrophages. J774 macrophages were incubated in the presence of 0.3 mmol/L cpt-cAMP (a and b). For the monolayers treated with probucol after the incubation time with cpt-cAMP (b), probucol (10 µmol/L) was added during the last 2 hours of equilibration phase. After this, incubation cells were washed with PBS, biotinylation assay was performed, biotinylated proteins were separated from nonbiotinylated proteins by using monomeric avidin column, and biotinylated proteins were analyzed by Western blot as described in Methods.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Even before the identification of ABCA1 as a mediator of lipid efflux, it was demonstrated that probucol treatment of cells would produce a marked inhibition of apolipoprotein-mediated lipid efflux from macrophages.^12,13 This inhibition of lipid efflux to apoproteins was accompanied by an inhibition of binding of the apoprotein to the probucol-treated cells.¹² In contrast to the apoprotein-mediated efflux, probucol was shown not to have an effect on FC efflux to LDL,¹² a process that results largely by unmediated or SR-BI–mediated cholesterol exchange.⁸ Using macrophages pretreated with cAMP, a procedure we now know up regulates ABCA1, Sakr et al¹³ demonstrated that exposure to probucol produced a rapid and marked inhibition of apo-AI–mediated efflux.

One of the most dramatic effects of probucol treatment of J774 macrophages is the rapidity (15 minutes) of the inhibition of lipid efflux to apo-AI in J774-expressing ABCA1. Because of the pronounced hydrophobicity of probucol, it can be anticipated that the compound rapidly partitions into the cell membrane. Both FC and phospholipid efflux are inhibited, and this inhibition appears to be specific to ABCA1-mediated efflux. Specificity is demonstrated by the lack of inhibition of efflux from Tangier fibroblasts and the observation that the treatment of Fu5AH rat hepatoma cells to the drug had no impact on the efflux of cholesterol to HDL, a process that has been shown to be largely mediated by SR-BI.²²

There are a number of mechanisms by which probucol could exert its inhibitory effect. Rapidity of probucol effect and Western blot analysis (Figure IB and Figure 1, inset) exclude an effect on ABCA1 expression or degradation. Studies by Vaughan and Oram²⁵ demonstrated that the presence of ABCA1 in the plasma membrane of baby hamster kidney cells resulted in an increase in the pool of cholesterol in the plasma membrane that became susceptible to oxidation by cholesterol oxidase. Here we demonstrated that upregulation of ABCA1 in J774 results in an increase in cholesterol oxidase-sensitive cholesterol pool and that probucol completely inhibited the formation of this lipid domain (Figure 3A). That this increase in sensitivity to cholesterol oxidase is an ABCA1-linked response is further demonstrated by the observation that treatment of normal human fibroblasts with LXR-RXR ligands resulted in an increase in the size of the oxidase-sensitive pool and elevated cholesterol efflux which was inhibited by probucol. A similar treatment of Tangier fibroblasts produced none of these responses (Figure 3B). It has been reported that cAMP-treated cells exposed to probucol exhibit reduced specific binding of apo-AI compared with control cells.¹² In this study, probucol largely inhibited cAMP-induced upregulation of binding, whereas in a previous study there was a somewhat less dramatic inhibition.¹⁶ Differences in the experimental conditions can account for the observed differences in binding. The previous study used cholesterol-loaded cells,¹⁶ whereas in the present protocol the cells were cholesterol-normal. Cell cholesterol enrichment may increase the binding of an extracellular acceptor independently of cellular metabolism,²⁶ an effect that might oppose the inhibitory activity of probucol. Neufeld et al²⁴ demonstrated that ABCA1 protein resides both on the cell surface and on intracellular vesicles and traffics between these 2 compartments. In this study, the reduced lipid efflux, cholesterol membrane pool, and apo-AI binding produced by probucol correlate well with the shift in the distribution of ABCA1 from the plasma membrane compartment to an internal location, as evidenced by confocal analysis and biotinylation assay (Figure 2, Figure III, and Figure 4, inset). Recently, an inhibitory effect of probucol on apo-AI binding was also reported in WI-38 human fibroblasts.²⁷ However, these authors did not observe any effect of probucol on ABCA1 protein or cholesterol cellular distribution. We do not have a clear explanation for this apparent discrepancy with our present data. The difference in cell lines and conditions of probucol presentation to cells may account for some of the different conclusions reached in this article.

Administration of probucol to both humans and animals has been shown to lower HDL levels.²⁸ This effect involves a probucol-related increase of plasma CE transfer protein and a production of HDL with an enhanced ability to promote cholesterol efflux.²⁹ We propose that in addition to this mechanism the drug may reduce hepatic HDL production by inhibiting the apo-AI interaction with ABCA1. Despite the reduction in HDL, probucol is still very effective in promoting the regression of xanthomas.^2,5 Also, a study by Braun et al⁴ demonstrated that administration of probucol to SR-BI/apoE double knockout mice reversed red blood cell abnormal morphology, restored normal FC/CE ratios, and blocked the development of coronary heart disease. A number of different mechanisms could be responsible for probucol-mediated regression of xanthomas and its antiatherogenic effects. A decrease in foam cell lipid content, and hence regression of xanthomas, could reflect either a reduction in cholesterol influx or stimulation of macrophage cholesterol efflux. Probucol is a potent antioxidant, and the presence of the drug in plasma has been shown to increase the resistance of LDL to oxidative modification and the subsequent uptake of modified LDL by scavenger receptors.² An alternative hypothesis is that enhanced efflux of cholesterol plays a role in xanthoma regression. The results of this study demonstrate that a probucol stimulation of macrophage cholesterol efflux would not occur through an ABCA1 pathway. In the absence of ABCA1-mediated efflux, passive diffusion or SR-BI appears to be a good candidate. However, it is unlikely that probucol is acting directly on cells to increase the level or activity of SR-BI, because drug treatment of SR-BI–rich Fu5AH cells failed to enhance cholesterol efflux to HDL, and feeding probucol to mice does not change the SR-BI mRNA¹⁴ or protein³⁰ in liver. Rather, it is possible that probucol may generate lipoprotein particles more active in promoting net cholesterol efflux from foam cells. In support of this model is the observation by Rinninger et al³⁰ that HDL from probucol-treated mice have increased CE selective uptake into Chinese hamster ovary cells compared with control mouse HDL. These modifications in HDL composition could also increase SR-BI bidirectional flux of FC and the net flux of cholesterol out of cholesterol-enriched foam cells.³¹

	Acknowledgments

 
This work was supported by grant No. QLGI-1999-01007 from the European Union; by grants from Compagnia di San Paolo, the Istituto Nazionale Ricerche Cardiovascolari, and the USA-Italy Agreement (to F.B.); and Heart, Lung, and Blood Institute (NHLBI) grants HL22633 and HL63768 (to G.H.R.). The authors would like to thank Prof S. Calandra of University of Modena, Italy, for providing the Tangier fibroblast. We thank Prof G. Orlandini, University of Parma, Italy, for his assistance on using the confocal microscopy.

Received February 14, 2004; accepted October 7, 2004.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Davignon J. Medical management of hyperlipidemia and the role of probucol. Am J Cardiol. 1986; 57: 22H–28H.[Medline] [Order article via Infotrieve]

2. Yamamoto A, Matsuzawa Y, Yokoyama S, Funahashi T, Yamamura T, Kishino B. Effects of probucol on xanthoma regression in familial hypercholesterolemia. Am J Cardiol. 1986; 57: 29H–36H.[CrossRef][Medline] [Order article via Infotrieve]

3. Franceschini G, Werba JP, Calabresi L. Drug control of reverse cholesterol transport. Pharmacol Ther. 1994; 6: 289–324.

4. Braun A, Zhang S, Miettinen HE, Ebrahim S, Holm TM, Vasile E, Post MJ, Yoerger DM, Picard MH, Krieger JL, Andrews NC, Simons M, Krieger M. Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse. Proc Natl Acad Sci U S A. 2003; 100: 7283–7288.[Abstract/Free Full Text]

5. Yamamoto A, Hara H, Takaku F, Wakasugi JI, Tomikawa M. Effect of probucol on macrophages, leading to regression of xanthomas and atheromatous vascular lesions. Am J Cardiol. 1988; 62: 31B–36B.[CrossRef][Medline] [Order article via Infotrieve]

6. Goldberg RB, Mendez A. Probucol enhances cholesterol efflux from cultured human skin fibroblasts. Am J Cardiol. 1988; 62: 57B–59B.[Medline] [Order article via Infotrieve]

7. Zambon S, Brazg R, Aviram M, Oram JF, Bierman EL. The effect of probucol on HDL-mediated sterol translocation and efflux from cells. Atherosclerosis. 1992; 94: 51–60.[Medline] [Order article via Infotrieve]

8. Yancey PG, Bortnick AE, Kellner-Weibel G, de la Llera-Moya M, Phillips MC, Rothblat GH. Importance of different pathways of cellular cholesterol efflux. Arterioscler Thromb Vascular Biol. 2003; 23: 712–719.[Abstract/Free Full Text]

9. Williams DL, Connelly MA, Temel RE, Swanakar S, Phillips MC, de la Llera-Moya M, Rothblat GH. Scavenger receptor BI and cholesterol trafficking. Curr Opin Lipidol. 1999; 10: 329–339.[CrossRef][Medline] [Order article via Infotrieve]

10. Oram JF, Vaughan AM. ABC1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Curr Opin Lipidol. 2000; 11: 253–260.[CrossRef][Medline] [Order article via Infotrieve]

11. Wang N, Tall AR. Regulation and mechanisms of ATP-binding cassette transporter AI-mediated cellular cholesterol efflux. Arterioscler Thromb Vasc Biol. 2003; 23: 1178–1184.[Abstract/Free Full Text]

12. Tsujita M, Yokoyama S. Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol. Biochemistry. 1996; 35: 13011–13020.[CrossRef][Medline] [Order article via Infotrieve]

13. Sakr SW, Williams DL, Stoudt GW, Phillips MC, Rothblat GH. Induction of cellular cholesterol efflux to lipid-free apolipoprotein A-I by cAMP. Biochim Biophys Acta. 1999; 1438: 85–98.[Medline] [Order article via Infotrieve]

14. Tsujita M, Tomimoto S, Okumura-Noji K, Okazaki M, Yokoyama S. Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice. Biochim Biophys Acta. 2000; 1485: 199–213.[Medline] [Order article via Infotrieve]

15. Takemura T, Sakai M, Matsuda H, Matsumura T, Biwa T, Anami Y, Nishikawa T, Sasahara T, Shichiri M. Effects of probucol on cholesterol metabolism in mouse peritoneal macrophages: inhibition of HDL-mediated cholesterol efflux. Atherosclerosis. 2000; 152: 347–357.[CrossRef][Medline] [Order article via Infotrieve]

16. Kellner-Weibel G, Luke SJ, Rothblat GH. Cytotoxic cellular cholesterol is selectively removed by apoA-I via ABCA1. Atherosclerosis. 2003; 171: 235–243.[CrossRef][Medline] [Order article via Infotrieve]

17. Bernini F, Calabresi L, Bonfadini G, Franceschini G. The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux. Biochim Biophys Acta. 1996; 1299: 103–109.[Medline] [Order article via Infotrieve]

18. Franceschini G, Vecchio G, Gianfranceschi G, Magani G, Sirtori CR. Apolipoprotein AIMilano. Accelerated binding and dissociation from lipids of a human apolipoprotein variant. J Biol Chem. 1985; 260: 16321–16325.[Abstract/Free Full Text]

19. Favari E, Lee M, Calabresi L, Franceschini G, Zimetti F, Bernini F, Kovanen PT. Depletion of PREbeta-HDL by human chymase impairs ATP-binding cassette transporter A1- but not SR-B1-mediated lipid efflux to HDL. J Biol Chem. 2003; 279: 9930–9936.

20. Yancey PG, Bielicki JK, Johnson WJ, Lund-Katz S, Palgunachari LM, Anantharamaiah GM, Segrest JP, Phillips MC, Rothblat GH. Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides. Biochemistry. 1995; 34: 7955–7965.[CrossRef][Medline] [Order article via Infotrieve]

21. Kellner-Weibel G, de la Llera-Moya M, Connelly MA, Stoudt G, Christian AE, Haynes MP, Williams DL, Rothblat GH. Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution. Biochemistry. 2000; 39: 221–229.[CrossRef][Medline] [Order article via Infotrieve]

22. Ji Y, Jian B, Wang N, Sun Y, de la Llera Moya M, Phillips MC, Rothblat GH, Swaney JB, Tall AR. Scavenger receptor B1 promotes high density lipoprotein-mediated cellular cholesterol efflux. J Biol Chem. 1997; 272: 20982–20985.[Abstract/Free Full Text]

23. Arakawa R, Yokoyama S. Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation. J Biol Chem. 2002; 277: 22426–22429.[Abstract/Free Full Text]

24. Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, Comly M, Dwyer NK, Zhang M, Santamarina-Fojo S, Brewer HB Jr, Blanchette-Mackie EJ. Cellular localization and trafficking of the human ABCA1 transporter. J Biol Chem. 2001; 276: 27584–27590.[Abstract/Free Full Text]

25. Vaughan AM, Oram JF. ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions. J Lipid Res. 2003; 44: 1373–1380.[Abstract/Free Full Text]

26. Bernini F, Bellosta S, Corsini A, Maggi FM, Fumagalli R, Catalano AL. Cholesterol stimulation of HDL binding to human endothelial cells EAhy 926 and skin fibroblasts: evidence for a mechanism independent of cellular metabolism. Biochim Biophys Acta. 1991; 1083: 94–100.[Medline] [Order article via Infotrieve]

27. Wu CA, Tsujita M, Hayashi M, Yokoyama S. Probucol inactivates ABCA1 in the plasma membrane with respect to its mediation of apolipoprotein binding and high density lipoprotein assembly and to its proteolytic degradation. J Biol Chem. 2004; 279: 30168–30174.[Abstract/Free Full Text]

28. Beynen AC. Mode of hypocholesterolemic action of probucol in animals and man. Artery. 1987; 14: 113–126.[Medline] [Order article via Infotrieve]

29. Chiesa G, Michelagnoli S, Cassinotti M, Gianfranceschi G, Werba JP, Pazzucconi F, Sirtori CR, Franceschini G. Mechanisms of high-density lipoprotein reduction after probucol treatment: changes in plasma cholesterol esterification/transfer and lipase activities. Metabolism. 1993; 42: 229–235.[CrossRef][Medline] [Order article via Infotrieve]

30. Rinninger F, Wang N, Ramakrishnan R, Jiang XC, Tall AR. Probucol enhances selective uptake of HDL-associated cholesteryl esters in vitro by a scavenger receptor B-I-dependent mechanism. Arterioscler Thromb Vasc Biol. 1999; 19: 1325–1332.[Abstract/Free Full Text]

31. de la Llera-Moya M, Connelly MA, Drazul D, Klein SM, Favari E, Yancey PG, Williams DL, Rothblat GH. Scavenger receptor, class B, type I (SR-BI) affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL. J Lip Res. 2001; 42: 1969–1978.[Abstract/Free Full Text]

This article has been cited by other articles:

				R. Arakawa, M. Tsujita, N. Iwamoto, C. Ito-Ohsumi, R. Lu, C.-A. Wu, K. Shimizu, T. Aotsuka, H. Kanazawa, S. Abe-Dohmae, et al. Pharmacological inhibition of ABCA1 degradation increases HDL biogenesis and exhibits antiatherogenesis J. Lipid Res., November 1, 2009; 50(11): 2299 - 2305. [Abstract] [Full Text] [PDF]

				H. Tanigawa, J. T. Billheimer, J.-i. Tohyama, I. V. Fuki, D. S. Ng, G. H. Rothblat, and D. J. Rader Lecithin: Cholesterol Acyltransferase Expression Has Minimal Effects on Macrophage Reverse Cholesterol Transport In Vivo Circulation, July 14, 2009; 120(2): 160 - 169. [Abstract] [Full Text] [PDF]

				G. L. Weibel, M. R. Joshi, E. T. Alexander, P. Zhu, I. A. Blair, and G. H. Rothblat Overexpression of Human 15(S)-Lipoxygenase-1 in RAW Macrophages Leads to Increased Cholesterol Mobilization and Reverse Cholesterol Transport Arterioscler Thromb Vasc Biol, June 1, 2009; 29(6): 837 - 842. [Abstract] [Full Text] [PDF]

				J. Stefulj, U. Panzenboeck, T. Becker, B. Hirschmugl, C. Schweinzer, I. Lang, G. Marsche, A. Sadjak, U. Lang, G. Desoye, et al. Human Endothelial Cells of the Placental Barrier Efficiently Deliver Cholesterol to the Fetal Circulation via ABCA1 and ABCG1 Circ. Res., March 13, 2009; 104(5): 600 - 608. [Abstract] [Full Text] [PDF]

				C. J. Delvecchio, P. Bilan, P. Nair, and J. P. Capone LXR-induced reverse cholesterol transport in human airway smooth muscle is mediated exclusively by ABCA1 Am J Physiol Lung Cell Mol Physiol, November 1, 2008; 295(5): L949 - L957. [Abstract] [Full Text] [PDF]

				H. H. Hassan, D. Bailey, D.-Y. D. Lee, I. Iatan, A. Hafiane, I. Ruel, L. Krimbou, and J. Genest Quantitative Analysis of ABCA1-dependent Compartmentalization and Trafficking of Apolipoprotein A-I: IMPLICATIONS FOR DETERMINING CELLULAR KINETICS OF NASCENT HIGH DENSITY LIPOPROTEIN BIOGENESIS J. Biol. Chem., April 25, 2008; 283(17): 11164 - 11175. [Abstract] [Full Text] [PDF]

				S. R. Bates, J.-Q. Tao, K. J. Yu, Z. Borok, E. D. Crandall, H. L. Collins, and G. H. Rothblat Expression and Biological Activity of ABCA1 in Alveolar Epithelial Cells Am. J. Respir. Cell Mol. Biol., March 1, 2008; 38(3): 283 - 292. [Abstract] [Full Text] [PDF]

				M. P. Adorni, F. Zimetti, J. T. Billheimer, N. Wang, D. J. Rader, M. C. Phillips, and G. H. Rothblat The roles of different pathways in the release of cholesterol from macrophages J. Lipid Res., November 1, 2007; 48(11): 2453 - 2462. [Abstract] [Full Text] [PDF]

				E. Favari, M. Gomaraschi, I. Zanotti, F. Bernini, M. Lee-Rueckert, P. T. Kovanen, C. R. Sirtori, G. Franceschini, and L. Calabresi A Unique Protease-sensitive High Density Lipoprotein Particle Containing the Apolipoprotein A-IMilano Dimer Effectively Promotes ATP-binding Cassette A1-mediated Cell Cholesterol Efflux J. Biol. Chem., February 23, 2007; 282(8): 5125 - 5132. [Abstract] [Full Text] [PDF]

				I. Zanotti, F. Poti, E. Favari, K. R. Steffensen, J.-A. Gustafsson, and F. Bernini Pitavastatin Effect on ATP Binding Cassette A1-Mediated Lipid Efflux from Macrophages: Evidence for Liver X Receptor (LXR)-Dependent and LXR-Independent Mechanisms of Activation by cAMP J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 395 - 401. [Abstract] [Full Text] [PDF]

				F. Zimetti, G. K. Weibel, M. Duong, and G. H. Rothblat Measurement of cholesterol bidirectional flux between cells and lipoproteins J. Lipid Res., March 1, 2006; 47(3): 605 - 613. [Abstract] [Full Text] [PDF]

				S. Yokoyama Assembly of High-Density Lipoprotein Arterioscler Thromb Vasc Biol, January 1, 2006; 26(1): 20 - 27. [Abstract] [Full Text] [PDF]

				M. Xia, M. Hou, H. Zhu, J. Ma, Z. Tang, Q. Wang, Y. Li, D. Chi, X. Yu, T. Zhao, et al. Anthocyanins Induce Cholesterol Efflux from Mouse Peritoneal Macrophages: THE ROLE OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {gamma}-LIVER X RECEPTOR {alpha}-ABCA1 PATHWAY J. Biol. Chem., November 4, 2005; 280(44): 36792 - 36801. [Abstract] [Full Text] [PDF]

				K.-i. Hirano, C. Ikegami, K.-i. Tsujii, Z. Zhang, F. Matsuura, Y. Nakagawa-Toyama, M. Koseki, D. Masuda, T. Maruyama, I. Shimomura, et al. Probucol Enhances the Expression of Human Hepatic Scavenger Receptor Class B Type I, Possibly Through a Species-Specific Mechanism Arterioscler Thromb Vasc Biol, November 1, 2005; 25(11): 2422 - 2427. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 24/12/2345    most recent 01.ATV.0000148706.15947.8av1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Favari, E. Articles by Rothblat, G. H. Search for Related Content PubMed PubMed Citation Articles by Favari, E. Articles by Rothblat, G. H. Related Collections Cardiovascular Pharmacology Cell biology/structural biology Lipid and lipoprotein metabolism Other Vascular biology