Macrophages are the major source of apolipoprotein E

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1 Distinct Cellular Loci for the ABCA1-Dependent and ABCA1-Independent Lipid Efflux Mediated by Endogenous Apolipoprotein E Expression Zhi H. Huang, Michael L. Fitzgerald, Theodore Mazzone Objective Macrophage expression of both apolipoprotein E (apoe) and ABCA1 have been shown to modulate lipid efflux from these cells and to play an important atheroprotective role in vivo. We evaluated the relationship between apoe and ABCA1 for regulating cellular sterol efflux. Methods and Results ApoE-mediated, but ABCA1-independent, lipid efflux was demonstrated in 3 model systems. First, adenoviral-mediated expression of apoe in dermal fibroblasts isolated from ABCA1 / mice significantly increased both sterol and phospholipid efflux. Second, expression of human apoe in a macrophage cell line increased sterol efflux, and this increment in efflux was not reduced by suppressing ABCA1 expression. Third, reduction of apoe expression using an apoe small interfering RNA significantly reduced sterol efflux from ABCA1 / mouse peritoneal macrophages. ApoE-mediated, but ABCA1-independent, lipid efflux could be differentiated from lipid efflux that was dependent on the extracellular accumulation of secreted apoe, because exogenous cell-derived apoe stimulated efflux only from cells expressing ABCA1. Sterol efflux was usually highest in cells expressing both ABCA1 and apoe, likely representing a summation of the ABCA1-dependent and -independent pathways for apoe-mediated sterol efflux. Conclusions ABCA1 expression is required for apoe-mediated efflux when endogenously synthesized apoe accumulates extracellularly. Our results, however, establish the existence of an ABCA1-independent pathway for lipid efflux that requires the intracellular synthesis and/or transport of apoe. (Arterioscler Thromb Vasc Biol. 2006;26: ) Key Words: apoe ABCA1 macrophage lipid efflux atherosclerosis Macrophages are the major source of apolipoprotein E (apoe) found in atherosclerotic lesions, 1 and the importance of macrophage-derived apoe for atheroprotection has been demonstrated in animal models. 2 4 Macrophage-derived apoe has been shown to have a number of properties that could contribute to an atheroprotective effect, including effects on platelet, arterial smooth muscle, and endothelial cell function. 5 However, given the observation that lipidladen macrophages are a hallmark of the atherosclerotic lesion, the ability of endogenously synthesized apoe to facilitate macrophage lipid efflux likely represents an important aspect of its atheroprotective function. 6,7 Facilitation of macrophage efflux would limit the conversion of macrophages to lipid-laden foam cells in the vessel wall and thereby prevent downstream deleterious effects. 8 Recently, an important role for the ATP-binding cassette (ABC) transporter family of proteins has been established for modulating lipid efflux from a number of cell types. 9 In macrophages, a primary role has been established for the ABCA1 transporter. 10 In vitro, the absence of the ABCA1 transporter, or inhibition of its function, eliminates sterol and phospholipid efflux from a number of cell types to extracellular lipid-free apolipoproteins. 11,12 Similar to what has been described for apoe, macrophage-specific expression of ABCA1 modulates atherosclerosis in vivo. 13,14 ABCA1 is an integral membrane protein, whereas 40% to 60% of macrophage apoe is secreted. Because apoe is secreted, it may facilitate sterol efflux from cells using autocrine or paracrine mechanisms. 15 Furthermore, autocrine stimulation of sterol efflux by endogenous expression of apoe could result from the interaction of secreted apoe with elements of the plasma membrane on the cell of origin or, alternatively, depend on its intracellular synthesis and movement through internal cellular membranes before secretion. We have previously reported the results of studies suggesting that apoe could mediate sterol efflux independent of ABCA1 expression. 16 For these studies we used camp to induce ABCA1 expression, and 4,4 diisothiocyanatostilbene-2,2 -disulfonic acid to inhibit ABCA1 function. However, recent reports emphasize the complexity of protein kinase A effects on cellular cholesterol metabolism, 17 and there is evidence regarding the Original received July 25, 2005; final version accepted October 17, From the Departments of Medicine (Z.H.H., T.M.) and Pharmacology (T.M.), University of Illinois at Chicago; and the Department of Medicine (M.L.F.), Massachusetts General Hospital, Harvard Medical School, Boston, Mass. Correspondence to Theodore Mazzone, Section of Diabetes and Metabolism (MC 797), University of Illinois at Chicago, 1819 W Polk St, Chicago, IL tmazzone@uic.edu 2005 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at DOI: /01.ATV d 157

2 158 Arterioscler Thromb Vasc Biol. January 2006 lack of specificity of 4,4 -diisothiocyanatostilbene-2,2 disulfonic acid for ABCA1. To deal with these issues, we used cell models with a genetic absence of ABCA1 to avoid confounding cellular effects of pharmacological inducers or inhibitors. In addition, using current models, we were also able to evaluate the relationship between apoe and ABCA1-mediated efflux in cholesterol-loaded cells. Furthermore, we evaluated macrophages in which we manipulated both apoe and ABCA1 expression using small interfering RNA (sirna) to evaluate the effect on efflux. The results of these investigations unequivocally demonstrate the existence of a sterol efflux pathway dependent on the intracellular synthesis and/or transport of apoe, but independent of ABCA1 expression. Methods Materials A polyclonal rabbit antiserum was prepared against amino acids 2071 to 2261 of ABCA1. The ABCA1 polypeptide was prepared as described previously. 18 All of the other materials were from previously described sources. 6,7,19,20 Cell Isolation and Culture ABCA1 / mice breeders were purchased from the Jackson Laboratory. Offspring were genotyped using the vendor s protocol. For establishing mouse dermal fibroblast (MDF) lines, full-thickness skin was harvested, minced, and cultured in DMEM with10% FBS for 3 to 5 days. Fibroblast outgrowths were collected by trypsinization and passaged 2 to 6 times. Mouse peritoneal macrophages (MPMs) were harvested by peritoneal lavage with sterile PBS using 10- to 12-week-old mice. MPMs were seeded into 12-well dishes and cultured for 10 days before use in experiments. To obtain RAW-E and RAW-C cells, RAW cells (which do not express endogenous apoe) were obtained from American Type Culture Collection and stably transfected to constitutively express a human apoe3 cdna controlled by the cytomegalovirus promoter in a neomycinresistance vector (RAW-E cells) or a resistance vector alone (RAW-C cells) as described previously. 19 Adenovirus Infection of MDFs MDFs were plated in 35-mm dishes and grown to 50% to 70% confluence. After 3 washes, cells were infected with a full-length human apoe3 adenovirus (kindly provided by Dr. Catherine Reardon, University of Chicago) or a LacZ control adenovirus in DMEM at a multiplicity of infection of 100 for 4 hours. Cells were washed, and fresh growth medium was added for an overnight incubation to allow for expression of apoe. MDF / and MDF / cells with equivalent levels of adenoviral apoe expression were used for evaluation of apoe-mediated efflux. Modulation of ABCA1 and ApoE Expression by sirna Cells were seeded in 12-well plates and grown to 50% confluence in DMEM and 10% FBS. sirnas to mouse ABCA1 (5 GUGGCCUG- GCCUCUCUUUAddT3 ), mouse apoe (5 GGCUUACAAAAAG- GAGCUGtt3 ), or control sirna were mixed with RNAiFect (Qiagen) or Trans IT-TKO (Takara Mirus Bio) to allow formation of a transfection complex. Cells were washed with DMEM and incubated with sirna complexes (1 nm of sirna) in DMEM for 6 hours. The efficacy of the sirna incubation for reducing apoe or ABCA1 protein expression was monitored by immunoblot. 20 Reduction in target protein expression after sirna incubations averaged 64%. Lipid Assays Cells were labeled with 3 [H]cholesterol (1 Ci/mL) and/or 14 [C]choline (0.5 Ci/mL) in DMEM with 10% FBS for 48 hour. 3 [H]cholesterol was added in ethanol vehicle, and the final ethanol concentration in the medium was 0.4%. After labeling, cells were washed 3 times with DMEM in 0.1% BSA and incubated with adenovirus or sirna (as indicated in the table or figure legends). Cells were washed, and fresh growth medium was added overnight to allow the cells to recover from the adenovirus or sirna incubation. Before the start of the efflux incubation, the cells were washed 3 times with DMEM plus 0.1% BSA, and the efflux incubation was initiated by the addition of fresh 0.1% BSA in DMEM. For efflux incubations with cholesterol-loaded cells, the efflux medium contained 5 g/ml S58035 to inhibit acyl CoA:cholesterol acyltransferase-mediated cholesterol esterification. Culture supernatants were harvested at time points indicated in each figure. Medium samples were centrifuged at 2500 rpm for 15 minutes at 4 C to pellet-detached cells, and radioactivity in the medium was measured by liquid scintillation counting. Radioactivity in cells was measured in hexane:isopropranol (3:2) extracts. The percentage of lipid efflux is calculated as the disintegrations per minute present in the medium divided by the disintegrations per minute in cells at the beginning of the efflux period. Cell protein was assayed using a DC protein kit (Bio-Rad). Cholesterol:methyl- -cyclodextrin (M CD) complexes were used in some experiments to increase cellular cholesterol content and were prepared in a 1:6 molar ratio. Cellular cholesterol mass was measured enzymatically in hexane/isopropanol extracts. ApoE Synthesis and Secretion MPMs were grown in 12-well plates for 10 days. After washing, cells were pulse labeled using methionine-free DMEM containing 100 Ci/mL 35 [S]methionine for 45 minutes. After washing, cells were chased in DMEM and 0.1% BSA with 500 mol/l cold methionine for 90 minutes. Cell lysate and medium samples were used for quantitative immunoprecipitation of apoe, as described previously. 19 The secreted apoe at the end of the 90-minute chase was calculated as a percentage of total cellular apoe present at the end of the pulse-labeling period. Statistical Analysis The results of representative experiments are expressed as mean SD of triplicate determinations for each group. The significance of differences was analyzed by ANOVA using SPSS. Results For the first series of experiments, MDFs were established from ABCA1 / or wild-type (WT) littermates. In initial studies, we demonstrated that there was no increase of sterol efflux to lipid-free exogenous apoa1 from MDFs isolated from ABCA1 / mice, confirming the complete absence of functional ABCA1 (data not shown). As an initial approach to determining the relationship between ABCA1 and endogenously expressed apoe for cellular lipid efflux, WT or ABCA1 / MDFs were transduced with an adenovirus specifying expression of LacZ (as a control) or apoe. Adenoviralmediated cellular apoe expression (Figure 1C) was equivalent in MDF / and MDF / cells. The endogenous expression of apoe significantly increased sterol and phospholipid efflux from both WT and ABCA1 / cells (Figure 1A and 1B); that is, apoe expression enhanced lipid efflux in an ABCA1-independent manner. The adenoviral expression of apoe in MDF cells leads to the accumulation of extracellular apoe in the medium over the 24-hour time course of efflux measurements. We, therefore, determined whether this extracellular apoe contributed to the apoe-dependent efflux observed in Figure 1 and how

3 Huang et al Macrophage ABCA1-Independent Efflux 159 Figure 2. Sterol efflux to cell-derived exogenous apoe. Conditioned medium from WT MDFs infected with an apoe or a control LacZ adenovirus were collected in 0.1% BSA over 24 hours. The conditioned medium from apoe-expressing cells (apoe CM) or the conditioned medium from LacZ-expressing cells (control CM) were then incubated with MDFs from WT or ABCA1 / mice, and sterol efflux was measured over 6 hours as described in Methods. The results are presented as mean SD from triplicate samples. P 0.02 (*) for the effect of apoe-containing vs control conditioned medium. Figure 1. Effect of apoe expression on cholesterol and phospholipid efflux from ABCA1 / or WT MDFs. MDFs from ABCA1 / or WT mice were labeled with 1 Ci/mL 3 [H]cholesterol and 0.5 Ci/mL 14 [C]choline in 10% FBS/DMEM for 48 hours. Cells were infected with an apoe or LacZ adenovirus, and lipid efflux was measured over 24 hours in 0.1%BSA/DMEM as described in Methods. A, Sterol efflux and (B) phospholipid efflux. Results are shown as mean SD from triplicate samples. P 0.01 (**) or 0.05 (*) for sterol or phospholipid efflux from apoe vs LacZ-expressing cells. C, Immunoblot showing cellular apoe after adenoviral expression of apoe in MDF / and MDF / cells. this related to the expression of ABCA1. ApoE-containing medium was collected from WT MDFs infected with an apoe-expressing adenovirus over 24 hours, or, as a control, from WT MDFs infected with a LacZ adenovirus. Each of these was then added to recipient WT or ABCA1 / MDFs. The exogenous addition of apoe-containing medium produced efflux from WT but not from ABCA1 / MDFs (Figure 2). These results indicate that the extracellular accumulation of apoe, produced by its endogenous expression, facilitates sterol efflux only via an ABCA1-dependent mechanism. Furthermore, they provide important evidence that the ABCA1-independent efflux that results from the endogenous expression of apoe (Figure 1) cannot be related to its extracellular accumulation, because extracellular apoe does not stimulate efflux from ABCA1 / cells. We have shown previously that loading cells with sterol substantially enhances the increment in sterol efflux produced by the endogenous expression of apoe. 7 We next evaluated whether this enhanced efflux response was dependent on the presence of ABCA1. Sterol content in WT or ABCA1 / MDFs was increased using cholesterol/m CD complexes, and the effect of endogenous apoe expression on sterol efflux was evaluated. Incubation with cholesterol/m CD complexes increased the total cholesterol content to and g/mg protein, respectively, in WT and ABCA1 / cells. These cellular sterol levels are similar to levels reported by others using similar sterol-loading conditions. 21,22 The expression of apoe significantly increased sterol efflux from both WT and ABCA1 / sterol-loaded cells (Table 1). The expression of endogenous apoe almost doubled sterol efflux from sterol-loaded ABCA1 / cells (from 6.5% to 11.0%) over 24 hours. We next performed experiments to compare the net reduction of cellular sterol mass produced by expressing apoe in ABCA1-deficient MDFs to that produced by adding exogenous apoa1 to WT MDFs. Before efflux incubations, cells were sterol-loaded using cholesterol:m CD complexes. As shown in Table 2, either the addition of exogenous apoa1 to WT MDF or the expression of apoe in ABCA1 / MDF produced substantial reduction of cell sterol mass. We next evaluated the interaction between apoe and ABCA1-dependent efflux in 2 complementary macrophage TABLE 1. Sterol Efflux From Cholesterol-Enriched MDF Variable WT MDF ABCA1 Knockout MDF Efflux (%)/24 Hours Control ApoE adenovirus * * MDF from ABCA1 knockout or WT mice were loaded with 10 g/ml cholesterol complexed to M CD and tranduced with an apoe-expressing or control adenovirus as described in Methods. Sterol efflux was measured for 24 h in 0.1% BSA/DMEM with 5 g/ml S *P 0.01 for the increase in sterol efflux produced by apoe expression.

4 160 Arterioscler Thromb Vasc Biol. January 2006 TABLE 2. Reduction of Cellular Sterol Mass by ApoE Expression or Incubation With ApoA1 Cell Cholesterol ( g/mg) WT baseline WT 24-h apoa ABCA1 knockout baseline ABCA1 knockout 24-h LacZ ABCA1 knockout 24-h ApoE MDFs from ABCA1 knockout or WT mice were loaded with 5 g/ml cholesterol complexed to M CD as described in Methods. ABCA1 knockout MDFs were transduced with LacZ or apoe adenovirus. For each cell type, the baseline total cholesterol mass is that amount present before a 24-h efflux incubation in 0.2% BSA with 5 g/ml S ApoA1 (5 g/ml) was added to WT MDF. The fall in cell sterol with addition of apoa1 to WT MDF compared with baseline is significant at P The fall in cell sterol with apoe expression in ABCA1 knockout MDF compared with baseline or LacZ expression is significant at P models. We first used the RAW mouse macrophage line that does not express its endogenous apoe gene and generated clones with constitutive expression of apoe by stable transfection. ApoE-expressing clones were selected that produced 0.8 to 1.2 g of apoe per milligram of cell protein over 24 hours in serum-free medium, similar to levels produced by mature human monocyte-derived macrophages. 6 These cells were then used to determine whether reduction of ABCA1 expression (using an sirna) would reduce the apoe-dependent increment in efflux. Expression of apoe in the RAW macrophage line produced a significant increment in sterol efflux (Figure 3A). Incubation with an ABCA1 sirna produced a significant reduction of apoa1- mediated efflux (compare hatched bars in Figure 3A) and ABCA1 expression (Figure 3B) but did not reduce the incremental increase in sterol efflux produced by apoe expression (compare solid bars in Figure 3A). The above experiment demonstrated that the increment in sterol efflux produced by macrophage apoe expression is not reduced by suppression of ABCA1 expression. We next took a complementary approach and determined whether suppressing apoe expression in the absence of ABCA1 would reduce sterol efflux. MPMs were harvested from WT or ABCA1 / mice to compare the effect of reducing apoe expression on efflux. Using biosynthetic labeling and quantitative immunoprecipitation, we first confirmed observations 23 that apoe secretion is reduced from ABCA1 / macrophages. In pulsechase experiments, apoe synthesis (measured after a 45- minute pulse-labeling period) was similar in WT versus ABCA1 / MPMs, but apoe secretion (measured after a 90-minute chase) was decreased by 49% (data not shown). WT and ABCA1 / macrophages were incubated with an sirna for apoe. This incubation reduced apoe present in cell lysates by 80% (Figure 4B) and reduced sterol efflux from both WT and ABCA1 / mouse peritoneal macrophages (Figure 4A). In ABCA1 / macrophages, suppressing apoe expression reduced sterol efflux from 4.0% to 2.8% over 6 hours (P 0.05). Therefore, in aggregate, the results in Figure 4A show that sterol efflux is lower in ABCA1 / macrophages compared with WT macrophages exposed to a control Figure 3. Effect of reduced ABCA1 expression on apoedependent sterol efflux from macrophages. A, RAW-C and RAW-E cells were labeled with 1 Ci/mL 3 [H]cholesterol for 48 hours in 10% FBS/DMEM. Cells were incubated with an sirna to ABCA1 or a control sirna, and sterol efflux was measured over 4 hours as described in Methods. ApoA1 (10 g/ml) was added where indicated. Results are mean SD from triplicate samples. *P 0.05 for the difference in efflux between RAW-C and RAW-E cells. B, Cell lysates from RAW cells treated with a control or ABCA1 sirna were used for ABCA1 immunoblot as described in Methods. sirna, likely related to both absent ABCA1 expression and reduced apoe secretion subsequent to absent ABCA1 expression. However, additional reduction of apoe expression in ABCA1 / macrophages produces an additional significant decrease in sterol efflux. Discussion The studies in this report clearly establish that the expression of endogenous apoe can mediate lipid efflux from cells independent of ABCA1 expression. Furthermore, these studies provide evidence that the sterol efflux produced by the extracellular accumulation of endogenously synthesized apoe is completely dependent on the expression of ABCA1. The ABCA1-independent efflux produced by endogenous cellular apoe expression, therefore, results entirely from the intracellular synthesis and transport of nascent apoe. Thus, apoeexpressing cells, most significantly macrophages, can utilize a novel efflux pathway that functions independently of ABCA1 expression to defend cell sterol balance. We have reported previously that the endogenous expression of apoe is more effective for stimulating macrophage sterol efflux compared with its exogenous addition, even when exogenous apoe is added at a final concentration that far exceeds that produced by the endogenous expression of apoe. 7 The results in this report provide a mechanism for this observation. The endogenous synthesis of apoe produces efflux by ABCA1-

5 Huang et al Macrophage ABCA1-Independent Efflux 161 Figure 4. Effect of reduced apoe expression on sterol efflux from ABCA1 / macrophages. A, MPMs from ABCA1 knockout (KO) or WT mice were labeled with 1 Ci/mL 3 [H]cholesterol in 10% FBS/ DMEM for 48 hours. Cells were incubated with sirna to apoe or with a control sirna, and sterol efflux was measured over 6 hours in 0.1% BSA/DMEM with 1 mg/ml phosphatidylcholine liposomes 7 as described in Methods. The results are presented as mean SD from triplicate samples. *P 0.05 for the decrease in sterol efflux produced by the apoe sirna. B, Cell lysates from mouse peritoneal macrophages treated with a control or apoe sirna were used for apoe immunoblot as described in Methods. independent and -dependent pathways, whereas its exogenous addition uses the ABCA1-dependent pathway only. In most experiments, lipid efflux was higher when ABCA1 expression was intact. In the case of macrophages or of MDFs infected with an apoe adenovirus, this could be explained by the ABCA1-dependent component of apoe-mediated efflux. We also noted, however, that lipid release was lower from ABCA1 / compared with WT MDFs (Figure 1) and from RAW cells incubated with an ABCA1 sirna (Figure 3), even in the absence of apoe expression. It has been shown that ABCA1 facilitates transport of lipid from internal membranes to the plasma membrane and also modulates the distribution of plasma membrane sterol between raft and nonraft domains. These effects of ABCA1 expression can be observed even in the absence of extracellular apolipoprotein acceptors. 24,25 It has also been shown that BSA can be a nonspecific, low-affinity but high-capacity acceptor for plasma membrane sterol. 26 Therefore, the reduction of efflux observed from ABCA1 / cells in the presence of BSA alone may be related to a reduced availability of plasma membrane lipid for nonspecific release to this high-capacity acceptor. Our results also show that sterol efflux mediated by endogenous apoe expression is substantially magnified in cells that are enriched with sterol (Tables 1 and 2). This occurs in both WT and ABCA1 / cells. In ABCA1 / cells, the increment in efflux cannot relate to any effect of sterol on ABCA1 expression, and we hypothesize that the effect of sterol loading to enhance sterol efflux in ABCA1 / cells could be mediated by sterol enrichment of cellular membranes used as a source for lipid efflux by endogenous apoe. What mechanisms can be considered for the lipid efflux facilitated by endogenous expression of apoe? Scavenger receptor (SR)-BI is expressed by macrophages and can, under some circumstances, facilitate sterol efflux However, SR-BI is not likely involved in the current observations, because we have shown previously that increased SR-BI expression in macrophages reduces the sterol efflux that is dependent on the endogenous expression of apoe. 29 Other laboratories have reported ABCA1-independent efflux to exogenous apoa1 or to exogenous synthetic amphipathic helical peptides; 30,31 and the involvement of other ABC transporters has been suggested. Macrophages have been shown to express ABCG1 and ABCA However, ABCG1 is most effective in promoting efflux to high-density lipoprotein particles and is ineffective when lipid-poor apolipoproteins are provided as acceptors. The role of ABCA7 for facilitating macrophage lipid efflux is less clear. 32,33 Cholesterol and phospholipid efflux do not differ in macrophages isolated from ABCA7 null mice compared with controls, suggesting that ABCA7 may not play an important role in modulating macrophage lipid flux. 36 The results in this report (Figure 2) argue that ABCA1 is the primary ABC transporter for mediating sterol efflux to cell-derived extracellular apoe. It is easy to envision that endogenously synthesized apoe may not require the function of a lipid transporter or of a plasma membrane receptor to facilitate lipid efflux from cells. The endogenous synthesis and movement of apoe through cellular membranes comprising the endoplasmic reticulum, Golgi, secretory vesicles, and, finally, the plasma membrane may facilitate solublization of membrane lipid by apoe COOH-terminal amphipathic -helical domains with subsequent assembly of a lipid-containing particle before release of apoe from the cell. The secretion of apoe already complexed to cellular membrane sterol and phospholipid would produce a net loss of lipid from macrophages. Consistent with this notion, we have shown previously that the lipid composition of intracellular membranes is an important factor for regulating the post-translational handling of apoe in the macrophage. 20,38 Our observations establish a novel pathway for sterol and phospholipid efflux that is available to cells that synthesize apoe. The cellular synthesis of apoe stimulates sterol efflux by both ABCA1-dependent and -independent pathways. For the former pathway, endogenously synthesized apoe accumulates in the pericellular space and acquires cellular lipid via an ABCA1-dependent pathway. This pathway could involve ABCA1-dependent internalization and recycling of pericellular apoe. 39 For the ABCA1-independent pathway, the intracellular synthesis and transport of apoe through internal membranes before secretion is required. In several of our experiments, it is possible to compare the magnitude of sterol efflux mediated by ABCA1-apoA1 interactions compared with that mediated by an apoe-dependent, but ABCA1-independent, pathway. In RAW cells, the addition of exogenous apoa1 (10 g/ml) increases sterol efflux from 2.5% to 15% (Figure 3). In the same experiment, the expression of endogenous apoe, after knockdown of ABCA1 expression, increases sterol efflux from 1.6% to 3.8%. In cholesterol-loaded MDFs (Table 2), the addition of exogenous apoa1 reduced cell sterol content by 19%. The expression of apoe in ABCA1 / MDFs reduced cell sterol by 23% (compared with LacZ expression). In these experimental

6 162 Arterioscler Thromb Vasc Biol. January 2006 models, therefore, the ABCA1-apoA1 pathway, and the apoe-dependent but A CA1-independent pathway, can each mediate significant sterol efflux from cells. The relative importance of each of these efflux pathways in vivo may be difficult to predict. Macrophage-specific deletion of either ABCA1 or apoe leads to increased atherosclerosis and macrophage-derived vessel wall foam cell formation. This observation suggests that each pathway plays an important and unique role in defending vessel wall lipid homeostasis. Acknowledgments This work was supported by grant HL (to T.M.) from the National Institutes of Health. We thank Stephanie Thompson for assistance with manuscript preparation. References 1. O Brien KD, Deeb SS, Ferguson M, McDonald TO, Allen MD, Alpers CE, Chait A. Apolipoprotein E localization in human coronary atherosclerotic plaques by in situ hybridization and immunohistochemistry and comparison with lipoprotein lipase. Am J Pathol. 1994;144: Hasty AH, Linton MF, Brandt SJ, Babaev VR, Gleaves LA, Fazio S. Retroviral gene therapy in apoe-deficient mice: apoe expression in the artery wall reduces early foam cell lesion formation. Circulation. 1999;99: Fazio S, Babaev VR, Murray AB, Hasty AH, Carter KJ, Gleaves LA, Atkinson JB, Linton MF. Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages. Proc Natl Acad Sci U S A. 1997;94: Curtiss LK, Boisvert WA. Apolipoprotein E and atherosclerosis. Curr Opin Lipidology. 2000;11: Mazzone T. ApoE secretion by macrophages; its potential physiological function(s). Curr Opin Lipidology. 1996;7: Mazzone T, Reardon C. Expression of heterologous human apolipoprotein E by J774 macrophages enhances cholesterol efflux to HDL 3. J Lipid Res. 1994;35: Lin C-Y, Duan H, Mazzone T. Apolipoprotein E-dependent cholesterol efflux from macrophages: kinetic analysis and divergent mechanism for endogenous versus exogenous apolipoprotein E. J Lipid Res. 1999;40: Yao PM, Tabas I. Free cholesterol loading of macrophages induces apoptosis involving the fas pathway. J Biol Chem. 2000;275: Dean M, Hamon Y, Chimini G. The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res. 2001;42: Tall AR, Costet P, Wang N. Regulation and mechanisms of macrophage cholesterol efflux. J Clin Invest. 2002;110: Krimbou L, Denis M, Haidar B, Carrier M, Marcil M, Genest J. Molecular interactions between apoe and ABCA1: impact on apoe lipidation. J Lipid Res. 2004;45: Fitzgerald ML, Morris AL, Chroni A, Mendez AJ, Zannis VI, Freeman MW. ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux. J Lipid Res. 2004;45: Aiello RJ, Brees D, Bourassa P-A, Royer L, Lindsey S, Coskran T, Haghpassand M, Francone OL. Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages. Arterioscler Thromb Vasc Biol. 2002;22: Van Eck M, Bos IST, Kaminski WE, Orso E, Rothe G, Twisk J, Bottcher A, Van Amersfoort ES, Christiansen-Weber TA, Fung-Leung W-P, Van Berkel TJC, Schmitz G. Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues. Proc Natl Acad Sci U S A. 2002;99: Dove DE, Linton MF, Fazio S. ApoE-mediated cholesterol efflux from macrophages: separation of autocrine and paracrine effects. Am J Physiol Cell Physiol. 2005;288:C586 C Huang ZH, Lin C-Y, Oram JF, Mazzone T. Sterol efflux mediated by endogenous macrophage apoe expression is independent of ABCA1. Arterioscler Thromb Vasc Biol. 2001;21: Kiss RS, Maric J, Marcel YL. Lipid efflux in human and mouse macrophagic cells: evidence for differential regulation of phospholipid and cholesterol efflux. J Lipid Res. 2005;46: Fitzgerald ML, Mendez AJ, Moore KJ, Andersson L, Panjeton H, Freeman M. ATP-binding cassette transporter A1 contains an NH2-termial signal anchor sequence that translocates the protein s first hydrophilic domain to the exoplasmic space. J Biol Chem. 2001;276: Mazzone T, Pustelnikus L, Reardon CA. Post-translational regulation of macrophage apoprotein E production. J Biol Chem. 1992;267: Zhao Y, Mazzone T. Transport and processing of endogenously synthesized apoe on the macrophage cell surface. J Biol Chem. 2000;275: Christian AE, Haynes MP, Phillips MC, Rothblat GH. Use of cyclodextrins for manipulating cellular cholesterol content. J Lipid Res. 1997; 38: Blom TS, Koivusalo M, Kuismanen E, Kostiainen R, Somerharju P, Ikonen E. Mass spectrometric analysis reveals an increase in plasma membrane polyunsaturated phospholipid species upon cellular cholesterol loading. Biochemistry. 2005;40: Von Eckardstein A, Langer C, Engel T, Schaukal I, Cignarella A, Reinhardt J, Lorkowski S, Li Z, Zhou X, Cullen P, Assmann G. ATP binding cassette transporter ABCA1 modulates the secretion of apolipoprotein E from human monocyte-derived macrophages. FASEB J. 2001;15: Vaughn AM, Oram JF. ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions. J Lipid Res. 2003;44: Orso E, Broccardo C, Kaminski WE, Bottcher A, Liebisch G, Drobnik W, Gotz A, Chambenoir O, Diederich W, Langmann T, Spruss T, Luciani M-F, Rothe G, Lackner KJ, Chimini G, Schmitz G. Transport of lipids from Golgi to plasma membrane is defective in Tangier disease patients and Abc1-deficient mice. Nature Genet. 2000;24: Zhao Y, Marcel YL. Serum albumin in a significant intermediate in cholesterol transfer between cells and lipoproteins. Biochemistry. 1996; 35: Williams DL, Connelly MA, Temel RE, Swarnakar S, Phillips MC, de la Llera-Moya M, Rothblat G. Scavenger receptor BI and cholesterol trafficking. Curr Opin Lipidology. 1999;10: Huang ZH, Gu D, Lange Y, Mazzone T. Expression of scavenger receptor BI facilitates sterol movement between the plasma membrane and endoplasmic reticulum in macrophages. Biochemistry. 2003;42: Huang ZH, Mazzone T. Apolipoprotein E-dependent sterol efflux from macrophages is modulated by scavenger receptor BI expression. J Lipid Res. 2002;43: Remaley AT, Thomas F, Stonik JA, Demosky SJ, Bark SE, Neufeld EB, Bocharov AV, Vishnyakova TG, Patterson AP, Eggerman TL, Santamarina-Fojo S, Brewer HB. Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1- independent pathway. J Lipid Res. 2003;44: Walter M, Forsyth NR, Wright WE, Shay JW, Roth MG. The establishment of telomerase-immortalized Tangier disease cell lines indicates the existence of an apolipoprotein A-I-inducible but ABCA1-independent cholesterol efflux pathway. J Biol Chem. 2004;279: Wang N, Lan D, Gerbod-Giannone M, Linsel-Nitschke P, Jehle AW, Chen W, Martinez LO, Tall AR. ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux. J Biol Chem. 2003;278: Abe-Dohmae S, Ikeda Y, Matsuo M, Hayashi M, Okuhira K-I, Ueda K, Yokoyama S. Human ABCA7 supports apolipoprotein-mediated release of cellular cholesterol and phospholipid to generate high density lipoprotein. J Biol Chem. 2004;279: Cignarella A, Engel T, Von Eckardstein A, Kratz M, Lorkowski S, Lueken A, Assmann G, Cullen P. Pharmacological regulation of cholesterol efflux in human monocyte-derived macrophages in the absence of exogenous cholesterol acceptors. Atherosclerosis. 2005;179: Wang N, Lan D, Matsuura F, Tall AR. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci U S A. 2004;101: Kim WS, Fitzgerald ML, Kang K, Okuhira K-I, Bell SA, Manning JJ, Koehn SL, Lu N, Moore KJ, Freeman MW. Abca7 null mice retain normal macrophage phosphatidylcholine and cholesterol efflux activity despite alterations in adipose mass and serum cholesterol levels. J Biol Chem. 2005;280: Schmitz G, Langmann T, Heimerl S. Role of ABCG1 and other ABCG family members in lipid metabolism. J Lipid Res. 2001;42: Duan H, Lin C-Y, Mazzone T. Degradation of macrophage apoe in a non-lysosomal compartment: regulation by sterols. J Biol Chem. 1997; 272: Hasty AH, Plummer MR, Weisgraber KH, Linton MF, Fazio S, Swift LL. The recycling of apolipoprotein E in macrophages: influence of HDL and apolipoprotein A-I. J Lipid Res. 2005;46: