Deletion of ABCA1 and ABCG1 Impairs Macrophage Migration Because of Increased Rac1 Signaling

Abstract

Rationale

Reduced plasma cholesterol and increased high-density lipoprotein (HDL) levels promote regression of atherosclerosis, in a process characterized by lipid unloading and emigration of macrophages from lesions. In contrast free cholesterol loading of macrophages leads to imbalanced Rac1/Rho activities and impaired chemotaxis.

Objective

To study the role of HDL and the ATP-binding cassette transporters ABCA1 and ABCG1 in modulating the chemotaxis of macrophages.

Methods and Results

Abca1^−/−Abcg1^−/− mouse macrophages displayed profoundly impaired chemotaxis both in a Transwell chamber assay and in the peritoneal cavity of wild-type (WT) mice. HDL reversed impaired chemotaxis in free cholesterol–loaded WT macrophages but was without effect in Abca1^−/−Abcg1^−/− cells, whereas cyclodextrin was effective in both. Abca1^−/−Abcg1^−/− macrophages had markedly increased Rac1 activity and increased association of Rac1 with the plasma membrane (PM). Their defective chemotaxis was reversed by a Rac1 inhibitor. To gain a better understanding of the role of transporters in PM cholesterol movement, we measured transbilayer PM sterol distribution. In WT macrophages, the majority of cholesterol was located on the inner leaflet, whereas on upregulation of transporters by liver X receptor activation, PM sterol was shifted to the outer leaflet, where it could be removed by HDL. Abca1^−/−Abcg1^−/− macrophages showed increased PM sterol content and defective redistribution of sterol to the outer leaflet.

Conclusions

Deletion of ABCA1 and ABCG1 causes an increased cholesterol content on the inner leaflet of the PM, associated with increased Rac1 PM localization, activation, and impairment of migration. ABCA1 and ABCG1 facilitate macrophage chemotaxis by promoting PM transbilayer cholesterol movement and may contribute to the ability of HDL to promote regression of atherosclerosis.

Raising high-density lipoprotein (HDL) levels has emerged as a potential therapeutic intervention in atherosclerotic cardiovascular disease. Clinical trials of reconstituted human HDL infusions in patients with established cardiovascular disease demonstrated a significant reduction in atheroma volume suggesting active regression.^1,2 Regression of atherosclerosis by lowering cholesterol or increasing HDL has also been shown in mouse models.^3–5 Using a surgical approach, aortic segments with established lesions from hypercholesterolemic Apoe^−/− mice were transplanted into wild-type (WT) and Apoe^−/− recipients.³ Transplantation of lesions into WT recipients resulted in depletion of foam cells and a marked regression of atherosclerotic plaques, whereas in Apoe^−/− recipients with high cholesterol and low HDL levels, further lesion progression was seen.^4,6 The reduction of plaque area was correlated with emigration of macrophages to regional and systemic lymph nodes. Laser capture microscopy analysis of foam cells from regressing lesions indicated up regulation of liver X receptor (LXR) and ABCA1.⁷ Furthermore, in Apoe^−/−Apoa1^Tg recipients with elevated HDL levels lipid unloading and depletion of macrophage foam cells from plaques were observed despite high cholesterol levels.⁵ This suggests that increased HDL perhaps acting in conjunction with ABC transporters promotes cholesterol efflux and stimulates emigration of macrophages from plaques.

In contrast to the beneficial effects of cholesterol efflux, plasma membrane (PM) free cholesterol loading caused impaired chemotaxis of macrophages, associated with increased levels of Rac-GTP and reduced RhoA activity.^8,9 A number of molecular events need to be integrated to allow a cell to become motile. Small Rho-GTPases play a pivotal role in actin filament organization and therefore in the coordination of cell migration.¹⁰ Rho-GTPases function as molecular switches in which the exchange of GDP for GTP triggers a conformational change that allows binding and activation of downstream effectors to direct cytoskeleton remodeling. To be in their active state, small GTPases translocate to the PM,¹¹ which is facilitated by post-translational isoprenylation and fatty acylation.

The ATP-binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux to apolipoprotein (apo)AI and HDL, respectively.^12,13 Hypercholesterolemic Ldlr^+/− mice transplanted with Abca1^−/−Abcg1^−/− bone marrow show significantly accelerated atherosclerosis and foam cell accumulation in various tissues.^14,15 Of particular interest is the ability of ABCA1 and ABCG1 to modulate PM cholesterol levels and lipid organization.¹⁶ PM staining with the ganglioside-binding cholera toxin B has suggested an increase of liquid ordered membrane domains (lipid rafts) in Abca1^−/− or Abcg1^−/− macrophages and an even more pronounced increase in Abca1^−/−Abcg1^−/− macrophages.^17–21 In this study, we initially explored the hypothesis that HDL and ABC transporters might act to facilitate chemotaxis of cholesterol-loaded macrophages. We further investigated the relationship of impaired chemotaxis of Abca1^−/−Abcg1^−/− cells to Rac1 activation, and related this to changes in PM lipid organization, using a novel fluorescence technique to monitor transbilayer sterol distribution.²²

Methods

An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.

Animals

WT and Abca1^−/−Abcg1^−/− littermates in a mixed C57BL/6×DBA background, as described previously, were used.¹⁴ All mice were housed at Columbia University Medical Center according to animal welfare guidelines. Animals had ad libitum access to food and water.

Sterol Transbilayer Distribution

As described previously²² cells were trace-labeled with cholestatrienol-methyl-β-cyclodextrin (CTL-MβCD) complexes (0.5 mmol/L) for 1 minute at 37°C, washed, and then incubated for 10 minutes at 37°C to reach a steady-state distribution of CTL. Fluorescence images were taken before and after addition of the membrane impermeable fluorescence quencher 2,4,6-trinitrobenzenesulphonic acid (TNBS) at 10 mmol/L. The PM associated CTL-fluorescence was measured before and after quenching and depicted as percentage quenching.

Results

Abca1^−/−Abcg1^−/− Macrophages Show Impaired Migratory Responses

The ability of WT and Abca1^−/−Abcg1^−/− macrophages (thioglycollate-elicited) to migrate toward the strong chemoattractant C5a was determined using a Transwell chamber. Chemotaxis was markedly reduced for Abca1^−/−Abcg1^−/− compared to WT macrophages (Figure 1). Similarly, migration of Abca1^−/−Abcg1^−/− macrophages was also impaired in response to MCP-1 and CCL21, a ligand of CCR7 that has been implicated in regression of lesions⁷ (Online Figure I). Therefore, downstream signaling and cellular responses for various chemokine receptors including atherorelevant CCR2 and CCR7 receptors are likely similar and thus we continued to use C5a for our studies.^23,24 Although combined deficiency of ABCA1 and ABCG1 dramatically reduced migration of macrophages, knock out of ABCG1 or ABCA1 alone did not significantly affect the migration of macrophages (Online Figure II, A and B), consistent with previous data indicating overlapping roles and mutual compensation by these two transporters.¹⁴

Figure 1. Abca1^−/−Abcg1^−/− macrophages show impaired Transwell migration not restorable by HDL.

Thioglycollate-elicited macrophages from WT or Abca1^−/−Abcg1^−/− mice were seeded on Transwell filters, and the migratory capacity was tested by stimulation with 10 nmol/L C5a for 2 hours. Where indicated, cells were treated with 5 mmol/L MβCD-C (6:1) with and without 100 μg/mL HDL₂ for 30 minutes before subjecting cells to migration. Migrated macrophages were quantified after fixation and DAPI staining. Results were normalized to the number of untreated WT macrophages that migrated. The results shown are means±SEM of 5 independent experiments performed in triplicates. ***P<0.001 vs WT–C5a or WT+C5a; **P<0.01 vs WT+C5a; ##P<0.01 vs WT cholesterol-loaded; §§§P<0.001 vs WT+C5a; §§P<0.01 vs WT cholesterol-loaded and HDL-treated.

Increased levels of membrane free cholesterol led to the abrogation of the migration responses in macrophages.⁸ Therefore, we treated WT and Abca1^−/−Abcg1^−/− macrophages with cyclodextrin complexed cholesterol and then carried out the Transwell migration assay (Figure 1). As reported,⁸ there was an almost complete abolition of chemotaxis in WT macrophages with no further impairment in Abca1^−/−Abcg1^−/− macrophage migration. The cholesterol mediated impairment of migration was partly reversed by pretreatment with 100 μg/mL HDL₂ in WT but not in Abca1^−/−Abcg1^−/− macrophages indicating the importance of ABCA1 and ABCG1 mediated cholesterol efflux in the restoration of migratory function. To further test if increased levels of PM cholesterol are the direct cause of impaired migration in Abca1^−/−Abcg1^−/− macrophages we treated control or cholesterol-loaded WT and Abca1^−/−Abcg1^−/− macrophages with cholesterol-poor MβCD before evaluation of migration behavior. Reduction of PM cholesterol completely restored the ability of Abca1^−/−Abcg1^−/− macrophages to migrate, with or without prior cholesterol loading, and reversed the effect of prior cholesterol loading in WT macrophages (Online Figure III).

To study the migratory behavior of Abca1^−/−Abcg1^−/− macrophages in vivo, we used a bead labeling assay. First, equal amounts of red and green labeled WT thioglycollate-elicited macrophages were injected into the peritoneal cavity of thioglycollate pretreated WT recipients. 12 hours after injection the amount of retained labeled macrophages was analyzed (Figure 2A). Similar amounts of WT red and green labeled macrophages were found indicating that the bead label does not influence macrophage behavior. Next, equal amounts of WT (red) and Abca1^−/−Abcg1^−/− (green) labeled macrophages were injected into the peritoneum of WT recipient mice (Figure 2B). Twelve hours after injection, 7-fold more Abca1^−/−Abcg1^−/− macrophages remained in the peritoneal cavity, indicating a profound defect in emigration compared to WT macrophages.

Figure 2. Abca1^−/−Abcg1^−/− macrophages in vivo show reduced emigration from the peritoneal cavity.

WT and Abca1^−/−Abcg1^−/− mice were injected with fluorescence-labeled beads into the peritoneal cavity 3 days after thioglycollate stimulation. Total macrophage population was harvested 1 hour after bead injection, and bead-labeled macrophages were sorted out identified by the bead label and F4/80 staining on a FACS sorter. Subsequently, equal amounts of red and green bead–labeled WT macrophages (A) or red bead–labeled macrophages from WT and green bead–labeled macrophages from Abca1^−/−Abcg1^−/− mice (B) were reinjected into the peritoneum of 3-day thioglycollate-stimulated WT recipient. The amount of labeled macrophages in the cavity was analyzed by FACS 12 hours after injection. Note that significant more Abca1^−/−Abcg1^−/− than WT macrophages remained in the cavity (B). Results are shown as means±SEM of 1 representative experiment, 2 with similar results (number of mice per experiment, n=3). **P<0.01 vs WT.

Increased Actin Polymerization, Membrane Ruffling, and Cell Spreading of Abca1^−/−Abcg1^−/− Macrophages

Cell motility is largely coordinated by the actin cytoskeleton, a key mediator of cell polarization and the directed migration of macrophages. Freshly isolated peritoneal macrophages were stained for F-actin to assess morphological and cytoskeletal changes. Abca1^−/−Abcg1^−/− macrophages exhibited increased ruffling of the cell membrane indicating F-actin polymerization (Online Figure IV). Extension of the cell membrane (lamellipodia formation) was observed surrounding cells (arrowheads). Increased ruffling at the dorsal side of the Abca1^−/−Abcg1^−/− macrophages (arrows) was also seen. Quantification of the cell adhesion area of WT and Abca1^−/−Abcg1^−/− macrophages furthermore showed that Abca1^−/−Abcg1^−/− macrophages are more spread out, with a 2-fold increase in the average cell area (Figure 3). Overall Abca1^−/−Abcg1^−/− macrophages show increased circumferential lamellipodia formation.

Figure 3. Abca1^−/−Abcg1^−/− macrophages show increased PM ruffling and cell spreading.

Thioglycollate-elicited macrophages from WT and Abca1^−/−Abcg1^−/− mice were seeded for 12 hours before fixation and staining for F-actin using TRITC-phalloidin. Quantification of cell adhesive area was done as described in the Online Data Supplement. The results shown are means±SEM of a total of 20 fields analyzed of 2 independent experiments. **P<0.01 vs WT.

Impaired Migration in Abca1^−/−Abcg1^−/− Macrophages Is Attributable to Increased Rac1-GTP Levels

Rho GTPases such as Rac, RhoA, and Cdc42 are well known to play a crucial role in cell migration. Rac1 especially has been linked to membrane ruffle/lamellipodia formation with the highest concentrations of active Rac located at the leading edge of motile cells.^11,25,26 Freshly isolated Abca1^−/−Abcg1^−/− macrophages showed a robust 1.4 fold increase in Rac1 activity as shown by Rac1-GTP levels (Figure 4A). Abca1^−/− or Abcg1^−/− macrophages did not have significantly altered Rac1-GTP levels (Online Figure V, A and B), consistent with their normal migratory ability. To further confirm a link between impaired migration of Abca1^−/−Abcg1^−/− macrophages and increased Rac1 activation we used an inhibitor to reduce Rac activation in a Transwell migration assay. The Rac inhibitor NSC23766 specifically and reversibly targets Rac1 GDP/GTP exchange activity thereby lowering Rac-GTP levels.²⁷ Reduction of Rac1-GTP levels slightly increased migration in WT macrophages and restored migration in Abca1^−/−Abcg1^−/− macrophages to WT levels (Figure 4B). As previous reports indicate a reduction of RhoA-GTP on increased activity of Rac1 we measured RhoA-GTP levels by ELISA (Online Figure VI). We did not find any significant changes in RhoA activity in Abca1^−/−Abcg1^−/− macrophages. However, impaired chemotaxis is thought to reflect an imbalance in Rac/Rho activity, consistent with our results. Rho-GTPases need to link to the PM to become biologically active. Therefore we studied the subcellular localization of Rac in WT and Abca1^−/−Abcg1^−/− macrophages. Western blot analysis showed a 2-fold increase in Rac in the PM fraction of Abca1^−/−Abcg1^−/− macrophages (Figure 5A and 5B).

Figure 4. Abca1^−/−Abcg1^−/− macrophages exhibit markedly increased Rac1 activation.

Thioglycollate-elicited macrophages from WT and Abca1^−/−Abcg1^−/− mice were serum-depleted for 24 hours before cell lysates were analyzed for Rac1-GTP levels using a commercially available ELISA kit (A). Thioglycollate-elicited macrophages from WT and Abca1^−/−Abcg1^−/− mice were subjected to 2 hours of Transwell migration as described in Figure 1. Where indicated, macrophages were treated with the Rac inhibitor NSC23766 (100 μmol/L) during the time of migration. The results shown are means±SEM of 3 independent experiments performed in triplicates. **P<0.05 vs WT (A); ***P<0.001 vs WT–C5a; #P<0.05 vs WT+C5a; ###P<0.001 vs Abca1^−/−Abcg1^−/−+C5a; §§§P<0.001 vs WT+C5a (B).

Figure 5. Increased Rac localization to the PM of Abca1^−/−Abcg1^−/− macrophages.

Thioglycollate-elicited macrophages from WT and Abca1^−/−Abcg1^−/− macrophages were plated overnight in regular growth media and subsequently serum-starved for 48 hours. Cells were then lysed in hypertonic buffer and subjected to ultracentrifugation to separate the PM from the cytosol fraction. An immunoblot for Rac1,2,3 was performed. Representative blot of Rac positive signal at 20 kDa is shown (A). Band densities normalized to β-actin are shown in a bar graph (B). Data represent means±SEM of a total of 4 to 5 mice per group in 2 independent experiments. *P<0.05 vs WT.

Abca1^−/−Abcg1^−/− Macrophages Show Increased PM Sterol and an Inability to Move Sterol From the Inner to the Outer Leaflet of the PM Bilayer

PM cholesterol levels have an important role in the regulation of cell migration.^8,28,29 To directly investigate whether excess levels of PM cholesterol are the underlying cause of the migratory dysfunction in Abca1^−/−Abcg1^−/− macrophages PM cholesterol levels were measured in freshly isolated WT and Abca1^−/−Abcg1^−/− peritoneal macrophages using filipin staining. Abca1^−/−Abcg1^−/− macrophages showed a 1.4-fold increase of filipin staining, suggesting increased free cholesterol in the PM (Figure 6), in agreement with previous data showing an increase in cholesterol content in freshly isolated Abca1^−/−Abcg1^−/− macrophages.¹⁴

Figure 6. Increased PM filipin staining in Abca1^−/−Abcg1^−/− macrophages.

Thioglycollate-elicited macrophages from WT and Abca1^−/−Abcg1^−/− mice were seeded for 4 hours and subsequently fixed and stained with filipin. Images were taken, and the average fluorescence intensity along the periphery indicating the PM was measured to determine the relative free cholesterol levels. Results are shown as means±SEM of a total of 30 fields analyzed per group in 2 independent experiments. ***P<0.001 vs WT.

To gain a better understanding of the role of ABCA1 and Abcg1 in PM sterol distribution across the PM bilayer we used a novel method using cholestatrienol (CTL) to measure sterol distribution between the inner and the outer leaflet of the PM.²² The proportion of CTL in the outer leaflet of the PM was measured by monitoring quenching of fluorescent CTL by the extracellular, membrane nonpermeable quencher TNBS (Figure 7A and 7B). Consistent with previous findings,²² approximately 20% of the PM sterol is quenched by TNBS in WT and Abca1^−/−Abcg1^−/− macrophages in the basal state (Figure 7C). On preloading cells with acetylated low-density lipoprotein (acLDL) and the LXR agonist T091317 overnight, WT macrophages increased the quenchable pool of sterol to 27%. Either treatment alone was insufficient to significantly increase this parameter (Online Figure VII). Strikingly, Abca1^−/−Abcg1^−/− macrophages were not able to increase the amount of sterol that was quenchable by extracellular TNBS. Treatment of cholesterol-loaded WT or Abca1^−/−Abcg1^−/− macrophage with HDL₂ led to a significant reduction of outer-leaflet sterol in WT cells, but to no change in Abca1^−/−Abcg1^−/− macrophages.

Figure 7. Abca1^−/−Abcg1^−/− macrophages are unable to move sterol from the inner to the outer PM leaflet.

Transbilayer PM cholesterol distribution was measured in macrophages from WT and Abca1^−/−Abcg1^−/− mice. Macrophages were trace-labeled with the fluorescent cholesterol analog MβCD-cholestatrienol for 1 minute, washed, and equilibrated for 10 minutes at 37°C. Subsequently, images were taken by focusing at the PM before the addition of the cell membrane nonpermeable quenching reagent TNBS (A). Distribution of CTL to PM, ER, and lipid droplets was observed (B). TNBS at 10 mmol/L was added on the microscope stage, and images of the same field were taken within 1 minute; note the decrease of fluorescence intensity after TNBS addition within the yellow circles (B). The quenching of exofacial incorporated CTL by TNBS was measured at multiple peripheral areas along the PM of each cell and depicted as percentage quenching (C). Where indicated, cells were treated with acLDL (50 μg/mL) and the LXR compound TO-901317 (3 μmol/L). To test the reversibility of this treatment, 100 μg/mL HDL₂ was added in a separate group. A total of 30 to 100 cells for each treatment condition were analyzed of 3 independent experiments. Data are shown as means±SEM. ***P<0.001 vs basal WT; #P<0.05 vs acLDL+T0 WT; §§§P<0.001 vs WT acLDL+T0.

In contrast, Abca1^−/− and Abcg1^−/− macrophages showed the same ratio of inner to outer leaflet cholesterol in basal and cholesterol loaded states as WT macrophages (Online Figure VIII), indicating mutual compensation of transporters in single knockout cells.

Discussion

Herein, we demonstrate the importance of the cholesterol transporters ABCA1 and ABCG1 in macrophage chemotaxis. Increased PM cholesterol content and an inability to move sterol across the PM bilayer in Abca1^−/−Abcg1^−/− macrophages are associated with increased PM localization and activation of Rac1, enhanced PM ruffling, cell spreading, and abrogation of migration toward various chemotactic stimuli including C5a, MCP-1, and CCL21. Abca1^−/−Abcg1^−/− myeloid cells show multiple abnormalities in cell motility, proliferation in response to growth factors,³⁰ and viability³¹ that likely contribute to accelerated atherosclerosis in Abca1^−/−Abcg1^−/− mice. The present study suggests that a fundamental underlying cellular mechanism may be altered transbilayer sterol distribution, formation of cholesterol-rich liquid ordered domains on the PM, and excessive activation of small GTPases such as Rac1 and Ras.³⁰

Although the detailed molecular mechanisms and sites of activity of ABCA1 and ABCG1 remain poorly understood, our findings show for the first time that both transporters promote the movement of sterol across the PM bilayer, where it becomes accessible to HDL for removal. Moreover, this activity is observed in the absence of HDL (Figure 7C) and probably explains the finding that transporter deficiency leads to suppression of endoplasmic reticulum (ER) sterol-regulated genes, because depletion of the inner leaflet of the PM of sterol by transporter activity should lead to rapid diffusional replenishment from other organelles such as ER.^32–36

Abca1^−/−Abcg1^−/− macrophages showed profoundly impaired chemotaxis both in a Transwell chamber assay and in vivo. During migration cells undergo the so called cell motility cycle starting with the polarization of the cell and resulting in extensions of lamellipodia at the leading edge.³⁷ This is followed by the formation of focal adhesions to the underlying substrate and ends in the contraction and detachment at the rear and the movement of the cell. These multiple steps depend on coordinated regulation by the Rho Family of small GTPases. We found an increase in the active form of the small GTPase Rac1 in double deficient macrophages. The role of increased Rac1-GTP for the observed migration defect in Abca1^−/−Abcg1^−/− macrophages was confirmed by the ability to restore migration by reducing Rac1-GTP levels. Active Rac at the leading edge of lamellipodia is thought to mediate actin polymerization, producing lamellar extension and forward cell movement.^38,39 Strong enhancement of Rac activity was shown to disrupt cell migration with a substantial decrease in velocity.⁴⁰ It seems that a tight regulation must be kept on the level of Rac activity and that substantial increase in Rac activity leads to increased attachment with the substratum and/or the loss of correct polarization keeping the cell fixed to the substratum and immobile.

Given that the total cholesterol content in the PM of Abca1^−/−Abcg1^−/− macrophages is increased, our findings indicate an increase in the sterol content and very likely an increase in formation of liquid ordered domains on the inner leaflet of the PM in Abca1^−/−Abcg1^−/− cells. Rac activation is accompanied by its translocation to the PM. The capacity of small GTPases like Rac to cycle on and off membranes is thought to be integral to their biological activity as they stay in their active GTP state as long as they are associated with the membrane. Consistent with this we found increased levels of Rac at the PM in Abca1^−/−Abcg1^−/− macrophages. Recently we showed that the combined deficiency of ABCA1 and ABCG1 in hematopoietic stem cells leads to an increase in Ras protein in the PM in vivo promoting hyper-proliferation in response to growth factors.³⁰ In addition, Abca1^−/−Abcg1^−/− macrophages show increased Nox2 assembly in the PM and in phagolysosomes as macrophages ingest apoptotic cells.³¹ Because the assembly of Nox2 complexes is dependent on the membrane recruitment of Rac1,^41–43 increased Rac1 activation may also be involved in increased Nox-2 assembly in Abca1^−/−Abcg1^−/− macrophages. Thus, HDL acting in conjunction with ABCA1/G1 may serve to limit formation of cholesterol-rich domains on the inner leaflet of the PM of cholesterol-loaded macrophages, and thus modulate signaling via small GTPases such as Ras and Rac1, affecting myeloid cell proliferation, as well as cell motility and reactive oxygen species formation during efferocytosis in macrophages.

The progression of atherosclerotic lesions is marked by an inability of macrophage foam cells to leave the artery. By using a well-described peritoneal cavity emigration model,^44–46 we showed that macrophages from Abcal^−/−Abcgl^−/− mice have a substantial defect in their migration behavior in vivo. In contrast to the present findings, studies in resident macrophages¹⁸ or in blood monocytes⁴⁷ show that cholesterol removal via the ABCA1/apoAI pathway can inhibit migration. Thus, ABC transporters and HDL may act to inhibit monocytosis³⁰ and monocyte entry, as well as macrophage retention in plaques. In addition to increased PM cholesterol levels, a variety of other factors may be involved, including altered expression or activity of chemokine receptors such as CCR2 or CCR7.^7,24 Exposure of macrophages to oxidized LDL or minimally modified LDL results in peripheral actin polymerization and lamellipodia formation similar to that described here, probably reflecting the effects of lipid hydroperoxides.⁴⁸ This suggests that multiple mechanisms in plaques may contribute to the immobilization of macrophages.

Novelty and Significance.

What Is Known?

• High-density lipoprotein (HDL) exerts antiatherogenic effects in part by promoting cholesterol efflux from macrophage foam cells via the ATP-binding cassette transporters ABCA1 and ABCG1.

• Increasing HDL-cholesterol concentrations promotes regression of atherosclerosis in mouse models, and this is associated with emigration of macrophages from lesions.

What New Information Does This Article Contribute?

• Abca1^−/−Abca1^−/− macrophages have severely impaired chemotaxis attributable to plasma membrane cholesterol accumulation and excessive Rac-1 activation.

• HDL can improve impaired chemotaxis in cholesterol loaded wild-type macrophages, but not in Abca1^−/−Abca1^−/− macrophages.

• ABCA1 and ABCG1 promote cholesterol movement from the inner to the outer leaflet of plasma membranes, and thus maintain correct localization and activity of Rac-1.

Therapeutic approaches to raising HDL-cholesterol concentrations are at the forefront of atherosclerosis clinical research. Recent studies in mice have shown that increased HDL may promote emigration of monocyte/macrophages from atherosclerotic plaques. A major property of HDL is its ability to mediate cholesterol efflux from macrophage foam cells in part via ABCA1 and ABCG1. We evaluated the hypothesis that macrophage chemotaxis might be promoted by ABCA1/G1 and HDL. We showed that HDL could reverse severely impaired chemotaxis of cholesterol-loaded wild-type macrophages, but not of macrophages lacking these ABC transporters. In further studies, we used a novel biophysical technique to demonstrate that ABCA1/G1 promote cholesterol movement from the inner to the outer leaflet of the plasma membrane. In the absence of transporters, sterol accumulation in the inner membrane leads to increased Rac-1 activity and thus impaired chemotaxis. The modulation of small GTPases that are membrane tethered appears to be a fundamental cellular effect of HDL and the ABC transporters that influences a variety of cellular processes including cell motility and proliferative responses. Thus, this study elucidates basic cellular mechanisms that are directly relevant to mechanisms by which increasing plasma HDL concentrations may produce regression of atherosclerotic plaques.

Non-standard Abbreviations and Acronyms

ABC

ATP-binding cassette transporter

acLDL

acetylated low-density lipoprotein

apo

apolipoprotein

CTL

cholestatrienol

ER

endoplasmic reticulum

HDL

high-density lipoprotein

LDL

low-density lipoprotein

LXR

liver X receptor

MβCD-C

methyl-β-cyclodextrin-cholesterol

OxLDL

oxidized low-density lipoprotein

PM

plasma membrane

ROS

reactive oxygen species

TNBS

2,4,6-trinitrobenzenesulphonic acid

WT

wild type