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【摘要】
Objective- During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within atherosclerotic lesions.
Methods and Results- We examined the effects of elevating membrane cholesterol on macrophage behavior. J774 macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-ß-cyclodextrin (chol-MßCD) to increase membrane cholesterol levels. Our results show that elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages was significantly inhibited when membrane cholesterol levels were raised.
Conclusions- These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.
【关键词】 actin atherosclerosis cholesterol macrophages migration
Introduction
Although it has long been accepted that elevated serum cholesterol levels correlate with development of atherosclerotic lesions, the reasons for this are imperfectly understood. It is known that the cholesterol carrier, low-density lipoprotein (LDL), normally found circulating in the blood, is deposited in the subendothelial space. 1,2 These LDL deposits undergo aggregation, association with extracellular matrix materials, and oxidation. 3,4 Monocytes that have traversed the endothelium then engulf the matrix-bound aggregated lipoprotein particles and develop into lipid-filled macrophages (known as foam cells). Macrophage lipid accumulation, and subsequent foam cell formation, promotes growth of the atherosclerotic plaque, whereas lesion regression, induced pharmacologically or through diet, may be attributed partly to foam cell migration out of the area. This synopsis illustrates the critical role of cell migration in the progression of atherosclerosis and suggests that understanding the causes of macrophage accumulation within the atherosclerotic lesion is important for developing strategies to curtail the progression and/or promote the regression of the disease. Currently, it is unclear why macrophages that enter atherosclerotic lesions remain there rather than depart with their lipid loads.
In vitro, treatment of macrophages with modified LDL caused alterations in actin organization, reduction of force generation, and inhibition of cell migration. 5,6 These findings suggest that the atherosclerotic environment, and specifically interactions with modified lipoproteins, can explain the retention of macrophages in lesions. However, it is still unclear how interactions with modified lipoproteins alter macrophage function: Can the changes in actin organization and migration be attributed to a signaling event initiated by binding of modified lipoproteins to their receptor(s)? Or are they a result of modulations in cellular cholesterol content after cholesterol transfer from the lipoproteins? One particularly interesting observation is that as macrophages begin to engulf aggregated matrix-bound LDL particles, they maintain prolonged contact with the LDL aggregates. 7 During this prolonged contact there can be rapid transfer of cholesterol and cholesteryl esters from the LDL aggregates to the macrophage, and transient increases in the cholesterol content of macrophage plasma membranes may result. Because reduction in membrane cholesterol levels has been shown to affect many cellular processes, 8-14 presumably by altering membrane lipid organization, we postulated that increasing membrane cholesterol content may likewise affect cell function. If elevated membrane cholesterol does indeed alter cell function, then this may explain why macrophages within atherosclerotic lesions are unable to egress from the affected area.
As a first step to test this hypothesis, we investigated how global increases in membrane cholesterol modify macrophage biology, using cholesterol delivery to cells via methyl-ß-cyclodextrin rather than delivery by lipoproteins. This allowed us to examine the effects of cholesterol elevation in the absence of possible effects from binding of apolipoproteins to their receptors. Interestingly, we found that elevated levels of membrane cholesterol affect F-actin organization and F-actin-dependent functions, but these changes in the actin cytoskeleton can be inhibited by blocking scavenger receptors even when cholesterol is delivered via methyl-ß-cyclodextrin.
Methods
Cells and Cell Culture
Mouse macrophage cell line J774A.1 was obtained from the American Type Culture Collection. See http://atvb.ahajournals.org for details on growth and maintenance of the cells.
Lipoproteins and Reagents
Human LDL was prepared as previously described. 15 Acetylated LDL (Ac-LDL) was prepared by acetylation of LDL with acetic anhydride as described previously. 16 ACAT inhibitor 58035, originally from Sandoz Inc, was kindly provided by Dr Ira Tabas. Methyl-ß-cyclodextrin (MßCD) and cholesterol-chelated methyl-ß-cyclodextrin (as "water-soluble cholesterol," molar ratio 1:6 cholesterol/MßCD) were purchased from Sigma. All treatment concentrations involving cholesterol-chelated-MßCD (chol-MßCD) were based on the weight of MßCD. Fucoidan and Clostridium difficile toxin B were purchased from Sigma and Calbiochem, respectively.
Phalloidin Staining and Filipin Staining
To visualize F-actin, J774 cells were simultaneously fixed, permeabilized, and labeled by incubation with 3.3% paraformaldehyde, 0.05% glutaraldehyde, 0.25 mg/mL saponin, and 2 U/mL AlexaFluor 488 phalloidin (Molecular Probes) in phosphate-buffered saline for 15 minutes at room temperature. Images were then acquired with a Zeiss LSM510 laser scanning confocal microscope, or a Leica DMIRB widefield microscope equipped with a Princeton Instruments cooled charge-coupled device camera driven by MetaMorph Imaging System software (Universal Imaging Corporation, Downingtown, Pa).
To quantify changes in cellular free cholesterol, cells were fixed with 3.3% paraformaldehyde for 15 minutes and then incubated with 50 µg/mL filipin (Sigma) for 1.5 hours at room temperature. Wide-field images were then obtained. Details on quantitative analysis of phalloidin- or filipin-stained cells can be found in the supplemental materials.
Macropinocytosis Assay
J774 cells, grown on polylysine-coated glass coverslip-bottom dishes for 16 hours, were first treated with 5 mmol/L chol-MßCD for indicated times or left untreated, then incubated with 1 mg/mL TRITC-conjugated Dextran (160 kDa; Sigma) for 15 minutes before fixation. 17 Wide-field fluorescence images of all samples were acquired under identical conditions for quantitative analysis using MetaMorph. The images were thresholded so that only dextran-positive endosomes were included, and all images were thresholded to the same scale. The integrated fluorescence in the thresholded area was measured and divided by the total number of cells to yield a measure of fluorescence intensity per cell.
Scanning Electron Microscopy
J774 cells in suspension were left untreated or treated with 5 mmol/L chol-MßCD for 30 minutes at 37°C, and then fixed for 10 minutes by addition of equal volume of 8% paraformaldehyde, 5% glutaraldehyde, and 0.04% picric acid in 0.1 mol/L sodium cacodylate buffer, pH 7.3. Samples were then prepared for scanning electron microscopy as described in the supplemental materials.
Immunofluorescence
For costaining of Rac and F-actin, J774 cells were fixed with 3.3% paraformaldehyde in the presence of 0.25 mg/mL saponin and 1 U/mL AlexaFluor 488 phalloidin for 15 minutes at room temperature. Excess AlexaFluor 488 phalloidin was included throughout the remaining labeling steps. Rac was visualized with a mouse monoclonal antibody against Rac1 (clone 102; BD Biosciences Pharmingen), followed by an AlexaFluor 546-conjugated goat anti-mouse secondary antibody (Molecular Probes). Images were then acquired with a Zeiss LSM510 laser scanning confocal microscope using a 63 x 1.4 numeric aperture objective.
Determination of Activated Rac
The PAK pull-down assay was performed using a Rac activation assay kit (Cytoskeleton) following the manufacturer?s instruction. Briefly, cells were grown to < 80% confluence on 100 mm tissue culture dishes (Falcon). After chol-MßCD treatment, cells were lysed in lysis buffer, clarified by centrifugation, and incubated with GST-PAK beads for 1 hour at 4°C. Bound Rac was analyzed by SDS-PAGE separation on a 4% to 12% polyacrylamide gel, and visualized by Western blotting with monoclonal anti-Rac1 antibody and HRP-conjugated goat anti-mouse IgG (Sigma) using the ECL system (Pierce). For normalization, whole cell lysates were run in parallel.
Macrophage Migration Assay
Migration of J774 cells was tested in a chemotaxis assay using Transwell Inserts (Costar, Cambridge, Mass) with 5-µm polycarbonate filter inserts. Further details regarding the conditions used for the migration assay are provided in the supplemental materials.
Results
Cholesterol Loading by Ac-LDL and ACAT Inhibitor Induces Membrane Ruffles and Stimulates Cell Spreading
To load cell membranes with cholesterol, we treated J774 macrophages with 50 µg/mL ac-LDL and 10 µg/mL ACAT inhibitor 58035 for 5 hours. Ac-LDL no longer binds to LDL receptors, but it is taken into cells by scavenger receptors, which are not subject to down regulation by the level of cellular cholesterol. 16,18 Ac-LDL uptake strongly triggers ACAT activity when cellular free cholesterol reaches certain threshold levels. 19 Thus, cotreatment with ac-LDL and ACAT inhibitor leads to the accumulation of free cholesterol in the cells. Under the conditions used in these experiments, the cellular free cholesterol level in treated cells was increased &2-fold over control cells, as determined by filipin staining (data not shown). To assess the validity of fluorescence image analysis of filipin-stained cells as a quantitative measure of cellular cholesterol content, the results from this technique were compared with those obtained by gas chromatographic analysis of cellular lysates. 20 Both techniques yielded similar results (Figure I, available online at http://atvb.ahajournals.org), demonstrating that quantitative fluorescence imaging of filipin-stained cells is an accurate method for evaluating cellular free cholesterol levels under conditions used in this study.
To observe the effects that treatment with ac-LDL and the ACAT inhibitor had on macrophage biology, treated and control cells were plated onto fibronectin-coated dishes for 10 minutes, fixed, and stained for F-actin. Figure 1 shows that cells treated with ac-LDL and the ACAT inhibitor become more spread and form more F-actin-rich membrane ruffles than untreated cells, whereas neither ac-LDL nor the ACAT inhibitor alone could cause these morphological changes (data not shown). These results suggest that raising the cholesterol content in the plasma membranes of macrophages alters their biology.
Figure 1. Loading plasma membranes of macrophages with cholesterol by ac-LDL and ACAT inhibitor treatment stimulated cell spreading and membrane ruffling. J774 macrophages were treated with 50 µg/mL ac-LDL and 10 µg/mL 58035 for 5 hours then plated onto fibronectin-coated dishes for 10 minutes before fixation and staining for F-actin. Arrows indicate membrane ruffles. Scale bar, 10 µm.
Increased Cell Spreading and Membrane Ruffling in Ac-LDL/ACAT Inhibitor-Treated Cells Can Be Attributed to Increases in Free Cholesterol
Although treatment with ac-LDL and the ACAT inhibitor induced a clear response in J774 macrophages, it was not clear if the spreading and ruffling were caused by signals initiated by ac-LDL, the ACAT inhibitor, or cholesterol loading. To determine whether raising membrane cholesterol levels, rather than signaling via the cholesterol delivery system (either the ac-LDL or the ACAT inhibitor), caused the observed effects, we used cholesterol-chelated methyl-ß-cyclodextrin (chol-MßCD) to acutely raise the cholesterol content of the plasma membrane. Treatment with 5 mmol/L chol-MßCD for either 15 or 30 minutes increased total cellular cholesterol levels by 1.5- to 2-fold ( Figure 2 A), which results in levels comparable to those obtained after treatment with ac-LDL and the ACAT inhibitor. These changes in membrane cholesterol stimulated cell spreading, with the average cell area for cells treated with chol-MßCD for 15 or 30 minutes increasing by 45% or 60%, respectively, compared with control cells ( Figure 2 A). The effects of cholesterol loading on cell spreading were readily reversed when membrane cholesterol was returned to starting levels by subsequent treatment with MßCD, and there is a clear correlation between the cellular cholesterol content and the extent of cell spreading ( Figure 2 A). As for cells treated with ac-LDL and the ACAT inhibitor ( Figure 1 ), J774 cells that were treated with chol-MßCD for 15 minutes and then plated onto fibronectin-coated dishes for 10 minutes exhibited F-actin-rich ruffles ( Figure 2 B). Confocal fluorescence images of the lower adherent surfaces of control and cholesterol-loaded cells show that F-actin is accumulated at the edges of the cholesterol-loaded J774 cells but not the control cells (Figure IIA, available online at http://atvb.ahajournals.org), and quantification of the F-actin at the cell edge shows that cholesterol-loaded cells have approximately twice the amount of F-actin near the cell edges compared with control cells (Figure IIB). Interestingly, although cholesterol loading caused increased membrane ruffling, there was no apparent increase in the total F-actin content (compare the intensity of the phalloidin staining in control versus treated cells in Figures 1 and 2 B). Quantification of the total F-actin content in control and chol-MßCD-treated J774 cells confirmed that there was no increase in the total amount of F-actin after chol-MßCD treatment, implying that there was a change in the organization and/or localization of F-actin but not an increase in actin polymerization ( Figure IIB). These results show that increasing membrane cholesterol levels causes morphological and cytoskeletal changes in macrophages.
Figure 2. Increasing membrane cholesterol reversibly induced cell spreading and membrane ruffling. (A) Modulation of membrane cholesterol caused corresponding changes in cell area. J774 cells, grown on polylysine-coated dishes overnight, were left untreated (control), treated with 5 mmol/L chol-MßCD (+), or treated sequentially with 5 mmol/L Chol- MßCD (+) and then 10 mmol/L MßCD (-) for indicated times before fixation. Cells were then stained with filipin to determine the relative free cholesterol levels per cell (gray bars) or labeled with a fluorescent conjugate of phalloidin to delineate the cell periphery for cell area measurements (white bars). Quantification of fluorescence images is described in supplemental materials. Data represent means±SD from at least 3 independent experiments. (B) Raising membrane cholesterol by treatment with chol-MßCD induced membrane ruffling (arrowheads). J774 macrophages were left untreated (control) or treated with 5 mmol/L chol-MßCD for 15 minutes (cholesterol-loaded) in suspension, and then plated onto fibronectin-coated dishes for 10 minutes before fixation and staining for F-actin. Projected confocal images formed from 20 0.7-µm confocal slices are presented. Scale bar, 10 µm.
Cholesterol Loading Induced Macropinocytosis Activity
Because one obvious effect of increasing membrane cholesterol levels on J774 macrophages was the induction of membrane ruffles, we predicted that macropinocytosis activity of J774 cells might also be induced on cholesterol loading. To test the effects of cholesterol loading on macropinocytosis, control or chol-MßCD-treated cells were incubated with TRITC-conjugated dextran for 15 minutes. The samples were then fixed, and fluorescence images were taken for quantitative analysis. As shown in Figure 3, the amount of fluorescent dextran taken up by the cells was dramatically increased when the J774 cells were cholesterol-loaded, indicating that increasing membrane cholesterol levels causes an increase in membrane pinocytic activity.
Figure 3. Increasing membrane cholesterol stimulated macropinocytosis. J774 cells were left untreated (control), or treated with 5 mmol/L chol-MßCD for indicated times (chol-loaded), then incubated with 1 mg/mL TRITC-Dextran for 15 minutes before fixation. Data shown are from 1 representative experiment of 3. Error bars represent SD.
Effects of Cholesterol Loading on Membrane Ruffling and F-Actin Organization Require Cell Adhesion
The results presented suggested that cholesterol itself might be acting as a signaling molecule. However, because our experiments were performed on adherent cells, it was also possible that cholesterol loading was potentiating signals initiated by adhesion molecules. To determine whether cell adhesion played a role in the cellular changes induced by cholesterol loading, we altered membrane cholesterol levels of J774 cells in suspension, and then fixed the samples for analysis by scanning electron microscopy or by confocal fluorescence microscopy after staining with fluorescent phalloidin ( Figure 4 A). We found no obvious differences in the surface morphologies or F-actin organization of control and chol- MßCD-treated cells, indicating that the effects of increased membrane cholesterol on membrane ruffling and F-actin reorganization required cell adhesion and increasing cellular cholesterol was not sufficient to induce this effect by itself.
Figure 4. Signaling via scavenger receptor-mediated adhesion or ligation is required for cellular responses to cholesterol loading. (A) J774 macrophages in suspension did not show morphological changes when treated with chol-MßCD. Cells were either left untreated (control) or treated with 5 mmol/L chol-MßCD for 30 minutes (cholesterol-loaded) before fixation. Scanning electron micrographs (SEM, top panels) and projected confocal images of F-actin staining (lower panels) are presented. (B) A general scavenger receptor inhibitor, fucoidan, blocked the effects of cholesterol loading. J774 cells were grown on polylysine-coated dishes overnight, in the presence (right panel) or absence (left and middle panels) of 250 µg/mL fucoidan. The cells were either left untreated (control), or treated with 5 mmol/L chol-MßCD for 30 minutes (chol-loaded) with or without fucoidan. Projected confocal images of phalloidin-stained cells are presented. Scale bar, 10 µm.
It has been shown that macrophage adhesion in vitro is mediated in large part by ß2 integrins and scavenger receptor A. 21 To test the role of these adhesion molecules, we used specific inhibitors to block their contribution during cholesterol loading. Our data suggest that RGD-binding integrins and ß2 family integrins do not contribute to the cellular changes induced by cholesterol loading (Figure III, available online at http://atvb.ahajournals.org). In contrast, the ruffling and spreading induced by cholesterol loading were inhibited by fucoidan ( Figure 4 B), indicating that cellular attachment via receptors other than scavenger receptors coupled with cholesterol-loading is not sufficient to mediate the observed cellular changes. To determine whether ligation of scavenger receptors and cholesterol loading would be sufficient to induce ruffling in the absence of cell attachment, we treated cells in suspension with fucoidan (a scavenger receptor ligand) and loaded them with cholesterol. This treatment did not induce any significant ruffling in suspended cells (Figure IV, available online at http://atvb.ahajournals.org), further indicating that signaling via scavenger receptor attachment and/or ligation of scavenger receptors by an unknown molecule in the substratum is involved.
Rac Is Activated and Recruited to Membrane Ruffles When Membrane Cholesterol Levels Are Increased
To begin to understand which signaling steps elevated membrane cholesterol levels affect, we investigated the effects of cholesterol modulation on Rac activation because it is well-established that Rho GTPases play important roles in actin polymerization. Rac activation has been particularly associated with the formation of membrane ruffles. 22,23
First, we used indirect immunofluorescence to localize endogenous Rac1 in control and chol-MßCD-treated macrophages using both J774 cells ( Figure 5 A), as well as primary human monocyte-derived macrophages (Figure V, available online at http://atvb.ahajournals.org). In control J774 cells, Rac 1 (shown in red) had a patchy distribution within the cell cytosol; Rac1 was found bounded within the F-actin (shown in green) and, even within the few visible membrane extensions (arrows), it did not colocalize with F-actin (note the lack of yellow color in "control" panel). In contrast, after cells were treated with chol-MßCD, Rac1 was found almost entirely in membrane ruffles (arrows), and it colocalized extensively with F-actin (note yellow color in "cholesterol-loaded" panel.) Similar results were observed in human monocyte-derived macrophages (Figure V). The membrane recruitment of Rac1 after chol-MßCD treatment suggests that elevating membrane cholesterol levels leads to Rac activation.
Figure 5. Rac GTPase was activated upon membrane cholesterol loading. (A) Rac (labeled in red) was recruited to ruffling membranes in cholesterol-loaded macrophages. J774 cells grown on polylysine-coated dishes were left untreated (control) or treated with 5 mmol/L chol-MßCD for 30 minutes (chol-loaded) before fixation and immunofluorescence. Green color represents F-actin. Projected confocal images are presented. (B) Rac was activated in cholesterol-loaded cells. J774 cells grown on tissue culture dishes (10 7 cells/dish) were left untreated (control) or treated with 5 mmol/L chol-MßCD (chol-loaded) for indicated times before harvest. A PAK pull-down assay was used to detect the amount of activated Rac under each treatment condition. A representative Western blot is shown. (C) Toxin B treatment completely abolished the ruffling stimulated by cholesterol-loading in J774 macrophages. J774 cells, grown on polylysine-coated dishes overnight, were left untreated (control), treated with 5 mmol/L chol-MßCD for 30 minutes (chol-loaded), or treated with 5 mmol/L chol-MßCD and 10 ng/mL toxin B for 30 minutes (chol-loaded+toxin B). The cells were then fixed and stained for F-actin. Projected confocal images are presented. Scale bar, 10 µm.
The activation of Rac was further confirmed using a pull-down assay with the Rac-GTP binding domain of PAK. 24 Figure 5 B shows that Rac1 was activated within 1 minute of chol-MßCD treatment, peaked at &15 minutes, and started to decrease at 30 minutes. In addition, treatment with Clostridium difficile toxin B completely abolished the ruffling stimulated by membrane cholesterol loading ( Figure 5 C). Toxin B specifically monoglucosylates and inactivates Rho-family GTPases, 25 and it has been a commonly used inhibitor to study the cellular function of Rac. 26-28 Taken together, these results suggest that Rac-mediated signaling pathways, possibly in concert with other Rho-family GTPases, are involved in the observed morphological changes induced by cholesterol loading.
Migration Is Inhibited in Macrophages With Elevated Membrane Cholesterol Levels
These results demonstrate that raising membrane cholesterol levels causes changes in cellular morphology and the organization of F-actin in macrophages. Because cell morphology and F-actin organization are vital components of cell migration, and because cell migration is an important factor in atherogenesis, we investigated the effects of cholesterol loading on macrophage migration. We used 3-dimensional Transwell migration assays to study the effects of cholesterol loading on J774 macrophage chemotaxis. J774 cells were first treated with 5 mmol/L chol-MßCD for 15 minutes, and then induced to migrate in response to 10 nM recombinant C5a. 29 After 2 hours, cells that had migrated through the Transwell filter were fixed and counted. Figure 6 shows that the migration of cholesterol-loaded J774 macrophages was inhibited by 60% compared with control cells, and this inhibition could be completely reversed when the membrane cholesterol of cholesterol-loaded cells was reduced by subsequent treatment with MßCD.
Figure 6. Elevated membrane cholesterol inhibited the Transwell migration of J774 cells. J774 cells were loaded with cholesterol by incubation with 5 mmol/L chol-MßCD for 15 minutes (chol-loaded). To test the reversibility of this treatment, an aliquot of cholesterol-loaded cells was subsequently treated with 10 mmol/L MßCD for 10 minutes to lower cholesterol levels (reversal). The migration of untreated, cholesterol-loaded, and cholesterol-reversed cells was tested in a Transwell migration assay in response to 10 nM C5a. Results were normalized to the number of untreated (control) cells that had migrated across the Transwell filter after incubation for 2 hours. Data shown are from one representative experiment. Error bars represent SD.
Discussion
Numerous studies have investigated the effects of reducing membrane cholesterol levels on signal transduction, but far fewer have looked at the effects of the potentially more physiologically relevant event of increasing membrane cholesterol levels. Here we have investigated the effects of raising cellular cholesterol on cellular functions related to the actin cytoskeleton. Specifically, we have shown that raising the cholesterol content in macrophage membranes affects Rac-mediated F-actin organization, increases membrane pinocytic activity, and decreases cell migration. These changes are similar to those observed when macrophages were treated with modified lipoproteins, 5,6 suggesting that the changes induced by modified lipoproteins can be attributed, in part, to alterations in cellular cholesterol levels.
A key pathogenic event in the development of atherosclerosis is the retention of lipoprotein particles in the subintima. 30 These lipoprotein particles subsequently undergo aggregation, association with extracellular matrix proteoglycans, and oxidative modification. 31 The physiological significance of the findings presented in this study becomes evident at the stage of atherosclerotic lesion formation when monocytes/macrophages first contact aggregated matrix-bound LDL. Previous studies have shown that macrophage interactions with aggregated, matrix-bound LDL particles initiate specific cellular events that can lead to increases in membrane cholesterol levels. 7,32 Specifically, macrophage uptake of aggregated matrix-bound LDL was found to be slow compared with aggregated lipoproteins that are not matrix-bound; during the slow internalization process, matrix-bound LDL was found to reside for extended times within deep invaginations in the macrophage cell surface. 7 Because the rate of cholesterol ester hydrolysis was found to greatly exceed the rate of protein degradation during the uptake of aggregated matrix-bound LDL, it is thought that the prolonged contact of the aggregates with the macrophage cell surface allows selective uptake of cholesterol ester from the extracellular lipoprotein. 7 In other words, there is transfer of cholesterol ester directly from LDL aggregates to the macrophage plasma membrane, rapid cholesterol ester hydrolysis, and a consequent increase in membrane cholesterol levels. Additional studies showed that uptake of aggregated matrix-bound LDL requires the actin-myosin cytoskeleton, presumably for extension of macrophage plasma membrane around the matrix-retained aggregates in a process reminiscent of phagocytosis. 32 We show here that when membrane cholesterol levels are elevated, there is an increase in actin-mediated membrane activities, including membrane ruffling and macropinocytosis. This observation is consistent with a recent finding that ACAT1-deficient peritoneal macrophages, which accumulate free cholesterol on ac-LDL treatment, appeared to have increased surface activities. 33 Based on these observations, one can propose a model for a pathogenic circle of events: First, macrophages come into contact with matrix-bound LDL aggregates, cholesterol ester transfer occurs, macrophage membrane cholesterol levels increase, and actin-mediated membrane extensions are stimulated. This causes a broader area of cell-surface contact with the aggregates, more cholesterol ester transfer, a further increase in membrane cholesterol levels, additional membrane extensions, greater cell-surface contact with the aggregates, and so on. This scenario could explain, in part, why macrophages within atherosclerotic lesions continue to take up retained lipoproteins and become foam cells.
How Does Cholesterol Induce Cellular Changes?
Cholesterol loading does not increase actin-based ruffling in suspended cells, and the ruffling induced by cholesterol loading of attached cells is blocked by incubation with fucoidan, a ligand for scavenger receptors. These data indicate that cholesterol itself does not serve as a signaling molecule to induce the effects observed in this study. Instead, increasing membrane cholesterol levels may alter plasma membrane organization in a way that potentiates signaling by adhesion molecules. Our data suggest that scavenger receptors contribute to the cholesterol-sensitive signals that lead to changes in cell function. In this regard it is interesting to note that scavenger receptors can activate signaling pathways leading to actin polymerization and focal adhesion formation, presumably by binding ligands in the extracellular matrix. 34
The Rho family GTPases, Rho, cdc42, and Rac, are known to be major mediators of signaling leading to actin reorganization. 35,36 The Rho GTPases are thought to regulate the formation of distinct actin filament-containing structures. Because the morphological responses in macrophages induced by membrane cholesterol loading resembled the effects of Rac activation, we investigated the involvement of Rac signaling in cholesterol-induced membrane ruffling. By each of 3 different methods (PAK pull-down assay, visualization of Rac recruitment to the plasma membrane, and Toxin B inhibition), we found that Rac was involved in the morphological and cytoskeletal changes induced by cholesterol loading. These findings are consistent with our previous ones in human neutrophils, namely, that when the levels of cholesterol in plasma membranes of neutrophils are reduced, both stimulated membrane ruffling and Rac recruitment to the plasma membrane are inhibited. 12
Membrane Cholesterol and Cell Migration
Despite having potentiating effects on membrane activity and F-actin reorganization, cholesterol loading caused the inhibition of macrophage migration. Cell migration is a highly integrated, multi-step process consisting of cell polarization, membrane extension at the front of the cell, regulated formation and release of adhesions along the length of the cell, and retraction of the cell rear (uropod). These steps are orchestrated in part by the interactive regulation of the Rho-family GTPases. 37-39 Our data clearly show that Rac activity and localization are altered after cholesterol loading, and it is likely that the activities of the other Rho family members are also affected by changes in membrane cholesterol levels. Misregulation of these signaling molecules could lead to the inability of the cells to correctly polarize, retract their uropods, or regulate their attachments with the substratum. Several studies have shown that plasma membrane organization is critical for cell migration, 11,12,40 and our previous work on neutrophils showed that depleting membrane cholesterol inhibited neutrophil migration because the cells were unable to form membrane extensions and polarize. 12 Further studies are required for understanding the exact defect in the migration of cholesterol-loaded macrophages.
The observations reported here indicate that increased membrane cholesterol causes dramatic effects on the actin cytoskeleton in macrophages associating with extracellular matrix. It is possible that after initial contact of a macrophage with lipoproteins in the vessel wall, cholesterol transfer to the cell causes changes similar to those observed in this study. The increased membrane ruffling and extensions could increase the contact of the macrophage with the lipoprotein deposit, leading to further cholesterol delivery. The decreased motility of the cell would keep it in the region of the lipoprotein deposit. Additional studies will be required to determine to what extent contact with lipoprotein particles causes local increases in membrane cholesterol levels and the consequences of these local changes for cell function. These findings should provide new insights into the early steps of atherogenesis and might eventually lead to innovative methods of preventing lesion formation or promoting lesion regression.
Acknowledgments
This work was supported by National Institutes of Health grants P01-HL072942. We thank Leona Cohen-Gould for technical help with the scanning electron microscopy experiment. We also thank Dr William Muller for helpful discussions.
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作者单位：Departments of Biochemistry (C.Q., T.N., I.G., F.R.M.) and Surgery (L.M.P.), Weill Medical College of Cornell University, New York, NY.