Uptake of modified low-density lipoproteins alters actin distribution and locomotor forces in macrophages

Celina V. Zerbinatti and Robert W. Gore

Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is postulated that macrophage-derived foam cells accumulate in the arterial wall because they lose the ability to migrate after excessive ingestion of modified forms of low-density lipoproteins (LDL). To assess changes in locomotor force generating capacity of foam cells, we measured isometric forces in J774A.1 macrophages after cholesterol loading with oxidized (Ox-LDL) or aggregated (Agg-LDL) LDL using a novel magnetic force transducer. Ox-LDL loading reduced the ability of J774A.1 macrophages to generate isometric forces by 50% relative to control cells. Changes in force frequency consistent with reduced motility were detected as well. Agg-LDL loading was also detrimental to J774A.1 motility but to a lesser extent than Ox-LDL. Ox-LDL loading significantly reduced total actin levels and induced changes in the F-actin to G-actin distribution, whereas Agg-LDL loaded cells had significantly increased levels of total actin. These data provide evidence that cholesterol loading and subsequent accumulation decreases macrophage motility by reducing the cells' force generating capacity and that Ox-LDL appears to be more effective than Agg-LDL in disrupting the locomotor machinery.

cell motility; cell force; actin cytoskeleton; J774A.1 macrophage


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EARLIEST STAGES OF ATHEROSCLEROSIS are characterized by the formation of a fatty streak where macrophages loaded with cholesterol accumulate in the intima of blood vessels and show a foamlike appearance (28). Monocyte-derived macrophages become foam cells in the intima by internalizing modified forms of low-density lipoprotein (LDL) through one or more cell surface scavenger receptors. Scavenger receptors, unlike classic LDL receptors, are not downregulated by intracellular cholesterol content, and internalization of LDL via this pathway can lead to uncontrolled cholesterol accumulation (2).

Oxidized and aggregated forms of LDL are the most probable sources of cholesterol that cause formation of foam cells in vivo (30, 34). Oxidized LDL (Ox-LDL) is recognized by one or more macrophage scavenger receptors and is not completely degraded in lysosomes. Foam cells derived from in vitro incubation of macrophage with Ox-LDL are known to esterify cholesterol poorly, so they accumulate high levels of free cholesterol (21, 27). In contrast, aggregated LDL (Agg-LDL) is recognized by the classic LDL receptor, but is internalized by phagocytosis instead of pinocytosis. Cholesterol accumulated during incubation with Agg-LDL is highly esterified in macrophages, and it is stored in lipid droplets (18).

One hypothesis to explain the accumulation of foam cells in the intima is that modified forms of LDL are internalized in sufficient quantity to cause reduction in the cells' normal ability to move and migrate within its environment (25, 26). It is further thought that the accumulation of cholesterol in macrophages disrupts the actin cytoskeleton and intracellular integrity and, hence, reduces the cells' ability to generate normal locomotor forces (25). Unfortunately, no quantitative studies have been done to demonstrate a clear connection between cholesterol accumulation, disruption of the cytoskeleton, and subsequent reduction in locomotor force generation. Indeed, without direct measurements of these parameters, one cannot conclude from simple qualitative observations whether foam cells become trapped in the intima because their ability to generate normal locomotor forces is altered or whether they accumulate such large quantities of cholesterol that their size severely limits their ability to migrate out of the intima.

A new isometric remote-sensing magnetic force transducer was developed in this laboratory to measure the forces produced by individual phagocytes during locomotion both in vitro and in vivo (15, 16). The purpose of the following study was to use this device to measure quantitatively the effect of cholesterol loading on locomotor force generation in macrophages. The macrophage-like cell line J774A.1 was used to investigate the effects of cholesterol loading with Ox-LDL and Agg-LDL on 1) the average and peak forces generated spontaneously by cells, 2) the frequency distribution (periodicity) of force generation, and 3) the distribution of the cytoskeleton components, G-actin and F-action, associated with locomotor force generation (5).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells. Monolayer cultures of the J774A.1 macrophage-like cell line (American Type Culture Collection, Rockville, MD), were maintained in alpha -minimum essential medium (GIBCO, Grand Island, NY) containing 5% heat-inactivated (56°C for 30 min) newborn calf serum (Hyclone, Logan, UT), 5 U/ml penicillin, and 5 µg/ml streptomycin (GIBCO). Cells were grown in plastic petri dishes at 37°C in 5% CO2 and were harvested by gentle scraping.

Modified lipoproteins. All modified lipoproteins were prepared from a stock reagent of sterile human LDL (density 1.019-1.063 g/ml) supplied in borate-buffered saline (pH 8.0) from Solomon Park Research Laboratory (Kirkland, WA). Ox-LDL was prepared by diluting stock LDL to a concentration of 0.5 mg protein/ml in phosphate-buffered saline (PBS, pH 7.4). The diluted stock was incubated for 24 h at 37°C in the presence of 10 µmol/l CuCl2. The resulting Ox-LDL was then dialyzed against PBS containing EDTA 0.01% for 24 h at 4°C and sterile filtered. Aggregated-LDL was produced by vortexing sterile, diluted LDL (1 mg protein/ml of PBS containing EDTA 0.01%) for 30 s in a bench-top vortex mixer at the fast-speed setting (18). All other reagents were purchased from Sigma Chemical (St. Louis, MO).

Cholesterol measurements. J774A.1 cells were plated into culture dishes (1.75 × 105 cells per 35-mm culture dish) in modified Eagle's medium (MEM) containing 5% newborn calf serum (NCS) and incubated overnight. Modified LDL was added to the culture dishes in 1 ml of fresh MEM containing 5% NCS in three different concentration aliquots of 100 µg of Ox-LDL protein/ml and 100 µg and 300 µg of Agg-LDL protein/ml, respectively. Controls were incubated with 1 ml of MEM containing 5% NCS. After incubation for 24 h at 37°C, the medium containing LDL was removed, and cells were washed with PBS and scraped in 0.5 ml of PBS with a Teflon policeman. Cell suspensions were sonified for 1 min (Sonic Dismembrator, Fisher Scientific), and 100-µl samples were assayed enzymatically for total and free cholesterol using the method described by Gamble et al. (11). Cholesterol oxidase, cholesterol ester hydrolase, horseradish peroxidase, and cholesterol standards (Preciset Cholesterol Calibrators, Boehringer Mannheim, Indianapolis, IN) were used. Fluorescence was measured in an F-2000 Hitachi fluorescence spectrophotometer (excitation = 315 nm, emission = 405 nm). Protein was detected in 200-µl samples with a modified Lowry protein assay (22) using bovine serum albumin as the standard. Cell cholesterol content was reported as micrograms of cholesterol per milligrams of cell protein.

Force measurements. J774A.1 cells were plated at 1.75 × 105 cells per 35-mm culture dish containing a 22-mm round, sterile Thermanox coverslip (Nunc, Fisher Scientific, Pittsburgh, PA). After incubation overnight, Ox-LDL (100 µg protein/ml) or Agg-LDL (100 or 300 µg protein/ml) was added in fresh medium and incubated for another 24 h. Subsequently, medium containing modified LDL was removed and replaced with 1.5 ml of fresh MEM containing 5% NCS. Random-sized iron-nickel microspheres (Hy-Mu 80, Carpenter Technology), manufactured by a procedure described previously (15, 16), were sterilized by dry heating (180°C for 2 h), suspended in cultured medium by sonication, and then dusted onto coverslips. Cells were then incubated overnight with the microspheres. Microspheres not internalized by cells were removed by washing with Hank's balanced salt solution (HBSS).

Force was measured using our remote-sensing isometric force transducer (15, 16). A cell with an internalized microsphere was centered between two electromagnets under a Zeiss ACM microscope equipped with a Leitz UO-55 water immersion objective (55×, NA = 0.85), and the force transducer was activated. Cells were maintained in HBSS at 37°C on a temperature-controlled stage throughout the measurements, and only cells containing one internalized bead were studied. A continuous video record of each experiment was recorded through the microscope on an S-VHS videocassette recorder (Mitsubishi) with a Dage VE-1000 charge-coupled device video camera. Individual video frames were also captured with a Scion ImageCapture 1000 board and NIH Image 1.52 software on a Macintosh IIfx microcomputer for subsequent analysis. A continuous record of isometric force, generated by the cell during its attempts to move out of the servocontrolled magnetic confinement trap, was measured as a function of the electric current delivered to the magnets to maintain the cell in the trap (15, 16). Force was expressed in units of nanoNewtons (nN). Complete details of the procedure for calibrating the force transducer are published elsewhere (15, 16). The measurements of interest, recorded from the force traces for the control cells, and the cells treated with different compositions of LDL were 1) average force, 2) peak force, and 3) long-frequency periodicity of force oscillations. The average force was the integrated mean of the forces generated by each cell throughout the recording period. The peak force was the maximum force (peak-to-peak) generated by each cell during the recording period. The long-period force-frequency oscillations provided a measure of the dynamics of force generation by the cells. Figure 1 illustrates the basic operating principles of the force transducer. Figure 2 is a confocal image of a J774A.1 cell showing an internalized magnetic bead surrounded by G-actin (red fluorescence) and F-actin (green fluorescence).


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Fig. 1.   Illustration of the remote-sensing isometric magnetic force transducer used to measure force in J774A.1 macrophages. A: a cell with an internalized microsphere (shown as a cutaway) was positioned between 2 electromagnets under a light microscope. The cell and the internalized microsphere were visualized with a video camera, and the position of the microsphere was monitored by edge detection. A servocontrol system modulated the current through the electromagnets to hold the microsphere stationary. Locomotor isometric force was related to the current through the electromagnets necessary to arrest the forward movement of the cell. B: an example trace showing the variations in force produced by a macrophage in vitro as a function of time. Positive and negative values on the force scale (1 nN = 0.1 µdyne) indicate force directed to the left or right, respectively. Notice that force oscillates.



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Fig. 2.   Confocal microscope image of an untreated J774A.1 macrophage showing F-actin (green fluorescence) and G-actin (red fluorescence) distribution around a phagocytized magnetic bead. Bead diameter = 8.7 µm.

Actin measurements. J774A.1 cells were plated at 1.75 and 3.5 × 105 cells per 35-mm culture dishes for measurements in control cells, and cells were incubated with modified LDL, respectively. Modified LDLs were added as described. After 24 h, LDL containing medium was removed and cells were washed twice with PBS. Cells were scraped in 0.25 ml of PBS and immediately assayed for G-actin and total actin using the FluoReporter actin assay kit from Molecular Probes (Eugene, OR). Briefly, a DNA/fluorescent dye complex was cleaved by addition of deoxyribonuclease I (DNase I), which resulted in decreased fluorescence emission. In the presence of G-actin, a stable DNase/G-actin complex was formed that prevented the action of the DNase I on the DNA/fluorescent dye complex (1, 33). The extent of the inhibition of the DNase I activity was a measure of the amount of G-actin present in the samples. Total actin was measured after treatment of the sample with an actin depolymerization buffer (2.5 mM Tris · HCl, pH 8.0, with 2% Triton X-100, 1 mM ATP, 1 mM CaCl2, 1 M guanidine hydrochloride, and 1 M sodium acetate), and F-actin was calculated as the difference between total and G-actin. Fluorescence was recorded at 520 nm using an F-2000 Hitachi fluorescence spectrophotometer set to excite at 480 nm.

Confocal microscopy. J774A.1 cells were plated at 1.75 × 105 cells per 35-mm culture dish containing a 22-mm square glass coverslip and incubated overnight. Modified LDL was added as described, and after 24 h incubation, cells were prepared for fluorescent staining. Cells were washed with prewarmed PBS (pH = 7.4) and fixed in 4% formaldehyde solution in PBS for 15 min at room temperature. Cells were then permeabilized by incubation with 0.1% Triton X-100 in PBS for 5 min. They were subsequently incubated for 20 min at room temperature with 0.25 ml of PBS containing 10 µg/ml of Texas red conjugate DNase I (G-actin staining) and 3 units of BODIPY-FL phallacidin (F-actin staining). The fluorescent labels were purchased from Molecular Probes. Coverslips were rinsed with PBS, air dried, and mounted on a glass slide with the cell side down using the acrylic-based resin Cytoseal-60 (Stephens Scientific, Riverdale, NJ). Specimens were stored in the dark at 4°C. Cells were examined in a Leica TCS-4D laser scanning confocal microscope using the Microsoft Windows program SCANWARE (version 4.0) for the acquisition and manipulation of images. Images were further postprocessed using NIH Image and Adobe Photoshop (Adobe Systems, San Jose, CA).

Statistical analysis. Results of cell cholesterol content (µg/mg cell protein), actin content (µg/105 cells), average and peak forces (nN), and force-frequency (s) were compared between groups by ANOVA (SigmaStat, SPSS, Chicago, IL). The Student-Newman-Keuls test was used for paired multiple comparisons of mean responses among different treatment groups. Statistical significance was assumed when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Uptake of modified LDL increased intracellular cholesterol content of J774A.1 macrophages. Table 1 shows the cholesterol content in J774A.1 macrophages after incubation for 24 h in medium containing modified Ox-LDL vs. Agg-LDL at different concentrations (100 and 300 µg protein/ml). Three times the loading concentration of Agg-LDL was required to achieve comparable amounts of total cholesterol when cells were incubated with 100 µg protein/ml of Ox-LDL for the same time interval. The total cholesterol content in cells after incubation with 100 µg protein/ml Ox-LDL and 300 µg protein/ml Agg-LDL was 3.8 times greater than in control cells but only 1.7 times greater after incubation with 100 µg protein/ml Agg-LDL. Also, the percentage of esterified intracellular cholesterol was much greater after incubation with Agg-LDL at both concentrations (100 and 300 µg protein/ml) than after incubation with Ox-LDL. The latter observation confirmed previous findings that cholesterol accumulated through incubation with Agg-LDL is highly esterified by J774A.1 macrophages and is therefore stored in lipid droplets, whereas Ox-LDL is poorly degraded in lysosomes because of its high content of oxidized products (18, 21, 27). The results in Table 1 demonstrate that our procedure for in vitro modification of LDL was satisfactory, producing the high intracellular levels of esterified cholesterol expected for incubation with Agg-LDL, and the relatively low levels of cholesterol esterification expected for incubation with Ox-LDL, as predicted from the work of others (18, 21, 27).

                              
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Table 1.   Cholesterol content in control untreated J774A.1 cells and after incubation for 24 h in media containing 100 µg protein/ml Ox-LDL and 100 and 300 µg protein/ml Agg-LDL

Uptake of modified LDL decreased magnitude of cell forces. Table 2 summarizes the results of measurements of spontaneously generated isometric forces in control and experimentally treated cells. The average force was the integrated mean of all forces generated throughout the record and the peak force was the highest force value achieved during that period. The peak force was recorded when the cell attempted to move in a straight line to either direction parallel to the servocontrolled magnetic confinement trap. In this situation, the current generated by the magnet to hold the bead (and the cell) was directly proportional to the force generated by the cell to move in the opposite direction. The peak force is the most meaningful measurement with respect to the locomotor capabilities of the cell. The data show clearly that loading J774A.1 cells with Ox-LDL at a medium concentration of 100 µg protein/ml reduced the magnitude of both the average and peak forces to nearly half the magnitude of these same forces generated by control cells. In contrast, cholesterol loading with Agg-LDL at the same medium concentration (100 µg protein/ml) had no statistically demonstrable effect on either average or peak forces relative to control cells. Whereas, cells loaded with Agg-LDL at medium concentrations of 300 µg/ml, which resulted in intracellular accumulation of comparable amounts of total cholesterol as observed with Ox-LDL loading at 100 µg/ml, did significantly reduce the peak force but had no statistically significant effect on average force.

                              
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Table 2.   Average and peak forces and average periodicity of force generated spontaneously by control J774A.1 macrophages and after incubation for 24 h in media containing 100 µg protein/ml Ox-LDL and 100 and 300 µg protein/ml Agg-LDL

Uptake of modified LDL affected time-dependent changes (periodicity) in cell force. Force periodicity is thought to be a sensitive predictor of a cell's movement strategy and its ability to translocate across an attachment surface (4, 6, 8), or through a three-dimensional matrix (9, 10). Movements of human neutrophils display oscillatory behaviors that separate into two different frequency domains, high-frequency short-period cycles (average periodicity ~8 s) and low-frequency long-period cycles (average periodicity ~60 s) (4, 6, 8). The long-period cycles correlated with cell translocation activities, whereas the short-period cycles correlated primarily with cell shape changes not necessarily associated with locomotion.

The low-frequency, long-period oscillations related to cell movement are summarized in the last column of Table 2. Normal, isometric forces generated by the control group of J774A.1 macrophage oscillated with a average periodicity of 80 s, and they were significantly altered by exposure to modified LDL. Incubation with 100 µg protein/ml of Ox-LDL and 300 µg protein/ml of Agg-LDL dramatically decreased the average periodicity of the low-frequency force component by 55% and 49%, from 80 to 36 and 41 s, respectively. Incubation with 100 µg protein/ml of Agg-LDL reduced the average periodicity of the low-frequency component by only 16%, from 80 to 67 s, which was not statistically different from control. The range of periodicities for the low-frequency force component for all the treatment groups were 16-190 s (control), 16-70 s (100 µg protein/ml Ox-LDL), 16-172 s (100 µg protein/ml Agg-LDL), and 16-86 s (300 µg protein/ml Agg-LDL). These results showed clearly that excessive cholesterol accumulation affected the oscillations in force generation by reducing the range of low-frequency, long-period components that have been implicated in cell translocation (4, 6, 8). Regardless of their treatment, all the cells showed the same high-frequency, short-period oscillations similar to frequencies associated with simple shape changes (6, 8). The average high-frequency, short periodicity for control cells and all treatment groups was 8 s (range 6-10 s).

Actin distribution and amount were altered after uptake of modified LDL. Figure 3 shows confocal images of J774A.1 cells stained for G-actin and F-actin before (Fig. 3A) and after cholesterol loading from medium containing 100 µg protein/ml of Ox-LDL (Fig. 3B) and 300 µg protein/ml of Agg-LDL (Fig. 3C). In control cells (Fig. 3A), G-actin was homogeneously distributed throughout the cell cytoplasm, and F-actin, associated with stress fibers, was restricted primarily to the cortical regions (periphery) of the cells. However, in both Oxi- and Agg-LDL-loaded cells (Fig. 3, B and C, respectively), stress fibers of F-actin were also distributed in bundles throughout the cytoplasm. Lipid droplets, seen clearly only in Agg-LDL-loaded cells, were extensively surrounded by G-actin (Fig. 3C).


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Fig. 3.   Confocal microscope image of J774A.1 macrophages showing F-actin (green fluorescence) and G-actin (red fluorescence) distribution in untreated and treated cells. A: untreated control cell. Note the clear separation between the pool of F-actin distributed in the outer, peripheral border, and the large central pool of G-actin. B: after incubation for 24 h in medium containing 100 µg protein/ml oxidized low-density lipoprotein (Ox-LDL). Compartmentalization and distribution of F-actin and G-actin between the peripheral border and the cell center, respectively, is less evident than in control cells (A). C: after incubation for 24 h in medium containing 300 µg protein/ml of aggregated LDL (Agg-LDL). Large lipid droplets (LD) at the interface between the peripheral border of F-actin and the large central pool of G-actin are evident (see higher magnification inset). A clear separation between the peripheral F-actin and the central G-actin pools is maintained, similar to control cells (A).

Table 3 summarizes the results of quantitative measurements of total actin, G-actin, and F-actin in cell samples taken from the same population of cells used to record the confocal images and previously to the incubation with beads. In J774A.1 control macrophages, only 34% of the total actin content was found as F-actin, with the major fraction of total actin residing in the G-actin pool. F-actin is associated with polymerized actin in stress fibers and is seen primarily in the cortical regions of cells, forming the leading edge of lamellipods (29).

                              
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Table 3.   Distribution of cytoskeletal actin in control J774A.1 macrophages and after incubation for 24 h in media containing 100 µg protein/ml Ox-LDL and 300 µg protein/ml Agg-LDL

A 30% decrease in J774A.1 total actin content, from 8.85 to 6.23 µg/105 cells, was observed after cholesterol loading from Ox-LDL. Curiously, Agg-LDL- (300 µg protein/ml) loaded cells had almost double the levels of total actin, which could be explained by the bigger overall size of individual cells in this experimental group. Incubation of J774A.1 macrophages with both forms of modified LDL caused a marked increase in the proportion of F-actin in the cytoskeleton. F-actin represented 61 and 50% of the total actin content in Ox-LDL- and Agg-LDL-loaded cells, respectively. The observation that the major fraction of total actin was shifted to the F-actin pool, particularly in Ox-LDL loaded cells, is consistent with the confocal images and correlates with the decrease in the ability of these cells to generate both static and dynamic forces associated with translocation movements (6, 9, 10, 16).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is generally accepted that foam cells accumulate in the intima as a result of reduced cell motility (26), even though migration of foam cells back into the arterial lumen appears to occur as suggested by electron microscopy studies (12, 13). In this study, we have shown that cholesterol loading with modified forms of LDL decreased macrophage ability to generate locomotor forces and changed actin cytoskeleton integrity and distribution in vitro.

Incubation with Ox-LDL at a concentration that has been previously shown to produce foam cells (100 µg protein/ml) reduced both average and peak forces generated by J774A.1 macrophages by ~50% of that observed in untreated control cells. In contrast, incubation with the same amount of Agg-LDL, which resulted in a much smaller intracellular cholesterol accumulation, did not significantly alter the ability of J774A.1 cells to generate locomotor forces. However, when the total intracellular cholesterol level was brought to levels equivalent to that in cells incubated with Ox-LDL, i.e., with Agg-LDL at 300 µg protein/ml, the peak force generated by J774A.1 was also significantly reduced by 40%. These results clearly indicated that intracellular cholesterol accumulation is a major determinant in the loss of motility in foam cells.

Coates and colleagues (4, 6, 8) observed that movements of human neutrophils display oscillatory behaviors that separate into high-frequency, short-period cycles (~8 s) and low-frequency, long-period cycles (~60 s). Whereas the low-frequency, long-period cycles correlated with cell translocation activities, the short-period cycles correlated primarily with cell shape changes not associated with locomotion. As predicted by their studies, our low-frequency periodicity of force generation was in accordance with the peak force results. The force produced by Ox-LDL and by high-Agg-LDL foam cells had decreased long-period frequency compared with that of controls and to that of macrophages loaded with low concentration of Agg-LDL, suggesting that the first two groups had reduced ability to move. Therefore, albeit the studies of Coates and colleagues did not include measures of force, our measurements indicate that their predictions about periodicity and locomotion were indeed correct.

Even though both forms of modified LDL decreased peak forces and long-period cycles of force generated by J774.A1 macrophages, our data also indicated a potential difference in the basic mechanisms of how they impaired cell movement. Although the average locomotor force was diminished by incubation with Ox-LDL, it was unaffected by incubation with a high concentration of Agg-LDL. In accordance, we found that cholesterol loading with Ox-LDL was more detrimental to the actin cytoskeleton integrity than cholesterol loading with high Agg-LDL. Incubation with modified forms of LDL and subsequent intracellular cholesterol accumulation increased the percentage of F-actin, in agreement with the reduced force measurements observed in cholesterol-loaded cells. However, parallel to the changes in the G- to F-actin ratio toward a less motile state, the total amount of intracellular actin was reduced in J774.A1 macrophages incubated with Ox-LDL. Impaired migration of mouse peritoneal macrophage by incubation with Ox-LDL in vitro has been previously reported and attributed to the disruption of the actin cytoskeleton (25). Also, a dose-dependent reduction in actin levels and changes in its distribution occurs upon chronic incubation with Ox-LDL in rabbit-derived vascular smooth muscle cells (23). Therefore, the differences in the average locomotor forces observed by us could have resulted from specific effects of these two modified forms of LDL on the actin cytoskeleton.

One explanation for the striking effects of cholesterol accumulation via Ox-LDL is that toxic oxidized lipids released intracellularly could directly damage the cytoskeleton proteins. Indeed, incubation of 73/73 endothelial cells with cholesterol oxides causes progressive disruption of actin filaments, with loss of stress fibers and marginalization and clustering of actin filaments to one edge of the cell (24). Alternatively, high levels of intracellular free cholesterol, which have been suggested as a cause of macrophage death by both necrosis and apoptosis (31), could be responsible for cytoskeletal collapse and decreased force generation.

In contrast to the results from the Ox-LDL treatment, Agg-LDL loaded macrophages had increased total actin levels compared with control cells. Previous findings show that actin cytoskeleton is important for cholesterol esterification (32). Because most of the cholesterol accumulated by incubation of macrophages with Agg-LDL was esterified, the increased levels of actin found in this group could be related to cholesterol esterification. Thus we speculate that actin involvement in cholesterol transport and esterification may also contribute to the reduction in force generation by competing with the pool of actin available for cell movement. This hypothesis is supported by studies using freeze-etch electron microscopy, showing that actin is associated with the subcellular compartments involved in the uptake and intracellular processing of Agg-LDL by macrophages (17).

The peak force measured by us is an indicator of a maximum force a cell could generate over a short period of time. Activated monocytes enter the intima by crossing the endothelium in areas of high permeability (14). However, once loaded with cholesterol, monocyte-derived macrophages are enlarged and, hence, have to break through the endothelium to leave the vessel and reenter the circulation (12). According to our findings, a macrophage exposed to Agg-LDL would have an increased probability of leaving the intima than one exposed to identical concentrations of Ox-LDL. The marked effects of Ox-LDL internalization on the ability of cells to move and, therefore, to remove cholesterol from the intima could explain how antioxidant drugs reduce the incidence of atherosclerotic lesions independently of their ability to reduce total cholesterol plasma levels (3, 19).

Although macrophage-derived foam cells appear to be the initiators of the cholesterol deposition in the atherosclerotic plaque, they have also been implicated in the regression of atherosclerosis (7, 20). By removing the highly atherogenic modified forms of LDL from the intima and migrating back into the circulation, foam cells can deliver excess cholesterol from the arterial wall to the liver for excretion in bile acids. The results presented here suggest that atherogenic forms of LDL, in particular Ox-LDL, have detrimental effects on cytoskeleton functions and could, therefore, interfere with macrophage-mediated regression of atherosclerosis.


    ACKNOWLEDGEMENTS

We are indebted to John S. Rozum for excellent technical assistance.


    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-13437.

Present address of C. V. Zerbinatti: Dept. of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8208, Saint Louis, MO 63110.

Address for reprint requests and other correspondence: R. W. Gore, Dept. of Physiology, College of Medicine, Univ. of Arizona, Tucson, AZ 85724 (E-mail: Gore{at}u.arizona.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 16, 2002;10.1152/ajpcell.00177.2002

Received 9 October 2002; accepted in final form 17 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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