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Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells
BBRC Biochemical and Biophysical Research Communications 338 (2005) 748–756 www.elsevier.com/locate/ybbrc
Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells Tong-le Deng, Lian Yu, Ya-kun Ge, Le Zhang, Xiao-xiang Zheng * Department of Biomedical Engineering, Zhejiang University (Yuquan Campus), Hangzhou 310027, PR China Received 28 September 2005 Available online 13 October 2005
Abstract The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca2+ plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca2+]i) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca2+ dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca2+]i in U937-derived macrophages in both acute and chronic treatments (P < 0.01). In particular, the increases of the induced [Ca2+]i were different in the presence or absence of extracellular Ca2+ under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24 h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca2+]i induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells. 2005 Elsevier Inc. All rights reserved. Keywords: Oxidized low-density lipoprotein; [Ca2+]i; F-actin; CD36; Macrophage foam cells; Atherosclerosis
Atherosclerosis is a multifactorial disease that is associated with many risk factors including dyslipidemia, diabetes, obesity, and hypertension. The accumulation of oxidized low-density lipoprotein (Ox-LDL) in the arterial wall constitutes a fundamental event in atherogenesis. Macrophages play unique and complex roles in arterial wall cholesterol metabolism. At early stages of lipid infiltration, macrophages may serve a protective role by internalizing Ox-LDL via the scavenger receptor (CD36) pathway. Macrophage foam cells are a characteristic feature of early atherosclerotic lesions [1–4]. One general signaling mechanism used to transfer the information delivered by agonists into appropriate intracellular compartments involves the rapid redistribution of ionized calcium throughout the cell, which results in transient elevations of the intracellular-free Ca2+ concentration
*
Corresponding author. Fax: +86 571 87951676. E-mail address: [email protected] (X. Zheng).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.010
([Ca2+]i). Various physiological stimuli increase [Ca2+]i transiently and, thereby, induce cellular responses. Under pathological conditions, changes of [Ca2+]i are generally more pronounced and sustained [5]. As an important messenger, Ca2+ cannot be metabolized like other second-messenger molecules, so cells regulate intracellular Ca2+ level mainly through Ca2+ transport systems on the plasma membrane and intracellular Ca2+ ‘‘pool’’ membranes [6]. However, from spatiotemporal view, the correlation between kinetic changes in [Ca2+]i and cellular dysfunctions, such as atherosclerosis, has not yet been studied in detail. It was reported that exposure of smooth muscle cells, macrophage, and endothelial cells to Ox-LDL could raise [Ca2+]i [7–10]. These findings suggested that Ca2+ antagonists are proven useful in the treatment of atherosclerosis [11–13]. The increase of [Ca2+]i by Ox-LDL may play a major role in the formation of macrophage foam cells. Calcium signaling would have a consistent effect on actin gelation. Calcium-calmodulin would also regulate this aspect of actin dynamics [14]. Moreover, it was known that
T. Deng et al. / Biochemical and Biophysical Research Communications 338 (2005) 748–756
F-actin was clearly linked to Ox-LDL uptake in vitro [15– 19]. The exact sequence of events responsible for the formation of macrophage foam cells is not to be defined until now and, in particular, the role of calcium remains unclear. This prompted us to investigate the effect of Ox-LDL on kinetic changes in [Ca2+]i and on polymerization of F-actin in CD36-mediated endocytosis of macrophages. In the current study, the human monocyte U937-derived macrophages by phorbol 12-myristate 13-acetate (PMA) were chosen as target cells [20,21]. The increase in [Ca2+]i and F-actin polymerization suggests that Ox-LDL may increase [Ca2+]i to modify cytoskeletal proteins, alter cell morphology, and hence affect endocytosis. Our present results suggest that Ox-LDL increase intracellular-free calcium in the formation of macrophage foam cells and its possible effect on F-actin polymerization in CD36-mediated endocytosis of macrophages. Materials and methods Materials. Culture media and reagents were purchased from Invitrogen (NY, USA), and tissue culture plates were from Corning (NY, USA). PMA and Oil red O were purchased from Sigma Chemical (St. Louis, USA). Fluo-3/AM and BODIPY FL phallacidin were obtained from molecular probes (USA), and monoclonal mouse anti-human CD36 (FITC) was purchased from CALTAG Laboratories (Netherlands). Total cholesterol and free cholesterol came from Daiichi Pure Chemicals (Japan). All other chemicals were of the highest grade of purity and were commercially available. All other chemicals were purchased from Sigma Chemical (St. Louis, USA) unless otherwise mentioned. Cells. The human monocyte line U937 was obtained from the cell bank in Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. U937 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM glutamine, and 12 mM sodium carbonate. Cell cultures were maintained and incubated in a humidified atmosphere containing 5% (v/v) CO2 at 37 C. In experiments, U937 cells were differentiated to resident macrophage-like by addition of 100 ng/ml PMA without changing the medium for 72 h. Cells were then cultured for another 48 h without PMA, plated at the indicated density, washed with serum-free medium or buffer as indicated to remove non-adherent cells, and then incubated with the respective stimuli for various time periods in serum-free medium. PKC depletion prior experimentation was achieved by PMA treatment (18 h, 200 nM) of differentiated, macrophage-like cells and was confirmed by the absence of PKCa and b isozymes as determined by Western blot analysis [20]. Preparation and oxidation of human low-density lipoprotein. Human Low-density lipoproteins (LDL, 1.019–1.063 g/ml) were prepared from different human healthy donors by density gradient ultracentrifugation in the presence of 1 mg/ml EDTA (pH 7.4). The isolated LDL was dialyzed to remove EDTA and filtered (0.22 lm pore size), and stored at 4 C. The LDL was analyzed for protein content by the Bradford method, using bovine serum albumin as standard. The purity and charge of the lipoproteins were evaluated by examining electrophoretic mobility in an agarose gel. Oxidation of LDL was carried out with copper sulfate (final concentration of 10 lM) at 37 C for 12 h. The degree of oxidation was determined by measuring the amount of thiobarbituric acid-reactive substances (TBARS). Ox-LDL had thiobarbituric acid-reactive substances of 18 nmol/mg. Ox-LDL was then dialyzed against PBS containing EDTA 0.01% for 24 h at 4 C and sterile filtered. Foam cell assay. U937 cells were plated at 1 · 105 cells per 35-mm culture dish containing glass coverslips and incubated overnight. Cells were stimulated with PMA (100 ng/ml) in RPMI 1640 medium (10% FBS) for 72 h. In parallel experiments, U937-derived macrophages were incu-
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bated in serum-free RPMI 1640 medium in the presence or absence of pretreatment with Ox-LDL (100 lg/ml/105 cells) for 12 and 24 h at 37 C. Cells were washed three times with PBS, fixed by 10% formalin in PBS for 1 h at room temperature, and then stained with 0.1 ml/ml Oil red O solution 2 h, washed three times with water, and vaporized all water (at 32 C for 45 min) [22]. Cells were viewed in situ in 35 mm diameter tissue culture plates under a bright-field microscope in 100· fields using an Olympus IX70 microscope (Olympus, Japan). Measurements of free and total cholesterol. U937-derived macrophages (5 · 105 cells/ml) were added to each well of a 24-well plate with or without Ox-LDL (100 lg/ml, replicates of six per treatment). The incubation at 37 C was lasted for 12 and 24 h. The U937 cells, were washed three times in PBS, then added 1 ml isopropyl alcohol, and sonified for 30 s (15 s intervals, 3 times) [23]. Total cholesterol and free cholesterol in cell extracts were determined by the cholesterol oxidase enzymatic method using a commercial kit by a Hitachi 7020 autoanalyser (Tokyo, Japan). Lipid-extracted cells were dissolved in 0.1% sodium dodecyl sulfate— 0.1 M NaOH for 30 min, and total cell protein was determined with a protein assay kit. Esterified cholesterol was calculated from (total cholesterol) (free cholesterol) values. Results were expressed in lg/mg protein. The hallmark of foam cells is their high cholesteryl ester content (P50% of total cellular cholesterol) [24]. The kinetic measurement of intracellular-free Ca2+ concentration. Kinetic study of the rise in intracellular Ca2+ was conducted as follows. To monitor the basis of the rise in [Ca2+]i changes in single cells undergoing Ox-LDL stimulation, U937-derived macrophages were plated on glass coverslips. The intracellular Ca2+ concentration ([Ca2+]i) was measured by Ca2+ imaging with a Ca2+-sensitive fluorescent dye, Fluo-3/AM. Cells were washed three times with serum-free RPMI 1640 medium and incubated with 5 lM Fluo-3/AM at 37 C for 30 min. Cells were washed free of extracellular Fluo-3/AM dye in Hanks balanced salt solution buffer (Hanks) (in mM) (137 NaCl, 5 KCl, 0.8 MgSO4 Æ 7H2O, 1.2 CaCl2, 0.6 KH2PO4, 0.4 Na2HPO4 Æ H2O, 4.2 NaHCO3, 5 glucose, and 10 Hepes, pH 7.4). In D-Hanks balanced salt solution buffer (D-Hanks) without Ca2+, Ca2+ was substituted with 0.5 mM EGTA. The coverslips with Fluo-3/AM-loaded cells were removed and attached to the coverslip clamp chamber for the Ca2+ imaging analysis. The temperature of the cells was maintained at 37 C. Fluorescence measurements of [Ca2+]i were performed using confocal laser scanning microscopy (Zeiss LSM510, Germany) in the presence and absence of Ca2+ in the bath. Excitation was set at 488 nm and emission was monitored between 500 and 550 nm. Images of 512 · 512 pixels were acquired with a 20· objective. Laser scanning was started to obtain a time series of images. Acquisition rate was 1 frame (512 · 512) per 15 s. Intracellular calcium was monitored for at least 600 s. The obtained images were quantitatively analyzed for changes in fluorescence intensities within ROIs (regions of interest) using the Zeiss LSM510 software. Increases of [Ca2+]i are expressed as the ratio of fluorescence intensity of Fluo-3/AM over baseline (FX/F0). This ratio method is used because it is independent of factors such as dye concentration, excitation intensity, and detector efficiency. The Ca2+ concentration curve was recorded during the experiment or images were saved for analysis after the experiment. After about 15 s of basal image acquisition, Ox-LDL was perfused into the chamber throughout the remaining observation time. Final concentration was 100 lg/ml. To exclude any effect of mechanical stimulation, control Hanks and D-Hanks buffer were perfused without Ox-LDL and the Ca2+ response was observed for the same time. In each cell well, at least 15 equivalent-sized ROIs were identified, monitored, and analyzed during the experimental period. For the measurement of cytoplasmic and nuclear [Ca2+], the optical slices were passed through the nuclei. Fluorescence intensity of Fluo-3/AM was represented by pseudo-color scale in arbitrary units. The flow cytometric kinetic assay of intracellular-free Ca2+ concentration. Ox-LDL was added in U937-derived macrophages as previously described, and after 12 and 24 h incubation, cells were prepared for fluorescent staining. Cells were harvested and suspended at a concentration of 1 · 106 cells/ml in PBS. Cells were washed with PBS containing 0.2% BSA and incubated with Fluo-3/AM (1 lM) for 30 min at 37 C in the dark [25]. After washing twice with PBS, cells were analyzed by flow
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cytometry (Becton–Dickinson, USA). The viability of cells was higher than 97% as determined by the trypan blue dye exclusion method. Fluo-3loaded cells were excited with an argon laser at 488 nm and fluorescence was measured at 525 nm. The Fluo-3 fluorescence of unstimulated cells (basal fluorescence) was set to one arbitrary unit. The increase in Fluo-3 fluorescence was determined accordingly. Approximately 5000 cells were analyzed per group. The analyses of cytoskeleton. U937-derived macrophages were plated on coverslips. Ox-LDL was added as previously described, and after 12 and 24 h incubation, cells were prepared for double fluorochrome-label staining. Cells were washed with prewarmed PBS and fixed in 3.7% (wt/ vol) formaldehyde solution in PBS for 10 min at room temperature. After washing in the same buffer, cells were permeabilized by incubation with 0.1% Triton X-100 in PBS for 5 min at room temperature and then blocked with 1% bovine serum albumin for 30 min at 37 C. For cytoskeletal analyses, cells were subsequently incubated for 20 min at room temperature with 50 ll PBS containing 10 lg/ml propidium iodide (PI) for nuclear staining and 1 U BODIPY FL phallacidin for F-actin staining. Afterwards, Cells were washed three times with Hanks buffer to remove excess dye. F-actin levels were detected by a Zeiss 510 confocal laser scanning microscope using Zeiss LSM software for the acquisition and manipulation of images. The measurement of CD36. Ox-LDL was added in U937-derived macrophages as previously described, and after 12 and 24 h incubation, cells were prepared for fluorescent staining. Cells were harvested and suspended at a concentration of 1 · 106 cells/ml in PBS. Cells were washed with PBS containing 0.2% BSA and incubated with mouse anti-human CD36 mAb alone for 30 min at 4 C. After washing twice with PBS, cells were analyzed by flow cytometry (Becton–Dickinson, USA). A control mouse IgG was used as a negative control. Approximately 10,000 cells were analyzed per group. Statistical analysis. All values were expressed as means ± SEM. Statistical comparisons were made using Students t test for paired multiple
A
comparison. Differences between means were considered significant when P < 0.05. All experiments were performed a minimum of three times.
Results Identification of foam cell from U937-derived macrophages induced by Ox-LDL The morphological features of U937-derived macrophages exposed to Ox-LDL (100 lg/ml) were demonstrated Table 1 Contents of free cholesterol (FC), total cholesterol (TC), and cholesterol esters (CE) within U937-derived foam cells (X ± SD, n = 6) Group
TC (mg g 1)
FC (mg g 1)
CE (mg g 1)
CE/TC (%)
Control Ox-LDL (12 h) Ox-LDL (24 h)
43.6 ± 6.2 63.7 ± 10.0* 80.8 ± 9.4**
29.7 ± 2.9 33.3 ± 3.8 31.7 ± 5.0
14.0 ± 4.3 30.4 ± 9.8* 49.1 ± 10.6**
31.4 ± 6.1 46.7 ± 8.6* 60.3 ± 7.8**
* **
P < 0.05. P < 0.01, vs control group.
B
Normal cell
Ox-LDL 12h
C
Ox-LDL 24h
Fig. 1. Micrographs of normal U937-derived macrophages and U937derived macrophage foam cells stained with the Oil red O by Ox-LDL for 12 h (B), 24 h (C), respectively. (A) Normal cell is shown; (B) less lipid droplets; (C) lots of lipid droplets (arrows). Bar = 50.0 lm.
Fig. 2. Effects of Ox-LDL on [Ca2+]i in single U937-derived macrophage (n = 12). (A) In the Hanks balanced salt solution; (B) in the D-Hanks balanced salt solution.
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in Figs. 1A–C by staining with Oil red O, respectively. After 12 and 24 h, the cells showed the typical morphology of foam-like cells which contain lots of lipid droplets. The amount of lipid droplets in 12 h was relatively less than in 24 h, but reproducibly increased in comparison to controls. The results suggested the effect of Ox-LDL on U937-derived macrophages was in a time-dependent manner. In Ox-LDLtreated cells, the total and free cholesterol increased significantly as compared with the control (Table 1). Table 1 showed the cholesterol content in U937-derived macrophages after incubation by Ox-LDL (100 lg/ml) for 12 and 24 h. The total cholesterol content in cells after incubation for 24 h was 1.8 times greater than in control cells but only 1.5 times greater than for 12 h. Simultaneously, the cholesteryl ester content at 24 h was 3.5 times greater than in control cells but only 2.4 times greater than at 12 h. It is noteworthy that the ratio of (cholesteryl ester)/(total cholesterol) at 24 h reached 62.3 ± 7.8%, which has gone beyond the level in the typical foam cells [24]. In contrast with control cells, the ratio of (cholesteryl ester)/(total cholesterol) was only 31.4 ± 6.1%. Thus, the morphological and biochemical results confirmed that U937-derived macrophages were transformed into typical foam cells in a time-response manner after incubation with Ox-LDL. The kinetic measurement of intracellular-free Ca2+ concentration and spatial Ca2+ gradient in U937-derived macrophage by Ox-LDL To explore the spatial and temporal changes of [Ca2+]i during the transformation of U937-derived macrophages
A 0min
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into foam cells, the average cytosolic Ca2+ concentration in single cells was determined by using fluorescent probes Fluo-3/AM, a cell-permeant calcium indicator. The study examined the effect of acute exposure of Ox-LDL on U937-derived macrophages intracellular-free calcium. To test if the application of Ox-LDL would alter [Ca2+]i as well as to assess the source of calcium ions entering the cytoplasm, Ox-LDL (100 lg/ml) was applied to the cells in the presence and absence of extracellular calcium. We first examined the effects of the concentration of Ox-LDL frequently employed to elicit a Ca2+ transient. Interestingly, in the presence of Ca2+ in the bath, application of Ox-LDL resulted in a very rapid increase of [Ca2+]i at the beginning (Fig. 2A), which was significantly different from control values (P < 0.01). The increase peaked at approximately 4.6 ± 0.1 (FX/F0 ratio) in 0.5 min followed by falling to 1.4 ± 0.4 at 2.5 min and then showed a gradual increase with time (Fig. 2A). The relative noise in the calcium signals increased beyond the level that was expected from the saturation of the dye. In contrast, When calcium was removed from the extracellular solution, Ox-LDL resulted in a gradual increase with time (Fig. 2B). To exclude the possibility that the vehicle solution (Hanks and D-Hanks) was responsible for the above-described effects, either the vehicle solution was also applied in the experiments presented. These treatments did not cause any measurable elevation in [Ca2+]i. The control cell ratios remained constant over the course of the experiment (Fig. 2). Controls showed no significant change in [Ca2+]i. The subsequent addition of Ox-LDL, however, resulted in a similar rapid increase in [Ca2+]i as shown in
B 0.5min
C
Fig. 3. Changes of Ca2+ gradient in U937-derived macrophage (Hanks balanced salt solution n = 6). (A) A normal U937-derived macrophage (0 min); (B) the same U937-derived macrophage by Ox-LDL (0.5 min) bar = 20 lm; (C) fluorescence changes of cell (0 and 0.5 min).
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Fig. 2. A significant proportion of the intracellular Ca2+ probably originated from subcellular compartments. The results suggested that, with external calcium, presenting Ox-LDL caused a remarkable increase of [Ca2+]i and the amplitude of the external calcium evoked calcium transient. Our data also implied that the rapid rise in [Ca2+]i was due to the Ca2+ influx from the medium. From these results, we hypothesize that Ox-LDL might cause the opening of Ca2+ channels, allowing rapid influx of extracellular Ca2+. These observations demonstrate the ability of Ox-LDL to induce in influx of calcium into cells, presumably through a non-specific pathway. Experiments conducted in Ca2+-free media proved that Ox-LDL also induced Ca2+-increase depending on the release of Ca2+ from intracellular Ca2+ stores. Both extracellular and Table 2 Changes of Ca2+ gradient in U937-derived macrophages (X ± SD, n = 6) Time (min)
0 0.5 **
Fluorescence intensity [Ca2+]C
[Ca2+]N
55.0 ± 7.1 99.5 ± 11.1
91.4 ± 17.5** 180.8 ± 51.5**
P < 0.01, vs [Ca2+]C.
[Ca2+]N/[Ca2+]C 1.7 1.8
intracellular Ca2+ involved in OxLDL-induced increase in [Ca2+]i. As shown in Fig. 2, the localized release of intracellular-free Ca2+ was the result of release of Ca2+ from localized Ca2+ stores in cells, and it continued in the absence and presence of extracellular Ca2+. The overload of Ca2+ in a time-dependent manner probably aggravated the formation of macrophage foam cells. To measure the spatial changes in intracellular Ca2+ gradient during the transformation of U937-derived macrophages into foam cells, cytoplasmic and nuclear Ca2+ concentrations within the cells were determined by LSM. According to the previous results, the Ca2+ in confocal images of single living cells was viewed at 0 and 0.5 min. The typical confocal images of normal macrophage (Fig. 3A) and cell exposed to Ox-LDL (Fig. 3B) were obtained from an optical section through the center of the nuclei. The fluorescence was measured by a single line through a cell and its nucleus is shown in Fig. 3C. The position of the nucleus was identified by viewing the cell simultaneously with LSM. The confocal image of macrophages had a characteristic gradient pattern of Ca2+ distribution, where the highest intensity of fluorescence appears to be located in the nucleus. Fluorescence intensity in the cytoplasmic region near the plasma membrane was
Fig. 4. Effects of Ox-LDL on cytosolic-free calcium in U937-derived macrophages (n = 7). **P < 0.01, vs control group;
##
P < 0.01, vs Ox-LDL (12 h).
T. Deng et al. / Biochemical and Biophysical Research Communications 338 (2005) 748–756
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remarkably lower than in the other areas, but significantly higher than background. After treatment with Ox-LDL, fluorescence intensity of both the cytoplasm and nucleus obviously increased, and the latter was larger than the former (Table 2). Moreover, it is noted that the change in Ca2+ fluorescence intensity of the perinuclear area is the greatest during the transformation of macrophages into foam cells (Fig. 3C). There was a transmembrane Ca2+ gradient across the nuclear membrane in macrophages following their transformation to foam cells and the [Ca2+]N/ [Ca2+]C ratio increases from 1.7 at 0 min to 1.8 at 0.5 min (Table 2). The measurement of intracellular-free Ca2+ concentration by flow cytometer To quantify [Ca2+]i at chronic exposure of cells to OxLDL (100 lg/ml), we, respectively, measured the fluorescence intensity of Ca2+ by flow cytometer at various times. As shown in the histogram of Fluo-3 fluorescence (Fig. 4), the mean fluorescence intensity (MFI) of intracellular-free calcium in cells was significantly increased at 12 and 24 h (from 71.9 ± 6.6 to 95.9 ± 8.9, P < 0.01). Simultaneously, it was significantly higher at 12 h in Ox-LDL group than in control group (71.9 ± 6.6 vs 9.6 ± 0.9, P < 0.01), meaning that the overload of intracellular-free Ca2+ by Ox-LDL was time-dependent. The distribution of F-actin in U937-derived macrophages by Ox-LDL To determine whether Ox-LDL affects actin polymerization or not in U937-derived macrophages, the fluorescence changes of F-actin at 12 h and 24 h were monitored. The relative distribution of macrophage cytoskeletal F-actin was monitored by BODIPY FL phalloidin. This molecule binds to F-actin and stabilizes microfilaments, providing detail of the cellular cytoskeleton. This enabled us to discern the kinetics of actin polymerization during the formation of macrophage foam cell. Fig. 5 showed fluorescence photomicrographs of the U937-derived macrophages with Ox-LDL bound at various times at 37 C. Fluorescent photomicrographs of control cells showed no discernable change in BODIPY FL phallacidin staining, implying F-actin filaments at the cytoplast polymerized less (arrowheads in Fig. 5A). The cell nucleus was stained with PI (arrowheads in Figs. 5B, D, and E). There was little or no change in F-actin content of control cells after warming to 37 C. The BODIPY FL phallacidin staining in control cells, which was restricted to the pericelluar membrane, was less than exposed to Ox-LDL. In contrast, by 12 h after the addition of Ox-LDL, early foam cell formation (Fig. 1B) and actin polymerization were evident (arrowheads in Fig. 5C). The actin webs at the cytoplast had thinned somewhat, compared with those at 24 h. But fluorescence intensity was more prominent
Fig. 5. Changes of F-actin in normal U937-derived macrophages and foam cells by Ox-LDL (100 lg/ml). Cells were double stained for F-actin with BODIPY FL phallacidin (A, C, and E) and for nuclei with PI (B, D, and F), with Ox-LDL for 12 h (C) and 24 h (E), respectively. (A,B) Normal cell is shown; (C,D) a less lesion with prominent microfilament bundles; (E,F) an intermediate lesion (arrows). Bar = 5 lm.
at 24 h (Fig. 5E). F-actin clustered in the cytoplastic region of the cells, indicating massive polymerization of F-actin, which implied that Ox-LDL caused a substantial polymerization of F-actin and altered cytoskeletal organization and rearrangement in U937-derived macrophages. The measurement of CD36 We next endeavored to determine which CD36 was associated with the inducible Ox-LDL uptake observed in U937-derived macrophages. The CD36-mediated endocytosis of modified LDL (including OxLDL and AcLDL) is the key step in the formation of foam cells. CD36 was clearly linked to Ox-LDL uptake in vivo [26–28]. The expression of CD36 on the surface of OxLDL-treated cells was quantified by flow cytometry. CD36 expression was
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very significantly higher at 24 h than at 12 h (68.3 ± 7.3 MFI vs 38.9 ± 2.3 MFI, P < 0.01, Fig. 6). On the other hand, CD36 expression increased significantly in OxLDLtreated cells at 12 h than in control cells (38.9 ± 2.3 MFI vs 7.4 ± 1.4 MFI, P < 0.01). The MFI of Fluo-3 among cells responding to Ox-LDL (100 lg/ml) was time-dependent (Fig. 6). Discussion In this study, we have observed an effect of Ox-LDL on [Ca2+]i and actin polymerization in CD36-mediated endocytosis of U937-derived macrophages. It was found that Ox-LDL induced the spatiotemporal changes of intracellular-free calcium in both acute and chronic treatments. Moreover, both in the presence and absence of extracellular Ca2+, acute exposure of Ox-LDL induced different increases of the [Ca2+]i. There was a significant alteration of actin polymerization and CD36 at various times. The polymerization and organization of F-actin led to substantial changes in the morphology of the macrophages, in
CD36-mediated endocytosis of macrophages. These data suggested that the actin polymerization and the increase of intracellular-free calcium have important effect on pathological atherosclerosis, particularly on the early phase of formation of macrophage foam cells. It is well known that Ox-LDL is generally believed to be an important agonist in pathological atherosclerosis [29]. There were substantial evidences that Ox-LDL increases its atherogenic potential by enabling its rapid cellular uptake by foam cell precursors through the scavenger receptor. The actin cytoskeleton plays an important role in the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages [13]. The present study demonstrates that CD36-mediated endocytosis in U937-derived macrophages was accompanied by a net increase in F-actin. Ox-LDL significantly induced the increase of CD36 at 12 and 24 h. This is consistent with the hypothesis that the localized accumulation of F-actin in the cytoplastic region (as seen in Figs. 5C and E) results from not only the redistribution of existing actin filaments, but also newly polymerized actin. In these respects, F-actin assembly dur-
Fig. 6. Effects of Ox-LDL on CD36 expression in U937-derived macrophages (n = 6). **P < 0.01, vs control group;
##
P < 0.01, vs Ox-LDL (12 h).
T. Deng et al. / Biochemical and Biophysical Research Communications 338 (2005) 748–756
ing CD36-mediated endocytosis of U937-derived macrophages resembles F-actin assembly after Ox-LDL stimulation of human umbilical vein endothelial cells (HUVECs) [9]. The fact that the F-actin polymerization in U937-derived macrophages was increased in a time-dependent manner might reflect Ox-LDL stimulation to macrophages affecting actin network formation. Increases of [Ca2+]i in endothelium as a result of exposure to Ox-LDL have been hypothesized as being a gradual process that may involve cellular uptake of Ox-LDL, which may target the cytoplasmic influx of Ca2+ from extracellular medium or from intracellular stores [9]. In agreement with these opinions, our results showed that Ox-LDL (100 lg/ml) was able to increase [Ca2+]i in U937-derived macrophages during the first 20 min of exposure. Our results also demonstrated that Ox-LDL induced a rapid transient increase in [Ca2+]i in the presence of extracellular Ca2+ between 0.5 and 2.5 min. In contrast to the data in Fig. 2A, in the absence of external calcium in the bath, Ox-LDL resulted in a gradual increase with time. This increase, although not much pronounced than in the presence of extracellular Ca2+ (Fig. 2B), demonstrates that the elicit of Ox-LDL, at least in part, was possible due to the mobilization of calcium from internal stores. The results were averaged from several independent experiments where more than 12 individual cells were studied and were statistically significant. The MFI of intracellular-free calcium in chronic exposure to Ox-LDL increased significantly in a time-dependent manner (Fig. 4), which caused a greater increase in the amplitude of the OxLDL-evoked calcium signal. It is unclear why there was a rapid increase [Ca2+]i in the presence of extracellular Ca2+ in OxLDL-stimulated U937-derived macrophages. Ox-LDL could also rapidly increase phosphoinositide turnover in vascular smooth muscle [30]. In the presence of extracellular calcium (Fig. 2A), Ox-LDL may activate Ca2+-gated ion channels during CD36-mediated endocytosis and sequentially induce an influx from the extracellular environment. It was shown that Ox-LDL was capable of mobilizing calcium from internal stores and inducing the influx of calcium through the surface membrane. When calcium influx was enabled, OxLDL did not significantly lower the content of the intracellular stores and could persistently result in the overload of Ca2+. By confocal laser scanning microscope, we found that [Ca2+]i of the perinuclear area was significantly higher than that of cytoplasm in cell space, regardless of exposure of Ox-LDL. In chronic exposure to Ox-LDL, a time-dependent increase of [Ca2+]i was detected. It was suggested that there was the overload of intracellular-free calcium in the formation of macrophage foam cells (Fig. 4). We have described dynamic [Ca2+]i changes and actin polymerization during Ox-LDL stimulation; however, the question of whether and how actin polymerization translates into cellular endocytosis as well as the contribution of Ca2+ is unknown. Further studies on this aspect are still in progress.
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Acknowledgments This study was supported by the Economic and Trade commission of Zhejiang Province, the Key Laboratory of Chinese Medicine Screening, Exploitation and Medicinal Effectiveness Appraise for Cardio-cerebral Vascular and Nervous System of Zhejiang Province, and the Key Laboratory for Biomedical Engineering of Ministry of China. References [1] D. Steinberg, S. Parthasarathy, T.E. Carew, J.C. Khoo, J.L. Witztum, Beyond cholesterol modifications of low-density lipoprotein that increase its atherogenicity, N. Engl. J. Med. 320 (1989) 915–924. [2] M.S. Brown, J.L. Goldstein, Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis, Annu. Rev. Biochem. 52 (1983) 223–261. [3] C.P. Sparrow, S. Parthasarathy, D.A. Steinberg, Macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein, J. Biol. Chem. 264 (1989) 2599–2604. [4] A. Ne`gre-Salvayre, R. Salvayre, UV-treated lipoproteins as a model system for the study of biological effects of lipid peroxides on cultured cells, Biochim. Biophys. Acta 1123 (1992) 207–215. [5] P. Nicotera, S. Orrenius, The role of calcium in apoptosis, Cell Calcium 23 (1998) 173–180. [6] E. Carafoli, Intracellular calcium Homeostasis, Annu. Rev. Biochem. 56 (1987) 395–433. [7] H. Massaeli, C. Hurtado, J.A. Austria, G. Pierce, Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein, Free Radic. Res. 34 (2001) 9–16. [8] X.Y. Yang, Y.Z. Zhang, Y.G. Huang, F.Y. Yang, Changes of transmembrane Ca2+ gradient in the formation of macrophagederived foam cells, Biosci. Rep. 20 (2000) 1–12. [9] A. Ne`gre-Salvayre, G. Fitoussi, V. Re´aud, M.T. Pieraggi, J.C. Thiers, R. Salvayre, A delayed and sustained rise of cytosolic calcium is elicited by oxidized LDL in cultured bovine aortic endothelial cells, FEBS Lett. 299 (1992) 60–65. [10] B. Zhao, W.D. Ehringer, R. Dierichs, F.N. Miller, Oxidized lowdensity lipoprotein increases endothelial intracellular calcium and alters cytoskeletal F-actin distribution, Eur. J. Clin. Invest. 27 (1997) 48–54. [11] D.B. Weinstein, J.G. Heider, Antiatherogenic properties of calcium antagonists, state of the art, Am. J. Med. 86 (1989) 27–32. [12] R.P. Hof, C. Tapparelli, D.B. Weinstein, Hemodynamic, antivasoconstrictor, and antiatherosclerotic effects of calcium antagonists in animal models of atherosclerosis, J. Cardiovasc. Pharmacol. 15 (1990) S7–S12. [13] T. Afroze, M. Husain, Cell cycle dependent regulation of intracellular calcium concentration in vascular smooth muscle cells: a potential target for drug therapy, Curr. Drug Targets Cardiovasc. Haematol. Disord. 1 (2001) 23–40. [14] F. Nakamura, J.H. Hartwig, T.P. Stossel, P.T. Szymanski, Ca2+ and calmodulin regulate binding of filamina to actin filaments, J. Biol. Chem. 280 (2005) 32426–32433. [15] I. Tabas, X.H. Zha, N. Beatini, J.N. Myers, F.R. Maxfield, The actin cytoskeleton is important for the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages, J. Biol. Chem. 269 (1994) 22547–22556. [16] H. Massaeli, C. Hurtado, J.A. Austria, G.N. Pierce, Oxidized lowdensity lipoprotein induces cytoskeletal disorganization in smooth muscle cells, Am. J. Physiol. Heart. Circ Physiol. 277 (1999) 2017– 2025. [17] C.V. Zerbinatti, R.W. Gore, Uptake of modified low-density lipoproteins alters actin distribution and locomotor forces in macrophages, Am. J. Physiol. Cell Physiol. 284 (2003) 555–561.
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Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells Tong-le Deng, Lian Yu, Ya-kun Ge, Le Zhang, Xiao-xiang Zheng * Department of Biomedical Engineering, Zhejiang University (Yuquan Campus), Hangzhou 310027, PR China Received 28 September 2005 Available online 13 October 2005
Abstract The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca2+ plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca2+]i) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca2+ dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca2+]i in U937-derived macrophages in both acute and chronic treatments (P < 0.01). In particular, the increases of the induced [Ca2+]i were different in the presence or absence of extracellular Ca2+ under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24 h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca2+]i induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells. 2005 Elsevier Inc. All rights reserved. Keywords: Oxidized low-density lipoprotein; [Ca2+]i; F-actin; CD36; Macrophage foam cells; Atherosclerosis
Atherosclerosis is a multifactorial disease that is associated with many risk factors including dyslipidemia, diabetes, obesity, and hypertension. The accumulation of oxidized low-density lipoprotein (Ox-LDL) in the arterial wall constitutes a fundamental event in atherogenesis. Macrophages play unique and complex roles in arterial wall cholesterol metabolism. At early stages of lipid infiltration, macrophages may serve a protective role by internalizing Ox-LDL via the scavenger receptor (CD36) pathway. Macrophage foam cells are a characteristic feature of early atherosclerotic lesions [1–4]. One general signaling mechanism used to transfer the information delivered by agonists into appropriate intracellular compartments involves the rapid redistribution of ionized calcium throughout the cell, which results in transient elevations of the intracellular-free Ca2+ concentration
*
Corresponding author. Fax: +86 571 87951676. E-mail address: [email protected] (X. Zheng).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.010
([Ca2+]i). Various physiological stimuli increase [Ca2+]i transiently and, thereby, induce cellular responses. Under pathological conditions, changes of [Ca2+]i are generally more pronounced and sustained [5]. As an important messenger, Ca2+ cannot be metabolized like other second-messenger molecules, so cells regulate intracellular Ca2+ level mainly through Ca2+ transport systems on the plasma membrane and intracellular Ca2+ ‘‘pool’’ membranes [6]. However, from spatiotemporal view, the correlation between kinetic changes in [Ca2+]i and cellular dysfunctions, such as atherosclerosis, has not yet been studied in detail. It was reported that exposure of smooth muscle cells, macrophage, and endothelial cells to Ox-LDL could raise [Ca2+]i [7–10]. These findings suggested that Ca2+ antagonists are proven useful in the treatment of atherosclerosis [11–13]. The increase of [Ca2+]i by Ox-LDL may play a major role in the formation of macrophage foam cells. Calcium signaling would have a consistent effect on actin gelation. Calcium-calmodulin would also regulate this aspect of actin dynamics [14]. Moreover, it was known that
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F-actin was clearly linked to Ox-LDL uptake in vitro [15– 19]. The exact sequence of events responsible for the formation of macrophage foam cells is not to be defined until now and, in particular, the role of calcium remains unclear. This prompted us to investigate the effect of Ox-LDL on kinetic changes in [Ca2+]i and on polymerization of F-actin in CD36-mediated endocytosis of macrophages. In the current study, the human monocyte U937-derived macrophages by phorbol 12-myristate 13-acetate (PMA) were chosen as target cells [20,21]. The increase in [Ca2+]i and F-actin polymerization suggests that Ox-LDL may increase [Ca2+]i to modify cytoskeletal proteins, alter cell morphology, and hence affect endocytosis. Our present results suggest that Ox-LDL increase intracellular-free calcium in the formation of macrophage foam cells and its possible effect on F-actin polymerization in CD36-mediated endocytosis of macrophages. Materials and methods Materials. Culture media and reagents were purchased from Invitrogen (NY, USA), and tissue culture plates were from Corning (NY, USA). PMA and Oil red O were purchased from Sigma Chemical (St. Louis, USA). Fluo-3/AM and BODIPY FL phallacidin were obtained from molecular probes (USA), and monoclonal mouse anti-human CD36 (FITC) was purchased from CALTAG Laboratories (Netherlands). Total cholesterol and free cholesterol came from Daiichi Pure Chemicals (Japan). All other chemicals were of the highest grade of purity and were commercially available. All other chemicals were purchased from Sigma Chemical (St. Louis, USA) unless otherwise mentioned. Cells. The human monocyte line U937 was obtained from the cell bank in Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. U937 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM glutamine, and 12 mM sodium carbonate. Cell cultures were maintained and incubated in a humidified atmosphere containing 5% (v/v) CO2 at 37 C. In experiments, U937 cells were differentiated to resident macrophage-like by addition of 100 ng/ml PMA without changing the medium for 72 h. Cells were then cultured for another 48 h without PMA, plated at the indicated density, washed with serum-free medium or buffer as indicated to remove non-adherent cells, and then incubated with the respective stimuli for various time periods in serum-free medium. PKC depletion prior experimentation was achieved by PMA treatment (18 h, 200 nM) of differentiated, macrophage-like cells and was confirmed by the absence of PKCa and b isozymes as determined by Western blot analysis [20]. Preparation and oxidation of human low-density lipoprotein. Human Low-density lipoproteins (LDL, 1.019–1.063 g/ml) were prepared from different human healthy donors by density gradient ultracentrifugation in the presence of 1 mg/ml EDTA (pH 7.4). The isolated LDL was dialyzed to remove EDTA and filtered (0.22 lm pore size), and stored at 4 C. The LDL was analyzed for protein content by the Bradford method, using bovine serum albumin as standard. The purity and charge of the lipoproteins were evaluated by examining electrophoretic mobility in an agarose gel. Oxidation of LDL was carried out with copper sulfate (final concentration of 10 lM) at 37 C for 12 h. The degree of oxidation was determined by measuring the amount of thiobarbituric acid-reactive substances (TBARS). Ox-LDL had thiobarbituric acid-reactive substances of 18 nmol/mg. Ox-LDL was then dialyzed against PBS containing EDTA 0.01% for 24 h at 4 C and sterile filtered. Foam cell assay. U937 cells were plated at 1 · 105 cells per 35-mm culture dish containing glass coverslips and incubated overnight. Cells were stimulated with PMA (100 ng/ml) in RPMI 1640 medium (10% FBS) for 72 h. In parallel experiments, U937-derived macrophages were incu-
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bated in serum-free RPMI 1640 medium in the presence or absence of pretreatment with Ox-LDL (100 lg/ml/105 cells) for 12 and 24 h at 37 C. Cells were washed three times with PBS, fixed by 10% formalin in PBS for 1 h at room temperature, and then stained with 0.1 ml/ml Oil red O solution 2 h, washed three times with water, and vaporized all water (at 32 C for 45 min) [22]. Cells were viewed in situ in 35 mm diameter tissue culture plates under a bright-field microscope in 100· fields using an Olympus IX70 microscope (Olympus, Japan). Measurements of free and total cholesterol. U937-derived macrophages (5 · 105 cells/ml) were added to each well of a 24-well plate with or without Ox-LDL (100 lg/ml, replicates of six per treatment). The incubation at 37 C was lasted for 12 and 24 h. The U937 cells, were washed three times in PBS, then added 1 ml isopropyl alcohol, and sonified for 30 s (15 s intervals, 3 times) [23]. Total cholesterol and free cholesterol in cell extracts were determined by the cholesterol oxidase enzymatic method using a commercial kit by a Hitachi 7020 autoanalyser (Tokyo, Japan). Lipid-extracted cells were dissolved in 0.1% sodium dodecyl sulfate— 0.1 M NaOH for 30 min, and total cell protein was determined with a protein assay kit. Esterified cholesterol was calculated from (total cholesterol) (free cholesterol) values. Results were expressed in lg/mg protein. The hallmark of foam cells is their high cholesteryl ester content (P50% of total cellular cholesterol) [24]. The kinetic measurement of intracellular-free Ca2+ concentration. Kinetic study of the rise in intracellular Ca2+ was conducted as follows. To monitor the basis of the rise in [Ca2+]i changes in single cells undergoing Ox-LDL stimulation, U937-derived macrophages were plated on glass coverslips. The intracellular Ca2+ concentration ([Ca2+]i) was measured by Ca2+ imaging with a Ca2+-sensitive fluorescent dye, Fluo-3/AM. Cells were washed three times with serum-free RPMI 1640 medium and incubated with 5 lM Fluo-3/AM at 37 C for 30 min. Cells were washed free of extracellular Fluo-3/AM dye in Hanks balanced salt solution buffer (Hanks) (in mM) (137 NaCl, 5 KCl, 0.8 MgSO4 Æ 7H2O, 1.2 CaCl2, 0.6 KH2PO4, 0.4 Na2HPO4 Æ H2O, 4.2 NaHCO3, 5 glucose, and 10 Hepes, pH 7.4). In D-Hanks balanced salt solution buffer (D-Hanks) without Ca2+, Ca2+ was substituted with 0.5 mM EGTA. The coverslips with Fluo-3/AM-loaded cells were removed and attached to the coverslip clamp chamber for the Ca2+ imaging analysis. The temperature of the cells was maintained at 37 C. Fluorescence measurements of [Ca2+]i were performed using confocal laser scanning microscopy (Zeiss LSM510, Germany) in the presence and absence of Ca2+ in the bath. Excitation was set at 488 nm and emission was monitored between 500 and 550 nm. Images of 512 · 512 pixels were acquired with a 20· objective. Laser scanning was started to obtain a time series of images. Acquisition rate was 1 frame (512 · 512) per 15 s. Intracellular calcium was monitored for at least 600 s. The obtained images were quantitatively analyzed for changes in fluorescence intensities within ROIs (regions of interest) using the Zeiss LSM510 software. Increases of [Ca2+]i are expressed as the ratio of fluorescence intensity of Fluo-3/AM over baseline (FX/F0). This ratio method is used because it is independent of factors such as dye concentration, excitation intensity, and detector efficiency. The Ca2+ concentration curve was recorded during the experiment or images were saved for analysis after the experiment. After about 15 s of basal image acquisition, Ox-LDL was perfused into the chamber throughout the remaining observation time. Final concentration was 100 lg/ml. To exclude any effect of mechanical stimulation, control Hanks and D-Hanks buffer were perfused without Ox-LDL and the Ca2+ response was observed for the same time. In each cell well, at least 15 equivalent-sized ROIs were identified, monitored, and analyzed during the experimental period. For the measurement of cytoplasmic and nuclear [Ca2+], the optical slices were passed through the nuclei. Fluorescence intensity of Fluo-3/AM was represented by pseudo-color scale in arbitrary units. The flow cytometric kinetic assay of intracellular-free Ca2+ concentration. Ox-LDL was added in U937-derived macrophages as previously described, and after 12 and 24 h incubation, cells were prepared for fluorescent staining. Cells were harvested and suspended at a concentration of 1 · 106 cells/ml in PBS. Cells were washed with PBS containing 0.2% BSA and incubated with Fluo-3/AM (1 lM) for 30 min at 37 C in the dark [25]. After washing twice with PBS, cells were analyzed by flow
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cytometry (Becton–Dickinson, USA). The viability of cells was higher than 97% as determined by the trypan blue dye exclusion method. Fluo-3loaded cells were excited with an argon laser at 488 nm and fluorescence was measured at 525 nm. The Fluo-3 fluorescence of unstimulated cells (basal fluorescence) was set to one arbitrary unit. The increase in Fluo-3 fluorescence was determined accordingly. Approximately 5000 cells were analyzed per group. The analyses of cytoskeleton. U937-derived macrophages were plated on coverslips. Ox-LDL was added as previously described, and after 12 and 24 h incubation, cells were prepared for double fluorochrome-label staining. Cells were washed with prewarmed PBS and fixed in 3.7% (wt/ vol) formaldehyde solution in PBS for 10 min at room temperature. After washing in the same buffer, cells were permeabilized by incubation with 0.1% Triton X-100 in PBS for 5 min at room temperature and then blocked with 1% bovine serum albumin for 30 min at 37 C. For cytoskeletal analyses, cells were subsequently incubated for 20 min at room temperature with 50 ll PBS containing 10 lg/ml propidium iodide (PI) for nuclear staining and 1 U BODIPY FL phallacidin for F-actin staining. Afterwards, Cells were washed three times with Hanks buffer to remove excess dye. F-actin levels were detected by a Zeiss 510 confocal laser scanning microscope using Zeiss LSM software for the acquisition and manipulation of images. The measurement of CD36. Ox-LDL was added in U937-derived macrophages as previously described, and after 12 and 24 h incubation, cells were prepared for fluorescent staining. Cells were harvested and suspended at a concentration of 1 · 106 cells/ml in PBS. Cells were washed with PBS containing 0.2% BSA and incubated with mouse anti-human CD36 mAb alone for 30 min at 4 C. After washing twice with PBS, cells were analyzed by flow cytometry (Becton–Dickinson, USA). A control mouse IgG was used as a negative control. Approximately 10,000 cells were analyzed per group. Statistical analysis. All values were expressed as means ± SEM. Statistical comparisons were made using Students t test for paired multiple
A
comparison. Differences between means were considered significant when P < 0.05. All experiments were performed a minimum of three times.
Results Identification of foam cell from U937-derived macrophages induced by Ox-LDL The morphological features of U937-derived macrophages exposed to Ox-LDL (100 lg/ml) were demonstrated Table 1 Contents of free cholesterol (FC), total cholesterol (TC), and cholesterol esters (CE) within U937-derived foam cells (X ± SD, n = 6) Group
TC (mg g 1)
FC (mg g 1)
CE (mg g 1)
CE/TC (%)
Control Ox-LDL (12 h) Ox-LDL (24 h)
43.6 ± 6.2 63.7 ± 10.0* 80.8 ± 9.4**
29.7 ± 2.9 33.3 ± 3.8 31.7 ± 5.0
14.0 ± 4.3 30.4 ± 9.8* 49.1 ± 10.6**
31.4 ± 6.1 46.7 ± 8.6* 60.3 ± 7.8**
* **
P < 0.05. P < 0.01, vs control group.
B
Normal cell
Ox-LDL 12h
C
Ox-LDL 24h
Fig. 1. Micrographs of normal U937-derived macrophages and U937derived macrophage foam cells stained with the Oil red O by Ox-LDL for 12 h (B), 24 h (C), respectively. (A) Normal cell is shown; (B) less lipid droplets; (C) lots of lipid droplets (arrows). Bar = 50.0 lm.
Fig. 2. Effects of Ox-LDL on [Ca2+]i in single U937-derived macrophage (n = 12). (A) In the Hanks balanced salt solution; (B) in the D-Hanks balanced salt solution.
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in Figs. 1A–C by staining with Oil red O, respectively. After 12 and 24 h, the cells showed the typical morphology of foam-like cells which contain lots of lipid droplets. The amount of lipid droplets in 12 h was relatively less than in 24 h, but reproducibly increased in comparison to controls. The results suggested the effect of Ox-LDL on U937-derived macrophages was in a time-dependent manner. In Ox-LDLtreated cells, the total and free cholesterol increased significantly as compared with the control (Table 1). Table 1 showed the cholesterol content in U937-derived macrophages after incubation by Ox-LDL (100 lg/ml) for 12 and 24 h. The total cholesterol content in cells after incubation for 24 h was 1.8 times greater than in control cells but only 1.5 times greater than for 12 h. Simultaneously, the cholesteryl ester content at 24 h was 3.5 times greater than in control cells but only 2.4 times greater than at 12 h. It is noteworthy that the ratio of (cholesteryl ester)/(total cholesterol) at 24 h reached 62.3 ± 7.8%, which has gone beyond the level in the typical foam cells [24]. In contrast with control cells, the ratio of (cholesteryl ester)/(total cholesterol) was only 31.4 ± 6.1%. Thus, the morphological and biochemical results confirmed that U937-derived macrophages were transformed into typical foam cells in a time-response manner after incubation with Ox-LDL. The kinetic measurement of intracellular-free Ca2+ concentration and spatial Ca2+ gradient in U937-derived macrophage by Ox-LDL To explore the spatial and temporal changes of [Ca2+]i during the transformation of U937-derived macrophages
A 0min
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into foam cells, the average cytosolic Ca2+ concentration in single cells was determined by using fluorescent probes Fluo-3/AM, a cell-permeant calcium indicator. The study examined the effect of acute exposure of Ox-LDL on U937-derived macrophages intracellular-free calcium. To test if the application of Ox-LDL would alter [Ca2+]i as well as to assess the source of calcium ions entering the cytoplasm, Ox-LDL (100 lg/ml) was applied to the cells in the presence and absence of extracellular calcium. We first examined the effects of the concentration of Ox-LDL frequently employed to elicit a Ca2+ transient. Interestingly, in the presence of Ca2+ in the bath, application of Ox-LDL resulted in a very rapid increase of [Ca2+]i at the beginning (Fig. 2A), which was significantly different from control values (P < 0.01). The increase peaked at approximately 4.6 ± 0.1 (FX/F0 ratio) in 0.5 min followed by falling to 1.4 ± 0.4 at 2.5 min and then showed a gradual increase with time (Fig. 2A). The relative noise in the calcium signals increased beyond the level that was expected from the saturation of the dye. In contrast, When calcium was removed from the extracellular solution, Ox-LDL resulted in a gradual increase with time (Fig. 2B). To exclude the possibility that the vehicle solution (Hanks and D-Hanks) was responsible for the above-described effects, either the vehicle solution was also applied in the experiments presented. These treatments did not cause any measurable elevation in [Ca2+]i. The control cell ratios remained constant over the course of the experiment (Fig. 2). Controls showed no significant change in [Ca2+]i. The subsequent addition of Ox-LDL, however, resulted in a similar rapid increase in [Ca2+]i as shown in
B 0.5min
C
Fig. 3. Changes of Ca2+ gradient in U937-derived macrophage (Hanks balanced salt solution n = 6). (A) A normal U937-derived macrophage (0 min); (B) the same U937-derived macrophage by Ox-LDL (0.5 min) bar = 20 lm; (C) fluorescence changes of cell (0 and 0.5 min).
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Fig. 2. A significant proportion of the intracellular Ca2+ probably originated from subcellular compartments. The results suggested that, with external calcium, presenting Ox-LDL caused a remarkable increase of [Ca2+]i and the amplitude of the external calcium evoked calcium transient. Our data also implied that the rapid rise in [Ca2+]i was due to the Ca2+ influx from the medium. From these results, we hypothesize that Ox-LDL might cause the opening of Ca2+ channels, allowing rapid influx of extracellular Ca2+. These observations demonstrate the ability of Ox-LDL to induce in influx of calcium into cells, presumably through a non-specific pathway. Experiments conducted in Ca2+-free media proved that Ox-LDL also induced Ca2+-increase depending on the release of Ca2+ from intracellular Ca2+ stores. Both extracellular and Table 2 Changes of Ca2+ gradient in U937-derived macrophages (X ± SD, n = 6) Time (min)
0 0.5 **
Fluorescence intensity [Ca2+]C
[Ca2+]N
55.0 ± 7.1 99.5 ± 11.1
91.4 ± 17.5** 180.8 ± 51.5**
P < 0.01, vs [Ca2+]C.
[Ca2+]N/[Ca2+]C 1.7 1.8
intracellular Ca2+ involved in OxLDL-induced increase in [Ca2+]i. As shown in Fig. 2, the localized release of intracellular-free Ca2+ was the result of release of Ca2+ from localized Ca2+ stores in cells, and it continued in the absence and presence of extracellular Ca2+. The overload of Ca2+ in a time-dependent manner probably aggravated the formation of macrophage foam cells. To measure the spatial changes in intracellular Ca2+ gradient during the transformation of U937-derived macrophages into foam cells, cytoplasmic and nuclear Ca2+ concentrations within the cells were determined by LSM. According to the previous results, the Ca2+ in confocal images of single living cells was viewed at 0 and 0.5 min. The typical confocal images of normal macrophage (Fig. 3A) and cell exposed to Ox-LDL (Fig. 3B) were obtained from an optical section through the center of the nuclei. The fluorescence was measured by a single line through a cell and its nucleus is shown in Fig. 3C. The position of the nucleus was identified by viewing the cell simultaneously with LSM. The confocal image of macrophages had a characteristic gradient pattern of Ca2+ distribution, where the highest intensity of fluorescence appears to be located in the nucleus. Fluorescence intensity in the cytoplasmic region near the plasma membrane was
Fig. 4. Effects of Ox-LDL on cytosolic-free calcium in U937-derived macrophages (n = 7). **P < 0.01, vs control group;
##
P < 0.01, vs Ox-LDL (12 h).
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remarkably lower than in the other areas, but significantly higher than background. After treatment with Ox-LDL, fluorescence intensity of both the cytoplasm and nucleus obviously increased, and the latter was larger than the former (Table 2). Moreover, it is noted that the change in Ca2+ fluorescence intensity of the perinuclear area is the greatest during the transformation of macrophages into foam cells (Fig. 3C). There was a transmembrane Ca2+ gradient across the nuclear membrane in macrophages following their transformation to foam cells and the [Ca2+]N/ [Ca2+]C ratio increases from 1.7 at 0 min to 1.8 at 0.5 min (Table 2). The measurement of intracellular-free Ca2+ concentration by flow cytometer To quantify [Ca2+]i at chronic exposure of cells to OxLDL (100 lg/ml), we, respectively, measured the fluorescence intensity of Ca2+ by flow cytometer at various times. As shown in the histogram of Fluo-3 fluorescence (Fig. 4), the mean fluorescence intensity (MFI) of intracellular-free calcium in cells was significantly increased at 12 and 24 h (from 71.9 ± 6.6 to 95.9 ± 8.9, P < 0.01). Simultaneously, it was significantly higher at 12 h in Ox-LDL group than in control group (71.9 ± 6.6 vs 9.6 ± 0.9, P < 0.01), meaning that the overload of intracellular-free Ca2+ by Ox-LDL was time-dependent. The distribution of F-actin in U937-derived macrophages by Ox-LDL To determine whether Ox-LDL affects actin polymerization or not in U937-derived macrophages, the fluorescence changes of F-actin at 12 h and 24 h were monitored. The relative distribution of macrophage cytoskeletal F-actin was monitored by BODIPY FL phalloidin. This molecule binds to F-actin and stabilizes microfilaments, providing detail of the cellular cytoskeleton. This enabled us to discern the kinetics of actin polymerization during the formation of macrophage foam cell. Fig. 5 showed fluorescence photomicrographs of the U937-derived macrophages with Ox-LDL bound at various times at 37 C. Fluorescent photomicrographs of control cells showed no discernable change in BODIPY FL phallacidin staining, implying F-actin filaments at the cytoplast polymerized less (arrowheads in Fig. 5A). The cell nucleus was stained with PI (arrowheads in Figs. 5B, D, and E). There was little or no change in F-actin content of control cells after warming to 37 C. The BODIPY FL phallacidin staining in control cells, which was restricted to the pericelluar membrane, was less than exposed to Ox-LDL. In contrast, by 12 h after the addition of Ox-LDL, early foam cell formation (Fig. 1B) and actin polymerization were evident (arrowheads in Fig. 5C). The actin webs at the cytoplast had thinned somewhat, compared with those at 24 h. But fluorescence intensity was more prominent
Fig. 5. Changes of F-actin in normal U937-derived macrophages and foam cells by Ox-LDL (100 lg/ml). Cells were double stained for F-actin with BODIPY FL phallacidin (A, C, and E) and for nuclei with PI (B, D, and F), with Ox-LDL for 12 h (C) and 24 h (E), respectively. (A,B) Normal cell is shown; (C,D) a less lesion with prominent microfilament bundles; (E,F) an intermediate lesion (arrows). Bar = 5 lm.
at 24 h (Fig. 5E). F-actin clustered in the cytoplastic region of the cells, indicating massive polymerization of F-actin, which implied that Ox-LDL caused a substantial polymerization of F-actin and altered cytoskeletal organization and rearrangement in U937-derived macrophages. The measurement of CD36 We next endeavored to determine which CD36 was associated with the inducible Ox-LDL uptake observed in U937-derived macrophages. The CD36-mediated endocytosis of modified LDL (including OxLDL and AcLDL) is the key step in the formation of foam cells. CD36 was clearly linked to Ox-LDL uptake in vivo [26–28]. The expression of CD36 on the surface of OxLDL-treated cells was quantified by flow cytometry. CD36 expression was
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very significantly higher at 24 h than at 12 h (68.3 ± 7.3 MFI vs 38.9 ± 2.3 MFI, P < 0.01, Fig. 6). On the other hand, CD36 expression increased significantly in OxLDLtreated cells at 12 h than in control cells (38.9 ± 2.3 MFI vs 7.4 ± 1.4 MFI, P < 0.01). The MFI of Fluo-3 among cells responding to Ox-LDL (100 lg/ml) was time-dependent (Fig. 6). Discussion In this study, we have observed an effect of Ox-LDL on [Ca2+]i and actin polymerization in CD36-mediated endocytosis of U937-derived macrophages. It was found that Ox-LDL induced the spatiotemporal changes of intracellular-free calcium in both acute and chronic treatments. Moreover, both in the presence and absence of extracellular Ca2+, acute exposure of Ox-LDL induced different increases of the [Ca2+]i. There was a significant alteration of actin polymerization and CD36 at various times. The polymerization and organization of F-actin led to substantial changes in the morphology of the macrophages, in
CD36-mediated endocytosis of macrophages. These data suggested that the actin polymerization and the increase of intracellular-free calcium have important effect on pathological atherosclerosis, particularly on the early phase of formation of macrophage foam cells. It is well known that Ox-LDL is generally believed to be an important agonist in pathological atherosclerosis [29]. There were substantial evidences that Ox-LDL increases its atherogenic potential by enabling its rapid cellular uptake by foam cell precursors through the scavenger receptor. The actin cytoskeleton plays an important role in the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages [13]. The present study demonstrates that CD36-mediated endocytosis in U937-derived macrophages was accompanied by a net increase in F-actin. Ox-LDL significantly induced the increase of CD36 at 12 and 24 h. This is consistent with the hypothesis that the localized accumulation of F-actin in the cytoplastic region (as seen in Figs. 5C and E) results from not only the redistribution of existing actin filaments, but also newly polymerized actin. In these respects, F-actin assembly dur-
Fig. 6. Effects of Ox-LDL on CD36 expression in U937-derived macrophages (n = 6). **P < 0.01, vs control group;
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P < 0.01, vs Ox-LDL (12 h).
T. Deng et al. / Biochemical and Biophysical Research Communications 338 (2005) 748–756
ing CD36-mediated endocytosis of U937-derived macrophages resembles F-actin assembly after Ox-LDL stimulation of human umbilical vein endothelial cells (HUVECs) [9]. The fact that the F-actin polymerization in U937-derived macrophages was increased in a time-dependent manner might reflect Ox-LDL stimulation to macrophages affecting actin network formation. Increases of [Ca2+]i in endothelium as a result of exposure to Ox-LDL have been hypothesized as being a gradual process that may involve cellular uptake of Ox-LDL, which may target the cytoplasmic influx of Ca2+ from extracellular medium or from intracellular stores [9]. In agreement with these opinions, our results showed that Ox-LDL (100 lg/ml) was able to increase [Ca2+]i in U937-derived macrophages during the first 20 min of exposure. Our results also demonstrated that Ox-LDL induced a rapid transient increase in [Ca2+]i in the presence of extracellular Ca2+ between 0.5 and 2.5 min. In contrast to the data in Fig. 2A, in the absence of external calcium in the bath, Ox-LDL resulted in a gradual increase with time. This increase, although not much pronounced than in the presence of extracellular Ca2+ (Fig. 2B), demonstrates that the elicit of Ox-LDL, at least in part, was possible due to the mobilization of calcium from internal stores. The results were averaged from several independent experiments where more than 12 individual cells were studied and were statistically significant. The MFI of intracellular-free calcium in chronic exposure to Ox-LDL increased significantly in a time-dependent manner (Fig. 4), which caused a greater increase in the amplitude of the OxLDL-evoked calcium signal. It is unclear why there was a rapid increase [Ca2+]i in the presence of extracellular Ca2+ in OxLDL-stimulated U937-derived macrophages. Ox-LDL could also rapidly increase phosphoinositide turnover in vascular smooth muscle [30]. In the presence of extracellular calcium (Fig. 2A), Ox-LDL may activate Ca2+-gated ion channels during CD36-mediated endocytosis and sequentially induce an influx from the extracellular environment. It was shown that Ox-LDL was capable of mobilizing calcium from internal stores and inducing the influx of calcium through the surface membrane. When calcium influx was enabled, OxLDL did not significantly lower the content of the intracellular stores and could persistently result in the overload of Ca2+. By confocal laser scanning microscope, we found that [Ca2+]i of the perinuclear area was significantly higher than that of cytoplasm in cell space, regardless of exposure of Ox-LDL. In chronic exposure to Ox-LDL, a time-dependent increase of [Ca2+]i was detected. It was suggested that there was the overload of intracellular-free calcium in the formation of macrophage foam cells (Fig. 4). We have described dynamic [Ca2+]i changes and actin polymerization during Ox-LDL stimulation; however, the question of whether and how actin polymerization translates into cellular endocytosis as well as the contribution of Ca2+ is unknown. Further studies on this aspect are still in progress.
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