Macrophages Stimulate Cholesteryl Ester Accumulation in Cocultured Smooth Muscle Cells Incubated With Lipoprotein-Proteoglycan Complex
Foam cells of atherosclerotic lesions originate from both macrophages and smooth muscle cells (SMCs). We explored the mechanism by which SMCs may become lipid laden. Confluent bovine aortic SMCs were cocultured with P388D1 macrophages, and the cocultures were incubated for various times with low-density lipoprotein (LDL), acetyl-LDL, or lipoprotein-proteoglycan (PG) complex isolated from human atherosclerotic lesions. Macrophages were then removed from the SMCs and the cholesteryl ester (CE) content of the SMCs was quantitated. Lipoprotein-PG complex but not LDL or acetyl-LDL produced a 6-fold to 9-fold stimulation of CE synthesis and a 4.4-fold increase in cellular CE mass in cocultured SMCs relative to control SMCs. In similar studies with human aortic SMC-macrophage cocultures, macrophages stimulated lipoprotein-PG complex–mediated CE synthesis 7-fold to 13-fold and CE mass 7.8-fold in cocultured SMCs compared with SMCs cultured alone. CE synthesis that was mediated by lipoprotein-PG complex was dose dependent and increased linearly with time. Incubation of lipoprotein-PG complex with SMC-macrophage cocultures but not with SMCs or macrophages alone resulted in aggregation of the complex and stimulation of cholesterol esterification in SMCs by the conditioned media containing the aggregated complex. Cytochalasin D, an inhibitor of phagocytosis, inhibited CE synthesis mediated by lipoprotein-PG complex by 73%, whereas polyinosinic acid, an inhibitor of the scavenger receptor, had no effect. Upregulation or downregulation of apolipoprotein B,E receptors did not affect the lipoprotein-PG complex–mediated CE synthesis by cocultured SMCs. Lipoprotein-PG complex did not stimulate CE synthesis in SMCs cocultured with aortic endothelial cells or macrophages cocultured with SMCs. These results indicate that macrophages can stimulate CE synthesis and accumulation in cocultured SMCs when incubated with lipoprotein-PG complexes isolated from atherosclerotic lesions. This could be a potential mechanism for myocyte foam cell formation.
- Received March 25, 1995.
- Revision received March 12, 1996.
- Accepted March 12, 1996.
Foam cells are the hallmarks of early atherosclerotic lesions and originate from SMCs and macrophages.1 2 3 4 It is known that CE, which accounts for most of the lipid in foam cells, is derived from plasma LDL. However, the in vivo mechanism of LDL-mediated foam cell formation is not known. The mechanism of conversion of macrophages into foam cells has been investigated extensively. The results indicate that in vitro, macrophages are converted into foam cells by uptake of several chemically and biologically modified forms of LDL.5 6 7 8 9 Foam cell formation also occurs in vitro when macrophages ingest complexes of LDL and artery wall PGs.10 11 12 13
The mechanism of conversion of SMCs into foam cells is less clear. Although SMCs express the acetyl-LDL (scavenger) receptor after treatment with phorbol ester, uptake of acetyl-LDL by these cells does not result in foam cell formation.14 Similarly, attempts by many other investigators to induce CE deposition in vascular SMCs have met with only limited success.15 16 17 18 19 20 A notable exception is the massive CE deposition in SMCs obtained by uptake of CE-rich droplets derived from lipid-laden macrophages.21 To induce cholesterol accumulation in SMCs, however, cholesterol-enriched macrophages must first be disrupted to release their lipid dispersions and then these dispersions must be placed in direct contact with the SMCs. Nevertheless, that study suggested the possibility that macrophages might play a significant role in facilitating lipid accumulation in SMCs.
Migraton of resident macrophages to the subendothelial space of the blood vessel occurs early during atherogenesis.2 Macrophage infiltration has been shown to be accompanied by the migration and proliferation of medial SMCs. In addition, the macrophage-SMC interaction could also regulate SMC metabolism.22 23 Our previous studies have shown that lipoprotein-PG complexes isolated from atherosclerotic lesions cause CE accumulation in human monocyte–derived macrophages.24 Therefore, in the present study we investigated whether macrophages modulate the uptake of such lipoprotein-PG complexes by vascular SMCs. The results indicate that macrophages can facilitate uptake of lipoprotein-PG complexes by SMCs, leading to intracellular CE deposition.
MEM, DMEM, RPMI 1640, and sera were obtained from GIBCO BRL, and human EC growth medium was from Clonetics. Cell culture dishes were from Costar. [1-14C]oleic acid (50 mCi/mmol) and sodium [125I]iodide were purchased from ICN Biomedicals, Inc. Collagenase (CLS 2, 197 U/mg) was obtained from Worthington Biochemical Corp. Elastase (type III) and SMC actin antibody were obtained from Sigma Chemical Co, anti-mouse IgG/Fc antibody from Pierce, and anti-human macrophage monoclonal antibody from Chemicon.
LDL (d=1.019 to 1.055 g/mL) was prepared by ultracentrifugation of pooled human serum by the method of Hatch and Lees.25 EDTA (0.05%), 10 μmol/L BHT, and 20 mmol/L PMSF were added to the blood immediately after collection. Acetyl-LDL and lipoprotein-deficient serum were prepared as described before.24 Acetylation modified 86% of the available free amino groups of LDL. Lipoproteins were stored at 4°C in 0.15 mol/L NaCl and 0.01% EDTA, pH 7.4, and used within 3 weeks. Before being used in cell culture studies, the lipoproteins were sterilized by membrane filtration.
Lipoprotein-PG complexes were isolated from human aorta fibrous plaque lesions and characterized as described before.24 In brief, aortas were obtained at autopsy and within 12 hours of death. Fibrous plaque lesions were dissected out and extracted with 0.15 mol/L NaCl and 0.05 mol/L Tris HCl (pH 7.4), containing protease inhibitors and antioxidants. The pooled extract from several aortas was fractionated on a column of Bio-Gel A 50m. The fraction that eluted in the inclusive volume of the column (ahead of the elution volume of LDL) was further purified by affinity chromatography on anti-apoB–Sepharose CL-4B. The lipoprotein-PG complex was eluted from the affinity column with 0.15 mol/L NaCl, pH 11, and dialyzed immediately against 0.15 mol/L NaCl and 0.05 mol/L Tris HCl (pH 7.4), containing protease inhibitors and antioxidants.24 ApoB was the only apoprotein identified in the affinity-purified material. Besides apoB lipoproteins, the fraction also contained hyaluronic acid, chondroitin 6-sulfate, dermatan sulfate, and heparin. The lipoprotein-PG complex was dialyzed against the appropriate medium and sterilized by membrane filtration prior to addition to cell cultures. Recently, we showed that the lipoprotein-PG complexes did not dissociate either during the affinity chromatography step or when incubated in the culture medium at 37°C for up to 40 hours.26
SMCs were cultured from bovine thoracic aortas by the explant technique.27 Bovine aortas were obtained from a local abbatoir. Explants of the vessel media were plated in sterile culture dishes in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μg/mL amphotericin B and incubated in a humidified CO2 (5%) incubator. Cells began to grow out of the explants in 5 to 7 days and became confluent in 2 to 3 weeks. The cells were characterized as SMCs by their morphology and staining with an anti–α-SMC actin antibody (Sigma). Cells at passages 5 to 8 were used in the study.
Bovine aortic ECs were isolated as described before.28 In brief, the EC layer was removed by scraping the intima with a sterile cell scraper. The cells were then dispersed by incubation in 0.1% collagenase for 20 minutes at 37°C, recovered by centrifugation, and cultured in the same medium that was used for SMC culture. The cells were identified as ECs by their “cobblestone” morphology at confluence and expression of factor VIII–related antigen. Cells between passages 4 to 6 were used for experiments.
Human aortic ECs and SMCs were cultured as described by Hosenpud et al.29 Aortic tissue for this purpose was obtained from the cardiac transplantation program at Ochsner Hospital, New Orleans, La. To isolate ECs, aortic tissue was digested with 0.1% elastase in RPMI 1640 supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C for 1 hour. ECs were then removed by gently swabbing the luminal surface of the aorta with a sterile cotton swab and recovered by swirling the swab in a sterile centrifuge tube containing culture medium (EC growth medium supplemented with 5% fetal bovine serum and antibiotics). The cells were pelleted by centrifugation (250g for 10 minutes), resuspended in culture medium, and plated in culture wells coated with fetal bovine serum. Cells at passage 3 were used in the study. ECs were identified by their immunofluorescent staining to factor VIII–related antigen.
After ECs were isolated from the aorta, the remaining tissue was digested in a mixture of 0.2% collagenase and 0.02% elastase for 1 hour at 37°C. SMCs were harvested by scraping the luminal surface with a sterile cotton swab as described above for ECs and cultured in gelatin-coated culture wells (1% gelatin) in MEM supplemented with 1% (vol/vol) nonessential amino acids, 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μg/mL amphotericin B. When confluent the cells were subcultured, and cells at passage 3 were used for experiments. SMCs were identified by positive staining with an SMC-specific antibody.
Mouse macrophage cell line P388D1 was obtained from American Type Culture Collection and grown in DMEM containing 10% fetal bovine serum and antibiotics. According to the vendor, these are well-differentiated cells.
Monocytes were isolated from heparinized human blood by the Ficoll-Hypaque method; details of the procedure have been described previously.24 The cells were plated in RPMI 1640 containing 20% (vol/vol) human serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured for 5 days before being used for experiments.
The metabolism of lipoprotein-PG complex by human monocyte–derived macrophages and P388D1 macrophages was comparable and led to stimulation of CE synthesis and accumulation to almost the same extent in both cell types.
Unless otherwise indicated, SMC-macrophage cocultures were routinely established in either 12-well cell culture plates or 35-mm dishes. SMCs were plated and allowed to grow to confluence. P388D1 macrophages (1 to 2×106 cells) or human monocytes (2 to 3×106 cells) were then seeded directly on top of the SMCs. After 2 hours, nonadherent cells were washed off with PBS and the cells were maintained in coculture for 24 hours (bovine SMC–P388D1) or 5 days (human SMC–human macrophages) before experiments were initiated.
For SMC-EC coculture, confluent SMCs in 12-well plates or 35-mm dishes were treated with 0.1% fibronectin for 5 minutes.30 ECs (4 to 6×105) were then added to the SMCs and cocultures were maintained for 4 days before they were used in the experiments. Control SMCs received similar treatment with 0.1% fibronectin.
At the end of the experiments, macrophages and ECs were selectively removed from the cocultures by incubation with 1% collagenase for 3 to 5 minutes at 37°C.30 Control SMCs were treated likewise.
P388D1 macrophages and monocyte-derived macrophages were cultured separately in 25-cm2 flasks for 2 days and 5 days, respectively. The medium was removed and the cells were washed twice with PBS. Fresh medium containing either no serum or 10% fetal bovine serum was then added to the flasks, and the cells were incubated for 48 hours at 37°C. The conditioned medium was centrifuged to remove cellular debris, filtered through a 0.45-μm filter, and stored at −20°C for up to 2 weeks. Media incubated similarly without cells served as controls.
In some experiments P388D1 macrophages were cocultured with bovine SMCs in medium containing no serum. After 24 hours the conditioned medium was harvested. We also harvested conditioned medium from SMC-macrophage cocultures after a 6-hour incubation in serum-free medium containing lipoprotein-PG complex (100 μg cholesterol per milliliter). Conditioned medium was also obtained from P388D1 macrophage cultures incubated with lipoprotein-PG complex (100 μg cholesterol per milliliter) for 24 hours. Alternatively, macrophages were cholesterol loaded by incubation with lipoprotein-PG complex (100 μg cholesterol per milliliter for 48 hours). The medium was removed and the cell layer washed and incubated in fresh DMEM containing 0.1% bovine serum albumin for 24 hours. The medium was then harvested.
CE synthesis was determined as described previously.24 In brief, the culture medium was removed from control SMC cultures and cocultures, and the cells were washed twice with PBS. DMEM containing 10% lipoprotein-deficient serum (medium A) was added to each culture well or dish, along with various ligands to be tested, and 0.2 mmol/L [14C]oleate-albumin. The concentration of individual ligands and the duration of incubation depended on the nature of the experiments and are indicated in “Results.” Alternatively, in some experiments CE synthesis was measured as described by Goldstein et al.31 In these experiments, the cultures were first incubated with various concentrations of each ligand in medium A for 16 hours. The medium was then removed and the cells were washed. Fresh medium A containing 0.2 mmol/L [14C]oleate-albumin was added to the culture wells and the plates were incubated for 4 hours. After incubation macrophages or ECs were removed from the cocultures in both procedures. Cellular lipids of SMCs were extracted with hexane/isopropanol (3:2, vol/vol) and separated by thin-layer chromatography. CE spots were scraped off the plates and quantified. Results were corrected for the percent recovery of an internal [3H]cholesteryl oleate standard. Control incubations were performed without cells, and these values were subtracted from the results. The solvent-extracted cells were dissolved in 0.2N NaOH, and an aliquot of the solution was used for protein assay.
Cultures were incubated in medium A containing 100 μg cholesterol per milliliter of various ligands. After 48 hours the medium was removed and cells were washed thoroughly. Macrophages or endothelial cells were then removed from the cocultures by treatment with collagenase. The SMCs were then extracted twice with hexane/isopropanol (3:2, vol/vol). To correct for loss during lipid extraction, an internal standard of [3H]cholesterol (10 000 disintegrations per minute) was added to each culture dish before solvent extraction. Cholesterol and CE were separated from the lipid extract by silica gel G–thin-layer chromatography and quantitated by the procedure of Bowman and Wolf,32 with the modification that the chromophore was measured spectrofluorometrically.
Protein was assayed by a modified Lowry procedure33 and DNA by a fluorometric procedure.34 Uronic acid was measured by the method of Blumenkrantz and Asboe-Hansen.35 Cholesterol content of lipoproteins and lpoprotein-PG complexes was determined with a commercial enzymatic reagent kit (Autoflow Cholesterol 236691, Boehringer Mannheim Diagnostics). Cellular total and free cholesterol contents were determined by the fluorometric assay of Gamble et al.36 Cell viability was determined by trypan blue dye exclusion.
Interexperimental variability was <10%. Statistical significance (P<.05) was analyzed by Student's t test.
Two types of cocultures were studied. In the SMC-macrophage cocultures, macrophages were seeded directly on top of confluent SMCs. About 85% to 92% of the cells became firmly attached to the SMCs within 2 hours of incubation. In contrast, as reported by others,30 in the SMC-EC cocultures the ECs became well attached to the underlying SMCs only if the SMCs were pretreated with 0.1% fibronectin.
A brief exposure to a 1% solution of collagenase was used to specifically remove the macrophages and ECs from their respective cocultures with SMCs. In addition to collagenase treatment, treatment with 1% neutral protease for 4 minutes or 0.2 mmol/L EDTA for 3 minutes was equally effective in removing macrophages from the cocultures. These treatments did not detach SMCs. Earlier, Hajjar et al30 used the enzyme procedure to remove ECs from underlying cocultured SMCs. Complete removal of macrophages and ECs from their cocultures was confirmed by cell count, DNA and protein determinations, histology, and immunohistochemistry. In a typical experiment, the bovine SMC-macrophage (P388D1) coculture contained 1.7±0.2×106 adherent macrophages. Collagenase treatment of the coculture for 4 minutes released 1.68±0.4×106 macrophages. The released cells showed uniform positive staining with anti-mouse IgG(Fc) and negative staining with an SMC-specific antibody. Table 1⇓ shows the DNA and protein contents of bovine SMCs before and after removal of macrophages and ECs. The DNA and protein contents of cocultures were significantly higher than those of SMC cultures. After collagenase treatment, these values were reduced to those of SMCs cultured alone. In the human SMC-macrophage coculture, after collagenase treatment the detached cells showed uniform staining for peroxidase and esterase and positive staining with anti-human macrophage monoclonal antibody. The DNA and protein contents of these cocultures before and after collagenase treatment followed a pattern similar to that described in Table 1 for bovine SMC-P388D1 macrophages (data not shown). Cells were also stained with antibodies specific for macrophages and ECs (data not shown). In the bovine SMC-macrophage (P388D1) cocultures, cells that remained in the dish after collagenase treatment did not stain with anti-mouse IgG(Fc) or anti-human macrophage antibody. Likewise, factor VIII was not detectable in SMC-EC cocultures after removal of ECs. These results indicated that the macrophages and ECs had been completely removed from their respective cocultures with SMCs after treatment with collagenase, thereby leaving the SMCs intact.
Coculturing of macrophages (P388D1 or human) with SMC (bovine or human) did not cause any lysis of macrophages, as determined by phase-contrast microscopy and trypan blue dye exclusion.
CE Synthesis and Accumulation
Fig 1 (A and B)⇓ shows the effect of various concentrations of LDL, lipoprotein-PG complex from fibrous plaques, and acetyl-LDL on the synthesis of CE by control bovine SMCs (Fig 1A) or SMCs cocultured with P388D1 macrophages (Fig 1B). In these experiments the cells were incubated with the ligands in the presence of [14C]oleate-albumin, and CE synthesis was then assayed. The results show that in control SMCs, LDL showed the highest stimulation of CE synthesis. Lipoprotein-PG complex also stimulated CE synthesis, but to a lesser extent, whereas acetyl-LDL had no effect. In contrast, when SMCs were cocultured with macrophages for 24 hours and then incubated with various ligands, lipoprotein-PG complex stimulated CE synthesis 6-fold to 9-fold above that of control SMCs. Coculture for 48 to 72 hours did not cause further stimulation of CE synthesis (data not shown). Compared with control SMCs, acetyl-LDL also produced a slight stimulation of CE synthesis in cocultured SMCs. At all concentrations, lipoprotein-PG complex was 5-fold to 8-fold more potent than acetyl-LDL in its ability to stimulate CE synthesis in cocultured SMCs. LDL had no stimulatory effect over control cells. The difference between LDL and lipoprotein-PG complex in their ability to stimulate CE synthesis in SMC-macrophage cocultures cannot be attributed to modification of the lipoprotein-PG complex during isolation. This notion is consistent with our earlier observation that LDL isolated by anti-apoB affinity chromatography did not stimulate cellular CE synthesis.24 Prior treatment of SMCs with collagenase did not stimulate CE synthesis when these cells were subsequently incubated in the presence of lipoprotein-PG complex (data not shown).
The aforementioned results indicated that lipoprotein-PG complex was significantly more effective than either native LDL or acetyl-LDL in stimulating CE synthesis in SMCs cocultured with macrophages. This phenomenon may be due to direct stimulation of lipoprotein-PG complex uptake in SMCs by macrophages. Alternatively, because the cells were incubated with various ligands together with radioactive oleate-albumin, the results may also suggest a greater association of [14C]oleic acid with the lipoprotein-PG complex. To resolve this question, we repeated the experiment by first incubating the cells with the ligands. The cells were then washed and incubated with radiolabeled oleate-albumin, and CE synthesis was then determined. The results presented in Fig 1C and 1D are similar to those reported in the previous experiment (Fig 1A and 1B). Thus, in control SMC cultures, LDL was the most effective in promoting CE synthesis (Fig 1C). On the other hand, in SMCs cocultured with macrophages, lipoprotein-PG complex was several-fold more potent than both LDL and acetyl-LDL in stimulating CE synthesis (Fig 1D). Therefore, these results confirm that the increased ability of lipoprotein-PG complex to stimulate CE synthesis in SMCs cocultured with macrophages is due to increased uptake of the complex by SMCs. Because the results of the two experiments were very similar, in all subsequent experiments CE synthesis was determined after incubation of the cells with the ligands and radioactive oleate-albumin.
Time-course experiments indicated that in control SMCs, CE synthesis increased very little with time. Contrary to this, in SMCs cocultured with macrophages, the lipoprotein-PG complex stimulated CE synthesis linearly up to 48 hours. LDL failed to produce any stimulation over control cells, whereas acetyl-LDL produced a slight increase.
Free and esterified cholesterol contents of bovine SMCs incubated with LDL, lipoprotein-PG complex, and acetyl-LDL are given in Table 2⇓. The free and esterified cholesterol contents of SMCs cocultured with P388D1 macrophages increased 2-fold and 4.4-fold, respectively, over control SMCs after incubation with the complex for 48 hours. LDL did not increase the cholesterol mass of SMCs cocultured with macrophages. Acetyl-LDL produced a 1.2-fold increase over control cells in the free cholesterol content and a 1.8-fold increase in CE mass.
To extend these studies to a homogeneous system, we investigated CE synthesis in cocultures of human aortic SMCs and monocyte-derived macrophages after their incubation with lipoprotein-PG complex. CE synthesis in SMCs was assayed after macrophages were removed. Fig 2⇓ shows that incubation with lipoprotein-PG complex resulted in slight stimulation of CE synthesis in control SMCs and a 7-fold to 13-fold stimulation over controls in SMCs that were cocultured with macrophages. This increase in CE synthesis in SMCs cocultured with macrophages was also reflected in significant intracellular accretion of free and esterified cholesterol. Thus, following a 48-hour incubation with 100 μg/mL of lipoprotein-PG complex, the free and esterified cholesterol contents of SMCs cocultured with macrophages increased by 1.4-fold and 7.9-fold, respectively, over that of SMCs incubated alone with the complex (free cholesterol in SMCs, 18.6±1.2; in SMCs+macrophages, 26.4±0.8; P<.005; CE in SMCs, 1.8±0.05; in SMCs+macrophages, 14.2±0.4; P<.0001; values in micrograms per milligram protein).
To determine whether lipoprotein-PG complex also stimulated cholesterol esterification in macrophages cocultured with SMCs, we compared the complex-mediated CE synthesis in macrophages cultured alone and macrophages cocultured with SMCs. There was no significant difference in CE synthesis between the two cultures when they were incubated with 100 μg cholesterol per milliliter of lipoprotein-PG complex (in nanomoles per milligram protein of cholesteryl [14C]oleate formed, mean±SEM: P388D1 macrophages, 26.8±4.4; P388D1 macrophages cocultured with bovine SMCs, 29.5±6.2; monocyte-derived macrophages, 21.2±2.6; monocyte-derived macrophages cocultured with human SMCs, 24.4±3.2).
Macrophage Stimulation of CE Synthesis in SMCs Requires Cell Contact
Studies were conducted to determine whether the lipoprotein-PG complex–mediated stimulation of CE synthesis observed in SMCs when cocultured with macrophages was due to the secretion of soluble factors by the macrophages. Undiluted, 1:1-diluted, or threefold-concentrated macrophage-conditioned media harvested in the presence or absence of serum did not stimulate the lipoprotein-PG complex–mediated CE synthesis in SMCs (data not shown). Similarly, conditioned medium obtained from SMC-macrophage coculture also was ineffective (data not shown).
The experiments discussed above indicated that soluble factors secreted by macrophages were not involved in the stimulation of CE synthesis in SMCs and that cell-cell contact was probably required. This hypothesis was confirmed by a different coculture experiment. Bovine SMCs grown in six-well Falcon tissue culture plates were cocultured with P388D1 macrophages that were grown in Transwell inserts, so that the cells were separated by a 0.45-μm membrane. After 24 hours in coculture, the effect of lipoprotein-PG complex on CE synthesis by the SMCs was determined. As shown in Fig 3⇓, in the absence of cell-cell contact, lipoprotein-PG complex did not stimulate CE synthesis in SMCs. Similar results were also obtained for human SMC-macrophage cocultures (data not shown).
In other studies addition of lipoprotein-PG complex to bovine SMCs that had been previously cocultured with P388D1 macrophages for 24 to 48 hours did not stimulate cellular CE synthesis. This finding again confirmed the requirement of cell-cell contact for the macrophages to stimulate CE synthesis in SMCs.
Mechanism of Uptake of Lipoprotein-PG Complex
Studies were conducted to elucidate the mechanism of uptake of lipoprotein-PG complex by SMCs cocultured with macrophages. Unless indicated otherwise, we used a bovine SMC-P388D1 macrophage coculture system in these studies. Fig 4⇓ shows the effect of several metabolic inhibitors on lipoprotein-PG complex–mediated CE synthesis in cocultured SMCs. The lysosomotropic agent chloroquine inhibited CE synthesis. Polyinosinic acid, an inhibitor of the acetyl-LDL (scavenger) receptor, had no effect. On the other hand cytochalasin D, an inhibitor of phagocytosis, inhibited CE synthesis by 73%. Cytochalasin D also inhibited lipoprotein-PG complex–mediated CE synthesis in human SMCs cocultured with monocyte-derived macrophages by 76% (data not shown). CE synthesis was also inhibited when the cocultures were incubated with lipoprotein-PG complex at 4°C (data not shown).
Macrophages and SMCs are capable of oxidizing lipoproteins,37 and oxidized LDL has been reported to enhance production of arachidonic acid in SMCs.38 Moreover, we reported earlier that prolonged incubation of lipoprotein-PG complex with macrophages caused oxidation of the complex.27 Studies were therefore conducted to determine whether oxidation of the lipoproteins in the fibrous plaque complex by macrophages or SMCs was responsible for increased uptake of these complexes by SMCs. Cocultures of SMCs and macrophages were incubated with lipoprotein-PG complex in the presence or absence of the antioxidant BHT, and CE synthesis was then determined. At the concentrations tested (25 and 50 μmol/L), BHT did not decrease CE synthesis (cholesteryl [14C]oleate formed: control 13.2±1.2; 50 μmol/L BHT, 12.8±0.9; in nanomoles per milligram protein, mean±SD). This observation indicated that although cells may oxidize the lipoprotein in the complex, cellular oxidation of lipoprotein-PG complex was not a factor in its uptake by SMCs.
Attempts to determine how macrophages affected the direct binding of 125I-LDL–PG complex to the SMCs were not successful. This was because collagenase treatment of cocultures to remove the macrophages after incubation with 125I-LDL-PG complex removed inconsistent quantities of 125I radioactivity from replicate cultures; subsequent binding data were therefore unreliable.
Stein et al17 reported that vascular SMCs accumulated CE when they were incubated with macrophage-conditioned medium containing acetyl-LDL. Therefore, experiments were performed to determine whether cellular CE mass increased in SMCs after exposure to macrophage-conditioned medium containing lipoprotein-PG complex from fibrous plaques. No stimulation of cholesterol esterification was observed over controls (data not shown). Similarly, conditioned medium from cholesterol-enriched macrophages also failed to stimulate cholesterol esterification in SMCs (data not shown).
We also determined whether lipoprotein-PG complex stimulated CE synthesis in SMCs cocultured with macrophages consequent to its uptake via the LDL receptor. Cocultures were preincubated for 24 hours in medium containing 10% lipoprotein-deficient serum to induce the LDL receptors. CE synthesis was then investigated after addition of lipoprotein-PG complex. Induction of LDL receptors did not result in additional stimulation of CE synthesis. Conversely, preincubation of cocultures with LDL (1 mg cholesterol per milliliter for 24 hours) did not suppress CE synthesis during subsequent incubation with lipoprotein-PG complex.
Inhibition of CE synthesis by cytochalasin D (Fig 4) indicated that uptake of lipoprotein-PG complex in SMCs cocultured with macrophages probably occurred via phagocytosis. This would suggest that under the conditions of coculture, the lipoprotein-PG complex must have undergone aggregation requiring phagocytosis for uptake by SMCs. To test this possibility, we incubated bovine SMCs and P388D1 macrophages (either alone or in coculture) in serum-free medium containing lipoprotein-PG complex (100 μg cholesterol per milliliter) for 6 hours at 37°C. We then harvested the media and compared elution profiles of the complexes in the various media on a column of Bio-Gel A-50m. Fig 5⇓ shows that after incubation with macrophages, the lipoprotein-PG complex eluted ahead of LDL. An identical elution profile was also obtained either after incubation of the complex with SMCs or in a cell-free medium (not shown). In contrast, after incubation with SMC-macrophage coculture, the elution position of the complex shifted toward the void volume of the column, indicating that the complex had undergone aggregation. In similar experiments we also observed aggregation of lipoprotein-PG complex after a 6-hour incubation with human SMC-macrophage coculture, but not with either cell type alone (data not shown).
Fig 6⇓ shows stimulation of CE synthesis in bovine SMCs by lipoprotein-PG complex that was previously incubated with bovine SMC-P388D1 macrophage coculture. After prior incubation with the coculture, the complex stimulated cholesterol esterification in SMCs to almost the same extent as did the complex-mediated CE synthesis in SMC-macrophage coculture (bars A and D). Lipoprotein-PG complex incubated with SMCs or macrophages alone did not stimulate CE synthesis in SMCs after its subsequent incubation with these cells (bars B and C). Likewise, prior incubation of LDL with SMC-macrophage coculture did not improve its ability to induce cholesterol esterification in SMCs (data not shown). Similar results were obtained when these studies were repeated in human SMC-macrophage cocultures (data not shown).
Lack of Stimulation of Lipoprotein-PG Complex–Mediated CE Synthesis in SMCs Cocultured With ECs
To determine whether stimulation of CE synthesis and accumulation induced in cocultures of SMCs and macrophages by lipoprotein-PG complex could be reproduced in similar cocultures of SMCs with other cells, studies were performed in cocultures of SMCs with vascular ECs. Table 3⇓ shows the effects of various ligands on the synthesis of CE by bovine SMCs cultured alone or in the presence of bovine ECs. Compared with controls, CE synthesis in coculture was significantly stimulated by LDL at both 24 and 48 hours. Although lipoprotein-PG complex and acetyl-LDL also stimulated CE synthesis in cocultures by 2.6-fold to 2.8-fold over controls at 48 hours, the absolute values were low (lipoprotein-PG complex, 4.5±0.5; acetyl-LDL, 5.2±0.6; in nanomoles per milligram protein, mean±SEM). These experiments thus showed that lipoprotein-PG complexes stimulated CE synthesis in bovine SMCs cocultured with bovine ECs to a much lesser degree than in SMCs cocultured with macrophages.
When similar experiments were performed with human cells, there was no significant difference in the lipoprotein-PG complex–mediated CE synthesis between SMCs cultured alone and SMCs that were cocultured with ECs.
Although the presence of SMC-derived foam cells is a common feature of atherosclerotic lesions,2 their mechanism of formation in vivo is not well understood. The results of this study show that in both homogeneous and heterogeneous cell culture systems in vitro, lipoprotein-PG complex isolated from atherosclerotic lesions can induce CE synthesis and accumulation in SMCs when these cells are cocultured with macrophages. The effect was specific for this complex because other ligands were either ineffective (LDL) or only minimally effective (acetyl-LDL). CE synthesis in cocultured SMCs was time and concentration dependent. Incubation of cocultured SMCs with lipoprotein-PG complex resulted in a significant increase in total and esterified cholesterol contents. Although the level of cholesterol loading obtained is much greater than that reported for SMCs in several other studies,14 15 16 17 18 19 it is far less than that obtained by incubating SMCs with macrophage-lipid inclusions.21
In our study, a 16-hour incubation with acetyl-LDL produced only a 1.8-fold increase in the intracellular CE mass of bovine SMCs cocultured with P388D1 macrophages (Table 2). In earlier studies, Stein et al17 reported that a 3-fold to 4-fold increase in CE mass occurred after a 48-hour incubation of bovine SMCs with macrophage media conditioned with acetyl-LDL. The difference in the extent of stimulation between the two studies is most likely due to differences in the experimental conditions.
The following results support the idea that cell-cell contact is essential for macrophages to stimulate lipoprotein-PG complex–mediated CE synthesis in SMCs: (1) Conditioned media from macrophage cultures or macrophage-SMC cocultures were unable to stimulate cholesterol esterification in SMCs; (2) Macrophages failed to stimulate CE synthesis when the cocultures were separated by a 0.45-μm membrane insert; and (3) Preincubation of SMCs with macrophages did not enhance CE synthesis in the SMCs during their subsequent exposure to lipoprotein-PG complex. Therefore, soluble factors secreted by macrophages could not replace the effect of cell-cell contact. Recently Zhang et al23 reported that macrophage-induced stimulation of prostanoid synthesis in SMCs also required cell-cell contact. Earlier studies of Hajjar et al32 also emphasized the importance of cell-cell contact in EC-mediated modulation of cholesterol metabolism in SMCs. Collectively, these results indicate that cell-cell interaction is an important regulator of the metabolic events in SMCs.
Lipoprotein-PG complex–mediated CE synthesis in cocultured SMCs did not occur at 4°C. This indicates that internalization of the complex is a prerequisite for stimulation of CE synthesis. Likewise, inhibition by choloroquine of CE synthesis mediated by the complex suggests that lysosomal hydrolysis of the complex is essential for stimulation of cholesterol esterification.
The mechanism of uptake of lipoprotein-PG complex in cocultured SMCs was investigated. The scavenger receptor has been recently described in SMCs.14 However, uptake of the lipoprotein-PG complex does not occur via the scavenger receptor in cocultured SMCs. This finding is consistent with the inability of polyinosinic acid, a specific inhibitor of the scavenger receptor, to suppress the CE synthesis that was mediated by lipoprotein-PG complex.
Our results also rule out the possibility that uptake of the lipoprotein-PG complex in cocultured SMCs occurs via the apoB,E receptor, because induction or downregulation of these receptors in SMCs did not affect CE synthesis.
The studies of Wolfbauer et al21 showed that uptake of lipid inclusions prepared from cholesterol-enriched macrophages caused CE accumulaton in SMCs. However, in the present study SMCs did not become cholesterol-enriched by this route because: (1) The cells did not accumulate CE when exposed to conditioned medium from cholesterol-loaded macrophages and (2) Coculture of macrophages with SMCs did not cause macrophage cell lysis, thereby preventing the release of lipid inclusions into the medium.
The results of experiments with cytochalasin D, a specific inhibitor of phagocytosis, indicate that lipoprotein-PG complex stimulates CE synthesis in cocultured SMCs following its uptake through phagocytosis. The phagocytic capability of SMCs has been well documented.39 40 Moreover, Wolfbauer et al21 reported that uptake of macrophage lipid inclusions by SMCs occurred via phagocytosis. Therefore, phagocytosis is a viable pathway for uptake of the lipoprotein-PG complex in SMCs cocultured with macrophages. Macrophages could enhance uptake by SMCs by modifying the complex so that its uptake via phagocytosis is enhanced. However, this possibility was excluded because conditioned medium from macrophage cultures incubated with lipoprotein-PG complex did not stimulate CE synthesis in SMCs. In contrast, the conditioned medium from SMC-macrophage cocultures incubated with lipoprotein-PG complex induced the same degree of cholesterol esterification in SMCs as that in SMCs cocultured with macrophages (Fig 6). This observation strongly suggests that the complex is induced to aggregate during its incubation with the coculture. An increase in size of the complex after incubation with the cocultures supports this idea. Although PG-bound LDL in vitro forms aggregates,41 42 in our study the failure of the lipoprotein-PG complex to increase cholesterol accretion in SMCs in the absence of macrophages implies that a certain level of ligand aggregation is necessary for uptake to occur via phagocytosis by SMCs. This is further supported by the failure of the conditioned medium from SMC-macrophage coculture incubated with LDL to induce cholesterol esterification in SMCs.
These results show that in SMCs cocultured with macrophages, uptake of the lipoprotein-PG complex occurs via phagocytosis. Since phagocytosis is not subject to feedback regulation, continued uptake of the complex by this process can lead to continued CE synthesis and accumulation in the SMCs and their eventual conversion into foam cells.
Unlike cocultured macrophages, cocultured ECs did not stimulate lipoprotein-PG complex–mediated CE synthesis in SMCs. ECs have been shown to increase uptake of LDL by cocultured SMCs and thus stimulate CE synthesis.43 The increased uptake of LDL is reported to be due to an EC-mediated increase in the number of receptors in SMCs. Since uptake of lipoprotein-PG complex in SMCs does not occur via the LDL receptor, an increase in the number of LDL receptors on SMC surfaces produced by ECs is not expected to enhance uptake of the complex. The lack of stimulation of cholesterol esterification by the complex in SMCs cocultured with ECs also indicates that the complex is not induced to aggregate by the coculture.
An interesting finding of the study is that whereas macrophages stimulated lipoprotein-PG complex–mediated CE accretion in cocultured SMCs, SMCs did not influence CE synthesis in macrophages. The reason for this is currently under investigation in our laboratory.
Several lines of evidence indicate that PGs play an important role in the retention of atherogenic lipoproteins in the artery wall.41 44 For example, Schwenke and Cerew45 reported that atherosclerotic lesion development in rabbits began only after focal retention of LDL in the extracellular compartment. Other studies have shown that the apoB-containing lipoproteins that accumulate in the artery wall are intimately associated with PGs.46 47 Additionally, lipoprotein-PG complexes that are either formed in vitro or isolated from atherosclerotic lesions are readily internalized by macrophages, thereby leading to foam cell formation.10 11 12 13 24 Our study provides an additional mechanism by which lipoprotein-PG complexes may contribute to atherogenesis. This study shows for the first time that macrophages can stimulate uptake of lipoprotein-PG complex by cocultured SMCs, leading to enhanced synthesis and deposition of CE in the SMCs.
Both macrophages and SMCs migrate into the subendothelial space of the blood vessel early during atherogenesis.2 Masuda and Ross48 observed the presence of lipid-laden macrophages in direct contact with SMCs and extracellular matrix in fatty streak lesions from pigtail monkeys. Since lipoprotein-PG complexes are already present in atherosclerotic lesions,24 49 macrophages could stimulate uptake of these complexes by SMCs, leading to myocyte foam cell formation.
Selected Abbreviations and Acronyms
|DMEM||=||Dulbecco's modified Eagle's medium|
|MEM||=||(Eagle's) minimal essential medium|
|SMC(s)||=||smooth muscle cell(s)|
This work was supported by grant HL42993 from the National Institutes of Health, Bethesda, Md. We thank Anne Compliment and Brenda Kuss for editorial and secretarial assistance.
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