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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:801-810

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:801-810.)
© 1995 American Heart Association, Inc.

Articles

Mast Cell Granule Remnants Carry LDL Into Smooth Muscle Cells of the Synthetic Phenotype and Induce Their Conversion Into Foam Cells

Yenfeng Wang; Ken A. Lindstedt; Petri T. Kovanen

From the Wihuri Research Institute, Helsinki, Finland.

Correspondence to Petri T. Kovanen, Wihuri Research Institute, Kalliolinnantie 4, SF-00140 Helsinki, Finland.


*    Abstract
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 *Abstract
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 down arrowResults
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Abstract We report the effect of mast cells on the uptake of LDL by smooth muscle cells (SMCs) and their conversion into foam cells in vitro. The mast cells were stimulated to exocytose their cytoplasmic secretory granules, and the granule remnants formed were recovered from the extracellular fluid and added to cultures of SMCs of either the synthetic or contractile phenotype in LDL-containing medium. In the presence but not in the absence of granule remnants, SMCs of the synthetic phenotype took up LDL with ensuing stimulation of intracellular cholesteryl ester synthesis and cytoplasmic accumulation of neutral lipid droplets. Using methylated LDL (mLDL), a modified species of LDL that binds to granule remnants but not to LDL receptors, we demonstrated that this uptake (leading to foam cell formation) occurred only when LDL was bound to granule remnants. After the addition of colloidal gold–LDL and granule remnants to the incubation system, electron microscopy revealed that within phagosomes of the SMCs there were granule remnants (diameter, 0.5 to 1 µm) coated with LDL, confirming that LDL had been carried into the cells with the remnants. SMCs of the contractile phenotype were less efficient than their synthetic counterparts at phagocytosing LDL-coated granule remnants and were not converted into foam cells. This difference in the rate of phagocytosis of granule remnants was present even in the absence of LDL, revealing that the more active phagocytosis by SMCs of the synthetic phenotype was not specifically related to uptake of lipids but rather reflected a general phenotype characteristic of these cells. These observations indicate a phagocytic mechanism by which SMCs of the synthetic phenotype are converted into cholesteryl ester–filled foam cells, and they also suggest that degranulation of mast cells plays a role in the development of fatty streak lesions.


Key Words: atherosclerosis • foam cells • LDL • mast cells • smooth muscle cells


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
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Human arterial intima, the site of atherogenesis, is composed of a smooth muscle cell (SMC) parenchyma1 in which are embedded three types of blood-borne cells: macrophages,2 T lymphocytes,3 and mast cells.4 5 The earliest recognizable gross lesion in atherogenesis, the fatty streak, is characterized by intracellular accumulation of LDL-derived cholesteryl esters in the affected sites of the arterial intima with formation of foam cells.6 Traditionally, it has been held that foam cells are mostly derived from circulating monocytes/macrophages,2 7 with a small fraction derived from SMCs that have migrated from the deep intima or the media, simultaneously changing their phenotype from contractile to synthetic.1 8 A recent report suggests that a significant fraction of the foam cells in the fatty streak lesions of the thoracic aorta may have arisen from SMCs.9

Since uptake of LDL through the classic LDL receptor pathway does not lead to accumulation of cholesteryl esters in cells,10 the mechanisms that govern foam cell formation must be different. Indeed, Goldstein et al11 were able to generate foam cells from human aortic SMCs by using positively charged LDL to bypass the LDL receptor system. They later showed12 that a specific chemical modification (acetylation) of the apoB-100 moiety of LDL can induce massive uptake of LDL by a receptor-mediated mechanism, the unregulated "scavenger receptor pathway." The presence of receptors of this type has been shown not only in macrophages10 12 13 14 but also in SMCs.15 16 17 18 Today, several biological modifications of LDL leading to foam cell formation have been recognized19 20 ; these modifications of LDL are generally held to be brought about locally in the arterial intima by the action of intimal cells, such as macrophages, SMCs, and endothelial cells.21 22 23 24 25 Another possible mechanism leading to foam cell formation is the "piggyback" system in which LDL is bound to negatively charged molecules (dextran sulfate) and then ingested by macrophages.26 Subsequently, it has been demonstrated that macrophages in vitro take up LDL particles bound to various components of the extracellular matrix of the arterial wall. These components include heparin-collagen complexes,27 heparin-fibronectin-collagen complexes,28 elastin and particles of collagenase-resistant matrix from human aorta,29 and proteoglycans from bovine atherosclerotic lesions.30 Accelerated uptake by SMCs of LDL bound to proteoglycans from injured rabbit aortas has also been described.31

We have investigated in vitro the possibility that mast cells play a role in foam cell formation. In light of these studies, we delineated a tightly regulated sequence of events in which degranulation of mast cells led to formation of macrophage foam cells.32 33 The cytoplasm of the rat serosal mast cell, the model cell in our studies, is filled with specific organelles, the secretory granules (diameter, 0.5 to 1.0 µm), which on stimulation of mast cells are expelled into the extracellular fluid. In the extracellular fluid, the soluble components of the granules, ie, histamine, chondroitin sulfate proteoglycans, and a fraction of their heparin proteoglycans, are solubilized and released from the granules. In contrast, two neutral proteases, chymase and carboxypeptidase A, and the major fraction of heparin proteoglycans remain tightly bound to each other, forming extracellular granule remnants. When the mast cells were cocultured with macrophages and stimulated to degranulate in the presence of LDL, the LDL particles bound avidly to the heparin proteoglycan component of the remnants, and the resultant LDL-coated remnants became phagocytosed by the macrophages, thus leading to massive uptake of LDL by the macrophages.34 35 36 For the present article, we studied whether SMCs, the other potential precursor of foam cells in the arterial intima, would also be capable of taking up LDL bound to exocytosed mast cell granules. More specifically, we examined whether SMCs of the synthetic, ie, the atherogenic, phenotype37 are prone to accumulate cholesteryl esters by this mechanism.


*    Methods
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 up arrowIntroduction
 *Methods
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Materials and Animals
Sodium [125I]iodide (13 to 17 mCi/µg), [1{alpha},2{alpha}(n)-3H]cholesteryl linoleate ([3H]CL; 30 to 60 Ci/mmol), and [1-14C]oleic acid (50 to 60 mCi/mmol) were from Amersham International. Eagle's basal medium with Earle's salts with 20 mmol/L HEPES (EBME) was from Flow Laboratories, RPMI-1640 culture medium supplemented with 25 mmol/L HEPES, Dulbecco's phosphate-buffered saline (PBS), fetal calf serum, penicillin, streptomycin, and L-glutamine were from GIBCO; bovine serum albumin (BSA), compound 48/80, soybean trypsin inhibitor, collagenase type I A, trypsin, protamine chloride, fluorescein isothiocyanate (FITC)–conjugated avidin, and anti-myosin (smooth and skeletal; smooth muscle from bovine uterus as immunogen) were from Sigma. FITC-conjugated anti-rabbit IgG (H+L) was from The Binding Site Ltd. Cholesterol ester transfer protein was a generous gift from Drs C. Ehnholm and M. Jauhiainen, National Public Health Institute, Helsinki, Finland. Male Wistar rats (300 to 500 g) and female New Zealand White rabbits (4 to 8 weeks) were purchased from the Laboratory Animal Center of the University of Helsinki.

Preparation and Phenotype Identification of SMCs
Arterial SMCs were isolated by carefully stripping pieces of the intima and media from the thoracic and upper parts of the abdominal aorta of 4- to 8-week-old female New Zealand White rabbits.38 The stripped segments were cut into 1-mm pieces, treated with 1 mg/mL collagenase for 1 hour to remove endothelial cells, washed once with medium, and then dispersed in a mixture of collagenase (1 mg/mL) and elastase (0.5 mg/mL) in RPMI-1640 culture medium containing 12.5% fetal calf serum. After incubation at 37°C for 2 hours with occasional gentle agitation, the cell suspension was centrifuged at 800g for 5 minutes. The cell pellet was washed and resuspended in medium A (RPMI-1640 culture medium containing 2 mmol/L L-glutamine, 20% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin). SMCs of the contractile phenotype were prepared by seeding cells at a density of 5x105 to 8x105 cells/mL and used in the experiment on day 5 of primary culture. To obtain their synthetic counterparts, the cells were inoculated at a density of 1x104 to 1x105 cells/mL and at confluence subcultured (1:2) for up to five to nine passages.39 SMCs of the contractile and synthetic phenotypes were identified with FITC-conjugated antibodies against myosin40 and photographed with the Olympus system microscope BH2-RFL using a combination of filters BP-490, DM-500, and B-460.

Isolation and Treatments of Mast Cell Granule Remnants
Remnants of the cytoplasmic secretory granules of mast cells were isolated from the extracellular medium of stimulated rat serosal mast cells as described by Lindstedt et al.41 The concentration of granule remnants used in experiments is expressed in terms of remnant protein. Granule remnants coated with FITC-avidin or protamine were obtained by incubating granule remnants with excess ligand at 0°C for 1 hour or at room temperature for 5 minutes.42 The volumes of the incubation mixtures containing the given amounts of granule remnants and the ligands were added to SMC monolayers. 125I-labeled granule remnants were prepared by incubating granule remnants with [125I]Bolton and Hunter reagent in 0.1 mol/L borate buffer, pH 8.3, at 4°C for 30 minutes. The specific activities of the labeled granule remnants ranged from 1500 to 2000 cpm/µg protein.

Isolation, Labeling, and Modification of LDL
Human LDL (d=l.019 to 1.050 g/mL) was isolated by sequential ultracentrifugation from blood plasma derived from single normolipidemic donors.43 [3H]CL was incorporated into the LDL by incubating LDL with solid dispersions of [3H]CL on Celite44 except that cholesteryl ester transfer protein (activity, 5 to 10 µmol cholesteryl ester transferred · mL-1 · hour-1) and isolated LDL were used instead of whole serum. The specific activity obtained varied from 20 to 60 dpm/ng LDL protein. Both LDL and [3H]CL-LDL, separately, were reductively methylated with formaldehyde according to the method of Mahley et al.45 The concentration of LDL is expressed in terms of its protein concentration.

Binding of LDL and Methylated LDL to Granule Remnants
Binding assays were conducted in 100 µL medium B (EBME containing 10 mg/mL BSA, 1.2 mg/mL soybean trypsin inhibitor, 100 U/mL penicillin, and 100 µg/mL streptomycin) containing either [3H]CL-labeled methylated LDL ([3H]CL-mLDL) or [3H]CL-LDL and granule remnants. Incubations were carried out at 0°C for 60 minutes. To determine the amount of labeled lipoproteins bound to the granule remnants, a portion of the reaction mixture was layered on 300 µL of 5 mmol/L Tris/HCl buffer containing 0.25 mol/L sucrose and 10 mg/mL BSA, pH 7.4. The tubes were then centrifuged at 12 000g for 10 minutes at 4°C, and the supernatant of each tube was removed by aspiration. The granule remnant–containing pellets were resuspended in Optiphase Hisafe II scintillation fluid and counted for their 3H radioactivity. The results are expressed as nanograms lipoprotein protein bound per microgram of granule remnant protein.

Uptake of 125I-Labeled, FITC-Avidin–Coated, or [3H]CL-mLDL–Coated Granule Remnants by SMCs
Monolayers of SMCs of the contractile or synthetic phenotype were prepared as described above. The monolayers were washed three times with PBS, and the indicated concentrations of either 125I-labeled, FITC-avidin–coated, or [3H]CL-mLDL–coated granule remnants in 300 µL medium C (EBME containing 10 mg/mL BSA, 100 U/mL penicillin, and 100 µg/mL streptomycin) were added to the wells. After incubation at 37°C for the indicated time periods, the medium was removed, and the cells were further incubated in 500 µL buffer A (PBS supplemented with 5 mg/mL heparin, 10 mg/mL BSA, and 1 mg/mL soybean trypsin inhibitor) at room temperature for 10 minutes. Finally, the cells were rinsed three times with PBS and, for the uptake of FITC-avidin–coated granule remnants, the cells were fixed and prepared for photography as described below; for the uptake of 125I-labeled and [3H]CL-mLDL–coated granule remnants, the cells were collected into 500 µL of 0.2 mol/L NaOH, and aliquots were removed for determination of radioactivity and protein content. The results are expressed as micrograms of granule remnants or mLDL protein per milligram of cell protein.

Electron Microscopic Examination of Uptake of LDL-Coated Granule Remnants by SMCs of the Synthetic Phenotype
Colloidal gold particles (5 nm) were prepared and conjugated with LDL as described by Robenek et al.46 The given amounts of gold-labeled LDL (gold-LDL) and granule remnants were preincubated at 0°C for 60 minutes to produce gold-LDL–coated granule remnants and then added to the monolayers of SMCs of the synthetic phenotype. After incubation at 37°C for 6 hours, the cells were treated with buffer A for 10 minutes and rinsed three times with PBS. The resulting cells were then fixed in pellets with 5% (vol/vol) glutaraldehyde for 1 hour. To preserve the lipid structures, the cell pellets were postfixed with successive treatments of osmium tetroxide, thiocarbohydrazine, and osmium tetroxide as described by Guyton et al.47 The samples were dehydrated and embedded in LX-112 embedding medium (Ladd Research Industries). Ultramicrotome sections were stained with uranyl acetate and lead citrate and viewed with a JEOL JEM-1200EX transmission electron microscope at the Department of Electron Microscopy, University of Helsinki.

Incorporation of [14C]Oleate Into Cholesteryl Esters by SMCs of the Synthetic Phenotype
The amount of [14C]oleate incorporated into cholesteryl esters by SMCs of the synthetic phenotype was determined according to the method of Brown et al.48 In a standard assay, each dish received the indicated amounts of mLDL, granule remnants, and [14C]oleate-albumin to give a final oleate concentration of 200 µmol/L. After incubation for the indicated time periods, the intracellular lipids were extracted in situ with hexane/isopropanol (3:2, vol/vol); the cholesteryl[14C]oleate was then separated by thin-layer chromatography, and its radioactivity was determined. The cells deprived of lipid were dissolved in 0.2 mol/L NaOH, and aliquots were removed for protein determination. The results are expressed as nanomoles [14C]oleate converted into cholesteryl[14C]oleate per milligram cell protein.

Oil Red O Staining of Lipid Droplets in SMCs
SMCs were sparsely inoculated onto slides and incubated with the indicated concentrations of either mLDL or mLDL-coated granule remnants at 37°C for 24 hours. After incubation, the cells were rinsed with PBS and fixed with a mixture of 4% paraformaldehyde and 0.05% glutaraldehyde. The fixed cells were stained with 0.5% oil red O and counterstained with Harris's hematoxylin.49

Other Assays
For measurement of the cellular cholesterol mass, SMC monolayers were extracted in situ with a mixture of hexane/isopropanol,48 and aliquots were taken for fluorometric assay of free and esterified cholesterol.50 The protein content was determined by the procedure of Lowry et al51 with BSA as standard.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
To obtain cultures of SMCs, strips of rabbit aortic media were treated with the enzyme dispersion method of Chamley et al.38 The isolated SMCs grown for 5 days in primary culture stained strongly with FITC-conjugated antibody against myosin, indicating the presence of SMCs and, more specifically, of SMCs of the contractile phenotype (Fig 1ADown). In contrast, SMCs that had been subcultured five times (grown for 20 to 40 days after the start of culture) stained only weakly with the myosin antibody (Fig 1BDown), reflecting dedifferentiation and showing that the cells were of the synthetic phenotype.40 Since the change in SMC phenotype from contractile to synthetic has been found to be irreversible after subculturing aortic SMCs for five times (corresponding to at least eight cell doublings),39 we adopted a standard protocol for studies on the synthetic phenotype in which SMCs were used for experiments after they had been subcultured at least five times.



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  		Figure 1. Photomicrographs showing smooth muscle cells (SMCs) of different phenotypes by staining myosin in situ with fluorescein isothiocyanate (FITC)–conjugated antibodies. SMCs were isolated from rabbit aortic media and stained with FITC-conjugated anti-myosin on day 5 of primary culture (A; contractile phenotype) or after they had been subcultured five times (B; synthetic phenotype) (original magnification x200).

If such actively dividing (subconfluent) SMCs of the synthetic phenotype were incubated in the presence of LDL (100 µg/mL) for 72 hours, only a slight increase in intracellular cholesteryl ester content could be observed (Fig 2ADown), a finding in accord with the notion that uptake of LDL is governed by a tightly regulated LDL receptor pathway. To investigate the effect of mast cell granule remnants on the uptake of LDL by SMCs of the synthetic phenotype, we next isolated mast cells from the peritoneal cavity of rats and stimulated them with compound 48/80 to induce exocytosis of their cytoplasmic secretory granules (see "Methods"). In sharp contrast to the above finding, the addition of granule remnants to the incubation mixture led to a significant increase in the content of intracellular cholesteryl esters in these cells (Fig 2ADown).



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  		Figure 2. Line graphs showing effect of mast cell granule remnants (GR) on the accumulation of free cholesterol and cholesteryl esters in smooth muscle cells of the synthetic phenotype (s-SMCs) (A) and on the uptake of LDL by s-SMCs before (B) and after (C) incubation of the cells with acetylated LDL. A, Monolayers of s-SMCs were loaded with 100 µg/mL LDL in medium C in the presence (+) or absence (-) of 5 µg/mL granule remnants. After incubation at 37°C for 72 hours, the cellular lipids were extracted and measured as described in "Methods." Control cells were analyzed at the start of the incubation. B, Monolayers of s-SMCs were incubated at 37°C for 6 hours in medium C containing increasing concentrations of [3H]cholesteryl linoleate ([3H]CL)–labeled LDL with or without 20 µg/mL granule remnants. The cells were collected after incubation, and the cellular contents of [3H]CL-LDL were determined. C, The same experiments as in B, except that cells were incubated with 100 µg/mL acetylated LDL at 37°C for 48 hours prior to the addition of [3H]CL-LDL and granule remnants.

We then determined the effect of granule remnants on the rate of LDL uptake by the SMCs of the synthetic phenotype by using various concentrations of LDL within the range of high-affinity uptake. Since the neutral proteases of granule remnants avidly degrade the apoB-100 of LDL in extracellular fluid,52 we used LDL particles in which the cholesteryl ester component had been labeled. Fig 2BUp shows the uptake of [3H]CL-LDL by the SMCs during a 6-hour incubation plotted as a function of the concentration of [3H]CL-LDL. The data show evidence for a saturable uptake process, with half-maximal uptake occurring at a [3H]CL-LDL concentration of {approx}15 µg/mL. This saturation curve is similar to the curve for receptor-mediated high-affinity LDL uptake in dividing human fibroblasts that have been induced to express a high level of LDL receptor activity53 and also accords with earlier observations that SMCs of the synthetic phenotype derived from rabbit aorta express the LDL receptor.54 If exocytosed mast cell granules (ie, granule remnants) were added to the incubation medium, the high-affinity component of the uptake process was replaced by a slower, linear type of uptake that showed no saturation at the LDL concentrations used. If the SMCs of the synthetic phenotype were first incubated for 48 hours in the presence of acetylated LDL, the rate of LDL uptake was decreased, a finding compatible with downregulation of LDL receptor activity. Under these conditions, addition of granule remnants to the incubation system significantly enhanced the rate of LDL uptake by the cells (Fig 2CUp).

The observations described above suggested that the granule remnants were able to induce uptake of LDL and subsequent accumulation of cholesteryl esters in SMCs of the synthetic phenotype through mechanisms differing from the classic LDL receptor pathway. Granule remnants can induce uptake of LDL by cultured mouse peritoneal macrophages when the apoB-100 of LDL binds to the heparin proteoglycan component of the remnants, and the granule remnant–LDL complexes so formed are taken up by macrophages through LDL receptor–independent phagocytosis ("granule remnant carrier mechanism").32 As a more direct test of whether granule remnants are capable of carrying LDL into SMCs of the synthetic phenotype without involving LDL receptors, we methylated the apoB-100 of LDL. mLDL does not bind to LDL receptors45 but retains its ability to bind to heparin. The magnitude of mLDL binding to mast cell granule remnants was similar to that of LDL (Fig 3ADown), but in contrast to untreated LDL, mLDL was not taken up by the SMCs of the synthetic phenotype (Fig 3BDown). In another experiment, however, after addition of granule remnants to the culture medium, the rate of uptake of mLDL by the SMCs of the synthetic phenotype was enhanced by about 10-fold (Fig 3CDown).



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  		Figure 3. Line graphs showing (A) binding of methylated LDL (mLDL) and LDL to mast cell granule remnants, (B) uptake of mLDL and LDL by smooth muscle cells of the synthetic phenotype (s-SMC), and (C) the effect of granule remnants on mLDL uptake as a function of time. A, Isolated mast cell granule remnants (2.5 µg) were incubated at 0°C for 60 minutes in 100 µL of medium B with the indicated concentrations of [3H]cholesteryl linoleate ([3H]CL)–mLDL or [3H]CL-LDL. B, Monolayers of s-SMCs were incubated at 37°C for 5 hours in 300 µL of medium C containing increasing concentrations of either [3H]CL-mLDL or [3H]CL-LDL. C, Monolayers of s-SMCs were incubated at 37°C for the indicated times in 300 µL of medium C containing 60 µg/mL [3H]CL-mLDL in the presence or absence of 20 µg/mL mast cell granule remnants. See "Methods" for determination of uptake and binding.

To demonstrate that the process of granule remnant–dependent LDL uptake involves both binding of LDL to the granule remnants and uptake of the remnants by the cells, the following experiments were conducted. First, we added to the incubation medium increasing concentrations of protamine, a molecule with strong positively charged domains that has been shown to inhibit binding of LDL to its cell surface receptors55 and that also could potentially compete with mLDL for binding to the granule remnants. Protamine effectively competed with mLDL for the binding of granule remnants (Fig 4ADown) and inhibited the granule remnant–mediated uptake of mLDL (Fig 4BDown). Second, using 125I-labeled granule remnants, we demonstrated the uptake of granule remnants by SMCs of the synthetic phenotype (Fig 5ADown). Cellular uptake of the granule remnants was also confirmed by visualizing ingested FITC-avidin–coated granule remnants in the perinuclear region of this cell (Fig 5BDown). Finally, as a direct demonstration that granule remnants carry LDL into the SMCs of the synthetic phenotype, the LDL particles were first allowed to attach to colloidal gold (5 nm)46 and subsequently to bind to the granule remnants. The SMCs of the synthetic phenotype were then incubated with these gold-LDL–coated granule remnants and prepared for electron microscopy. Fig 6Down shows an electron photomicrograph in which a granule remnant coated with gold-LDL has been ingested by an SMC of the synthetic phenotype and can be seen within a phagosome.



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  		Figure 4. Line graphs showing ability of protamine (A) to compete with [3H]cholesteryl linoleate ([3H]CL)–labeled methylated LDL (mLDL) for binding to granule remnants and (B) to inhibit the granule remnant–mediated uptake of [3H]CL-mLDL by smooth muscle cells of the synthetic phenotype (s-SMC). A, Mast cell granule remnants (20 µg/mL) were preincubated at room temperature for 5 minutes with the indicated concentrations of protamine chloride in medium B and then incubated with 30 µg/mL [3H]CL-mLDL at 0°C for 60 minutes. B, Monolayers of s-SMCs were incubated at 37°C for 10 hours in 300 µL of medium B containing 30 µg/mL [3H]CL-mLDL in the presence of 20 µg/mL of either untreated mast cell granule remnants or granule remnants pretreated at room temperature for 5 minutes with increasing concentrations of protamine chloride. See "Methods" for determination of binding and uptake.



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  		Figure 5. Uptake of (A) 125I-labeled and (B) fluorescein isothiocyanate (FITC)–avidin–coated mast cell granule remnants by smooth muscle cells of the synthetic phenotype (s-SMCs). A, Line graph. Monolayers of s-SMCs were incubated at 37°C for 6 hours in 300 µL of medium C in the presence of increasing concentrations of 125I-labeled granule remnants. After incubation, the cells were rinsed extensively, and the radioactivity associated with the cells was determined. B, Photomicrograph. Mast cell granule remnants (6 µg) and FITC-avidin (18 µg) were preincubated at 0°C for 60 minutes in 300 µL of medium B; the incubation mixture was then added to the s-SMC monolayers. After incubation at 37°C for 5 hours, the cells were rinsed extensively and prepared for fluorescence microscopy (original magnification x400). Control cells were analyzed without granule remnants; no fluorescence was observed in these cells (data not shown).



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  		Figure 6. Electron photomicrograph showing uptake of a granule remnant coated with gold-labeled LDL by a smooth muscle cell of the synthetic phenotype (s-SMC). Mast cell granule remnants (60 µg/mL) and LDL particles (100 µg/mL) attached to colloidal gold (5 nm) were preincubated at 0°C for 60 minutes in medium C, and the incubation mixture was added to s-SMC monolayers. After incubation at 37°C for 6 hours, the cells were prepared for electron microscopy. Gr-R indicates gold-LDL–coated granule remnant; N, nucleus; arrow, cell membrane; and arrowhead, phagosomal membrane. Bar=200 nm (original magnification x30 000).

Uptake of mLDL-coated granule remnants also leads to massive uptake of the heparin proteoglycans contained in the granule remnants. It has been suggested that heparin, by means of its negative charge, inhibits lysosomal (phagosomal) hydrolysis of other ingested molecules.56 To study whether the cholesteryl esters of mLDL were hydrolyzed and reesterified in the SMCs of the synthetic phenotype, we measured the rate of conversion of [14C]oleate into cholesteryl[14C]oleate by the cells that had been induced to take up mLDL-coated granule remnants. The incorporation rate was greatly enhanced when the incubation medium contained granule remnants in addition to mLDL (Fig 7ADown). By measuring the cholesterol mass, we showed that the granule remnant–induced activation of cellular cholesterol esterification was associated with a steady increase in the cellular content of cholesteryl esters over the 24-hour incubation period without any increase in the cellular content of unesterified cholesterol (Fig 7BDown and 7CDown). Staining such cholesteryl ester–containing SMCs for neutral lipids with oil red O revealed the presence in the cytoplasm of the numerous lipid droplets typical of foam cells (Fig 8ADown). If granule remnants were omitted, no lipid droplets were present in the cells (Fig 8BDown).



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  		Figure 7. Line graphs showing formation and accumulation of cholesteryl esters in smooth muscle cells of the synthetic phenotype (s-SMCs) incubated with granule remnants and methylated LDL (mLDL). A, Monolayers of s-SMCs were incubated at 37°C for 15 hours in 300 µL of medium C containing increasing concentrations of mLDL and 200 µmol/L [14C]oleate-albumin (10 400 dpm/nmol) in the presence or absence of mast cell granule remnants (20 µg/mL). After incubation, the lipids in the cells were extracted, and the radioactivity associated with the cholesteryl ester fraction was determined. B and C, s-SMC monolayers were incubated with 100 µg/mL mLDL as described in A but without [14C]oleate-albumin. After incubation, the lipids were extracted, and the amounts of cholesteryl esters and free cholesterol were determined.



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  		Figure 8. Light microscopic appearance of smooth muscle cells of the synthetic phenotype (s-SMCs) incubated with methylated LDL (mLDL). Monolayers of s-SMCs were incubated at 37°C for 24 hours with 200 µg/mL mLDL in the presence (A) or absence (B) of 20 µg/mL of mast cell granule remnants in medium C and then fixed and stained with oil red O and hematoxylin (original magnification x100).

The above series of experiments was conducted using SMCs of the synthetic phenotype. To study the effect of mast cell granule remnants on the uptake of mLDL by SMCs of the contractile phenotype, we prepared primary cultures of SMCs and measured the granule remnant–mediated uptake on day 5 of culture, when the cells were still contractile (Fig 1AUp). More specifically, we compared the SMCs of the two phenotypes for their ability to take up granule remnants in the absence of LDL and to take up mLDL in the absence and presence of remnants. The cells of the synthetic phenotype ingested more granule remnants than their contractile counterparts, the difference being fourfold at the maximum remnant concentration used (Fig 9ADown). In the absence of granule remnants, uptake of mLDL by SMCs of both phenotypes was low (Fig 9BDown). Consistent with the observation that the uptake of granule remnants by SMCs of the synthetic phenotype was higher than by their contractile counterpart was the finding that the granule remnant–mediated uptake of mLDL was also significantly higher by SMCs of the synthetic phenotype (Fig 9CDown). If SMCs of the contractile phenotype were incubated for 24 hours with 200 µg/mL mLDL in the presence of 20 µg/mL granule remnants (as in the experiment with SMCs of the synthetic phenotype shown in Fig 8Up), no lipid droplets accumulated in the cells (not shown).



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  		Figure 9. Line graphs showing uptake of (A) granule remnants, (B) methylated LDL (mLDL), and (C) mLDL in the presence of granule remnants by smooth muscle cells (SMCs) of both the contractile and synthetic phenotypes. SMCs of both phenotypes were incubated in 300 µL of medium C with increasing concentrations of (A) 125I-labeled granule remnants or increasing concentrations of [3H]cholesteryl linoleate ([3H]CL)–mLDL in (B) the absence or (C) presence of unlabeled granule remnants (20 µg/mL). After incubation at 37°C for 15 hours, the cellular contents of 125I or 3H radioactivity were determined.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study reveals a novel mechanism for the formation of smooth muscle foam cells, ie, the mast cell–induced accumulation of cytoplasmic cholesteryl ester droplets derived from LDL. In this process, LDL is carried into the SMC on the surface of the mast cell granule remnants by a phagocytic mechanism. The precise mechanism that triggers the uptake of LDL-coated granule remnants by SMCs is currently under investigation. Campbell et al,54 57 working with rabbit SMCs, have suggested that in contrast to the SMCs of the contractile phenotype in the arterial media, the synthetic counterparts in atherosclerotic plaques accumulate LDL and that this accumulation is LDL receptor–mediated and depends on reduced cellular catabolism of LDL. Ismail et al,31 using rabbit aortic SMCs of the synthetic phenotype (derived from explants), have reported that the phagocytic uptake of lipoprotein-proteoglycan complexes obtained from injured rabbit aortas is mainly LDL receptor–dependent and that scavenger receptors play only a minor part in this process. The present results suggest that the granule remnant–mediated uptake of LDL and ensuing cholesterol accumulation in these cells are largely independent of LDL receptor activity. In mouse peritoneal macrophages uptake of LDL-coated granule remnants does not depend on LDL receptor activity (P.T. Kovanen, K.A. Lindstedt, unpublished data, 1992). This finding was not unexpected, since the mouse macrophages used in those studies, in contrast to rabbit aortic SMCs of either phenotype, express LDL receptors at very low levels.10 On the other hand, the levels of scavenger receptors expressed by mouse peritoneal macrophages are high, yet these receptors are not involved in granule remnant phagocytosis by these cells (P.T. Kovanen, K.A. Lindstedt, unpublished data, 1992). It remains to be seen whether these receptors are involved in the phagocytic uptake of the granule remnants by SMCs of the synthetic phenotype with ensuing foam cell formation. The presence of negative charges (heparin proteoglycans) in the granule remnants, a prerequisite for recognition by scavenger receptors, and the increased expression of scavenger receptors in SMCs of the synthetic phenotype16 58 appear to favor this possibility.

We noticed an increase in the phagocytic capacity of the SMCs that had been induced to change their phenotype from contractile to synthetic in vitro. This increase was observed on adding exocytosed mast cell granules to the SMCs (in the absence of LDL). Phagocytosis of latex and carbon particles has been observed in several types of muscle cells. Of the three types of muscle cells compared by Garfield et al,59 the aortic SMCs of the contractile phenotype were more active than skeletal or cardiac muscle cells as judged by their ability to take up latex particles. Of the two cell types compared by Blaes et al,60 the SMCs of the synthetic phenotype from rat aorta phagocytosed carbon particles more actively than did fibroblasts derived from rat skin. The current observation that SMCs of the synthetic phenotype derived from rabbit aorta phagocytose mast cell granule remnants more actively than do their contractile counterparts suggests that among muscle cells, aortic SMCs of the synthetic phenotype have the highest phagocytic capacity. However, these cells ingested LDL-coated granule remnants at a rate that was about 20% that observed in mouse peritoneal macrophages,34 the known specialized phagocytes.

The high phagocytic capacity of SMCs of the synthetic phenotype appears likely to be relevant to foam cell formation in the arterial intima in atherogenesis. Indeed, numerous observations indicate that the conversion of SMCs from the contractile into the synthetic phenotype occurs in vivo when these cells migrate from the medial layer into the intimal space of atherosclerosis-prone arteries.1 8 61 Moreover, within the intima there appears to be a continuous shift of the SMC phenotype toward the synthetic type as the cells move from the deep intimal layer toward the more superficial subendothelial layer7 ; in the fatty streaks most of the foam cells are in this superficial layer.62 Presumably, then, the increased phagocytic capacity of SMCs of the synthetic phenotype contributes to foam cell formation. Indeed, it is likely that such intimal SMCs readily phagocytose various complexes of LDL formed not only with exocytosed mast cell granules but also with other extracellular components, such as proteoglycans, collagen, and fibronectin.31 63 LDL aggregates (in the absence of any carriers) can also be avidly phagocytosed by human aortic intimal SMCs.64 Besides LDL, other lipoproteins could also form complexes with extracellular particulate matter and so be subjected to phagocytic removal mechanisms. Indeed, the "granule remnant carrier mechanism" is not specific for LDL42 ; granule remnants can bind and carry lipoprotein particles that contain either apoB-100 or apoE (or both) but not lipoproteins lacking these apolipoproteins, such as human HDL3. In addition, the extracellular lipid droplets that appear during the initial stage of atherogenesis65 and then disappear as foam cells are formed66 may also be subject to the phagocytic removal mechanism. Interestingly, Wolfbauer et al67 , using an in vitro model, have demonstrated the ability of SMCs of the synthetic phenotype (derived from explants of rabbit aorta) to phagocytose large macrophage-derived lipid droplets.

One important regulator of the SMC phenotype is heparin.68 69 Since heparin proteoglycans are the major component of mast cell granule remnants, heparin-like effects of the remnants on the SMC phenotype have to be taken into account. In the present study, we could not detect any inhibition of phagocytic activity when SMCs of the synthetic phenotype were incubated with granule remnants for up to 18 hours. The time may have been too short for a regulatory effect. Alternatively, it has been suggested that many features of the phenotypic modulation of SMCs are regulated independently. This seems also to apply to the actions of heparin. In an animal model, heparin selectively modulates the composition of the extracellular matrix produced by SMCs of the synthetic phenotype by decreasing the contents of elastin and collagen and increasing the content of proteoglycans,70 but it does not prevent connective tissue formation.71 Analogously, the ability to phagocytose, another trait of SMCs of the synthetic phenotype, might well remain uninfluenced by heparin and reflect the general refractoriness of these cells against reversal of phenotypic change in the conditions used.

When mast cells induce the uptake of LDL by other cells, such as SMCs of the synthetic phenotype, the pathway that LDL particles then follow is initiated when the mast cell expels a granule and ends when a phagocyte engulfs the LDL-coated granule remnant. How is the action of this exocytosis-phagocytosis pathway regulated in vivo? As discussed above, conditions suitable for efficient phagocytosis appear to prevail in the areas of the arterial intima where foam cell formation occurs. The condition necessary for granule exocytosis, ie, mast cell stimulation, also appears to prevail in these atherosclerosis-prone areas. Thus, we have observed that human aortic and coronary fatty streak lesions contain not only mast cells but also T lymphocytes and macrophages,4 5 both of which may secrete factors that can stimulate mast cells to degranulate.72 73 Indeed, the number of degranulated mast cells was much higher in coronary fatty streaks than in unaffected areas of the same region.5 Support for the claim that mast cells are actually involved in the generation of foam cells in human fatty streaks was recently obtained when electron microscopic studies of human aortic fatty streaks revealed an SMC adjacent to a degranulated mast cell in the process of phagocytosing an exocytosed granule remnant (M. Kaartinen, A. Penttilä, P.T. Kovanen, unpublished data, 1993). The current in vitro demonstration of the unabated ability of SMCs of the synthetic phenotype to phagocytose mast cell granule remnants provides a potential mechanism for the generation of lipid-filled SMCs in the human arterial intima in vivo, not only when SMCs are located next to stimulated mast cells, but also, more generally, when they are surrounded by large-sized aggregates of LDL formed with other components of the arterial intima.

Received January 11, 1995; accepted March 3, 1995.


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