© 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
From the Wihuri Research Institute, Helsinki, Finland.
Correspondence to Petri T. Kovanen, Wihuri Research Institute, Kalliolinnantie 4, SF-00140 Helsinki, Finland.
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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
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Introduction |
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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.
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Methods |
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Materials and Animals
Sodium [125I]iodide (13 to 17 mCi/µg), [1
,2(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.
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Results |
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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 1A
). 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 1B), 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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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 2A), 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 2A).
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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 2B
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 
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 2C).
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 3A), but in contrast to untreated LDL, mLDL was not
taken up by the SMCs of the synthetic phenotype (Fig 3B). 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 3C).
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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 4A) and inhibited the granule remnant–mediated uptake
of mLDL (Fig 4B). Second, using 125I-labeled granule
remnants, we demonstrated the uptake of granule remnants by SMCs of the
synthetic phenotype (Fig 5A). 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 5B). 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 6 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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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 7A). 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 7B and 7C). 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 8A). If granule remnants were omitted,
no lipid droplets were present in the cells (Fig 8B).
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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 1A). 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 9A). In the absence of
granule remnants, uptake of mLDL by SMCs of both phenotypes was low
(Fig 9B). 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 9C). 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 8), no lipid droplets
accumulated in the cells (not shown).
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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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Kaartinen M, Penttilä A, Kovanen PT. Mast
cells of two types differing in neutral protease composition in the
human aortic intima: demonstration of both tryptase- and
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