Heparin Proteoglycans Released From Rat Serosal Mast
Cells Inhibit Proliferation of Rat Aortic Smooth Muscle
Cells in Culture
Yenfeng Wang, Petri T. Kovanen
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Abstract—Mast cells are present in the human arterial intima. To study whether mast-cell degranulation influences the rate
of proliferation of smooth muscle cells, we cocultured sensitized (IgE-bearing) rat serosal mast cells and rat aortic
smooth muscle cells (SMCs). When sensitized mast cells were stimulated to degranulate with antigen, the rate of
proliferation of the cocultured SMCs decreased sharply. This inhibitory effect was found to be due mainly to the very
high molecular weight (Mr) heparin proteoglycans (average Mr 750 000) released from the stimulated mast cells. When
the heparin proteoglycans were purified from mast-cell granule remnants and added to the SMC culture, they were found
to block the cell cycle at the G03 S transition and the exit from the G2/M phase, their inhibitory effect resembling that
of commercial heparin. However, in contrast to the reported dependence of the inhibitory effect of commercial heparin
on the release of transforming growth factor-b from serum, the inhibitory effect of the mast cell– derived heparin
proteoglycans in the presence of serum was not transforming growth factor-b dependent. Moreover, the effect of the
mast cell– derived heparin proteoglycans was more efficient than that of commercial heparins of high (average Mr
15 000) and low (average Mr 5000) molecular weight. We also purified heparin glycosaminoglycans (average Mr 75 000)
from the mast cell– derived heparin proteoglycans and found that they also inhibited SMC growth efficiently, although
less strongly than their parent heparin proteoglycans. These results reveal, for the first time, that mast cells are able to
regulate SMC growth. Thus, activated mast cells, by releasing heparin proteoglycans, possibly participate in the
regulation of SMC growth in the human arterial intima, the site of atherogenesis. (Circ Res. 1999;84:74-83.)
Key Words: atherosclerosis n heparin proteoglycan n mast cell n proliferation n smooth muscle cell
A
ccumulation of smooth muscle cells (SMCs) in the
human arterial intima has been recognized as an important feature of atherosclerosis.1,2 Especially during the late
stages of atherogenesis, the SMCs, which originate either in
the intima or in the underlying media, may undergo abnormally regulated proliferation.3–5 However, compared with the
SMCs in restenotic lesions, the rate of proliferation of the
SMCs in atherosclerotic lesions is low. Accordingly, as
reported recently, the fraction of the SMCs that bear markers
of ongoing cell proliferation in advanced human atherosclerotic lesions is 3.6% (versus 15.2% in restenotic lesions),
when detected with an antibody against the proliferating cell
nuclear antigen, or 7.2% (versus 20% in restenotic lesions),
when the mRNA of proliferating cell nuclear antigen is
detected with an antisense probe.5 Such a relatively low rate
of growth, together with the observation that a fraction of the
SMCs in the lesions undergo apoptotic cell death,6 is consistent with the clinical observation that atherosclerosis usually
takes several decades to cause clinically significant obstruction of the arteries involved. Similarly, angiographic and
ultrasound studies have shown that plaques, once established,
often remain unchanged over several years of observation.7,8
The low rate of SMC growth in atherosclerotic lesions
likely results from the net effect of growth-stimulating and
growth-inhibiting factors acting locally on the SMCs. Wellknown stimulating factors include platelet-derived growth
factor, basic fibroblast growth factor, epidermal growth
factor, and interleukin-1.9 On the other hand, heparan sulfate
proteoglycan secreted by endothelial cells has been said to be
an important inhibitor of SMC growth,10,11 although recently
this opinion has been challenged.12 Finally, transforming
growth factor-b (TGF-b) exerts stimulatory or inhibitory
effects on the SMCs, depending on the subtype of TGF-b
receptor expressed by these cells.13
The human arterial intima also contains mast cells. In fatty
streaks and in atheromas, the number of mast cells is
increased,14,15 and in atherosclerotic lesions a substantial
fraction of these cells are seen to be degranulated, reflecting
their ongoing stimulation.15,16 We previously studied the
interactions between rat serosal mast cells and rabbit aortic
SMCs in vitro and found that, when stimulated, the mast cells
induced intracellular accumulation of LDL cholesterol in the
SMCs.17 On stimulation, rat serosal mast cells exocytose their
Received April 13, 1998; accepted October 20, 1998.
From the Wihuri Research Institute, Helsinki, Finland.
Reprint requests to Petri T. Kovanen, MD, PhD, Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki, Finland. E-mail
[email protected]
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
74
Wang and Kovanen
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cytoplasmic secretory granules and an array of soluble
mediators of inflammation, including preformed mediators
such as histamine, tumor necrosis factor-a, TGF-b, and
newly formed mediators such as prostaglandin D2 and leukotriene C4.18 The main components of the granules are heparin
proteoglycans, a fraction of which, after exocytosis, become
solubilized and are released into the extracellular fluid. The
residues, known as “granule remnants,” are globular particles
(0.5 to 1.0 mm in diameter) composed solely of proteoglycans
of the heparin type to which are tightly bound 2 neutral
proteases, chymase and carboxypeptidase A (CPA).19
The heparin proteoglycans of rat serosal mast-cell granules
are called “native heparin” or “macromolecular heparin.”
They have an average molecular weight (average Mr) of
750 000 (range 500 000 to 1 000 000), each monomer comprising, on average, 10 heparin glycosaminoglycan chains
with average Mr values of 75 000 (range 50 000 to
100 000).20,21 Structural analysis of the heparin proteoglycans
shows that their disaccharide units have the composition
typical of heparin (J.-p. Li, P. Kovanen, and U. Lindahl,
unpublished results, 1995), thus differing from those of the
heparan sulfate secreted by many other cell types, including
the endothelial cells and SMCs of the arterial intima.11,22,23
Indeed, mast cells are the only cells in the mammalian body
that synthesize heparin.24 Commercial heparin, when given
intravenously to a rat or a rabbit before arterial injury, has
been found to inhibit the rate of proliferation of SMCs in the
neointima, leading to decreased growth of the neointima.25–27
This inhibitory effect of heparin on human SMCs has also
been observed in culture.28 –30 Since mast cells are the
endogenous source of arterial heparin,16,31 the above observations with commercial heparin raise the possibility that
mast cells, when stimulated to secrete their macromolecular
heparin, may also participate in local regulation of SMC
proliferation in the arterial intima. In the present investigation, we used an experimental model to study the effect of rat
mast cells on rat aortic SMC proliferation in vitro.
Materials and Methods
Materials and Animals
Sodium [3H]thymidine (53 mCi/mg) was from Amersham International; RPMI 1640, Dulbecco’s PBS, FCS, penicillin, streptomycin,
and L-glutamine were from GIBCO; BSA, compound 48/80,
N-benzoyl-tyrosine ethyl ester, phenylmethylsulfonyl fluoride, collagenase type IA, trypsin, heparinase I, RNase, ovalbumin, and high
molecular weight (HMW) heparin (H-3393) were from Sigma; low
molecular weight (LMW) heparin (Fragmin) was from Kabi Pharmacia; Sephacryl S-200 HR and Superdex 75 PC 3.2/30 column
were from Pharmacia; recombinant human TGF-b1 (rhTGF-b1),
anti–TGF-b neutralizing antibody (chicken IgY, Chinese hamster
ovary cell– derived rhTGF-b1 as immunogen), and an ELISA kit for
TGF-b1 were from R&D Systems; anti–TGF-b1 antibody (rabbit
IgG, a peptide corresponding to amino acids 328 to 353 mapping at
the carboxyl terminus of human TGF-1 as immunogen) used in
Western blot was from Santa Cruz Biotechnology; and biotinylated
goat anti-rabbit immunoglobulins and horseradish peroxidase conjugated streptavidin were from DAKO. The male Wistar rats (300 to
500 g) used in this study were from the Laboratory Animal Center of
the University of Helsinki, Finland.
January 8/22, 1999
75
Preparation of Rat Aortic SMCs
Aortic SMCs were prepared from male Wistar rats as described
previously.17,32 Briefly, the intima and media were dissected out
from the thoracic aorta and cut into 1-mm pieces, which were first
treated with 1 mg/mL collagenase for 1 hour to remove the
endothelial cells, then washed with medium, and dispersed in a
mixture containing 1 mg/mL collagenase and 0.5 mg/mL elastase in
RPMI 1640 with 12.5% of FCS. 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 containing 2 mmol/L L-glutamine, 100
U/mL penicillin, and 100 mg/mL streptomycin) containing 20%
FCS. The cells were seeded at a density of 13105 cells/mL;
incubated in the same medium; and, at confluency, subcultured (1:2)
up to 9 times. The SMCs of 5 to 9 passages were used for the
experiments.17,33
Growth Arrest of SMCs
To arrest the growth of SMCs, sparsely plated cultures (13104 to
53104 cells/mL) were washed and placed in medium A containing
0.4% FCS for 72 hours, as described by Castellot et al.34 Flow
cytometric analysis showed that .80% of the cells had been arrested
in the G0/G1 phase.
Isolation and Stimulation of Mast Cells
Serosal mast cells were isolated from the pleural and peritoneal
cavities of rats as described previously.35 The mast cells were then
stimulated to degranulate in culture medium or in PBS, ie, to trigger
exocytosis of their cytoplasmic secretory granules. As stimulating
agents, we used either compound 48/80,21 or a specific antigen
(ovalbumin), if the mast cells had first been passively sensitized with
hyperimmune serum containing anti-ovalbumin IgE.36 After stimulation, the mast cells were sedimented by centrifugation at 800g for
5 minutes. The supernatant, which contains all the material released
from the stimulated mast cells, will be referred to in the text as
“mast-cell releasate.” This releasate contains mainly the cytoplasmic
secretory granules and histamine released from the granule compartment.19 The degree of mast-cell degranulation was determined by
measuring the histamine content of the releasate and is expressed
as mmol/L.37
Preparation of Granule Remnants and Granule
Remnant–Free Releasate
To sediment the granule remnants, the mast-cell releasate was
centrifuged at 13 000g for 10 minutes. After centrifugation, the
supernatant was collected and used as granule remnant–free releasate. The sedimented granule remnants were then washed twice with
water, resuspended in PBS, and used as granule remnants. The
concentration of the granule remnants is expressed in terms of their
protein content or of their content of Alcian blue–reactive material,
which reflects their content of negatively charged sulfate groups. The
ratio of Alcian blue–reactive material (mg) to protein (mg) in the
remnants was 0.3560.19 (mean6SD) in the 6 remnant preparations.
The concentrations of the soluble heparin proteoglycans and chondroitin sulfate proteoglycans (“heparin-chondroitin sulfate proteoglycans”) in the granule remnant–free releasates are given in terms of
their content of Alcian blue–reactive material.
Preparation of Purified Heparin Proteoglycans
and Heparin Glycosaminoglycans From Mast-Cell
Granule Remnants
Granule remnants of rat serosal mast cells contain only heparin
proteoglycans (but not chondroitin sulfate proteoglycans) and neutral
proteases (chymase and CPA). To obtain protease-free heparin
proteoglycans from granule remnants, the neutral proteases were
dissociated from the remnant heparin proteoglycans by incubating
the remnants in 10 mmol/L phosphate buffer supplemented with 2
mol/L KCl, pH 7.0. The mixture containing the solubilized remnants
(heparin proteoglycans and proteases) was then applied to a
76
Mast Cells Inhibit SMC Growth
Sephacryl S-200 HR column (103600 mm) and eluted with the same
buffer as was used for dissociation. Fractions containing Alcian
blue–reactive material were collected, dialyzed extensively against
water, concentrated with a Centricon 10 filter (Amicon), and used in
the experiments.20,38 These fractions were devoid of any protease
activity, as determined by a sensitive method involving analysis of
the proteolytic products of angiotensin I by reverse-phase high-performance liquid chromatography39 and will be referred to in the text
as mast cell– derived “heparin proteoglycans.” Heparin glycosaminoglycans were prepared from such heparin proteoglycans by incubating the latter for 14 hours in 0.5 mol/L NaOH at 25°C to
hydrolyze the core protein of the proteoglycans.20 The sample was
then lyophilized to 80 mL, and its heparin glycosaminoglycans
(average Mr 75 000) and the protein hydrolysates (peptides and
amino acids) were separated on a Superdex 75 PC 3.2/30 column
with 150 mmol/L NaCl in 5 mmol/L Tris-HCl buffer, pH 7.4. The
fractions containing Alcian blue–reactive material were collected
and used for the experiments.
Separation of LMW and HMW Substances
Present in Granule Remnant–Free Releasate
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To separate the soluble LMW substances (histamine; also prostaglandins, leukotrienes, and cytokines) from the soluble HMW
substances (heparin-chondroitin sulfate proteoglycans; average Mr
750 000), a granule remnant–free releasate was prepared and applied
to a Centricon 10 filter (Amicon) with a Mr 10 000 cutoff. The
releasate was then centrifuged at 3000g until the flow through the
filter ceased.35 The fractions of the releasate retained by the filter,
which contained the HMW substances (Mr .10 000), were washed
off with PBS to the original volume, while the ultrafiltrates, which
contained the LMW substances (Mr ,10 000), were used as such.
Determination of DNA Synthesis
Mast cell– derived products were usually collected in PBS. Purified
heparin proteoglycans were reconstituted for bioassay by exhaustive
dialysis against distilled water and diluted at least 20-fold with
culture medium before being added to the SMCs. Aortic SMCs,
13104 to 53104 cells/mL, were seeded, and when they reached
subconfluency, the growth of the SMC monolayers was arrested, and
they were incubated with the various mast cell– derived products for
16 hours or as indicated in the figure legends. After incubation, the
cells were released from the G0 block by addition of FCS (final
concentration: 20%, vol/vol), and incubation was continued for 26
hours. The rate of DNA synthesis was then determined by measuring
the incorporation of [3H]thymidine into the trichloroacetic acid
(TCA)–precipitable material of the SMCs.34 For this purpose, 2
mCi/mL [3H]thymidine was added to the culture medium, followed
by incubation for an additional 2 hours. At the end of the labeling
period, the cells were detached with 0.25% trypsin and collected in
tubes. A 10-mL sample of the cell suspension was used for counting
the cell number. The remaining cells were washed 3 times with cold
PBS and incubated in 1 mL of cold 0.61 mol/L (10%) TCA solution
for 30 minutes at 4°C. The TCA solution was then discarded, and the
cells were washed 3 times with 0.61 mol/L (10%) TCA solution. The
residual TCA-precipitated label was extracted with 0.2 mol/L NaOH,
and the radioactivity of the extract was determined with a WinSpectral 1414 liquid scintillation counter (Wallac). [3H]Thymidine incorporation into the DNA of SMCs is expressed in terms of dpm/104
cells.
Flow Cytometric Analysis of the Cell Cycle
The cell-cycle distribution of SMCs was determined by flow
cytometric analysis of propidium iodide–labeled cells.40 Briefly, the
cells were collected and fixed in 90% methanol on ice for 10
minutes. After fixation, the cells were washed twice with PBS,
resuspended in RNase solution in PBS (100 U/mL), and incubated at
37°C for 30 minutes. The RNase-treated cells were then stained with
propidium iodide (20 mg/mL) and analyzed by FACScan (Becton
Dickinson). Cells having lower DNA content than G1/G0 cells
(hypodiploidy) were considered apoptotic.41
Determination of TGF-b1 in SMC
Culture Medium
Cultures of SMCs were treated with mast cell– derived heparin
proteoglycans. After incubation, the culture media received the
following protease inhibitors: phenylmethylsulfonyl fluoride (final
concentration 1 mmol/L), aprotinin (2 mg/mL), leupeptin (2 mg/mL),
and pepstatin A (2 mg/mL). The medium was then collected and
centrifuged at 800g for 5 minutes. The quantity of TGF-b1 in the
supernatant was determined directly by an ELISA kit as recommended by the manufacturer (R&D systems) or concentrated with a
Centricon 10 filter (Amicon; Mr 10 000 cutoff) for immunoblotting
of active TGF-b1, as described by Taipale et al.42
Other Assays
Granule remnant protein was determined by the procedure of Lowry
et al,43 with BSA as standard. The glycosaminoglycan content of
proteoglycans was determined by assaying Alcian blue–reactive
material with commercial heparin as standard.44 Chymase activity
was determined spectrophotometrically with N-benzoyl-tyrosine
ethyl ester as substrate.45 The activities of chymase and CPA in the
purified heparin proteoglycans were determined by analyzing the
proteolytic products of angiotensin I with reverse-phase high-performance liquid chromatography.39
Statistical Analysis
Data, shown as mean6SD, were analyzed with Student’s t test for
determination of the significance of differences, which were considered to be statistically significant at a P value ,0.05.
Results
Effect of Mast-Cell Degranulation on
SMC Proliferation
To study the effect of activated mast cells on proliferation of
SMCs, we prepared monolayers of subconfluent SMCs from
rat aortic media and arrested them at the G0 phase with 0.4%
FCS medium. Rat serosal mast cells were then isolated and
passively sensitized with rat serum containing anti-ovalbumin
IgE. The sensitized mast cells (ie, mast cells with IgE bound
to their high-affinity IgE receptors) were added to the SMCs
and stimulated to degranulate in the coculture system by
adding ovalbumin (the antigen) to the incubation medium.
The degree of degranulation of the sensitized mast cells was
ascertained by measuring the concentration of histamine in
the culture medium. As shown in Figure 1A, in dishes
containing both sensitized mast cells and antigen (column b),
the amount of histamine in the culture medium was higher,
revealing degranulation of the mast cells. Sixteen hours after
mast cell degranulation, the growth-arrested SMCs were
stimulated to proliferate with 20% FCS, and their proliferation was monitored by determining the amount of [3H]thymidine incorporated into the DNA of the cells. In the dishes in
which mast cells had been stimulated to degranulate, as
shown in Figure 1B, incorporation of [3H]thymidine by the
SMCs was strongly inhibited as compared with control SMCs
that had been cultured without mast cells (b versus a,
P,0.0001). No such inhibition was observed when only the
antigen was added, without mast cells (c versus a; P.0.05).
Furthermore, when the coculture system included sensitized
mast cells without the antigen (column d) or with nonsensitized mast cells and the antigen (column e), there was only
slight release of histamine (because of spontaneous degranulation; panel A), and the effect on SMC proliferation was
also slight (d versus a and e versus a, P,0.05 for both).
Wang and Kovanen
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Figure 1. Effect of activated mast cells on [3H]thymidine incorporation into DNA of SMCs in coculture. Serosal mast cells
were isolated and passively sensitized with IgE-containing
serum as described in Materials and Methods. The sensitized
cells (13105) were then washed with PBS 3 times and added to
subconfluent monolayers of growth-arrested SMCs with or without stimulation with the IgE-specific antigen (89 nmol/L ovalbumin; 4 mg/mL). Antigen, sensitized mast cells, and unsensitized
mast cells plus antigen were used as controls. After incubation
at 37°C for 16 hours, the cocultured SMCs were stimulated to
proliferate by addition of FCS (final concentration: 20%, vol/vol),
and [3H]thymidine incorporation into the DNA of the SMCs was
determined as described in Materials and Methods (B). Histamine in the culture medium was determined to quantify the
degranulation of mast cells (A). Values are mean1SD of triplicate incubations. Similar results were obtained in 2 other independent experiments.
Similar results were observed when the cells were counted
after coculture of SMCs and degranulating mast cells for 72
hours (data not shown). To relate the degree of mast-cell
degranulation (determined as release of histamine) to the
inhibitory effect on SMC proliferation, mast cells were
stimulated to degranulate with a commercial noncytotoxic
mast cell–specific agent, compound 48/80, with respect to the
compound concentrations. We found that the concentration of
histamine increased rapidly as the concentration of compound
48/80 increased. Most importantly, incorporation of [3H]thymidine into the DNA of the SMCs was inversely related in a
dose-dependent manner to the degree of mast-cell degranulation, half-maximal inhibition being achieved with releasate
containing 30 mmol/L histamine (data not shown).
Heparin Proteoglycans Are the Main Inhibitors of
SMC Proliferation in the Mast-Cell Releasate
The inhibitory effect of commercial heparin on the growth of
vascular SMCs is well established.46 – 49 Mast-cell granule
January 8/22, 1999
77
Figure 2. Kinetics of granule remnant–mediated inhibition of
SMC proliferation. Growth-arrested SMCs were incubated at
37°C for the indicated time periods in medium A containing
20% FCS with or without 2 mg heparin/mL of granule remnants.
After incubation, [3H]thymidine incorporation into the DNA of the
SMCs was determined. For comparison, [3H]thymidine incorporation by SMCs in G0 block (0.4% FCS) was also determined
(A). In parallel dishes, the cell number was counted (B). Values
are mean6SD of triplicate incubations.
remnants contain “native” or “macromolecular” heparin,
which is a heparin proteoglycan of very high molecular
weight (average Mr 750 000). We isolated granule remnants
from mast-cell releasate, added them to SMCs, and found that
incorporation of [3H]thymidine into the DNA of SMCs was
gradually inhibited as the concentration of the granule remnants in the incubation medium increased. Half-maximal
inhibition was achieved with 2 mg/mL remnant heparin
proteoglycans (data not shown).
We next studied the effect of granule remnants on [3H]thymidine incorporation into the DNA of SMCs after the release
of the SMC from the G0 phase. In this case, FCS was added
to the cultures (final concentration: 20%, vol/vol) to release
growth-arrested SMC from the G0 block in the absence or
presence of granule remnants. As shown in Figure 2A, in the
absence of granule remnants, [3H]thymidine incorporation
into the DNA of the SMCs started between 8 and 12 hours
after release of the cells from the G0 phase and peaked at 24
hours. Correspondingly, between 30 and 48 hours after
release from G0, the numbers of cells started to increase
(Figure 2B). But when granule remnants (2 mg/mL heparin
proteoglycans) were added, both DNA synthesis (panel A)
and cell proliferation (panel B) were inhibited. To find out
how long after release from the G0 block the addition of
78
Mast Cells Inhibit SMC Growth
TABLE 1. Effect of Mast-Cell Granule Remnants and the
Remnant-Derived Heparin Proteoglycans on Cell Cycle
Distribution of SMCs
Proportion of Cells, %
Experiment
G0/G1
S
G2/M
0
52.861.5
33.661.5
13.661.4
2.5
52.561.9
30.461.5*
17.162.3†
5
55.161.7†
27.761.8‡
17.262.3†
10
57.161.1‡
25.861.4‡
17.860.7‡
A. Granule remnants,
mg heparin/mL
B. Heparin proteoglycans,
mg heparin/mL
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Figure 3. Sensitivity of SMC proliferation to inhibition with granule remnants at different time points after release from G0.
Growth-arrested SMCs were released from G0 block with 20%
FCS (0 hour). After incubation for the indicated time periods, 2
mg heparin/mL of granule remnants was added, and incubation
was continued for up to 26 hours. [3H]thymidine incorporation
into the DNA of the SMCs was then determined during an additional 2-hour period. Values are mean6SD of triplicate
incubations.
granule remnants would still inhibit SMC proliferation, the
remnants were added to the cultures at various time points
after addition of FCS. As shown in Figure 3, granule
remnants could be added as much as 16 hours after addition
of serum with little loss of their antiproliferative effect, but
this effect was progressively lost when the remnants were
added 20 or more hours after the cells had been released from
the G0 phase. In accord with the observations shown in Figure
2A, that the SMCs started to enter the S-phase of the cell
cycle at 8 to 12 hours, granule remnants may block the G03 S
transition or early S-phase events. Therefore, in an additional
experiment (not shown), we examined how long the inhibitory effect would last after removal of the granule remnants.
For this purpose, SMCs were kept growth arrested by
incubating them in the presence of 0.4% FCS with or without
granule remnants (2 mg/mL heparin proteoglycans). After
incubation for 48 hours, any granule remnants were removed,
and fresh medium containing 20% FCS was added to release
the cells from G0 and stimulate them to proliferate. We found
that the control SMCs (not exposed to granule remnants)
began to incorporate [3H]thymidine into their DNA between
8 and 12 hours and peaked at 26 hours, similarly to those
shown in Figure 2A. In contrast, the SMCs exposed to
granule remnants showed only a slow increase in the rate of
[3H]thymidine incorporation into the DNA within the period
of observation (26 hours), revealing that after removal of the
granule remnants, their inhibitory effect persisted for at least
this length of time.
We also studied the effect of mast-cell granule remnants on
the cell-cycle distribution by flow cytometric analysis. As
shown in Table 1 (experiment A), when the concentration of
granule remnants increased, the proportion of SMCs in the
0
52.461.7
33.161.5
14.560.2
20
57.962.0†
26.061.2*
16.561.1†
Growth-arrested SMCs were incubated for 16 hours with indicated concentrations of mast-cell granule remnants and the remnant-derived heparin
proteoglycans. After incubation, the cells were stimulated to proliferate by
addition of FCS (final concentration: 20%, vol/vol), and incubation was
continued for another 26 hours. The cell-cycle distribution of the SMCs was
examined by flow cytometric analysis as described in Materials and Methods.
Values shown in experiment A are mean6SD of 2 separate experiments, each
performed in triplicate incubations, and those shown in experiment B are
mean6SD of triplicate incubations of a single experiment.
*P,0.01; †P,0.05; ‡P,0.001.
G0/G1 phase and in the G2/M phase gradually increased, while
the proportion of SMCs in the S phase gradually decreased,
suggesting an inhibition or a delay of the G03 S transition
and an extension of the G2/M phase of the cell cycle in the
SMCs. When considered together with the result from the
[3H]thymidine incorporation assay shown in Figure 2A, an
inhibition rather than a delay of the G03 S transition of SMCs
by mast-cell granule remnants is likely.
To further establish that the proteoglycans in the granule
remnants were responsible for the inhibitory effect, we
purified heparin proteoglycans from the remnants by sizeexclusion chromatography. The purified proteoglycans were
extensively dialyzed and added to SMC cultures. As shown in
Table 1, experiment B, when SMCs were treated with the
purified heparin proteoglycans, the proportions of the cells in
the G0/G1 and G2/M phases increased, and the proportion in
the S-phase decreased, as found with native granule remnants
(experiment A). If cell proliferation was measured by
[3H]thymidine incorporation into the DNA of SMCs, the
same amount of the purified heparin proteoglycans inhibited
the incorporation of [3H]thymidine into the DNA of the
SMCs by 68% (P,0.005; n56; data not shown).
As described in Materials and Methods, we prepared
mast-cell releasates by stimulating mast cells to degranulate
and then removing the cells by centrifugation. The releasates
contained all of the material released from the stimulated
mast cells. We next separated the releasates by centrifugation
into granule remnants (sediment) and granule remnant–free
releasate (supernatant). Granule remnants of rat serosal mast
cells are composed, in addition to the heparin proteoglycans,
of 2 neutral proteases, chymase and CPA. CPA, when
purified and added to SMC culture, had no effect on the cell
Wang and Kovanen
TABLE 2. Soluble Heparin Proteoglycans Are the Only Inhibitor
of SMC Growth in Granule Remnant–Free Releasate
[3H]Thymidine Incorporation
Into the DNA of SMCs
Experiment and Additions
104 dpm/104
Cells
Percentage
of Control
A. Fractions of granule remnant-free
releasate
Control (no addition)
17.5364.73
Granule remnant-free releasate
10.1261.17*
100
58
LMW substances
16.0064.34†
91
HMW substances
10.2762.00*
59
Control (no addition)
7.7961.71
100
HMW substances
3.6761.37‡
47
Heparinase-treated HMW substances
7.8061.85†
100
B. Effect of heparinase on the inhibitory
effect of HMW substances
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Growth-arrested SMCs were incubated for 16 hours with indicated mast-cell
products (100 mL/mL in experiment A; 10 mg heparin/mL in experiment B).
After incubation, the cells were stimulated to proliferate with 20% FCS, and
[3H]thymidine incorporation into the DNA of the cells was determined as
described in Materials and Methods. Each microliter of the product was derived
from 13104 mast cells. Heparinase-treated HMW substances were produced
by incubation of 10 mg Alcian blue–reactive material of HMW substances for 6
hours with 34 units of heparinase at 37°C in 50 mmol/L Tris-HCl containing
1 mmol/L calcium acetate and 100 mg/mL trypsin inhibitor, pH 7.4. Data shown
are from 3 separate experiments. Values of experiment A are means6SD of 6
incubations, and that of experiment B, mean6SD of 9 incubations.
*P,0.01; †P.0.05; ‡P,0.001.
growth, whereas purified chymase, when added to SMC
culture, did inhibit SMC growth (data not shown).
When granule remnant–free releasate was added to the
SMC culture (see Table 2, experiment A), [3H]thymidine
incorporation into the DNA of the cells was inhibited by 42%
(P,0.01). We then separated by molecular sieve filtration
(cutoff, Mr 10 000) the HMW substances (ie, the soluble
heparin-chondroitin sulfate proteoglycans; average Mr
750 000) and the LMW substances (histamine, prostaglandins, leukotrienes, and cytokines) present in the granule
remnant–free releasate and added them to SMC cultures
separately. It was found that only the HMW substances
inhibited SMC growth. To study whether the inhibitors
present in the HMW fraction are the heparin chains of the
soluble proteoglycans present in the granule remnant–free
releasate, we treated the HMW substances (the soluble
heparin-chondroitin sulfate proteoglycans) with heparinase
and studied their effect on [3H]thymidine incorporation by
SMCs. As shown in Table 2, experiment B, after treatment
with heparinase, the inhibitory effect of the proteoglycans
was completely blocked, revealing that the inhibitory capacity of the granule remnant–free releasate was due to heparin
chains (rather than to chondroitin sulfate chains).
The growth-inhibiting effects of mast cell– derived heparin
proteoglycans appeared not to be due to toxicity, as judged by
viewing the granule remnant– and heparin proteoglycan–
treated cells under a phase-contrast microscope with or
without trypan blue staining (data not shown). Granule
January 8/22, 1999
79
TABLE 3. Effect of Mast-Cell Granule Remnants on Growth
and Apoptosis of SMCs
Addition
Apoptosis, %
Cell Number,
3105/well
Control
0.3560.07
6.1860.50 (100%)
Granule remnants
2.3361.30*
4.4160.20 (71%)*
Growth-arrested SMCs were incubated for 16 hours with 5 mg heparin/mL
mast-cell granule remnants. After incubation, the cells were stimulated to
proliferate for 2 cell cycles by addition of FCS (final concentration: 20%,
vol/vol). At the end of the incubation, the number of SMCs was counted, and
the percentage of the apoptotic cells was determined by flow cytometric
analysis as described in Materials and Methods. Values are mean6SD of
quadruplicate incubations.
*P,0.001.
remnants, but not isolated heparin proteoglycans derived
from them, induced apoptosis of SMCs. As shown in Table 3,
when growth-arrested SMCs were cultured in the presence of
mast-cell granule remnants, the percentage of apoptotic cells
was significantly higher than that of the control cells
(P,0.001). If the cells were counted at the end of the
incubation, the number of granule remnant–treated SMCs
was 29% less than in control cultures. The low percentage of
apoptotic cells (2.33%) contrasts with the 29% reduction in
the final number of SMCs after the 42-hour (total duration)
incubation with granule remnants. Therefore, we infer that
the apoptotic cell death cannot explain the observed effect of
granule remnants on SMC number. Apoptosis of the SMCs in
granule remnant– containing cultures was also identified by
observing the condensation of the cytoplasm, compaction of
the chromatin, and fragmentation of the nucleus into discrete
masses scattered throughout the cell cytoplasm when the cells
were stained with May-Grünwald Giemsa stain (data not
shown).
Mast Cell–Derived Heparin Proteoglycans Are
More Potent Inhibitors of SMC Proliferation Than
Commercial Heparins
We next compared the inhibitory effects of the purified
heparin proteoglycans obtained from mast-cell granule remnants and of commercial heparin of 2 types, LMW heparin
(LMW heparin; average Mr 5000) and HMW heparin (HMW
heparin; average Mr 15 000). As shown in Figure 4A, for
equal concentrations of heparin (0220 mg/mL Alcian blue–
reactive material), the mast cell– derived heparin proteoglycans had a stronger inhibitory effect on [3H]thymidine incorporation by SMCs than either of the 2 commercial heparins
(P,0.05). This difference was even more pronounced when
the data were considered on the basis of molarity rather than
mass. Thus, in a separate experiment (panel B), mast cell–
derived heparin proteoglycans (average Mr 750 000) at a
concentration of 27 nmol/L (corresponding to 20 mg/mL
heparin proteoglycans, 0.4 mg/mL HMW heparin, and 0.13
mg/mL LMW heparin) exhibited an inhibitory activity that
was '20 times that of either commercial heparin (95%
inhibition versus 5% inhibition). We next hydrolyzed the core
protein of the heparin proteoglycans and purified heparin
glycosaminoglycans from the hydrolysates. It was found that
the purified heparin glycosaminoglycans efficiently inhibited
80
Mast Cells Inhibit SMC Growth
TABLE 4. Effect of Mast Cell–Derived Heparin Proteoglycans
on the Quantity of Active and Total TGF-b1 in SMC
Culture Medium
Addition
Active TGF-b1,
pg/well
Total TGF-b1,
pg/well
Control
58610
1288655
Heparin proteoglycans
58617*
10646189*
Growth-arrested SMCs were incubated for 16 hours with 20 mg/mL mast
cell– derived heparin. After incubation, the cells were stimulated to proliferate
by addition of FCS (final concentration: 20%, vol/vol), and incubation was
continued for another 26 hours. At the end of the incubation, cell culture
medium was collected and the amount of TGF-b1 in the medium was
determined by ELISA, as described in Materials and Methods. Values are
mean6SD of quadruplicate incubations.
*NS, vs control.
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Figure 4. Comparison of inhibitory effects of mast cell– derived
heparin proteoglycans and LMW and HMW commercial heparins on the proliferation of SMCs. Growth-arrested SMCs were
prepared as described previously, and the effect of purified heparin proteoglycans obtained from mast-cell granule remnants,
LMW commercial heparin, and HMW commercial heparin on
SMC proliferation was studied as described in Table 2. Values
are mean6SD of triplicate incubations.
[3H]thymidine incorporation into SMCs. This inhibitory effect was weaker than that of intact heparin proteoglycans but
stronger than that of either LMW or HMW commercial
heparins (each P,0.05, n53; data not shown).
Effect of Mast Cell–Derived Heparin
Proteoglycans on SMC Proliferation Is Not
TGF-b Dependent
Commercial heparin has been reported to release TGF-b from
serum, and this in turn may inhibit the proliferation of
SMCs.50,51 To study whether the inhibitory effect of the mast
cell– derived heparin proteoglycans on SMC proliferation is
TGF-b dependent, we first tested the effects of commercial
rhTGF-b1 on SMC proliferation under the conditions used in
this study. As shown in Figure 5A, 50 ng/mL rhTGF-b1
inhibited the synthesis of DNA by SMCs by 48% (b versus a,
P,0.05). This inhibitory effect was blocked by 65% in the
presence of 100 mg/mL anti–TGF-b neutralizing antibody (c
versus b, P,0.05). Incubation of SMCs with mast cell–
derived heparin proteoglycans in medium containing 20%
FCS inhibited the synthesis of DNA by SMCs by 80% (panel
B, b versus a, P,0.0005). This inhibitory effect was not
counteracted even when increasing concentrations of anti–
TGF-b neutralizing antibody were present in the culture
medium (c through f versus b, each P.0.05). In addition, we
determined by ELISA the amount of TGF-b1 in medium of
mast-cell heparin proteoglycan–treated SMC culture in the
presence of 20% FCS. As shown in Table 4, compared with
the amount of TGF-b1 in the control culture medium, mastcell heparin proteoglycans did not significantly increase the
quantity of either active or total TGF-b1 in the culture
medium. Furthermore, we failed to detect the presence of
active TGF-b1 by immunoblotting (detection limit, 0.5
ng/lane) in 25-fold concentrated SMC culture medium, in
which 20% FCS and 20 mg/mL mast cell– derived heparin
proteoglycans were included (data not shown).
Discussion
The present study revealed a novel property of mast cells,
namely, that they are capable of inhibiting SMC growth. The
main inhibitory factor was identified as the heparin proteoglycans secreted by these cells. This applies to both the
insoluble heparin proteoglycans of the granule remnants and
the soluble heparin proteoglycans released from the exocytosed granules. The mast cell– derived heparin proteoglycans,
also called “macromolecular heparin,” were much more
efficient than their commercial counterparts; on a molar basis,
Figure 5. Blocking of the effect of rhTGF-b1 (A)
and mast cell– derived heparin proteoglycans (B)
by anti–TGF-b neutralizing antibody. The study
was conducted as described in Table 2, except
that SMCs were incubated with either 50 ng/mL
rhTGF-b1 or 20 mg/mL purified heparin proteoglycans obtained from mast-cell granule remnants in
the presence of the indicated concentrations of
anti–TGF-b neutralizing antibody. Values are
mean1SD of triplicate incubations.
Wang and Kovanen
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their inhibitory effect on SMC proliferation was at least
20-fold that of the commercial heparins. In a structural
analysis of the heparin proteoglycans, the composition of the
disaccharide units was found to be typical of heparin (J.-p. Li,
P. Kovanen, and U. Lindahl, unpublished results, 1995).
Therefore, the observed differences in inhibitory potential
between the heparin proteoglycans and commercial heparins
must depend on factors other than the composition of the
individual glycosaminoglycan chains. The ability of intact
heparin proteoglycans (average Mr 750 000) to inhibit SMC
growth was greater than that of the heparin glycosaminoglycan chains (average Mr 75 000) prepared from the heparin
proteoglycans, which, again, was greater than that of the 2
types of commercial heparin (HMW heparin, average Mr
15 000, and LMW heparin, average Mr 5000). Accordingly,
the most important factors contributing to the observed
inhibition are the large size of the mast cell– derived heparin
chains and their attachment to a core protein (creating
“macromolecular” heparin). The same principle of size dependency was recently observed to apply also to the ability of
heparins of various types to prevent collagen-induced platelet
aggregation.52 It is of interest that endothelial heparan sulfate
proteoglycans, with an overall size of average Mr '1 000 000
and with heparan sulfate chains of average Mr 60 000 (ie,
closely resembling in size the mast cell– derived heparin
proteoglycans used in this study), are much more potent
inhibitors of SMC growth than is commercial heparin.11
Stimulated mast cells also secrete factors that could potentially act as stimuli of SMC growth. For example, histamine,
which is released by mast cells on their activation, is known
to stimulate SMC proliferation when added to cultured
SMCs.53 Nevertheless, in the present experiments, the net
effect of stimulated mast cells on SMC proliferation was
always (irrespective of the degree of mast-cell stimulation)
inhibitory. The failure of the released histamine to counteract
the effect of the released heparin proteoglycans was found to
be due to its low concentration. In the incubation media of
maximally stimulated mast cells, this was in the micromolar
range (maximally 100 mmol/L), whereas, with commercial
histamine, the concentration required for stimulation of SMC
growth has been found to be in the millimolar range
(100 mmol/L).53 The fraction of mast-cell releasate containing the LMW substances (including histamine) was without
any significant effect on SMC growth even in the absence of
heparin proteoglycans (see Table 2). This conclusion regarding the physiological effects of degranulating rat mast cells
also applies to the other LMW substances, such as TGF-b,
present in the mast-cell releasate and known to inhibit or
stimulate the growth of rat aortic SMCs in culture.54,55
What could be the mechanism by which the mast cell–
derived heparin proteoglycans inhibit the growth of SMCs in
response to serum in vitro? In principle, the heparin proteoglycans could activate inhibitory factors present in the serum
or exert their effects directly on the SMCs. Previous reports
with commercial heparin have provided evidence for actions
of both types. Regarding the first type of action, Grainger et
al50 reported that the heparin-induced inhibition of SMC
growth in response to FCS could be due to release of active
TGF-b from the added FCS by the heparin. We found that
January 8/22, 1999
81
proliferation of cultured rat aortic SMCs in response to FCS
was inhibited when commercial TGF-b was added to the
incubation medium, revealing that, under the culture conditions used, the rat aortic SMCs were responsive to TGF-b–
induced growth inhibition. However, in contrast to the findings reported with commercial heparin, the mast cell– derived
heparin proteoglycans appeared to inhibit SMC growth independently of TGF-b, since addition of anti–TGF-b neutralizing antibody to the FCS-containing culture medium failed
to block the inhibitory effect of the added heparin proteoglycans (see Figure 5B). In addition, the heparin proteoglycans
did not increase the quantity of active TGF-b1 in the culture
medium (see Table 4). Regarding a possible direct inhibitory
effect, binding and uptake of heparin mediated by both
receptor-dependent and receptor-independent endocytic pathways was required for its inhibitory effect on rat aortic
SMCs.56,57 The conclusion that heparin uptake by SMCs is
essential for the inhibitory action of heparin was also reached
in a study with heparin-sensitive and heparin-resistant SMCs,
in which it was found that upregulation of heparin binding to
the SMCs was strongly linked to subsequent internalization
and degradation of heparin and was required for the antiproliferative effect of heparin.46,47 We have previously shown by
electron microscopy and by fluorescent microscopy that
exocytosed mast-cell granule remnants are phagocytosed by
SMCs in culture17 and are also phagocytosed by the SMCs in
the atherosclerotic human arterial intima in vivo.16 In vitro
studies revealed that this phagocytosis was mediated by the
scavenger receptors of the SMCs, the negatively charged
heparin proteoglycans of the remnants being responsible for
receptor recognition.58 In the present study, we found that
maleylated albumin, a compound able to block scavenger
receptors,59 was without effect on SMC growth, but when
added together with granule remnants, it counteracted their
growth-inhibitory effect (data not shown). Taken together,
the present and previous observations with mast cell– derived
heparin proteoglycans strongly suggest that they directly
inhibit the SMC growth response to serum. Regarding the
intracellular regulatory events of SMC proliferation, we
found that the heparin proteoglycans of rat serosal mast cells
blocked the G03 S–phase transition and the exit from the
G2/M phase of the cell cycle. The precise mechanism by
which heparin proteoglycans act on the intracellular signaling
pathways regulating cell proliferation remains to be studied.
The rate of proliferation of the SMCs in the human arterial
intima is controlled by a multitude of factors present in the
blood or produced locally in the vessel wall.60 – 62 The present
findings reveal a new potential source of local growth
regulators, ie, activated mast cells. The number of mast cells
and the degree of their degranulation are known to be
increased in atherosclerotic lesions.14,15,63 On the basis of the
present in vitro findings, we propose that heparin proteoglycans secreted by activated mast cells in the arterial intima
tend to locally inhibit SMC growth.
Acknowledgment
We are grateful to Monica Schoultz (Transplantation Laboratory,
University of Helsinki) and Markus Leskinen for their help with the
82
Mast Cells Inhibit SMC Growth
flow cytometric analysis and to Hua Ma for her donation of the
anti-ovalbumin IgE-containing rat serum.
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Heparin Proteoglycans Released From Rat Serosal Mast Cells Inhibit Proliferation of Rat
Aortic Smooth Muscle Cells in Culture
Yenfeng Wang and Petri T. Kovanen
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Circ Res. 1999;84:74-83
doi: 10.1161/01.RES.84.1.74
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