© 2001 American Heart Association, Inc.
Vascular Biology |
Mast Cell Chymase Induces Apoptosis of Vascular Smooth Muscle Cells
From the Wihuri Research Institute (M.L., Y.W., K.A.L., P.T.K.) and the STUK-Radiation and Nuclear Safety Authority (D.L.), Helsinki, Finland.
Correspondence to Petri T. Kovanen, MD, PhD, Wihuri Research Institute, Kalliolinnantie 4, 00140 Helsinki, Finland. E-mail petri.kovanen{at}wri.fi
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Abstract—In human coronary atheromas, the numbers of degranulated mast cells and of apoptotic smooth muscle cells (SMCs) are increased. Accordingly, the possibility exists that mast cells participate in the regulation of SMC apoptosis in the lesions. Mast cells isolated from the serosal cavities of rats were stimulated to release their secretory granules. The neutral protease chymase, present in the exocytosed granules, was found to induce apoptosis when added to rat aortic SMCs in culture. The chymase-induced apoptosis of SMCs was detected by flow cytometry, microscopic analysis of cellular morphology, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL), and electrophoretic demonstration of DNA laddering. Chymase induced SMC apoptosis in a dose- and time- dependent manner, and its proteolytic activity was essential for the proapoptotic effect. In addition to rat chymase, recombinant human chymase was also found to induce apoptosis of human coronary artery SMCs in culture. These results suggest that mast cells may participate in the apoptotic regulation of SMCs in atherosclerotic lesions.
Key Words: apoptosis • smooth muscle • atherosclerosis • mast cells • chymase
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Introduction |
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Smooth muscle cells (SMCs) are the main cellular component in atherosclerotic lesions, and their number and activity largely determine the thickness of the intima.1 During the final stages of atherosclerosis, the clinical relevance of the thickness of the intima is apparent: a thick intima (fibrous cap) overlying a lipid-rich core of an atheroma protects it from rupturing, whereas a thin cap predisposes it to rupture.2 The concept has been put forward that the fibrous cap of an atheroma is a dynamic structure in which connective tissue matrix is produced and maintained by SMCs.3 Accordingly, reduced production of the extracellular matrix due to decreased SMC number, either by necrotic or apoptotic cell death, would weaken the cap and potentiate the risk of cap rupture.
Recent findings have demonstrated that SMCs of human
coronary and carotid atheromas undergo
apoptosis.4 5 6 7
Interestingly, it has been shown that SMCs isolated from human
coronary atherosclerotic plaques are more prone to
apoptosis than are SMCs from normal
vessels.8 The SMCs undergoing
apoptosis are localized mainly in the fibrous caps of the
atheromas and are accompanied by the presence of
inflammatory
cells.6 9 In the
inflammatory infiltrates, mast cells are also
present.10 11 12
Our previous investigations have used isolated rat serosal mast cells,
which, like human mast cells, release on stimulation an array of
vasoactive mediators, proteoglycans, and neutral
proteases.13 14 The
rat mast cells have been shown to be a useful model for studying
various molecular aspects relevant to human
atherogenesis.15 Chymase is
contained in the cytoplasmic granules of rat serosal mast cells and, on
their degranulation, is secreted within expelled
granules.14 In the
extracellular fluid, the soluble components of the exocytosed granules
(ie, histamine, chondroitin sulfate proteoglycans, and a fraction of
heparin sulfate proteoglycans) are solubilized and released from the
granules. In contrast, the major fraction of heparin proteoglycans and
the 2 neutral proteases, chymase and carboxypeptidase A (CPA), remain
tightly bound to each other in the form of insoluble granule
remnants.15 In addition, on
appropriate activation, cytokines, such as tumor necrosis
factor-
and transforming growth factor-ß, and newly formed lipid
mediators, prostaglandin D2 and
leukotriene C4, are released from
the mast
cells.13 16
We have recently shown that mast cell–derived heparin proteoglycans are capable of inhibiting the proliferation of cultured SMCs.17 In that study, we also found that mast cell–derived granule remnants, consisting of heparin proteoglycans and 2 neutral proteases, chymase and CPA, could induce SMC apoptosis.17 However, the component in the granule remnants responsible for the apoptotic effect was not determined, and the mechanism for it was not studied. In the present study, we investigated the effect of mast cell stimulation and degranulation on SMC apoptosis and found that mast cell–derived chymase was responsible for vascular SMC apoptosis in vitro.
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Methods |
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Materials and Animals
Human coronary artery SMCs and SmGM-3 medium were from Clonetics. Compound 48/80 and trypsin inhibitor type I-S from soybean were from Sigma Chemical Co. Caspase inhibitor I (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) were from Calbiochem. The ApoTag In Situ Apoptosis Detection Kit was from Oncor Inc. Sephacryl S-200 HR columns and HiTrap Heparin-Sepharose columns were from Pharmacia. Male Wistar rats (300 to 500 g) were from the Laboratory Animal Center of the University of Helsinki and were treated in accordance with institutional guidelines. Recombinant human chymase was kindly provided by Dr Takashi Kamimura, Teijin Institute for Biomedical Research, Teijin Limited, Tokyo, Japan.
Culture of Rat Aortic SMCs and Human
Coronary Artery SMCs
Aortic SMCs were prepared from male Wistar rats as
described previously.17 The
cells were seeded into 25-cm2 Falcon
polystyrene tissue culture flasks at a density of
2x104 cells/cm2
in 5 mL of RPMI 1640 culture medium supplemented with 2 mmol/L
L-glutamine, 100 U/mL
penicillin, 100 µg/mL streptomycin, and 10% FCS and were, at
confluence, subcultured (1:2) up to 9 times. SMCs of the fifth to ninth
passages were used for the experiments. Human coronary artery
SMCs were cultured in SmGM-3 medium and were, at confluence,
subcultured up to 3 times, as recommended by the
manufacturer.
Growth Arrest of SMCs
To obtain growth-arrested SMCs, sparsely seeded SMCs
(1.6x104
cells/cm2) were cultured to a subconfluent
cell density (1.3x105
cells/cm2) in 12-well tissue culture plates
in 2 mL of the above culture medium containing 10% FCS, after which
they were growth-arrested in 0.4% FCS–containing medium for 48 hours
(rat SMCs).18 Human
coronary artery SMCs were growth-arrested in growth
factor–free SmBM-3 for 24 hours.
Isolation and Stimulation of Mast Cells
Serosal mast cells were isolated from pleural and
peritoneal cavities of rats, as described
previously,19 and stimulated
with compound 48/80, a noncytotoxic mast cell–specific
stimulator.20 After
stimulation, the 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." The degree of mast cell degranulation was determined by
measuring the histamine content of the
releasate.19
Preparation of Granule Remnants and Granule
Remnant–Free Releasate
To sediment granule remnants, the mast cell releasate
was centrifuged at
13 000g for 10 minutes. After
centrifugation, the supernatant, which contains all the
soluble material of the mast cell releasate, was collected and used as
"granule remnant–free releasate." The sedimented granule remnants
were washed twice with water, resuspended in PBS, and used as
"granule remnants." The concentration of the granule remnants is
expressed in terms of their total protein content or of their chymase
activity, with the use of
N-benzoyl-tyrosine ethyl ester
(BTEE) as substrate.21 In our
9 remnant preparations, the ratio of BTEE unit to total protein (in
micrograms) was 1.47±0.64 (mean±SD).
Purification of Heparin Proteoglycans, Chymase,
and CPA
To dissociate chymase and CPA, 2 neutral proteases,
from heparin proteoglycans, isolated granule remnants were incubated in
high-salt buffer consisting of 10 mmol/L phosphate buffer
supplemented with 2 mol/L KCl, pH 7.0. The mixture was then applied to
a Sephacryl S-200 HR column and eluted with the same high-salt buffer.
Fractions containing Alcian blue–reactive material were collected,
dialyzed extensively against water, concentrated with a Centricon 10
filter (Amicon), and used in the
experiments.22 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
chromatography.23
Fractions containing chymase or CPA activity were collected separately
and further purified on a HiTrap Heparin-Sepharose column. The purity
of chymase and of CPA was determined by SDS-PAGE and as described
above.23 In our preparation,
1 µg of granule remnant total protein contained 147 ng chymase (1.47
BTEE units), 51 ng CPA, and 0.35 µg heparin proteoglycans. The
purified heparin proteoglycans, chymase, and CPA were stored at
-70°C until use.
Microscopic Analysis of
Apoptosis
Cell smears were stained by 3 methods: (1) the
standard May-Grünwald-Giemsa (MGG) method, after which
apoptotic cells were identified by the following criteria:
condensation of cytoplasm, cell surface blebbing, compaction of
chromatin, and fragmentation of the nucleus into discrete masses
scattered throughout the cell
cytoplasm24 ; (2) propidium
iodide (PI) staining with 25 µg/mL PI, after which the cells were
viewed under fluorescence microscope, and cells with fragmented
nuclei were considered as apoptotic; and (3) terminal
deoxynucleotidyl transferase–mediated dUTP nick
end-labeling (TUNEL) with an ApoTag In Situ Apoptosis Detection
Kit as recommended by the manufacturer (Oncor Inc). Briefly,
apoptotic cells were labeled with digoxigenin-conjugated dUTP
and terminal deoxyribonucleotide transferase, and the
labeled DNA fragments were stained with anti-digoxigenin monoclonal
antibody linked with fluorescein and viewed under
fluorescence microscope.
Flow Cytometric Analysis of
Apoptosis
Cellular DNA content was determined by flow
cytometric analysis of PI-labeled
cells.25 Briefly, SMCs
attached to culture dishes were detached with trypsin and combined with
the cells floating in the culture medium. The cells were then fixed
with methanol, incubated with 100 U/mL RNase, and, finally, stained
with PI (final concentration 25 µg/mL). After they were stained, the
cells were analyzed by FACScan (Becton Dickinson) by using
CellFit 2.02 software. Fluorescence was measured from 10 000
events in list mode, and gating on FL2-A versus FL2-W was used
to remove doublets. Apoptotic nuclei were identified as a
subgenomic DNA peak and were distinguished from cell debris on the
basis of forward light scatter and fluorescence of
PI.
DNA Fragmentation Analysis
DNA of SMCs (5 to 10x106)
was extracted by a standard
method,26 and equal amounts
of DNA from each sample were subjected to electrophoresis on a 1.5%
agarose gel for 3 hours at 80 V in TBE buffer (45 mmol/L
Tris-borate and 1 mmol/L EDTA, pH 8.0). The gel was stained with
ethidium bromide and photographed under UV light.
Other Assays
Protein was determined by the standard Lowry
procedure.27 The
glycosaminoglycan content of heparin proteoglycans
was determined by assaying Alcian blue–reactive material with
commercial heparin as
standard.28
Statistical Analysis
Data, shown as mean±SEM, were analyzed by
the Student t test for
determination of the significance of differences, which were considered
to be statistically significant at a value of
P<0.05.
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Results |
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To investigate whether activated rat serosal mast cells induce the apoptosis of rat aortic SMCs, growth-arrested SMCs were exposed to increasing concentrations of mast cell releasate for 24 hours, and their DNA content was measured by flow cytometry. Cells having DNA content lower than that of G0/G1 cells (hypodiploidy) were considered apoptotic (Figure 1A
, right, asterisk). As the concentrations of mast
cell releasate increased, the percentage of apoptotic SMCs
increased in a dose-dependent manner, reaching 11.4±1.7% with
releasate derived from 3.6x105 mast cells
(Figure 1A, left). The presence of apoptotic SMCs was
further confirmed by microscopic analysis of MGG-stained cell
smears (data not shown). To identify the factors responsible for
inducing apoptosis of SMCs, we further separated the mast cell
releasate into granule remnants and granule remnant–free releasate. As
shown in
Figure 1B, the granule remnants induced apoptosis of
SMCs in a manner similar to that of the total mast cell releasate,
whereas the granule remnant–free releasate had no significant effect
(Figure 1C).
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Next, we purified the 3 main components of the granule
remnants (ie, heparin proteoglycans, chymase, and CPA) and examined
their effects on growth-arrested SMCs. As shown in
Figure 2, chymase induced the apoptosis of SMCs to a
degree similar to that of its parent granule remnants (both
P<0.001 versus control). In
sharp contrast, CPA and heparin proteoglycans were without effect on
SMC apoptosis.
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The ability of chymase to induce apoptosis of SMCs
was further confirmed by 4 independent methods. As shown in
Figure 3A, control SMCs showed intact nuclei and were TUNEL
negative, whereas chymase-treated SMCs showed nuclear fragmentation, as
detected by MGG staining, PI staining, and the appearance of
TUNEL-positive staining. In addition, agarose gel electrophoresis of
the DNA extracted from SMCs treated with chymase revealed a
"ladder" of DNA fragments that consisted of integral multiples of
180 to 200 bp, typical of apoptosis
(Figure 3B, right lane).
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The time and dose dependence of chymase-induced SMC
apoptosis was examined by flow cytometry. As the concentration
of chymase increased, the percentage of apoptotic SMCs
increased in a dose-dependent manner up to a chymase level of 20 BTEE
units/mL, corresponding to the chymase activity present in
1.3x105 mast cells
(Figure 4A). Exposure of SMCs to 20 BTEE units/mL of chymase
for various periods of time demonstrated that the induction of
apoptosis by chymase was evident as early as 2 hours after
exposure and that the number of apoptotic cells increased in a
time-dependent manner
(Figure 4B). We also counted cell numbers in several
experiments and found a clear concentration-dependent decrease in the
chymase-treated samples (data not shown).
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To study whether the proteolytic activity of chymase is
responsible for the induction of SMC apoptosis, we incubated
the cells in the presence of soybean trypsin inhibitor
(TRINH), a commercially available serine protease
inhibitor. Indeed, purified chymase (20 BTEE units/mL) was
fully blocked (0 BTEE units/mL) by 100 µg/mL TRINH, and the
proportion of apoptotic cells (18.4±1.4% in chymase-treated
cells) was reduced to the basal level (4.2±1.3% versus 3.2±0.5% in
control cells). The TRINH alone was without any effect on SMC
apoptosis (3.8±1.0% versus 3.2±0.5% in control cells).
Furthermore, 10% FCS, which contains several natural protease
inhibitors, such as
1-antitrypsin,
2-macroglobulin, and
1-antichymotrypsin,29
also completely inhibited the proteolytic activity of purified chymase
and, thus, the chymase-induced SMC apoptosis (data not shown).
Interestingly, in contrast to purified chymase, chymase in its natural
form, ie, bound to the heparin proteoglycans of mast cell granule
remnants, retained 
20% of its activity in the presence of 10% FCS
and induced a low but significant degree of SMC apoptosis
(0.98±0.12% versus 0.45±0.09%). These results indicate that the
proteolytic activity was essential for the chymase-mediated
apoptosis.
Next, we investigated the significance of the findings with
the rat system by using recombinant human chymase (rH-chymase) and SMCs
derived from human coronary arteries. As shown in
Figure 5, exposure of growth-arrested coronary
artery SMCs to 20 BTEE units/mL rH-chymase for 48 hours induced
apoptosis of the human cells (16.7±3.7% in rH-chymase-treated
cells versus 0.9±0.1% in control cells), as measured by flow
cytometry and confirmed by TUNEL staining.
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Finally, to obtain information about the intracellular
mechanisms of chymase-induced apoptosis, SMCs were treated with
chymase in the presence of caspase inhibitor I (Z-VAD-FMK,
100 µ mol/L), a broad spectrum inhibitor that has been
shown to efficiently inhibit effector caspases, such as
caspase-330 31 and
caspase-7.32 Addition of this
inhibitor to the SMCs strongly decreased the
chymase-dependent increase of the number of apoptotic cells
(Figure 6). We next tested whether inhibiting caspase-8 or
-9, 2 well-known initiator caspases, would have an effect on the
chymase-induced apoptosis. Caspase-8 inhibitor
reduced the apoptotic rate by 60%, whereas caspase-9
inhibitor was without effect
(Figure 6). These results suggest that the chymase-mediated
SMC apoptosis proceeds through the classical pathway of
caspases and, more specifically, through the mitochondria-independent
pathways.
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Discussion |
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The present results show that chymase, a neutral serine protease secreted by activated rat serosal mast cells, is capable of inducing apoptosis of rat aortic SMCs. In addition, we found that rH-chymase was capable of inducing significant apoptosis in cultured human coronary artery SMCs, indicating that the observation is not restricted to rodents but is also potentially relevant in humans.
The chymase-mediated apoptotic effect was completely
dependent on its proteolytic activity, inasmuch as the inhibition of
purified chymase with TRINH abolished the induction of SMC
apoptosis. Also, the serum, which contained several natural
protease inhibitors, notably, members of the serpin
superfamily,29 completely
blocked the proteolytic activity of purified chymase and, thus,
prevented it from inducing apoptosis. However, chymase in its
natural form, ie, bound to the heparin proteoglycans of granule
remnants, induced the apoptosis of SMCs even in the presence of
serum (data not shown). Indeed, it has been shown that heparin
proteoglycans can protect chymase from inactivation by its natural
inhibitors, such as
1-antitrypsin,
2-macroglobulin, and
1-antichymotrypsin.33 34
Additionally, it has recently been shown that chymase and
interstitial collagenase, known to be
present in atherosclerotic
lesions,35 36 37
can avidly hydrolyze and inactivate their natural
inhibitors,38
suggesting that the amount of functional inhibitors are
decreased in the atherosclerotic arterial
intima.39
Protease-induced apoptosis is not a specific
phenomena for chymase and SMCs, inasmuch as elastase and proteinase
3 (neutrophil serine proteases) can induce the apoptosis of
endothelial
cells,40 -chymotrypsin and
trypsin can induce the apoptosis of
neutrophils,41 and chymase
can induce the apoptosis of
cardiomyocytes.42
However, the observation that granule-bound natural chymase can induce
apoptosis, even in the presence of natural
inhibitors, might support a role for mast cell–derived
granule-bound chymase compared with other neutral proteases at the site
of atherogenesis in inducing apoptosis in vivo. Interestingly,
peripheral blood monocytes, lymphocytes, several human
leukemic cell lines (THP-1, HL-60, and K562), murine L929 fibroblasts,
mouse peritoneal
macrophages,41 and
myocardial fibroblasts42
failed to undergo apoptosis after treatment with proteases,
suggesting that cells also differ in their susceptibility to undergo
protease-induced apoptosis.
The mechanism of protease-induced apoptosis is still unknown, albeit several mechanisms have been proposed. Among them are (1) degradation of extracellular matrix components, (2) activation of a receptor, (3) stimulation of the uptake of proapoptotic factors by target cells, and (4) degradation of factors that inhibit apoptosis.43 44 However, because the chymase-mediated apoptotic process is rapid, being significant after incubating SMCs with chymase for 2 hours, the chymase-induced apoptosis differs from the classical process of "anoikis," which is a slowly occurring (24- to 36-hour) apoptotic process induced by denied adhesion to the extracellular matrix.45 Furthermore, the chymase-mediated apoptotic process is also clearly different from the process of cell detachment induced by trypsin treatment, which, like anoikis, requires 24 to 36 hours in suspension before the apoptotic process starts.46 Chymase-mediated SMC apoptosis is also different from granzyme-mediated apoptosis, because in spite of similarities in cleavage specificity, chymase is not known to be taken up by SMCs, and mast cells have not been shown to express perforin, a T-lymphocyte–derived cytolytic molecule necessary for granzyme-mediated apoptosis.44 These results suggest that different proteases might induce apoptosis through various mechanisms depending on their substrate specificities and on the presence or absence of proapoptotic cofactors, such as perforin.
SMCs are the major cell population in the cap of the
atherosclerotic plaques and the main cells that secrete extracellular
matrix to stabilize the
plaque.3 Accordingly,
apoptotic death of SMCs can be speculated to have a role in
destabilization of the atherosclerotic plaques by decreasing the
cellularity and the extracellular collagen content of the fibrous cap.
Interestingly, SMCs derived from atherosclerotic plaques have an
increased expression of BAX, a proapoptotic oncoprotein of the
BCL-2 family,6 and are more
liable to die by apoptosis than are their counterparts obtained
from the arterial
media.8 Although the actual
triggers of apoptosis of SMCs in atherosclerotic plaques are
presently unknown, a multitude of possible proapoptotic
stimulants have been identified in
vitro.47 Most of them are
products of inflammatory cells (such as
macrophages48 and T
lymphocytes49 ) that
colocalize with the apoptotic SMCs in atherosclerotic lesions.
Mast cells, like macrophages and T lymphocytes, are also an
integral component of the inflammatory
infiltrate11 and may
participate in the regulation of SMC apoptosis in
atherosclerotic lesions. Indeed, it is conceivable that
macrophages, T lymphocytes, and mast cells may act in concert
to induce apoptosis of SMCs in the inflamed areas of
atherosclerotic lesions. We have recently determined the mast
cell–to–SMC ratio to be 1:5 at the immediate site of erosion and
rupture,11 and we have
estimated the concentration of chymase to be 0.6 µg/mL in the intimal
fluid of such vulnerable
areas.33 This concentration
of chymase is equivalent to 6 BTEE units/mL. Thus, the cell and
chymase concentrations used in this in vitro study reflect the
presumptive in vivo situation. Taken together, the present findings
suggest an additional mechanism by which apoptosis of SMCs
might be triggered in atherosclerotic lesions, namely, the action of
the chymase secreted by mast
cells.
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Acknowledgments |
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We are grateful to Monica Schoultz (Transplantation Laboratory, University of Helsinki) for skillfully performing the flow cytometric analysis.
Received October 22, 2000; accepted December 20, 2000.
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