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Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:516-522

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:516.)
© 2001 American Heart Association, Inc.

Vascular Biology

Mast Cell Chymase Induces Apoptosis of Vascular Smooth Muscle Cells

Markus Leskinen; Yenfeng Wang; Dariusz Leszczynski; Ken A. Lindstedt; Petri T. Kovanen

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


*    Abstract
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 *Abstract
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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


*    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-{alpha} 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.


*    Methods
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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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
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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 1ADown, 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 1ADown, 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 1BDown, 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 1CDown).



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  		Figure 1. Mast cell releasate and granule remnants induce apoptosis of SMCs. Growth-arrested SMCs were incubated at 37°C for 24 hours in serum-free medium containing increasing concentrations of mast cell releasate (A), granule remnants (B), or granule remnant–free releasate (C). After incubation, floating and attached cells were collected, and their DNA content was analyzed with flow cytometry as described in Methods and expressed as percent apoptotic cells (graphs on left). Graphs on the right show flow cytometric images of SMCs treated with material released from the highest number (3.6x105) of mast cells. Note the appearance of apoptotic cells (the subgenomic DNA peak, sub-G1, labeled with an asterisk), with smaller amounts of DNA than cells in the G0/G1 phase in SMC cultures exposed to mast cell releasate (panel A, right) and granule remnants (panel B, right). Data shown are mean±SEM of triplicate incubations. Similar results were obtained in 2 other independent experiments.

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 2Down, 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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  		Figure 2. Effect of different components of granule remnants on SMC apoptosis. Growth-arrested SMCs were incubated at 37°C for 24 hours with granule remnants (13.6 µg/mL, 20 BTEE units/mL) or with components derived from 13.6 µg granule remnants, ie, 2 µg/mL of chymase (20 BTEE units/mL), 0.7 µg/mL CPA, or 4.8 µg/mL heparin proteoglycans. Apoptotic SMCs were quantified by flow cytometry. Data shown are mean±SEM of triplicate incubations. A similar result was obtained in another independent experiment. *P<0.001 vs control cells with no additions.

The ability of chymase to induce apoptosis of SMCs was further confirmed by 4 independent methods. As shown in Figure 3ADown, 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 3BDown, right lane).



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  		Figure 3. Mast cell chymase induces apoptosis of SMCs. A, Microscopic analysis: photographs of control (left) and chymase-treated (20 BTEE units/mL, 24 hours; right) rat aortic SMCs. Apoptotic cells (arrowheads) show chromatin condensation and nuclear fragmentation. Original magnification x200. B, DNA fragmentation analysis. Note the 200-bp ladder formation (arrows) in DNA extracted from SMCs exposed to chymase (20 BTEE units/mL, 24 hours).

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 4ADown). 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 4BDown). 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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  		Figure 4. Concentration and time dependence of SMC apoptosis induced by mast cell chymase. A, Growth-arrested SMCs were exposed for 24 hours to increasing concentrations of mast cell chymase, as described in the legend to Figure 1Up. After incubation, the percentage of apoptotic cells was measured by flow cytometry. Data shown are mean±SEM of triplicate incubations. Similar results were obtained in 3 other independent experiments. B, Growth-arrested SMCs were incubated in serum-free medium in the absence or presence of 20 BTEE units/mL of mast cell chymase for the indicated times. Data shown are mean±SEM of triplicate incubations. Similar results were obtained in another independent experiment.

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 {alpha}1-antitrypsin, {alpha}2-macroglobulin, and {alpha}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 {approx}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 5Down, 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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  		Figure 5. Recombinant human chymase induces apoptosis of human coronary artery SMCs. Growth-arrested human coronary artery SMCs were exposed for 48 hours to 20 BTEE units/mL rH-chymase. Apoptotic coronary artery SMCs were detected by flow cytometry and TUNEL staining. Data shown are mean±SEM of triplicate incubations. Similar results were obtained in 2 other independent experiments. *P<0.05 vs control cells with no additions.

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 6Down). 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 {approx}60%, whereas caspase-9 inhibitor was without effect (Figure 6Down). 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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  		Figure 6. Caspase inhibitors reduce chymase-induced apoptotic cell death. Growth-arrested rat SMCs were exposed for 24 hours to rat chymase in the absence or presence of caspase inhibitor I, caspase-8 inhibitor, or caspase-9 inhibitor (100 µmol/L each). After incubation, the percentage of apoptotic cells was measured by flow cytometry. Data shown are mean±SEM of triplicate incubations. A similar result was obtained in another independent experiment. *P<0.05 vs chymase-treated cells.


*    Discussion
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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 {alpha}1-antitrypsin, {alpha}2-macroglobulin, and {alpha}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 {alpha}-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 {approx}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.


*    Acknowledgments
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
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 *References
 

  1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
  2. Falk E. Why do plaques rupture? Circulation. 1992;86(suppl III):III-30–III-42.
  3. Lee RT, Libby P. The unstable atheroma. Arterioscler Thromb Vasc Biol. 1997;17:1859–1867.[Free Full Text]
  4. Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. 1995;91:2703–2711.[Abstract/Free Full Text]
  5. Geng Y, Libby P. Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1ß-converting enzyme. Am J Pathol. 1995;147:251–266.[Abstract]
  6. Kockx MM, De Meyer GRY, Muhring J, Jacob W, Bult H, Herman AG. Apoptosis and related proteins in different stages of human atherosclerotic plaques. Circulation. 1998;97:2307–2315.[Abstract/Free Full Text]
  7. Bauriedel G, Schluckebier S, Hutter R, Welsch U, Kandolf R, Lüderitz B, Prescott MF. Apoptosis in restenosis versus stable-angina atherosclerosis. Arterioscler Thromb Vasc Biol. 1998;18:1132–1139.[Abstract/Free Full Text]
  8. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995;95:2266–2274.
  9. Crisby M, Kallin B, Thyberg J, Zhivotovsky B, Orrenius S, Kostulas V, Nilsson J. Cell death in human atherosclerotic plaques involves both oncosis and apoptosis. Atherosclerosis. 1997;130:17–27.[Medline] [Order article via Infotrieve]
  10. Kaartinen M, Penttilä A, Kovanen PT. Accumulation of activated mast cells in the shoulder region of human coronary atheroma, the predilection site of atheromatous rupture. Circulation. 1994;90:1669–1678.[Abstract/Free Full Text]
  11. Kovanen PT, Kaartinen M, Paavonen T. Infiltrates of activated mast cells at the site of coronary atheromatous erosion or rupture in myocardial infarction. Circulation. 1995;92:1084–1088.[Abstract/Free Full Text]
  12. Jeziorska M, McCollum C, Woolley DE. Mast cell distribution, activation, and phenotype in atherosclerotic lesions of human carotid arteries. J Pathol. 1997;182:115–122.[Medline] [Order article via Infotrieve]
  13. Galli SJ. New concepts about the mast cell. N Engl J Med. 1993;328:257–265.[Free Full Text]
  14. Schwartz LB, Austen KF. Structure and function of the chemical mediators of mast cells. In: Ishizaka K, ed. Mast Cell Activation and Mediator Release. Basel, Switzerland: S Karger; 1984:271–321.
  15. Kovanen PT. Role of mast cells in atherosclerosis. In: Marone G, ed. Human Basophils and Mast Cells: Clinical Aspects. Basel, Switzerland: S Karger; 1995:132–170.
  16. Kaartinen M, Penttilä A, Kovanen PT. Mast cells in rupture-prone areas of human coronary atheromas produce and store TNF-{alpha}. Circulation. 1996;94:2787–2792.[Abstract/Free Full Text]
  17. Wang Y, Kovanen PT. Heparin proteoglycans released from rat serosal mast cells inhibit proliferation of rat aortic smooth muscle cells in culture. Circ Res. 1998;84:74–83.[Abstract/Free Full Text]
  18. Castellot JJ Jr, Pukac LA, Caleb BL, Wright TC Jr, Karnovsky MJ. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells. J Cell Biol. 1989;109:3147–3155.[Abstract/Free Full Text]
  19. Kokkonen JO, Kovanen PT. Low density lipoprotein degradation by rat mast cells: demonstration of extracellular proteolysis caused by mast cell granules. J Biol Chem. 1985;260:14756–14763.[Abstract/Free Full Text]
  20. Lindstedt KA, Kokkonen JO, Kovanen PT. Soluble heparin proteoglycans released from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor-mediated phagocytosis. J Lipid Res. 1992;33:65–75.[Abstract]
  21. Woodbury RG, Everitt MT, Neurath H. Mast cell proteases. Methods Enzymol. 1981;80:588–609.
  22. Yurt RW, Leid RW Jr, Austen KF. Native heparin from rat peritoneal mast cells. J Biol Chem. 1977;252:518–521.[Abstract/Free Full Text]
  23. Kokkonen JO, Saarinen J, Kovanen PT. Regulation of local angiotensin II formation in the human heart in the presence of interstitial fluid: inhibition of chymase by protease inhibitors of interstitial fluid and of angiotensin converting enzyme by Ang-(1–9) formed by heart carboxypeptidase A-like activity. Circulation. 1997;95:1455–1463.[Abstract/Free Full Text]
  24. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306.[Medline] [Order article via Infotrieve]
  25. Leszczynski D. Regulation of cell cycle and apoptosis by protein kinase C in rat myeloid leukemia cell line. Oncol Res. 1995;7:471–480.[Medline] [Order article via Infotrieve]
  26. Strauss WM. Preparation of genomic DNA from mammalian tissue. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. New York, NY: John Wiley & Sons; 1987:2:2.1–2.2.3.
  27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]
  28. Bartold PM, Page RC. A microdetermination method for assaying glycosaminoglycans and proteoglycans. Anal Biochem. 1985;150:320–324.[Medline] [Order article via Infotrieve]
  29. Travis J, Salvesen GS. Human plasma proteinase inhibitors. Ann Rev Biochem. 1983;52:655–709.[Medline] [Order article via Infotrieve]
  30. Kawasaki M, Kuwano K, Hagimoto N, Matsuba T, Kunitake R, Tanaka T, Maeyama T, Hara N. Protection from lethal apoptosis in lipopolysaccharide-induced acute lung injury in mice by a caspase inhibitor. Am J Pathol. 2000;157:597–603.[Abstract/Free Full Text]
  31. Slee EA, Zhu H, Chow SC, MacFarlane M, Nicholson DW, Cohen GM. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem J. 1996;315:21–24.
  32. Chandler JM, Cohen GM, MacFarlane M. Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver. J Biol Chem. 1998;273:10815–10818.[Abstract/Free Full Text]
  33. Lindstedt L, Lee M, Kovanen PT. Chymase bound to heparin is resistant to its natural inhibitors and capable of proteolyzing high density lipoproteins in aortic intimal fluid. Atherosclerosis. 2001;155:87–97.[Medline] [Order article via Infotrieve]
  34. Pejler G, Berg L. Regulation of rat mast cell protease 1 activity: protease inhibition is prevented by heparin proteoglycan. Eur J Biochem. 1995;233:192–199.[Medline] [Order article via Infotrieve]
  35. 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 chymase-containing mast cells in normal intimas, fatty streaks, and the shoulder region of atheromas. Arterioscler Thromb. 1994;14:966–972.[Abstract/Free Full Text]
  36. Nikkari ST, O’Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE, Clowes AW. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation. 1995;92:1393–1398.[Abstract/Free Full Text]
  37. Johnson JL, Jackson CL, Angelini GD, George SJ. Activation of matrix-degrading metalloproteinases by mast cell proteases in atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 1998;1707–1715.
  38. Schechter NM, Sprows JL, Schoenberger OL, Lazarus GS, Cooperman BS, Rubin H. Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors. J Biol Chem. 1989;264:21308–21315.[Abstract/Free Full Text]
  39. Desrochers PE, Jeffrey JJ, Weiss SJ. Interstitial collagenase (matrix metalloproteinase-1) expresses serpinase activity. J Clin Invest. 1991;88:2258–2265.
  40. Yang JJ, Kettritz R, Falk RJ, Jennette JC, Gaido ML. Apoptosis of endothelial cells induced by the neutrophil serine proteases proteinase 3 and elastase. Am J Pathol. 1996;149:1617–1626.[Abstract]
  41. Trevani AS, Andonegui G, Giordano M, Nociari M, Fontán P, Dran G, Geffner JR. Neutrophil apoptosis induced by proteolytic enzymes. Lab Invest. 1996;74:711–721.[Medline] [Order article via Infotrieve]
  42. Hara M, Matsumori A, Ono K, Kido H, Hwang MW, Miyamoto T, Iwasaki A, Okada M, Nakatani K, Sasayama S. Mast cells cause apoptosis of cardiomyocytes and proliferation of other intramyocardial cells in vitro. Circulation. 1999;100:1443–1449.[Abstract/Free Full Text]
  43. Altieri DC. Proteases and protease receptors in modulation of leukocyte effector functions. J Leukoc Biol. 1995;58:120–127.[Abstract]
  44. Smyth MJ, Trapani JA. Granzymes. exogenous proteinases that induce target cell apoptosis. Immunol Today. 1995;16:202–206.[Medline] [Order article via Infotrieve]
  45. Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol. 1994;124:619–626.[Abstract/Free Full Text]
  46. Brassard DL, Maxwell E, Malkowski M, Nagabhushan TL, Kumar CC, Armstrong L. Integrin alpha(v)beta(3)-mediated activation of apoptosis. Exp Cell Res. 1999;251:33–45.[Medline] [Order article via Infotrieve]
  47. Bennett MR, Boyle JJ. Apoptosis of vascular smooth muscle cells in atherosclerosis. Atherosclerosis. 1998;138:3–9.[Medline] [Order article via Infotrieve]
  48. Han DKM, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995;147:267–277.[Abstract]
  49. Björkerud S, Björkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol. 1996;149:367–380. [Abstract]



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