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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1928.)
© 2001 American Heart Association, Inc.

Vascular Biology

Mast Cell Chymase Inhibits Smooth Muscle Cell Growth and Collagen Expression In Vitro

Transforming Growth Factor-ß1-Dependent and -Independent Effects

Yenfeng Wang; Naotaka Shiota; Markus J. Leskinen; Ken A. Lindstedt; Petri T. Kovanen

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 petri.kovanen{at}wri.fi

	Abstract

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In the vulnerable areas of fibrous caps of advanced atherosclerotic lesions, chymase-containing mast cells are present. In such areas, the numbers of smooth muscle cells (SMCs) and the content of collagen are reduced. In this in vitro study, we found that the addition of chymase, isolated and purified from rat serosal mast cells, to cultured rat aortic SMCs of the synthetic phenotype (s-SMCs) inhibited their proliferation by blocking the G₀/G₁S transition in the cell cycle. Rat chymase and recombinant human chymase inhibited the expression of collagen type I and type III mRNA in s-SMCs and in human coronary arterial SMCs. The growth-inhibitory effect of chymase was partially reversed by addition to the culture medium of an antibody capable of neutralizing the activity of transforming growth factor-ß1 (TGF-ß1). Immunocytochemistry showed that the s-SMCs expressed and synthesized extracellular matrix-associated TGF-ß1. On exposure to mast cell chymase, the extracellular matrix-associated latent TGF-ß1 was released and activated, as demonstrated by immunoblotting and by an ELISA with TGF-ß1 type II receptor for capture. When added to s-SMCs, such chymase-released TGF-ß1 was capable of inhibiting their growth. In contrast, the inhibitory effect of chymase on collagen synthesis by s-SMCs did not depend on TGF-ß1. Taken together, the findings support the hypothesis that chymase released from activated mast cells in atherosclerotic plaques contributes to cap remodeling.

Key Words: atherosclerosis • smooth muscle cells • transforming growth factor-ß • collagen • chymase

	Introduction

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In atherosclerotic lesions, locally synthesized growth factors, such as transforming growth factor-ß (TGF-ß), regulate the differentiation, migration, and proliferation of smooth muscle cells (SMCs) as well as the synthesis and secretion of extracellular matrix (ECM) by the SMCs.^1–4 In normal coronary arteries, TGF-ß1 is the dominant isoform of the TGF-ß family, whereas different isoforms of TGF-ß, notably TGF-ß1 and TGF-ß3, are found to be highly expressed in fatty streaks/fibrofatty lesions.³ In fibrous plaques, TGF-ß1 is again the dominant isoform, whereas TGF-ß3 immunoreactivity varies from low to undetectable levels.³ Thus, in the fibrous cap of an atherosclerotic lesion, TGF-ß1 appears to be the major active member of the TGF-ß family, whereas the potential roles of TGF-ß2 and TGF-ß3 in the atherosclerotic process remain to be determined.

TGF-ß1 is known to be secreted by many cell types in the latent form as a complex known as small latent TGF-ß1, in which TGF-ß1 is noncovalently associated with its propeptide homodimer, the latency-associated peptide. An additional high molecular weight protein, termed the latent TGF-ß1-binding protein (LTBP), is bound to the small latent TGF-ß1 complex and mediates binding and retention to the ECM of the large latent TGF-ß1 complex thus formed.⁵ To become biologically active, TGF-ß1 must be released from the ECM through proteolytic processing of LTBP and then further activated by selective removal of the latency-associated peptide. The active TGF-ß1 that is generated is a homodimer (25 kDa) capable of binding to specific high-affinity TGF-ß receptors and of triggering local autocrine and/or paracrine cellular responses.⁵

Among several possible mechanisms of TGF-ß activation in atherosclerotic lesions, the roles of proteases (notably plasmin) produced and secreted by the cell types also present in the lesions, eg, SMCs, macrophages, and T lymphocytes, have received attention.^6–8 Mast cells are also present in atherosclerotic lesions⁹ and are known to contain exceptionally large amounts of neutral proteases, such as chymase. Therefore, mast cells may also be involved in the extracellular metabolism of TGF-ß. In fact, chymase isolated from human skin was earlier shown to release latent TGF-ß from the ECM of cultured epithelial and endothelial cells in vitro.¹⁰ Moreover, we have recently shown that rat and human chymase are capable of activating latent TGF-ß1.¹¹ In the present report, we show that mast cell-derived chymase inhibits growth of SMCs of the synthetic phenotype (s-SMCs) by activating s-SMC-derived ECM-bound latent TGF-ß1. Furthermore, we show that chymase suppresses the mRNA expression of collagen type I and type III in s-SMCs through a TGF-ß1-independent mechanism.

	Methods

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Materials and Animals
Sodium [³H]thymidine (53 mCi/mg) and enhanced chemiluminescence reagent were from Amersham International; RPMI 1640 culture medium, FCS, TRIzol reagent, and the Superscript preamplification system were from GIBCO-BRL; recombinant human TGF-ß1 (rhTGF-ß1), anti-TGF-ß neutralizing antibody, rhTGF-ß1-soluble receptor II, and biotinylated anti-human TGF-ß1 antibody were from R&D Systems; TGF-ß1 polyclonal antibody was from Santa Cruz; biotinylated goat anti-rabbit immunoglobulins and horseradish peroxidase-conjugated streptavidin were from DAKO; and male Wistar rats (300 to 500 g) were purchased from the Laboratory Animal Center of the University of Helsinki, Helsinki, Finland.

Culture and Growth Arrest of Rat Aortic SMCs
Aortic SMCs were prepared from male Wistar rats and cultured as described previously.¹² To obtain s-SMCs, SMCs beyond the fifth passage were used for the experiments.¹² To obtain growth-arrested SMCs, sparsely plated cell cultures (1 to 5x10⁴ cells per milliliter) were incubated in medium containing 0.4% FCS for 72 hours. Flow cytometric analysis showed that >80% of the cells had been arrested in the G₀/G₁ phase.

Isolation and Purification of Mast Cell Chymase
Serosal mast cells were isolated from the pleural and peritoneal cavities of rats and stimulated to exocytose their chymase-containing cytoplasmic granules, as described previously.¹³ Rat chymase 1, which is referred to in the text as "chymase," was purified from the isolated granule remnants to apparent homogeneity, as described previously.^11,14 The specific activity of the purified chymase was 10 N-benzoyl-L-tyrosine ethyl ester units/µg when measured spectrophotometrically.¹⁴

Preparation of Preconditioned Medium and ECM From s-SMC Monolayers
Subconfluent rat aortic SMCs were washed extensively in PBS and incubated in serum-free RPMI 1640 culture medium for 24 hours to remove traces of serum proteins. The SMCs were then incubated at 37°C for 16 hours in the presence of chymase, and the thus-formed "preconditioned medium" was collected by centrifugation at 800g for 5 minutes. To inhibit chymase activity, the preconditioned medium was supplemented with either a mixture of protease inhibitors (ELISA, immunoblotting, and reverse transcription [RT]-polymerase chain reaction [PCR]: 1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 2 µg/mL leupeptin, and 2 µg/mL pepstatin A; all final concentrations) or with FCS (cell culture experiments: 10% final concentration). The quantity of TGF-ß1 was determined directly by ELISA, as recommended by the manufacturer (R&D Systems), or concentrated with a Centricon 10 filter (10-kDa cutoff, Amicon) for immunoblotting of active TGF-ß1, as described by Taipale et al.¹⁵ To prepare ECM, s-SMC monolayers were cultured as described above. The cells were removed with 0.5% sodium deoxycholate in the presence of the mixture of protease inhibitors, and the residual ECM was obtained according to Hedman et al.¹⁶

Polyacrylamide Gel Electrophoresis and Immunoblotting of TGF-ß1
Gradient (4% to 20%) sodium deoxycholate-polyacrylamide gel electrophoresis for detection of active TGF-ß1 was carried out as described.¹⁷ Active TGF-ß1 was detected with a polyclonal antibody against TGF-ß1, with the use of biotin-streptavidin amplification and enhanced chemiluminescence detection.¹⁵

Immunocytochemical Staining of TGF-ß1 in s-SMC Monolayers and ECM
s-SMC monolayers and ECM were prepared as described above, except that the cells were cultured on Thermanox coverslips. The samples were fixed with 4% paraformaldehyde; endogenous peroxidase activity was inhibited with 0.8% hydrogen peroxide; and nonspecific binding was blocked with PBS containing 1% skimmed milk, 3% goat serum, and 1% saponin. TGF-ß1 in the s-SMC monolayer and in the ECM was then detected with a polyclonal antibody against TGF-ß1 with the use of biotin-streptavidin amplification and peroxidase staining, as recommended by the manufacturer (Santa Cruz).

Competitive RT-PCR Analysis
Total RNA was extracted from s-SMC monolayers by use of an ultrapure TRIzol reagent; 1 µg of RNA was then reverse-transcribed into cDNA by using a Superscript preamplification system and was further amplified by PCR with specific oligonucleotides as follows: TGF-ß1 5-CAGACATTCGGGAAGCAGTG (sense), 5'-GTTCA-TGTCATGGATGGTGC (antisense); plasminogen activator inhibitor (PAI)-1 5'-ACCCTCAGCATGTTCATTGC (sense), 5'-CTCGTTCACCTCGATCTTGAC (antisense); TGF-ß receptor type I (TßRI) 5'-TTGCCCATCTTCACATGGAG (sense), 5'-CATTGCATAGATGTCAGCACG (antisense); TGF-ß receptor type II (TßRII) 5'-CTGTCTGTGGATGACCTGGC (sense), 5'-CTGGTGGTTGAGCCAGAAGC (antisense); GAPDH 5'-ACCAC-AGTCCATGCCATCAC (sense), 5'-TCCACCACCCTGTTGCTGTA (antisense); collagen type I 5'-GACCGATGGATTCCAGTTCG (sense), 5'-TGTGACTCGTGCAGCCATCG (antisense); and collagen type III 5'-AGATGTCTTTGATGTGCAGC (sense), 5'-CCACCAATGTCATAGGGTGC (antisense). The PCR fragments were verified to represent the corresponding targets by specific restriction enzyme treatment and quantified with a Gel Doc 2000 gel documentation system (Bio-Rad). For competitive RT-PCR, competitor DNAs were prepared by insertion of a 125-bp external DNA fragment at the PstI site for TGF-ß1, a 129-bp fragment at the StuI site for collagen type I, and a 133-bp fragment at the BamHI site for collagen type III. The PCR products for the target and its competitor were 350 and 475 bp for TGF-ß1, 266 and 395 bp for collagen I, and 312 and 445 bp for collagen type III, respectively.

Determination of DNA Synthesis
Growth-arrested s-SMCs were incubated with or without mast cell chymase in medium containing 0.4% FCS. After incubation for 16 hours, the cells were released from the G₀ block by addition of FCS (final concentration 10% [vol/vol]), and incubation was continued for the times indicated in figures. The rate of DNA synthesis was then determined by measuring the incorporation of [³H]thymidine (1 µCi/mL) into the trichloroacetic acid-precipitable material of the s-SMCs.¹² Cell viability was monitored by viewing the chymase-treated s-SMCs under a phase-contrast microscope with and without trypan blue.

Flow Cytometric Analysis of the Cell Cycle and Apoptosis of s-SMCs
The cell cycle distribution and the percentage of apoptotic s-SMCs were determined by flow cytometric analysis (FACScan, Becton Dickinson) of propidium iodide-labeled cells.¹⁸ 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 propidium iodide.¹⁹

Statistical Analysis
Data, shown as mean±SD, were analyzed with 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

Top Abstract Introduction Methods Results Discussion References

 
Mast Cell Chymase Inhibits s-SMC Proliferation by Blocking the G₀/G₁S Transition
As shown in Figure 1A, incorporation of [³H]thymidine by growth-arrested s-SMCs was strongly inhibited in the presence of increasing amounts of chymase activity. In addition, counting the cells at the end of the incubation revealed a dose-dependent decrease in the cell number (Figure 1B). When growth-arrested s-SMCs were preincubated with mast cell chymase (20 BTEE units/mL) for 16 hours, we observed a small reduction in cell number (see Figure 1D, 0 time point), which was due to chymase-mediated apoptosis and disruption of cell-matrix interactions with ensuing loss of adhering s-SMCs. Interestingly, when FCS was added to the incubation medium to inhibit chymase activity and to stimulate s-SMC proliferation, the rate of incorporation of [³H]thymidine by the s-SMCs was found to be significantly reduced (Figure 1C). The chymase-mediated growth-inhibitory effect lasted for up to 72 hours, after which there was a rapid increase in [³H]thymidine incorporation to a level comparable to that of untreated cells. A similar effect was observed on analyzing the number of s-SMCs (Figure 1D). In a parallel experiment, we found only low percentages of apoptotic cells (analyzed by flow cytometry) during the exponential growth phase: 2% in the chymase-treated s-SMC monolayer and 0.4% in the untreated cells. Moreover, only a few trypan blue-positive cells were observed in either the chymase-treated or the untreated s-SMC monolayers. By using flow cytometric analysis, we found that the proportion of s-SMCs in the G₀/G₁ phase was significantly higher in the presence of chymase (54.6±2.5% versus 47.3±0.9% for no treatment). In addition, the proportion of cells in the S phase was significantly lower in the presence of chymase (32.1±3.0% versus 38.0±0.7% for no treatment), suggesting inhibition or delay of the G₀/G₁S transition in the chymase-treated s-SMCs.

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of mast cell chymase on s-SMC growth. Growth-arrested s-SMCs were incubated with mast cell chymase at 37°C for 16 hours. After incubation, FCS was added to achieve a final concentration of 10% (vol/vol), and incubation was continued. A and B, Incubation with different amounts of chymase, followed by 48-hour incubation in medium containing 10% FCS. C and D, Incubation with 20 BTEE units/mL chymase. Incubation times are indicated. [3H]Thymidine incorporation into the DNA of s-SMCs and cell numbers were determined as described in Methods. Cell numbers were counted before and after incubation with mast cell chymase. Values are mean±SD of triplicate incubations. The results shown represent 3 independent experiments.

Anti-TGF-ß Neutralizing Antibody Partially Blocks the Inhibitory Effect of Preconditioned Medium
Because s-SMCs are known to produce and secrete TGF-ß1 avidly and to respond to it by changing their rates of growth,³ we tested whether TGF-ß1 was involved in the observed chymase-mediated inhibition of growth. As shown in Figure 2, in the absence of anti-TGF-ß neutralizing antibodies, the preconditioned medium induced a 50% decrease in the rate of [³H]thymidine incorporation (from 4.9x10⁵ to 2.4x10⁵ dpm per well, panel A) and in the cell number (from 15x10⁴ to 7x10⁴ cells per well, panel B). In the presence of increasing concentrations of anti-TGF-ß neutralizing antibody, the rate of [³H]thymidine incorporation and the cell number correspondingly increased, suggesting that TGF-ß1 was responsible for the chymase-mediated inhibition of s-SMC growth. Addition of increasing concentrations (0, 25, 50, and 100 µg/mL) of anti-TGF-ß neutralizing antibody to control s-SMCs, ie, without preconditioned medium, did not increase their growth rate (data not shown), indicating that the observed effect was due to the presence of active TGF-ß1 in the preconditioned medium. Furthermore, as shown in Figure 2C and 2D, similar results were obtained with equivalent amounts of rhTGF-ß1. Interestingly, inhibition of chymase activity in the preconditioned medium completely abolished the observed effects on DNA synthesis and cell growth in the treated s-SMCs (Figure 2C and 2D).

View larger version (33K): [in this window] [in a new window]   Figure 2. Blocking of the inhibitory effect of preconditioned medium on s-SMC growth by anti-TGF-ß neutralizing antibody. The preconditioned medium was incubated with the indicated concentrations of anti-TGF-ß neutralizing antibody at 37°C for 1 hour before it was added to growth-arrested s-SMCs. After incubation at 37°C for 48 hours, cellular DNA synthesis (A) and cell numbers (B) were determined as described in Methods. The effect of preconditioned medium on cellular DNA synthesis (C) and cell numbers (D) was further compared with equivalent amounts of rhTGF-ß (2 ng/mL) in the presence and absence of anti-TGF-ß neutralizing antibody (anti-TGF-ß Ab, 100 µg/mL). Cultures with medium only (no chymase added) were used as controls. Values are means of duplicate incubations, and similar results were obtained in another independent experiment.

Mast Cell Chymase Releases Active TGF-ß1 From ECM of s-SMCs
To verify that rat aortic s-SMCs produce TGF-ß1, we analyzed s-SMC monolayers by immunocytochemistry. Figure 3A (left panel) shows, at high magnification, 1 s-SMC (arrow) stained positively (red) with a polyclonal antibody that recognizes active and latent forms of TGF-ß1. To separate between intracellular and extracellular TGF-ß1, the cells were gently removed with 0.5% sodium deoxycholate. As shown in the right panel of Figure 3A, the s-SMC-derived ECM was also stained positively for TGF-ß1 (arrow, red). As shown in Figure 3B, the amount of active TGF-ß1 (25 kDa) in the culture medium gradually increased in the presence of increasing concentrations of chymase. By performing an ELISA assay with TGF-ß1 type II serine/threonine kinase receptor (TßRII) as capture, we found that chymase significantly increased the amounts of biologically active TGF-ß1 released from the s-SMC monolayer (653±77 versus 282±85 pg/10⁶ cells) and the s-SMC-derived ECM (140±28 versus 0 pg/10⁶ cells). Furthermore, as shown in Figure 4A, the expression of PAI-1, a good indicator of active TGF-ß1, was significantly induced (4.6-fold) in the presence of chymase. No effect was observed in the expression of TßRI or TßRII. In contrast, as shown in Figure 4B, in the chymase-treated s-SMCs, the ratio (target/competitor) of the specific TGF-ß1 signal was decreased (2.4-fold).

View larger version (71K): [in this window] [in a new window]   Figure 3. Effect of mast cell chymase on TGF-ß1 expression and activation. A, Immunostaining of s-SMCs and ECM. Monolayers (A, left) and ECM (A, right) of rat aortic s-SMCs were stained with the use of polyclonal antibodies against TGF-ß1. The arrows point toward TGF-ß1-positive areas (red). Original magnification x200. B, Immunoblotting of active TGF-ß1 in preconditioned medium. Active TGF-ß1 in preconditioned medium was detected by immunoblotting with a polyclonal antibody against TGF-ß1. The amounts of protein loaded per lane were equal as determined by Lowry analysis. rhTGF-ß1 was used as a standard. The photographs shown are representatives of 4 independent experiments.

View larger version (36K): [in this window] [in a new window]   Figure 4. Effect of mast cell chymase on the mRNA expression of TGF-ß1, PAI-1, TßRI, and TßRII. s-SMC monolayers were incubated at 37°C for 16 hours in serum-free medium with or without mast cell chymase (20 BTEE units/mL) and subjected to standard RT-PCR (A) and competitive RT-PCR (B). s-SMCs treated with rhTGF-ß1 (100 ng/mL) were used as positive controls. The expression of GAPDH, a housekeeping gene, was used as a control of cDNA quantity. DNA markers were 644, 404, 279, and 187 bp. TGF-ß1 competitor was 1x10-7 µg. DNA markers were 644, 404, 279, and 187 bp. The photographs shown are representatives of 3 independent experiments.

Mast Cell Chymase Inhibits the Expression of Collagen Type I and Type III in a TGF-ß-Independent Manner
Incubation of s-SMCs with chymase, as shown in Figure 5A, resulted in reduced mRNA expression of collagen type I (69%) and collagen type III (79%). Furthermore, addition of an anti-TGF-ß neutralizing antibody to the chymase-containing culture medium did not block the inhibitory effect of chymase on collagen expression in s-SMCs (Figure 5B). These results suggest that the observed chymase-mediated inhibition of collagen mRNA expression had occurred independently of chymase-mediated TGF-ß1 activation. Indeed, the preconditioned medium, which was found to contain 2 ng/mL of active TGF-ß1, had no effect on the collagen expression in the s-SMCs. Similarly, addition of rhTGF-ß1 (2 ng/mL final concentration) had no effect on the collagen expression in the s-SMCs. However, when s-SMCs were treated with high concentrations of rhTGF-ß1 (200 ng/mL), the expression of collagen type I was increased (2-fold). Taken together, the above results are compatible with the notion that the chymase-induced decrease in s-SMC collagen expression did not depend on the presence of active TGF-ß1. Furthermore, incubation of human coronary arterial SMCs with recombinant human chymase for 48 hours resulted in reduced expression of collagen I (74%) and collagen III (23%) in the treated human SMCs (Figure 5C).

View larger version (39K): [in this window] [in a new window]   Figure 5. Effect of mast cell chymase on the mRNA expression of collagen types I and III. A, s-SMC monolayers were incubated with mast cell chymase (20 BTEE units/mL) at 37°C for the indicated times. B, s-SMC monolayers were incubated at 37°C for 16 hours with mast cell chymase (20 BTEE units/mL), preconditioned medium (prepared with 20 BTEE units/mL chymase), or rhTGF-ß1. Ab indicates antibody. C, Human coronary arterial SMCs were incubated at 37°C for 16 hours with recombinant human chymase (20 BTEE units/mL). Competitive RT-PCR was performed in the presence of a collagen type I competitor (1x10-7 µg) and a collagen type III competitor (1x10-7µg). DNA markers were 644, 404, 279, and 187 bp. The photographs shown are representatives of 2 independent experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study revealed a novel mast cell chymase-dependent paracrine growth-inhibitory effect on s-SMCs, mediated by TGF-ß1. As determined by immunoblotting with a polyclonal antibody against TGF-ß1 and by ELISA with TßRII for capture, the ECM-associated latent TGF-ß1 was released and activated on exposure to mast cell chymase. This activated TGF-ß1, when added to fresh s-SMC cultures, inhibited their growth by blocking the G₀/G₁S transition in the cell cycle. Furthermore, chymase-mediated activation of TGF-ß1 induced the expression of PAI-1, a sensitive bioindicator of TGF-ß1-mediated cellular responses,²⁰ in the s-SMCs. We hypothesize that, analogously, activated mast cells, by releasing chymase, may affect s-SMCs through activation of TGF-ß1 in tissues in which mast cells and SMCs coexist.

Numerous experimental approaches have been carried out to identify natural mechanisms for TGF-ß activation,²¹ and many physiologically relevant proteases have been analyzed for their capacity to activate latent forms of TGF-ß1. The first successful one, the wide-spectrum protease plasmin,²² can activate latent TGF-ß1, as later shown in numerous experimental settings. However, in contrast to TGF-ß1 knockout mice, which die shortly after weaning as a result of a massive invasion of inflammatory cells,²³ loss of plasminogen activator (urokinase or tissue plasminogen activator) function in mice does not severely affect the immune system.²⁴ These observations suggest that other mechanisms besides plasmin are responsible for the in vivo activation of TGF-ß1. Indeed, recently, gelatinases A and B (matrix metalloproteinase-2 and -9, respectively)²⁵ and collagenase 3 (matrix metalloproteinase-13)²⁶ have also been observed to activate TGF-ß1.

Furthermore, other proteases, such as neutrophil elastase and human mast cell chymase, can degrade LTBP-1 and release the truncated large latent TGF-ß1 from the extracellular matrix.^10,17 However, chymase purified from human skin, in contrast to plasmin, was unable to activate purified recombinant large latent TGF-ß1.¹⁰ We have recently shown that when rat chymase 1 and TGF-ß1 were allowed to remain in their natural forms, ie, bound to the heparin proteoglycan matrix of the granule remnants, rat chymase 1 was able to activate TGF-ß1.¹¹ Furthermore, we also found that recombinant human chymase was capable of activating recombinant human latent TGF-ß1.

Another major finding in the present study is that mast cell chymase, although releasing active TGF-ß1 (a known collagen-producing factor) from the ECM of the s-SMCs, has a net inhibitory effect on the expression of collagens of type I and type III in vascular s-SMCs. Indeed, in our in vitro conditions, TGF-ß1 was either without effect or increased the expression of type I collagen of s-SMCs, depending on its concentration. Importantly, when the concentration of TGF-ß1 was 2 ng/mL (the actual concentration found in the culture medium derived from chymase-treated s-SMCs), no effect on collagen expression was observed. Therefore, we infer that the inhibitory effect of chymase on collagen mRNA expression in the s-SMC cultures was not TGF-ß1 dependent.

Under what circumstances may chymase-dependent inhibition of s-SMC growth and inhibition of collagen synthesis by s-SMCs be of relevance in vivo? The fibrous cap of an atheroma is composed of a dense connective tissue matrix containing different types of collagen secreted by the s-SMCs present in the cap,²⁷and a thick cap overlying a lipid-rich core of an atheroma is not prone to rupture, whereas a thin cap is liable to rupture. Mast cell activation with the subsequent release of chymase in the cap of an atheroma^28,29 could contribute to the numerous processes that tend to render the plaque unstable by inhibiting the expression of collagen types I and III in the s-SMCs and also by reducing s-SMC proliferation. Furthermore, targeted delivery of chymase-containing and heparin proteoglycan-containing granules from activated mast cells to neighboring s-SMCs³⁰ in the fibrous cap could result in their growth inhibition¹² and in inhibition of their collagen synthesis. The relative importance of these various putative inhibitory actions of chymase in vivo is not known. However, considering the remarkably low rate of SMC proliferation in primary atherosclerotic lesions,³¹ the inhibition of collagen expression is likely to be of greater importance than the inhibition of SMC proliferation. Recent findings demonstrating very low expression of the TßRI and TßRII in fibrous plaques support this conclusion.³ Moreover, a subset of SMCs in atherosclerotic lesions may show a profibrotic response to TGF-ß1, which would counteract the antiproliferative action of TGF-ß1.³² In any case, the present and previous data suggest that mast cells may participate in the remodeling of the fibrous caps of advanced lipid-rich lesions by multiple mechanisms.³³

	Acknowledgments

 
This study was supported in part by grants from the Aarne Koskelo Foundation (Y.W., K.A.L.) and the Paavo Nurmi Foundation (K.A.L.). We are grateful to Dr Kamimura, Teijin Limited, Japan, for kindly providing us with recombinant human chymase.

Received July 2, 2001; accepted September 17, 2001.

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