Collagens are key components in the extracellular framework of artery media and major constituents of human atherosclerotic plaques, in which the most abundant collagens are types 1 and 3. Matrix metalloproteinases (MMPs) specialize in resorption of extracellular collagens and are responsible for vascular matrix remodeling during atherosclerosis (1-4). Matrix metalloproteinase-1 (MMP-1), also called interstitial metalloproteinase, is a member of the MMP family characterized by its distinctive ability to degrade collagen types 1 and 3 (5).
In human atherosclerosis, unstable atherosclerotic plaque ruptures trigger acute coronary syndromes. Unstable plaques exhibit a large core of lipid covered by a thin fibrous cap with an increased monocyte/macrophage density and decreased collagen and vascular smooth muscle cell (VSMC) content (4,6-9). Recent findings have demonstrated that MMP-1 activity is elevated in vulnerable regions of plaques and contributes to the weakening of plaque caps by degrading extracellular collagen (2,10). Conversely, increased MMP-1 activity in atherosclerotic plaques allows the migration and proliferation of VSMCs from the media into the intima, where they form the major components of the restenotic lesions (11). The MMP-1 levels influence plaque stability as well as disease progression.
Monocytes/macrophages and VSMCs are major components of atherosclerotic plaques (7-9,12). It has been reported that VSMCs can synthesize MMPs, but in nonatherosclerotic arteries, only MMP-2 (gelatinase A) has been shown to be expressed (1,11-14). In contrast, atherosclerotic lesions are immunoreactive for all MMPs tested, and VSMCs in lesions have been shown to contain MMP-1 (10,12). Monocytes/macrophages in various states of differentiation and activation, especially lipid-laden macrophages, also may be an important source of MMP-1 (12, 15-18), but freshly isolated monocytes or unstimulated monocytes did not express MMP-1 (15,19,20).
Interactions between monocytes and other cells that are components of vascular walls and atherosclerotic plaques have been reported to play an important role in the expression of various vasoactive substances under pathophysiologic conditions (21,22). In this study, we used coculture systems to elucidate the mechanisms responsible for the induction of MMP-1 expression resulting from cell-to-cell adhesive interactions.
Phorbol 12-myristate 13-acetate (PMA) and H-7 were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.), genistein from Wako Chemicals Inc. (Tokyo, Japan), herbimycin A from Alexis Co. (San Diego, CA, U.S.A.), and PD98059 from Biomol Research Lab. Inc. (Plymouth Meeting, PA, U.S.A.). Calphostin C and KT 5720 were gifts from Kyowa Hakko Co. (Tokyo, Japan). Monoclonal antibodies against human interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β were purchased from Genzyme (Cambridge, MA, U.S.A.). Mouse monoclonal anti-human MMP-1 antibody (clone 41-1E5) was obtained from Sigma Chemical Co., and nonimmune rabbit immunoglobulin G (IgG) was purchased from Cedarlane (Hornby, Ontario, Canada).
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. VSMCs prepared from human aorta were purchased from Clonetics Corp. (San Diego, CA, U.S.A.) and cultured in smooth muscle basal medium (SMBM; Clonetics) supplemented with 5% fetal calf serum, 5 μg/ml insulin, 2 pg/ml human basic fibroblast growth factor, 50 μg/ml gentamicin, and 50 pg/ml amphotericin B on plastic culture dishes or 24-well plates (Falcon, Franklin Lakes, NJ, U.S.A.). Cells between passages six to nine were used in the experiments. We confirmed the α-actin staining in VSMCs by immunohistochemistry. The cultures were incubated until confluency at 37°C in humidified 5% CO2 atmosphere.
THP-1 cells (human acute monocytic leukemia cells) were obtained from American Type Culture Collection (Rockville, MD, U.S.A.), cultured in RPMI medium 1640 supplemented with 10% bovine serum albumin (BSA), and 5 × 10−5M 2-mercaptoethanol, and maintained at a cellular density of 2 × 105 to 106 cells/ml, as described previously (23).
Human mononuclear cells were prepared from heparinized venous blood of healthy adults, as described previously (24,25). Monocytes were then purified from mononuclear cells by centrifugal elutriation with a Hitachi SRR6Y elutriation rotor (Hitachi Ltd., Tokyo, Japan).
Human embryonic lung fibroblasts were purchased from Clonetics Corp. and cultured in 24-well plates in fibroblast basal medium (FBM; Clonetics) containing 20% fetal calf serum, 10 pg/ml human fibroblast growth factor, 5 μg/ml insulin, and 50 μg/ml gentamicin. Cells between passages three and four were used in the experiments.
VSMCs were incubated in SMBM supplemented with 0.1% BSA, 50 μg/ml gentamicin, and 50 pg/ml amphotericin B at 37°C for 24 h before the experiments. Confluent monolayers of VSMCs (1.0 × 105 cells) on 24-well plates were washed twice with 500 μl phosphate-buffered saline (PBS; 0.01 M sodium phosphate, 0.14 M NaCl, pH 7.2). The cell numbers per well of 24-well plates were confirmed before starting each experiment.
THP-1 cells were exposed to 200 nM PMA for 12 h before they were used for the experiments. This treatment was reported to cause THP-1 cells to acquire macrophage-like characteristics (26,27). Phenotypic characterization of macrophages in stimulated THP-1 cells was confirmed by immunohistochemical staining with specific antibody. Cell viability of THP-1 cell was not changed after stimulation. Then THP-1 cells were washed 3 times with PBS to remove the PMA to avoid contaminating the coculture wells. THP-1 cells or human monocytes were resuspended in 500 μl of the VSMC growth medium described earlier and cocultured with VSMCs at 37°C in a 5% CO2 humidified incubator for the indicated periods.
Each well of the 24-well plates was divided into two compartments using an inner well (Cell culture insert; Falcon), and coculture was carried out under four sets of conditions (i.e., direct coculture, THP-1 cells and VSMCs were cocultured in the lower chamber; separated coculture, THP-1 cells (1.0 × 105 cells) were cultured in the upper chamber and VSMCs (1.0 × 105 cells) in the lower chamber; VSMCs alone, VSMCs were cultured alone in the lower chamber; THP-1 cells alone, THP-1 cells were cultured alone in the upper chamber. All the cells were incubated at 37°C in a 5% CO2 humidified incubator for 24 h. The upper and lower chambers contained 250 μl and 1 ml growth medium containing 0.1% BSA, respectively. The supernatant in each chamber was collected, and its MMP-1 levels were determined.
Isolation of cell membrane fraction
Cell membrane fractions of THP-1 cells and VSMCs were isolated as described elsewhere (28). Cell membrane fractions obtained from VSMCs (1.0 × 105 cells) were coincubated with THP-1 cells (1.0 × 105 cells) for 24 h on 24-well plates. Conversely, cell membrane fractions from THP-1 cells (1.0 × 105 cells) were coincubated with VSMCs (1.0 × 105 cells). MMP-1 levels in the supernatant were determined.
Measurement for MMP-1, TIMP-1, IL-1β, IL-6, and TNFα concentrations
The MMP-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) concentrations of the culture media were determined using the appropriate enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (Amersham International plc, Buckinghamshire, U.K.). The MMP-1 assay kit recognizes total MMP-1 (proMMP-1, free MMP-1, and MMP-1-TIMP complex). The lower limits of detection of MMP-1 and TIMP-1 were 6.25 and 3.13 ng/ml, respectively. Concentrations of IL-1β and TNF-α in the culture media were determined using ELISA kits (Amersham). The levels of IL-6 were determined by chemiluminescent enzyme immunoassay kits (Fuji Rebio Co., Tokyo, Japan). The lower limits of detection of IL-1β, TNF-α, and IL-6 were 10, 25, and 0.1 pg/ml, respectively.
Assay for collagenolytic activity
Collagenolytic activity, which reflects mainly MMP-1 activity as well as MMP-2, MMP-8, and MMP-13 activity, was measured by fluorescent-labeled collagen digestion with a type 1 collagenase activity assay kit (Yagai Research Center, Yamagata, Japan). In brief, fluorescent-labeled collagen solution (50 μg/50 μl), after adding 50 μl buffer solution (0.1 M Tris-HCl, pH 7.5, containing NaCl, CaCl2, and NaN3), was mixed with 100-μl samples or with double-diluted buffer solution as the blank and the total. Samples and the blank were incubated at 37°C for 3 h, whereas the total was treated at 100°C for 1 min. After placement at 4°C for 5 min, the reaction was stopped by adding 200 μl of enzyme stop reagent/extraction solution (0.005 M Tris HCl, pH 9.5/ethanol containing NaCl, o-phenanthroline). The fluorescence in the supernatants was measured at 520 nm with excitation at 495 nm using a fluorescence spectrometer (Nihon Bunko Corp., Tokyo, Japan). Collagenolytic activity was calculated according to the equation supplied in the manufacture's instructions.
Human VSMCs were cultured on gelatin-coated glass slides and incubated in growth medium containing 0.1% BSA at 37°C for 24 h before coculture with THP-1 cells. Immunohistochemistry was performed as described previously (21). In brief, after rinsing with PBS, the cells were fixed with 4% paraformaldehyde in PBS containing 0.02% Triton X-100 for 20 min at room temperature. Before staining, nonspecific binding reactions were blocked with 1% BSA in PBS. Then slides were rinsed with 0.1% Triton X-100/PBS, incubated with 10 μg/ml monoclonal anti-MMP-1 antibody (Oncogene Research Products, Cambridge, MA, U.S.A.; clone 41-1E5) or control nonimmune IgG at room temperature for 1.5 h, rinsed again with PBS, incubated with biotinylated goat anti-mouse IgG, incubated for 20 min, and rinsed with PBS. Finally the slides were treated with peroxidase-conjugated streptavidin reagent for 15 min at room temperature and visualized with a solution of 0.05% 3,3′-diaminobenzidine tetrahydrochloride in 0.05 M Tris buffer (pH 7.6) containing 0.005% H2O2 for 5 min at room temperature.
All the values are expressed as mean ± SEM of four to eight samples, which represent at least three separate experiments. The significance of the difference between two groups was determined using one-way analysis of variance (ANOVA) combined with Scheffé's test. Differences of p < 0.05 were considered to be significant.
Expression of MMP-1 by coculture of VSMCs and monocytes
We investigated whether MMP-1 production was induced by interaction between monocytes and VSMCs, by measuring the MMP-1 levels of their culture media. As shown in Fig. 1A, after incubation for 48 h, human monocytes alone did not produce MMP-1, whereas VSMCs alone produced small amounts of MMP-1 (30.0 ± 5.1 ng/ml). In contrast, the MMP-1 levels of the coculture medium of these cells increased in a time-dependent manner and reached 363 ± 46.2 ng/ml after incubation for 48 h, 12 times higher than those produced by VSMCs alone. Similarly, after incubation for 24 h, PMA-stimulated THP-1 cells (human monocytoid cells) alone did not produce MMP-1 (Fig. 1B). The MMP-1 levels of the coculture medium of PMA-stimulated THP-1 cells and VSMCs increased in a time-dependent manner and reached 1,575 ± 35 ng/ml after incubation for 48 h, ∼50 times higher than those produced by VSMCs alone.
We also investigated MMP-1 expression by cocultures of human monocytes and fibroblasts. A very small amount of MMP-1 was detected in the culture medium of fibroblasts alone (4.8 ± 0.4 ng/ml), and as a result of coculture of monocytes and VSMCs, the MMP-1 levels of the coculture medium of PMA-stimulated THP-1 cells and fibroblasts increased with time (Fig. 2A). We also investigated MMP-1 expression by cocultures of PMA-stimulated THP-1 cells and fibroblasts. As a result of coculture of PMA-stimulated THP-1 cells and VSMCs, the MMP-1 levels of the coculture medium of PMA-stimulated THP-1 cells and fibroblasts increased markedly with time (Fig. 2B).
We next investigated the effect of increasing the number of PMA-stimulated THP-1 cells relative to that of VSMCs or fibroblasts on MMP-1 production. As shown in Fig. 3, the MMP-1 levels in the coculture medium increased as the number of added PMA-stimulated THP-1 cells increased and reached a peak at a THP-1/VSMC ratio of 1.0 (panel A), and a THP-1/fibroblast ratio of 5.0 (panel B).
TIMP-1 expression and collagenolytic activity
Because MMP-1 is tightly regulated by its specific inhibitor (TIMP-1) in vivo, a high level of expression does not always result in high enzyme activity. Therefore, to assess the role of the enzymes and the possible mechanisms involved, it is necessary to measure TIMP-1 levels and collagenolytic activity. Figure 4 shows the time course of TIMP-1 levels in supernatants of culture medium. As shown in Fig. 4B, fibroblasts, monocytes, and PMA-stimulated THP-1 cells produced small amounts of TIMP-1, whereas cocultures of fibroblasts and monocytes or PMA-stimulated THP-1 cells had markedly enhanced TIMP-1 production. Conversely, as shown in Fig. 4A, coculture of VSMCs and monocytes or PMA-stimulated THP-1 cells had relatively lower levels of TIMP-1 production after 48-h incubation.
Figure 5 shows the collagenolytic activity in the culture media. Coculture of VSMCs and PMA-stimulated THP-1 cells significantly increased collagenolytic activity in a time-dependent manner (Fig. 5A), whereas coculture of fibroblasts and PMA-stimulated THP-1 cells did not cause a significant increase in collagenolytic activity in the culture medium (Fig. 5B).
Immunohistochemical staining of MMP-1-producing cells
Next we investigated which types of cells in the coculture produced MMP-1 by carrying out immunohistochemical staining using an anti-MMP-1 antibody. As shown in Fig. 6A, both PMA-stimulated THP-1 cells and VSMCs in the coculture showed cytosolic staining for MMP-1, indicating that both types of cells produce MMP-1. No cytosolic staining of the cocultured cells incubated with nonimmune IgG as the primary antibody was observed (Fig. 6B).
MMP-1 production by separated cocultures
We analyzed the possible mechanism responsible for MMP-1 production by coculture of PMA-stimulated THP-1 cells and VSMCs. To determine whether direct cell-to-cell contact is necessary for MMP-1 production by such coculture, we set up separated cocultures using an inner well system that physically separated the PMA-stimulated THP-1 cells in the upper compartment from the VSMC layer in the lower compartment, but allowed soluble mediators to diffuse freely. As shown in Fig. 7, the MMP-1 levels of the direct cocultures were significantly higher than those of the separated cocultures (37.7 ± 2.7 vs. 16.8 ± 2.1 ng/well; p < 0.001). The amount of MMP-1 produced by the separated cocultures was still significantly higher than that produced by VSMCs (9.0 ± 0.4 ng/well,) or PMA-stimulated THP-1 cells (1.9 ± 0.1 ng/well) cultured alone.
Involvement of direct contact of cell membranes in MMP-1 production
Addition of cell membrane fractions obtained from 1 × 105 PMA-stimulated THP-1 cells to 1 × 105 VSMCs in culture induced significant increases in MMP-1 levels in the culture medium after incubation for 24 h (from 38.3 ± 2.3 to 362 ± 65.1 ng/ml; p < 0.01). Conversely, addition of cell membrane fractions obtained from 1 × 105 VSMCs to 1 × 105 PMA-stimulated THP-1 cells in culture did not cause significant changes in MMP-1 levels (MMP-1 was not detected).
Involvement of cytokines in MMP-1 production
These results indicated that soluble factors, as well as direct contact between VSMCs and PMA-stimulated THP-1 cells, contributed to MMP-1 production by the cocultures. MMP-1 is produced by various cells in response to proinflammatory cytokines. As shown in Fig. 8, substantial amounts of IL-1β, IL-6, and TNF-α accumulated in the supernatants of the PMA-stimulated THP-1 cell and VSMC cocultures. Thus, MMP-1 production by coculture may have been mediated by these soluble factors.
To explore this hypothesis, we used antibodies against these cytokines. First, PMA-stimulated THP-1 cells or VSMCs were exposed to the conditioned media from PMA-stimulated THP-1 cells and VSMC 4-h cocultures for 16 h. Exposure of 1.0 × 105 PMA-stimulated THP-1 cells to coculture conditioned medium for 16 h evoked significant increases in MMP-1 production (from 1.40 ± 0.10 to 5.57 ± 0.34 ng/ml; p < 0.001). The addition of 25 μg/ml of IL-1α, IL-1β, IL-6, or TNF-α antibody significantly inhibited conditioned medium-induced MMP-1 production by PMA-stimulated THP-1 (Fig. 9A). Addition of anti-IL-6 or TNF-α antibody to the cultures also significantly inhibited conditioned medium-induced MMP-1 production by VSMCs (Fig. 9B). However, direct addition of these antibodies to the coculture system had no significant effect on MMP-1 production.
Involvement of protein kinases in MMP-1 production
To investigate further the intracellular signal pathways involved in MMP-1 production by coculture of PMA-stimulated THP-1 cells and VSMCs, the effects of various protein kinase inhibitors on MMP-1 production were examined. PMA-stimulated THP-1 cells and VSMCs were pretreated with protein kinase inhibitors for 1 h and then cocultured for another 16 h. As shown in Fig. 10, addition of the tyrosine kinase inhibitors genistein (10−5M) or herbimycin A (10−6M), the protein kinase C (PKC) inhibitors H-7 (10−5M) or calphostin C (10−7M), or the mitogen-activated protein (MAP) kinase kinase inhibitor PD98059 (10−5M) significantly inhibited MMP-1 production by the cocultures. These inhibitors did not affect the lactic dehydrogenase levels of the supernatants (data not shown), suggesting that reduced MMP-1 production was not due to inhibitor cytotoxicity. Furthermore, the numbers of nonadherent THP-1 cells in the presence and absence of these inhibitors did not differ significantly (data not shown), indicating that reduced MMP-1 production was not due to disturbance of the adhesion of THP-1 cells to VSMCs.
In this study, we demonstrated that MMP-1 is expressed at very low levels by VSMCs, fibroblasts, and human monocytes individually under basal culture conditions, whereas expression was markedly induced by coculture of these two types of cells. Similar and more pronounced changes in MMP-1 levels were observed when activated THP-1 cells were used instead of human monocytes (Fig. 1). We used PMA-stimulated THP-1 cells as the model of activated monocytes/macrophages to investigate cell-to-cell interaction in atherosclerotic plaques, although it is possible that there are some differences between THP-1 cells and human macrophages.
Collagenolytic activity was increased only in the coculture of THP-1 cells and VSMCs. We also confirmed that direct adhesion of VSMCs and THP-1 cells was more effective for MMP-1 production than was separated coculture of VSMCs and THP-1 cells. The mechanism responsible for enhanced MMP-1 production resulting from the interaction between THP-1 cells and VSMCs is incompletely understood. It is possible that direct contact between the cell membranes of the two different types of cells stimulates a cellular mechanism responsible for MMP-1 production and that a paracrine effect of soluble factors including IL-1β, TNF-α, and IL-6 is augmented when the two types of cells are very close together. Our results suggest that direct adhesion of the cell membrane fraction of THP-1 cells to VSMCs induced increases in MMP-1 production. Direct contact of cell-surface antigens between THP-1 cells and VSMCs may trigger cellular mechanisms for MMP-1 production by VSMCs.
MMP-1 is not detectable in uninjured arteries, but was detected in atherosclerotic lesions (12,14). Under our coculture conditions, both VSMCs and THP-1 cells produced MMP-1, as determined by immunohistochemical analysis. Such in vitro phenomena also may occur in vivo, and indeed MMP-1 production has been detected in atherosclerotic lesions in humans. We speculate that both contact of monocytes and VSMCs and paracrine interactions between two types of cells are important signals in the pathogenesis of atherosclerosis by promoting MMP-1 production.
The MMP-1 production by coculture of PMA-stimulated THP-1 cells and VSMCs reached a peak at a THP-1/VSMC ratio of 1.0. We observed that increasing the number of THP-1 cells relative to VSMCs did not affect the cell viability by THP-1/VSMC ratio of 10. Why MMP-1 levels in the coculture medium decreased at a THP-1/VSMC ratio of 10 was still unclear. It is possible that effect of some humoral factors that suppress MMP-1 production in the coculture is dominant at a THP-1/VSMC ratio of 1.0.
To assess the role of active MMP-1, it is important to look also at the production of TIMP-1. TIMP-1 is synthesized by most types of cells, including macrophages and VSMCs (13), and acts against all members of MMPs but has a particular affinity for MMP-1. Its production also is induced by cytokines such as IL-1 and IL-3 (29). In this study, the high concentration of TIMP-1 in the coculture medium containing fibroblasts and monocytes or THP-1 cells suggests the existence of a mechanism inhibiting MMP-1 activity. If MMP-1 is not tightly regulated, matrix degradation could proceed rapidly. This fact, combined with the measurement of MMP-1 activity, suggests that the net effect of MMP-1 produced by coculture of fibroblasts and THP-1 cells remains unaffected. However, the significantly elevated MMP-1 activity found in the coculture medium of VSMCs and THP-1 cells suggests that the balance of matrix metabolism may favor matrix degradation when VSMCs adhere to monocytes. Our results suggest that adhesive interactions between monocytes and VSMCs play an important role in atherosclerotic plaque rupture by enhancing local destruction of extracellular matrix. We used a fluorescent-labeled collagen digestion method for measurement of MMP-1 activity; however, it is possible that other MMPs such as MMP-2 or MMP-13 are involved in the digestion of collagen.
Previously several researchers, such as Galis et al. (13) and Lee et al, (20,30), reported that TNF-α or IL-1 enhances MMP-1 expression in VSMCs. Pickering et al. (1) and Yanagi et al. (31) demonstrated that fibroblast growth factor or platelet-derived growth factor upregulates MMP-1 expression in VSMCs. Monocytes/macrophages produce MMP-9 (92-kDa gelatinase) when stimulated by TNF-α or IL-1β (32). Thus, it is possible that several cytokines regulate MMP expression in monocytes and VSMCs. In our study, exposure of monocytes or VSMCs alone to coculture-conditioned medium resulted in elevated MMP-1 levels in the supernatant, suggesting that a soluble factor(s) induces MMP-1 production by both types of cells. Neutralizing anti-TNF-α and IL-6 antibodies significantly inhibited coculture-conditioned medium-induced MMP-1 production by both VSMCs and THP-1 cells, and anti-IL-1α and IL-1β antibodies inhibited that MMP-1 production by THP-1 cells. These results suggest that several cytokine-mediated mechanisms are involved at least partially in MMP-1 production in cocultures of monocytes and VSMCs. We speculate that activated monocytes, through a cell-to-cell interaction, release several inflammatory cytokines such as IL-1, TNF-α, and IL-6 (33), which in turn induce neighboring VSMCs and/or macrophages to produce MMPs through an endocrine-paracrine pathway. The effect of these inflammatory cytokines is very similar, suggesting that synergistic responses might be included.
To elucidate the intracellular mechanisms that regulate MMP-1 production, we examined the effects of several protein kinase inhibitors on MMP-1 production by coculture of monocytes and VSMCs. The results indicate that PKC, tyrosine kinases, and MAP kinase-mediated pathways are involved in MMP-1 production by these cocultures. However, the signals that elicit expression of MMP-1 remain to be further investigated. Experiments using genetic manipulation of MAP or tyrosine kinase function may elucidate the mechanism.
In conclusion, interactions between monocytes and VSMCs markedly induced MMP-1 levels and activity, and the mechanism responsible is mediated, at least partially, by cytokines, suggesting that MMP-1 produced locally as a result of monocyte-VSMC adhesive interactions plays an important role in the pathogenesis of atherosclerotic plaques and their rupture.
Acknowledgment: We thank Toshiko Kambe for her excellent technical assistance. This study was supported by the Ministry of Education, Science, Sports and Culture of Japan, and the Takeda Medical Research Foundation.
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