The HASMCs and smooth muscle cell medium (SMCM) were purchased from ScienCell (Carlsbad, CA, USA). Recombinant human TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). The antibodies against cyclin D1 (#2926), cyclin D3 (#2936), cyclin-dependent kinase (CDK)4 (#2906), CDK6 (#3136), p15 (#4822), p21 (#2946), p27 (#2552), p65 (#4764), phosphorylated (p-)p65 (#3033), IκBα (#4814), c-Jun N-terminal kinase (JNK; #9258), p-JNK (#4668), p38 (#9212), p-p38 (#9211), extracellular signal-regulated kinase (ERK; #4695) and p-ERK (#4370) were obtained from Cell Signaling Technology (Danvers, MA, USA) and the antibodies against TATA-binding protein (TBP; ab818) were purchased from Abcam (Cambridge, MA, USA). Recombinant human platelet-derived growth factor (PDGF) and antibodies against β-actin (A1978) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HCA and MCA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Unless otherwise mentioned, all other chemicals were purchased from Sigma-Aldrich.

The HASMCs were maintained at 37°C under 5% CO₂ in SMCM supplemented with the reagents provided with the medium. The cells were treated with various concentrations (1-50 µM) of MCA or HCA (dissolved in 0.1% DMSO) for the indicated periods of time.

The HASMCs were seeded on 12-well plates at a density of 1.5×10⁵ cells/ml. The cells were cultured overnight and treated with various concentrations of MCA or HCA for 24 h. Cell proliferation was evaluated by MTT assay according to a previously described method (22). Briefly, each well was washed twice with PBS, and 0.5 ml of cell culture medium and 50 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg/ml in PBS) were added. After 3 h of incubation, the medium was removed and 250 µl of acid-isopropanol (0.04 mol/l HCl in isopropanol) were added. The absorbance was measured at 570 nm using a Microplate reader (iMarkTM; Bio-Rad, Hercules, CA, USA).

For the cell cytotoxicity assay, the cells were treated with various concentrations of MCA or HCA for 24 h. Cell toxicity was evaluated by measuring the lactate dehydrogenase (LDH) activity using the CytoTox 96^® non-radioactive assay kit (Promega, Madison, WI, USA) in accordance with the manufacturer's instructions. In brief, the supernatant of the cell media was transferred to a 96-well plate. An equal volume of CytoTox 96^® reagent was then added and incubated for 30 min at room temperature. After adding stop solution, the absorbance was measured at 490 nm using a Microplate reader (iMarkTM; Bio-Rad).

The HASMCs were exposed to TNF-α (10 ng/ml) alone or together with MCA for 24 h. The cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer (PBS supplemented with 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, 1 µg/ml aprotinin, and 1 mM sodium orthovanadate). The cell lysates were then incubated at 4°C for 30 min, followed by centrifugation at 10,000 × g for 10 min. Alternatively, the cytoplasmic and nuclear fractions were obtained using a subcellular fractionation method that has been described previously (23). The protein samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. The blots were blocked and then incubated with primary antibodies. The immunoreactive bands were detected using the Immobilon™ Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).

The cells were exposed to TNF-α alone or together with various concentrations of MCA for 24 h. Total RNA was isolated using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA), and semi-quantitative RT-PCR was conducted using the One-Step RT-PCR PreMix kit (Intron Biotechnology, Seongnam, Korea) according to the manufacturer's instructions. The specific primers used for RT-PCR are shown in Table I. RT-PCR was performed under the following conditions: 1 cycle of 30 min at 45°C, 1 cycle of 5 min at 94°C, and 25 to 30 cycles of 30 sec at 94°C, 30 sec at 55°C, and 40 sec at 72°C, with a final extension at 72°C for 5 min. The PCR products were electrophoresed on a 1.7% agarose gel and visualized by ethidium bromide staining.

The cell culture supernatants were resuspended in sample buffer (60 mM Tris-Cl, pH 6.8, 15% glycerol, 2% SDS and 0.001% bromophenol blue) and loaded, without boiling, onto a 0.1% gelatin gel containing 10% acrylamide. Following electrophoresis, the gels were washed twice with 0.25% Triton X-100 solution for 30 min/wash and then incubated in incubation buffer (50 mM Tris-Cl, pH 7.6, 5 mM CaCl₂, 20 mM NaCl) at 37°C for 18–24 h to allow for proteolysis of the gelatin. The gels were stained with Coomassie Brilliant Blue R. Proteolysis can be detected as a white zone in a dark blue field.

A 0.71 kb segment at the 5′-flanking region of the human MMP-9 gene, corresponding to GenBank^® accession number D10051 was amplified by PCR using genomic DNA from 293 cells (American Type Culture Collection, Manassas, VA, USA) as a template. The luciferase reporter vector for the MMP-9 promoter was created by inserting the MMP-9 promoter DNA fragment into the 5′ SacI and 3′ HindIII sites of the pGL3-Basic vector. The constructs were confirmed by DNA sequencing. To assess the effects of MCA on MMP-9 promoter activity, the cells were co-transfected with pGL3-MMP-9-Luc and pCH110 using the Neon Transfection system (Invitrogen) according to the manufacturer's instructions. After 30 h, the cells were exposed to TNF-α and/or various concentrations of MCA for 24 h. The cells were then lysed and luciferase activity was measured using the Luciferase assay system (Promega). The luciferase activity was normalized to the β-galactosidase activity.

The HASMCs were seeded at a density of 1.5×10⁵ cells/ml. The cells were cultured overnight and exposed to TNF-α and/or MCA for 3 h. The cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 min. After being washed twice with PBS, the cells were incubated with methanol for 2 min. The immunostaining was performed as previously described (24). The fluorescence analysis was performed by conventional fluorescence microscopy (Axio Observer D1; Carl Zeiss, Oberkochen, Germany).

Cell migration assay was performed using the Transwell^® system (Corning Inc., Corning, NY, USA). Briefly, the cells were seeded at a density of 3×10⁴ cells/100 µl on a 0.1% gelatin-coated upper chamber. A 500 µl aliquot of serum-free SMCM with hPDGF (10 ng/ml) and/or MCA was added to the lower compartment of the invasion chamber. Following 24 h of incubation, the filter insert within the upper chamber was removed. The cells on the upper side of the filter were removed using cotton swabs, and the cells that had migrated to the underside of the filter were stained with hematoxylin and eosin (H&E). The migrated cells were observed under a light microscope (Olympus CKX41; Olympus, Tokyo, Japan).

All experiments were performed at least 3 times and the data are expressed as the means ± SD. ANOVA and the Student's t-test were applied to determine the statistical significance. P-values <0.01 were considered to indicate statistically significant differences.

In an effort to find possible anti-atherosclerotic agents, we first evaluated the effects of MCA and HCA, natural derivatives of cinnamaldehyde, on HASMC proliferation (25). Instead of a hydrogen, HCA has a hydroxy group and MCA has a methoxy group at the 2′ site of cinnamaldehyde (Fig. 1). We, as well as others have demonstrated that HCA markedly inhibits the proliferation and induces the apoptosis of various human cancer cells (16,18–20). Consistent with these studies, in this study, HCA significantly decreased cell proliferation in a dose-dependent manner. Treatment with 50 µM HCA reduced cell proliferation by 62.7% compared with the untreated control cells (Fig. 2A). Furthermore, HCA markedly increased cytotoxicity in a dose-dependent manner at a concentration of up to 30 µM (Fig. 2B). We could not obtain reasonable cytotoxicity data for concentrations >50 µM as HCA was highly toxic. On the other hand, MCA only slightly decreased cell proliferation, by 13.3%, at a concentration of 50 µM and did not result in any cytotoxicity up to a concentration of 50 µM (Fig. 2B). Therefore, we used MCA for the subsequent experiments.

We then assessed the effects of MCA on TNF-α-induced cell proliferation. The HASMCs were exposed to 10 ng/ml TNF-α alone or together with 50 µM MCA for the indicated periods of time, and cell proliferation was monitored by MTT assay. TNF-α increased cell proliferation by approximately 19.2% at day 3 compared with the untreated control cells, while MCA decreased cell proliferation by approximately 31.1% (Fig. 3A). More importantly, MCA completely abolished the TNF-α-induced increase in HASMC proliferation (Fig. 3A). To confirm the inhibitory effects of MCA on TNF-α-induced cell proliferation, we then examined the expression levels of cell cycle regulatory proteins. The cells were exposed to TNF-α alone or with a combination of various concentrations of MCA for 24 h. TNF-α increased the levels of cyclin D1 and cyclin D3 and markedly increased the levels of CDK6 (Fig. 3B). Of note, MCA attenuated the TNF-α-induced increase in the levels of cyclin D1, cyclin D3 and CDK6 in a dose-dependent manner compared with the control levels of these proteins. Additionally, the levels of the CDK inhibitor (CDKI) proteins, p21 and p27, were increased by MCA. These results suggest that MCA effectively suppresses TNF-α-induced cell proliferation through the regulation of cell cycle regulatory proteins.

MMP-9 plays an important role in VSMC proliferation and migration, and the expression of MMP-9 can be induced by TNF-α (9,10). Thus, to assess whether MCA affects TNF-α-induced MMP-9 expression, a gelatin zymography assay was performed. While no MMP-9 activity was detected in the medium from the untreated control cells, exposure to TNF-α markedly enhanced the secretion of MMP-9, and MCA significantly inhibited the secretion of MMP-9 in a dose-dependent manner (Fig. 4A). Unlike MMP-9, MMP-2 exhibited high proteolytic activity in the medium from the untreated control cells. Although TNF-α did not affect the level of MMP-2, MCA slightly reduced the level of MMP-2. Compared with the TNF-α-exposed controls, MCA inhibited MMP-9 and MMP-2 secretion by approximately 91.5 and 48.8%, respectively (Fig. 4A).

We then examined whether the MCA-dependent decrease in MMP-9 secretion is caused by the transcriptional regulation of the MMP-9 gene. The cells were exposed to TNF-α alone or together with various concentrations of MCA for 24 h. Total RNA was then isolated, and semi-quantitative RT-PCR was performed. Consistent with the results from zymography assay, TNF-α markedly increased the mRNA level of MMP-9, and the TNF-α-induced increase in the MMP-9 mRNA levels was attenuated by MCA in a dose-dependent manner, with a 78.7% inhibition observed in the cells treated with MCA. The constitutive mRNA expression of MMP-2 was also down-regulated, exhibiting a 48% inhibition in the MCA-treated cells. However, MCA did not affect the transcription of tissue inhibitor of metalloproteinases (TIMP)1 (Fig. 4B).

To further confirm the transcriptional regulation of MMP-9 by MCA, a luciferase reporter gene assay was performed. The cells were transfected with the pGL3-MMP-9-Luc reporter vector and then exposed to TNF-α alone or together with various concentrations of MCA. TNF-α enhanced MMP-9 promoter activity up to 5-fold compared with the untreated controls (Fig. 4C). However, when the cells were treated with MCA in the presence of TNF-α, MMP-9 promoter activity was reduced in a dose-dependent manner, suggesting that MCA inhibits TNF-α-induced MMP-9 transcriptional activity.

To further understand the molecular mechanisms through which MCA alters MMP-9 expression, the effects of MCA on mitogen-activated protein kinase (MAPK) signaling pathways were examined. Although TNF-α induced the activation of JNK and ERK, MCA did not affect the JNK, p38 and ERK signaling pathways, suggesting that MCA functions through another signaling pathway (Fig. 5).

As MCA did not exert any significant effects on the TNF-α-induced activation of MAPK signaling pathways, we examined the activity of the transcription factor, NF-κB, a well-known effector of TNF-α. As the nuclear translocation of NF-κB is an important indicator of its activation, we evaluated whether MCA affects NF-κB subcellular translocation. For this experiment, the HASMCs were exposed to TNF-α and/or MCA for 24 h. The nuclear proteins were separated from the cytosolic proteins through fractionation, and the distribution of NF-κB was evaluated. As expected, TNF-α markedly enhanced the nuclear localization of the NF-κB p65 subunit. However, MCA significantly inhibited the TNF-α-induced nuclear localization of p65. We also demonstrated that the TNF-α-induced nuclear p65 was phosphorylated, which is required for NF-κB activation (Fig. 6A). It is well known that the nuclear translocation of p65 is caused by the degradation of IκBα. Thus, to confirm the effects of MCA on the activation of NF-κB, the levels of IκBα were evaluated. Exposure to TNF-α decreased the level of IκBα compared with the untreated controls (Fig. 6B). However, MCA effectively inhibited the TNF-α-induced IκBα degradation. The effects of MCA on NF-κB activation were also assessed by immunofluorescence staining. Under the control conditions, NF-κB was mainly localized in the cytoplasm, and exposure to TNF-α induced the nuclear translocation of NF-κB. However, co-treatment with MCA and TNF-α resulted in the cytoplasmic localization of NF-κB, suggesting that MCA inhibits the TNF-α-induced NF-κB translocation (Fig. 6C).

Our results demonstrated that MCA effectively inhibited the TNF-α-induced HASMC proliferation and migration by inhibiting the activation of the NF-κB signaling pathway. To further evaluate the potential of MCA as an anti-atherosclerotic agent, a migration assay was performed. For this experiment, HASMC migration was induced by treating the cells with PDGF. PDGF is the strongest chemoattractant that contributes to the progression of atherosclerosis by inducing the proliferation and migration of VSMCs (2,26). Treatment with PDGF (10 ng/ml) markedly enhanced HASMC migration (Fig. 7). However, combined treatment with PDGF and MCA significantly inhibited the PDGF-induced cell migration, suggesting that MCA may be a potential therapeutic agent for atherosclerosis. The TNF-α-induced cell migration was also effectively inhibited by MCA (data not shown).