All of the chemicals used in this study were purchased from Sigma (St. Louis, MO, USA) unless otherwise specified. The stock solution of N-acetyl-l-cysteine (NAC, 500 mM) and ethylene glycol tetraacetic acid (EGTA, 50 mM) was prepared in distilled water. Nicotine (100 mM) and 1,2-Bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM, 10 mM) were dissolved in ethanol and dimethyl sulfoxide (DMSO), respectively. The stock solutions of chemicals were diluted in culture medium to a working concentration. When ethanol or DMSO was used as a solvent, a solution containing an equivalent concentration was used as a control.

CSE was prepared using a previously published method [22]. To produce 100% CSE, one unfiltered Camel cigarette (R.J. Reynolds, Winston-Salem, NC, USA) was passed through 10 ml of phosphate-buffered saline (PBS) using a vacuum pump. The 100% CSE solution was adjusted to a pH of 7.4 and filtered through a 0.2-µm-pore filter (Minisart®, Sartorius, Goettingen, Germany) to remove particles and bacteria. The CSE was diluted to the appropriate concentration and was added to HBSMCs within 10 min of preparation.

Normal HBSMCs were purchased from PromoCell GmbH (Heidelberg, Germany). The HBSMCs were cultured in smooth muscle cell basal medium supplemented with 0.5 ng/ml epidermal growth factor, 2 ng/ml basic fibroblast growth factor, 5µg/ml insulin, 5% fetal calf serum, 50µg/ml amphotericin B, and 50 ng/ml gentamycin based on the manufacturer's recommendations (PromoCell). Cells were incubated at 37℃ in humidified air containing 5% CO₂. The medium was replaced every two days.

Cell viability was determined colorimetrically using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent (Duchefa, Haarlem, Netherlands). Cells at the exponential phase were seeded (4×10⁴ cells/ml) in a 24-well plate. After 24 h of extract or chemical treatment, 20µl of 5 mg/ml MTT solution was added to each well (0.1 mg/ml) and incubated for 4 h. The supernatants were aspirated, the formazan crystals in each well were dissolved in 200µl DMSO for 30 min at 37℃, and the 24-well plates were read at 570 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).

The HBSMCs were cultured with or without 3 mM NAC while exposed to 10% CSE for 4 h. The HBSMCs were loaded with 5µM of dichlorodihydrofluorescein diacetate (H2DCFDA, Calbiochem, San Diego, CA, USA) for 30 min in the dark. After incubation, the cells were washed three times with PBS and immediately analyzed for fluorescence intensity using a confocal laser scanning microscope equipped with a fluorescence system (IX70 Fluoview, Olympus, Tokyo, Japan). For the detection of green fluorescence (H₂DCFDA), cells were illuminated with 488- and 519-nm laser lines. The fluorescent images were saved in the tagged image file format and analyzed using the Fluoview software program (version 2.0, Olympus).

For the two-dimensional (2-D) migration assay, cells were seeded in a µ-dish (ibidi GmbH, Munich, Germany) at a density of 1×10⁶ cells/ml and treated with 8µg/ml mitomycin C in culture media for 2 h to eliminate proliferation. Photomicrographs were taken after incubation. Relative cell migration distance (closure) was determined by measuring the gap width on the monolayer under 10× magnification (Axiovert 40C, Carlzeiss MicroImaging, Göttingen, Germany) and by subtracting this value from the initial value (i.e., the initial width at 0 h). The transwell (three-dimension, 3-D) migration assay was performed using the CytoSelect™ Cell Migration Assay Kit containing polycarbonate membrane inserts (8-µm-pore membrane; Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. A cell suspension containing 1×10⁵ cells/ml in serum-free medium was prepared, and 300µl of the cell suspension was added to the upper chamber of each insert. The lower chamber contained medium with 10% FBS to allow cell migration toward the lower face of the transwell culture inserts. Cells were incubated for 12 h at 37℃ in a 95% air-5% CO₂ gas mixture. Non-migrating cells on the inner side of the transwell culture inserts were gently removed with a cotton-tipped swab. Migrated cells remaining on the bottom surface were stained with a cell-staining solution (1% methylene blue) for 40 min at room temperature. Photomicrographs of five individual fields per insert were taken using a microscope (Axiovert 40C), and cells were enumerated to calculate the average number of cells that had migrated. The stained insert was washed thoroughly and dissolved by incubating for 10 min with extraction solution (1% Triton X-100). Each sample was transferred to a 96-well microtiter plate, and the absorbance at 560 nm was measured using a plate reader (Infinite® F200, Tecan, Männedorf, Switzerland).

The changes in [Ca²⁺]_i was measured using the Ca²⁺-sensitive fluorescent indicator fluo 3-AM (Molecular probe, Eugene, Oregon, USA) and a confocal laser scanning microscope (IX70 Fluoview). The HBSMCs were incubated with 5µM fluo 3-AM in a coverglass-bottom dish (SPL, Pocheon, South Korea) for 45 min and washed three times with serum-free medium. Fluorescent images were scanned every 5 sec at 488 nm with an excitation argon laser and 530 nm longpass emission filter. All scanned images were processed to analyze changes in [Ca²⁺]_i at the single-cell level.

The contraction assay was performed using a cell contraction assay kit (Cell Biolabs). Briefly, HBSMCs (5×10⁵ cells/ml) were cultured for 2 days; the cultured HBSMCs were resuspended in culture media; and the cell suspension was mixed with collagen gel solution (bovine Type I). The cell-collagen mixture was placed in a 24-well plate and incubated for 1 h at 37℃. After collagen polymerization, culture medium without the growth factor supplement was added onto the collagen gel lattice. Cells in collagen gels were allowed to equilibrate for 2 days, the gels were released from the culture plate, and various agents were added to the media 1 h prior to release. The control for CSE and NAC was serum free media, and the control for 2, 3-butanedione monoxime (BDM) was 0.1% DMSO in medium. BDM was used as a contraction inhibitor (negative control). Images of the gels were captured with a camera (Canon, Tokyo, Japan), and the gel size change was measured at the indicated times with a ruler and quantified with image analysis software (Fluoview software program, Olympus). For each condition, collagen contraction was determined in at least quadruplicate.

The HBSMCs were treated with 10% CSE for 24 h in the presence or absence of NAC in 96-well plates. The cell culture media was collected and stored at -20℃ prior to the analysis. The amounts of secreted TNF-α in the media were measured using TNF-α ELISA kits (BioSource; Camarillo, CA, USA) according to the manufacturer's instructions.

The HBSMCs were homogenized in a protein extraction solution (PRO-PREP™, iNtRON Biotechnology Inc, Seongnam, Korea) containing 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.5% nonyl phenoxylpolyethoxylethanol (NP-40), 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 1µM leupeptin, 1µM pepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1µM aprotinin. After the addition of the protein extraction solution, the cells were incubated for 30 min on ice with intermittent vortexing. The extracts were clarified by centrifugation at 13,000 rpm (16,609×g; Micro 17TR, Hanil, Korea) for 20 min at 4℃. The resulting supernatant was separated with 8% SDS-polyacrylamide gel and transferred to PVDF membrane for 15 min using a semi-dry transfer (Bio-Rad, CA, USA). The membranes were blocked with 5% fat-free dry milk and then incubated with NF-κB p65 polyclonal (1:1,000 dilution) (Cell Signaling, Danvers, MA, USA), Phospho-NF-κB p65 (Ser536) rabbit monoclonal (Cell Signaling, 1:1,000 dilution), and α-tubulin monoclonal antibodies (1:10,000 dilution). The primary antibody incubation was followed by incubation with a secondary peroxidase-conjugated anti-rabbit or anti-mouse antibody at 1:10,000. Immuno-positive bands were visualized by enhanced chemiluminescence (ECL Plus kit, ELPIS, Taejon, Korea) following the manufacturer's instructions.

A one-way ANOVA (SPSS18, Chicago, IL, USA) was used with p<0.05 as the threshold for significance. Data are represented as mean±SD.

The cytotoxicity of CSE was examined in HBSMCs using an MTT assay. The HBSMCs were cultured in the presence of various concentrations of CSE (0.01~30%). When the cells were treated with CSE for 24 h, the cell viability significantly decreased by approximately 38% in the 10% CSE treatment compared with the control (Fig. 1A). As shown in Fig. 1B, ROS was detected using H₂DCFDA that has been widely used as a detector of ROS in many kinds of cells. The dye becomes fluorescent after cellular oxidation. CSE significantly increased ROS levels by 2.5-fold compared with the control (p<0.05, n=5). CSE-induced ROS generation was attenuated by pretreatment for 1 h with 3 mM NAC. The CSE-induced cell death was also reduced by NAC treatment (Fig. 1C).

To test the migration ability of HBSMC, the cells were incubated in a µ-dish (ibidi GmbH). However, the 10% CSE treatment reduced cell migration ability by 57% compared with the control (Fig. 2A). The cells' migration ability was determined using a 3-D migration assay. The 3-D migration assay revealed that CSE treatment significantly inhibited cell migration (p<0.05, Fig. 2B), reducing migration by 75% in the CSE treatment (Fig. 2C). The combination of NAC and CSE offset the CSE-induced reduction in cell migration (Fig. 2C). NAC alone also enhanced cell migration, suggesting that regulation of ROS concentration might play a role in migration of HBSMCs. NAC treatment may reduce endogenous ROS generation in HBSMCs.

The TNF-α concentration was analyzed in the supernatants obtained after CSE treatment of HBSMCs. CSE had no significant effect on TNF-α concentration compared with that in the control (Fig. 3A, 15.9±3.4 pg/ml in CSE versus 19.5±8.4 pg/ml in control), and CSE (10%) failed to induce NF-κB activation (Fig. 3B). Nicotine (10µM), a major component of CSE, also failed to increase TNF-α concentration and NF-κB activation (Fig. 3). CSE (0.01~1%) reduced NF-κB activation compared with the control (data not shown).

To identify CSE-induced changes in [Ca²⁺]_i, a calcium image analysis was performed. The addition of 10% CSE to the normal physiological solution transiently increased [Ca²⁺]_i in the HBSMCs. The application of the 10% CSE produced different patterns of transient Ca²⁺ spikes in the HBSMCs (Fig. 4A). The 10% CSE treatment induced a low repetitive Ca²⁺ spike in some HBSMCs while some cells showed a high Ca²⁺ spike with or without a plateau in response to CSE treatment. These Ca²⁺ responses were observed in 128 of 200 HBSMCs. However, the rest of the cells (72 of 200, 34.6±11.3%) did not produce Ca²⁺ peaks or showed reduction in response to 10% CSE (Fig. 4A). The combined treatment with both CSE and NAC did not show significant increase in the levels of intracellular Ca²⁺ (CSE: 1.6±1.4 in 200 of 200; CSE+NAC: 2.1±1.1 in 66 of 66, Fig. 4B). The combined treatment with CSE and NAC also showed similar patterns of transient Ca²⁺ spike to CSE treatment (data not shown). As shown in Fig. 4A, the changes in [Ca²⁺]_i were represented as fluorescence intensity (F), and the F was normalized to basal F before CSE treatment (F/F0). The net changes in F shown in Fig. 4B were calculated with subtraction of F0 from the maximum level of F showing after the addition of 10% CSE divided by F0 ((Fmax-F0)/F0). The data shown in Fig. 4B were obtained from average of all data tested in this study.

Cell contraction is a result of cell proliferation, migration, Ca²⁺ changes, or cytokine release. To test whether CSE modulates the contractility of HBSMCs, a gel contraction assay was performed. The addition of 10% CSE to the HBSMC gels reduced contractility by 32% compared with the control (Fig. 4C), and the area of the treated gels was larger than that of the control gels (Fig. 4D). However, the treatment combination of CSE with NAC increased contraction compared with the cells treated only with CSE (Fig. 4C and D). The results of combined treatment with both CSE and Ca²⁺ chelator (EGTA or BAPTA) could not be clearly analyzed because the gel's properties were transformed (e.g. gel is torn easily) (data not shown).