Cigarette Smoke Extract-induced Reduction in Migration and Contraction in Normal Human Bronchial Smooth Muscle Cells

Abstract

The proliferation, migration, cytokine release, and contraction of airway smooth muscle cells are key events in the airway remodeling process that occur in lung disease such as asthma, chronic obstruction pulmonary disease, and cancer. These events can be modulated by a number of factors, including cigarette smoke extract (CSE). CSE-induced alterations in the viability, migration, and contractile abilities of normal human airway cells remain unclear. This study investigated the effect of CSE on cell viability, migration, tumor necrosis factor (TNF)-α secretion, and contraction in normal human bronchial smooth muscle cells (HBSMCs). Treatment of HBSMCs with 10% CSE induced cell death, and the death was accompanied by the generation of reactive oxygen species (ROS). CSE-induced cell death was reduced by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, CSE reduced the migration ability of HBSMCs by 75%. The combination of NAC with CSE blocked the CSE-induced reduction of cell migration. However, CSE had no effect on TNF-α secretion and NF-κB activation. CSE induced an increase in intracellular Ca(2+) concentration in 64% of HBSMCs. CSE reduced the contractile ability of HBSMCs, and the ability was enhanced by NAC treatment. These results demonstrate that CSE treatment induces cell death and reduces migration and contraction by increasing ROS generation in normal HBSMCs. These results suggest that CSE may induce airway change through cell death and reduction in migration and contraction of normal HBSMCs.

Keywords: Bronchiole; Cell migration; Cigarette smoke extract; Reactive oxygen species; Smooth muscle.

Fig. 1

CSE-induced cell death in HBSMCs. (A) The effect of CSE on HBSMC viability. HBSMCs were exposed to 0 to 30% CSE for 24 h. (B) CSE-induced ROS generation in HBSMCs. Representative photomicrographs of HBSMCs labeled with H₂DCFDA to evaluate ROS generation (left panel, fluorescent image; right panel, phase contrast). The ROS levels in the cells were quantified by fluorescence microscopy after 4 h of CSE treatment. NAC was pretreated 1 h before CSE treatment. The 10% CSE and 3 mM NAC were applied to HBSMCs. The scale bar represents 100µm. (C) The effect of NAC on HBSMC viability. HBSMCs were exposed to 10% CSE and/or NAC (3 mM) for 24 h. Each bar is the mean±SD of five independent experiments. The plus and minus signs (+ and -) represent conditions with and without each treatment, respectively. ^*p<0.05 compared with the corresponding control (0% or -/-), ^†p<0.05 compared with the CSE treatment.

Fig. 2

CSE-induced reduction of cell migration. (A) Migration ability of HBSMCs by CSE treatment. Phase-contrast images of wound areas at 0, 6, 12, and 24 h after 10% CSE treatment. (B) Representative microscopic images of migrating cells by 3-D migration assays. HBSMCs were seeded onto the transwell culture inserts and incubated at 37℃ for 12 h. Migrated cells remaining on the bottom surface were stained with 1% methylene blue. (C) Summary of effect of CSE on migration ability of HBSMCs. Each bar is the mean±SD of five independent experiments. The scale bar represents 100µm. ^*p<0.05 compared with the control (-/-), ^†p<0.05 compared with the CSE treatment.

Fig. 3

Effect of CSE on TNF-α secretion and NF-κB activation in HBSMCs. (A) TNF-α concentration secreted from HBSMCs by CSE treatment. HBSMCs were treated with 10% CSE and/or 3 mM NAC for 24 h. Nicotine (10µM) effect was compared with the control. The supernatants were collected and measured using a TNF-α ELISA kit. Each bar is the mean±SD of seven independent experiments. (B) No effect of CSE on NF-κB activation. HBSMCs were treated with CSE (10%) or nicotine (10µM) for 24 h. Total protein was extracted and subjected to western blot analysis using anti-NF-κB and anti-phospho-NF-κB antibodies. Lipopolysacarride was used as a positive control for NF-κB activation [25]. The time of chemiluminescent reaction was controlled to show strong signals.

Fig. 4

Effect of CSE on intracellular Ca²⁺ increase and cell contraction. (A) CSE-induced Ca²⁺ wave patterns in HBSMCs. CSE (10%) was applied to the bath medium. Arrowheads indicate the addition of CSE and/or NAC. (B) Effect of NAC on the CSE-induced Ca²⁺ increase. The net change in Ca²⁺ levels was normalized to F0 (Fmax-F0/F0), and the data obtained from all cells were averaged. Fmax and F0 represent the maximum fluorescence level and initial fluorescence intensity of a cell. (C) Representative photographs of collagen matrices in a collagen gel contraction assay. Cells were exposed to control medium or medium containing 10% CSE for 24 h. N/C represents negative control containing BDM (a contraction inhibitor). ACh represents acetylcholine, which was used as a positive control. (D) Summary of the evaluation of assay results of cell contraction. Each bar is the mean±SD of five independent experiments. ^*p<0.05 compared with the control, ^†p<0.05 compared with the CSE treatment.