Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco Life Technologies (Carlsbad, CA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise specified.

Primary rat PASMCs were isolated and cultured, as previously described (26). Briefly, 20 male Sprague-Dawley rats were obtained from the Animal Experiment Center of Wenzhou Medical University (Wenzhou, China) and were housed alone with a 12:12 h light/dark cycle with free access to water and food at room temperature. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Wenzhou Medical University and were performed in accordance with the Wenzhou Medical University Guidelines for the Ethical Care of Animals (WYDW2012-0032). At the age of 12 weeks, the rats were anesthetized with 100 mg/kg intraperitoneal ketamine and 5 mg/kg intraperitoneal xylazine. Subsequently, the distal pulmonary arteries were isolated and immersed immediately in Hanks' solution containing collagenase (1.5 mg/ml) for 20 min. Following incubation at 37°C, a thin layer of adventitia and endothelium were carefully stripped off. The PASMCs were then harvested by digesting the remaining smooth muscle with collagenase (2.0 mg/ml) and elastase (0.5 mg/ml) for 40 min, and were cultured in DMEM, containing 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a 5% CO₂ atmosphere for 5~7 days. The cells were identified using immunochemistry and immunofluorescent staining the of α-SM-actin antibody (AA132; 1:100; Beyotime Institiute of Biotechnology, Jiangsu, China). For all experiments, PASMCs between passages three and six were used, which were made quiescent by replacing the medium with FBS-free DMEM for 24 h prior to treatments.

CSE was prepared freshly for each experiment, as described previously (27). Briefly, the smoke derived from one Double Happiness cigarette (Shanghai Tobacco Corporation, Shanghai, China) was drawn slowly into a 50 ml syringe, passed through 30 ml DMEM and was repeated for five draws of 50 ml of the syringe. The resulting solution, which was expressed as '100%' final concentration, was then adjusted to pH 7.4 using concentrated NaOH, filtered (0.25 µm size) for sterilization and diluted in DMEM to the required concentration (1, 2, 5, 10 and 20%) for the experiment.

Cell proliferation was assessed using a Cell Counting kit-8 (CCK-8; Dojindo, Tokyo, Japan), according to the manufacturer's instructions. The PASMCs were plated into 96-well plates at a density of 1×10⁴ cells/ml/well and 10 µl CCK-8 reagent was added and incubated for 2 h at 37°C. The absorbance was measured at 450 nm using a microplate reader (Multiskan Spectrum; Thermo Fisher Scientific, Pittsburgh, PA, USA).

The present study also examined the BrdU incorporation to measure DNA synthesis. The cells (2×10⁵) were incubated with 50 mM BrdU for 4 h at 37°C and subsequently fixed with 4% paraformaldehyde in 0.01 M cold phosphate buffer saline (PBS; pH 7.4). The cells were permeabilized with 2% HCI containing 0.4% Triton X-100 for 15 min. Following incubation with the monoclonal mouse anti-BrdU monoclonal antibody (1:50; ab12219; Abcam, Cambridge, MA, USA) at 4°C overnight, the samples were treated with biotinylated goat anti-mouse immunoglobulin G antibody (1:100; #7056; Cell Signaling Technology, Inc., Danvers, MA, USA) for 60 min and subsequently stained with diaminobenzidene. PBS was used as a negative control by replacing the primary antibodies. The percentage of positively stained cells was determined by counting the numbers of stained cells and the total cells using a Nikon Eclipse E600FN microscope (Nikon, Tokyo, Japan).

The PASMCs were harvested by centrifugation at 200 × g for 5 min at 4°C. The pellets were washed with PBS and fixed in 70% ethanol overnight at −20°C. The samples were subsequently resuspended in PBS, containing propidium iodide (50 µg/ml), DNase-free RNase (10 µg1ml), 0.1% sodium citrate and 0.1% TritonX-100. The DNA content was analyzed using a Beckman EPICS XL flow cytometer (Beckman Coulter, Miami, FL, USA).

DNA fragmentation, a marker of apoptosis, was assessed using a TUNEL assay, as previously described (28). The cells (2×10⁵) were fixed with 4% paraformaldehyde and permeabilized for 30 min using 70% ethanol on ice. Cell apoptosis was measured using an In situ Cell Death Detection kit^® (Roche, Basel, Switzerland), according to the manufacturer's instructions. The positive cells were visualized with the Nikon Eclipse E600FN microscope and the percentage of positive cells was determined by counting the numbers of TUNEL positive cells and the total cells.

The sequence of the stealth siRNA duplex oligoribonucleotides against the rat FHL1 gene (GenBank accession no. NM_001033926.2) was 5′-UGCCAAGCAUUGCGUGAAA-3′, and its corresponding complementary strand was 5′-CUAAGGAGGUGGACAUAA-3′ (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The siRNA were transiently transfected using Lipofectamine RNAi max reagent (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions, and a negative stealth siRNA sequence was used as a control. Briefly, the siRNA and Lipofectamine RNAi max reagent were mixed and diluted in FBS-free DMEM. Following coincubation for 15 min at room temperature, the mixture was added to the PASMCs in a quiescent state and gently agitated to ensure uniform distribution. The transfection mixture was removed following incubation for 6 h at 37°C and complete medium was added for further incubation at 37°C for 48 h.

Full-length FHL1 cDNA was amplified and cloned into a pAdTrack-CMV plasmid (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. The recombinant pAdTrack-CMV shuttle plasmids containing the FHL1 gene were linearized by PacI digestion and were transfected into 293A cells using Lipofectamine RNAi max reagent to produce a recombinant adenovirus. Viruses were packaged and amplified in 293A cells and purified using CsCl (Sigma-Aldrich) banding followed by dialysis against 10 mmol/l Tris-buffered saline with 10% glycerol. The titers of virus were assayed using a p24 ELISA kit (Cell Biolabs, San Diego, CA, USA). An adenovirus bearing LacZ was obtained from Clontech (Mountain View, CA, USA) and was used as the negative control. For overexpression of FHL1, 2×10⁵ PASMCs were cultured in FBS-free DMEM, containing the appropriate multiplicity of infection of adenovirus vectors for 6 h at 37°C. The cells were then transferred into complete medium and cultured for 48 h.

The total protein was harvested for western blot analysis, as described previously (29). Briefly, the cells were washed with cold PBS three times and subsequently lysed in radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology), containing 1% protease inhibitor cocktail (Merck, Darmstadt, Germany). Following determination of the protein content using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, USA), the proteins were resolved on 8–10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were incubated with blocking buffer for 1 h and subsequently with the following primary antibodies at 4°C overnight: Mouse-anti-FHL-1 monoclonal antibody (sc-374246), mouse-anti-PCNA monoclonal antibody (sc-25280) and mouse-anti-β-actin monoclonal antibody (sc-8432) (1:1,000; Santa Cruz Biotechnology, Inc.); rabbit-anti-cyclin D1 polyclonal antibody (#2922), rabbit-anti-Ki67 monoclonal antibody (#9129) and rabbit-anti-p27 monoclonal antibody (#3686) (1:500; Cell Signaling Technology, Inc.). Following washing and incubation with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (A0216) or HRP-labeled goat anti-rabbit IgG (A0208) secondary antibodies (1:1,000; Beyotime Institute of Biotechnology) for 1 h at room temperature, the membranes were detected by a chemiluminescence system (Cell Signaling Technology, Inc.). Image quantification was performed using ImageJ software, version 1.37 (NIH, Bethesda, MD, USA).

The total RNA was isolated from the PASMCs using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. A total of 1 µg RNA was reverse transcribed using the SuperScript III First-Strand Synthesis system (Invitrogen Life Technologies). A Fast SYBR^® Green Master Mix kit (Applied Biosystems) was used for qPCR on a Fast RT-PCR system (ABI 7300; Applied Biosystems). A total of 40 cycles were performed as follows: 95°C for 15 sec, 60°C for 1 min and 72°C for 30 sec. Relative expression was determined using GAPDH as an internal control and was reported using the 2^−ΔΔCT method. The specific primer sequences used for amplification were as follows: FHL1, forward 5′-GTGCCCTTGTACTCCACGTT-3′ and reverse 5′-GTGTCCAAGGATGGCAAGAT-3′ and GAPDH, forward 5′-ATGAGCCCCAGCCTTCTCCAT-3′ and reverse 5′-GGTCGGAGTCAACGGATTTG-3′.

All data are expressed as the mean ± standard error of mean. A one-way analysis of variance or an unpaired two-tailed Student t-test was used to analyze the differences between groups. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS16.0 software. (SPSS, Inc., Chicago, USA).

To investigate the effects of CSE on the proliferation of PASMCs, PASMCs were treated with 0, 1, 2, 5, 10 or 20% CSE for 48 h and cell proliferation was determined using CCK-8 and BrdU incorporation assays. As shown in Fig. 1A and B, CSE at low concentrations of 1% significantly increased the number of cells by 19% and BrdU incorporation by 21%, compared with the control (0%). The peak increase in cell number (69%) and BrdU incorporation (78%) were observed at a concentration of 5%. Notably, CSE at high concentrations (20%) exhibited a significant inhibition on cell proliferation. Furthermore, no significant effect on cell apoptosis was observed at any of the CSE concentrations (Fig. 1C). In addition, the PASMCs were treated with 5% CSE for different durations, and cell proliferation was examined. The results from the CCK-8 and BrdU incorporation assays revealed that cell proliferation was triggered from 12 h and reached a peak at 48 h (Fig. 1D and E). Although incubation of CSE for 72 h marginally increased the percentage of apoptotic cells, no significant difference was observed compared with the control (0 h; Fig. 1F), indicating that CSE has no toxic effect on PASMCs. These data suggested that CSE induced the PASMC proliferation.

To investigate whether the expression of FHL1 correlated with the rate of cell proliferation, the effect of CSE on the expression of FHL1 was determined. As shown in Fig. 2A, 5% CSE induced the protein expression of FHL1 in a time-dependent manner. Compared with the control (0 h), the protein expression of FHL1 at 12, 24, 48 72 h was increased 1.24±0.13, 1.57±0.21, 2.45±0.34 and 2.32±0.32-fold, respectively. The protein expression of FHL1 reached the maximal expression level at 48 h, which paralleled with CSE-induced cell proliferation. To further confirm the effect of CSE on cell proliferation, the expression levels of two proliferation markers, PCNA and Ki67, were assessed. As expected, the expression levels of PCNA and Ki67 were increased following treatment with CSE in a time-dependent manner. Notably, the protein expression of FHL1 was positively correlated with the protein expression levels of PCNA and Ki67 (Fig. 2B and C). These data suggested that increased expression of FHL1 may be involved in the CSE-induced proliferation of PASMCs.

To determine whether FHL1 is involved in the CSE-induced proliferation of PASMCs, the effect of FHL1 knockdown on PASMC proliferation was determined. The silencing efficiency of the siRNA was detected using western blot and RT-qPCR analyses. Compared with the control, 20 nM FHL1 siRNA significantly decreased the protein expression of FHL1 by 81% and its mRNA expression by 75%, while the negative siRNA (Neg) caused no change in the mRNA or protein expression levels of FHL1 (Fig. 3A and B). FHL1 deficiency significantly decreased the cell number at the basal level and inhibited the increase of cell number induced by CSE (Fig. 3C). The result of BrdU incorporation revealed a similar tendency (Fig. 3D). The effect of FHL1 overexpression on cell proliferation was assessed to confirm the role of FHL1 in proliferation. Following the successful overexpression of FHL1, confirmed using western blotand RT-qPCR analyses (Fig. 3E and F), the proliferation of the PASMCs was markedly increased at the basal level and following CSE stimulation (Fig. 3G and H). Negative siRNA and Lacz transfection caused no significant alteration to the rate of PASMCs proliferation at the basal level or following treatment with CSE.

The effect of FHL1 on cell cycle status was also investigated in the present study. According to flow cytometric analysis, 5% CSE reduced the proportion of cells in the G₀/G₁ phase between 69.3 and 48.9%, and simultaneously increased the proportion of cells in the S phase between 21.3 and 38.7%, compared with the control (Fig. 4A), which suggested that CSE promoted cell cycle progression and an increase in cells entry into the S phase. However, inhibition of FHL1 significantly decreased the percentage of cells in S phase and resulted in G₀/G₁ cell cycle arrest at the basal level and following CSE stimulation, suggesting that the deficiency of FHL1 arrested the cell cycle in G₀/G₁ phase by preventing entrance into the S phase in the PASMCs. By contrast, FHL1 overexpression decreased the percentage of cells in the G₁ phase and increased the percentage in the S phase, compared with the corresponding control (Fig. 4B). To investigate the molecular mechanisms by which FHL1 knockdown arrests cells at the G₁/S transition, the expression levels of cyclin D1 and p27, which are involved in the regulation of the G₁ to S phase transition checkpoint were investigated. The results demonstrated that silencing of FHL1 markedly decreased the expression of cyclin D1 at the basal level and inhibited the CSE-induced increase of the expression of cyclin D1. By contrast, the expression of p27 was increased at the basal level and restored following CSE stimulation (Fig. 4C). The inverse results were obtained in the group overexpressing FHL1 (Fig. 4D).

To further investigate the mechanism by which CSE induced the expression of FHL1, the mRNA expression level of FHL1 was determined. RT-qPCR analysis revealed that the mRNA expression of FHL1 remained unchanged following treatment with CSE (Fig. 5A), indicating that a post-transcriptional mechanism may be involved. Since the predominant mechanism involved in the modulation of protein expression is degradation, the present study determined whether CSE altered the protein stability of FHL1. The PASMCs were treated with or without 5% CSE for 48 h and cycloheximide (CHX; 10 µg/ml), a protein synthesis inhibitor, was added at various time points (0, 16, 32 and 48 h). The results revealed that treatment of the PASMCs with CHX led to a time-dependent decrease in the protein expression of FHL1 in the two groups. Notably, the combination of CHX and CSE significantly changed the rate of FHL1 degradation, increasing the FHL1 half-life by >12 h compared with the control (Fig. 5B). These results suggested that CSE upregulates FHL1, at least in part, at the post-translational level, rather than the transcriptional level.