Ambient particulate matters and preparation of organic components
Collection of ambient particulate matter (PM) at Seoul metropolitan area during summer (SPM) and winter (WPM) season and particle preparation was described in the previous study [52], which showed the detailed process for the sampling of PM10 and extraction of organic matter from the PM. The analyzed organic compounds including PAHs and oxy-PAHs and their concentrations were presented in the previous study [20].
Isolation of VSMCs
All animal experiments for the isolation of rat aortic VSMCs were conducted in accordance with the International Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Research Committee of the Chosun University School of Medicine (Protocol No. CIACUC2020-S0032). Rat aortic VSMCs were isolated from 6-week-old Sprague-Dawley rats as described previously [53]. Briefly, the removed aorta was freed from connective tissues and blood clots, then severed and transferred into a tube containing a mixture of collagenase type I (1 mg/mL, Sigma, St. Louis, MO, US) and elastase (0.5 mg/mL, Worthington, NJ, US) and incubated for 30 min at 37°C. The aorta was placed into a 100-mm cell culture dish and the adventitia was stripped with forceps under a binocular microscope. Each piece of the aorta was transferred into a tube containing 5 mL of enzyme dissociation mixture (containing collagenase and elastase), and the tubes were incubated for 2 h at 37°C. The dispersion of the tissue was accomplished by slow pipetting. The suspension was centrifuged (1600 ×g for 5 min), and the obtained pellet was resuspended in DMEM with 10% fetal bovine serum (FBS, WelGENE, KOREA). This dissociation step was repeated until tissues were completely dispersed. The cells were cultured at 37°C in an incubator with a humidified atmosphere of 95% air and 5% CO2 and used for up to 10 passages in this study.
Measurement of cytotoxicity and cell proliferation
Cytotoxicity was determined by MTT assay using a CellTiter 96 Assay kit (Promega) according to the manufacturer’s instructions. Briefly, VSMCs were seeded into a 96-well plate in triplicate and cultured for 12 h, and then cells were treated with PAHs or oxy-PAHs for 24 h. The absorbance was measured at 490 nm using an ELISA reader (TECAN, infinite M200 PRO). The proliferative rates of VSMCs were evaluated by determining BrdU incorporation using the BrdU Cell Proliferation Assay kit (Cell signaling). VSMCs were treated with seasonal ambient PM, PDGF-BB (20 ng/ml), PAHs, or oxy-PAHs for 24 h. Then cells were incubated with BrdU for 4 h and subsequent procedure was performed according to the manufacturer’s instructions. The absorbance was measured at 450 nm using an ELISA reader (TECAN, infinite M200 PRO).
Cell migration assay
Cell migration was examined by three-dimensional Boyden chamber assay and two-dimensional wound healing assay. For Boyden chamber assays, cells (5×104 cells in 100 μl) were placed in the upper compartment of the transwell chambers coated with collagen I on the lower surface. Ambient PM or PAHs were treated with the designated concentration in each upper compartment with designated concentration. After incubation for 16 h at 37°C, cells on the lower surface of the filter were fixed and stained, and five random fields/membranes were counted at ×200 magnifications. For wound healing assay, a rectangular lesion was created using a cell scraper, and then the cells were incubated with a designated concentration of ambient PM or PAHs for designated times. The distance from the margin of the lesion to the most migrated cells was measured, and the mean value of the distances was taken as the mobility of cells in each culture dish.
Measurement of intracellular reactive oxygen species (ROS)
Intracellular ROS were measured using the fluorescent dye technique. VSMCs were seeded into a 24well plate with glass coverslips at a density of 5×104 cells/ml and cultured for 24h. Then, cells were treated with negative control (DMSO), positive control (200 nM of H2O2), ambient PM, or PAHs in a dose dependent manner for 1 h. Then, cells were washed twice with calcium-free PBS (PBSc) and loaded with 2’,7’-dichlorofluorescein diacetate (H2DCF-DA, Invitrogen, USA) and 4’,6-diamidino-2-phenylindole (DAPI) diluted with calcium-free warm PBS to a final concentration of 10 μM and 50 μg/ml, respectively. After, cells were incubated for 10 min at 37℃ in dark. The probe H2DCF-DA (10 μM) entered into the cells, and the acetate groups on the H2DCF-DA were cleaved by cellular esterases, trapping the nonfluorescent 2’,7’-dichlorofluorescein (DCFH) within the cells. Subsequent oxidation by reactive oxygen species yielded the fluorescent product DCF. Then, cells were gently washed the coverslips three times in warm PBS and the coverslips were placed in the chamber, which was mounted on the stage of an inverted microscope equipped with a confocal laser-scanning system. The dye, when exposed to an excitation wavelength of 480 nm, emitted light at 535 nm only when it had been oxidized. Fluorescence images were collected using a confocal microscope (Fluoview FV1000 confocal system, Olympus) by excitation at 488 nm and emission greater than 500 nm with a long-pass barrier filter. The fluorescence intensity of an equivalent field size (3×3 mm) in the plate was measured using the Image J quantification software.
Zymography
Briefly, aliquots of the control and test media were electrophoresed on a 10% SDS-polyacrylamide gel containing 0.8% gelatin or 0.3 mg/ml collagen. Gels were washed with 2.5% Triton X-100 to remove SDS for 1 h, washed with D.W for 1hr and then incubated at 37°C for 48 h in developing buffer (gelatin incubation buffer : 50 mM Tris-HCL, pH 7.5, 5 mM CaCl2, 1 mM ZnCl2, 0.02% sodium azide, 1% Triton X-100 or collagen incubation buffer : 50 mM Tris-HCL, pH 7.5, 10 mM CaCl2, 50 mM NaCl, 0.05% Brij35, pH 7.6). After 48 h, the gel was stained with 1% Coomassie blue for 1 h and later, then destained until there is a good resolution between the bands and blue background. The gel was photographed and zymolytic area was quantified using Image J software (NIH, Bethesda, MD, US).
MMPs promoter luciferase activity assay
For the luciferase assay, the reporter constructs of rat MMP2 (GenBank: DQ915967.1) and rat MMP9 (GenBank: AF148065.1) promoter were cloned into the pGL3 basic vector (Promega). VSMCs were transiently transfected with a total of 100 ng each of the luciferase reporter constructs using Lipofectamine 3000 (Invitrogen). To ensure efficient transfection, all wells were also co-transfected with a Renilla luciferase vector (pRL-TK; Promega). After 24h transfection, the cells were treated with either negative control (DMSO), SPM, WPM, PAHs, or oxy-PAHs for 12h. Luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) as described in the manufacturer’s protocol. Data were normalized by internal Renilla luciferase activity.
Quantitative real-time PCR (qRT-PCR)
The expression levels of the various genes were analyzed by qRT-PCR. Cells were seeded into a 6well plate with glass coverslips at a density of 5×105 cells/ml and cultured for 24 h. Cells were treated with negative control (DMSO), ambient PM, or PAHs for 12 h. Total RNA was extracted using TRIzol lysis reagent (QIAGEN) according to the instructions provided by the manufacturer. The RNA concentration of each sample was measured by a spectrophotometer (Eppendorf) at 260 nm. Total RNA was subjected to reverse transcription using HelixCript™ 1st-Strand cDNA Synthesis Kit (NanoHelix). Real-time quantitative PCR with realHelix™ qPCR kit (NanoHelix) was performed by the SYBR Green method using an Applied Rotor-Gene 3000TM. Gene expression was normalized to GAPDH. The relative mRNA expression levels were quantified and analyzed using Rotor-Gene 6 software (Corbett-research) using △△Ct methods. Table 1 was primer sequences for qPCR.
Immunoblot analysis
VSMCs were seeded into a 6well plate at a density of 5×105 cells/ml and cultured for 24 h. Cells were treated with negative control (DMSO), ambient PM, or PAHs in a dose dependent manner for 24 h. Cells were washed once in PBS and lysed in RIPA buffer containing PMSF and phosphatase inhibitor. Protein concentrations were determined using the Bradford protein Assay. Proteins were separated in a 6-10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). After membrane blocking with Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween 20) containing 5% skim milk for 1 h at room temperature, the membrane was incubated with primary antibody overnight at 4°C. The primary antibodies were used at the following dilutions in blocking buffer: MMP2 (1:4000), MMP9 (1:1000) (Abcam, Cambridge, MA, US), FAK (1:1000), p-FAK(Y397) (1:500), p-FAK(Y925) (1:500), Src (1:4000), p-Src (1:2000), Akt (1:4000), p-Akt (1:2000), ERK (1:3000), p-ERK (1:5000) (Cell Signaling, Beverly, MA, US), MMP13 (Novus, Centennial, CO, US, 1:1000), β-actin (Sigma, MO, US, 1:5,000,). The membrane was washed five times with TBS-T for 5 min and incubated for 1h at room temperature with secondary antibodies. After extensive washing, bands were detected by enhanced chemiluminescence reagent (ECL, BIONOTE, Animal Genetics Inc.). Band intensities were quantified using the Image J quantification software.
Statistical analysis
All quantified data from at least triplicate samples were analyzed with GraphPad Prism 8.0 software. Data are expressed as mean ± SD. Statistical comparisons between the two groups were performed using Student's t-test. Statistical comparisons among multiple groups were performed using one-way ANOVA followed by Bonferroni post hoc test when the F statistic was significant. A two-tailed P < 0.05 was considered statistically significant.