5.10 Toxicity of nanofiber mats
The nanofiber mats were implanted under the abdominal skin of C57 male mice. Tissue specimens were collected 1 and 3 months after implantation and were stained with hematoxylin and eosin (H&E) to observe the inflammatory reaction of the mats on the tissue.
5.11 Viabilities of PDGF-BB-induced inflammatory, rosuvastatin-treated, and rosuvastatin-treated inflammatory SMCs
PDGF-BB was used to establish an inflammatory-SMC model. PDGF-BB was dissolved in sterilized H2O and diluted with DMEM to 0, 1, 10, 20, 50, 100, and 1000 ng/mL. Rat aortic SMCs were seeded onto 96-well plates at a density of 3 × 103 cells/well in triplicate and cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Epizyme, China). When the cells reached 50–60% confluence, SMCs were incubated in the serum-starvation condition for 24 h. Cell viability was measured using the CCK-8 assay. The absorbance was measured at 450 nm using a spectrometer.
The toxicity of rosuvastatin on contractile SMCs was further evaluated. Similarly, cells were seeded into 96-well plates at 3 × 103/well in triplicate and further subjected to starvation conditions for 24 h. Rosuvastatin was dissolved in dimethyl sulfoxide (DMSO; Simga-aldrich, Merck, Germany) and diluted with DMEM to 0, 0.01, 0.1, 1, 10, 20, and 100 µM. Cell viability was measured with the CCK-8 assay, as previously described in this section.
The effective rosuvastatin concentration that inhibited PDGF-BB was further explored. Cells were seeded and starved under the same conditions previously described in this section. After 24 h, cells were treated with a mixed solution of 10 ng/mL PDGF-BB and different doses of rosuvastatin (0, 0.01, 0.1, 1, 5, 10, 20, and 100 µM for 24 or 48 h. Cell viability was again measured using the CCK-8 assay kit.
5.12 Phalloidin assay
SMCs were stimulated with 10 ng/mL PDGF-BB for 24 h, seeded on nanofiber mats at a density of 2 × 104/well, and cultured for 48 h. The cultured cells were fixed with 4% PFA overnight and stained with phalloidin solution for 60 min according to the manufacturer’s instructions. The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Bioss, China) for 10 min. Similarly, the SMCs were seeded into 24-well plates at a density of 1.5 × 104/well and were cultured for 24 h. The cultured SMCs were then starved for 24 h. The cells were fixed and stained with phalloidin solution (Meilunbio, China) and DAPI, as previously described in this section. A confocal fluorescence microscope was used to observe the cytoskeleton.
5.13 Cell proliferation assay
SMCs were seeded into 24-well plates at a density of 2 × 104/well in triplicate. The cells were starved prior to the intervention and were divided into groups as follows: the control and SMCs stimulated with 10 ng/mL PDGF-BB (PDGF), treated with 10 µM Rosuvastatin (Rosu), and cotreated with 10 ng/mL PDGF-BB and 10 µM rosuvastatin (PDGF + Rosu). Each group was cultured for 24 h, after which an EdU cell proliferation detection kit (Beyotime, China) was used to evaluate the proliferation rate, according to the manufacturer’s instructions. The cells were counterstained with Hoechst 33342 (Beyotime, China) for 10 min and then observed with a focused fluorescence microscope. Hoechst 33342 and EdU showed blue and green fluorescences, respectively. ImageJ software used to count the Hoechst-33342- and EdU-labeled cells, and the EdU/Hoechst-33342 ratio was then calculated.
5.14 Scratch test
Rat aortic SMCs were seeded into six-well plates at a density of 1 × 105/well in triplicate. When reaching 80% density, the cells were cultured in serum-free DMEM for 24 h. Pipette tips were used to scratch the cell monolayer across the center of each well. The detached cells were washed away with sterile PBS. The cells were divided into control, PDGF, Rosu, and PDGF + Rosu groups. Each group was cultured for 24 h. Three images were photographed for each well and were processed with ImageJ software. The areas in the images where cells remained after 24 h were compared.
5.15 Transwell tests
SMCs were seeded into a 12-well plate at a density of 5 × 104/well in triplicate. The cells were starved prior to the intervention. The cells were then digested and seeded into 8-µm-pore transwell chambers (Corning, NY) at a density of 1 × 104/well. Then, 600 µL of DMEM containing 10% FBS was added to the lower chamber in which the cells were incubated at 37°C for 6 h. The upper chambers were replaced with serum-free DMEM and incubated for another 18 h. The cells were then fixed with 4% PFA for 30 min at room temperature and then with crystal violet (Beyotime, China) for 5 min. The cells attached to the interior of the upper chamber were removed with a cotton swab, and the chambers were placed on a new 24-well plate and were air-dried for 15 min. The number of cells on the surface of the lower chamber were both calculated and photographed under a microscope.
5.16 Cell apoptosis of rosuvastatin-treated contractile SMCs
An Annexin V-FITC cell apoptosis detection kit (Beyotime, China) was used to detect the effect of rosuvastatin on contractile SMCs, according to the manufacturer’s instructions. Flow cytometry (BD, New Jersey, USA) was used to detect Annexin V-FITC and propidium iodide (PI) showing green and red fluorescences, respectively.
5.17 RNA extraction and real-time polymerase chain reaction
The cellular ribonucleic acid (RNA) was isolated using an RNA purification kit (Yishan, China) according to the manufacturer’s instructions. The RNA integrity was quantified using a NanoDrop™ 1000 spectrophotometer (ThermoFisher Scientific™, UT, USA). The reverse transcription (RT) reaction was performed using a fast all-in-one RT kit (Yishan, China) according to the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was performed using the Hieff® qPCR SYBR® Green master mix (Yeasen, China) and was detected with a real-time PCR system (7900HT, ABI). No nonspecific amplification was observed based on the dissociation curve. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sangon biotech, China) was used as an internal control. The data were further analyzed by the comparison Ct (2−ΔΔCT) method and were expressed as a fold change relative to the respective control. The sequences used for the qPCR primers are listed in Table S1.
5.18 Western blot
Proteins were lysed from the rat aortic SMCs after the intervention. The cells were divided into 4 groups, as described in above experiments, and 40 µg of protein per lane was separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; Epizyme, China). The proteins were electrotransferred onto a polyvinylidene difluoride (PVDF; Millipore Sigma, Billerica, MA, USA) membrane and blocked with a western quick-blocking buffer (Beyotime, China) for 15 min at room temperature. The blocked PVDF membranes were incubated overnight with primary antibodies including goat anti-SM22α (1:500 dilution; Abcam, England), rabbit anti-OPN (1:1000 dilution; Abcam, England), and rabbit anti-MMP9 (1:1000 dilution; Abcam, England). After 12–14 h, the PVDF membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody(Huabio, Chia) for 1 h at room temperature. The immunoblots were probed using an enhanced chemiluminescence (ECL; Thermo, Rockford, IL, USA) substrate. An imaging system (Bio-Rad, Hercules, CA, USA) was used to detect the blots, and the chemiluminescence levels were recorded. The results were normalized to that of GAPDH, and the experiments were replicated three times.
5.19 Cytokine and chemokine analyses
SMCs were seeded into a 24-well plate containing PBS, Rosu 50, Rosu 75, and Rosu 100 nanofiber mats at a density of 1 × 104/well. The cells were cultured for 48 h, and the cellular supernatants were collected and used for interleukin-1 β (IL-1β ), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), fractalkine (CX3CL1), and RANTES (CCL5) testing with a MILLIPLEX® MAP Rat cytokine/ chemokine factor panel (Millipore, Billerica, MA). Similarly, SMCs were transferred into a 24-well plate at a density of 2×104 cells/well, and the cells were treated as previously described in this section. DMEM was used as a control. At 24 h, the culture supernatants were collected and tested for IL-1β , IL-6, MCP-1, and TNF-α using a MILLIPLEX® MAP Rat CVD Panel (Millipore, Billerica, MA).