Animals
Ninety male Sprague-Dawley (SD) rats (200 to 250 g) were used in the current study. All animal care and experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Xi'an Jiaotong University Animal Experiment Centre. All protocols used in this study were approved by the Laboratory Animal Care Committee of Xi'an Jiaotong University. The rats with similar baseline characteristics were randomly divided into six groups: control group (Con group, n = 15), monocrotaline (MCT)-treated group (MCT group, n = 15), MCT and vehicle (dimethylsulfoxide, DMSO)-treated group (MCT + vehicle group, n = 15), MCT and thiostrepton (TRT)-treated group (MCT + TRT group, n = 15), MCT and 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)-treated group (MCT + Y15 group, n = 15), and MCT and chloroquine phosphate (CQ)-treated group (MCT + CQ group, n = 15). All rats were housed in a 12h light-dark cycle at 22 ± 2°C and free accessed to food pellets and tap water.
Generation of PAH models and drug treatment
MCT (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.1 mol/L HCl, the solution was then titrated to pH 7.4 with 0.1 mol/L NaOH with the final concentration of 30 mg/mL. Thiostrepton (Selleckchem, USA) were dissolved in DMSO, and then diluted with 0.9% NaCl to a final concentration of 10 mg/mL. DMSO was diluted with 0.9% NaCl to a final concentration of 0.5% as a vehicle for thiostrepton. Y15 (Selleckchem, USA) and CQ (Aladdin Bio-Chem Technology Co., Shanghai, China) was dissolved in 0.9% NaCl with the final concentration of 7.5mg/mL and 20 mg/mL.
The PAH model was established by intraperitoneally (IP) injection of MCT (60 mg/kg) on day 1. Once PAH was established 2 weeks post MCT injections, FOXM1 inhibitor thiostrepton (20 mg/kg) or its vehicle was administrated to the rats by IP injection every day for 2 weeks. After the establishment of MCT-induced PAH rat model, FAK inhibitor Y15 (15 mg/kg) or CQ (40 mg/kg) were daily administered to the rats by IP. The healthy control rats were IP injected with the same volume of vehicle solution (DMSO or 0.9% NaCl).
Measurement of RVSP and RVH
At the endpoint of the study, all survived rats were anesthetized by a spontaneous inhalation of isoflurane. After stable anesthesia, we isolated the right internal jugular vein of rats, and then inserted a polyethylene catheter into the right ventricle (RV). A Grass polygraph was used to detect the right ventricle systolic pressure (RVSP). Then hearts and lungs of rats were excised. The RV was divided into the left ventricle (LV) and interventricular septum (S), and then each part was weighed separately. The ratio of the weight of RV to the LV plus S [RV/ (LV+S)] was calculated to assess the index of right ventricular hypertrophy (RVH).
Histologic analysis
Harvested pulmonary tissues from marginal right lower lobes were immersed in 4% paraformaldehyde, and then embedded in paraffin. Tissue blocks were cut in a thickness of 5 mm, and then stained with hematoxylin and eosin (HE). The medial wall thickness (%MT) of vessels (20–70 μm diameters, n = 15 per rat) was observed using a light microscope to assess pulmonary arterioles vascular remodeling. The %MT was calculated as follows: %MT = (2 × medial wall thickness) ×100/external diameter.
Immunohistochemistry
To further assesse the pulmonary arterial muscularization and cell proliferation, we used immunofluorescence. Lung sections of rats from each group were dewaxed and dehydrated, followed by antigen retrieval and proteinase digestion. And then, the sections were incubated with anti-α-SMA (BM0002, Boster, CA, USA, 1:200 dilution), anti-Ki67 (YM3064, Immunoway, TX, USA, 1:200 dilution) and anti-LC3B antibodies (18725-1-AP, Proteintech, Chicago, IL, USA, 1:200 dilution). Lung tissue sections were soaked in fluorescence-conjugated secondary antibodies, and then mounted with neutral resin.
Western blot analysis
Harvested lung tissues were immersed in RIPA lysis buffer (HEART, Xi'an, China), and the concentrations of protein were measured by BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Equal amounts of protein were resolved and separated on 8%–15% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked with nonfat dry milk (5%, w/v) for 1h, and then incubated overnight at 4°C with the specific primary antibodies against β-actin (YM3028, Immunoway, TX, USA, 1:1000 dilution), FOXM1 (no 32671, Signalway Antibody, Pearland, TX, 1:1,000 dilution), LC3B (18725-1-AP, Proteintech, Chicago, IL, USA, 1:800 dilution), FAK (12636-1-AP, Proteintech, Chicago, IL, USA, 1:800 dilution) and p‐FAK (sc‐374668; Santa Cruz, 1:600 dilution). After washing blots three times in PBST, membranes were soaked in horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (AP156P or AP127P, Sigma-Aldrich, St. Louis, MO, USA, 1:5000) for 1 hours. Blots were visualized by the enhanced chemiluminescence detection system (Amersham Bioscience), and the band densities were measured using Quality One software (Bio-Rad).
Statistical analysis
Experimental data are presented as means ± standard deviation. Group comparisons were analyzed using one-way analysis of variance followed by a Tukey post hoc test. P < 0.05 was considered statistically significant.