PASMC isolation and culture
Primary PASMC was isolated from small pulmonary arteries with a diameter of 500-1500μm from healthy donors according to previous reports. Briefly, the pulmonary arterioles were separated from the healthy donors and the surrounding connective tissue was removed. The arterioles were cut along the longitudinal axis of the blood vessels, and the intima and outer membrane tissues were gently scraped with a blade. The fragments were cut into 1-3mm2, and the inner side was attached to the cell culture dish at 37°C and 5% CO2. 1.5 hours later, smooth muscle cell culture medium (SMCM) (ScienCell, Rockville, MD, USA) contained fetal bovine serum (3%) and 100 U/mL penicillin/streptomycin was added, and when the cells covered the whole dish, then the cells were digested and transferred to a new culture dish. These cells were detected by α-SMA staining. The cells were used from 5 to 8 passages. Cells were treated with PCSK9 monoclonal antibody or 3% oxygen concentration
Experimental PAH modeling
This research protocol was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University and was conducted by the Guidelines for The Care and Use of Laboratory Animals (National Research Council). Mice (8-10 weeks, 20-25g, male) were given a single intraperitoneal injection of SU5416 (VEGF receptor inhibitor, 20mg/kg, weekly, subcutaneously) and then exposed to a hypoxic environment (10% O2 and 90% N2) on 4 weeks to establish a hypoxic PAH model. The cabin is open once a week for cleaning and changing food and water. Mice were exposed to normoxic conditions (21% oxygen) with the same 12-hour light / 12-hour dark cycle as hypoxic mice. The PAH mice were subcutaneously injected with PCSK9 monoclonal antibody (alirocumab, 20mg/Kg, once two days, Sanofi, France).
Western blotting
Western blotting follows the previous study. In simple terms, cells and tissues are cleaved in lysis buffers (RIPA buffers contained protease inhibitor complexes and phosphatase inhibitors (Beyotime, China)). After determination of protein concentration, these proteins were separated by SDS‐PAGE and transferred to the PVDF membrane. PVDF membranes containing proteins were incubated with primary antibodies overnight, followed by secondary antibodies for 2 hours. Finally, the PVDF membranes were visualized using chemiluminescence and Syngene bioimaging equipment (Syngene, Cambridge, UK), and the immune response band density was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
Immunofluorescence assay
Frozen lung tissue sections and cells were washed with frozen PBS three times. Then, they were immobilized with 0.5% Triton X-100 at room temperature with 4% permeability for 15 min. The non-specific sites were sealed with PBS containing 5% bovine serum albumin (BSA) for 30 min at room temperature and then incubated with PCSK9 primary antibody (Santa Cruz, 1:200), Notch3(Abcam, 1:50), and α-SMA antibody (1:200) at 4℃. After incubation overnight, the slices were washed and incubated at room temperature for 2 hours with their respective secondary antibodies. The nucleus is restained with DAPI (blue). Photographs were taken with a fluorescence microscope (original magnification 400×, Zeiss, Germany)
Histological analysis
Animal lung tissue was fixed in 4% paraformaldehyde solution for 48 hours and embedded in paraffin. Paraffin-embedded lung tissue was cut into 5 μm thick sections and stained with hematoxylin and eosin (H&E). Photographs were taken with a light microscope (original magnification 200 ×, Nikon, Tokyo, Japan)
PASMC proliferation and migration assay
PASMC migration was assessed by wound healing test and PASMC proliferation (Haimen Beyotime, China) was assessed by 5-ethynyl-2 '-deoxyuridine (EdU) test, according to manufacturer's protocol.
Echocardiogram
After successful modeling, mice were anesthetized with isoflurane. To evaluate cardiac function, the Vevo 2100 system (Fujifilvisualsonics, Inc., Toronto, Canada) and high frequency (30mhz) ms-400 transducer were used in the animal center laboratory of Nanjing medical university. Right ventricular diastolic diameter (RVID, D), right ventricular anterior wall (RVAW), and pulmonary artery velocity time integral (PAVTI) were measured.
Right ventricular systolic pressure and right ventricular hypertrophy index measurement
Right ventricular systolic pressure (RVSP) was measured by right heart catheterization as described earlier. Mice were anesthetized by inhalation of 1% isoflurane. Under ventilator anesthesia, mice with left anterior cardiac region ribs were cut to expose the heart. The probe is then inserted into the right ventricle to record the waveform. Right ventricular Hypertrophy Index (RVHI) is defined as the ratio of Right ventricular weight to left ventricular and interventricular septum weight.
Statistical analysis
All continuous variables were expressed as mean ± standard deviation (SD). Student paired or unpaired T-tests were used to assess the statistical significance of differences between the two groups. Univariate analysis of variance (ANOVA) was used to compare differences between groups. Graphpad Prism 8.0 software was used for statistical analysis. P < 0.05 was considered statistically significant.