Animal studies
The study was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by Animal Care and Use Committee of Gansu Province people’s hospital (No. 202004FT). A total of 48 adult male C57BL/6 mice (4weeks) obtained from Shanghai SLAC Laboratory Animal Co.,Ltd (Shanghai, China) were housed under pathogen-free conditions with 12-h light/dark cycle and 22 ± 1°C and a humidity of 40–60%. Experimental PAH Model was performed as previously described [23]. The mice were randomly divided into normal group (saline) and PAH group (exosomes form vehicle, exosomes form HIF-1α). Then 50 µl exosomes (from 5 × 107 ADSCs) were intravenously injected. The mice in all the groups were killed after intraperitoneal injection with 10% chloral hydrate, and samples of lung tissues were removed histopathological examinations.
Preparation and characterization of exosomes
Exosomes were isolated from ADSCs using ExoQuick-TC (SBI, USA) according to the manufacturer’s instructions. Western blotting was performed to detect the expression of known exosomal markers anti-CD63 (#ab59479, Abcam, Cambridge, MA, USA) and anti-CD81 (#ab79559, Abcam) and the exosome morphology were observed by transmission electron microscopy (TEM) (Hitachi, H7500 TEM, Tokyo, Japan).
Adipose-derived stem cell isolation and culture
The groin adipose tissue was harvested from C57BL/6 mice ageing 4 weeks, after clearing and 0.075% collagenase digestion, then centrifugation removed the supernatant and undigested fat. The cells were resuspended in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin (P/S) at 37°C with 5% CO2. The first change of medium took place 8 h later and the medium was replaced every 2–3 days. Cells were digested and passaged after reaching 80% confluency.
Establishment of HIF-1α stable overexpression ADSCs
Full-length HIF-1α coding sequence was subcloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro (System Biosciences, Beijing, China). Lentivirus was generated in HEK293T cells and filtration with 0.22 µm filter membrane. To construct the stable cell lines with target gene overexpression, ADSCs were infected with Lentivirus for 24h, 2 µg/mL puromycin (Sigma-Aldrich, St Louis, MO, USA) was added to culture medium after transducing with lentivirus 48h later to continuously screen the stable cells for 10 days. The small interfering RNA (siRNA) targeting SIRT3 were obtained from Shanghai GenePharma Co., Ltd (Shanghai, China) and transfected using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. The in vitro preconditioning hypoxia model was established by flooding the chamber with 95% N2 and CO2 using a ProOx model 110 oxygen regulator purchased from BioSpherix (New York, NY, USA).
Cell viability assay
Cells of each group were planted in 96-well microculture plates with 1 × 104/well and cultured at 37°C with 5% CO2 overnight. 10 µl of CCK-8 solution (TAKARA, USA) was added to each well and incubated for 2 h at 24, 48, 72, and 96 h. Finally, absorbance was assessed using a microplate reader (Bia-rad, Hercules, CA, USA) at a wavelength of 450 nm.
Cell apoptosis assay
To evaluate cell apoptosis, apoptotic ratios were evaluated by the Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's recommendations. The stained cells were analyzed were analyzed by FACScan flow cytometer (Becton Dickinson, USA) and evaluated using the ModFit program software.
Evaluation of lung histological pathology and pulmonary vascular permeability
To assess the severity of lung injury, the lung tissues were harvested and fixed with 4% paraformaldehyde and embedded in paraffin and then cut into 4 µm sections. Then, the sections were deparaffinized and stained with hematoxylin-eosin (H&E). The degrees of inflammation, congestion, and edema were evaluated using lung injury score as previously described [24].
For arterial blood gas studies, partial pressure of oxygen (PaO2) and partial pressure of carbon dioxide (PaCO2) were measured from blood samples obtained from the femoral artery. The dry weight was determined, and the lung wet-to-dry weight (W/D) ratio was measured to assess the pulmonary vascular permeability. For hemodynamic studies, the PICCO system was used to evaluate the mean arterial pressure, heart rate, stroke volume variation, and cardiac output.
Measurement of Inflammatory Cytokines
Mouse blood was centrifuged at 4°C and 3000 r/min for 10 minutes, and the serum was harvested to measure the levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), vascular endothelial growth factor (VEGF) and monocyte chemotactic protein 1 (MCP-1) using ELISA kits in accordance with the manufacturer's instructions (eBioscience, San Diego, CA, USA).
Transwell invasion assays
Invasion assays of HUVECs in vitro were performed using transwell chambers with 8 µm pores (Corning Incorporated, Corning, NY, USA) coated with Matrigel (BD Bioscience, Bedford, MA, USA). Cells were harvested after the specific treatment and resuspended in serum-free medium. About 4×105 cells in 100 µl serum-free medium was added into the upper chamber, 600 µl complete medium was added into lower chamber. After incubation for 24 h, the cells were fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 15 min. The invaded cells were imaged and counted in five randomly selected fields at 200× magnification using an Olympus optical microscope (Tokyo, Japan).
Tube formation assay
After reaching 80% cell confluence, ADSCs cells were cultured with serum-free DMEM for another 24 h, and the supernatant was collected as a conditioned medium. 100 µL Matrigel was added into 24-well plates to polymerize for 2 h at 37°C. Then 2 × 105 HUVECs were seeded into coated wells with conditioned medium. Images were taken by an Olympus optical microscope 6 h later, and the numbers of tubes formed were counted to measure tubule-forming ability.
Western Blot Analysis
Total protein was extracted from cells were RIPA buffer (Sigma-Aldrich) containing Protease Inhibitor Cocktail (Cell Signaling Technology, Inc., Beverly, MA, USA). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Thermofisher, Carlsbad, CA, USA). Equal amounts of protein extracts (40 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bia-Rad). Then the membranes were blocked and incubated overnight with rabbit anti-HIF-1α (Cell Signaling Technology), rabbit anti-SIRT3 (Cell Signaling Technology), rabbit anti-SDF-1α (Cell Signaling Technology), rabbit anti-VEGF (Cell Signaling Technology), rabbit anti-Rac1 (Proteintech Group. Inc, Rosemont, IL, USA), rabbit anti-Rac2 (Proteintech Group. Inc), and mouse anti-GAPDH (Cell Signaling Technology) antibodies, respectively. The expression levels of protein were measured by enhanced chemiluminescence reagents (Millipore, Plano, TX, USA).
Statistical Analysis
Data are expressed as means ± standard deviation (SD). Each experiment was performed independently at least three times. Student’s t-test was used to compare the difference between two groups and the difference among multiple groups were determined based on one-way analysis of variance. Statistical analyses were performed using the SPSS 21.0 statistical package (SPSS Inc., Chicago, IL, USA).