Animal
All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Also, all experiments were approved by the Institutional Animal Care and Use Committee of Shandong University. The monocrotaline-induced PH (MCT-PH) rat model has been well-established extensively. All animals were anaesthetized by isoflurane inhalation (1.5-2%) and then euthanized by cervical dislocation.
Preparation of exosomes
Healthy pregnant women umbilical cord collection from obstetrics of our hospital. We stated that the experiment process conform to the principles outlined in the declaration of Helsinki and investigators have obtained the informed written consent before enrolling participants in clinical trials. Isolation of MSCs from human umbilical cord (hUCMSCs) Wharton’s Jelly with some modification[10].The immunotyping characterization of hUCMSCs occurred at passage 3-4 using human specific antibodies CD34, CD45, CD73, CD90, CD105, HLA-DR (BD Biosciences Pharmingen, San Diego, CA) by a fluorescence-activated cell sorter (FACS, BD FACSAria II). Adipogenic, osteogenic and chondrogenic differentiation capacity ofT of hUCMSCs was performed by special medium (Cyagen US Inc), and then stained with Alizarin Red-S,Toluidine blue and Oil Red-O, respectively. The plates were seen using microscope, and images were taken from them.
When reached 90% confluences, the adherent cells were incubated in DMEM with 5% exosome-depleted FBS for 24 h, and 5-8 passages hUC-MSC were used for experiments. The conditioned medium was centrifuged at 4°C at 300 g for 10 min at 2000 g for 10 min and finally at 10 000 g for 30 min to remove the cells and debris, followed by centrifugation of the supernatant at 100 000 g at 4°C for 1 h. hUC-MSC-EXO was resuspended in PBS and filtered with a 0.22 µm microfiltration membrane, centrifuged again in PBS at 100,000 g for 1 h to collect the exosomes. The protein concentration of hUC-MSC-EXO was determined using a bicinchoninic acid (BCA) assay kit.
The morphology of MSC-EXO was examined using a transmission electron microscope (TEM) according to the manufacturer’s instructions. Briefly, the prepared exosomes were stained with phosphotungstic acid solution and then performed under a Hitachi-9000 TEM system. Western blotting was used to detect the protein markers of hUC-MSC-EXO, such as CD63, CD81, TS101 and ALIX.
Animals experimental design
Sprague-Dawley rats PH model was established by a single subcutaneous injection of MCT(60 mg/kg; Sigma, St. Louis, MO, USA) [15]. Three weeks later, the animals received were given at a dose of 50 µg/day hUC-MSC-EXO or an equal volume of culture medium (hUC-MSC-CM) via tail vein injection once daily for 3 days, starting 1 weeks after the last series MCT injections[5]. The protein concentration of hUC-MSC-EXO was determined by a BCA assay kit, and 50 μg protein in 100 μl PBS was used. The rats were randomly divided into 4 groups (n=10): Control, MCT-PH, hUC-MSC-EXO, and hUC-MSC-CM groups. The animals were evaluated at 4 weeks after MCT injection.
Hemodynamic and the right ventricular hypertrophy assessment
Post-operation, all animals were anaesthetized by isoflurane inhalation (1.5-2%) and then euthanized by cervical dislocation. Hemodynamic data were recorded eight weeks after operation as previously described with some modifications. Via femoral vein access, a 5F Swan-Ganz catheter (Edwards Lifesciences Corp, Irvine, CA) was advanced into the pulmonary artery for determination of heart rate (HR), systemic blood pressure (SBP) and right ventricular systolic pressure (RVSP). For assessment of right ventricular hypertrophy, the left ventricle (LV) plus the septum (LV+S) were harvested, and the weight ratio of the RV to LV+S weight calculated to quantify the right ventricular hypertrophy. The index by the formula: RV/(LV+S)×100.
Histology and immunology analysis
Post-operation, the lung and heart were quickly harvested and fixed in 4% paraformaldehyde and embedded in paraffin, the serially sectioned at a thickness of 4-5 µm were stained with hematoxylin-eosin (H&E). To evaluate pulmonary artery structural remodeling, the vascular wall thickness (WT), vascular external diameter (ED), vascular wall area (WA) and total vascular area (TA) to calculate WT% (WT/ED) and WA% (WA/TA) were measured as our previously[6,7]. Fibrosis area of heart and lung were analyzed by Masson’s trichrome staining, and then the sections were captured as digital images. Images were taken with an Eclipse 90i microscope (Nikon, Tokyo, Japan).
Immunofluorescence were used to analysis the expression of CD31 and a-SMA. Briefly, after blocking with 5% bovine serum albumin for 30 min at room temperature, the lung sections were incubated overnight at 4°C with anti-CD31 (AF3628) and α-SMA (ab21027) antibodies. Then, sections were further incubated with a antibody for 2 h at room temperature. Subsequently, the sections were followed by 1-h incubation in the dark with florescence isothiocyanate-conjugated secondary antibody. Images were taken with ZEISS LSM800 confocal microscope (Tokyo, Japan). All experiments were performed by two examiners blinded to treatment assignment.
Cell experiment
Rats PASMCs were purchased from Procell Life Science&Technology Co,.Ltd. (Wuhan, China), and cultured in special culture medium (Procell, China) supplemented with 100 Ug/ml of penicillin, 100 IU/ml streptomycin, and 10% (vol/vol) fetal bovine serum (FBS) at 37°C in a humidified normoxia condition (21% O2, 5% CO2,74% N2) or a hypoxic cells incubator in hypoxia condition (3% O2, 5% CO2, 92% N2). At different time points, hypoxia-induced pulmonary vascular smooth muscle cells (H-PASMCs) were treatment with hUC-MSC-EXO (100 μg/ml).The experimental were randomly divided into 3 groups: Normal, H-PASMCs, and hUC-MSC-EXO groups.
Cell proliferation assays
Cell proliferation was monitored using a cell counting kit (CCK)-8 assay, Briefly, PASMCs were seeded into 96-well plates at about at a density of 5×103 cells/well. The cells were subjected to hypoxia with or without hUC-MSC-EXO at different time points, and then CCK-8 reagent (10 µl) was added to each each well and further incubated for 3 h. The absorbance was measured at 450 nm in a spectrophotometer. Analysis of cellular deoxyribonucleic acid (DNA) content discloses frequencies of cells in G0/1, S and G2/M phase by Flow Cytometry used Cell Cycle and Apoptosis Analysis Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions.
Real-time PCR analysis
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to detect the relative expression of CD31 and a-SMA using genespecific primers as described previously. Briefly, total RNA in lung tissues or PAEC was extracted using RNeasy kit (Qiagen, Valencia, CA). ABI Prism 7900 sequence detection system software (version 2.2) was used to analysis the data were, and β-actin was used as an internal control for input RNA. The primers were designed by the Primer Express software package.
Western blot analysis
The tissues and cells protein concentration was detected using a BCA assay kit, lysates were separated by polyacrylamide gel electrophoresis (PAGE) and electro-transferred onto a polyvinylidene fluoride (PVDF), the embranes were blocked in 5% skimmed milk-Tris-buffered saline plus Tween-20 solution and incubated with primary antibodies, respectively, overnight at 4°C. The primary antibody-labeled membranes were then treated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody to IgG at room temperature for 1.5h. The bound antibodies were visualized by using an enhanced chemiluminescence reagent (Millipore, Billerica, Ma, USA) followed by Bio-Rad Image Lab™. Data was expressed as the relative density of the protein normalized to GAPDH. Primary antibodies of CD9 (ab92726), CD63 (Invitrogen,10628D), CD81(MA5-32333), TSG101(MA5-32463), ALIX(ab186429), α-SMA (ab21027), BMPR2 (ab170206), NF-κB-p65 (ab16502), p-NF-κB-p65 (ab86299), BMP4 (ab39973), BMP9 (ab35088), ID1(ab168256), Gremlin (sc-18274), PCNA (ab92552), Cyclin D1(MA5-15512), P27Kip1(ab32034), and GAPDH (ab181602) were used, respectively.
Statistical analysis
All data are expressed as mean±SD. Comparisons of parameters between 2 groups were made with unpaired Student t test. Comparisons of parameters among 3 groups were made with one-way analysis of variance (ANOVA), followed by the Scheffe post hoc test. Statistical analysis was carried out by using the SPSS 19.0 software. P<0.05 was regarded as significant statistical difference.