Culture of endothelial cells and IH exposure
Brain microvascular endothelial cells (Bend 3, B3) were purchased from the China Infrastructure of Cell Line Resource (Beijing, China), and cultured in DMEM high glucose supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO2.
B3 cells were exposed to IH until they reached approximately 80% confluence. The IH cell model was established according to our previously described protocol. Briefly, the culture medium was changed to fresh serum-free medium before the cells were exposed to IH. B3 cells grown in culture plates were placed in a Plexiglas exposure chamber, which was alternately flushed with a hypoxia gas mixture (1.5% O2, 5% CO2, and balanced N2, hypoxia phase, 600 s) or normoxia gas mixture (21% O2, 5% CO2, and balanced N2, reoxygenation phase, 300 s). The chamber was equipped with a humidifier, thermostat, and molecular sieve to maintain an inner temperature of 37℃, humidity of 45%, and relatively germ-free conditions..
EMV generation and isolation
EMV were isolated from the medium of cultured B3 treated with normoxia, intermitent hypoxia (21% O2, 1.5% O2).Samples were centrifuged at 120 g for 20 minutes at 20℃ to obtain cell-free media. MPs were then pelleted from cell-free media by centrifugation at 1500 ×g for 20 minutes at 20℃. Then samples were centrifuged at 13000 g for 3 minutes at 20℃ to obtain endothelial MVs. EMVs were confirmed and quantified by flow cytometry.
Detection of microvesicles by flow cytometry
To label EMV, resuspension solution containing MVs was incubated with PE-Annexin V (BD Biosciences, USA) for 30 minutes. As a negative control, a subpopulation of MVs was resuspended in Annexin V binding buffer lacking calcium. MVs were centrifuged and resuspended in PBS for cell treatment studies.
Treatment of PC12 cells With Endothelial Microvesicles
Microvesicles were isolated from cultured B3 in basal conditions or intermitent hypoxia conditions as described above and quantified by flow cytometry (annexin V positivity). PC12 were treated with microparticles (105 microvesicles/mL).
Western blot analysis
PC12 were harvested after co-cultured with N-EMVs or IH-EMVs, B3 were harvested for tight junction protein expression detection. Proteins were extracted from B3 cells using a protein extraction kit (Promega, Madison, WI) in accordance with the manufacturer’s instructions. Protein concentrations were measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA). Protein sample and prestained molecular weight markers (Thermo Fisher Scientific) were applied to 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA). The membranes were blocked by 5% non-fat dry milk in Tris-buffered saline and Tween 20 for 2 h at room temperature, and then incubated with primary antibodies at 4℃ overnight. The appropriate horseradish peroxidase-conjugated secondary IgG was incubated with the membranes for 1 h at room temperature. Chemiluminescence was imaged in a Chemi Doc XRS+ WB molecular imager using Image Lab software (Bio-Rad Laboratories, Herculaes, CA), and band intensity was measured by ImageJ software. GAPDH (1:1000, CST, USA ) was used to normalize protein loading. The following primary antibodies were used: Fas (1:1000, Abcam, USA), FasL (1:1000, Abcam, USA) and Caspase-8 (1:1000, Abcam, USA), Bax (1:200, Santa, USA) and Bcl-2(1:200, Santa, USA).
Immunofluorescence assay for tight junction proteins
After the experimental conditions were established, the B3 cells were cultured on coverslips, and then cultured with N-EMVs and IH-EMVs for 8h. B3 cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 15 min. Then, cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 3% bovine serum albumin in buffer for 30 min at room temperature. Subsequently, cells were incubated with primary antibodies(Claudin-5, 1:50;occludin-1, 1:50) over night at 4 °C. The appropriate FITC-labeled secondary antibody was incubated with the slides for 1 h at room temperature. Apoptosis was assessed with a TdTdUTP nick-end labeling (TUNEL) apoptosis assay kit (Roche, Indianapolis, IN). The slides were incubated with TUNEL reaction mixture for 1 h at 37°C. Finally, slides were washed and nuclei were stained with DAPI. Images were obtained using a Fluorescence microscope and Zen software.
Overexpression of miR-126 in B3 cells
The lentivirus containing murine miR-126 (Lenti-miR126) and lentivirus containing green fluorescence protein (Lenti-GFP) were obtained from GenePharma (GenePharma Co, Ltd, Shanghai, China). B3 cells were seeded in 6-well plates (1 × 105/well) for a confluence of 70%; then, lentivirus was added (at 5 × 106 infectionforming units) for 6 h, after which the serum-free medium was changed by 10% (v/v) FBS medium for 72 h. The level of miR-126 in B3 cells and EMVs were analysed by real-time PCR (RT-PCR).
Gene expression analysis
The levels of miR-126 in EPCs and EPC-MVs were analysed. Briefly, total RNA was extracted by using miRcute miRNA isolation kit according to the manufacturer’s instructions. cDNA was synthesized using miRcute miRNA First-Strand cDNA Synthesis kit. RT-PCR was conducted with miR-126 specific primers and miRcute miRNA qPCR Detection Kit (SYBR Green) (Tiangen, Beijing, China). Small nuclear RNA U6 was used as an internal control. Real-time PCR was performed with a LightCycler® 480ⅡReal-Time thermal cycler (Roche, Basel, Swiss). The cycling condition was carried out as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5s and 60°C for 20 s. The relative quantification of gene expression was presented with the values of 2–ΔΔCT method.
All statistical tests were performed with SPSS 16.0 (SPSS Inc., Chicago, IL). All data are based on at least three independent experiments and are expressed as the mean ± standard error of the mean (SEM). Results between groups were compared by Student’s t test or one-way analysis of variance. A p-value less than 0.05 was considered significant.