Cell culture
Human ADSCs and dermal lymphatic endothelial cells (LECs) were obtained from Guangzhou Cyagen Biology (Huangpu District, Guangzhou, China). ADSCs were cultured in DMEM medium (Gibco, Waltham, MA, USA) containing 10% FBS (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) under hypoxia (5% O2) or normoxia conditions (21% O2). LECs were maintained in endothelial growth medium-2-MV (EGM-2-MV, Lonza) that contains endothelial basal medium-2 (EBM-2, Lonza) and SingleQuots kit (Lonza).
Exosome isolation
ADSCs were cultured under hypoxia or normoxia conditions for 6 days and the medium was harvested for exosome isolation. The medium was centrifuged at 3,000 g for 15 min and then 20,000 g for 45 min at 4°C. The supernatant was filtered and further centrifuged at 110,000 g for 70 min at 4°C. The sample were washed once with PBS and then recentrifuged at 110,000 g for another 70 min at 4°C.
Transmission electron microscopy
Exosomes isolated from ADSCs were resuspended in PBS. A drop of exosomes was placed on the top of a copper grid. After washing once with PBS, exosomes were fixed with 3% glutaraldehyde for 15 min. The sample was washed again with PBS and incubated with 2% uranyl acetate for 5 min. Then, exosomes were visualized under a transmission electron microscope. Dynamic light scattering was used to determine the exosome size.
Uptake of exosomes
ADSC-derived exosomes were labelled with KH67 dye (Sigma, USA). LECs were seeded in 6-well plates and 2 μg KH67-stained exosomes were added. After incubation for 24 h, LECs were fixed with 4% paraformaldehyde. After stained with DAPI, LECs were analyzed under a confocal microscope (Carl Zeiss, Germany), and the location of exosomes was visualized.
CCK-8 assay
LECs (2,000 cells/well) in 100 μl medium were seeded in 96-well plate. For detecting the viability of LECs, 10 μl Cell Counting Kit-8 (CCK-8) reagent (Seyotin, China) was added to each well. After incubation for 1~4 h, the absorbance at 450 nm was determined by a plate reader.
Transwell migration assay
LECs (1× 105) in 100 μl serum-free medium were added into Transwell inserts (Corning, USA), and 600 μl completed medium with 10 μg/ml exosomes was added to the lower compartment of the chamber. After incubation for 24 h, LECs were fixed with methanol for 10 min and stained with 0.0.5% crystal violet for 15 min. The non-migrating cells were scraped with a cotton swab and the migrated cells were visualized under an inverted microscope (Carl Zeiss, Germany).
Tube formation analysis
Twenty-four-well plates were precoated with Matrigel (BD Sciences, USA) for 30 min at 37°C prior to cell seeding. LECs (2×104 cells/well) were seeded in 24-well plates and 10 μg/ml ADSC-derived exosomes were added. After incubation for 24 h, the tube number at three random field was counted using a light microscope.
Quantitative RT-PCR
LECs were treated with ADSC-derived exosomes (10 μg/ml) for 24 h. LECs were collected and used for RNA isolation. Total RNA was extracted using TRIzol reagent (Life Science, Waltham, MA, USA) and was reverse transcribed to cDNA using the PrimeScript RT-PCR kit (Seyotin, China). qPCR was performed with the qPCR Mix (Seyotin, china) on the Applied Biosystems 7300 system (Applied Biosystems, USA). The experiments were conducted in triplicate. Gene (or miRNA) expression was normalized to GAPDH (or U6) using the 2-ΔΔCT formula.
DNA and RNA transfection
miR-129 mimics and control mimics (NC mimics) were purchased from RiboBio (Guangzhou, China). HMGB1 expression plasmid and control plasmid were obtained from Hanbio (Shanghai, China). miRNA mimics and HMGB1 expression plasmid were transfected into LECs using Lipofectamine 2000 (ThermoFisher, USA) according to the manufacturer’s instructions.
Luciferase reporter assay
The wild type and mutated HMGB1 3’UTR were cloned into pGL3 plasmid to form recombinant plasmids pGL3-HMGB1-3’UTR-wt and pGL3-HMGB1-3’UTR-mut. LECs (2×105 cells/well) were seeded in 24-well plates and were cotransfected with pGL3-HMGB1-3’UTR-wt (or pGL3-HMGB1-3’UTR-mut), miR-129 mimics (or NC mimics), and pRL-TK (Promega, USA). After transfection for 48 h, the luciferase activity was determined by using a Dual-Luciferase Reporter Assay System (Promega).
Western blot
LECs or ADSC-derived exosomes were lysed using RIPA lysis buffer. The sample was separated by SDS-PAGE gel and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the PVDF membrane was incubated with primary antibody at 4°C overnight. After washing three times with TSB-Tween 20 (TBST), the membrane was incubated with the secondary antibody for 1.5 h at room temperature. After three washes with TBST, the protein bands were detected by using the Western Blot Detection Kit (Seyotin, China). The antibodies used were as follows: CD9 (ab59479; Abcam, UK), CD63 (ab59479; Abcam), TSG-101 (ab125011; Abcam, UK), HMGB1 (ab18256; Abcam, UK), LYVE-1 (ab14917; Abcam, UK), PROX1 (ab101851; Abcam, UK), GAPDH (#2118; Cell Signaling Technology, USA), AKT (#9272; Cell Signaling Technology, USA), p-AKT (#4060; Cell Signaling Technology, USA).
Statistical analysis
The data were represented as the mean ± standard deviation (SD) from three independent experiments. Statistical comparisons were performed with Student t-test (two groups) or one-way ANOVA (more than two groups). A P value lower than 0.05 was considered as statistically significant.