Clinical specimens and cell lines
A total of 25 patients with CRSwNP were enrolled in this prospective clinical study from the Affiliated Hospital of Nantong University (Nantong, China). The diagnosis of CRSwNP was made to the criteria established by the European Position Paper on rhinosinusitis and nasal polyps guidelines (1). Each patient underwent routine workup before operation, including a medical interview, a physical examination, anterior rhinoscopy, nasal endoscopy, a computerized tomography (CT) scan, blood tests, and skin prick tests (SPT). No patient had a history of allergic rhinitis, asthma, or aspirin sensitivity, and none had received systemic and/or topical nasal steroids treatment at least 3 weeks before surgery. A total of 15 healthy volunteers were recruited to collect their NLF.
HUVECs (ScienCell Research Laboratories, Inc., San Diego, CA, USA) were cultured in DMEM low glucose (HyClone, Logan, UT, USA) and incubated at 37°C containing 5% CO2. Written informed consent was obtained from all the NPs patients and healthy volunteers, and the study was approved by the Medical Ethics Committee of the Affiliated Hospital of Nantong University.
NLF collection
NLF was collected from NP patients and healthy volunteers respectively. We used an established method with minor adjustments (27). In brief, 5 ml of 0.9% saline was instilled in right nostril of the person while the head leaned back at an angle about 30° and the soft palate closed. The NLF was obtained by bending the head forward and passively collecting the fluid. And then the procedure was repeated in the left nostril. The NLF we collected was then passed through a cell strainer (100 µm) and promptly centrifuged (3,000 × g, 20 min, 4°C) to remove crust and debris. The supernatant of NLF was stored at −80°C before exosomes isolation.
Exosomes isolation and purification
Exosomes were isolated from NLF of NP patients (NPs exosomes) and healthy volunteers (normal exosomes) using a previously described method (28,29) with the modifications that included differential centrifugation of NLF (6,000 × g for 30 min at 4°C and 10,000 × g for 60 min at 4°C) followed by ultrafiltration (0.2-µm filter; Sarstedt, Nümbrecht-Rommelsdorf, Germany) and qEV size-exclusion columns (iZON Science, Christchurch, New Zealand). The supernatant of NLF was then ultracentrifuged (100,000 × g) for 60 min at 4°C (Type 90 Ti Rotor; Beckman Coulter, Inc., Brea, CA, USA) to pellet the exosomes. The exosome pellets were then washed once with PBS.
Electron microscopy
The exosome pellets were fixed with 2.5% glutaraldehyde and centrifuged at 100,000 × g to remove the glutaraldehyde. Afterwards, the pellets were stained by 3% aqueous phosphotungstic acid and fixed on copper mesh formvar grids. Samples were examined in a JEOL Transmission Electron Microscope (JEM-1230; JEOL, Ltd., Tokyo, Japan).
Nanoparticle tracking analysis (NTA)
NTA utilizes the properties of both light scattering and Brownian motion in order to obtain the particle size distribution of samples in liquid suspension. The NanoSight range of instruments provides high resolution particle size, concentration and aggregation measurements. Appropriate exosomes concentration were used to assess the size distribution by Zeta View (Particle Metrix GmbH, Meerbusch, Germany).
In vitro tube formation assay
Matrigel tube formation assays using growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). HUVECs were seeded at 1.5×104 cells/well of 96-well plates precoated with Matrigel and cocultured with exosomes for 6 h at 37°C. Images of the tube formation were obtained using an inverted microscope. Tubes were defined as elongated connecting branches between 2 identifiable HUVECs.
In vitro permeability assay
HUVECs were seeded on transwell chamber (0.4-µm pore size; Costar, Corning, NY, USA) in 24-well plates and grown until reaching confluence. Fluorescein isothiocyanate (FITC)-dextran (1 mg/ml) (M, 40 000; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was then added to the upper chamber. At the indicated time points 50-µl samples were taken from the lower chamber and replaced with the same volume of culture medium. A fluorescence plate reader (Lambda Fluoro 320; MWG Biotech, Ebersberg, Germany) was using to measure the fluorescent content of samples.
Cell viability assay
Cells were seeded into 96-well plates and assessed by CCK-8 assay (Beyotime Institute of Biotechnology, Haimen, China). The absorbance of each well was read on a microplate reader (F-2500 Fluorescence Spectrophotometer; Hitachi, Ltd., Tokyo, Japan) at 450 nm.
Uptake experiment
According to the manufacturer's instructions, NLF-derived exosomes labeled by PKH67 (Sigma-Aldrich, St. Louis, MO, USA) were added into HUVECs. HUVECs were then fixed and processed an immunocytochemical analysis. Photographs were acquired using a TCS SP-5 confocal microscope (Leica Microsystems, Wetzlar, Germany), captured under 400 Hz with an image resolution of 512 ×512 pixels, and then analyzed by Leica Application Suite 2.02.
Western blot analysis
Protein samples were separated using 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes, and then blocked with 5% non-fat milk. The PVDF membranes were incubated with primary antibody against ADAM10 (1:300; D221496-0100; Sangon Biotech Co., Ltd., Shanghai, China), β-actin (1:2,000; sc-47778) and CD63 (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), CD9 (Abcam, Cambridge, MA, USA), respectively, overnight at 4°C. Afterwards, the membranes were incubated with horseradish peroxidase-linked immunoglobulin G (1:5,000; sc-2374; Santa Cruz Biotechnology, Inc.). Gray-scale analysis was performed using Image J Software to examine the protein bands.
Statistical analysis
Statistical analysis was performed using SPSS 19.0. software (IBM Corp., Armonk, NY, USA). The results were presented as mean ± standard deviation, and the statistical significance was evaluated using analysis of variance with a Bonferroni post hoc test. P-value <0.05 was considered as a statistically significant difference.