Cell culture.
Human umbilical vein endothelial cells (HUVECs) were purchased from Zhejiang Meisen Cell Technology (Zhejiang, China). HUVECs were used in passage 3-6. The cells were cultured in ECM (ScienCell, US) in a 37℃incubator with humidified atmosphere of 5%CO2/95% air. Cells were digested to conventional passage or cryopreservation when the cells grew to 70% confluence.
Preparation and identification of Exosomes from HUVEC culture medium.
The FBS was ultracentrifuged at 100,000g for 16 h to deplete FBS-derived exosomes before experiments. We isolated HUVEC-derived exosomes by total Exosome isolation kit (Thermo Scientific US) .The HUVECs were cultured in T75 for 48-72 h and then the culture medium was collected and centrifuged at 2000g for 30min to remove cells and debris. The supernatant was subjected to incubation with 1/2 volume of total exosome isolation kit (Thermo Scientific, US) overnight at 4℃ as the kit introduction illustrates. Then the mixture was centrifuged at 10000g for 1 h at 4℃ to pellet HUVEC-exosomes. The pelleted exosomes that each milliliter of supernatant generated were resuspended with 10ul normal saline and aliquoted for nanoparticle tracking analysis (Malvern, Nanosight NS300, UK), transmission electron microscopy (TEM, Leica JEM-1400, Germany) and western blot. The protein concentration of extraction was about 2ug/ul by BCA (Beyotime Biotechnology, China).
Preparation and identification of EXO-KV11
The KV11 sequence was YTMNPRKLFDY, the KV11(CP05) sequence was YTMNPRKLFDYCRHSQMTVSRL. KV11 and KV11(CP05) were synthesized as a single fusion peptide via peptide bond without space by ChinaPeptides (Shanghai, CHINA) with 98% of purity.
We aimed to combining Exosomes with FITC-labeled (CP05) KV11 in order to evaluate the binding rate. For this, (CP05)KV11 was incubated with exosomes overnight at 4℃. Analyses of labeled samples were performed on an Apogee A-50 Micro flow cytometer (FC) (Apogee Flow Systems, UK) equipped with 488-nm(green) that were dedicated to KⅤ11(FITC) detection. Flow cytometry performance was first verified on each day of measurement using a reference bead mix (ApogeeMix, Apogee Flow Systems composed of a mixture of non-fluorescent silica beads with diameters of 180 nm, 240 nm, 300 nm, 590 nm, 880 nm, and 1300 nm with a refractive index (RI) of 1.42 and 110-nm and 500-nm green fluorescent (excited using a blue laser) latex spheres with an RI of 1.59. Acquisition was performed in duplicate at a sample flow rate 0.75 µl/min and a 150-µl sample volume for 180 s.
The exosome concentration (number of Exosomes per µl of prepared purified MVs) was measured automatically based on the sample volume, cytometer flow rate and the number of fluorescence-positive events(n). Exosomes were gated using medium angle light scatter (MALS) and fluorescence triggering channel and defined as KV11(FITC)-positive events as observed in color fluorescence plots .
CCK-8 assay
The CCK-8 (Cell Counting Kit-8, Dojindo, Japan) assay was utilized to test the influence of EXO-KV11 on HUVECs proliferation ability contrast to KV11. HUVECs were cultured as set forth, and were starved for 24h in FBS-free ECM, and each group respectively were subjected to incubation with EXO-KV11(6.25µg/ml+25µg/ml),KV11༈25µg/ml༉, EXO༈6.25µg/ml༉, normal saline of the same volume or Avastin༈10µg/ml༉.After 24h, cell suspension of every group was prepared and diluted to the concentration of 5×104cells/ml. 100µl diluted cell suspension of each group was seeded into 96-well plates. Each well contained 20ng/ml VEGF (hVEGF165, Proteintech, China) ECM and was treated separately with EXO-KV11༈6.25µg/ml+25µg/ml༉,KV11༈25µg/ml༉, EXO༈6.25µg/ml༉,normal saline of the same volume or Avastin༈10µg/ml, Roche, Switzerland༉. Then HUVECs were cultured for 24h. After that the CCK-8 solution (10µl) was added to each well and incubated for 2h at 37℃. The optical density (OD) was read at 450 nm on a microplate reader (Molecular Devices, SpectraMax 190, China). Cells from 3-5 wells were counted at each time point, and the experiment was repeated 3 times.
Tube formation assay
We evaluated the ability of tube formation contrast by utilizing the capacity of endothelial cells formatting tube on Matrigel (Corning, US). HUVEC was cultured as set previously mentioned. Before the experiment, we put the Matrigel at 4℃ overnight and precooled the pipette and 96-well plates. 60 µl Matrigel was put onto the 96-well plate on ice without air bubbles and then put into 37℃ cell incubator for 30min to be solidified. HUVEC was digested to cell supernatant and diluted to 15×104 cells/ml. 100µl diluted cell suspension of each group was put into Matrigel-covered 96-well plates. Each well was treated separately with EXO-KV11(6.25µg/ml+25µg/ml),KV11༈25µg/ml༉, EXO༈6.25µg/ml༉,normal saline of the same volume or Avastin༈10µg/ml༉under the stimulation of 20ng/ml VEGF. Each group had 3-5 replicates and then continued to culture for 6h. Photographs were taken under microscope (Olympus BX41, Japan). We utilized software image j to analyze the amount of meshes to evaluate the capacity of HUVEC tube formation in vitro.
Transwell migration assay
Transwell (Corning, US) consisted of upper and lower chambers separated by microporous polycarbonate membranes. We put HUVEC in the upper chamber containing our treatment and put ECM containing 20ng/ml VEGF in the lower chamber to test the ability of migration. HUVEC was cultured as set previously mentioned, and then HUVEC was digested to cell supernatant and diluted to 15×104 cells/ml. we added 600ul ECM containing 20ng/ml VEGF in the lower chamber and 100µl cell supernatant in the upper chamber with EXO-KV11(6.25µg/ml+25µg/ml),KV11༈25µg/ml༉, EXO༈6.25µg/ml༉,normal saline of the same volume or Avastin༈10µg/ml༉in each group. And the plates were set in the cell incubator for 24h. Then we pipetted out the fluid in the upper chamber and wipe the cells on the upper chamber surface softly by wet cotton swab. The chamber was fixed by 4% paraformaldehyde for 30 min at room temperature. Cells were stained with crystal violet (Beyotime, China) and photographed under microscope. The results were analyzed by software image j.
Western blot
The protein of ECs and cornea tissue was extracted with RIPA lysis buffer (Thermo Scentific, USA) with protease inhibitor. Protein lysates were electrophoresed through SDS-PAGE gels and transferred onto NC membranes (Millipore, Germany). The membranes were blocked with TBS blocking buffer (LI-COR, US) for 1.5 h and incubated with primary antibodies at 4℃ overnight. Target proteins were detected by specific primary antibodies including CD63 (cat.no. 25682, 1:1000, Proteintech, China), VDAC1 (cat.no.55259, 1:1000, Proteintech, China), PERK(C33E10) (cat.no.3192, 1:1000, Cell Signaling Technology, US) ,SQSTM1/p62 (cat.no.5114, 1:1000, Cell Signaling Technology, US), LC3B (cat.no.ab192890, 1:1000, Abcam), caspase-3 (cat.no.19677, 1:1000, Proteintech, China), claudin-5 (cat.no.bs-10296R, 1:500, Bioss Antibodies, China) or β-actin (cat.no.66009, 1:1000, Proteintech, China)-were used as loading control.
The bound primary antibody was detected by mouse anti-rabbit IgG-CFL 790 (cat.no.sc-516253 1:200, Santa Cruz, US) and m-IgGκBP-CFL 790 (cat.no.sc-516181 1:1000, Santa Cruz, US) respectively. The Odyssey CLx western blotting analysis system (LI-COR, US) was applied. Quantification of WB signal was done by Image J software. Protein expression values were divided by their β-actin respectively. Each experiment was performed at least three times.
Rat model of alkali burn injury-induced corneal neovascularization
100 Sprague Dawley (SD) rats with a mean weight of 200 g (Jiangsu Huafukang Biotechnology, China) were used in the study. All animal studies were approved by the Peking People’s Hospital Ethics Committee (No.20210019) and in accordance with the regulations on the management of laboratory animals (the national science and technology commission).
Rats were anesthetized by exposure to 2.5% isoflurane. The center of the right cornea was cauterized by pressing a filter paper (3 mm, soaked in 1 mol/L NaOH solution and strained off any excess liquid) for 30 s (day 0). After that, 15 mL normal saline was used to fully clean the NaOH solution. Each injured rat eye received a subconjunctival injection of 100µL of EXO-KV11(25µg), KV11(25µg) and normal saline every other day. The cauterized corneas were monitored under slit-lamp biomicroscopy (TOPCON, 75-1, Hasunnuma-cho, Itabashi-ku, Tokyo, Japan) on day 1,4,7,14.
Analyses of corneal neovascularization
The area of corneal neovascularization was determined as previously described.[49] Each photograph was analyzed at the same magnification using Image J software. Through the use of a reticule, the vessel length(L) from the limbus and the number of clock hours (C) of the cornea involved were measured, and the vascularized area=C/12×3.1416[r2-(r-L)2], where r=3 mm and is the radius of the rat cornea. Investigators who performed this assay were not blinded to the treatment.
Furthermore, the area of corneal neovascularization was also analyzed by intracardial injection performed as previously described[50] and the protocol was modified moderately. In brief, rats were anesthetized by ketamine (80mg/kg)/xylazine(10mg/kg). And then they were perfused with 25mg/ml fluorescein isothiocyanate (FITC)-labeled dextran (MW 2000,000, Sigma). After 1 min, the eyes were enucleated and fixed in paraformaldehyde solution (4.0%) for 30 min. After the posterior segment of the eyeball was removed, the anterior segment was mounted on a glass slide and observed using a fluorescence microscope (Olympus AX80, Japan). Corneal neovascularization was quantitatively evaluated by detecting the ratio of the fluorescent area of neovascularization to the area of total cornea using the image pro plus software.
H&E staining and immunostaining of corneal neovascularization
Corneal were carefully dissected under microscopy and rinsed in normal saline. Paraformaldehyde-fixed corneas were embedded in paraffin and sectioned at 4µm for histological and immunohistological analysis. For the visualization of three layers of cornea structure, three non-consecutive sections were stained with hematoxylin and eosin (H&E) in each cornea.
To directly observe CoNV, section were incubated with primary antibodies to rat CD31 (1:500, ab281583, Abcam, US) at 4℃ overnight. This was followed by rinse 3 times in PBS. The staining was visualized through the use of horseradish peroxidase (HRP) detection system (Servicebio, China), followed by counterstaining with hematoxylin.
Statistical analysis
All data were expressed as mean±standard deviation(SD) for the respective groups. Statistical analyses were performed with GraphPad Prism software. The Student’s t test, two-way ANOVA followed by a Least Significant Distance (LSD) post hoc test were used by SPSS 23.0 statistical software for analyzing the data. For all measurements, a p < 0.05 was considered statistically significant. The following designations for the p value were used in the manuscript figures: ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.