Animals
C57BL/6J mice had been bought from particular pathogen-free Biotechnology Co., Ltd. (Beijing, China). All procedures involving mice had been authorized by the Animal Care and Use Committee of Tianjin Medical College Eye Hospital and conformed to the ARVO Assertion for the Use of Animals in Ophthalmic and Imaginative and prescient Analysis (Allow Quantity: TJYY2019091225).
Isolation and identification of MSCs
Human umbilical twine MSCs had been obtained from Beijing Beilai Organic Co., Ltd. (China). Recent human umbilical cords had been obtained and enzyme-digested as beforehand described to isolate MSCs [27]. Briefly, the umbilical cords had been washed twice, reduce into roughly 1–3 mm3 sections and digested with 0.1% collagenase kind II (17,101,015, Gibco, USA) at 37 °C for 1 h on a shaker. The cell suspension was then filtered by a mesh and centrifuged. The cell precipitate was re-suspended in full Dulbecco’s modified Eagle’s medium/nutrient combination F12 (C11330500BT, DMEM/F12; Gibco) full medium. The medium contained 10% foetal bovine serum (16,000,044, FBS; Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells had been seeded in T175 flasks and cultured at 37 °C in a 5% CO2 incubator. The adipogenic, osteogenic, and chondrogenic differentiation skills of MSCs had been evaluated utilizing stem cell differentiation kits based on the producer’s directions (90,021, 90,031, and 90,041, respectively; OriCell, China).
Isolation and identification of sEVs
Passage 3–5 MSCs had been cultured with 25 mL tradition medium containing 10% sEV-free FBS (ready utilizing ultracentrifugation at 110,000 × g at 4 °C in a single day) for 48 h. Supernatants had been collected from MSC tradition and sEVs had been remoted by ultracentrifugation. The supernatants had been centrifuged at 2000 × g for 10 min after which at 10,000 × g for 30 min to take away mobile particles and lifeless cells. This was adopted by two rounds of ultracentrifugation (Beckman Coulter, USA) at 110,000 × g for 70 min at 4 °C. The pellets had been resuspended in 300 μL PBS and sterilised by filtration by a pre-rinsed 0.22 mm filter. Protein concentrations within the remoted sEVs had been quantified utilizing a Bicinchoninic Acid (BCA) protein assay package (PC0020, Solarbio, China). The scale distribution of sEVs derived from MSCs was decided utilizing nanoparticle monitoring evaluation (NTA) (NanoSight NS300, Malvern, UK). The morphology of sEVs was visualised utilizing high-resolution transmission electron microscopy (TEM; Hitachi HT7700, Japan). Twenty milligrams of sEVs had been used for western blotting to detect the standard sEV markers, together with CD9, CD63, and tumour susceptibility 101 (TSG101).
Preparation of PEDF-sEVs
PEDF-sEVs had been ready by sonication. PEDF (1177-SF-025, R&D SYSTEMS, USA) was blended with sEVs at a 1:5 focus ratio. The combination was incubated for 10 min and sonicated utilizing an ultrasonic cell crusher (Fisher Scientific* Mannequin 120, Hampton, NH, USA) on ice (20% energy, 45 cycles of a 4 s pulse/2 s pause). The combination was incubated at 37 °C for 1 h to permit the restoration of the sEV membrane. The unloaded PEDF was eliminated by centrifugation at 14,000 × g for 20 min in a 100 kDa diafiltration tube (UFC510096, Millipore, Germany). The ultimate resolution was collected by reversing the tube and centrifuging at 2000 × g for two min, and was used to check the loading capability and encapsulation effectivity utilizing a PEDF ELISA package (RD191114200R, R&D SYSTEMS) following the producer’s directions. The drug-loading capability (LC) was calculated based on the next components: Loading Capability (%) = (Drug entrapped)/(Drug entrapped + Whole mass of sEVs) × 100. The encapsulation effectivity (EE) was calculated as follows: Encapsulation effectivity (%) = (Drug entrapped)/(Whole quantity of drug) × 100. All experiments had been carried out in triplicate.
The remoted sEVs had been labelled with DiD (V22887, Invitrogen) following the producer’s directions. FITC-labelled PEDF was synthesised by CLOUD-CLONE CORP (RPB972Hu01, Wuhan, China). PEDFFITC-sEVs ready utilizing sonication had been used to measure the loading effectivity on a FACSCalibur move cytometer (BD Biosciences, USA), and the info had been analysed utilizing move cytometry software program (FlowJo, USA). All experiments had been carried out in triplicate.
Endothelial cell proliferation assay
Human retinal endothelial cells (HRECs), bought from the American Sort Tradition Assortment (ATCC), had been cultured in Endothelial Cell Medium (1001, ECM, ScienCell, USA) containing 5% FBS, 1% endothelial cell development dietary supplements, and 1% antibiotic resolution. All cells had been used from passage 2 to six. For stimulation, the cells had been starved in ECM supplemented with 2.5% FBS, 0.5% endothelial cell development dietary supplements, and 1% antibiotic resolution.
A cell proliferation assay was carried out utilizing the Cell Counting Package-8 (CCK-8; HY-K0301, MedChemExpress, USA). HRECs had been seeded at 4 × 103 cells/properly in 96-well plates, allowed to stick for 12 h, after which incubated with VEGF (10 ng/mL; 48,143, Cell Signalling Know-how, UK), PEDF (400 ng/mL), sEVs (10 µg/mL), a combination of PEDF (400 ng/mL) and sEVs (10 µg/mL) with out sonication, or PEDF-sEVs (10 µg/mL) in hunger medium. After 24 h of incubation, cells had been incubated with a combination containing 90 µL ECM and 10 µL CCK-8 reagent for two–4 h at 37 °C. Absorbance was measured at 450 nm utilizing a multimode microplate reader (Infinite M200 Professional, Thermo Fisher Scientific). All experiments had been carried out in triplicate.
Transwell assay
Transwell chambers (3422, Corning Integrated, USA) had been used for the migration assay. HRECs (8 × 104) had been seeded within the higher chamber in FBS-free medium in 24-well plates and incubated with VEGF, PEDF, sEVs, a combination of PEDF and sEVs with out sonication, or PEDF-sEVs. The underside of the chamber contained the ECM medium supplemented with 10% FBS. After the cells had migrated for twenty-four h, they had been mounted and stained with crystal violet. The migrated HRECs had been counted underneath an inverted mild microscope. The variety of migrated HRECs was quantified by counting the cells in 5 random fields. All experiments had been carried out in triplicate.
Scratch assay
HRECs (1.5 × 105) had been seeded in 12-well plates. After 12 h, a wound was made by scraping the cell monolayer with a 1 mL pipette tip, and cells had been stimulated with VEGF, PEDF, sEVs, a combination of PEDF and sEVs with out sonication, or PEDF-sEVs for twenty-four h within the hunger medium. Pictures had been acquired at 0 and 12 h after incubation at 37 °C. The realm of wound closure between 0 and 12 h was analysed utilizing ImageJ software program. All experiments had been carried out in triplicate.
Tube formation assay
Forty-eight-well plates had been pre-coated with 150 µL Matrigel (Bedford, USA) for 30 min at 37 °C. Then, 3 × 104 HRECs per properly had been seeded on Matrigel (354,234, Biocoat, USA) and handled with VEGF, PEDF, sEVs, a combination of PEDF and sEVs with out sonication, or PEDF-sEVs within the hunger medium. The pictures had been acquired after 4 h. The nodes, grasp conjunctions, and lengths of the tubes had been measured utilizing ImageJ software program. All experiments had been carried out in triplicate.
Evaluation of VEGF downstream signalling in HRECs
To detect VEGF downstream signalling, 8 × 104 HRECs had been seeded in 12-well plates for 12 h after which handled with PEDF, sEVs, or PEDF-sEVs for twenty-four h. The HRECs had been then stimulated with or with out VEGF (10 ng/mL) for 20 min. The cells had been washed with PBS and lysed in chilly RIPA lysis buffer supplemented with 1% PMSF (P0100, Solarbio) and 1% phosphatase inhibitor (P1260, Solarbio) for western blotting to detect the relative expression of ERK, p-ERK, AKT, and p-Akt. All experiments and analyses had been carried out in triplicate.
Software of MSC-sEVs in oxygen-induced retinopathy mouse mannequin
An oxygen-induced retinopathy mouse mannequin (OIR) was established, as beforehand described [34]. Briefly, neonatal C57BL/6J mice and nursing moms had been uncovered to hyperoxia (75% O2) on postnatal day 7 (P7) for five days and returned to room air on P12. The variety of neonatal mice assigned to every nursing mom was the identical. The mice had been supplied with an ordinary weight-reduction plan and water and randomly assigned to every of the remedy teams. 0.4% oxybuprocaine hydrochloride eye drops had been used for topical anesthesia of OIR mouse eyes earlier than intravitreal injection. The OIR mice had been handled with 1 µL of PBS, PEDF (40 µg /mL), sEVs (1 mg/mL), a combination of PEDF (40 µg /mL) and sEVs (1 mg/mL) with out sonication, anti-VEGF drug (10 mg/mL, Ranibizumab, Genentech, USA) or PEDF-sEVs (the protein focus of sEVs was 1 mg/mL) by intravitreal injection on P12. A ten µL 34G Hamilton syringe (Hamilton, Reno, NV, USA) was used for intravitreal injections. The mice had been euthanised on P17, and the retinas had been dissected alongside the cornea–sclera divide. The iris, cornea, lens, and vitreous had been eliminated and discarded. Retinas had been peeled off, instantly flash-frozen in liquid nitrogen, after which saved at − 80 °C for western blot and PCR analyses.
Quantification of avascular space and neovascularisation
OIR mice had been sacrificed on P17. The eyeballs had been enucleated and glued in 4% paraformaldehyde (p1110, PFA, Solarbio) for 30 min. Retinas had been dissected, washed with PBS, permeabilised with PBS containing 1% TritonX-100 for 30 min, after which blocked in PBS containing 2% BSA, 0.3% TritonX-100 at 4 °C for two h. Subsequently, flat-mounted retinas had been stained with isolectinGS-IB4 (121,411, 1:500, Thermo Fisher Scientific) in a single day at 4 °C at nighttime for visualization of retinal vasculature in OIR mice. Retinal vascular buildings had been noticed utilizing a confocal fluorescence microscope (LSM 800; Carl Zeiss, Germany). In response to beforehand described strategies [34], the areas of vaso-obliteration and retinal neovascular tufts had been quantified utilizing the Adobe Photoshop software program (Adobe, USA).
Immunofluorescence
On P17, OIR mice had been sacrificed. Eyeballs from every group had been enucleated, transferred to an optimum slicing temperature compound (4583, OCT, Solarbio) and frozen at − 80 °C. Sections had been reduce at a thickness of 8 μm and glued in 4% PFA for 30 min. For immunofluorescence staining of glial fibrillary acidic protein (GFAP), the sections had been incubated in a single day in a blocking resolution with the anti-GFAP antibody (ab7260, 1:500, Abcam, UK) in a humidified chamber at 4 °C. After washing, the sections had been incubated with Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G (ab150080, 1:2000, Abcam) at room temperature (RT) for 3 h at nighttime. The nuclei of the retinal cells had been stained with DAPI (C0065, Solarbio). Lastly, the sections had been noticed underneath a confocal fluorescence microscope (LSM 800; Carl Zeiss), and the fluorescence depth was analysed utilizing ImageJ software program.
Optical coherence tomography imaging
Spectral area optical coherence tomography (OCT; Heidelberg Engineering) imaging was carried out on P25 (± 1 d) and P42 (± 1 d) to judge the retinal thickness. Mice had been anaesthetised, and their pupils had been dilated utilizing Tropicamide Eye Drops (Alcon, Belgium). The retinas had been scanned utilizing an OCT digital camera positioned in entrance of the cornea, with the optic disc positioned on the centre of the picture. OCT picture knowledge had been collected utilizing a pc and retinal thickness was robotically quantified utilizing the OCT software program.
Electroretinogram
Electroretinogram (ERG) was carried out on P25 (± 1 d) and P42 (± 1 d) to judge retinal perform of OIR mice utilizing Ganzfeld Electroretinogram (Phoenix Micron IV, Phoenix Applied sciences, USA). The mice had been subjected to darkish adaptation in a single day and had been anaesthetised earlier than the ERG. Corneas had been anaesthetised with 0.4% oxybuprocaine hydrochloride, and Gatifloxacin Eye Gel was utilized to the ocular floor to stop tissue dryness. The reference electrode was inserted underneath the scalp, near the midline between the ears. One other electrode was inserted into the mouse tail. Pupils had been centred to the digital camera and a spread of sunshine depth stimulation parameters of − 1.1, 0.1, 1.0, and three.0 log (cd•s/ m2) had been set for waveform detections. Every mouse was examined thrice to acquire a median worth. The a-wave amplitude was measured from the baseline to the trough of the a-wave, and the b-wave amplitude was measured from the trough of the a-wave to the height of the b-wave, which was robotically quantified utilizing the ERG software program.
Haematoxylin-eosin staining
Haematoxylin-eosin (H&E) staining was carried out on P25 and P42 to judge retinal buildings in OIR mice. Eyes from every group had been mounted, dehydrated, and embedded in paraffin. Then, the paraffin-embedded eyes had been sectioned at a thickness of 4 μm and stained with H&E (G1120, Solarbio). The stained sections had been examined underneath a microscope.
Toxicity evaluation
OIR mice had been sacrificed on P17. The eyeballs from every group had been enucleated and transferred to an optimum slicing temperature compound. Sections had been reduce at a thickness of 8 μm and glued in 4% PFA for 30 min. A FragEL™ DNA Fragmentation Detection Package (11,684,817,910, Roche Molecular Biochemicals, Germany) was used to judge the toxicity of PEDF-sEVs on the retina. DAPI was used to stain cell nuclei. Lastly, sections had been noticed underneath a confocal fluorescence microscope (LSM 800, Carl Zeiss). Terminal deoxynucleotidyl transferase-mediated dUTP nick finish labelling (TUNEL)-positive cells within the retina had been counted to quantify toxicity [35].
PEDF-sEVs uptake by HRECs
DiD-labelled sEVs and FITC-labelled PEDF had been used to check the mobile uptake of sEVs by HRECs. Synthesised FITC-labelled PEDF had been obtained from CLOUD-CLONE CORP. HRECs (1.5 × 104) had been seeded in 24-well plates for 12 h, after which handled with 10 µg/mL PEDFFITC-sEVs or a combination of PEDF (400 ng/mL) and sEVs (10 µg/mL) for twenty-four or 48 h. The focus of PEDF was according to that of the PEDF-sEVs, as measured utilizing ELISA. Subsequently, the cells had been washed with PBS, mounted with 4% PFA at RT for 30 min, and nuclei had been stained with DAPI. The cells had been imaged utilizing a confocal microscope (LSM800, Carl Zeiss). All experiments had been carried out in triplicate.
To match the effectivity of PEDF supply in HRECs, cells from every group had been harvested. Circulate cytometry knowledge had been collected utilizing a FACSCalibur move cytometer (BD Biosciences, USA) and analysed utilizing move cytometry software program (FlowJo, USA). All experiments had been carried out in triplicate.
Detection of PEDF focus in supernatants collected from HRECs
HRECs (4 × 103) had been seeded in 96-well plates for 12 h. Subsequently, they had been handled with PEDF (400 ng/mL), sEVs (10 µg/mL), or PEDF-sEVs (10 µg/mL) for six, 24, 48, or 72 h. The supernatants had been collected and lysed in chilly RIPA lysis buffer with 1% PMSF (P0100, Solarbio) and 1% phosphatase inhibitor (P1260, Solarbio) to measure the focus of PEDF utilizing a PEDF ELISA package (R&D SYSTEMS), following the producer’s directions. All experiments had been carried out in triplicate.
Ocular distribution of PEDF
FITC-labelled PEDF was used to detect the distribution of PEDF within the retina. OIR mice had been handled with PEDF, sEVs, or PEDF-sEVs through intravitreal injections on P12. Mice had been sacrificed on P13 or P17. The eyes from every group had been enucleated and transferred to an optimum slicing temperature compound and frozen at − 80 °C. Sections had been reduce at a thickness of 8 μm and glued in 4% PFA for 30 min. Then the sections had been stained with DAPI for five min and noticed underneath a confocal fluorescence microscope (LSM 800, Carl Zeiss). The fluorescence depth of FITC was analysed utilizing ImageJ software program.
RNA extraction and quantitative real-time PCR evaluation
Whole RNA was extracted from HRECs and retinas utilizing a common RNA Purification Package (B004, EZ Bioscience, USA) following the producer’s protocol. The focus and high quality of RNA had been examined utilizing Nanodrop 2000 (Thermo Fisher Scientific). RNA samples had been reverse-transcribed to complementary DNA (cDNA) utilizing a Color Reverse Transcription Package (A0010CGQ, EZBioscience). Quantitative polymerase chain response (qPCR) was carried out utilizing SYBR Inexperienced Grasp Combine (A0012, EZBioscience). GAPDH was used as an inner reference for every response. The relative expression was calculated utilizing the next equation: Relative gene expression = 2[△Ct(control)–△Ct(target)]. The sequences of the primers are listed in Extra file 1: Desk S1.
Western blot evaluation
Cells or retinal tissues had been lysed in chilly RIPA lysis buffer supplemented with 1% PMSF (P0100, Solarbio) and 1% phosphatase inhibitor (P1260, Solarbio) for 10 min, and the overall protein focus was measured utilizing a BCA protein assay package (PC0020, Solarbio). Proteins (20 µg for every pattern) had been subjected to electrophoresis on a ten% or 12.5% SDS-polyacrylamide gel for 1 h. The electrophoresed proteins had been transblotted to a polyvinylidene fluoride membrane (RF1136, Millipore). Subsequent, 5% non-fat milk or 5% BSA in Tris-buffered saline with Tween (TBST, T1082, Solarbio) buffer was used to dam the membranes for two h at RT. The membranes had been then incubated in a single day with the next main antibodies at 4 °C: anti-CD9 (ab92726, 1:1000, Abcam), anti-CD63 (ab216130, 1:2000, Abcam), anti-TSG101 (ab125011, 1:1000, Abcam), anti-pAkt (4060, 1:2000, Cell Signaling Know-how), anti-Akt (4691, 1:2000, Cell Signaling Know-how), anti-ERK (4370, 1:2000, Cell Signaling Know-how), anti-pERK (4695, 1:2000, Cell Signaling Know-how), anti-VEGF (ab46154, 1:1000, Abcam), anti-PEDF (ab180711, 1:1000, Abcam), anti-GFAP (ab7260, 1:5000, Abcam), anti-ICAM-1 (10831-1-AP, 1:1000, Proteintech), or anti-GAPDH (60004-1, 1:1000, Proteintech), which was used as an inner reference. The membranes had been washed and incubated with the suitable secondary antibody for two h at RT. Sure main antibodies had been detected utilizing horseradish peroxidase-conjugated goat anti-mouse IgG (7076, 1:2000, Cell Signaling Know-how) and goat anti-rabbit IgG (7074, 1:2000, Cell Signaling Know-how). The processed blots had been developed utilizing Immobilon ECL reagent (RPN2232, Cytiva) and imaged utilizing a transilluminator (Tanon, China). The membranes had been then stripped in a Stripping Buffer (CW0056M, Cwbiotech, Shanghai, China) and re-probed with one other main antibody. Pixel densities of the protein bands had been calculated utilizing the ImageJ software program, and protein expression values had been divided by these of GAPDH.
Statistical evaluation
Knowledge are introduced as imply ± customary deviation (SD). To calculate statistical significance, a Pupil’s t-test was used for two-group comparisons, and one-way evaluation of variance was used for multigroup comparisons. The GraphPad Prism 9.4 software program (GraphPad Software program, USA) was used for statistical evaluation and mapping charts. A p-value ≤ 0.05 was thought of to point a statistically important distinction.
