Patients and tissue samples
To investigate the role of M2 microglia in DR, 20 proliferative membranes were collected from 20 patients with proliferative diabetic retinopathy (PDR) and 15 from 15 patients with proliferative vitreoretinopathy (PVR) (as controls). None of the patients were treated with anti-VEGF medications. All proliferative membranes were collected intraoperatively. The patients were enrolled from 2019 to 2020 at the First Affiliated Hospital of Nanjing Medical University (Additional file 3: Table S1).
This study was conducted in accordance with the Declaration of Helsinki and was approved by The Ethics Committee of the Faculty of Medicine, Nanjing Medical University (2017-SR-283). All patients provided informed consent to participate in the study.
Cell culture
Human primary endothelial cells (hRECs) used in the experiments were purchased from the American Type Culture Collection. Two to three generations of hRECs were used. Primary cultures of mouse microglia were obtained according to an established protocol with minor modifications, and their purity was verified using flow cytometry [17]. Subsequently, primary extraction and culture of pericytes from mouse retina were performed based on a previous protocol, and the purity of cultured pericytes was verified by immunofluorescence [18]. HRECs were cultured in endothelial cell medium (5.5 or 30 mM glucose), and pericytes were cultured in pericyte medium (5.5 or 30 mM glucose). To investigate the effect of exosomes from M2 microglia on DR, hRECs and pericytes were cultured in a medium containing normal glucose (5.5 mM) or high glucose (30 mM), as an osmotic pressure control.
Exosome isolation and identification
The extraction of exosomes from the culture medium supernatant of microglia with and without IL-4 pretreatment. Briefly, microglial cells were cultured with and without IL-4 for 48 h. After incubation, the microglia were washed twice with phosphate-buffered saline (PBS). The PBS was replaced with an exosome-free medium and then the medium was collected for further analysis. Subsequently, the collected medium was centrifuged at 300×g for 10 min and then at 2000×g for 10 min. The cell supernatant obtained was filtered through a 0.22-μm filter to remove cellular debris and then transferred to an Amicon Ultra-15 centrifuge filter (Millipore, USA) and centrifuged at 4000×g until the volume of the upper chamber was reduced to approximately 200 μL. To purify the exosomes, the liquid from the upper chamber was loaded on 30% sucrose/D2O pads and then subjected to ultracentrifugation at 100,000 g (Beckman Coulter) for 60 min at 4 °C. The pellet obtained was suspended in PBS for further analysis.
The exosomes obtained were morphologically characterized using transmission electron microscopy (TEM; Tecnai 12; Philips, Best, The Netherlands). The diameter and number of exosomes were determined using nanoparticle tracking analysis (NTA, Nanosight Ltd., Navato, CA). Western blot analysis was performed to examine the surface biomarkers of exosomes.
Uptake of exosomes by hRECs and pericytes
Briefly, 4 mg/mL of Dil solution (Molecular probe, USA) was incubated with PBS-containing exosomes for fluorescent labeling. Excess dye was removed by centrifugation at 100,000×g at 4 °C, and the labeled exosomes were washed thrice with PBS. These Dil-labeled exosomes were then co-cultured with hRECs and pericytes for 37 °C 24 h. Subsequently, the cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min. The uptake of exosomes by cells was observed using laser confocal microscopy (Carl Zeiss Microscopy GmbH, Germany) (Additional file 1: Video S1).
Cell proliferation assay
Cell proliferation was assessed using the EdU Cell Proliferation Kit and the Alexa Fluor 596 Imaging Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, hRECs in normal (5.5 mM glucose) or high-glucose medium (30 mM glucose) were seeded into 96-well plates at an initial density of 5 × 103 cells/well and were co-cultured with 100 mg/mL M0-exo or M2-exo for 24 h. Subsequently, 50 mM EdU medium was added to each well and incubated for 2 h at 37 °C. The cells were then washed twice with PBS for 10 min each. They were then fixed with 4% paraformaldehyde (PFA) for 15 min, neutralized with 2 mg/mL glycine, and then washed with PBS before permeabilization with 0.5% Triton X-100 for 10 min. Finally, the hRECs obtained were labeled with Apollo-596 stain; excess stain was removed by washing thrice with 0.5% Triton X-100. EdU assays were performed three times independently, and a total of three areas were randomly selected for confocal fluorescence microscopy imaging under both glucose conditions (MIC00223 LSM5 Live). The percentage of EdU-positive cells (red markers) was calculated and analyzed using the ImageJ software.
Transwell assay
The Transwell assay was conducted using a 24-well chamber containing a membrane filter insert. After 48 h of treatment, each set of transpore chambers was removed, and the cells on the surface of the apical chamber were removed using a cotton swab. Only the cells migrating to the lower side of the membrane were collected and fixed with 4% PFA for 15 min, followed by staining with 0.5% crystal violet solution for 15 min and washing thrice with PBS.
The experiments were repeated thrice independently. A total of three areas were randomly selected for imaging under each condition. Micrographs were obtained using a DP71 digital camera (Olympus).
Tube formation assay
Briefly, 96-well plates were coated with 50 µL of basement membrane matrix (Matrigel; BD Biosciences, USA), followed by seeding hRECs at a density of 1 × 106 cells/well. The cells were then treated with endothelial cell medium for 8 h at 37 °C. The capillary structures formed by hRECs on Matrigel were photographed using a DP71 digital camera (Olympus) and were analyzed using the ImageJ software.
Tube formation assay was performed thrice independently, and a total of three areas were randomly selected for imaging under each condition.
Blood–retinal barrier permeability of hRECs and pericytes in co-culture model
Briefly, pericytes were seeded in inserts (Corning, USA) with a pore size of 0.4 μm and cultured until confluence. hRECs were then spread evenly over the upper layer of pericytes and cultured until confluence. The culture medium was changed every alternate day. After the cells attained confluence and reached the upper chamber, PBS, M0-exo, or M2-exo was added to the upper chambers and allowed to incubate for 48 h. Then, FITC-dextran (70 kDa, 0.5 mg/mL) was added to the upper chamber for 1 h. Subsequently, the medium was collected from the bottom chamber and the fluorescence intensity of FITC-dextran was measured at 538 nm (Fig. 6E).
Transepithelial electrical resistance (TEER) measurement
TEER was assessed to evaluate the barrier function between cells. Briefly, the cells were pre-equilibrated with Hank’s balanced salt solution for 30 min, followed by measurement of TEER using Millicell® ERS (Millipore, USA). TEER values were calculated using the following formula:
$${\text{TEER}}\;\left( {\Omega \cdot\;{\text{cm}}^{2} } \right) = \left[ {{\text{TEER}}\;{\text{total}} – {\text{TEER}}\;{\text{blank}}} \right] \times {\text{membrane}}\;{\text{area}}.$$
Immunofluorescence assay
Cell or tissue sections were fixed with 4% polyoxyethylene and permeabilized with 0.3% Triton X-100, then blocked with 5% bovine serum albumin (BSA) and finally incubated overnight at 4 °C with the corresponding primary antibody for 24 h. Then, the sections were treated with corresponding secondary antibodies and DAPI reagents to assess immunoreactivity using fluorescence microscopy.
Western blot assay
M0-exo and M2-exo concentrations were determined by BSA (Beyotime, China) according to the manufacturer’s instructions. Proteins from cells or tissues were extracted by RIPA lysis (Beyotime, China). Then, the proteins were denatured, followed by separation on SDS-PAGE gels. Subsequently, the proteins were transferred to PVDF membranes, which were then blocked using a blocking buffer (Beyotime, China) for 1 h at room temperature, followed by overnight incubation at 4 °C with the primary antibody. After washing with Tris-buffered saline with 0.1% Tween® 20 detergent, the membrane was incubated with the corresponding secondary antibody for 2 h. The protein bands obtained were visualized using a PowerOpti-ECL detection system (ThermoFisher Scientific, USA).
In vivo EdU proliferation assay
EdU (50 μg per day at a concentration of 125 μg EdU per 100 μL PBS) was intraperitoneally administered to mice with OIR on 5 consecutive days from p17 to p21. Mice were sacrificed at p21. Eye fixation and flat-mount preparation were performed. Retinal was stained by the EdU Kit (Beyotime, China) according to the manufacturer’s protocol.
Flow cytometry
Eyecups of PBS-perfused mice were dissected to separate the retina from adjacent tissue. Subsequently, the retinal tissue was dissociated by resuspension, and dead cells were excluded by incubating in the fixable viability dye 450 (Thermo Fisher Scientific). Cells were stained with anti-CD45, anti-CD11b, anti-F480, and CD206 (1:200; Thermo Fisher Scientific) at 4 °C for 30 min. Then, the cells were washed and analyzed using CytoFlEX (Beckman Coulter, USA) to sort retinal cells. Flow results data were obtained using the CytExpert software (Beckman Coulter, USA).
Apoptosis was examined by flow cytometry analysis. For apoptosis analysis, cells were collected, washed with PBS, and incubated with Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme) for 15 min. Apoptosis was then analyzed by flow cytometry CytoFlEX (Beckman coulter, USA) according to the manufacturer’s instructions. The CytExpert software (Beckman coulter, USA) was used to analyze flow cytometry data. Results are expressed as means ± standard deviation of three independent experiments.
Establishment of the OIR mouse model
All the animal experiments were approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University.
OIR mouse model has been widely used for the mechanism research of retinal proliferative microvascular disease [19, 20]. Mice and their lactating dams were housed in an oxygen supply chamber (oxygen concentration 75% ± 3%) from p7 to p12 and were subsequently returned to room air, as previously reported [21]. All mice were housed and maintained at a constant temperature with a 12-h light/dark cycle. The weight of each pup was closely monitored to determine adequate metabolic health. Mice in the control group were housed in room air and sacrificed at their specified time points.
Establishment of STZ-induced DR mouse model
After 2 weeks of adaptive feeding, 30 C57BL/6 mice (weight 18–22 g) were intraperitoneally administered 55 mg/kg STZ for 5 consecutive days. In contrast, the other 30 mice were treated with normal, while the other 16 mice were treated with normal saline and served as the control group. Serum glucose concentrations were monitored 7 days after treatment initiation, and mice with glucose levels > 16.7 mmol/L were considered diabetic. Mice with diabetes received an intravitreal injection of either PBS (n = 10), M0-exo (n = 10), or M2-exo (n = 10). In the STZ group, six mice from each group were administered Evans Blue (2% saline, 200 μL; tail vein injection) for disrupting the blood–retinal barrier (BRB) (Sigma) 3 months after STZ injection. After 3 h, mice were sacrificed and their retinas were removed and homogenized in a trichloroacetic acid and ethanol solution (1:2). The homogenized tissue was then incubated at 60 °C for 24 h, followed by centrifugation at 10,000×g for 10 min. The supernatant obtained was collected, and fluorescence was quantified using a spectrophotometer at an excitation wavelength of 620 nm and an emission wavelength of 680 nm (Fig. 4A).
Trypsin-digested vessel preparation
At autopsy, each eye was removed and then fixed in 4% paraformaldehyde for 24 h. The retinas were isolated under a dissecting microscope and were then washed completely with copious amounts of PBS. The retinas were then incubated in 3% trypsin in sodium phosphate buffer for 1 h. After completion of digestion, the isolated retinal vessels were stained with periodic acid-Schiff hematoxylin (PAS) and were visualized for.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., USA). Student’s t-test was used for the comparison of data between two groups. One-way or two-way analysis of variance tests were used for multivariate analysis. Data were expressed as means ± standard deviations. Results with a P-value < 0.05 were considered significant.