All animal experimental protocols in this study were reviewed and approved by the Institute of Animal Care and Use Committee of Capital Medical University Affiliated Beijing Chaoyang Hospital. All animal procedures were performed according to NIH guidelines for the care and use of laboratory animals. The animal were maintained under a conventional state and fed with standard laboratory food and water. The study was conducted in accordance with the ethical principle of the World Medical Association Declaration of Helsinki, and local legislation. This study was carried out in compliance with the ARRIVE guidelines.
Rat bone marrow derived-EPCs cultures
Rat bone marrow mononuclear cells (BM-MNCs) were collected and isolated as previously reported. Briefly, after centrifugation, the cells were divided into four layers. The second layer was MNCs. The MNCs were suspended in EGM-2MV medium (containing 20% fetal bovine serum, vascular endothelial growth factor, recombinant basic fibroblast growth factor, insulin-like growth factor, recombinant epidermal growth factor, hydrocortisone and ascorbic acid, etc.) and inoculated with 1×106/cm2 in a 25 cm2 culture bottle with FN (2ug/cm2). Then the cells were cultured in a 5% CO2 saturated humidity cell incubator at 37 ℃. When cells fusion over 80%, cells were harvested with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) and cultured passage continuously. Cells growth was observed by fluorescent microscope every day.
Identification of EPCs
EPCs were identified by DIL-AC-LDL and FITC-UEA-l uptake test and flow cytometry. Flow cytometry was used to identify the expression of CD31, CD34, CD133, VEGER-2 and CD45 on EPCs surface.
The primary passage EPCs were digested with trypsin and inoculated into a 12 well plate, after cells adhered to the wall, DIL-AC-LDL(10μg/ml; Molecular Probes, Carlsbad, Calif) and FITC-UEA-l (10μg/ml; Sigma) was added into each well plate and incubated in 5% CO2 incubator at 37 ℃ for 4 hours.
After EPCs fusion over 90%, 0.25% trypsin was performed to digest the cells. 100μl of FACS buffer solution suspended 5×105 Of EPCs, add different fluorescent labeled monoclonal antibodies (FITC labeled mouse anti human CD31, CD45, PE labeled mouse anti human CD133, VEGFR-2 APC labeled mouse anti human CD34) and well mixed, incubation at 4℃ for 15 minutes, and then detected by flow cytometry (FACScan, Becton Dickinson).
Purification of EPC-exo
2-3 passages EPCs fusion over 80%, EGM-2 MV medium was washed with PBS for 3 times, and new EGM-2 MV medium replaced and cultured go on for 48 hours under hypoxic conditions. The supernatants were collected and centrifuged at 300×g for 5 minutes at 4℃, then the precipitate was discarded, and centrifuged at 1000×g for 15 minutes at 4℃ to remove cell debris, and then filtered through a 0.22 μm filter.
The supernatants were transfered to the ultrafiltration tube and centrifuged at 10,000 ×g at 4℃ for 1 hour. Subsequently, the remaining supernatant was purified after 2 centrifugations at 100,000 ×g at 4 °C for 2 hours at 4 °C. The pellets were resuspended with PBS and stored at −80°C until use. The morphology of exosomes were observed by transmission electron microscopy.
Western blot analysis
Western blot analysis was used to identify the following surface markers of EPC-exo: CD9, CD63 and CD81. Protein extracted from the exosomes samples was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), and then incubated with CD9, CD63 and CD81 antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies.
Rat chronic hindlimb ischemia model and intramuscularly injection
3 months old SD rats, weighting 175-210g, were anesthetized by inhalation of 2% isoflurane. Under sterile conditions, a 1.5cm skin incision was made overlying the middle portion of the left hindlimb of each rat, and was pulled away with a small retractor. The adipose tissue around thigh muscle was pushed with cotton swab to expose the vascular and nerve bundles including femoral artery, femoral vein and femoral nerve. The branches of superficial circumflex iliac artery, deep femoral artery, abdominal pudendal artery and descending genicular artery were ligated with 7-0 silk suture. After temporary blocked with vascular clamp, a small transverse incision was made in the anterior wall of femoral artery. A silicone tube was inserted into the femoral artery through the small transverse incision and fixed with 7-0 silk suture. The process of chronic thrombosis in silicone tube was used to simulate the pathology of peripheral artery occlusion.
7 days later after the surgery, the rats were divided randomly into three groups. The experimental group (n=5) was intramuscularly injected with 30ul EPC-exo through four points in ischemic hindlimb, while the EPC group (n=5) was injected with 30ul EPCs, and the control group (n=5) was injected with 30ul PBS.
Laser doppler perfusion imaging (LDPI)
Laser doppler perfusion imaging (LDPI) (Moor Instruments, Devon, UK) , as a non-invasive measurement, used to assessment blood flow perfusion of rat hindlimb. Rats were anesthetized with 2% isoflurane and subjected to LDPI measurement at baseline, 7 day, 28 day after intramuscularly injection. At each time, both ischemic limb and non-ischemic limb should be measured simultaneously. The images were analyzed to compare the blood flow ratio of the ischemic limb vs. the non-ischemic limb expressed as percentage perfusion.
Immunofluorescence analysisevaluation of capillary density
Capillary density of muscle tissue was evaluated by immunofluorescence analysis, 28 day after intramuscularly injection, rats were sacrificed to harvest muscle tissue. Muscle samples were fixed in methanol, paraffin embedded, and cross-sectioned (5μm), and monoclonal antibody against mouse CD31 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used. The number of CD-31-positive per high power field (x400) was represents capillary density.
Quantitative reverse-transcriptase polymerase chain reaction
Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression change of vascular related genes in ECs induced by EPC-exo. 5×105 ECs were seeded in a six well plate, when the cell adhesion and fusion degree was about 80%, the cells culture medium was changed. The experimental group (containing 100ug/mL EPC-exo), the EPC group (containing 100ug/mL EPCs) and the control group (containing 100ug/mL PBS), were cultured in the cell incubator with 5% CO2 at 37°C for 24 hours.
Total RNA was extracted with Trizol reagent (Invitrogen) following the manufacturer’s protocol, and cDNA was synthesized from 1 μg of extracted total RNA using the PrimeScript RT reagent kit (TAKARA, Tokyo, Japan). Then, 1μl of cDNA sample was used as a template for qRT-PCR using an ABI PRISM 7900HT System with SYBR Premix ExTaq II (Takara Biotechnology). The following human primers were used: VEGFA, VEGFR2, HIF-1a, eNOS, ANG1, ANG2, CXCL-16. The primer sequences used in the study are summarized in Table 1. Relative gene expression data were analyzed with the 2−ΔΔCt method.
The data were shown as the means ± SEM and analyzed using GraphPad Prism 7.0 software. Unpaired Student’s t test was used for statistical comparison of the data. Differences between groups were considered statistically significant at p < 0.05.