Transplantation of endothelial progenitor cells derived exosomes improve angiogenesis in a rat hindlimb ischemia model

Objective: We investigated whether endothelial progenitor cells (EPCs) derived exosomes (EPC-exo) could improve angiogenesis in a rat hindlimb ischemia model to evaluate whether the EPC-exo may represent a potential therapeutic strategy for lower limb ischemia (LLI). Methods: EPCs were obtained from SD rats using the method of density gradient centrifugation, and identified by DIL-AC-LDL and FITC-UEA-l uptake test and flow cytometry. EPC-exo was isolated and purified from EPCs using ultracentrifugation method, and identified by transmission electron microscopy and western blot analysis. Rat chronic hindlimb ischemia model 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) was used to assessment blood flow perfusion of rat hindlimb at baseline, 7 day, 28 day after intramuscularly injection. Capillary density of muscle tissue was evaluated by immunofluorescence analysis at 28 day. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression change of vascular related genes in endothelial cells (ECs) induced by EPC-exo. Results: DIL-AC-LDL and FITC-UEA-l uptake test showed that more than 90% of the EPCs were double-positive. Flow cytometry showed that in our cultured EPCs, the positive rates of CD31, CD34, CD133 and VEGFR-2 were 97.8%, 85.3%, 83.2% and 91.3%, respectively, but the positive rate of CD45 was only 1.3%. EPC-exo with a size ranging from 30 to 100 nm and western blot analysis confirmed the presence of exosomes markers, including CD9, CD63 and CD81. Compared with non ischemic limbs, EPC-exo group recovered about 80% of the blood perfusion after 28 days, while the EPC group had a recovery rate of about 60%, and the control group had a recovery rate of about 40% only. Capillary density of muscle tissue in EPC-exo group increased significantly than EPC group and control group. qRT-PCR detected the expression of vascular related genes (VEGFA, VEGFR2, HIF-1a, eNOS, ANG1, ANG2, CXCL-16) increased significantly in EPC-exo group than control group. Conclusion: The study results suggest that transplantation of EPC-exo could significantly improve angiogenesis in a rat hindlimb ischemia model, which may be a potential therapeutic strategy for LLI.