Role Of HucMSC-ex Delivered Ang-2 In Angiogenesis Promotion And Cutaneous Regeneration

Objective:Burn injury is a common skin damage,it causes physical and emotional suffering in affected individuals.Human umbilical cord mesenchymal stem cells（hucMSCs）and their exosomes（hucMSC-Ex）can promote skin wound healing;however,its underlying mechanisms are still not well known.The aim of this study was to investigate the role of hucMSC-Ex-delivered angiopoietin-2（Ang-2）in promoting skin angiogenesis and repairing deep second-degree skin burns,and to construct hucMSC-Ex with more significant function in wound healing that modified with Ang-2.Methods:HucMSCs were isolated and cultured,and then hucMSC-Ex was extracted by ExoQuick-TCTM precipitation method.The morphology and particle size of exosomes were analyzed using transmission electron microscope（TEM）and nanoparticle tracking analysis（NTA）.The Ang-2 protein in hucMSC-Ex and the CD9,CD63 and CD81 molecules which frequently located on the surface of exosomes were analyzed using Western blot.Human umbilical vein endothelial cells（HUVECs）were co-cultured with hucMSC-Ex.Then the expression level of CD63 and Ang-2 in HUVECs were detected by Western blot,real-time fluorescence quantitative PCR and cellular immunofluorescence staining.Through the CCK8,transwell and tube formation experiments,we observed the promotion of hucMSC-Ex on HUVECs proliferation,migration and tube formation ability.Ang-2 in hucMSC-Ex was overexpressed or knocked down(Ex^Ang-2 or Ex^shAng-2)by using recombinant lentivirus transfection and then verified by Western blot.After being co-cultured with Ex^Ang-2 or Ex^shAng-2,the expression of Ang-2 protein in HUVECs and proliferation,migration and tube formation abilities of HUVECs were analyzed.The SD rat deep second-degree burn injury model was established by 80℃hot water scald,and then hucMSC-Ex,Ex^Ang-2 or Ex^shAng-2hAng-2 was injected for treatment.The repair effect was evaluated by observing the gross view of the wound,HE staining and tissue immunochemical staining of CD31.Results:The extracted hucMSC-Ex had the typical characterization of exosomes and expressed Ang-2 protein.HucMSC-Ex treatment could increase the expression level of Ang-2 protein in HUVECs,and promote the proliferation,migration and tube formation abilities of HUVECs.Ex^Ang-2 or Ex^shAng-2hAng-2 was successfully constructed.The overexpression of Ang-2 in hucMSC-Ex further enhanced the migration and tube formation abilities of HUVECs,while the knockdown of Ang-2 in hucMSC-Ex weakened the promotion effect on HUVECs migration and tube formation ability.In rat deep second-degree burn injury model,hucMSC-Ex promoted the formation of CD31 positive blood vessels,and accelerated wound healing,the regeneration of epidermal and subcutaneous glandular structures in wound areas.Compared with hucMSC-Ex,Ex^Ang-2 had a more pronounced pro-angiogenic effect,and the therapeutic effects on wound healing and the regeneration of epidermal and subcutaneous accessory gland structures in wound areas were stronger.The angiogenic effect of Ex^shAng-2 was weakened compared to hucMSC-Ex,and its therapeutic effects on wound healing and the regeneration of epidermal and subcutaneous accessory gland structures in wound areas were reduced.Conclusion:Ang-2 was the key active molecule for hucMSC-Ex to promote angiogenesis and accelerate wound healing.HucMSC-Ex that modified with Ang-2 had more positive effects on pro-angiogenesis and skin damage repair.