Exosomes From Adipose Stem Cells Promote Diabetic Wound Healing Through Modulating Oxidative Stresses Via HSP90/LRP1/AKT Axis

Objective: Diabetic wounds remain a great burden to global healthcare systems.Currently,there is no satisfactory treatment for these hard-to-heal-wounds.Recent studies demonstrated that exosomes from various stem cells could promote wound healing without the risks of immunorejection and teratomas.Oxidative damages are recognized as a critical cause of diabetic wounds.Previous studies mainly focused on the effect of exosomes in optimizing cellular functions.This study will investigate whether exosomes from adipose stem cells(ADSC-EXOs)could promote diabetic wound healing through modulating oxidative stress,and unveil the underlying mechanisms.Thus,this project will improve the existing mechanisms of exosomes-based therapy,and provide a promising therapeutic strategy for wound healing in diabetes.Methods: Human ADSCs were identified by flow cytometry analysis and evaluation of their osteogenic and adipogenic differentiation potential.ADSC-EXOs were isolated by differential ultracentrifugation,stained by PKH26,and identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and western blot.The ability of ADSC-EXOs in modulating cellular function were assessed by Ed U,transwell and in vitro tube formation assays.H2O2 and Co Cl2 were used to conduct oxidative stress and hypoxia models separately.The capacities of ADSC-EXOs in modulating oxidative stress and cell protection were verified by CCK8,reactive oxygen species(ROS)assay and flow cytometry analysis.The signaling pathways activated by ADSC-EXOs were confirmed by western blot.Primary anti-hsp90 antibody was used to neutralize the function of hsp90 in ADSC-EXOs’ surface.LY294002 and U0126 were used to inhibit the activation of AKT and ERK signaling pathways separately.The expression level of LRP1 was knockdown by the sh RNA lentivirus vector,and further confirmed by real-time quantitative polymerase chain reaction(RT-PCR)and western blot.Finally,we assessed the therapeutic effect of ADSC-EXOs in a diabetic skin injury model.Hematoxylin-eosin(HE)staining,Masson’s trichrome staining,Immunohistochemical staining,Immunofluorescence staining and so on were performed to evaluate the re-epithelialization,collagen deposition,neo-vascularization,cell proliferation rate and death rate of skin wounds.Results: We have successfully isolated and purified human ADSCs and ADSC-EXOs.ADSC-EXOs could promote the proliferation,migration and angiogenesis of Ha CAT,fibroblasts and human umbilical vein endothelial cells(HUVECs).Besides,ADSC-EXOs could reduce the ROS level in these cells and protect them from hypoxia and oxidative stress injuries.Further research found that the function of ADSC-EXOs highly relied on their surface heat shock protein 90(hsp90),and anti-hsp90 antibody could inhibit their abilities.Hsp90 in ADSC-EXOs surface could bind the LRP1 receptor in cell membrane,and then activate AKT signaling pathway.Knockdown of LRP1 in fibroblasts and inhibition of AKT signaling pathway by LY294002 could impair the beneficial effect of ADSC-EXOs.Finally,the local injection of ADSC-EXOs at wound sites could significantly increase re-epithelialization,collagen deposition and neo-vascularization,reduce the level of ROS and cell death,and thus led to accelerated diabetic wound closure.Conclusion: Our data suggest that ADSC-EXOs therapy can significantly accelerate diabetic wound healing through modulating oxidative stresses via HSP90/LRP1/AKT axis.This illustrates the therapeutic potential of ADSC-EXOs which may become a novel treatment paradigm for diabetic wound healing.