The Function And Mechanism Of HucMSC-EVs Facilitating Wound Healing And Angiogenesis In Diabetic Mice

ObjectiveEndothelial dysfunction caused by persistent hyperglycemia in diabetes is responsible for a lack of angiogenesis in diabetic wounds,which brings about defective and prolonged wounds.EVs are considered as potential therapeutic tools for diabetic wound healing.The aims of this study were to investigate the effects of human umbilical cord mesenchymal stem cell-derived EVs（huc MSC-EVs）on diabetic wound healing and angiogenesis and to elucidate the cellular and molecular mechanisms of huc MSC-EVs improving angiogenesis through high glucose-induced endothelial cell-aging model.Methods1.Huc MSC were isolated and cultured with exosome-free fetal bovine serum.Supernatants were collected.Huc MSC-EVs were extracted by ultracentrifugation from the supernatants and identified by transmission electron microscopy,western blots and nanoparticle tracking analysis technology.Full-thickness wounds were established on the back of diabetic mice.Huc MSC-EVs were injected around the wounds of diabetic mice.Gross views of wounds were observed at day 4,8 and 12 after operation to calculate wound healing rate.Hematoxylin and eosin staining（H&E）was employed for histological observation and extents of re-epithelization were examined.Masson staining was applied to observe deposition of collagen.The effects of huc MSC-EVs on diabetic wound healing were evaluated by the above parameters.Newly-formed blood vessels were examined under skin.Doppler ultrasound was used to observe the local blood flow and CD31 protein was used to evaluate the neovascularization.2.Human umbilical vascular endothelial cells（HUVECs） were incubated with 35 m M high glucose（HG）for 7-9 days to establish endothelial cell-aging model in vitro.Huc MSC-EVs were added into HG treated HUVECs and EVs internalization assay was performed to examine whether huc MSC-EVs could enter HUVECs.The intracellular reactive oxygen species（ROS）content was detected via DCFH-DA probe.The expressions of SA-β-gal,senescence related proteins including p21,p16 and p53 were examined to evaluate the senescent status of endothelial cells.Cell proliferation was evaluated by CCK-8 and flow cytometry.Cell migration was evaluated by scratch test and transwell assay.Angiogenesis ability of endothelial cells was evaluated by tube formation assay.3.Western blot was used to detect the expression of key proteins in PTEN/AKT/HIF-1α/VEGF signaling pathway,and RNAi was used to further determine the regulatory role of key proteins.Next,the miRNAs in huc MSC-EVs were identified by miRNA microarray and further confirmed by quantitative real-time PCR（q RT-PCR）assay.miRNA mimics and miRNA inhibitors were used to verify the regulating role in the signaling pathway by western blots.Meanwhile,the effects of candidate miRNAs on abilities of proliferation,migration and tube formation of high glucose-induced endothelial cells were evaluated by cck-8 assay,wound scratch assay,transwell assay and tube formation assay.Furthermore,the roles of candidate miRNAs on wound healing in diabetic mice were also examined by evaluating wound closure rates,blood perfusion and CD31 expression.Results:1.The ultrastructure of the huc MSC-EVs was lipid bilayer structure,with a diameter of approximately 100 nm and being cup or ball shaped.Markers including CD9,CD63,and TSG101 were expressed by the huc MSC-EVs and Calnexin,as a negative protein marker of EVs,was not detectable in our study.After treatment with huc MSC-EVs,the wound healing was accelerated in diabetic mice,the epithelialization was more rapid and the collagen fibers were arranged regularly.Furthermore,we can find a lot of neovascularization in the wound and the blood perfusion was better than that in untreated group.CD31 expression of huc MSC-EVs treated group is higher than that of the control.2.After internalization of huc MSC-EVs by senescent endothelial cells induced by high glucose,cell senescence was significantly improved.The ROS generation and the expression of SA-β-gal,p21、p53 and p16 were significantly reduced.The results of CCK-8 assay showed the enhanced cell proliferation and more cells were in the S and G2/M phases after treatment with huc MSC-EVs.The results of scratch test and transwell assay showed that huc MSC-EVs enhanced the migration ability of senescent endothelial cells.In addition,the numbers of vascular branches and nodes were significantly increased by huc MSC-EVs.3.In order to explore the regulatory mechanism of huc MSC-EVs improving the function of senescent endothelial cells,the expression of key proteins in PTEN/AKT/HIF-1α/VEGF signaling pathway was detected.Western blot analysis showed that the expression of PTEN increased significantly after high glucose treatment and the expression of p-Akt,HIF-1α and VEGF decreased significantly.But huc MSC-EVs treatment could reverse the expression of PTEN,p-Akt,HIF-1αand VEGF.We used si RNA PTEN（si PTEN）to inhibit the expression of PTEN and found si RNA PTEN can imitate the function of huc MSC-EVs to increase the proliferation,migration and tube formation of senescent endothelial cells.4.In order to analyze the components of huc MSC-EVs,miRNA expression profiles were detected by micro RNA chip.The top four miRNAs were miR-17-5p,miR-3960,miR-4497and miR-let-7d-5p,which was confirmed by q RT-PCR.Followingly,we investigated the potential targets of miR-17-5p and found 3’-UTR of human PTEN was composed of a putative region.By transfecting miR-17-5p inhibitor into hucMSCs,EVs without miR-17-5p(EVs-inhibitor^miR-17-5p)was obtained.EVs-inhibitor^miR-17-5p,EVs-inhibitor^NC,miR-17-5p agomirs and NC agomirs were transfected into high glucose-induced endothelial cells. miR-17-5p agomirs could effectively mimic the function of huc MSC-EVs to downregulate the expression of PTEN and activate AKT/HIF-1α/VEGF signaling pathway,but EVs- inhibitor ^miR-17-5phad no effect on the expression of these genes.5.We further observed the effect of miR-17-5p in huc MSC-EVs on the biological function of high glucose-induced endothelial cells.CCK-8 results showed that EVs-inhibitor^NCand miR-17-5p agomirs significantly increased the proliferation of senescent endothelial cells and miR-17-5p agomirs could partially simulate the effect of huc MSC-EVs on cell proliferation.The results of scratch test and transwell assay showed that EVs-inhibitor^NC and miR-17-5p agomirs enhanced the migration ability of senescent endothelial cells.Tube formation assay showed the increased numbers of vascular branches and nodes in miR-17-5p agomir group.6.In vivo,EVs-inhibitor^miR-17-5p,EVs-inhibitor^NC,miR-17-5p agomirs and NC agomirs were injected into diabetic wounds in mice.Compared with EVs-inhibitor^miR-17-5pand NC agomir groups,EVs-inhibitor^NC and miR-17-5p agomirs improved the wound healing,enhanced the blood perfusion,and increased the number of new blood vessels.ConclusionBy downregulating the expression of PTEN and activating the AKT/HIF-1α/VEGF signaling pathway,miR-17-5p in huc MSC-EVs improved the function of high glucose-induced endothelial cells,enhanced neovascularization,and increased the blood perfusion,thereby accelerating diabetic wound healing in mice.huc MSC-EVs and miR-17-5p are expected to be important means to promote vascular regeneration in high glucose microenvironment and to be the effective treatment for diabetic wounds.