| Background and objective: The exosomes are derived from mesenchymal stem cell(MSC)and may be potentially used as an alternative for cell therapy,for treating diabetic wounds,and aid in angiogenesis.This study,aimed to investigate whether exosomes originated from bone marrow-derived MSCs(BMSCs)preconditioned by a low dose of deferoxamine(DFO-Exos)exhibited superior proangiogenic properties in wound repair,and to explore the underlying mechanisms involved.To investigate the effects of these exosomes,human umbilical vein endothelial cells(HUVECs)were used for assays involving tube formation,scratch wound-healing,and cell proliferation.To test the effects in vivo,streptozotocin-induced line of diabetic rats was established.Two weeks after the procedure,histological/histomorphometrical analysis was used to measure wound healing effects,and micro-CT was used to measure the neovascularization.Our findings demonstrated that DFO-Exos activate the PI3K/AKT signaling pathway via mi R-126 to stimulate angiogenesis in HUVECs.This contributed to wound healing and angiogenesis in streptozotocin-induced diabetic rats in vivo.Our results suggest that in cell-free therapies,exosomes may be induced by DFO at low doses to manifest increased proangiogenic ability.Method: 1.Collection and identification of the exosomes 1.1 the collection of exosomes: Exosomes were isolated from the conditioned medium by ultracentrifugation and ultrafiltration.1.2 the identification of exosomes: we use transmission electron microscopy(TEM)to observe the morphologies of the exosomes.We use Tunable resistive pulse sensing(TRPS)to analyse the size distribution and concentration of the exosomes.We use Western Blot to detect the surface markers on the surface of the exosomes.2.DFO-Exos promoted angiogenesis in vitro.The effects of different exosomes on HUVECs' proliferation,tube formation,and migration were evaluated by CCK-8 assay,Matrigel-based tube formation assay,and scratched assay,respectively.3.The STZ-induced diabetic rats show a faster cutaneous wound healing by DFO-Exos 3.1 We made the diabetic skin wound rat model first,and then we administrate exosomes or an equal volume of PBS in multi-points around the scars.3.2 We evaluated the wound healing effects of different exosomes and calculated the reduction of the wound-size at day 4,and 14 post-wounding.3.3 We assessed the extent of re-epithelialization and scar formation by hematoxylin and eosin(H&E)staining;Masson's trichrome staining was set to determine the fact of the collagen regeneration.4.The effects of DFO-Exos on angiogenesis around the wound sites 4.1 We perfuse the Blood vessels of the rat were with the microfilm.And then we reconstructed the blood vessels by micro-CT.4.2 Immunostaining and double-staining for CD31 and ?-SMA were done to identify respectively both numbers and blood vessels that were mature in wound sites that received various treatments.5.The mechanism for the promotion of angiogenesis by DFO-Exos 5.1 We utilize real-time PCR(q RT-PCR)to examine the molecules that were involved in the mediation of the pro-wound healing effects of DFO-Exos.5.2 We co-culture HUVECs with green dye Di O-labeled DFO-Exos.The uptake of DFO-Exos by HUVECs was analyzed with a fluorescence microscope.5.3 The PI3K/AKT signaling circuit,is recognized to be targeted by mi R-126,is vital in controlling migration,proliferation and survival of cells.Western Blot were used to verify the alteration of PI3K/AKT signaling-related proteins in HUVECs after DFOExos stimulation.6.DFO-Exos stimulation causes PI3K/AKT signaling to activate in HUVECs.6.1 Analysis by western blot in HUVECs 6.2 The transwell assay and tube formation assay was done to observe the function exosomes of derived MSCs stimulated by DFO.Results: 1.Characterization of exosomes 1.1 The presence of exosomal marker proteins CD9,CD63,TSG101,and GM130 was detected in both Exos and DFO-Exos.1.2 Analysis using TEM analysis revealed that these exosomes were vesicles bound by a membrane,round in shape and had a diameter of 30-100 nm.1.3The size of the exosomes ranged from 18 nm to 182 nm,with a mean size of 75 nm,6.65×108 particles/m L was predicted as the concentration.2.Exosomes promoted angiogenesis in vitro 2.1The Exos and DFO-Exos increased the proliferation of endothelial cell significantly compared to the control group,at 24 and 48 hours.2.2The scratch wound-healing assay shows the HUVEC co-cultured with DFO-Exos were seen to migrate at rates more rapid than the other two groups.? 2.3 The transwell assay that finds wide application to study cell migration led to an additional confirmation of the pro-migratory ability of DFO-Exos.2.4 In the assay for tube formation,it was observed that a greater number of cord-like structures were formed on Matrigel by HUVEC co-cultured with DFO-Exos in comparison to the Exos group and controls.3.The cutaneous wound healing of the STZ-induced diabetic rats 3.1 On days 7,and 14 post-wounding,wound closure was accelerated in animals that received Exos in comparison to control PBS,and a higher rate of wound closure was observed in rat treated with DFO-Exos compared with that treated with common Exos.3.2 Following 14 days after causing wounds,the DFO-Exos treated wounds showed increased re-epithelialization in comparison to those treated with Exos and PBS.Additionally,the scars were narrower and collagen deposition areas were larger in the wound treated with DFO-Exos in comparison with that of the controls.4.The diabetic rats show increased angiogenesis on the wound sites 4.1 In the 14 th day post-wounding,the 3D images were reconstructed by micro-CT that showed an increase in density of blood vessels by administration of Exos and DFOExos when compared to controls.Additionally,the DFO-Exos treated wound sites exhibited a greater degree of formation of blood vessels.4.2 The quantification of average density of the blood vessels as well as a count of blood vessels that were mature was done.According to the results,the number of overall as well as mature blood vessels in the wound sites that received DFO-Exos treatment was enhanced remarkably compared to that of Exos and the controls.5.DFO-Exos shuttle mi R-126 into HUVECs 5.1 The expression of several mi RNAs involved in angiogenesis regulation in exosomes: mi R-214,mi R-21,mi R-126,mi R-125 b mi R-27 b,and mi R-19 b were detected.Among these mi RNAs detected,mi R-126 was found to display maximum expression.5.2 Fluorescence microscopy confirmed this uptake of exosomes by HUVECs.More than 90% HUVECs showed positive staining for DAPI after 8 hours that is indicative of uptake of exosomes labeled by DIO and their transfer to compartments of the cytoplasm.6.DFO-Exos stimulation causes PI3K/AKT signaling to activate in HUVECs.6.1 Analysis by western blot in HUVECs showed an evident reduction of phosphoinositol-3 kinase regulatory subunit 2(PIK3R2)and an increase in the amount of phosphorylation of AKT(p AKT)subjected to DFO-Exos treatment.The results also are indicative of the possibility of transfer of mi R-126 into target cells to cause regulation of gene expression.6.2 There was an upregulation of PIK3R2 and a downregulation of p AKT in the 126IDFO-Exos group.The migration of cells was lower in the 126I-DFO-Exos group,as seen in the transwell assay.The length of tubes and branch numbers were lowered by a significant degree in the 126I-DFO-Exos group as observed in the tube formation assay.These observations are indicative of an important function of mi R-126 in the features of proangiogenesis of exosomes of derived MSCs stimulated by DFO. |
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