| Background:Poor wound healing,chronic ulcers,and resulting amputations are serious complications of diabetes.In the process of skin wound healing,The accumulation of advanced glycation end product(AGE)caused by long-term high systemic glucose level and high glucose environment has been confirmed to cause poor wound healing,resulting in chronic wounds that are difficult to heal and show repeated inflammation and infection.However,the pathophysiological mechanism of chronic diabetic wound formation is multifactorial and complex,among which microvascular neovasculariz-ation disorder is one of the main causes of poor healing.Studies have found that the hypoxic microenvironment after wound formation is the key initiating factor mediating angiogenesis,and the transcription factor hypoxia-inducible factor-1(HIF-1)regulates the main response mechanism of tissues and cells to hypoxia.HIF-1has been shown to be closely related to angiogenesis during wound healing,and the low expression of HIF-1 in diabetic wounds is considered to be one of the key factors leading to delayed wound healing.However,there is a lack of biological agents targeting HIF-1 in clinical practice.Adipose-derived stem cells(ADSCs)are the most abundant and easily available type of mesenchymal stem cells(MSCs)in the human body.In the past decade,adipose-derived stem cells(ADSCs)have been reported to play a significant role in tissue repair and regeneration.With the discovery of extracellular vesicles and exosomes,paracrine vesicles secreted by adipose-derived stem cells have also been confirmed to have similar functions to their parent cells.However,whether it can promote diabetic wound healing and what is the key molecular mechanism remain to be proved.Therefore,this study intends to confirm the regulatory effect of human adipose-derived stem cell extracellular vesicles(h ADSC-EVs)on angiogenesis and reveal its potential molecular mechanism by cell proliferation,migration,tube formation,detection of key signal molecules expression,and monitoring of wound healing in a cell model of cultured vascular endothelial cells in vitro and a diabetic rat wound model.Methods:Part 1: Clinical specimens were collected and tested.A small amount of waste wound tissue and full-thickness normal skin tissue at the edge of the wound were collected from 3 diabetic patients during surgery.Immunofluorescence staining,real-time fluorescent quantitative PCR and Western blot were used to detect the microvessel density,the expression of phosphatidylinositol 3 kinase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(m TOR),and the expression of HIF-1α and its downstream cytokines in the tissues.Part 2: To investigate the effect of h ADSC-EVs on the function of vascular endothelial cells induced by AGE.The waste adipose tissue of patients who underwent autologous liposuction was collected.Adipose-derived stem cells were extracted by enzymatic digestion and identified by flow cytometry and oil red O staining.Adipose stem cells of 3 to 5 generations were amplified and cultured,supernatant was collected,and ADSC-EVs were obtained by ultra-high speed centrifugation.ADSC-EVs were identified by nanoparticle size analysis,transmission electron microscopy,and Western blot of extracellular vesicle molecular markers.Different concentrations of ADSC-EVs were co-cultured with AGE-treated human umbilical vein endothelial cells,and the cell proliferation,migration and tube differentiation ability were evaluated by CCK-8 assay,scratch test and matrigel tube formation test.The expression of HIF-1αand its downstream cytokines was detected by real-time fluorescent quantitative PCR and Western blot.Part 3: To investigate the upstream signaling pathway of h ADSC-EVs in improving the functional damage of vascular endothelial cells induced by AGE.The hypoxia-treated human umbilical vein endothelial cell transcriptome data(GSE1041,GSE36837,GSE12546)were screened in GEO Datasets for KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis.The possible key signaling pathways were identified.Human umbilical vein endothelial cells were induced by AGE and co-cultured with ADSC-EVs.Real-time fluorescence quantitative PCR and Western blot were used to evaluate whether PI3K-AKT-m TOR,the upstream signaling pathway of HIF-1α,was regulated by ADSC-EVs.Part 4: To verify the mechanism of h ADSC-EVs in the rat diabetic wound model.Streptozotocin injection via caudal vein and high-sugar-fat feeding to construct a diabetic rat model and make a round full-thickness skin defect on the back.By injecting ADSC-EVs and combined PI3 K inhibitor PI3K-IN-1,Masson staining,immunofluorescence staining and real-time fluorescence quantification PCR were used to evaluate the effect and molecular mechanism of ADSC-EVs on wound healing.Results:Part 1: The results of immunofluorescence staining showed that the microvessel density in diabetic wound tissue was significantly lower than that in normal skin tissue.The results of real-time fluorescent quantitative PCR and immunofluorescence staining showed that PI3 K,AKT,and m TOR were down-regulated in diabetic wound tissue.At the same time,the results of Western blot showed that the protein levels of HIF-1α,vascular endothelial growth factor(VEGF),chemokine 12(CXCL12),and chemokine receptor 4(CXCR4)were lowly expressed in diabetic wound tissue.Part 2: The expression of CD49 d,CD90,and CD105 in the third passage of human adipose stem cells was positive,while CD34,CD45,and CD106 were negative.After 14 days of adipogenic induction,clear round lipid droplets were observed and stained red with oil red O(positive).Under the transmission electron microscope,ADSC-EVs were round membranous vesicles with uniform size and uniform morphology,and highly expressed the marker proteins TSG101,CD63 and CD9,with an average particle size of 198.1±91.5 nm.h ADSC-EVs can be taken up and internalized by human umbilical vein endothelial cells when co-cultured with them.CCK-8 assay,scratch test and tube formation assay confirmed that ADSC-EVs could alleviate the inhibitory effects of AGE on the proliferation,migration and tube formation of human umbilical vein endothelial cells and promote the expression of HIF-1α,VEGF,CXCL12 and CXCR4 in a concentration-dependent manner when the concentration was not more than 100μg/ml.Part 3: KEGG enrichment analysis was performed on three sets of human umbilical vein endothelial cell transcriptome data(GSE1041,GSE36837,GSE12546)from GEO Datasets after hypoxia treatment,and the results suggested that PI3K-The AKT pathway is significantly enriched in the hypoxic response of vascular endothelial cells,and may be a key pathway.Combined with the differential expression of PI3 K,AKT and m TOR in diabetic wound tissue and normal skin tissue in the previous experiment,we further confirmed that h ADSC-EVs can up-regulate the expression of PI3K-AKT-m TOR in vascular endothelial cells induced by AGE through real-time fluorescent quantitative PCR and Western blotting.And this effect can be partially blocked by PI3 K inhibitor PI3K-IN-1,suggesting that the molecular mechanism of ADSC-EVs improving the function of vascular endothelial cells may be PI3K-AKT-dependent.Part 4: In animal experiments,the hyperglycemia rat model was successfully constructed by tail vein injection of STZ.After the wound model was established,local injection of h ADSC-EVs showed that ADSC-EVs could significantly improve the wound healing rate of diabetic rats,promote angiogenesis,and up-regulate the expression of PI3K-AKT-m TOR-HIF-1α/VEGF.The addition of PI3 K inhibitor PI3K-IN-1 can partially block the promotion of wound healing and HIF-1α/VEGF expression by ADSC-EVs,suggesting that the mechanism of ADSC-EVs promoting diabetic wound healing may be PI3K-AKT dependent.Conclusion:(1)Compared with normal skin,the microvessel density and the expressions of PI3 K,AKT,and m TOR on microvessels in diabetic wound tissue were significantly decreased,and the expressions of HIF-1α and downstream angiogenic factors VEGF,CXCL12,and CXCR4 in wound tissue were also significantly decreased.(2)Human adipose-derived stem cell extracellular vesicles can alleviate AGE-induced vascular endothelial cell injury,improve cell activity,enhance cell migration and tube formation ability,and up-regulate the expression of HIF-1α and its downstream pro-angiogenic factors VEGF,CXCL12 and CXCR4.(3)Human adipose-derived stem cell extracellular vesicles can improve AGE-induced vascular endothelial cell injury by activating PI3K-AKT-m TOR-HIF-1α/VEGF pathway.(4)Extracellular vesicles of human adipose-derived stem cells can improve the healing rate of diabetic wounds in rats in a PI3K-Ak T-dependent manner,and promote angiogenesis and the expression of HIF-1α/VEGF... |
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