Effect of Extracellular Vesicles in Adipose-derived Mesenchymal Stem Cells on Human Microvascular Endothelial Cells Injury by Increasing LC3B Expression
Objective:To investigate the protective effect of extracellular vesicles(Evs)in adipose-derived mesenchymal stem cells(ADSCs)induced by hypoxic microenvironment on human microvascular endothelial cells injury by increasing the expression of microtubule-associated protein light chain 3B(LC3B).Methods:The rat ADSCs were isolated and identified by osteogenic differentiation,lipogenic differentiation and expression of cell surface markers.Normoxic Evs and hypoxic Evs were obtained under normal oxygen(21％O2)and hypoxic oxygen(1％O2)conditions respectively,and identified by the expression of morphology and surface markers.The oxygen-glucose deprivation/reperfusion(OGD/R)model of cardiac microvascular endothelial cells(CMECs)was constructed,and CMECs were divided into Control group,OGD/R group,N-Evs group,hypoxic Evs group(H-EVS group,hypoxic Evs culture for 24 h),hypoxic Evs+autophagy inhibitor group(H-EVS+3-MA group,hypoxic Evs and 3-MA 5 mmol/L culture for 24 h).The proliferation,migration,invasion,angiogenesis and autophagy of CMECs were detected by cell counting kit(CCK-8),5-acetylidene-2-deoxyuracil nucleoside(EdU)staining,scratch test,Transwell test,tubular formation test and transmission electron microscopy.The expression of proliferating cell nuclear antigen(PCNA),Ki67,matrix metalloproteinase(MMP)-2/MMP-9,hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),LC3B and recombinant human autophagy effect protein 1(Beclin1)were detected by enzyme-linked immunosorbent assay.Results:Compared with the Control group,the proliferation,migration,invasion,angiogenesis and autophagy of CMECs in the OGD/R group were reduced(P＜0.05),and the expressions of PCNA,Ki67,MMP-2/MMP-9,HIF-1α,VEGF,LC3B-Ⅱ/Ⅰ and Beclin1 decreased(P＜0.05).Compared with the OGD/R group,the proliferation,migration,invasion,angiogenesis and autophagy of CMECs in the N-Evs group and H-Evs group were enhanced(P＜0.05),and the expressions of PCNAKi67,MMP-2/MMP-9,HIF-1α,VEGF,LC3B-Ⅱ/Ⅰ and Beclin1 increased(P＜0.05).These indexes of H-Evs group were better than those of N-Evs group(P＜0.05).Compared with the H-Evs group,proliferation,migration,invasion,angiogenesis and autophagy of CMECs in the H-Evs+3-MA group reduced(P＜0.05),while expressions of PCNA,Ki67,MMP-2/MMP-9,HIF-1α,VEGF,LC3B-Ⅱ/Ⅰ and Beclin1 decreased(P＜0.05).Conclusion:ADSCs-Evs induced by hypoxic microenvironment could promote proliferation,migration,invasion,angiogenesis and autophagy of OGD/R injured CMECs by increasing LC3B expression.
cardiac microvascular endothelial cellsadipose-derived mesenchymal stem cellsexternal vesicleshypoxic microenvironmentratsexperimental study
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