| Research BackgroundSenescence is a natural process that all living mammals go through.Senescence leads to a specific set of changes in cellular function,morphology,gene expression and senescence-related β-galactosidase-positive staining.Senescent endothelial cells(ECs)compromise the integrity of the vascular endothelium,which leads to vascular aging and a range of diseases such as atherosclerosis and diabetes.When ECs exhibit a senescent phenotype,the homeostasis and function within them are impaired,and these alterations result in endothelial dysfunction,vascular inflammation,vascular neovascularization and impaired vascular repair.Senescence of ECs may be induced by multiple factors,among which oxidative stress plays an important role.Despite the fact that oxidative stress is one of the key causes of aging,the mechanisms of oxidative stress-induced senescence in ECs are yet unknown.Preventing or slowing down the senescence of ECs may become one of the promising therapeutic modalities for many diseases associated with vascular aging.MSCs(mesenchymal stem cells)are progenitor cells found in a range of tissues,including adipose and bone marrow.with functions including angiogenesis promotion,anti-apoptosis and immunomodulation.MSCs also have the ability to differentiate to multiple directions,such as lipogenesis and osteogenesis.Many clinical trials have also investigated the use of MSCs in a variety of diseases.Extracellular vesicles are vesicles produced by a wide range of cells,including MSCs.Small extracellular vesicles(sEV)are the type of vesicles generated by the fusion of cell membranes in multivesicular bodies.They are between 30-200 nm in diameter and contain abundant functional components,such as proteins and micro RNAs.In addition,sEV are able to transport functional components such as DNA,proteins,mRNA,micro RNA,lipids and organelles from the source cells to the recipient cells,which is an important mechanism for intercellular communication.Recent studies have shown that sEV have the potential to transfer bioactive molecules to recipient cells and induce phenotypic changes.Therefore,sEV have emerged as sophisticated means used to regulate various cellular processes.Moreover,MSCs have long been considered as important source cells for regenerative medicine,and MSCs in turn are among the most potent sEV-producing cells of different cell types.Therefore,studying the biological functions and mechanisms of MSC-derived extracellular vesicles(MSC-sEV)for ECs aging is not only instructive and informative for the treatment of vascular aging-related diseases,but also these findings will provide new ideas and directions to encourage the development of therapeutic techniques to decrease the progression of human aging and age-related disorders.Research objectivesWhile studies have shown that MSC-sEVs have beneficial effects on tissue repairing and the treatment of various diseases,the biological effects of MSC-sEVs on the aging of ECs are still unclear,and the mechanism underlying this biological action is still unknown.The purpose of this paper is to investigate the relevant role of MSC-sEV in the aging of ECs and its potential molecular mechanisms.Research methodsTo investigate whether MSC-sEV have a rescue effect on ECs senescence,we first treated ECs with different concentrations of hydrogen peroxide(H2O2)and identified various senescence markers by different experiments,such as immunoblotting,to establish an efficient senescence model of ECs.Then we treated senescent ECs with MSC-sEV and identified senescence.After that,we used MSC-sEV to treat senescent ECs and identified the changes of senescence markers by immunoblotting and a series of functional experiments to determine whether they have an effect on ECs’ functions,including cell migration ability,in vitro and in vivo vasculogenic ability,and proliferation ability.We then performed wound healing experiments using naturally aged mice and type 2 diabetic model mice to observe the effect of MSC-sEV on wound healing and neovascularization in mice.We next performed a study on the molecular mechanisms of MSC-sEV intervention in the aging of ECs.A miRNA microarray was used to analyze the expression profiles of MSC-sEV and endothelial cells before and after its intervention,and miRNA that might play a role were selected for further molecular and functional validation.After that,we used phosphokinase microarray to detect the signaling pathways with altered expression before and after the intervention,and selected the key signaling pathways for inhibitor intervention to study their effects on ECs senescence.Finally,we investigated whether the screened miRNA in MSC-sEVs had corresponding effects on the gene expression changes of ECs.Results of the studyThe findings of in vitro tests revealed that MSC-sEV could reduce the expression of biomarkers of senescence in ECs,decrease the senescence-associated secretory phenotype,and rescue oxidative stress-induced angiogenesis,migration and other dysfunctions in senescent ECs.In natural aging and type 2 diabetic mice wound healing experiments,we found that MSC-sEV could promote wound closure and neointima formation.Microarray expression profiling revealed that miR-146a was highly expressed in MSC-sEVs and in ECs after MSC-sEV treatment.miR-146a inhibitor blocked the restorative effect of MSC-sEVs on ECs senescence.In addition,we found that miR-146a inhibited phosphorylation of Src and activation of the downstream molecules VEcadherin and Caveolin-1.ConclusionMSC-sEV could rescue oxidative stress-induced senescence in ECs at the molecular level and functional level,including changes in various senescence markers,restoration of cell migration capacity,angiogenic capacity,and proliferative capacity.Meanwhile,MSC-sEV could promote wound healing capacity and angiogenic capacity in naturally senescent and type 2 diabetic mice.Overall,our data suggested that MSC-sEV slowed down ECs senescence and promote angiogenesis via miR-146a/Src. |
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