The Effects Of Serum-derived Exosomes From Patients With Stable Coronary Artery Disease On The Biological Function Of Endothelial Cells And The Underlying Mechanisms

Stable coronary artery disease(SCAD)is the most common form of CAD,which accounting for the majority of deaths worldwide.Atherosclerosis(AS)is the pathological basis of CAD,and its main initial factor is endothelial dysfunction.Exosomes are nanoscale vesicles secreted by cells,which contain bioactive molecules such as RNA and protein,and widely participate in the pathophysiological process of the body.Previous studies have shown that the abnormal expression of the contents in circulating exosomes,especially miRNAs,is closely related to cardiovascular and cerebrovascular diseases induced by AS such as CAD and cerebral infarction.However,there are few reports on the role and mechanism of serum-derived exosomes and their contents in the occurrence and development of AS.The aim of this study was to investigate the effects of serum-derived exosomes from SCAD and their differentially expressed miRNA on the biological function of endothelial cells including cell proliferation,migration,tube formation and inflammation and the underlying mechanisms.In order to investigate whether the serum-derived exosomes from SCAD affect the biological function of endothelial cells,we used an ExoQuick reagent to extract the serum-derived exosomes from SCAD group and control group.Then the extracted exosomes from the two groups were co-cultured with human umbilical vein endothelial cells(HUVECs),respectively.Laser confocal microscopy and flow cytometry analysis showed that there was no difference in the uptake of serum-derived exosomes between the two groups.CCK-8 assay,scratch and Transwell assay,tubule formation assay and qPCR assay showed that the serum-derived exosomes from SCAD could significantly reduce cell proliferation activity,migration ability and tubule formation ability,while up-regulate the expression of inflammatory factors IL-1?,TNF-? and ICAM-1,which indicated that the serum-derived exosomes from SCAD could induce endothelial cell injury and inflammatory response.In order to identify the differentially expressed miRNAs in the serum-derived exosomes from SCAD and explore whether they are involved in regulating the biological function of endothelial cells,we used high-throughput sequencing to analyze the miRNAs expression profiles of serum-derived exosomes from the SCAD group and the control group,and selected the miRNAs with higher expression level and more than two-fold change for qPCR verification.qPCR assay showed that the expression levels of miR-942-5p,miR-149-5p and miR-32-5p were significantly increased in the serum-derived exosomes from SCAD.Moreover,bioinformatics analysis showed that these miRNAs were involved in regulating the biological function of endothelial cells.Notably,miR-32-5p has been reported to be associated with endothelial cell injury and inflammatory response in CAD,but its role and mechanism remain unclear.In order to further explore the role and mechanism of miR-32-5p in endothelial cell injury and inflammatory response,the endothelial cell injury and inflammatory response were induced by the treatment of HUVECs with oxidized low density lipoprotein(oxLDL).The putative binding sites of miR-32-5p and AIDA 3'UTR were predicted by TargetScan software and verified by luciferase reporter assay.We found that the expression of miR-32-5p was decreased while the expression of AIDA was increased in HUVECs induced by oxLDL,and miR-32-5p could inhibit the expression of Aida.What's more,overexpression of miR-32-5p or knockdown of AIDA could significantly improve the proliferation activity,migration ability and tube formation ability of HUVECs induced by oxLDL,as well as inhibit the expression of inflammatory factors IL-1 ?,IL-6,TNF-?,ICAM-1 and VC AM-1 and the activity of NF-?B signaling pathway.These results suggest that miR-32-5p may alleviate oxLDL-induced endothelial cell injury and inflammation through regulating AIDA/NF-?B pathway.In conclusion,the serum-derived exosomes from SCAD and their differentially expressed miR-32-5p play important roles in regulating the biological function of endothelial cells.The serum-derived exosomes from SCAD could induce endothelial cell injury and inflammatory response,and their differentially expressed miR-32-5p might play an inhibitory role in this process.This work provides new insights on the potential role of the serum-deri ved exosomes and their contents in the pathophysiological process of AS,and helps to provide novel diagnostic markers and therapeutic targets for SCAD.