EPSTI1 Promotes Monocyte Adhesion To Endothelial Cells Via VCAM-1 And ICAM-1 Upregulation In Atherosclerosis

Background and aimAtherosclerosis is a chronic inflammatory disease of the arterial wall.Endothelial injury is a crucial step in the initiation of atherosclerosis.The phenotypic characteristics of endothelial injury include excessive secretion of chemokines and adhesion molecules,which leads to the recruitment and firm adhesion of circulating monocyte to endothelial cells.Increasing studies have revealed that the enhanced adhesion and migration of circulating monocyte to vascular endothelial cells are typical features of the inflammatory response during atherosclerosis formation.In this study,differentially expressed genes in human atherosclerotic plaques were screened by RNA-seq,and it was found that epithelial stromal interaction 1(EPSTI1)was highly expressed.Although EPSTI1 has been well studied in the development of breast cancer,the association between EPSTI1 and atherosclerosis has not been reported.The purpose of this study is to explore the expression pattern of EPSTI1 in atherosclerotic plaques and its role and mechanism in monocyte-endothelial cell adhesion,so as to provide a new scientific basis for the prevention and treatment of atherosclerosis.Method1.The expression of EPSTI1 in human atherosclerotic plaques was detected by qPCR,Western blot and immunohistochemistry and its expression in endothelial cells of human and mouse atherosclerotic plaques was analyzed by double immunofluorescence staining;2.Primary human umbilical vein endothelial cells(HUVECs)were isolated,and the EPSTI1 overexpression recombinant plasmid and small interference RNA targeting EPSTI1 were constructed and transfected into HUVECs.The effect of EPSTI1 on monocyte-endothelial cell adhesion was observed by adhesion experiment;3.qPCR and Western blot were used to detect the effect of EPSTI1 on the expression of VCAM-1 and ICAM-1;4.qPCR and Western blot were used to detect EPSTI1,VCAM-1 and ICAM-1 expression of HUVECs stimulated by LPS at different concentrations and times,and the effect of EPSTI1 knockdown on LPS induced VCAM-1 and ICAM-1 expression and monocyte-endothelial cell adhesion.5.The NF-κB p65 overexpression recombinant plasmid and small interference RNA targeting NF-κB p65 were constructed and transfected into HUVECs.qPCR and Western blot were used to explore the mechanism of LPS regulating the expression of EPSTI1.Result1.The expression of EPSTI1 was elevated in human atherosclerotic plaques and highly expressed in endothelial cells of human and mouse atherosclerotic plaques;2.Overexpression of EPSTI1 promoted the adhesion of THP-1 cells to HUVECs,while knockdown of EPSTI1 inhibited its adhesion;3.Overexpression of EPSTI1 upregulated the mRNA and protein expression of VCAM-1 and ICAM-1,while knockdown of EPSTI1 downregulated their expression;4.Knockdown of VCAM-1 and ICAM-1 significantly inhibited the effect of EPSTI1 promoting the adhesion of THP-1 cells to HUVECs;5.LPS induced EPSTI1 expression at both mRNA and protein levels in a dose-and time-dependent manner;6.Knockdown of EPSTI1 significantly inhibited LPS-induced VCAM-1 and ICAM1 expression and the adhesion of THP-1 cells to HUVECs;7.Overexpression of NF-κB p65 upregulated the mRNA and protein expression of EPSTI1,while knockdown of NF-κB p65 downregulated its expression;8.Knockdown of NF-κB p65 significantly inhibited LPS-induced EPSTI1 expression.Conclusion1.EPSTI1 is highly expressed in endothelial cells of human and mouse atherosclerotic plaques;2.EPSTI1 promotes monocyte-endothelial cell adhesion by upregulating the expression of VCAM-1 and ICAM-1;3.EPSTI1 is involved in LPS-induced expression of VCAM-1 and ICAM-1 and monocyte-endothelial cell adhesion;4.LPS induces the expression of EPSTI1 through NF-κB p65.