The Role And Mechanism Of Pim1 In The Regulation Of ENOS Ser633、Ser1177 And VCAM1 Expression In Vascular Endothelial Cells By Shear Stress

Background: The incidence of atherosclerosis（AS）and its related cardiovascular and cerebrovascular diseases is increasing year by year,and vascular endothelial cell dysfunction is the initial link of the pathogenesis of AS.Blood flow shear stress is one of the important factors regulating endothelial cell function,but the specific regulatory mechanism remains unclear.The blood flow shear stress is roughly divided into two modes,one is laminar shear stress（LS）and the other is oscillatory shear stress（OS）.Different blood flow shear stress has different effects on endothelial cell function and gene expression.LS promotes nitric oxide（NO）secretion by activating endothelial nitric oxide synthase（eNOS）phosphorylation and inhibits the expression of inflammationrelated genes,such as vascular cell adhesion molecule 1（VCAM1）,maintains vascular homeostasis and health,and inhibits the development of AS,while OS causes endothelial dysfunction and promotes the development of AS.Pim1 is a serine/threonine protein kinase involved in regulating endothelial cell adhesion phenotype,eNOS phosphorylation,and angiogenesis.However,whether Pim1 regulates endothelial cell eNOS Ser1177,Ser633 phosphorylation and VCAM1 expression under shear stress and its mechanism remain unclear.Objective:（1）To explore the effect of different shear stress on the expression of Pim1 in endothelial cells;（2）To explore whether LS promotes the phosphorylation of eNOS Ser1177 and Ser633 of endothelial cells through Pim1 and its mechanism;（3）To explore whether LS inhibits the expression of VCAM1 in endothelial cells through Pim1 and its mechanism.Methods: The aortic arch and thoracic aortic vessel wall tissues of C57BL/6 mice were collected,and qPCR and Western blot were used to detect the differences in the expression of Pim1 in the vessel wall at different shear stress locations in vivo.Primary cultured human umbilical vein endothelial cells（HUVECs）were loaded with LS（15 dyn/cm2）,OS（0.5 ± 4 dyn/cm2）or static conditions（static,ST）culture;specific small interfering RNA（siRNA）transfection technology was used to silence Pim1,Akt,PFKFB3 genes;HUVECs were transfected with pc DNA3.1-Pim1 to overexpress Pim1;PI3K specific inhibitor Wortmannin was used to inhibit PI3 K expression;Western blot was used to detect the protein expressions of Pim1,PFKFB3 and VCAM1,as well as the phosphorylation of PI3 K p85α,Akt Ser473,eNOS Ser1177 and Ser633 phosphorylation.The lactate content in HUVECs culture medium was determined by colorimetry.Results:（1）In C57BL/6 mice,the expression of Pim1 protein and m RNA in the thoracic aorta（the site of action of LS）was significantly higher than that of the aortic arch（the site of action of OS）;（2）Compared with the ST group,LS promoted the expression of Pim1 in HUVECs and inhibited OS;（3）Silencing Pim1,LS-induced Akt Ser473 phosphorylation,eNOS Ser1177 and Ser633 phosphorylation expression decreased;（4）Akt silencing also significantly inhibited Akt Ser473 phosphorylation,eNOS Ser1177 and Ser633 phosphorylation expression,but had no effect on LS-induced Pim1 expression;（5）Inhibiting PI3 K expression,LS-induced Pim1,Akt Ser473 phosphorylation,eNOS Ser1177 and Ser633 phosphorylation expression decreased;（6）Silencing PFKFB3,OS-induced lactate content and VCAM1 expression decreased;（7）After silencing Pim1,the expression of PFKFB3 was up-regulated,accompanied by an increase in lactate content;after overexpression of Pim1,the expression of PFKFB3 was down-regulated,accompanied by a decrease in lactate content.Conclusion:（1）The expression of Pim1 is regulated by different shear stress,and LS significantly up-regulates the expression of Pim1;（2）Pim1 promotes the phosphorylation of eNOS Ser1177 and Ser633 of endothelial cells by LS through Akt;（3）LS regulates the expression of Pim1 through PI3K;（4）Shear stress regulates the expression of VCAM1 in endothelial cells through Pim1 and PFKFB3;（5）Pim1 has a negative regulatory effect on PFKFB3.