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Shear stress regulates eNOS signaling in human umbilical vein endothelial cells via SRB1-PI3KAP1 pathway

Ying Zhang*, Ya-Hui Dong*, Bin Liao, Yong-Mei Nie, Ju-yi Wan, Liu-Lin Xiong, Yong Fu, Xiao-Jun Xie, and Feng-Xu Yu

Background: The occurrence and development of atherosclerosis are closely related to shear stress on the endothelial cells. However, related molecular mechanisms of controlling mechanical signal transduction in endothelial cells remain largely unknown. Aim of the Study: To obtain this goal, here, a parallel-plate flow chamber was used to impose laminar shear stress (8.4 dyne/cm2 ) on endothelial cells for 0 minute (min), 10 min, 30 min, 60 min, and 120 min. Subjects and Methods: The RNA interference was employed to down-regulate scavenger receptor class B1 (SRB1) and LY294002 was performed to block the phosphorylation status of the phosphoinositide 3-kinase pathway. Then, the cell status was observed under the microscope, followed by detection of phosphorylated phosphoinositide 3-kinase (PI3K), p38, extracellular signal-regulated protein kinases 1 and 2 and the binding activities of activator protein 1, nuclear factorκB and endothelial nitric oxide synthase (eNOs) by western blot. The results showed that shear stress (8.4 dyne/cm2 ) for 60 min induced obvious cell morphologic changes. Meanwhile, PI3K phosphorylation and eNOS expression were significantly increased by shear stress while reversed by SRB1 si-RNA and LY294002. Additionally, shear stress caused the enhanced binding activity of nuclear transcription factor activator protein 1 (AP-1) to DNA from 10 to 120 min, while only at 30 and 60 min for NF-κB. After treatment with SRB1 si-RNA and LY294002 inhibitor, the binding activities of AP-1 and DNA was significantly decreased, however, there was no effect on NF-κB binding activity. Results: Therefore, SRB1 plays a crucial role in mediating shear stress-induced eNOS activation in endothelial cells via PI3K-AP1 pathway. These findings will provide an important understanding of shear stress-induced cell alterations in clinic detection