β2AR-dependent signaling contributes to in-vivo reendothelialization capacity of endothelial progenitor cells by shear stress

BACKGROUND: Endothelial progenitor cells (EPCs) play a crucial role in the endothelial repair after arterial injury. Shear stress has a beneficial effect on modulating EPC functions. The molecular mechanism underlying the influence of EPCs on the endothelial integrity and shear stress effects on EPC regulation remained unclear. Herein, we investigated the influence of beta2 adrenergic receptor (beta2AR)-dependent signaling on in-vitro shear stress-mediated function and in-vivo reendothelialization capacity of human EPCs. METHOD: The human EPCs from healthy population were exposed to in-vitro 5, 10, and 20 dyn/cm shear stress for 15 h, and 10 dyn/cm for 5, 10, and 15 h, respectively. The in-vitro proliferation was assessed by CCK8 and BrDU tests. The migration and adhesion were evaluated by Transwell system and human umbilical vein endothelial cells (HUVECs) incorporation assays. Meanwhile, the angiogenic cytokine stromal derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) concentration of supernatant were tested by ELISA. Phosphorylated beta2AR, Akt, and eNOS were detected by western blot. In an in-vivo study, mice carotid injury models were produced through denuding the endothelium with a curved flexible wire, and thereafter CM-Dil-labeled EPCs were injected intravenously. After 3 days, cells recruited to the injury sites were detected by fluorescent microscopy, and the in-vivo reendothelialization capacity was assessed by Evans blue dye. RESULTS: Shear stress improved in-vitro functions and in-vivo reendothelialization capacity of human EPCs. In parallel, shear stress up-regulated the phosphorylation of beta2AR, Akt, and eNOS, and promoted vascular endothelial growth factor (VEGF) secretion of human EPCs. With ICI118,551 (a beta2AR inhibitor) treatment, shear stress-induced Akt and eNOS phosphorylation as well as VEGF secretion were suppressed. After beta2AR/PI3K/Akt/eNOS pathway of EPCs was blocked, the effects of shear stress on in-vitro functions and in-vivo reendothelialization capacity of EPCs were inhibited. CONCLUSION: The present study provided the novel data that shear stress-induced beta2AR/Akt/eNOS pathway enhanced reendothelialization capacity of EPCs. Shear stress-induced beta2AR-dependent pathway may be a novel and important therapeutic target for endothelial repair.