Abstract
Recent studies have indicated that protease-activated receptor-1 (PAR-1) is involved in cytoprotective and anti-inflammatory responses in endothelial cells (ECs). However, the role of PAR-1 in laminar flow-mediated atheroprotective responses remains unknown. Herein, we investigated whether PAR-1 regulates laminar flow-mediated mechanotransduction in ECs. Confocal analysis showed that PAR-1 was internalized into early endosomes in response to laminar flow. In addition, flow cytometry analysis showed that cell surface expression of PAR-1 was reduced by laminar flow, suggesting that PAR-1 was activated in response to laminar flow. Depletion of PAR-1 using human PAR-1 siRNA inhibited unidirectional laminar flow-mediated actin stress fiber formation and cellular alignment as well as atheroprotective gene expressions in HUVECs. Moreover, PAR-1 knockdown inhibited laminar flow-stimulated eNOS phosphorylation, and inhibited the phosphorylations of Src, AMPK, ERK5 and HDAC5. Furthermore, PAR-1 depletion inhibited laminar flow-mediated anti-inflammatory responses as demonstrated by reduced TNFα-induced VCAM-1 expression and by monocyte adhesion to HUVECs, and prevented laminar flow-mediated anti-apoptotic response. An investigation of the role of PAR-1 in vasomotor modulation using mouse aortic rings revealed that acetylcholine-induced vasorelaxation was diminished in PAR-1 deficient mice compared to littermate controls. Taken together, these findings suggest that PAR-1 be viewed as a novel pharmacologic target for the treatment of vascular diseases, including atherosclerosis.
Highlights
Atherosclerosis preferentially involves regions prone to low and/or disturbed blood flow at vessel branch points and the outer walls of bifurcations, and regions exposed to steady, high levels of laminar blood flow are relatively protected from atherosclerosis[1,2,3,4]
In the present study, we investigated whether protease-activated receptor-1 (PAR-1) acts as a mechano-sensor in the laminar flow-mediated atheroprotective signaling pathway
After cell had been exposed to laminar flow, ATAP2 binding affinity to surface PAR-1 was found to be significantly down-regulated in a time-dependent manner like as activated protein C (APC) which is an endogenous agonist of PAR-1 (Fig. 1C)
Summary
Atherosclerosis preferentially involves regions prone to low and/or disturbed blood flow at vessel branch points and the outer walls of bifurcations, and regions exposed to steady, high levels of laminar blood flow are relatively protected from atherosclerosis[1,2,3,4]. In a recently study, it was shown laminar flow-mediated signalings are negatively regulated by histone deacetylase 520 (HDAC5; a member of the class IIa histone deacetylases and negative regulator of myocyte enhancer factor-2 (MEF2), a downstream target of the laminar flow-induced ERK5 activating pathway)[21]. One study demonstrated that laminar flow reduced the cell surface expression of PAR-132, but the mechanism whereby PAR-1 acts as a mechano-sensor in the laminar flow-mediated atheroprotective signaling pathway has not been studied. We found that PAR-1 is activated by laminar flow and regulates laminar flow-induced atheroprotective gene expression dependent on Src, AMPK, ERK5, HDAC5 and eNOS in EC. We demonstrated that acetylcholine-induced vasorelaxation was diminished in aortic rings of PAR-1 KO compared to littermate controls These data provide evidence that PAR-1 is a mechano-sensor for laminar flow which mediates anti-atherosclerotic responses in EC
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