Vascular endothelial cells play an important role in the regulation of vascular function in response to mechanical stimuli in both healthy and diseased states. Prostaglandin I2 (PGI2) is an important anti-atherogenic prostanoid and potent vasodilator produced in endothelial cells through the action of cyclooxygenase (COX) isoenzyme COX-2. However, the mechanisms involved in sustained, shear-induced production of COX-2 and PGI2 have not been elucidated.
To address this question, we use cultured endothelial cells exposed to steady fluid shear stress (FSS) of 10 dyn/cm2 for 5-hrs to examine shear stress induced induction of COX-2/PGI2. Our results demonstrate the relationship between mechanosensor platelet endothelial cell adhesion molecule-1 (PECAM-1) and intracellular mechansensing molecules phosphatidylinositol 3-kinase (PI3K), focal adhesion kinase (FAK), and mitogen-activated protein kinase p38, in the FSS induction of COX-2 expression and PGI2 release. Knockdown of PECAM-1 (small interference RNA) expression inhibited FSS-induced activation of α5β1 integrin, upregulation of COX-2 and release of PGI2 in both bovine aortic endothelial cells (BAECs) and human umbilical cord endothelial cells (HUVECs). Furthermore, inhibition of the PI3K pathway (LY294002) completely inhibited FSS activation of α5β1 integrin, upregulation of COX-2 gene and protein expression and release of PGI2 in BAECs. Finally, we showed that inhibition of integrin associated FAK (PF573228), and MAPK p38 (SB203580) inhibited the shear induced up-regulation of COX-2.
The results of the present study demonstrate for the first time that PECAM-1 is the primary mechanosensor responsible for the FSS up-regulation of COX-2 and PGI2 at longer times as summarized in the figure below. Integrin α5β1 is activated by a PI3K mediated “inside out” signaling cascade leading to early activation of FAK/MAP:p38, sustained transcription of COX-2 mRNA and translation of COX-2 protein expression and PGI2 production. However, further studies will be required to determine the mechanism by which mechanical forces applied to the cell surface lead to the activation of cell junction protein PECAM-1 in confluent ECs.