Introduction Regions of the arterial tree exposed to disturbed blood flow with low wall shear stress (WSS) are prone to atherosclerotic plaque development, whereas areas under undisturbed flow with high WSS are considered protected. In response to shear stress, endothelial cells release ATP extracellularly, which activates nearby cell surface receptors. The endothelium expresses the ATP-gated cation channels P2X4 and P2X7, which previous studies have shown to be activated by shear stress-induced ATP release in the endotheliumand osteoblasts respectively. As shear stress influences atherosclerotic plaque development, and the release of ATP, we hypothesised that P2X4 and P2X7 receptors are differentially regulated by shear stress and thereby contribute to the focal nature of atherosclerosis.
Methods Human umbilical vein endothelial cells (HUVECs) were exposed to different levels of shear stress for 72 h using either an orbital shaker or an ibidi flow pump system to mimic atheroprone or atheroprotective flow. Transcripts of P2X4 and P2X7 splice variants were identified using reverse transcriptase-PCR, quantitative real time-PCR or western blotting. Immunocytochemistry was used to identify subcellular localisation of P2X7 receptors and calcium imaging was used as a functional readout of P2X receptor activation. P2X7 receptor activation was modulated using BzATP, a P2X7 agonist, and A438079 hydrochloride, a potent antagonist of P2X7 receptors.
Results We found that the endothelium expresses several P2X4 and P2X7 splice variants. Expression of P2X7 splice variants was significantly increased under high shear undisturbed flow relative to low shear disturbed flow. Immunocytochemistry confirmed that P2X7 was expressed on the surface of HUVECs, as well as a population in the ER/golgi. mRNA expression of P2X4 splice variants significantly increased under disturbed flow, but protein levels remained unchanged. Calcium imaging on static HUVEC showed the presence of functional P2X4 and P2X7 channels, which could be modulated pharmacologically. The P2X7 agonist BzATP induced calcium responses in endothelial cells that were modulated according to pre-conditioning with either high WSS, low WSS or oscillating low WSS, indicating that P2X7 responses may be altered after exposure to different WSS.
Conclusions These studies show that P2X7 expression is augmented under high shear undisturbed flow. Our results also indicate that P2X7 activity may be differentially regulated in endothelial cells conditioned to different WSS conditions. Thus P2X7 may contribute to atheroprotection at sites of high shear by controlling downstream calcium signalling pathways. Our future studies will test this concept and determine how shear stress conditions alter P2X receptor trafficking and regulatory interacting proteins.
- Shear stress
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