Abstract 198: Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathway in Human Aortic Endothelial Cells Exposed to Periodic Acceleration (pGz).
Background: Periodic acceleration (pGz), motion of the whole body in a head-foot direction in the spinal axis produced by a motion platform, produces pulsatile shear stress on the vascular endothelium. In models of whole body and focal ischemia reperfusion injury, pGz provides cardioprotection. In isolated perfused vessles pGz induces NO production, while in vivo pGz increases expression of eNOS and eNOS phosphorylation in heart and vasculature. Signal transduction pathway for pGz upregulation of eNOS and consequently NO are unknown. Using an in-vitro model of endothelia-coated tubing, we mimicked realistic conditions of blood vessels exposed to pGz.
Hypothesis: pGz increases the NO-producing capacity by increasing eNOS expression and phosphorylation.
Methods: Human aortic endothelial cells (HAoEC) were cultured in tubings and perfused with DMEM. Coated tubings were exposed to either; a) P-Flow (pulsatile flow), perfused with a flow rate of 560ml/min and pulsatility of 90 beat/min only or b) pGz, tubes exposed to the same pulsatile flow as P-Flow in addition to pGz at a frequency of 150 cycles/min and Gz of ± 3.5m/sec2. To study PI3K-Akt and MEK-ERK1/2 pathways, Wortmanin (PI3K inhibitor) or PD98059 (MEK inhibitors) were preincubated with HAoEC-coated tubing for 30 min prior to the treatment and throughout the experiment. Endothelial cells were harvested after the 6-hour treatment to determine eNOS, phospho-eNOS (p-eNOS), Akt, phospho-Akt (p-Akt), ERK1/2 and phospho-ERK1/2 (p-ERK1/2) by immunoblotting analysis.
Results: Compared to P-Flow, pGz increased total eNOS level by 30–35% (p<0.05), and the p-eNOS (Ser-1177) level by 65% (p<0.05). pGz also increased the ratio of p-Akt (Thr-308) to total Akt by 50% (p<0.05), and the ratio of p-ERK1/2 to total ERK1/2 by 80% (P<0.05). Wortmanin inhibited phosphorylation of eNOS and Akt but not eNOS upregulation. PD98059 inhibited ERK1/2 phosphorylation and eNOS upregulation, but not eNOS phosphorylation.
Conclusions: pGz increased eNOS expression and phosphorylation. Based on the inhibitor studies, pGz upregulates eNOS expression through MEK-ERK1/2 pathway, and increases eNOS phosphorylation (Ser-1177) through PI3K-Akt pathway in isolated endothelial cells.
- © 2010 by American Heart Association, Inc.