ENOS Phosphorylation Under Flow-Mediated Shear Stress: Effects of Flow Pattern and Ambient Oxygen Concentration

Abstract

Vascular endothelial cells (EC) are continuously exposed to flow-mediated shear stress, a friction force generated by flowing blood, which is increased during aerobic exercise. The flow-mediated shear stress is the most important physiological stimulus for the formation of nitric oxide (NO) via activation of endothelial nitric oxide synthase (eNOS). It has been demonstrated that flow-mediated shear stress modulates vascular structure and function, and is a major determinant for vascular remodeling and arterial tone, which depends primarily on NO produced by EC in response to shear stress. NO plays many essential roles including vessel relaxation, inhibition of apoptosis, and anti-inflammation. However, effects of flow patterns and ambient oxygen concentration on the eNOS activation and NO production are largely unknown. PURPOSE: To determine the effects of flow patterns (low and high) and ambient oxygen concentration (5%, 10% and 20% O2) on eNOS activation and NO production in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were grown in M199 supplemented with 20% FBS, Endothelial Cell Growth Supplement, Heparin, P/S and Fungizone. At 100% confluency, HUVECs were subjected to physiological level of low (l-flow) or high (h-flow) shear stress (5 or 20 dyne/cm2) using a cone-and-plate shear apparatus under 21% O2 (atmospheric oxygen), 10% O2 (arterial blood level), or 5% O2 (venous blood level). Protein samples were analyzed by western blotting. RESULTS: The morphology of HUVECs was not different among the ECs grown under different oxygen concentrations. However, the growth rate was slightly higher at 10% O2 at low confluency (< 30%) while it was similar at high confluency (60-100%). During 5-60 mins of h-flow, eNOS phosphorylation at the Ser633 residue was similar. However, under 24 hrs of h-flow, the levels of eNOS phosphorylation at both Ser1177 and Ser633 were 2-3 fold higher in 10% O2 compared to that of 21% O2. NO production level was also significantly higher in 10% O2 than in 21% O2 under 24 hrs of h-flow. CONCLUSION: Physiological level of h-flow under 10% O2 increases eNOS phosphorylation and NO production greater than that of under 21% O2 in human endothelial cells. The present study may provide a better in vitro model for the area of vascular mechanotransduction research.