Effects of Oxygen Tension in Shear Stress-Induced eNOS Activation in Human Endothelial Cells

Abstract

As a signaling molecule, mitochondrial nitric oxide (NO), which diffuses mainly from the cytosolic nitric oxide synthase (NOS) isoforms into the matrix, regulates cellular respiration via inhibition of ETC complexes. Studies have shown that cultured vascular endothelial cell exposure to laminar shear stress (LSS) results in downregulation of oxygen consumption by accelerating peroxinitrite formation, in part, due to the increased NO production. However, these studies have been carried out under ambient oxygen condition (21% O2) which is well above the physiological level (5-13% O2). Knowledge underscoring the effects of oxygen tension on eNOS activation and endothelial phenotype will help to better understand the physiological implication of in vitro endothelial cell responses to LSS. PURPOSE: To determine the effect of physiological levels of oxygen tension (5% and 10% O2), compared with more conventional hyperoxic ambient oxygen tension (21% O2), in eNOS activation under LSS in human endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were grown in M199 supplemented with 20% FBS, ECGS, Heparin, P/S and Fungizone, and at 100% confluency, HUVECs were subjected to physiological level of LSS (15 dyne/cm2) using a cone-and-plate shear apparatus under 21% O2 (ambient oxygen), 10% O2 (simulating arterial blood), or 5% O2 (simulating venous blood). HUVECs were maintained for three passages under the designated O2 conditions to prevent a potential acute hypoxic effect. Protein samples were collected in ice-cold RIPA lysis buffer and resolved by SDS-PAGE, and transferred to PVDF membrane for standard western blotting. RESULTS: HUVECs presented typical cobble-stone shapes, and the size and morphology were indistinguishable among the cells grown under different oxygen tensions. The growth rate was slightly higher at 10% O2 at low confluency (< 30%) but similar at higher confluency (60 - 100%). Basal eNOS levels were marginally higher in 5% and 10% O2 compared to those in 21% O2. In all oxygen conditions, we observed eNOS phosphorylation via Ser1177, a prototypic regulatory phosphorylation site, as early as 5 min after shear stimulation, which indicated an adequate shear response. During the early shear response (5, 15, 30, 60 min), the pattern of eNOS phosphorylation at the Ser633 residue was comparable. However, after 24 hrs of LSS, the levels of eNOS protein expression and eNOS phosphorylation at both Ser1177 and Ser633 were 2-3 fold higher in 10% O2 compared to that of 21% O2. CONCLUSIONS: Our results suggest a potential role of oxygen tension in shear-induced NO and eNOS bioactivity and the signal transduction under LSS.