Effect of Nitric Oxide Synthase and growth conditions on hydrogen peroxide production in cultured endothelial cells during shear stress

Abstract

Endothelial cells release nitric oxide (NO) and reactive oxygen species (ROS) during acute shear stress (SS). We hypothesized that chronic SS attenuates the production of H2O2 during acute SS.Human microvascular endothelial cells (HMVEC) were exposed to one hour of laminar SS, either with or without a preceding 24 hour SS exposure (chronic SS). Cells were studied either in full serum media (5%) or after serum‐starving (to increase ROS) overnight (0.5% serum). 2′,7′‐dichlorofluorescein (DCF) assessed H2O2 formation with flow cytometry (N=3–7).Acute SS increased H2O2 release compare to static conditions (4.4±0.5 fold increase (F.I.) for 0.5% serum and 1.7±0.2 F.I for 5% serum (p<0.05)). Presence of L‐Nitro‐Arginine Methyl Ester (L‐NAME; nitric oxide synthase inhibitor) during acute shear reduced production of H2O2 in serum starved cells (2.7±0.2 F.I. vs. 4.4±0.5 F.I. (p<0.05)), but not in full serum. Acute SS after chronic SS reduced H2O2 in cells grown in low (1.9±0.8 F.I.) and full serum (0.9±0.1 F.I.). This reduction in H2O2 was reversed by L‐NAME in full serum (2.2±0.5 F.I. vs. 0.9±0.1 F.I. (p<0.05)), but not in low serum.Preconditioning with 24 hrs of SS inhibits generation of H2O2 during acute SS. We conclude that NOS modulates H2O2 production differentially in high and low serum conditions.