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

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 H 2 O 2 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 H 2 O 2 formation with flow cytometry (N=3–7). Acute SS increased H 2 O 2 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 H 2 O 2 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 H 2 O 2 in cells grown in low (1.9±0.8 F.I.) and full serum (0.9±0.1 F.I.). This reduction in H 2 O 2 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 H 2 O 2 during acute SS. We conclude that NOS modulates H 2 O 2 production differentially in high and low serum conditions.