β2-adrenergic agonist protects human endothelial cells from hypoxia/reoxygenation injury in vitro

Objective: Circulatory shock results in hypoxia/reoxygenation processes that lead to the release of reactive oxygen species, endothelial injury, and multiple organ failure. Previous data suggest that β2-adrenergic agonists prevent endothelial dysfunction. The study aimed at determining whether the β2-adrenergic agonist formoterol protects endothelial cells against hypoxia/reoxygenation injury in vitro. Design: Prospective controlled trial. Setting: University hospital research laboratory. Subjects: Cultured human umbilical vein endothelial cells (HUVECs). Interventions: Confluent HUVECs were sealed in a flow-through chamber mounted on an inverted microscope and perfused with a constant flow of Krebs medium. After 1 hr of equilibration, HUVECs underwent 2 hrs of hypoxia and 1 hr of reoxygenation. Cell death at the end of reoxygenation and reactive oxygen species formation were assessed with fluorescent probes propidium iodide and 2′,7′-dichlorodihydrofluorescein diacetate, respectively. The effects of the β2-adrenergic agonist formoterol, the β2-adrenergic antagonist ICI 118,551 and the nitric oxide synthase inhibitor L-NNA were investigated. Statistical analysis was performed with analysis of variance followed by post hoc Fisher’s test. Measurements and Main Results: Hypoxia/reoxygenation increased cell death (hypoxia/reoxygenation 29 ± 4% vs. control 1 ± 5%, p < .05) and endothelial reactive oxygen species production (hypoxia/reoxygenation 126 ± 4% vs. control 108 ± 4%, p < .05). Formoterol reduced cell death in a concentration-dependent manner (EC95 = 10−7 mol/L) and reduced endothelial reactive oxygen species production (hypoxia/reoxygenation + formoterol EC95 109 ± 4% vs. hypoxia/reoxygenation 126 ± 4%, p < .05). When added to formoterol EC95, ICI 118,551 and L-NNA abolished the formoterol-induced cell protection and reduced reactive oxygen species production. Conclusions: These results indicate that formoterol reduces endothelial cell death and reactive oxygen species production in this in vitro hypoxia/reoxygenation model. These effects are β2-adrenergic specific and are partially mediated by nitric oxide synthase.