Increased endothelial ICAM-1 expression is found in normal aging and in atherosclerosis and is related to the chronic effects of oxidative stress. We examined the Ca2+-dependence of ICAM-1 mRNA expression in human aortic endothelial cells (HAEC) exposed to hypoxia/reoxygenation (H/R) as a model of oxidative stress. HAEC were exposed to glucose-free hypoxia (95% N-2/5% CO2) for 60 min and were then reoxygenated (21% O-2/15% CO2) and observed for up to 6 h. Reactive oxygen species (ROS) generation was measured by dichlorofluorescein fluorescence and ICAM-1 mRNA was assessed by Northern blot. Upon reoxygenation after hypoxia, ROS production occurred in HAEC and was inhibited by diphenyleneiodonium and by polyethylene glycol-catalase, suggesting the involvement of NADPH oxidase-derived hydrogen peroxide. Hypoxia alone did not increase either ROS production or ICAM-1 mRNA levels, but a 2.5-fold increase in ICAM-1 mRNA was noted by 30 min of reoxygenation. This was not observed in Ca2+-free buffer or in cells treated with diphenyleneiodonium. Thus, H/R upregulates ICAM-1 mRNA in HAEC by a Ca2+- and ROS-dependent mechanism. Characterizing the signaling pathways involved in H/R-induced adhesion molecule expression may result in a better understanding of the vascular biology of normal aging and the pathobiology of atherosclerosis. (C) 2004 Elsevier Inc. All rights reserved.