Alterations of cellular redox balance in microvascular endothelium results in changes of essential cell functions. These alterations may arise, in part, due to modifications in the pattern of gene expression produced by transcription factor activation. Endothelium subjected to hypoxia/reoxygenation becomes redox imbalanced, thereby leading to activation and perhaps production of a proinflammatory state. A human dermal microvascular endothelial cell line (HMEC-1) was exposed to 6 h of hypoxia (3% O(2)) followed by return to normoxia atmospheric conditions. Reactive oxygen species (ROS) generation (dichlorofluoroscein epifluorescence) was immediate and significant following reoxygenation. Electrophoretic mobility shift assays revealed activation of the oxidant sensitive transcription factors NFkappaB and AP-1, though importantly, peak activation of each factor was separated temporally by greater than 60 min. NFkappaB activation occurred without degradation of the inhibitory protein IkappaBalpha. Reoxygenating HMEC-1 exhibited a greater than 500-fold increase in polymorphonuclear neutrophil (PMN) adhesion when compared to normoxic controls. Exposure of reoxygenating HMEC-1 to the antioxidant pyrrolidine dithiocarbamate produced complete abrogation of NFkappaB activation and the intensive PMN adhesion observed in untreated, posthypoxic HMEC-1. Though rexoygenation stress induced significant upregulation of PMN adhesion, no upregulation of interleukin-8 production was observed. Our results suggest that ROS generation occurring in endothelium following onset of reoxygenation stress signals activation of key transcription factors and that their activation takes place in a temporal fashion. The temporal feature of transcription factor activation may be key to production of a postischemic proinflammatory state.