Nitric oxide attenuates reoxygenation-induced ICAM-1 expression in coronary microvascular endothelium: Role of NFκB

Enhanced leukocyte adhesion has been shown to occur in post-ischemic reperfused hearts due to the upregulation of specific cell-surface adhesion molecules. Therefore, we investigated the influence of 4 h of reoxygenation after 20 h of hypoxia on ICAM-1 induction in primary cultures of rat coronary microvascular endothelial cells (CMEC). ICAM-1 surface expression as well as oxygen free radical formation were measured by flow cytometry. Changes in ICAM-1 mRNA levels were assessed by Northern blot and activation of NFκB and AP-1 signalling were analysed by electrophoretic mobility shift assays (EMSA) in CMEC lysates. Although hypoxia alone did not affect cell-surface ICAM-1 expression, 4 h of reoxygenation induced a significant upregulation of ICAM-1. ICAM-1 mRNA could not be found after hypoxia alone, but could be detected as early as 1 h following reoxygenation. Unlike AP-1, the activation of which could be detected in CMEC lysates following hypoxia alone, NFκB binding activity was induced only following reoxygenation, concurrent with an increase in the formation of reactive oxygen species (ROS). A proteasome inhibitor, nor-Leu (25 μM) inhibited NFκB activation by reoxygenation and ICAM-1 expression. Blockade of endogenous nitric oxide (NO) synthesis in CMEC with L-nitroarginine (10 μM) accentuated post-reoxygenation ICAM-1 expression. Finally, an exogenous NO donor, s-nitrosoacetyl-penicillamine (SNAP, 100 μM), suppressed the generation of ROS upon reoxygenation, and blocked the activation of NFκB and the upregulation of ICAM-1. Thus, ICAM-1 upregulation in CMEC primary cultures is not induced by hypoxia alone, but appears shortly after reoxygenation in the absence of exogenous cytokines or inflammatory cells. Because upregulation of AP-1 through hypoxia alone did not affect ICAM-1 expression, we conclude that redox-sensitive NFκB activation triggers ICAM-1 upregulation, NO inhibits reoxygenation-specific ICAM-1 upregulation, most likely by diminishing oxidative stress that leads to NFκB activation.