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

Enhanced leukocyte adhesion has been shown to occur in postischemic 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 now cytometry. Changes in ICAM-1 mRNA levels were assessed by Northern blot and activation of NF kappa B 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, NF kappa B 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 mu M) inhibited NF kappa B activation by reoxygenation and ICAM-1 expression. Blockade of endogenous nitric oxide (NO) synthesis in CMEC with L-nitroarginine (10 mu M) accentuated post-reoxygenation ICAM-1 expression. Finally an exogenous NO donor, s-nitrosoacetyl-penicillamine (SNAP 100 mu M), suppressed the generation of ROS upon reoxygenation, and blocked the activation of NF kappa B 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 NF kappa B activation triggers ICAM-1 upregulation. NO inhibits reoxygenation-specific ICAM-1 upregulation, most likely by diminishing oxidative stress that leads-to NF kappa B activation. (C) 1997 Academic Press Limited.