We show here, for the first time, that NO delivered at a steady state of 20 ppm inhibits hyperoxia-induced NF-κB activation. We chose 20 ppm as this is the maximum dose administered in recent randomized control trials evaluating the role of iNO in preventing BPD and appears to limit the adverse effects of the gas (18
). Additionally, NO donors have variable half-lives, making it difficult to deliver a continuous steady-state in cell culture (19
). We found that cells exposed to NO plus hyperoxia expressed less ICAM-1 and exhibited less death compared to cells exposed to hyperoxia alone. Thus, NO may help prevent inflammatory cell recruitment and prevent hyperoxic lung injury.
For this study, we chose to evaluate the effect of NO on hyperoxic injury in primary endothelial cells derived from human neonates. Our interest in evaluating this specific cell type stems from previous work showing that the pulmonary endothelium is uniquely sensitive to hyperoxic injury. These cells are the first to demonstrate morphologic changes in response to hyperoxia and contribute to the early inflammatory stage of injury by binding neutrophils and platelets (20
). Pulmonary endothelial cells of newborn mice exposed to hyperoxia demonstrate structural alterations including polymorphonuclear leukocyte and platelet adhesion (21
). Fewer inflammatory cells were seen in newborn rats exposed to NO plus hyperoxia when compared to oxygen exposed controls (22
). Thus, it appears that NO acts in part by limiting inflammatory cell recruitment in the newborn lung, thus limiting the extent of hyperoxic lung injury. Our study supports these findings and defines a mechanism by which this occurs.
One important mediator of inflammation in the lung is NF-κB. This transcription factor regulates the expression of adhesion molecules, including ICAM-1 (23
). ICAM-1 mediates interactions between endothelial cells and cells expressing β2 integrins, including neutrophils, monocytes, lymphocytes and natural killer cells. Following exposure to inflammatory stimuli, including TNF-α, endothelial cells increase ICAM-1 expression via NF-κB activation (23
). Hyperoxia increases ICAM-1 expression in pulmonary endothelium in neonatal models (24
). To this point, only one report has linked hyperoxia-induced NF-κB activation and ICAM-1 expression. In that study, inhibiting hyperoxia-induced NF-κB activation with glucocorticoids prevented ICAM-1 upregulation in cultured human adult pulmonary artery endothelial cells (25
). Hyperoxia-induced NF-κB activation is maturationally regulated (12
), with a more robust response occurring in neonatal animals. Our report shows NO inhibits hyperoxia-induced NF-κB activation in a neonatal model. Our data suggest that NO, through inhibition of hyperoxia induced-NF-κB upregulation of ICAM-1 expression, could decrease inflammatory cell influx into the neonatal lung and prevent injury. In vivo
studies are needed to confirm these findings.
Previous work has shown that NO inhibits NF-κB regulated adhesion cell molecule expression in endothelial cells following inflammatory stimuli (26
). This inhibition has been attributed to stabilization of the inhibitory protein IκBα (27
) and multiple post-translational modifications of proteins in the NF-κB activation cascade. These modifications include s-nitrosylation of NF-κB subunits (7
). To this point, much of the work on how NO affects NF-κB activation has focused on canonical signaling. However, oxidant-stress induced NF-κB activation occurs via the atypical pathway, distinct from the canonical pathway responsible for inflammatory induction of NF-κB activation (10
). Previous work from our laboratory has identified a role for phosphorylation of IκBα on tyrosine 42 in mediating hyperoxia-induced NF-κB activation (13
). In the current study, we asked whether post-translational modifications of IκBα, specifically nitrotyrosine, accounted for the inhibition of hyperoxia-induced NF-κB activation seen in cells exposed to NO plus hyperoxia. However, there was no evidence of nitrotyrosine modifications of IκBα (via immunoprecipitation, data not shown), nor of a global increase in nitrotyrosine post-translational modifications in cells exposed to NO plus hyperoxia. Furthermore, we could not find evidence to support the effect of NO on hyperoxia-induced NF-κB signaling occurred due to increases in cGMP or decreased ROS formation. We speculate that NO causes other post-translational modifications of the proteins in the pathway leading the hyperoxia-induced NF-κB activation. Further work to identify the exact mechanism of inhibition of hyperoxia-induced NF-κB activation needs to be performed.
Many studies have raised the concern of potential synergistic toxicity of hyperoxia and NO. Fetal type II pneumocytes and lung fibroblasts demonstrate increased cell death with concurrent NO and hyperoxic exposure (28
). Here, cultured primary neonatal pulmonary endothelial cells showed no evidence of synergistic toxicity between hyperoxia and NO. Rather, these cells had preserved cellular proliferation, less cell death, and inhibition of apoptosis with the dose of NO we used for up to 48 hours. We speculate that the response to NO plus hyperoxia is cell type specific and depends on how NO is delivered, and accounts for these differences. In studies using other cell culture models, NF-κB activation has been shown to inhibit angiogenesis (17
). We speculate the increased cellular proliferation seen in cells exposed to NO plus hyperoxia may be due in part to inhibition of NF-κB activity, but other factors may play a role. The physiologic implications of these findings must be evaluated using in vivo
models. Furthermore, we limited our exposures to hyperoxia and NO to 48 hours. Toxicity may increase with longer exposures, and this may have clinical relevance for premature infants with hypoxic respiratory failure treated with NO and oxygen for prolonged periods of time.
In summary, we report that hyperoxia-induced NF-κB activation is inhibited by NO resulting in decreased ICAM-1 expression in endothelial cells. Furthermore, NO has protective effects against hyperoxia-induced cell death and inhibition of cellular proliferation. We speculate that the ability of NO to inhibit hyperoxia-induced NF-κB activation may explain in part the protective effect of iNO in preventing BPD in some preterm infants.