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Inhaled nitric oxide may be protective against hyperoxic injury in the premature lung, but the mechanism is unknown. We hypothesized that nitric oxide (NO) would prevent hyperoxia-induced NF-κB activation in neonatal pulmonary microvascular endothelial cells (HPMEC) and prevent the upregulation of target genes. Following hyperoxic exposure (O2> 95%), nuclear NF-κB consensus sequence binding increased and was associated with IκBα degradation. Both of these findings were prevented by exposure to NO. Furthermore, ICAM-1 mRNA and protein levels increased in cells exposed to hyperoxia, an effect abrogated by NO. To evaluate the potentially toxic effect of NO plus hyperoxia, cell viability and proliferation were assessed. Cells exposed to NO plus hyperoxia demonstrated improved survival as measured by trypan blue exclusion when compared to cells exposed to hyperoxia alone. These differences in cell death could not be attributed to apoptosis measured by caspase-3 activity. Finally, cellular proliferation inhibited by hyperoxia was rescued by concurrent exposure to NO. These data demonstrate that NO prevents hyperoxia-induced NF-κB activation in HPMEC and results in decreased expression of adhesion molecules and decreased cellular toxicity. This may help explain the protective effects of NO on hyperoxic injury in the developing lung vasculature.
Oxidative and inflammatory stimuli encountered after preterm birth injure the immature lung and contribute to the pathogenesis of bronchopulmonary dysplasia (BPD) (1). Strategies aimed at preventing BPD, including inhaled nitric oxide (iNO), have been minimally successful thus far (2).
Studies using animal models have shown nitric oxide (NO) is protective against hyperoxic lung injury (3). However, NO can react with oxygen to form NO2 or with superoxide (O2.-) to form peroxynitrite (ONOO-), both of which are toxic to the respiratory system (4). A better understanding of the mechanism by which NO protects the neonatal lung from hyperoxia could lead to a more targeted use of this therapy.
The transcription factor NF-κB activates genes in response to inflammatory and oxidative stress (5). In preterm infants, NF-κB activation has been linked to BPD (6). However, whether this increased activation is a protective response or whether it is causative to the injury remains unexplored.
Following inflammatory stimuli such as TNF-α, NO can inhibit canonical NF-κB signaling in a cell type and concentration dependent manner (5). Post-translational modifications of proteins in the NF-κB activation cascade, including s-nitrosylation of NF-κB subunits (7, 8) and s-nitrosylation of IKK (9), account for this inhibition. In contrast, oxidant-stress induced NF-κB activation occurs via the atypical pathway and involves unique kinases, including PI3 kinase and c-Src, which are not involved in the canonical pathway (10). Less is known regarding the effect of NO on NF-κB activation following oxidant stress. In adult rats exposed to hyperoxia, iNO inhibited NF-κB activation in the lung (11). However, hyperoxia-induced NF-κB activation is maturationally regulated (12), and it is not known whether NO has a similar inhibitory effect in the newborn lung.
In this study, primary neonatal pulmonary microvascular endothelial cells (HPMEC) were exposed to hyperoxia and NO and NF-κB activation was evaluated. Hyperoxia-induced NF-κB activation was inhibited by concurrent exposure to NO. In addition, NO prevented NF-κB regulated intracellular adhesion molecule-1 (ICAM-1) expression. Oxygen toxicity, manifesting as increased cell death following 48 hours of exposure, was limited by concurrent exposure to NO. Furthermore, in contrast to hyperoxia, NO plus hyperoxia preserved HPMEC proliferation. Overall, the data provides new insights on the potential mechanism by which NO limits hyperoxic injury in the newborn lung.
HPMEC (ScienCell, San Diego, CA), primary cells derived from human neonatal lung, were exposed to hyperoxia (95% O2/5% CO2), NO alone (5% CO2, 95% room air, 20 ppm NO) or hyperoxia plus NO (5% CO2, 95% O2, 20 ppm NO) in a C-Chamber (Biospherix, Redfield, NY). NO was delivered via an iNOvent (Ikaria, Clinton, NJ) using a gas flow of 3.5 liters per minute to prevent the accumulation of nitrogen dioxide within the C-Chamber. Levels of NO2 were checked at the expiratory port on the C-chamber. Canonical NF-κB signaling was induced with TNF-α (13) and NF-κB activity was inhibited by using BAY 11-7082 as previously described (12). To evaluate the effect of cGMP on NF-κB activity, the cell permeable and phosphodiesterase resistant cGMP analog 8-bromo-cGMP (1 mM, Sigma, USA) was added to cells 1 hour prior to exposures.
Nuclear and cytosolic fractions were extracted and protein content determined as previously described (13).
EMSA was performed as detailed elsewhere (13).
Immunoblotting was performed as previously described (13). The following antibodies were used: anti-IκBα (catalog no. sc-371, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ICAM1 antibodies (catalog no. ab2213, Abcam, Cambridge, CA), anti-Caspase-3 (Calbiochem, USA), anti-nitrotyrosine (609, kind gift of Dr. Harry Ischiropolous), anti-calnexin (Stressgen, Ann Arbor, MI) or anti-TBP (Aviva, San Diego, CA).
Cells were incubated with 10µM CM-H2DCFDA in serum-free medium for one hour. Medium was replaced prior to exposures. Cell lysate was prepared and fluorescence measured (excitation 492 nm, emission wavelength 525nm) using the SpectraMax Gemini Spectrophotometer and analyzed using Softmax Pro Software (Molecular Devices, Sunnyvale, CA).
Total RNA was isolated using the RNeasy Kit (Quiagen, USA). Reverse transcription was performed with Superscript IITM Reverse Transcriptase (Invitrogen, USA). ICAM-1 mRNA (#Hs00164932_m1, Applied Biosystems, USA) levels were analyzed with the Taqman Gene Expression Assay according to the manufacturer’s instructions, and normalized to 18s rRNA (Hs99999901.s1, Applied Biosystems, USA).
The trypan blue exclusion assay, as a measure of cell viability, was performed as described previously (12).
Caspase-3 activity, as a measure of apoptosis, was performed as described previously (13).
A cellular proliferation ELISA kit was used according to the manufacturer’s instructions (Roche, USA). Light emitted by the chemiluminescent reaction was quantified using the In Vivo Imaging System (Xenogen, Alameda, CA).
For comparison between treatment groups, the null hypothesis that no difference existed between treatment means was tested by ANOVA for multiple groups or t-test for 2 groups (InStat, GraphPad, San Diego, CA). Results are given as mean ± SEM.
Nuclear extracts from HPMEC exposed to 24 hours of hyperoxia demonstrated significantly increased NF-κB consensus sequence binding when compared to control (Figure 1A and 1B). The band that appeared after exposure to hyperoxia was completely dissipated by an unlabeled oligonucleotide, but was not affected by the addition of a mutant oligonucleotide, suggesting this newly appearing band represented hyperoxia-induced NF-κB DNA binding. In contrast, no increased binding was observed in HPMEC exposed to NO plus hyperoxia or NO alone at this same time point. Densitometric analysis revealed a statistically significant increase in NF-κB consensus sequence binding only in cells exposed to hyperoxia, with no differences in any group at 4 or 8 hours of exposure (data not shown). The increased binding was similar in pattern but lower in intensity to binding induced by TNF-α, used here as a positive control. Differences between inflammatory and oxidant stress induced NF-κB consensus sequence binding is demonstrated by the modest binding in response hyperoxia when compared to TNF-α, and is consistent with previous reports (13). Furthermore, a supershift after incubation with both anti-p50 and -p65 antibodies was observed, indicating activation of the p50-p65 heterodimer (Figure 1C). Supporting this data, significant increases in nuclear p65 occurred only after exposure to hyperoxia (Figure 1D and 1E). These results suggest that NO inhibits hyperoxia-induced NF-κB activation prior to nuclear translocation of activated subunits.
To further define the signaling events leading to NF-κB activation, levels of cytoplasmic immunoreactive IκBα were evaluated (Figure 2A and 2B). HPMEC exposed to hyperoxia showed significantly decreased IκBα levels after 4 hours of hyperoxia that remained low throughout the 24-hour exposure. In contrast, levels of IκBα returned to baseline by 24 hours in cells exposed to NO plus hyperoxia. Cells exposed to NO alone showed no change in the level of immunoreactive IκBα. These data suggest that NO prevents sustained hyperoxia-induced degradation of IκBα and that NO may inhibit hyperoxia-induced NF-κB activation by stabilizing the inhibitory protein IκBα.
To better understand the consequences of hyperoxia-induced NF-κB activation in this primary endothelial cell model, the expression of the adhesion molecule ICAM-1 was evaluated. NF-κB regulates the expression of ICAM-1 (14), an adhesion molecule that mediates neutrophil adhesion and transmigration across the endothelium. Hyperoxic exposure for 24 hours resulted in a significant increase in the amount of ICAM-1 protein expressed by HPMEC when compared to control (Figure 3A and 3B). In contrast, no increase ICAM-1 was observed in cells exposed to NO plus hyperoxia. To demonstrate ICAM-1 expression is dependent on NF-κB in our model system, Bay 11-7082, an inhibitor of IκBα phosphorylation, was added to cells prior to exposure with TNF-α. Addition of Bay 11-7082 completely prevented IκBα degradation following a 30-minute exposure to TNF-α (Figure 3C). Furthermore, Bay 11-7082 prevented increased ICAM-1 expression following a 4-hour exposure to TNF-α, and had no effect on hyperoxia-induced ICAM-1 expression. These results show that hyperoxia-induced NF-κB activation occurs via a pathway independent from canonical NF-κB signaling. Further supporting the finding that ICAM-1 expression is dependent on NF-κB activation, modest but significant increases in ICAM-1 mRNA was observed in cells exposed to hyperoxia (Figure 3D). In contrast, cells exposed to NO plus hyperoxia showed no increase in ICAM-1 mRNA. These data suggest that NO inhibits hyperoxia-induced NF-κB regulated ICAM-1 expression.
To determine whether the effect of NO on hyperoxia-induced NF-κB activation was mediated through increased levels of cGMP, cells were treated with to 8-bromo-cGMP (1 mM) one hour prior to exposure to hyperoxia. This cell permeable and phosphodiesterase-resistant cGMP analog was unable to replicate that ability of NO to prevent IκBα degradation (Fig 4A). This suggests that the effect of NO does not occur due to an increase in intracellular levels of cGMP. To further define the events leading to inhibition of hyperoxia-induced NF-κB activation by NO, levels of immunoreactive nitrotyrosine post-translational modifications were evaluated. No difference in the global amount of nitrotyrosine modifications in cells exposed to hyperoxia or NO plus hyperoxia for 24 hours was observed (Figure 4B). Human nitrated fibrinogen, isolated as described elsewhere, was used as a positive control (15). This indicates the effect of NO on NF-κB signaling was independent of this specific protein modification. Finally, as NO itself is a free radical and thus can act to scavenge other free radicals (16), ROS generation following exposures to hyperoxia and NO plus hyperoxia were evaluated. After 24 hours, the time point at which cells exposed to hyperoxia or NO plus hyperoxia demonstrate different NF-κB activation, there was a 1.2 fold increase in ROS formation in cells exposed to hyperoxia (Figure 4C). In contrast, there was a 2-fold increase in ROS in cells exposed NO plus hyperoxia. This data suggests that NO does not inhibit hyperoxia-induced NF-κB activation by acting as an anti-oxidant.
To assess the potential effect of NO plus hyperoxia on endothelial cell death, trypan blue exclusion assays were performed. No significant difference in the percentage of dead cells was found between HPMEC exposed to room air, hyperoxia or NO plus hyperoxia following 24 hours of exposure (data not shown). In contrast, HPMEC exposed to 48 hours of hyperoxia demonstrated 60% mortality, while cells exposed to hyperoxia plus NO showed no difference from control (Figure 5). Using this assay, control cells demonstrated 25% mortality, an effect attributed to use of a primary cell culture for this study. This suggests that NO delivered at a constant level of 20 ppm does not increase oxygen toxicity in neonatal pulmonary endothelial cells, and in fact protects cells from the toxic effects of hyperoxia.
To evaluate whether the increase in cell death following exposure to hyperoxia was due to apoptosis, caspase-3 activity was assessed. A significant decrease in caspase-3 activity was found in cells exposed to hyperoxia for 24 hours, with a return to baseline by 48 hours (Figure 6A). In contrast, while cells exposed to NO plus hyperoxia also demonstrated a significant decrease in caspase-3 activity at 24 hours, by 48 hours this activity was inhibited even further. Western analysis of pro-caspase-3 performed on cells exposed to room air, hyperoxia or NO plus hyperoxia for 48 hours supported these findings (Figure 6B). Densitometric analysis revealed there was no significant change in pro-caspase-3 following any exposure, thus no cleavage and caspase activation, supporting the data found by caspase-3 activity assay (Figure 6C). These data suggest that neither hyperoxia nor NO plus hyperoxia induce apoptosis in HPMEC, implicating necrosis as the cause of cell death.
As inhibition of NF-κB activity has been shown to promote angiogenesis (17), BrdU labeling was assessed to determine whether the inhibition of hyperoxia-induced NF-κB activity by NO affected cellular proliferation. Decreased proliferation was seen in cells exposed to hyperoxia for 24 hours (Figure 7). In contrast, cells exposed to NO plus hyperoxia had increased cellular proliferation when compared to hyperoxia alone (Figure 7). Exposure to NO alone resulted in no changes in proliferation when compared to room air control (data not shown). Thus, NO abrogates the effect of hyperoxia on endothelial cell proliferation. As inhibition of NF-κB activation has been implicated in stimulating angiogenesis (17), we speculate the effect observed here may be related to the effect of NO on NF-κB activity.
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, 8). 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, 29). 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.
Patrick Fernando provided technical assistance for completion of this manuscript.
Supported by the Pediatric Scientist Development Program, grant K12-HD00850 [ to C.J.W.], the iNO Therapeutics Advancing Newborn Medicine Fellowship Grant [to C.J.W.], the Marshall-Klaus Perinatal Research Award [to C.J.W.] and RO-1 HL-58752 [to P.A.D.].
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