Sustained hyperoxia-induced NF-κB activation improves survival and preserves lung development in neonatal mice

Abstract

Oxygen toxicity contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Neonatal mice exposed to hyperoxia develop a simplified lung structure that resembles BPD. Sustained activation of the transcription factor NF-κB and increased expression of protective target genes attenuate hyperoxia-induced mortality in adults. However, the effect of enhancing hyperoxia-induced NF-κB activity on lung injury and development in neonatal animals is unknown. We performed this study to determine whether sustained NF-κB activation, mediated through IκBβ overexpression, preserves lung development in neonatal animals exposed to hyperoxia. Newborn wild-type (WT) and IκBβ-overexpressing (AKBI) mice were exposed to hyperoxia (>95%) or room air from day of life (DOL) 0-14, after which all animals were kept in room air. Survival curves were generated through DOL 14. Lung development was assessed using radial alveolar count (RAC) and mean linear intercept (MLI) at DOL 3 and 28 and pulmonary vessel density at DOL 28. Lung tissue was collected, and NF-κB activity was assessed using Western blot for IκB degradation and NF-κB nuclear translocation. WT mice demonstrated 80% mortality through 14 days of exposure. In contrast, AKBI mice demonstrated 60% survival. Decreased RAC, increased MLI, and pulmonary vessel density caused by hyperoxia in WT mice were significantly attenuated in AKBI mice. These findings were associated with early and sustained NF-κB activation and expression of cytoprotective target genes, including vascular endothelial growth factor receptor 2. We conclude that sustained hyperoxia-induced NF-κB activation improves neonatal survival and preserves lung development. Potentiating early NF-κB activity after hyperoxic exposure may represent a therapeutic intervention to prevent BPD.

Keywords: IκBβ hyperoxic lung injury; NF-κB; bronchopulmonary dysplasia; lung development.

Fig. 1.

Neonatal AKBI mice are resistant to hyperoxia-induced mortality. A: schematic of IκB expression patterns in wild-type (WT) and AKBI mice. The IκBα gene has been replaced by IκBβ cDNA. The IκBα promoter controls the expression of the IκBβ transgenic loci. Thus AKBI overexpress IκBβ, without expressing IκBα. B: representative Western analysis of the NF-κB subunit p65 and NF-κB inhibitory proteins IκBα and IκBβ from WT and AKBI whole lung homogenate. Calnexin is shown as loading control. Densitometric evaluation of protein expression normalized to loading control (calnexin) and expressed as a ratio to WT is provided. C: Kaplan-Meier survival analysis of WT and AKBI neonatal mice exposed to hyperoxia (>95%, 14 days). Values are expressed as the percentage of surviving animals. n = 30/group. *P < 0.05 vs. WT.

Fig. 2.

Impaired alveolarization induced by short-term exposure to hyperoxia is attenuated in AKBI mice. Radial alveolar counts (A) and mean linear intercept (B) at 3 days in WT and AKBI mice in room air (RA) or 95% O₂. *P < 0.05 vs. genotype control, †P < 0.05 vs. paired WT exposure. C–F: representative hematoxylin and eosin-stained photomicrographs of lung tissue from WT exposed to RA (C). AKBI exposed to RA (D), WT exposed to O₂ (E), and AKBI exposed to O₂ (F). All images were obtained using the ×10 objective lens, internal scale bar 100 = μM. DOL, days of life.

Fig. 3.

Impaired alveolarization caused by prolonged exposure to hyperoxia is attenuated in AKBI mice. Radial alveolar counts (A) and mean linear intercept (B) at 28 days in WT and AKBI mice in RA or 95% O₂ for 14 days followed by 14 days of RA (O₂). *P < 0.05 vs. air exposed genotype control, †P <0.05 vs. paired WT exposure. C–F: representative hematoxylin and eosin-stained photomicrographs of lung tissue from WT exposed to RA (C). AKBI exposed to RA (D), WT exposed to O₂ and RA recovery (E), and AKBI exposed to O₂ and RA recovery (F). All images were obtained using the ×10 objective lens, internal scale bar = 100 μM. Pulmonary surface area per high-powered field (in μm) (G), airspace volume (in μm²) (H), and average alveolar size (in μm²) (I) at 28 days in WT and AKBI mice in RA or 95% O₂ for 14 days followed by 14 days of RA (O₂). *P < 0.05 vs. air exposed genotype control, †P < 0.05 vs. paired WT exposure.

Fig. 4.

Hyperoxia-induced nuclear NF-κB translocation is sustained in AKBI mice. A: representative Western blot showing p65 in lung nuclear extracts from WT and AKBI mice exposed to RA or 8 h of hyperoxia (O₂ >95%). B: representative Western blot showing p65 and IκBβ in lung nuclear extracts from WT and AKBI mice exposed to RA or 24 h of hyperoxia (O₂ >95%, 24 h), with lamin B as loading control. Densitometric evaluation of nuclear p65 at 8 and 24 h of exposure (C) and nuclear IκBβ at 24 h of exposure (D). Values are means ± SE (n = 3/time point); *P < 0.05 vs. unexposed control; †P < 0.05 vs. paired WT exposure. Dividing lines in A and B indicate that lanes are noncontiguous. Time point 0 (AKBI) presented in the p65 and lamin B blots is the same control for both the 8-h (A) and 24 h (B) time points. E: representative EMSA of lung nuclear extracts from neonatal AKBI mice exposed to hyperoxia (24 h). F: darker contrast of lanes 4–9. Bands representing NF-κB consensus sequence binding, nonspecific binding, free probe, and super shift (SS) bands are labeled. Lane 1, dye; lane 2, hyperoxia; lane 3, hyperoxia, no DTT; lanes 4–6, hyperoxia with p50 supershift; lane 4, Abcam; lane 5, Calbiochem; lane 6, Santa Cruz Biotechnology; lanes 7–8, hyperoxia with p65 supershift; lane 7, Abcam; lane 8, Calbiochem; lane 9, hyperoxia with p65-phosphorylated serine 276, Cell Signaling; lane 10, cold, 50-fold excess of unlabeled oligonucleotide added to hyperoxia sample; lane 11, mutant, 50-fold excess of mutated oligonucleotide added to hyperoxia sample.

Fig. 5.

Hyperoxia-induced IκBβ degradation is sustained in AKBI mice. A: representative Western blot showing IκBα and IκBβ in lung cytosolic extracts from WT and AKBI mice exposed to RA or 24 h of hyperoxia (O₂ >95%), with calnexin as loading control. Densitometric evaluation of cytosolic IκBα (B) and cytosolic IκBβ (C) at 24 h of exposure. Values are means ± SE (n = 4/time point); *P < 0.05 vs. unexposed genotype control.

Fig. 6.

Sustained NF-κB activation induces NF-κB target gene expression in AKBI mice. A: pulmonary proinflammatory mRNA expression. B: pulmonary antioxidant mRNA expression. MnSOD, manganese superoxide dismutase; RANTES, regulated on activation, normal T cell expressed, and presumably secreted; MMP, matrix metalloproteinase. C: pulmonary pro- and antiapoptotic mRNA expression. Values are means ± SE (n = 3–4/time point); *P < 0.05 vs. unexposed genotype control; †P <0.05 vs. paired WT exposure. BIRC, bacloviral IAP repeat-containing. D: representative Western blot showing Bax and Bcl-xL from lung cytosolic extracts from WT and AKBI mice exposed to RA or hyperoxia (O₂ >95%, 0–24 h), with calnexin as a loading control. Densitometric evaluation of Bax and Bcl-xL is provided. Values are means ± SE (n = 3–4/time point); *P < 0.05 vs. unexposed genotype control.

Fig. 7.

Sustained NF-κB activation induces protective gene expression in AKBI mice. A: representative Western blot showing VEGFR2 from lung cytosolic extracts from WT and AKBI mice exposed to RA or hyperoxia (O₂ >95%, 0–72 h), with calnexin as a loading control. B: densitometric evaluation of VEGFR2. C: pulmonary VEGFR2 mRNA expression. D: pulmonary VEGF mRNA expression. E: representative Western blot showing cleaved poly(ADP-ribose) polymerase (PARP) from lung cytosolic extracts from WT and AKBI mice exposed to RA or hyperoxia (O₂ >95%, 0–72 h), with calnexin as a loading control. F: densitometric evaluation of cleaved PARP. Values are means ± SE (n = 3–4/time point); *P < 0.05 vs. unexposed genotype control; †P <0.05 vs. paired WT exposure.

Fig. 8.

Pulmonary vessel density is preserved in AKBI mice exposed to hyperoxia followed by RA recovery. A: pulmonary vessel density at 28 days in WT and AKBI mice in RA or 95% O₂ for 14 days followed by 14 days of RA (O₂). *P < 0.05 vs. air exposed control, †P < 0.05 vs. paired WT exposure. Number represents an average in 5 high-power fields in 3 separate animals per condition. B–E: representative images of on Willebrand Factor -stained lung tissue from WT exposed to RA (B), AKBI exposed to RA (C), WT exposed to O₂ and RA recovery (D), and AKBI exposed to O₂ and RA recovery (E). All images were obtained using the ×10 objective lens, internal scale bar = 100 μM.