Murine Neonatal Oxidant Lung Injury: NRF2-Dependent Predisposition to Adulthood Respiratory Viral Infection and Protection by Maternal Antioxidant

NRF2 protects against oxidant-associated airway disorders via cytoprotective gene induction. To examine if NRF2 is an important determinant of respiratory syncytial virus (RSV) susceptibility after neonate lung injury, Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice neonatally exposed to hyperoxia were infected with RSV. To investigate the prenatal antioxidant effect on neonatal oxidative lung injury, time-pregnant Nrf2−/−and Nrf2+/+mice were given an oral NRF2 agonist (sulforaphane) on embryonic days 11.5–17.5, and offspring were exposed to hyperoxia. Bronchoalveolar lavage and histopathologic analyses determined lung injury. cDNA microarray analyses were performed on placenta and neonatal lungs. RSV-induced pulmonary inflammation, injury, oxidation, and virus load were heightened in hyperoxia-exposed mice, and injury was more severe in hyperoxia-susceptible Nrf2−/− mice than in Nrf2+/+ mice. Maternal sulforaphane significantly alleviated hyperoxic lung injury in both neonate genotypes with more marked attenuation of severe neutrophilia, edema, oxidation, and alveolarization arrest in Nrf2−/− mice. Prenatal sulforaphane altered different genes with similar defensive functions (e.g., inhibition of cell/perinatal death and inflammation, potentiation of angiogenesis/organ development) in both strains, indicating compensatory transcriptome changes in Nrf2−/− mice. Conclusively, oxidative injury in underdeveloped lungs NRF2-dependently predisposed RSV susceptibility. In utero sulforaphane intervention suggested NRF2-dependent and -independent pulmonary protection mechanisms against early-life oxidant injury.

Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification

Background: Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. Objective: In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. Design/Methods: Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Results: Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. Conclusion(s): These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.