168 - Nox4 gene deletion in AT2 cells protects against hyperoxia- induced neonatal bronchopulmonary dysplasia (BPD) in the murine model.

Background: Bronchopulmonary dysplasia (BPD) is an illness affecting preterm neonates, leading to poor lung development. This condition is multifactorial in nature. However, exposure to oxygen constitutes a major factor contributing to the pathogenesis of BPD. Hyperoxia-based rodent models of BPD are well-established. Hyperoxia (HO) leads to reactive oxygen species (ROS) generation and this, in turn, triggers the pathological processes contributing to alveolar simplification which is a hallmark of BPD. NADPH oxidase enzyme systems play an important role in the generation of ROS under hyperoxia. NOX4 is an isoenzyme of NOX which is constitutionally active but gets induced under HO and tends to produce excess ROS, of which H₂O₂ is the major product. NOX4 is present in the mitochondria (mt) as well as the nucleus. NOX4 has been shown to interact with the electron transport chain proteins (ETC). We noted that deletion of Nox4 in the AT2 cells of lung protected adult mice from HO-induced acute lung injury.

Objective:

Hypothesis: NOX4 is a predominant isoenzyme of NOX in lung AT2 cells, producing ROS under HO contributing to the pathogenesis of BPD.

Design/Methods:

Methods: Nox4 gene was selectively knocked out in AT2 cells using Nox4 flox/flox and Tamoxifen inducible SPC Cre mice. Tamoxifen was injected on postnatal day 1 (PN1), and the experiments exposing the neonatal mice to normoxia (NOR) or HO was conducted from PN 5-15. HO experiments were conducted in MLE12 cells treated with scrambled RNA or siNox 4. MitoSOX was used to study the impact of siNox4 in the generation of mitochondrial ROS. Bioenergetics studies were performed with Agilent Seahorse XF96 system.

Results:

Results: Deletion of Nox4 in AT2 cells on PN5 was confirmed by flow cytometry. Compared to the Nox4 flox/flox controls exposed to HO, the Nox4^-/-SPC Cre pups in whom Nox4 was deleted in the AT2 cells showed significant protection against BPD. This was evidenced by reduced mean linear intercept in Nox4^-/-SPC Cre compared to Nox4 flox/flox exposed to HO. MLE12 cells exposed to HO, but pretreated with siNox 4, showed less mt ROS production and cellular apoptosis compared to scrambled RNA controls. Mitochondrial oxygen consumption rate was reduced upon exposure of MLE12 cells to HO which was restored by inhibition of NOX4.

Conclusion(s):

Conclusion: NOX4 serves as a therapeutic target for neonatal BPD, inhibition of which enables lung development under HO by restoring mitochondrial function, thus alleviating experimental BPD.