For patients with ARDS, a ventilator is essential for supplying oxygen, but it can also cause lung injury. In this study, we developed a VILI model. Then, we examined the role of NOX4 and the effects of NOX4 inhibition and as the blockade of EphA2 using KO mice in this VILI model. We also examined NOX4 levels in patients with respiratory diseases according to their use of a ventilator.
NOX4 acts as an oxygen sensor for catalysis of molecular oxygen in various reactive oxygen species (ROS), and the resulting ROS undergo many biological reactions, including those involved in signal transduction, cell differentiation, and tumor cell growth.(M Skonieczna et al., 2017) Eph-ephrin signaling, as part of receptor tyrosine kinase (RTK) pathways, have been implicated in many cellular processes, including vasculogenesis, angiogenesis, cell migration, axon guidance, embryonic development, fluid homeostasis, and injury repair.(A Barquilla and EB Pasquale, 2015; JY Hong et al., 2015)
Palumbo et al.(S Palumbo et al., 2017) reported the role of NOX4 in the lungs and showed that ALI is more severe in aged mice than in young mice because of increased vascular permeability, albumin influx, and BAL neutrophils and proteins. In addition, they showed that ROS levels are elevated in aged or injured mice, suggesting that lung injury is associated with NOX4, a ROS-generating enzyme. They detected the senescence of endothelial cells based on β-galactosidase activity and an increase in p16 level and studied the regulation of the endothelial cell barrier in human lungs. Their results showed that membrane permeability caused by LPS is increased in senescent endothelial cells compared to that in young endothelial cells, and the expression of NOX4 is rapidly induced by LPS challenge via a proteasome/ubiquitin system. They found that pharmacological inhibition of NOX4 reduced the change in membrane permeability due to LPS. Our study showed that NOX4 was involved in VILI, similar to its involvement in lung injury by LPS. Our results also demonstrated lung injury prevention by NOX4 inhibition.
Hong et al.(JY Hong et al., 2016) reported downregulation of PI3K 110γ, phospho-Akt, phospho-NF-κB p65, phospho-Src, and phospho-S6K via EphA2 antibody in an LPS-induced lung injury model. The above two studies showed that NOX4 and EphA2 are involved in lung injury due to LPS and that NOX4 inhibition and EphA2 receptor inhibition are effective in limiting such lung injury. Therefore, with VILI, it can be inferred that the NOX4 and EphA2 pathways function similarly to that in LPS-induced lung injury, and that there is a therapeutic effect from NOX4 or EphA2 inhibition.
A study of the EphA2/ephrinA1 signaling pathways in a VILI model was performed by Park et al.(BH Park et al., 2017) Placing mice in a prone position reduced lung injury, and EphA2 antagonism downregulated the expression of PI3Kγ, Akt, and NF-κB, which resulted in lung protective effects. EphA2/ephrinA1 is shown to be a VILI therapeutic target in their study. Similarly, Leem et al.(AY Leem et al., 2017) reported that EphA2/ephrinA1 is elevated by bleomycin in a bleomycin-induced lung injury model, that elevation of IL-6 and TNF-a in PI3K-Akt leads to lung injury, and that elevation of EphA2-ephrin A1 could be blocked by all-trans retinoic acid. As indicated by the studies described above, EphA2 levels are closely related to the extent of lung injury in patients undergoing ventilator care in ICU, and, in our study, we found that EphA2 levels were higher in the severe lung injury group. Che et al.(H Chu et al., 2017) showed that bleomycin-induced lung injury is caused by TGF-β/Smad3 signaling and oxidative stress, which can be regulated by a Chinese medicine called Shenks. This medicine increases the expression of the antioxidant-related genes Gclc and Ec-sod, both in vivo and in vitro, by increasing the transcription of oxidative-related genes, including Rac1 and Nox4. Zhang et al.(D Zhang et al., 2017) also showed the overexpression of NOX4 in a bleomycin-induced lung injury model and reported that schizandrin B and glycyrrhizic acid are effective inhibitors of the TGF-β1/Smad3 signaling pathway in this model.
Lee et al.(SH Lee et al., 2017) measured plasma EphA2 levels in a prospective study of patients who were admitted to an ICU because of sepsis and had their disease severity determined based on acute physiology and chronic health evaluation (APACHE) II and sequential organ failure assessment (SOFA) scores to examine the correlation between SOFA score and EphA2 level. They confirmed a positive correlation between serum levels of the EphA2 receptor and severity of sepsis in ICU patients. In addition, when the area under the curve of EphA2 receptor levels is measured, it is 0.690 higher than that of the APACHE II scores. Additionally, they showed that EphA2 receptor levels are associated with sepsis severity and 28-day mortality. These results suggest that EphA2 levels are associated with severity of VILI in ICU patients.
NOX4 is also expressed in the pulmonary endothelium, which acts as a barrier that prevents plasma exudate from entering the interstitium and alveolar space, and plays an important role in regulating lung inflammation, apoptosis, and permeability along with NOX2 in pneumonia caused by Pseudomonas aeruginosa(K Bernard et al., 2014).
In our study, NOX4 levels were also high in patients with pneumonia. In particular, NOX4 levels were significantly higher in the group of pneumonia patients using ventilators in the ICU, matching the data derived from the mouse experiments.
In summary, we found that a signaling pathway with NOX4, EphA2, and PI3K is associated with VILI and that there are several potential mechanisms by which NOX4 inhibition may affect VILI. In addition, we showed the potential for a NOX4 inhibitor to decrease VILI through EphA2 and PI3k 110λ signaling (Fig S2).