Acute lung injury and ARDS are serious conditions; both can be life-threatening, yet there is no effective treatment. This ex-vivo study confirmed previously known activations in TGF-β and Wnt signaling pathways in adult lung injury. It also showed that hyperoxia-induced activation in these pathways in adult lung injury was effectively blocked by combined treatment with PGZ and B-YL. The PGZ + B-YL combination also attenuated the hyperoxia-induced increase in inflammatory cytokines, cellular apoptosis, myogenic protein levels, and alterations in endothelial injury markers. Additionally, PGZ + B-YL treatment increased protein levels of PPARγ, SP-C, and CCTα in normoxia, indicating its pro-homeostatic effect. Although PGZ alone also had significant lung protectant and pro-homeostatic effects, overall, these effects were more pronounced with the PGZ + B-YL combination versus PGZ alone. These data suggest possible effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult lung injury, likely mediated via blockage of TGF-β and Wnt signaling activations, both of which are critical mediators of adult lung injury.
As supported by multiple human and animal studies, TGF-β activation plays a central role in injury repair in almost all organs, including in the lung [11–13]. In addition, activation of Wnt signaling in adult lung injury is supported by numerous experimental models of ALI and adult patients with ARDS [14–16]. Importantly, TGF-β activation and its downstream interaction with canonical Wnt pathway intermediates result in disrupted homeostatic epithelial-mesenchymal interactions [6, 17–21]. Although a complex network of signaling molecules is involved in these interactions, the primary TGF-β signaling intermediates include ALK5 and SMAD 3 and SMAD 7. The major Wnt signaling pathway intermediates include Wnt receptors, Frizzled and LRP 5/6, and intracellular mediators such as Disheveled, Axin, APC, and GSK-3β. Interactions of these intermediates lead to β-catenin stabilization, resulting in its nuclear translocation where it binds to LEF-1 to induce target gene transcription. Therefore, in this study, we examined key signaling molecules of TGF-β (ALK 5, SMAD 3 and SMAD 7) and Wnt (β-catenin and LEF-1) pathways and their downstream targets, including cellular apoptosis (BCL-2 and BAX), inflammatory markers (IL-6, IL-1β, MCP, TNFα), myogenic markers (fibronectin and calponin) and endothelial (VEGF-A, PECAM-1, FLT, FLK) markers. In addition, expression of key Wnt pathway antagonists PPARγ and C/EBPα was determined.
As in our prior study that examined neonatal rat lung injury , in this study, using adult mouse lung explants, we observed that hyperoxia-induced TGF-β activation (upregulation of ALK5 and SMAD 3 protein levels) was effectively blocked by PGZ + B-YL combination. In contrast to our previous study, an increase in SMAD 7 protein level, which is an autoinhibitory response to TGF-β activation, was not observed; this is likely due to a relatively shorter duration of hyperoxia exposure (72-hours) in this study versus the 7-day exposure in the previous study.
Interestingly, in contrast to hyperoxia-induced PPARγ downregulation in the developing lung rat model, in adult mouse lung explants, PPARγ protein levels were unaffected at both timepoints examined, suggesting that alterations in TGF-β and Wnt signaling pathways likely play a more dominant role in the adult lung injury than in the neonatal lung injury. Nevertheless, the evidence from numerous adult organ injury models including the adult lung clearly reflects antagonistic effects of PPARγ and Wnt signaling pathways [10, 22, 23]. Therefore, it is not surprising that upregulation of PPARγ signaling (increase in PPARγ and C/EBPα protein levels) with both PGZ alone and PGZ + B-YL combined treatments was associated with blockage of hyperoxia-induced activation in Wnt signaling (increased β-catenin and LEF-1 protein levels) and increase in its downstream myogenic targets (fibronectin and calponin). Lastly, although molecular mechanisms involved remain incompletely understood and were not the focus of this study, hyperoxia-induced increases in inflammatory cytokines and apoptosis were effectively blocked by the PGZ + B-YL combination consistently at 72h but not at 24h, which may be a function of the shorter duration of treatment. Regarding the effects of hyperoxia on pulmonary endothelial markers, unlike our findings, hyperoxia has been shown to decrease VEGF and VEGF receptor (FLT-1) levels in other models. This difference also might be due to a relatively shorter duration of hyperoxia exposure and the use of lung explants in this study versus other in-vivo studies [24, 25]. However, similar to our observation, upregulation of PECAM-1 has been reported with hyperoxia-induced lung injury in an adult mouse model .
Although both surfactant and PPARγ agonists treatment have been tried individually in various ARDS models, benefits of either drug alone are limited. For example, exogenous surfactant treatment in patients with ALI/ARDS significantly lowered inspired oxygen need; however, there was no significant decrease in mortality . The lack of mortality benefit may be explained at least partially due to only limited surfactant reaching the distal lung in these studies . In addition, although PPARγ agonist administration has shown benefits in ameliorating pulmonary inflammation, e.g., a decrease in neutrophil influx and tissue injury in a LPS-induced lung injury model , sole treatment with PGZ did not have any benefit on endotoxin induced lung inflammation in humans [29, 30]. To promote injury repair and block lung surfactant dysfunction and oxidant damage simultaneously, we combined PGZ with a novel synthetic surfactant B-YL. This combined PGZ + B-YL approach targets key signaling pathways disrupted in ALI/ARDS and provides an oxidant-resistant surfactant B-YL. B-YL is an innovative surfactant developed to resist hyperoxia-induced inactivation; it is relatively stable and highly active in its native form. We previously demonstrated that combining B-YL with PGZ does not affect B-YL’s surfactant bioactivity, and B-YL does not affect PGZ’s PPARγ agonist activity. Moreover, when delivered via nebulization, combining B-YL with PGZ improved PGZ’s delivery by about 30% versus PGZ delivered alone .
In summary, this study, using the adult mouse lung explant model, demonstrates that combined PGZ and B-YL treatment mitigates hyperoxia-induced activation of TGF-β and Wnt signaling and the accompanying increase in inflammatory cytokines, myogenic proteins, and cellular apoptosis. Although PGZ alone also exhibited significant protective effects, overall, the effects were more pronounced with PGZ + B-YL combination versus PGZ alone. The effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult lung injury ex-vivo encourages testing this promising therapeutic approach in-vivo.