Abstract Introduction: Acute lung injury and acute respiratory distress syndrome are characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. We previously reported that a combination of aerosolized pioglitazone (PGZ) and synthetic lung surfactant B-YL peptide as surfactant protein B mimic prevents hyperoxia-induced neonatal rat lung injury, but this has not been tested in adult lung injury. Method: We characterize effects of hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of hyperoxia-induced lung injury, 2) aberrations of lung homeostasis and injury repair following 24 or 72-hour exposure of adult mice lung explants to hyperoxia, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination. Results: Our study reveals that hyperoxia exposure of adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, MCP-1, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1 and PECAM-1) markers. All of these changes were largely prevented by PGZ+B-YL combination. Conclusions: The effectiveness of the PGZ+B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo sets the stage to test this promising therapeutic approach for adult lung injury in vivo.

Background

Despite improvements in perinatal care, bronchopulmonary dysplasia (BPD) in extremely premature infants has not decreased. Postnatal surfactant therapy provides symptomatic relief from respiratory distress syndrome, but does not translate into a reduction in BPD. Therefore, the search for effective interventions to prevent BPD continues.

Objectives

Since PPAR-γ agonists have been demonstrated to promote neonatal lung maturation and injury repair, we hypothesized that a formulation of a PPAR-γ agonist, pioglitazone (PGZ) and a synthetic lung surfactant (a surfactant protein B peptide mimic, B-YL) combined would stimulate lung maturation and block hyperoxia-induced neonatal lung injury more effectively than either modality alone.

Methods

One-day-old Sprague-Dawley rat pups were administered PGZ + B-YL via nebulization every 24 h for up to 72 h. The pups were exposed to either 21 or 95% O2, and then sacrificed. Their lungs were examined for markers of lung maturation (levels of PPAR-γ, SP-C and choline-phosphate cytidylyltransferase [CCT-α] and [3H]triolein uptake) and injury repair (bronchoalveolar lavage cell count and protein content, and levels of LEF-1, fibronectin, ALK5, and β-catenin) by Western blot analysis.

Results

Markers of alveolar epithelial/mesenchymal maturation (PPAR-γ, SP-C, CCT-α, and triolein uptake) increased significantly in the PGZ + B-YL group, more than with either drug alone. Similarly, markers of hyperoxia-induced lung injury were blocked effectively with PGZ + B-YL treatment.

Conclusions

Nebulized PPAR-γ agonist PGZ with a synthetic lung surfactant accelerates lung maturation and prevents neonatal hyperoxia-induced lung injury more than either modality alone, with the potential to provide more effective prevention of BPD.

Introduction

Hyperoxia-induced lung injury is characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. Although a combination of aerosolized pioglitazone (PGZ) and a synthetic lung surfactant (B-YL peptide, a surfactant protein B mimic) prevents hyperoxia-induced neonatal rat lung injury, whether it is also effective in preventing hyperoxia-induced adult lung injury is unknown.

Method

Using adult mice lung explants, we characterize the effects of 24 and 72-h (h) exposure to hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of lung injury, 2) aberrations of lung homeostasis and injury repair pathways, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination.

Results

Our study reveals that hyperoxia exposure to adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1, and PECAM-1) markers. All of these changes were largely mitigated by the PGZ + B-YL combination.

Conclusion

The effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo is promising to be an effective therapeutic approach for adult lung injury in vivo.

The three-dimensional structure of the synthetic lung Surfactant Protein B Peptide Super Mini-B was determined using an integrative experimental approach, including mass spectrometry and isotope enhanced Fourier-transform infrared (FTIR) spectroscopy. Mass spectral analysis of the peptide, oxidized by solvent assisted region-specific disulfide formation, confirmed that the correct folding and disulfide pairing could be facilitated using two different oxidative structure-promoting solvent systems. Residue specific analysis by isotope enhanced FTIR indicated that the N-terminal and C-terminal domains have well defined α-helical amino acid sequences. Using these experimentally derived measures of distance constraints and disulfide connectivity, the ensemble was further refined with molecular dynamics to provide a medium resolution, residue-specific structure for the peptide construct in a simulated synthetic lung surfactant lipid multilayer environment. The disulfide connectivity combined with the α-helical elements stabilize the peptide conformationally to form a helical hairpin structure that resembles critical elements of the Saposin protein fold of the predicted full-length Surfactant Protein B structure.