Combination of pioglitazone, a PPARγ agonist, and synthetic surfactant B-YL prevents hyperoxia-induced lung injury in adult mice lung explants

Abstract

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.

Keywords: Hyperoxia; Lung injury; PPARγ; Pioglitazone; Surfactant.

Fig. 1.. Effect of PGZ, B-YL, and PGZ + B-YL on lung homeostasis.

Adult mice lung explants were cultured in normoxia for a) 24 h or b) 72 h in either control medium or medium treated with B-YL (100 mg/kg), PGZ (1 mg/kg), or B-YL (1 mg/kg) + PGZ (100 mg/kg) to determine effects on epithelial (SP–C and CCTα) and mesenchymal (PPARγ) markers of lung homeostasis. There was an increase in SP-C and CCTα, as determined by Western analysis when treated with PGZ + B-YL for 24 or 72 h. With PGZ-only treatment, the SP-C protein level was not affected at 24 h but increased at 72 h; CCTα protein levels increased at both 24 and 72 h time points. Although PPARγ protein was not affected at 24 h, at 72 h, it increased with either PGZ-only or PGZ + B-YL treatment. Values are means ± SE (*p < 0.05 vs control: N = 3).

Fig. 2.. Effect of PGZ, B-YL, and PGZ + B-YL on hyperoxia-induced activation of Wnt (β-catenin and LEF-1) and TGF-β (ALK5 and SMAD 3) signaling pathways.

Adult mice lung explants were cultured in normoxia or hyperoxia ± B-YL, PGZ, or PGZ + B-YL for 24 or 72 h. a) Western analysis was used to measure β-catenin levels, which were unchanged in all groups at 24 h but increased with hyperoxia and this increase ameliorated with PGZ-only and PGZ + B-YL treatments at 72 h. LEF-1 protein level was not affected following 24 h exposure to hyperoxia, but was decreased in PGZ-only and PGZ + B-YL treated groups. At 72 h, the LEF-1 protein level increased in the hyperoxia exposure group, and this increase was blocked in all treatment groups. b) ALK5 and SMAD 3 protein levels increased in the hyperoxia exposure groups, and this effect was ameliorated by concurrent treatment with PGZ + B-YL for 24 and 72 h. However, at 72 h, with PGZ-only treatment, ALK5 protein level decreased, while no effect was seen on SMAD 3 protein levels. There was an increase in SMAD 7 protein level with hyperoxia exposure at 24 h, an effect that was not impacted with any treatment. c) Using qRT-PCR, TGF-β1 and β3 mRNA expression was determined. TGF-β1 expression increased at both 24 h and 72 h time-points following exposure to hyperoxia; however, TGF-β3 expression was increased only at the 24 h time-point, which was decreased in treatment groups. However, there was a decrease in TGF-β3 with hyperoxia at 72 h time-point with no changes in the treated groups. Representative protein bands and densitometric values relative to the GAPDH of each group are shown. Values are means ± SE (*p < 0.05 vs 21% O2, and #p < 0.05 vs 95% O2 control: N = 3).

Fig. 3.. Effect of PGZ, B-YL, and PGZ + B-YL on hyperoxia-induced changes in lung myogenic proteins.

Western analysis was used to measure calponin and fibronectin protein levels. Calponin was increased on exposure to hyperoxia, and this increase was blocked with PGZ-only and PGZ + B-YL treatment groups at both 24 h and 72 h time points. Fibronectin was increased on exposure to hyperoxia at 24 h and 72 h time points. The hyperoxia-induced increase in fibronectin protein level was ameliorated with PGZ-only and PGZ + B-YL treated groups at both time points. Representative protein bands and densitometric values relative to the GAPDH of each group are shown. Values are means ± SE (*p < 0.05 vs 21% O₂, and #p < 0.05 vs 95% O₂ control: N = 3).

Fig. 4.. Effect of PGZ, B-YL, and PGZ + B-YL on hyperoxia-induced changes in PPARγ and C/EBPα protein levels.

Exposure to hyperoxia did not alter PPARγ protein levels at either 24 or 72 h timepoint, but these levels increased with PGZ + B-YL treatment at both time points. C/EBPα protein level decreased on exposure to hyperoxia, and this decrease was blocked by PGZ and PGZ + B-YL treatments at 24 and 72 h. Representative protein bands and densitometric values relative to the GAPDH of each group are shown. Values are means±SE; n = 3. (*p < 0.05 vs 21% O2, and #p < 0.05 vs 95% O2 control; N = 3).

Fig. 5.. Effect of PGZ, B-YL, and PGZ + B-YL on hyperoxia-induced changes in inflammatory and apoptosis markers.

Adult mice lung explants were exposed to hyperoxia ± B-YL, PGZ, or PGZ + B-YL for 24 h or 72 h. qRT-PCR was used to measure inflammatory cytokines and Western analysis for apoptosis. a) Following 72 h exposure to hyperoxia, expression of all inflammatory cytokines examined was significantly increased, and this increase was either blocked (IL-6, IL-1β in PGZ only and PGZ + B-YL treated groups) or showed an improving trend (TNFα in the PGZ + B-YL treated group). b) Hyperoxia-induced decrease in BCL-2/BAX ratio at 24 h and 72 h was blocked by PGZ only and PGZ + B-YL treatments. c) mRNA expression of VEGF-A, VEGF-receptor FLT-1, and PECAM-1 increased following 24 h exposure to hyperoxia; this increase was blocked in all treatment groups with the most pronounced effect observed in the PGZ + B-YL treated group. However, at 72 h, the effect of hyperoxia on endothelial markers was Variable, i.e., an increase in VEGF-A, a decrease in FLT-1, and no change in PECAM mRNA expression, with an inconsistent effect of interventions. Values are means ± SE (*p < 0.05 vs 21% O₂, and #p < 0.05 vs 95% O₂ control: N = 3).