nature publishing group Basic Science Investigation Articles Activin A contributes to the development of ­ hyperoxia-induced lung injury in neonatal mice Rebecca Lim1,2, Ruth Muljadi1, Eugenia Koulaeva2, Patricia Vosdoganes2, Siow Teng Chan1, Rutu Acharya1, Seshini Gurusinghe1, Olli Ritvos3, Arja Pasternack3 and Euan M. Wallace1,2 BACKGROUND: Bronchopulmonary dysplasia (BPD) is one of There is currently no cure for BPD and asides from ventilatory the leading causes of morbidity and mortality in babies born support, clinical management involves the use of steroids, which prematurely, yet there is no curative treatment. In recent years, is associated with an increased incidence of cerebral palsy. There a number of inhibitors against TGFβ signaling have been tested is thus a need for new therapeutic approaches. for their potential to prevent neonatal injury associated with In the past decade, much light has been shed on the contri- hyperoxia, which is a contributing factor of BPD. In this study, bution of TGFβ signaling to neonatal lung development and we assessed the contribution of activin A—a member of the injury. TGFβ1 has been shown to be important for the sur- TGFβ superfamily—to the development of ­hyperoxia-induced vival and repair of type II alveolar epithelial cells (AEC2) (4). lung injury in neonatal mice. Specifically, TGFβ1 treatment of hyperoxia damaged AEC2 METHODS: We placed newborn C57Bl6 mouse pups in con- improved cell survival, migration, and secretion of proan- tinuous hyperoxia (85% O2) to mimic many aspects of BPD giogenic ligands such as vascular endothelial growth factor including alveolar simplification and pulmonary inflamma- (VEGF) and matrix proteins such as fibronectin. However, tion. The pups were administered activin A receptor type IIB-Fc inhibition of TGFβ signaling by curcumin and PPARγ agonist, antagonist (ActRIIB-Fc) at 5 mg/kg or follistatin at 0.1 mg/kg on rosiglitazone, were protective against hyperoxia-induced lung postnatal days 4, 7, 10, and 13. injury in neonatal rat models of BPD (5,6). These reports point RESULTS: Treatment with ActRIIB-Fc and follistatin protected to the need for better understanding of TGFβ signaling within against hyperoxia-induced growth retardation. ActRIIB-Fc also the context of BPD. reduced pulmonary leukocyte infiltration, normalized tissue: Activin A, a member of the TGFβ superfamily, has been airspace ratio and increased septal crest density. These findings reported to contribute to adult lung disease. In adult animals, were associated with reduced phosphorylation of Smad3 and overexpression of activin A in the lungs results in an alteration decreased matrix metalloproteinase (MMP)-9 activity. in the composition of the lung epithelial cell populations where CONCLUSION: This study suggests that activin A signal- ciliated cells dominate, with fewer AEC2 (7). These changes ing may contribute to the pathology of bronchopulmonary result in a compromise to lung compliance and a significant dysplasia. reduction in surfactant protein C-expressing AEC2. In another study, blocking activin A using follistatin following in bleomy- cin challenge, prevented lung inflammation and fibrosis (8). remature birth refers to birth before 37 wk of gestation. There While these studies support a role for activin A in adult inflam- Pare a large number of medical complications associated with matory and fibrotic lung injury, its role in neonatal lung injury, being born prematurely, many of which are a consequence of repair and development has never been previously studied. life-saving interventions, including bronchopulmonary dyspla- In this study, we exposed newborn mice to hyperoxia (85% sia (BPD). Historically, BPD was originally described in 1967, O2) and administered either soluble ActRIIB-Fc (5 mg/kg) or as a form of chronic lung disease observed in premature babies, follistatin (0.1 mg/kg) twice weekly to determine the contribu- resulting from the injurious effects of high oxygen tension and tion of activin A signaling to the pulmonary phenotype. We airway pressure used during mechanical ventilation (1). This assessed changes to lung remodeling, activin A levels, Smad form of lung disease is commonly referred to as “old BPD”. signaling and markers of lung injury including leukocyte infil- Today, this is rare due to gentler ventilation strategies and the tration, cell death, and proliferation. use of surfactant and antenatal steroids. Instead, a “new BPD” has emerged, defined as the requirement of supplemental oxy- RESULTS gen at 36 wk postconceptional age (2). The pathological hall- ActRIIB-Fc and Follistatin Treatment Improved Bodyweights marks are suggestive of arrested septation and alveolarisation Hyperoxia reduced bodyweights of pups as previously­ triggered by inflammation, corticosteroids, and hyperoxia (3). reported. The bodyweights of animals from the hyperoxia 1The Ritchie Centre, MIMR-PHI Institute of Medical Research, Victoria, Australia; 2Department of Obstetrics and Gynecology, Monash University, Victoria, Australia; 3Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland. Correspondence: Rebecca Lim ([email protected]) Received 11 March 2014; accepted 22 November 2014; advance online publication 15 April 2015. doi:10.1038/pr.2015.46 Copyright © 2015 International Pediatric Research Foundation, Inc. Volume 77 | Number 6 | June 2015 Pediatric REsEarcH 749 Articles Lim et al. ab † 8 ‡ 800 ** * 6 600 4 400 Body weight (g) Activin A (pg/ml) 2 200 0 0 Saline ActRII-Fc FS288 Saline ActRII-Fc FS288 Saline ActRII-Fc FS288 Saline ActRII-Fc FS288 Normoxia Hyperoxia Normoxia Hyperoxia Figure 1. Hyperoxia reduced bodyweights of neonatal mouse pups. Hyperoxia reduced bodyweights of developing pups. They are significantly lighter at postnatal day day 14 compared to normoxia pups (a, *P < 0.05). Administration of ActRIIB-Fc and FS288 significantly increased bodyweights of ­hyperoxia-exposed pups (†P < 0.001, ‡P < 0.0001). Activin A levels were elevated in hyperoxia animals, which was reduced following FS288 treatment (b, *P < 0.05). + saline cohort were significantly lower compared to the a 2.0 ** normoxia + saline cohort at postnatal day 14 (Figure 1a). * Treatment with ActRIIB-Fc and follistatin significantly ** improved the bodyweights of pups subjected to hyperoxia such 1.5 that they were not significantly different to the normoxia treat- ment groups. Hyperoxia was associated with increased activin levels (Figure 1b, P < 0.05), which remained unchanged by 1.0 ActRIIB-Fc treatment but significantly reduced by follistatin (P < 0.05). pSmad3 vs total Smad 3 0.5 ActRIIB-Fc Treatment Improves Septation Hyperoxia increased Smad3 phosphorylation as previously reported by Dasgupta et al. (9) (Figure 2a, P < 0.05). This was 0.0 significantly reduced by ActRIIB-Fc and follistation (P < 0.01). Saline ActRII-Fc FS288 Saline ActRII-Fc FS288 Representative western blots are provided in Figure 2b. Typical Normoxia Hyperoxia of hyperoxia exposure, we saw a reduction in tissue: airspace b Normoxia Hyperoxia Normoxia Hyperoxia Normoxia Hyperoxia ratio (Figure 3a, P < 0.01) as increased oxygen tension induced + Saline + Saline + ActRII-Fc + ActRII-Fc + FS288 + FS288 alveolar simplification. Treatment with ActRIIB-Fc signifi- cantly increased tissue: airspace ratio in hyperoxia-treated ani- Total Smad 3 mals (P < 0.0001), suggesting that there was some prevention Phospho Smad 3 of alveolar simplification. Smad 2/3 signaling is increased in mouse models of Figure 2. Treatment with ActRIIB-Fc and follistatin reduced hyperoxia-induced lung injury (5) as well as sheep mod- ­hyperoxia-induced Smad 3 phosphorylation. Hyperoxia significantly els of antenatal inflammation-associated lung injury (10). increased Smad 3 phosphorylation (a, *P < 0.05). This was reduced in Given the effects of ActRIIB-Fc treatment on improving tis- ActRIIB-Fc- and FS288-treated animals (**P < 0.01). Representative western sue: airspace ratio and reducing phosphorylation of Smad 3, blots are shown (b). we sought to assess the effects of ActRIIB-Fc and follistatin on septation. Secondary septal crest density was thus deter- ActRIIB-Fc Treatment Reduced Pulmonary Leukocyte Infiltration mined by morphometric analysis. We confirmed that hyper- and IL-6 Production oxia significantly reduced septal crest density (Figure 3a, Unsurprisingly, hyperoxia increased the recruitment of leu- P < 0.001). Administration of ActRIIB-Fc increased septa- kocytes in the lungs as determined by CD45 immunohisto- tion in the hyperoxia-treated animals (P < 0.0001) while chemistry (Figure 4a, P < 0.05). This was significantly reduced follistatin treatment had no significant impact on septation. by ActRIIB-Fc treatment (P < 0.01) where the percentage of Representative images of septal crest imaging are shown in CD45+ cells was reduced to numbers comparable to that of Figure 3c. normoxia + saline animals. Given that ActRIIB-Fc treatment 750 Pediatric REsEarcH Volume 77 | Number 6 | June 2015 Copyright © 2015 International Pediatric Research Foundation, Inc. Activin contributes to hyperoxic injury Articles ab‡ 0.8 ** 0.20 0.6 † 0.15 ‡ 0.4 0.10 % Septal crests Tissue space: air space 0.2 0.05 0.0 0.00 Saline ActRII-Fc FS288 Saline ActRII-Fc FS288 FS288 ActRII-Fc Saline Saline ActRII-Fc FS288 Normoxia Hyperoxia Normoxia Hyperoxia c Saline ActRII-Fc FS288 Normoxia Hyperoxia Figure 3. Changes in tissue: airspace ratio and secondary septation. Tissue: airspace ratio was reduced by hyperoxia (a, **P