| ObjectiveExcessive localization of elastin from the septal tips to the alveolar walls is a key feature of bronchopulmonary dysplasia (BPD). The abnormal accumulation of lung elastin, involving the structural and functional interaction of a series of proteins, is remains poorly understood. To further investigate the mechanisms accounting for the abnormal accumulation of elastin in the lungs of newborn mice with BPD. We evaluated elastin synthesis, distribution and its interaction with proteins involved in its aberrant localization in lungs of BPD mice. To determine if airway instillation treatment of newborn mice with the neutrophil elastase inhibitor (NEI) Elafin would prevent 85% O2 long-time exposure induced lung elastin degradation and the ensuing cascade of events causing lung growth arrest. Furthermore, we investigated the role of transforming growth factor-β (TGF-β) signaling in the inhibition of the terminal stage of lung development and the effect of Elafin on TGF-β activation.MethodDue to the similarity of lung growth between human beings and mice, BPD mice model was established by newborn C57BL/6 mice exposed to normobaric 85% O2 (hyperoxia) soon after birth (PO) for up to 21 days (P21, the period of postnatal lung growth), while mice were exposed to 21% O2 (air) from after birth as control.1. To investigate the effect of hyperoxia on the elastin system, eight mice were euthanized using ether to obtain lungs for histopathology on postnatal days (P) 1,3,7,14, and 21. Endotracheal intubation was utilized to maintain the lung inflation states. The right lobe was fixed in 4% paraformaldehyde for hematoxylin and eosin staining (H&E) to observe the morphological structure of the lung tissue. All of the fixed tissue samples were embedded in paraffin and sectioned at 4 μm for histochemical analysis. To assess the amount and localization of elastin deposition in the lung tissue, the sections were stained using Gomori’s aldehyde-fuchsin method. Alveolar development was evaluated by radial alveolar counts (RAC) on lung sections. Dual immunofluorescence was applied to evaluate the distribution and localization of integrin av to investigate whether the expression of integrin av was altered by O2 exposure. Additional mice were evaluated for tidal volume (TV) and frequency of breathing (F) at different time points to evaluate the lung function. The left lobe was frozen in liquid nitrogen for quantitative real-time PCR (Q-RT-PCR) and western blotting (WB). After the extraction of RNA from lung and reverse transcription, Q-RT-PCR were performed to identify the gene expression of tropoelastin and related genes, including integrin av, integrin (33, fibrillin-1, fibrillin-2, Fibulin-4, fibulin-5, Neutrophil elastase (NE), Lysyl oxidase (Lox) and Lysyl oxidase-like 1 (Loxl-1). Western blot was used to test tropoelastin, NE protein expression.2. In this part of study, newborn mice were randomly assigned to one of three groups:(1) Air (untreated, exposed to 21% O2); (2) Hyperoxia+ lactated-ringer solution (L/R, airway instillation of vehicle alone,10 μl/g body weight, bw, exposed to 85% O2); (3) Hyperoxia+Elafin (airway instillation treatment of the NEI Elafin (recombinant human elafm,40 ng/g bw, in 10 μl/g bw of L/R, exposed to 85% O2) at P3, P6 and P10. Lung tissues were harvested at P14 (the period of maximal alveolarization development). H&E staining was used to observe the morphological structure of the lung tissue after Elafin treatment. Gomori’s aldehyde-fuchsin staining was applied to assess the amount and localization of elastin fiber deposition in the lung tissue. The ultrastructure of elastin fiber was observed by transmission electron microscopy (TEM). NE activity in lung tissue and desmosine concentration in urinary were detected by enzyme-linked immunoabsorbent assay (ELISA).3. To further assess the role of endogenous TGF-β in the inhibition of lung development and the effect of airway instillation Elafin on TGF-β1/Smad2 signal pathway, the localization of pSmad2 was investigated by IHC and the ratio of pSmad2 and Smad2/3 was assessed by WB. To complement immunohistochemical assessment of pSmad2 in lung as an index of TGF-P activation, which was inhibited by elafin during hyperoxia-exposure, we applied an ELISA assay to measure active TGF-β1 and total TGF-β1 in whole lung homogenates obtained from the three groups of mice, respectively. Q-RT-PCR was performed to identify the genes expression of transcription factors including TTF-1 and HGF-3β.Result1. In this study, histological measurements revealed that lung septation or elastin accumulation was affected in O2-exposed pups, suggesting that elastin deposition and remodeling were arrested. Elastin metabolism was dysregulated in the 85% O2-injured developing mouse lungs, and this dysregulation was associated with altered elastin turnover. We found that elastin was predominantly deposited at the tips of short, thickened, secondary septal crests in the developing lungs, whereas O2 exposure caused disordered lung elastin in the distal lung, failed alveolar formation and excess elastin in neonatal mice.2. Lung morphometry showed that distal airspace size was greater and RAC were reduced in L/R-treated mice after hyperoxia for 14 days when compared with air-exposed control mice. However, Elafin treatment could attenuate the above-mentioned changes in lung structure and block the increased NE activity.3. We assessed the expression of pSmad2 in lung sections, a marker of TGF-P activity, in newborn mice exposed to hyperoxia for 14 days. The sevenfold to eightfold increase in pSmad2-expressing cells observed in lungs of L/R-treated pups was completely suppressed in elafin-treated mice after hyperoxia for 14 days. Consequently, lung abundance of pSmad-2-expressing cells was similar in hyperoxia-exposed pups treated with elafin compared with air-exposed without treatment. Active TGF-β1 was significantly increased after hyperoxia for 14 days and decreased after the treatment of Elafin, while total TGF-β1 in whole lung homogenates showed no significance in the three groups of mice.Conclusion1. We demonstrated the up-regulation of several elastin synthesis, assembly and degradation genes, such as integrin av, fibulin-5, fibrilin-1, and NE, in newborn mice with hyperoxic lung injury dysregulated elastin localization and arrested lung development.2.85% O2 long-time exposure of neonatal mice could increase the lung NE activity, which would caused redistribution of elastin from the tips of secondary septa, resulting in elastic fibers being scattered throughout the walls of distal airspaces. After airway instillation treatment of Elafin, the increased NE Activity was blocked, thereby helping to preserve the normal distribution of elastic fibers at the tips of secondary septa in lungs and preventing elastin degradation and dispersion of lung elastin fiber induced by hyperoxia injury.3.85% O2 long-time exposure increased the active TGF-β ratio in the newborn lung, as an index of TGF-β activation, which was consistent with the result of previous studies. We demonstrated in this study that elafin inhibited TGF-β activation during hyperoxia injury. And it is likely that TGF-β inhibition in response to elafin treatment played a key role in preventing at least some of the adverse pulmonary effects of hyperoxia injury, namely dysregulated elastin production, which can contribute to defective alveolar septation and lung growth. Thus, suppression of TGF-P activation may account, at least in part, for the beneficial effects of elafin treatment in stabilizing lung elastin and enabling alveolar septation in newborn mice during 85% O2 long-time exposure. |
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