Z. D. Fox1,2, G. Jiang2, K. A. Walker2, K. Ho3, A. P. Liu3, S. M. Kunisaki1,2 1University Of Michigan Medical School,Ann Arbor, MI, USA 2Michigan Medicine,Pediatric Surgery Section, Department Of Surgery,Ann Arbor, MICHIGAN, USA 3University Of Michigan,Mechanical Engineering,Ann Arbor, MICHIGAN, USA
Introduction: The molecular mechanisms associated with potentially lethal pulmonary hypoplasia and pulmonary hypertension in congenital diaphragmatic hernia (CDH) remain poorly understood. Periostin (POSTN) has been shown to be an important stress response molecule during the saccular stage of lung development and is a critical regulator of alveolar septation in association with α-smooth muscle actin (SMA) myofibroblasts. This study employed an ex vivo model to determine whether mechanical compression affects POSTN and other pulmonary transcripts during fetal CDH lung development.
Methods: Sprague-Dawley dams were gavaged with nitrofen (100 mg) at E9.5 gestation to induce fetal CDH pulmonary hypoplasia. Whole fetal rat lungs from nitrofen-exposed and control (vehicle only) dams at E15.5 were explanted and cultured ex vivo in customized chambers under static mechanical compression (0.2 or 0.4 kPa, n=8/group) for 12 hrs to mimic physiologic compression forces that occur in CDH in vivo. Lungs were evaluated for mesenchymal (POSTN, SMA, TGF-β) and epithelial (SP-C) expression by qPCR and immunohistochemistry (IHC). Statistical comparisons normalized to lungs at 0 kPa were made by analysis of variance with significance set at p<.05.
Results: Control lungs exposed to 0.2 and 0.4 kPa showed significant increases in POSTN expression (1.79±.10; 2.12±.39, respectively; both p<.001). In contrast, compressed nitrofen-exposed lungs revealed significant decreases in POSTN expression (0.4 kPa: 0.67±0.15, p<.001). IHC confirmed increased presence of POSTN in control lungs, but not in nitrofen-exposed lungs (Figure). TGF-β was significantly increased in nitrofen-exposed lungs (1.39±.12, p=.045) and control lungs under increased compression (0.2 kPa: 1.33±.08, p=.036). Compression alone did not alter SMA expression in control lungs, but nitrofen-exposed lungs revealed significantly increased SMA at both 0.2 and 0.4 kPa (2.04±.15; 2.22±.11; both p<.001, respectively). Control lungs exposed to 0.4 kPa showed significant increases in SP-C (1.20±.20, p<.001). Conversely, nitrofen-exposed lungs had a significant reduction in SP-C expression at 0.2 and 0.4 kPa (0.53±.04, p<.01; 0.69±.23, p<.001; respectively).
Conclusion: Collectively these data suggest that mechanical compression induces a distinct transcriptome pattern within the nitrofen CDH fetal lung characterized by downregulation of POSTN and SP-C and upregulation of TGF-β and SMA. This ex vivo compression system may serve as a novel functional platform to better understand the impact of mechanical stress on the complex genetic control of matricellular dynamics during CDH lung development.
