O2137: Development of a Novel Ex Vivo Model of Congenital Diaphragmatic Hernia Lung Hypoplasia by Mechanical Compression and YAP Inhibition

Background: Rodent models of the congenital diaphragmatic hernia (CDH) fetal lung have traditionally relied on exposure to maternal nitrofen, a teratogen that has never been linked to the human CDH phenotype. Our laboratory has previously shown that mechanical compression of the fetal mouse lung is associated with reduced nuclear YAP (nYAP) signaling and impaired early epithelial differentiation resembling CDH lung hypoplasia. In this study, we sought to develop an ex vivo, nitrofen-free model of the fetal CDH lung to better understand the mechanistic role of nYAP inhibition in disease pathogenesis.

Methods: After animal welfare approval (MO20M150), timed pregnant CD1 mice were gavaged with oil alone (normal) or nitrofen/4-biphenyl carboxylic acid (nitrofen) at E8.5 to induce CDH fetal lung hypoplasia. Dams were sacrificed for whole fetal lung harvesting at E15.5 (canalicular lung stage). For 48 hours following harvest, lungs were cultured ex vivo in control media (n=24) or one of three treatment conditions: 10µM verteporfin (nYAP inhibitor, n=18), mechanical compression (n=18), or a combination of these conditions (n=15). Fetal lungs exposed to compression were subjected to dynamic compression forces (range, 0-7.7cmH2O) within microdevices (Compression Plus FX-5000C) to mimic the in-utero hernia forces encountered in CDH. Immunofluorescence staining for YAP proteins were performed, and whole mount branching was assessed by lung epithelial progenitor Nkx2.1. Quantitative expression of Ctgf (downsteam nYAP marker) as well as lung alveolar type I (Pdpn) and mesenchymal (Vim) genes were measured. Normalized values are reported as mean±SEM and analyzed for significance by 2-way ANOVA(p < 0.05).

Results: Whole mount immunofluorescence revealed less complex branching patterns among lungs exposed to compression, verteporfin, and in combination when compared to normal lungs (Figure 1). Immunofluorescence staining and gene expression data showed reduced nYAP and increased cytoplasmic (inactivated) YAP in all compression and verteporfin-exposed lung groups (Figure 2). Both Vim (normal: 1.045±0.114, compression: 0.896±0.171, verteporfin: 1.176±0.124, compression+verteporfin:0.410±0.065; p=0.0003) and Pdpn (normal: 1.063±0.138, compression: 0.601±0.255, verteporfin: 0.645±0.064, compression+verteporfin:0.388±0.060; p=0.029) were significantly reduced in lungs exposed to both compression and verteporfin. Compared to fetal lungs harvested after maternal nitrofen administration, normal lungs exposed to both compression and verteporfin showed no difference in expression of Nkx2.1 (p=0.980) and Pdpn (p=0.299) but had significantly reduced Vim (p=0.004), suggesting a less differentiated mesenchyme.

Conclusion: For the first time, our data shows that ex-vivo exposure of fetal canalicular lungs to a combination of mechanical compression and an nYAP inhibitor induces substantial hypoplasia analogous to that observed following nitrofen exposure. Further work using this novel nitrofen-free modeling platform is warranted given its potential for (1) providing important mechanistic insights into the role of YAP as a key mechanosensory molecule regulating pulmonary development, and (2) the study of human CDH lung tissues and organoids as a prelude to pharmacologic testing and other clinical applications.