The Pulmonary Mesenchymal Tissue Layer Is Defective in an in Vitro Recombinant Model of Nitrofen-Induced Lung Hypoplasia

Despite modern treatments, congenital diaphragmatic hernia (CDH) remains associated with variable survival and significant morbidity. The associated pulmonary hypoplasia is a major determinant of outcome. To develop better treatments, improved comprehension of the pathogenesis of lung hypoplasia is warranted. We developed an in vitro cell recombinant model to mimic pulmonary hypoplasia and specifically to investigate epithelial-mesenchymal interactions and to decipher which tissue layer is primarily defective in nitrofen-induced CDH-associated lung hypoplasia. Epithelial cells (E) and fibroblasts (F) were isolated from E19 control (C) and nitrofen-induced hypoplastic rat lungs (N). Cells were recombined and cultured as either homotypic [(FC)(EC) and (FN)(EN)] or heterotypic [(FC)(EN) and (FN)(EC)] recombinants. Recombinants containing FN fibroblasts had a thickened fibroblast tissue layer and there were fewer organized alveolar-like epithelial structures compared with those in control (FC)(EC) recombinants. These FN recombinants exhibited a decrease in terminal deoxynucleotidyl transferase dUTP nick end labeling and cleaved caspase-3 positive cells. Cell proliferation was arrested in recombinants containing FN fibroblasts, which also exhibited increased p27Kip1 and p57Kip2 expression. In conclusion, fibroblasts, and not epithelial cells, appear to be the defective cell type in nitrofen-induced hypoplastic lungs due to a decreased ability to undergo apoptosis and maintain overall proliferation. This may explain the characteristic pulmonary interstitial thickening and hypoplasia observed in both nitrofen-induced hypoplastic lungs as well as human hypoplastic CDH lungs.