Aberrant pulmonary lymphatic development in the nitrofen mouse model of congenital diaphragmatic hernia

Abstract Purpose Many infants develop a postsurgical chylothorax after diaphragmatic hernia repair. The pathogenesis remains elusive but may be owing to dysfunctional lymphatic development. This study characterizes pulmonary lymphatic development in the nitrofen mouse model of CDH. Methods CD1 pregnant mice were fed nitrofen/bisdiamine (N/B) or olive oil at E8.5. At E14.5 and E15.5, lung buds were categorized by phenotype: normal, N/B without CDH (N/B − CDH), or N/B with CDH (N/B + CDH). Anti-CD31 was used to localize all endothelial cells, while anti-LYVE-1 was used to identify lymphatic endothelial cells in lung buds using immunofluorescence. Differential protein expression of lymphatic-specific markers was analyzed. Results Lymphatic endothelial cells localized to the mesenchyme surrounding the airway epithelium at E15.5. CD31 and LYVE-1 colocalization identified lymphatic endothelial cells. LYVE-1 expression was upregulated in N/B + CDH lung buds in comparison to N/B − CDH and normal lung buds by immunofluorescence. Western blotting shows that VEGF-D, LYVE-1, Prox-1, and VEGFR-3 expression was upregulated in N/B + CDH lung buds in comparison to N/B − CDH or control lung buds at E14.5. Conclusions Lung lymphatics are hyperplastic in N/B + CDH. Upregulation of lymphatic-specific genes suggests that lymphatic hyperplasia plays an important role in dysfunctional lung lymphatic development in the nitrofen mouse model of CDH.