Reduced KLF4 expression in endothelial cells triggers pulmonary vascular abnormalities associated with experimental congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a rare anomaly with an incidence of 1 in 3000 live births and characterized by defective closure of the diaphragm, lung hypoplasia, and pulmonary vascular remodeling. Although several genes associated with the occurrence of CDH, no clear genetic component has been identified. Previously, we showed that CDH patients have vascular abnormalities already early in development, and that perivascular cells are affected in CDH. In the current study, we focused on early abnormalities in the nitrofen-induced CDH mouse model. Transcriptome analysis of FACS-sorted perivascular and endothelial cell populations from lungs of control and experimental mouse CDH revealed a high similarity in GO terms of differentially expressed genes (DEGs) between these populations, suggesting a disturbed interaction between endothelial cells and pericytes. The disturbed interaction is the basis for the aberrant development of the pulmonary vasculature in CDH. Furthermore, the RNA sequence data revealed reduced expression of the Kruppel like factor 4 (Klf4) gene in endothelial cells of CDH, which was confirmed with protein expression analysis. Furthermore, we show that KLF4 is an important up stream regulator of genes associated with vascular development and confirm that members of the NOTCH signaling pathway are differentially expressed, indicating that NOTCH signaling is disturbed in CDH. Collectively, our data support the importance of KLF4 in pulmonary angiogenesis and contribute to our previous data that pulmonary vessels in CDH patients are already affected before birth, which in turn may impact therapeutic strategies to reduce pulmonary hypertension associated with CDH.