Increased pulmonary vascular expression of Krüppel-like factor 5 and activated survivin in experimental congenital diaphragmatic hernia

The high morbidity and mortality in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). PH is characterized by increased pulmonary artery smooth muscle cell (SMC) proliferation, suppressed apoptosis as well as endothelial dysfunction. Krüppel-like factor 5 (KLF5) belongs to a family of transcription factors that has diverse functions during cell differentiation and embryonic development. KLF5 is preferentially expressed in proliferating SMCs but reduced in differentiated cells. KLF5 induces the expression of Survivin, a 16.5 kDa protein overexpressed in almost all malignancies but hardly detected in normal differentiated tissues. Survivin has been shown to inhibit apoptosis, promote cell proliferation, and enhance angiogenesis. Recent studies have implicated activation of KLF5 and Survivin in the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that KLF5 and Survivin expression are increased in nitrofen-induced CDH.

Pregnant rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen (n = 16) and control group (n = 16). Quantitative real-time PCR, western blotting, and confocal immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of KLF5, Survivin, and phosphorylated Survivin (p-Survivin).

Confocal microscopy revealed markedly increased pulmonary vascular KLF5 and p-Survivin expression in lungs of nitrofen-exposed fetuses compared to controls. These results were confirmed by western blotting, showing increased pulmonary expression of KLF5 and p-Survivin. Furthermore, the relative pulmonary gene expressions of KLF5 and Survivin were significantly increased in the CDH group compared to controls (p < 0.005 rsp. p < 0.01).

This study provides striking evidence of increased gene and protein expression of KLF5 and activated Survivin in the pulmonary vasculature of nitrofen-induced CDH, suggesting that increased expression of KLF5 may activate p-Survivin expression and play an important role in the pathogenesis of PH in nitrofen-induced CDH.