Purpose: The high mortality in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PPH). The nitrofen-induced CDH model is an established model to study the pathogenesis of PPH in CDH. Bone-morphogenetic-protein-receptor-2 (BMPR-2) plays a key role in pulmonary vasculogenesis during the late stage of fetal lung development. BMPR-2 is essential for the modulation of differentiation, proliferation and the fibrous matrix production of both endothelial and smooth muscle cells (SMCs). Mutations of BMPR-2 have been identified in 50-60% of patients with familial pulmonary arterial hypertension (PAH). Furthermore, patients with primary and secondary PAH show reduced pulmonary expression of BMPR-2. In mice, conditional knock out of BMPR-2 in SMCs resulted in the pulmonary hypertensive phenotype and increased pulmonary expression of monocyte-chemoattractant-protein-1 (MCP-1) - a chemokine that plays a major role in the development of PAH by inducing vascular remodeling. MCP-1, which is inhibited by BMPR-2 signaling, is consistently found to be increased in patients with PAH and several animal models of PAH/PPH. Both experimental BMPR-2 gene therapy and anti-MCP-1 therapy have been reported to attenuate PAH in rodents. We designed this study to investigate the hypothesis that BMPR-2 signaling is disrupted in nitrofen-induced CDH.
Methods: Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9) (Ethics: B100/4360). Fetuses were sacrificed on D21 and divided into control (n=15) and CDH (n=16). Quantitative real-time polymerase chain reaction (RT-PCR) was performed to determine pulmonary gene expression levels of BMPR-2 and MCP-1. Western blotting was used to evaluate protein expression of BMPR-2 (pulmonary) and MCP-1 (pulmonary/systemic). Serum MCP-1 levels were determined by enzyme-linked immunosorbent assay. Confocal microscopy with immunofluorescence double staining and 3D-reconstruction were performed to investigate pulmonary vascular BMPR-2 and MCP-1 expression.
Results: There was marked increase in medial and adventitial thickness in pulmonary arteries of all sizes in CDH compared to controls. Pulmonary BMPR-2 gene expression levels were significantly decreased in nitrofen-induced CDH compared to controls (Figure 1A). Western blotting revealed decreased pulmonary BMPR-2 protein expression in CDH compared to controls (Figure 1A). Confocal-microscopy showed markedly decreased vascular BMPR-2 expression in lungs of nitrofen-exposed fetuses compared to controls (Figure 2A). These results were accompanied by increased pulmonary MCP-1 gene/protein expression as well as increased circulating MCP-1 in nitrofen-induced CDH compared to controls (Figures 1B, 2B).
Conclusion: The observed disruptions of the BMPR-2 signaling pathway may lead to extensive vascular remodeling, contributing to persistent pulmonary hypertension in the nitrofen-induced CDH model. Activation of BMPR-2 and/or inhibition of MCP-1 may therefore represent a potential therapeutic approach for the treatment of PPH in CDH.