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Disruption of the Bone-Morphogenetic-Protein-Receptor-2 Pathway In Nitrofen-Induced Congenital Diaphragmatic Hernia

Sunday, October 21, 2012: 7:30 AM
Versailles Ballroom (Hilton Riverside)
Jan-H. Gosemann1, Florian Friedmacher1, Naho Fujiwara1, Nicolae Corcionivoschi1, Luis A. Alvarez2 and Prem Puri1, (1)Our Lady's Children's Hospital, National Children's Research Centre, Dublin, Ireland, (2)University College Dublin, Conway Institute of Biomedical Research, Dublin, Ireland

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).

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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.