Eur J Pediatr Surg 2015; 25(01): 15-19
DOI: 10.1055/s-0034-1386644
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Lysyl Oxidase Expression Is Decreased in the Developing Diaphragm and Lungs of Nitrofen-Induced Congenital Diaphragmatic Hernia

Toshiaki Takahashi
1   National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
,
Florian Friedmacher
1   National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
,
Hiromizu Takahashi
1   National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
,
Alejandro Daniel Hofmann
1   National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
,
Prem Puri
1   National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
2   Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland
› Author Affiliations
Further Information

Publication History

15 May 2014

23 June 2014

Publication Date:
11 August 2014 (online)

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Abstract

Introduction Malformation of the nonmuscular tissue components in congenital diaphragmatic hernia (CDH) is thought to underlie the diaphragmatic defect, causing intrathoracic herniation of abdominal viscera and thus disturbing normal lung development. It has been shown that diaphragmatic and pulmonary morphogeneses require the structural integrity of connective tissue, and developmental mutations that inhibit the formation of extracellular matrix (ECM) result in CDH with hypoplastic lungs. Lysyl oxidase (lox), an extracellular enzyme that catalyzes the cross-linking of ECM proteins, plays an essential role during diaphragmatic and pulmonary development by controlling the formation of connective tissue. Furthermore, lox −/− knockouts exhibit abnormal connective tissue with diaphragmatic defects and impaired airway morphogenesis. We designed this study to investigate the hypothesis that diaphragmatic and pulmonary lox expression is decreased in the nitrofen-induced CDH model.

Materials and Methods Timed-pregnant Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time points D15 and D18. The micro-dissected fetal diaphragms (n = 48) and lungs (n = 48) were divided into two groups: control and nitrofen-exposed samples (n = 12 per specimen and time point, respectively). Diaphragmatic and pulmonary gene expression levels of lox were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemical staining was performed to evaluate lox protein expression in diaphragms and lungs.

Results Relative mRNA expression of lox was significantly reduced in diaphragms and lungs of nitrofen-exposed fetuses on D15 (0.29 ± 0.08 vs. 0.12 ± 0.05; p < 0.05 and 0.52 ± 0.44 vs. 0.20 ± 0.04; p < 0.05) and D18 (0.90 ± 0.25 vs. 0.57 ± 0.23; p < 0.05 and 0.59 ± 0.26 vs. 0.35 ± 0.09; p < 0.05) compared with controls. Diaphragmatic and pulmonary immunoreactivity of lox was markedly decreased in nitrofen-exposed fetuses on D15 and D18 compared with controls.

Conclusions Decreased lox expression during diaphragmatic development and lung branching morphogenesis may interfere with normal cross-linking of ECM proteins, disrupting the integrity of connective tissue, and contributing to the diaphragmatic defect and impaired airway formation in the nitrofen-induced CDH model.