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.
Keywords
lysyl oxidase - diaphragm - lung - nitrofen - congenital diaphragmatic hernia
