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

[congenital diaphragmatic hernia]

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 (D 9 ), and fetuses were harvested on selected time points D 15 and D 18 . 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 D 15 (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 D 18 (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 D 15 and D 18 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 .