Expression of Nitric Oxide Synthase Interacting Protein (NOSIP) is Decreased in the Pulmonary Vasculature of Nitrofen-Induced Congenital Diaphragmatic Hernia.

INTRODUCTION: Persistent pulmonary hypertension (PPH) is a major cause of morbidity and mortality in newborns with congenital diaphragmatic hernia (CDH). PPH is characterized by increased vascular resistance and smooth muscle cell (SMC) proliferation, leading to obstructive changes in the pulmonary vasculature. Nitric oxide (NO), generated by endothelial NO synthase (eNOS), is an important regulator of vascular tone and plays a key role in pulmonary vasodilatation. NO synthase interacting protein (NOSIP), which is strongly expressed by pulmonary SMCs, has recently been identified to reduce the endogenous NO production by interacting with eNOS. We designed this study to investigate the pulmonary vascular expression of NOSIP in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and lung specimens divided into CDH and control (n = 6 for each group). Quantitative real-time polymerase chain reaction and Western blotting were performed to analyze pulmonary gene and protein expression of NOSIP. Immunofluorescence double staining for NOSIP was combined with a specific SMC marker to evaluate protein expression in the pulmonary vasculature. RESULTS: Relative messenger ribonucleic acid and protein expression of NOSIP was significantly decreased in nitrofen-exposed CDH lungs compared with controls. Confocal laser scanning microscopy revealed markedly diminished NOSIP immunofluorescence in nitrofen-exposed CDH lungs compared with controls, mainly in the muscular and endothelial components of the pulmonary vasculature. CONCLUSION: This study demonstrates for the first time decreased NOSIP expression in the pulmonary vasculature of the nitrofen-induced CDH. These findings suggest that NOSIP underexpression may interfere with NO production, contributing to abnormal vascular remodeling and PPH.

Down-regulation of N-deacetylase-N-sulfotransferase-1 signaling in the developing diaphragmatic vasculature of nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) has been attributed to various developmental abnormalities of the underlying tissue components. N-deacetylase-N-sulfotransferase-1 (Ndst1) is a strongly expressed biosynthetic enzyme in endothelial cells, which has recently been identified as an important factor during diaphragmatic vascularization. Loss of endothelial Ndst1 has been demonstrated to cause angiogenic defects in the developing diaphragm and disrupt normal diaphragmatic development. Furthermore, deficiency of Ndst1 diminishes the expression of slit homolog 3 (Slit3), a known CDH-related gene that has been associated with reduced vascular density and muscle defects in the diaphragm of Slit3-/- mice. We hypothesized that expression of Ndst1 and Slit3 is decreased in the diaphragmatic vasculature of fetal rats with nitrofen-induced CDH. METHODS: Time-mated rats received either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15 and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Ndst1 and Slit3 were assessed using qRT-PCR. Immunofluorescence-double-staining for Ndst1 and Slit3 was performed to evaluate protein expression and localization. RESULTS: Relative mRNA expression of Ndst1 and Slit3 was significantly decreased in pleuroperitoneal folds (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal-laser-scanning-microscopy revealed markedly diminished Ndst1 and Slit3 expression in endothelial cells within the diaphragmatic vasculature on D13, D15 and D18 compared to controls. CONCLUSIONS: Down-regulation of Ndst1 signaling in the developing diaphragm may impair endothelial cell migration and angiogenesis, thus leading to defective diaphragmatic vascular development and CDH. LEVEL OF EVIDENCE: Ib.

Elastase and matrix metalloproteinase activities are associated with pulmonary vascular disease in the nitrofen rat model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Pulmonary vascular disease (PVD) is a leading cause of congenital diaphragmatic hernia (CDH) mortality. Progression of PVD involves extracellular matrix remodeling by elastases and matrix metalloproteinases (MMP), concomitant with proliferation of smooth muscle cells in a growth factor-enriched environment. Blockade of this pathway reversed primary pulmonary hypertension and improved survival. This study was designed to determine whether a similar pathway is induced in PVD secondary to CDH. METHODS: Fetal rats exposed to nitrofen at gestational day 9 developed left-sided CDH and were compared at term to their non-CDH littermates by assessing histologic and biochemical features of PVD. RESULTS: Rats with CDH displayed right ventricle hypertrophy, increased pulmonary artery medial wall thickness and muscularization, and decreased lumen size. As revealed by in situ zymography and immunohistochemistry, this was associated with an induction of elastolytic and MMP activities as well as an elevation of epidermal growth factor and osteopontin levels in the diseased lung vasculature. CONCLUSIONS: CDH-associated PVD involves an induction of elastase and MMP activities and increased osteopontin deposition in an epidermal growth factor-rich environment. Inhibition of this pathway may thus represent a novel therapeutic approach for the treatment of CDH-associated PVD. LEVEL OF EVIDENCE: Level I (Basic Science Study).

Decidual ß-carotene-15,15'-oxygenase-1 and 2 (BCMO1,2) expression is increased in nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: Retinoids are essential for fetal and lung development. Beta-carotene(BC) is the main dietary retinoid source and beta-carotene-15,15'-oxygenase-1 and 2 (Bcmo1,2) is the primary enzyme generating retinoid from BC in adult mammalian tissues. Placenta has a major role in the retinol homeostasis in fetal life: Since there is no fetal retinol synthesis, maternal retinol has to cross the placenta. It has been recently shown that BC can be converted to retinol by Bcmo1,2 in placenta for retinol transfer and moreover, BC can cross the placenta intact. The placental Bcmo1,2 expression is tightly controlled by placental retinol level. In severe retinol deficiency it has been shown that placental Bcmo1,2 expression are increased for generating retinol from dietary maternal BC even when the main retinol transfer is blocked. In recent years, low pulmonary retinol levels and disrupted retinoid signaling pathway have been implicated in the pathogenesis of pulmonary hypoplasia and congenital diaphragmatic hernia (CDH) in the nitrofen model of CDH. Recently, it has been demonstrated that the main retinol transfer in the placenta is blocked in the nitrofen model of CDH causing increased placental and decreased serum retinol level. The aim of our study was to determine maternal and fetal ß-carotene levels and to investigate the hypothesis that placental expression of BCMO1 and BCMO2 is altered in nitrofen-exposed rat fetuses with CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Maternal and fetal serum, placenta, liver and left lungs were harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Immunochistochemistry was performed to evaluate trophoblasts by cytokeratin expression and placental Bcmo1,2 expression. Expression levels of Bcmo1,2 genes in fetal lungs and liver were determined using RT-PCR and immunohistochemistry. BC level was measured using HPLC. RESULTS: Markedly increased decidual Bcmo1,2 immunoreactivity was observed in CDH group compared to controls. There was no difference neither in the trophoblastic Bcmo1,2 immunoreactivity nor in the pulmonary and liver Bcmo1,2 expression compared to controls. There was no significant difference in maternal serum BC levels between control and CDH mothers (2.14 ± 0.55 vs 2.56 ± 1.6 µM/g, p = 0.8). BC was not detectable neither in the fetal serum nor liver or lungs. CONCLUSIONS: Our data show that nitrofen increases maternal but not fetal Bcmo1,2 expression in the placenta in nitrofen-induced CDH group. The markedly increased decidual Bcmo1,2 expression suggests that nitrofen may trigger local, decidual retinol synthesis in the nitrofen model of CDH.

Lysyl oxidase expression is decreased in the developing diaphragm and lungs of nitrofen-induced 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 (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.