Yes-associated protein is dysregulated during nitrofen-induced hypoplastic lung development due to congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a birth defect associated with abnormal lung development. Yes-associated protein (YAP) is a core kinase of the Hippo pathway, which controls organ size during development. The absence of YAP protein during lung development results in hypoplastic lungs comparable to the lung phenotype in CDH (Mahoney, Dev Cell 30(2):137-150, 2014). We aimed to describe the expression of YAP during normal and nitrofen-induced abnormal lung development. METHODS: Intra-gastric administration of dams with 100 mg of nitrofen was used to induce CDH and abnormal lung development in the embryos. Immunofluorescence was performed to visualize the localization of YAP and p-YAP during lung development (E15, E18, E21). Western Blotting was used to determine the abundance of YAP and p-YAP in E21 control and nitrofen-induced hypoplastic CDH lungs. RESULTS: Immunofluorescence demonstrated cytoplasmic localization of YAP protein in airway epithelial and mesenchymal cells of nitrofen-induced hypoplastic lungs compared to nuclear localization in control lungs. Western Blotting showed a decrease (p = 0.0188) in abundance of YAP (active form) and increase in p-YAP (inactive form) in hypoplastic lungs compared to control lungs. CONCLUSION: Our results demonstrate that YAP protein is mostly phosphorylated, inactive, and expressed in the cytoplasm at the later stages of nitrofen-induced hypoplastic lung development indicating that the alteration in regulation of YAP can be associated with the pathogenesis of abnormal lung development in experimental CDH.

miR-200 family expression during normal and abnormal lung development due to congenital diaphragmatic hernia at the later embryonic stage in the nitrofen rat model.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is a life-threatening disease associated with pulmonary hypoplasia. CDH occurs approximately 1 in every 2000-3000 live births, and the pathophysiology is unknown. MicroRNAs are short, non-coding RNAs that control gene expression through post-transcriptional regulation. Based on our previous work, we hypothesized that the miR-200 family is differentially expressed in normal and abnormal lung development. We aimed to examine the expression of the miR-200 family during normal and hypoplastic lung development due to CDH. METHODS: We performed reverse transcriptase polymerase chain reaction (RT-qPCR) and fluorescent in situ hybridization (FISH) to study the expression levels and distribution of the miR-200 family members on embryonic day 21 (E21) rat control and nitrofen-induced hypoplastic CDH lungs. RESULTS: RT-qPCR showed up-regulation of miR-200a in hypoplastic CDH lungs. FISH showed contrasting expression patterns for miR- 200a, miR-200c, and miR-429 between control and hypoplastic CDH lungs, while we could not detect miR-141 in control and hypoplastic CDH lungs. CONCLUSION: We demonstrate a specific expression pattern of miR-200 family members in hypoplastic CDH lungs different from control lungs. This study suggests that disruption of miR-200 family expression plays a role in the pathogenesis of pulmonary hypoplasia associated with CDH.

Prenatal microRNA miR-200b Therapy Improves Nitrofen-induced Pulmonary Hypoplasia Associated With Congenital Diaphragmatic Hernia.

OBJECTIVE: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). BACKGROUND: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. METHODS: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. RESULTS: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-ß signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. CONCLUSIONS: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH.

MicroRNAs in Lung Development and Disease.

MicroRNAs (miRNAs) are small (∼22 nucleotides), non-coding RNA molecules that regulate gene expression post-transcriptionally by inhibiting target mRNAs. Research into the roles of miRNAs in lung development and disease is at the early stages. In this review, we discuss the role of miRNAs in pediatric respiratory disease, including cystic fibrosis, asthma, and bronchopulmonary dysplasia.

Unique Tracheal Fluid MicroRNA Signature Predicts Response to FETO in Patients With Congenital Diaphragmatic Hernia.

OBJECTIVE AND BACKGROUND: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. METHODS: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-ß2 in postnatal lung sections. We investigated miR-200b effects on TGF-ß signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. RESULTS: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-ß2 expression was lower in CDH lungs. miR-200b inhibited TGF-ß-induced SMAD signaling in cultures of human bronchial epithelial cells. CONCLUSIONS: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-ß/SMAD signaling.

Antenatal Administration of Extracellular Vesicles Derived From Amniotic Fluid Stem Cells Improves Lung Function in Neonatal Rats With Congenital Diaphragmatic Hernia.

BACKGROUND: The severity of pulmonary hypoplasia is a main determinant of outcome for babies with congenital diaphragmatic hernia (CDH). Antenatal administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) has been shown to rescue morphological features of lung development in the rat nitrofen model of CDH. Herein, we evaluated whether AFSC-EV administration to fetal rats with CDH is associated with neonatal improvement in lung function. METHODS: AFSC-EVs were isolated by ultracentrifugation and characterized by size, morphology, and canonical marker expression. At embryonic (E) day 9.5, dams were gavaged with olive oil (control) or nitrofen to induce CDH. At E18.5, fetuses received an intra-amniotic injection of either saline or AFSC-EVs. At E21.5, rats were delivered and subjected to a tracheostomy for mechanical ventilation (flexiVent system). Groups were compared for lung compliance, resistance, Newtonian resistance, tissue damping and elastance. Lungs were evaluated for branching morphogenesis and collagen quantification. RESULTS: Compared to healthy control, saline-treated pups with CDH had fewer airspaces, more collagen deposition, and functionally exhibited reduced compliance and increased airway resistance, elastance, and tissue damping. Conversely, AFSC-EV administration resulted in improvement of lung mechanics (compliance, resistance, tissue damping, elastance) as well as lung branching morphogenesis and collagen deposition. CONCLUSIONS: Our studies show that the rat nitrofen model reproduces lung function impairment similar to that of human babies with CDH. Antenatal administration of AFSC-EVs improves lung morphology and function in neonatal rats with CDH. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Abnormal lung development in congenital diaphragmatic hernia.

The outcomes of patients diagnosed with congenital diaphragmatic hernia (CDH) have recently improved. However, mortality and morbidity remain high, and this is primarily caused by the abnormal lung development resulting in pulmonary hypoplasia and persistent pulmonary hypertension. The pathogenesis of CDH is poorly understood, despite the identification of certain candidate genes disrupting normal diaphragm and lung morphogenesis in animal models of CDH. Defects within the lung mesenchyme and interstitium contribute to disturbed distal lung development. Frequently, a disturbance in the development of the pleuroperitoneal folds (PPFs) leads to the incomplete formation of the diaphragm and subsequent herniation. Most candidate genes identified in animal models have so far revealed relatively few strong associations in human CDH cases. CDH is likely a highly polygenic disease, and future studies will need to reconcile how disturbances in the expression of multiple genes cause the disease. Herein, we summarize the available literature on abnormal lung development associated with CDH.

MicroRNA-200b regulates distal airway development by maintaining epithelial integrity.

miR-200b plays a role in epithelial-to-mesenchymal transition (EMT) in cancer. We recently reported abnormal expression of miR-200b in the context of human pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Smaller lung size, a lower number of airway generations, and a thicker mesenchyme characterize pulmonary hypoplasia in CDH. The aim of this study was to define the role of miR-200b during lung development. Here we show that miR-200b-/- mice have abnormal lung function due to dysfunctional surfactant, increased fibroblast-like cells and thicker mesenchyme in between the alveolar walls. We profiled the lung transcriptome in miR-200b-/- mice, and, using Gene Ontology analysis, we determined that the most affected biological processes include cell cycle, apoptosis and protein transport. Our results demonstrate that miR-200b regulates distal airway development through maintaining an epithelial cell phenotype. The lung abnormalities observed in miR-200b-/- mice recapitulate lung hypoplasia in CDH.

MicroRNAs and lung development.

MicroRNAs (miRNAs) constitute a large group of small (∼22 nucleotides), non-coding RNA sequences that are highly conserved among animals, plants and microorganisms, suggesting that microRNAs represent a highly conserved and important regulatory mechanism. They have been demonstrated to play an important role in gene regulation. Recently, miRNAs have become a major focus of interest for research in organ development. Research focusing on the potential role of microRNAs during lung development is slowly starting to emerge. A number of miRNAs have been demonstrated to play important roles during early and late lung development. Several studies have begun to profile miRNA expression at various stages of lung development and this article provides an overview of the various miRNAs that have been implicated in lung organogenesis.

Cysteine proteinase type III is protective against Leishmania infantum infection in BALB/c mice and highly antigenic in visceral leishmaniasis individuals.

Visceral leishmaniasis is the most acute form of leishmaniasis and vaccination is the best approach to control it. One of the major groups of virulence factors in Leishmania belongs to cysteine proteinase family. In this study, for the first time, the protective potential of Leishmania infantum cysteine proteinase type III (CPC) by using a prime-boost strategy is evaluated in BALB/c mice. The experiment was carried out in three groups of mice. Vaccinated group was primed with pcDNA-cpc and boosted with rCPC-DHFR in combination with CpG motif and Montanide 720 as adjuvant. Control groups received pcDNA and rDHFR or PBS. The ratio of IgG2a/IgG1, nitric oxide concentration and IFN-gamma induction in vaccinated group is significantly higher than controls. Furthermore, the parasite load of vaccinated group is significantly lower than controls. In addition, sera reactivity of visceral leishmaniasis individuals was examined and showed considerable reactivities toward rCPC in comparison with cutaneous leishmaniasis. The achieved result is highly encouraging the use of cysteine proteinases types I, II and III as vaccine candidate against visceral leishmaniasis.