Experimental congenital diaphragmatic hernia features an alteration of DNA sensing targets cGAS and STING.

BACKGROUND: The pathogenesis of congenital diaphragmatic hernia (CDH) depends on multiple factors. Activation of the DNA-sensing cyclic-GMP-AMP-synthase (cGAS) and Stimulator-of-Interferon-Genes (STING) pathway by double-stranded DNA (dsDNA) links environmental stimuli and inflammation. We hypothesized that nitrofen exposure alters cGAS and STING in human bronchial epithelial cells and fetal rat lungs. METHODS: We used the Quant-IT™-PicoGreen™ assay to assess dsDNA concentration in BEAS-2B cells after 24 h of nitrofen-exposure and performed immunofluorescence of cGAS/STING. We used nitrofen to induce CDH and harvested control and CDH lungs at embryonic day E15, E18 and E21 for cGAS/STING immunofluorescence, RT-qPCR and RNA-Scope™ in-situ-hybridization (E18, E21). RESULTS: We found a higher concentration of dsDNA following nitrofen treatment. Nitrofen-exposure to BEAS-2B cells increased cGAS and STING protein abundance. cGAS abundance was higher in nitrofen lungs at E15, E18 and E21. RNA-Scope in-situ-hybridization showed higher cGAS and STING expression in E18 and E21 lungs. RT-qPCR revealed higher mRNA expression levels of STING in E21 nitrofen-induced lungs. CONCLUSION: Our data suggest that nitrofen-exposure increases dsDNA content which leads to stimulation of the cGAS/STING pathway in human BEAS-2B cells and the nitrofen rat model of CDH. Consequently, DNA sensing and the cGAS-STING-pathway potentially contribute to abnormal lung development in CDH. IMPACT STATEMENT: We found an alteration of DNA sensing targets cGAS and STING in human BEAS-2B cells and experimental congenital diaphragmatic hernia with higher protein abundance and mRNA expression in cells and lung sections of nitrofen-treated rat pups. This is the first study to investigate DNA sensing, a potential link between environmental stimuli and inflammation, in experimental CDH. Our study extends the knowledge on the pathogenesis of experimental CDH.

A Tracheal Aspirate-derived Airway Basal Cell Model Reveals a Proinflammatory Epithelial Defect in Congenital Diaphragmatic Hernia.

Rationale: Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. Objectives: To establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. Methods: We developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from patients with CDH and preterm and term non-CDH control subjects were derived and analyzed by bulk RNA sequencing, assay for transposase accessible chromatin with sequencing, and air-liquid interface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main Results: Transcriptomic and epigenetic profiling of CDH and control BSCs reveals a proinflammatory signature that is manifested by hyperactive nuclear factor kappa B and independent of severity and hernia size. In addition, CDH BSCs exhibit defective epithelial differentiation in vitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, blockade of nuclear factor kappa B hyperactivity normalizes epithelial differentiation phenotypes of human CDH BSCs in vitro and in nitrofen rat tracheas in vivo. Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

The RNA-binding protein quaking is upregulated in nitrofen-induced congenital diaphragmatic hernia lungs at the end of gestation.

BACKGROUND: The RNA-binding protein Quaking (QKI) increases during epithelial-to-mesenchymal transition and its expression is controlled by microRNA-200 family members. Here, we aimed to describe the expression of QKI in the developing lungs of control and nitrofen-induced congenital diaphragmatic hernia lungs (CDH). METHODS: To investigate the expression of QKI, we dissected lungs from control and nitrofen-induced CDH rats on embryonic day 15, 18, 21 (E15, E18, E21). We performed immunofluorescence (IF) and quantitative reverse transcription PCR (RT-qPCR) for QKI expression. Additionally, we assessed Interleukin-6 (IL-6) abundance using IF. RESULTS: On E21, IF showed that the abundance of all three QKI isoforms and IL-6 protein was higher in CDH lungs compared to control lungs (QKI5: p = 0.023, QKI6: p = 0.006, QKI7: p = 0.014, IL-6: p = 0.045, respectively). Furthermore, RT-qPCR data showed increased expression of QKI5, QKI6, and QKI7 mRNA in E21 nitrofen lungs by 1.63 fold (p = 0.001), 1.63 fold (p = 0.010), and 1.48 fold (p = 0.018), respectively. CONCLUSIONS: Our data show an increase in the abundance and expression of QKI at the end of gestation in nitrofen-induced CDH lungs. Therefore, a disruption in the regulation of QKI during the late stage of pregnancy could be associated with the pathogenesis of abnormal lung development in CDH.

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

Overactivated Epithelial NF-κB Disrupts Lung Development in Congenital Diaphragmatic Hernia.

Abnormal lung development is the main cause of morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH), a common birth defect (1:2,500) of largely unknown pathobiology. Recent studies discovered that inflammatory processes, and specifically NF-κB-associated pathways, are enriched in human and experimental CDH. However, the molecular signaling of NF-κB in abnormal CDH lung development and its potential as a therapeutic target require further investigation. Using sections and hypoplastic lung explant cultures from the nitrofen rat model of CDH and human fetal CDH lungs, we demonstrate that NF-κB and its downstream transcriptional targets are hyperactive during abnormal lung formation in CDH. NF-κB activity was especially elevated in the airway epithelium of nitrofen and human CDH lungs at different developmental stages. Fetal rat lung explants had impaired pseudoglandular airway branching after exposure to nitrofen, together with increased phosphorylation and transcriptional activity of NF-κB. Dexamethasone, the broad and clinically applicable antiinflammatory NF-κB antagonist, rescued lung branching and normalized NF-κB signaling in hypoplastic lung explants. Moreover, specific NF-κB inhibition with curcumenol similarly rescued ex vivo lung hypoplasia and restored NF-κB signaling. Last, we showed that prenatal intraperitoneal dexamethasone administration to pregnant rat dams carrying fetuses with hypoplastic lungs significantly improves lung branching and normalizes NF-κB in vivo. Our results indicate that NF-κB is aberrantly activated in human and nitrofen CDH lungs. Antiinflammatory treatment with dexamethasone and/or specific NF-κB inhibition should be investigated further as a therapeutic avenue to target lung hypoplasia in CDH.

MicroRNA-200b deficiency is not sufficient to increase susceptibility to allergen-induced airway inflammation and dysfunction in mice.

MicroRNA-200b (miR-200b) has emerged as a therapeutic option for reducing inflammation and airway dysfunction in asthma. miR-200b belongs to a family of miRNAs that regulate epithelial-to-mesenchymal (EMT) transition and IL-33 abundance. In asthma, miR-200b abundance is reduced in the airways and is correlated with disease severity. In addition, prophylactic treatment with a miR-200b mimetic reduces airway inflammation and airway dysfunction in a mouse model. However, it is unclear whether miR-200b deficiency is sufficient to drive airway dysfunction and airway inflammation in asthma. Here, we show that male and female mice deficient in miR-200b do not display heightened airway inflammation or alterations in lung function that are characteristic of asthma. Following sensitization with house dust mite (HDM), female miR-200b knockout (KO) mice have elevated total lung resistance and male miR-200b KO have increased airway resistance. However, neither male nor female miR-200b mice display any changes in methacholine sensitivity or responsiveness and do not have enhanced HDM-induced airway inflammation. Collectively, these findings suggest that loss of miR-200b does not drive airway inflammation and airway dysfunction in mice. Thus, although treatment with exogenous miR-200b may ameliorate inflammation in asthma, deficiency of miR-200b is not likely driving pathobiology in asthma.NEW & NOTEWORTHY MicroRNA-200b regulates the abundance of key asthma-related genes. However, loss of miR-200b does not potentiate allergic asthma in a mouse model, suggesting that miR-200b deficiency may not be sufficient to drive of asthma pathogenesis.

Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To understand the underlying pathobiology, we compared the proteomic profiles of fetal rat lungs at the alveolar stage (E21) that were either exposed to nitrofen in utero (CDH lungs, n=5) or exposed to vehicle only (non-CDH control lungs, n=5). Pathway analysis of proteomic datasets showed significant enrichment in inflammatory response proteins associated with cytokine signaling and Epstein Barr Virus in nitrofen CDH lungs. Among the 218 significantly altered proteins between CDH and non-CDH control lungs were Tenascin C, CREBBP, LYN, and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen rat lungs and human CDH lungs, obtained from stillborn fetuses that did not receive pre- or postnatal treatment. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially rescued the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3-associated cytokines (IL-15, IL-9, andIL-2) are increased in fetal tracheal aspirates of CDH survivors compared with nonsurvivors after fetoscopic endoluminal tracheal occlusion. With our unbiased proteomics approach, we showed for the first time that downstream inflammatory processes are likely involved in the pathogenesis of abnormal lung development in CDH.

Skin Lesions as a Presenting Feature of Dimorphic T-Lymphoblastic Leukemia in an Adolescent Male.

Cutaneous involvement is rare in acute lymphoblastic leukemia/lymphoma, particularly within the T-cell lineage. Review of the literature for cutaneous involvement in T-cell lymphoblastic lymphoma/leukemia identifies mostly case reports, with the majority of cases involving adults. We describe an adolescent male presenting with cervical lymphadenopathy and skin lesions leading to a diagnosis of early T-cell precursor lymphoblastic leukemia. Unique to this case is the age of the patient, presence of a dimorphic blast population, and the skin lesions preceding other signs of disease by at least 1 month.

Post-discharge follow-up of congenital duodenal obstruction patients: a systematic review.

PURPOSE: Long-term follow-up of congenital duodenal obstruction patients often falls on care providers with little experience of this condition. We performed a systematic review of the long-term outcomes of duodenal obstruction and provide a summary of sequelae care providers should anticipate. METHODS: In 2022, after registering with PROSPERA, Medline (Ovid), EMBASE, PSYCHINFO, CNAHL and SCOPUS databases were searched using the title keyword 'intestinal atresia'. Abstracts were filtered for inclusion if they included the duodenum. Papers of filtered abstracts were included if they reported post-discharge outcomes. Methodological Index for Non-Randomized Studies was used to grade the papers. RESULTS: Of the 1068 abstracts were screened, 32 papers were reviewed. Eleven studies were included. Thirty additional papers were included after reviewing references, for a total of 41 papers. The average MINORS was 7/16. CONCLUSION: There is good evidence that children with congenital duodenal obstruction do well in terms of survival, growth and general well-being. Associated cardiac, musculoskeletal and renal anomalies should be ruled-out. Care providers should be aware of anastomotic dysfunction, blind loop syndrome, bowel obstruction and reflux. Reflux may be asymptomatic. Laparoscopic repair does not change long-term outcomes, and associated Trisomy 21 worsens neurodevelopmental outcomes.

Effect of trisomy 21 on long-term gastrointestinal outcomes in duodenal atresia.

PURPOSE: We aimed to determine if Trisomy 21 (T21) affected gastrointestinal outcomes for children with duodenal atresia (DA). METHODS: We identified children born with DA between 1991 and 2017. Cases were divided into DA with T21 and DA without T21. Ten healthy controls per case were included. Esophageal, ulcerative, obstructive and stomach complaints were assessed. Risk ratios (RR), rate ratios (RaR) and Cox models were constructed. Analyses were performed for cases versus controls, and for T21 cases versus non-T21 cases. RESULTS: DA cases totaled 52: 22 had T21 and 30 did not. There were 520 controls. DA cases had more gastrointestinal complaints than controls. T21 cases were at greater risk and frequency of esophageal disease than non-T21 cases (RR = 4.08, p = 0.002, RaR = 69.8, p < 0.001). T21 and non-T21 cases were equally likely to present with obstruction (RR = 0.91, p = 1), but T21 cases complained of obstructive symptoms less (RaR = 0.57, p = 0.003). T21 and non-T21 cases had the same risk of stomach diseases, but T21 cases complained more frequently (RaR = 6.20, p < 0.001). Cox models supported these observations. T21 did not affect ulcerative diseases. CONCLUSION: DA cases had more gastrointestinal problems than controls. T21 increased esophageal and gastric complaints in DA cases but did not affect ulcerative and obstructive complaints.

The impact of intestinal atresia on educational and mental health outcomes in school-aged children: A case-control cohort study.

PURPOSE: To assess real-world educational outcomes, neurodevelopmental disorders and mental health disorders in patients with intestinal atresia (IA) and compare these to outcomes to age-matched controls. METHODS: We performed a retrospective case-control study of children with IA born between 1991 and 2017. We evaluated educational outcomes using an Early Developmental Instrument, Grades 3, 7 and 8 assessments, Grade 9 completion and performance, high school graduation, and neurodevelopmental and mental health disorders using International Classification of Diseases codes available from a population-based dataset. RESULTS: We identified a total of 94 IA cases and 940 date-of-birth-matched controls. Cases with chromosomal anomalies were more likely to fail the Early Developmental Instrument (OR = 2.88, p = 0.037), Grade 8 reading (OR = 2.94, p = 0.043) and Grade 12 (OR = 3.25, p = 0.036); cases without chromosomal anomalies were at no greater risk than their peers of failing to meet expectations and did not have an increased risk of anxiety (OR = 0.48, p = 0.12), depression (OR = 0.74, p = 0.6) or hyperactivity disorder (OR = 1.92, p = 0.087). CONCLUSIONS: Our study demonstrated that IA patients without chromosomal anomalies are not at higher risk of neurodevelopmental and mental health disorders than controls. These results will offer more accurate outcome predictions for parents of children with IA.

Placental Nanoparticle Uptake-On-a-Chip: The Impact of Trophoblast Syncytialization and Shear Stress.

The placenta is a dynamic and complex organ that plays an essential role in the health and development of the fetus. Placental disorders can affect the health of both the mother and the fetus. There is currently an unmet clinical need to develop nanoparticle-based therapies to target and treat placental disorders. However, little is known about the interaction of nanoparticles (NPs) with the human placenta under biomimetic conditions. Specifically, the impact of shear stress exerted on the trophoblasts (placental epithelial cells) by the maternal blood flow, the gradual fusion of the trophoblasts along the gestation period (syncytialization), and the impact of microvilli formation on the cell uptake of NPs is not known. To this end, we designed dynamic placenta-on-a-chip models using BeWo cells to recapitulate the micro-physiological environment, and we induced different degrees of syncytialization via chemical induction with forskolin. We characterized the degree of syncytialization quantitatively by measuring beta human chorionic gonadotropin (ß-hCG) secretion, as well as qualitatively by immunostaining the tight junction protein, ZO-1, and counter nuclear staining. We also characterized microvilli formation under static and dynamic conditions via F-actin staining. We used these models to measure the cell uptake of chondroitin sulfate a binding protein (CSA) conjugated and control liposomes using confocal microscopy, followed by image analysis. Interestingly, exposure of the cells to a dynamic flow of media intrinsically induced syncytialization and microvilli formation compared to static controls. Under dynamic conditions, BeWo cells produced more ß-hCG in conditions that increased the cell exposure time to forskolin (p < 0.005). Our cell uptake results clearly show a combined effect of the exerted shear stress and forskolin treatment on the cell uptake of liposomes as uptake increased in forskolin exposed conditions (p < 0.05). Overall, the difference in the extent of cell uptake of liposomes among the different conditions clearly displays a need for the development of dynamic models of the placenta that consider the changes in the placental cell phenotype along the gestation period, including syncytialization, microvilli formation, and the expression of different transport and uptake receptors. Knowledge generated from this work will inform future research aiming at developing drug delivery systems targeting the placenta.

Antenatal management of congenital diaphragmatic hernia: What's next ?

Congenital diaphragmatic hernia can be diagnosed in the prenatal period and its severity can be measured by fetal imaging. There is now level I evidence that, in selected cases, Fetoscopic Endoluminal Tracheal Occlusion with a balloon increases survival to discharge from the neonatal unit as well as the risk for prematurity. Both effects are dependent on the time point of tracheal occlusion. Fetoscopic Endoluminal Tracheal Occlusion may also lead to iatrogenic death when the balloon cannot be timely retrieved. The implementation of the findings from our clinical studies, may also vary based on local conditions. These may be different in terms of available skill set, access to fetal therapy, as well as outcome based on local neonatal management. We encourage prior benchmarking of local outcomes with optimal postnatal management, based on large enough numbers and using identical criteria as in the recent trials. We propose to work further on prenatal prediction methods, and the improvement of fetal intervention. In this manuscript, we describe a research agenda from a fetal medicine perspective. This research should be in parallel with innovation in neonatal and pediatric (surgical) management of this condition.

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.

Tenascin C is dysregulated in hypoplastic lungs of miR-200b-/- mice.

PURPOSE: We previously demonstrated that absence of miR-200b results in abnormal lung development in congenital diaphragmatic hernia due to imbalance between epithelial and mesenchymal cells. Tenascin C is a highly conserved extracellular matrix protein involved in epithelial to mesenchymal transition, tissue regeneration and lung development. Considering the involvement of Tenascin C and miR-200b and their potential interaction, we aimed to study Tenascin C during lung development in the absence of miR-200b. METHODS: We collected lungs of miR-200b-/- mice (male, 8 weeks). We performed Western blot (WB) analysis (N = 6) and immunofluorescence (N = 5) for Tenascin C and alpha smooth muscle actin and RT-qPCR for Tenascin C gene expression (N = 4). RESULTS: Using WB analysis, we observed a decreased total protein abundance of Tenascin C in miR-200b-/- lungs (miR-200b+/+: 3.8 × 107 ± 1 × 107; miR-200b-/-: 1.9 × 107 ± 5 × 106; p = 0.002). Immunofluorescence confirmed decreased total Tenascin C in miR-200b-/- lungs. Tenascin C was significantly decreased in the mesenchyme but relatively increased in the airways of mutant lungs. Total lung RNA expression of Tenascin C was higher in miR-200b-/- lungs. CONCLUSION: We report dysregulation of Tenascin C in lungs of miR-200b-/- mice. This suggests that absence of miR-200b results in abnormal Tenascin C abundance contributing to the lung hypoplasia observed in miR-200b-/- mice.

Musculoskeletal deformities in children with congenital thoracic malformations: a population-based cohort study.

PURPOSE: It is unclear if musculoskeletal deformities observed in patients with congenital diaphragmatic hernia (CDH), congenital lung lesion (CLL) and esophageal atresia/tracheoesophageal fistula (EA/TEF) are associated with the anomaly or are a result of the surgery required to treat the anomaly. This study compared the prevalence of musculoskeletal deformities for: (1) children with congenital thoracic anomalies to controls; (2) CLL to EA/TEF both repaired via thoracotomy; and (3) CLL and EA/TEF to CDH repaired via laparotomy. METHODS: We performed a retrospective study of children with CLL, CDH or EA/TEF between 1990 and 2016. Date-of-birth-matched control groups were generated from a population-based dataset. International Classification of Disease codes were used to identify scoliosis and pectus anomalies. We determined Hazard ratios (HR) for cases versus controls. RESULTS: We included 167 cases (CDH n = 82; CLL n = 29; EA/TEF n = 56) and 1670 controls. EA/TEF had a greater risk of scoliosis (HR 5.52, 95%CI 1.49,13.73) and pectus deformities (HR 4.07, 95%CI 1.96,8.45). CDH showed more scoliosis (HR 5.03, 95%CI 1.99,12.74) but not pectus anomalies. Musculoskeletal deformities were not more common in CLL. CONCLUSION: Children born with CDH or EA/TEF, but not CLL, had more musculoskeletal deformities than controls. The inconsistent association between musculoskeletal deformities and the surgical approach suggested a congenital predisposition.

The effect of tracheal occlusion in congenital diaphragmatic hernia in the nitrofen rat lung explant model.

PURPOSE: Here, we establish a tracheal occlusion (TO) model with rat lung explants in nitrofen-induced pulmonary hypoplasia in the congenital diaphragmatic hernia (CDH). METHODS: We extracted lungs from rats on an embryonic day 18. We mimicked TO in the lung explants by tying the trachea. We assessed lung weight, morphometry, and abundance of Ki-67, Active caspase-3, and Prosurfactant Protein C (proSP-C) with immunofluorescence. RESULTS: Lung weight was higher in TO + than TO - on day 1. Abundance of Ki-67 was higher in TO + than TO - (0.15 vs. 0.32, p = 0.009 for day 1, 0.07 vs. 0.17, p = 0.004 for day 2, 0.07 vs. 0.12, p = 0.044 for day 3), and Active caspase-3 was higher in TO + than TO - on day 2 and day 3 (0.04 vs. 0.03 p = 0.669 for day 1, 0.03 vs. 0.13 p < 0.001 for day 2, 0.04 vs. 0.17 p = 0.008 for day3). However, proSP-C protein abundance was lower in TO + than TO - (67.9 vs. 59.1 p = 0.033 for day 1, 73.5 vs. 51.6 p = 0.038 for day 2, 83.1 vs. 56.4 p = 0.009 for day 3). CONCLUSIONS: The TO model in lung explants mimics the outcomes of current surgical models of TO and further studies can reveal the cellular and molecular effects of TO in CDH lungs.

The RNA-binding protein Quaking regulates multiciliated and basal cell abundance in the developing lung.

RNA-binding proteins (RBPs) form complexes with RNA, changing how the RNA is processed and thereby regulating gene expression. RBPs are important sources of gene regulation during organogenesis, including the development of lungs. The RBP called Quaking (QK) is critical for embryogenesis, yet it has not been studied in the developing lung. Here, we show that QK is widely expressed during rat lung development and into adulthood. The QK isoforms QK5 and QK7 colocalize to the nuclei of nearly all lung cells. QK6 is present in the nuclei and cytoplasm of mesenchymal cells and is only present in the epithelium during branching morphogenesis. QK knockdown in embryonic lung explants caused a greater number of multiciliated cells to appear in the airways, at the expense of basal cells. The mRNA of multiciliated cell genes and the abundance of FOXJ1/SOX2+ cells increased after knockdown, whereas P63/SOX2+ cells decreased. The cytokine IL-6, a known regulator of multiciliated cell differentiation, had increased mRNA levels after QK knockdown, although protein levels remained unchanged. Further studies are necessary to confirm whether QK acts as a blocker for the IL-6-induced differentiation of basal cells into multiciliated cells, and a conditional QK knockout would likely lead to additional discoveries on QK's role during lung development.

Epithelial cell-adhesion protein cadherin 26 is dysregulated in congenital diaphragmatic hernia and congenital pulmonary airway malformation.

BACKGROUND: Congenital diaphragmatic hernia (CDH) and congenital pulmonary airway malformation (CPAM) are two inborn pathologies of the lung of unknown origin. Alterations of gene expression in airway epithelial cells are involved in the pathobiology of both diseases. We previously found decreased expression of the epithelial cell adhesion protein cadherin 26 (CDH26) in hypoplastic mice lungs. Here, our objective was to describe the expression and localization of CDH26 in hypoplastic CDH lungs and hyperproliferative CPAM tissues. METHODS: After ethical approval, we used human lung tissues from CDH and CPAM cases and age-matched control samples from a previously established biobank. Furthermore, lungs from the nitrofen rat model of CDH were included in the study. We performed immunohistochemistry and western blot analysis with antibodies against CDH26 to examine protein localization and abundance. Statistical analysis was performed using Mann-Whitney U test with significance set at p < 0.05. RESULTS: We observed an overexpression of CDH26 within the epithelium of cystic CPAM lesions compared to normal airways within the same lung and compared to control lungs. Western blot demonstrated a downregulation of CDH26 in the nitrofen rat model of CDH compared to healthy controls. Immunohistochemistry could not show consistent differences between CDH and control in human and rat lungs. In the studied human lung samples, CDH26 was localized to the apical part of the airway epithelial cells. CONCLUSION: CDH26 is differentially expressed in human CPAM lung tissues and may be downregulated in nitrofen-induced hypoplastic rat lungs compared to control lungs. Disruption of CDH26 associated pathways in lung development may be involved in the pathogenesis of lung hypoplasia or cystic lung disease.

Misoprostol attenuates neonatal cardiomyocyte proliferation through Bnip3, perinuclear calcium signaling, and inhibition of glycolysis.

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.