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Background: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied., Methods: Epitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting., Results: An overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases., Conclusion: Missense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV., (© 2024. The Author(s).)

Background: Gastroesophageal reflux disease (GERD) is a common comorbidity associated with congenital diaphragmatic hernia (CDH), with reported cases of Barrett's esophagus (BE) and esophageal adenocarcinoma before the age of 25. The prevalence and natural course of GERD in CDH survivors remain uncertain due to variations in diagnostic methods. We aimed to analyse the GERD prevalence from infancy through young adulthood., Methods: We retrospectively analyzed pH-impedance measurements and endoscopic findings in 96 CDH survivors evaluated as routine care using well established clinical protocols. GERD was defined as an abnormal acid exposure time for pH-MII measurements and as presence of reflux esophagitis or BE at upper endoscopy. Clinical data including symptoms at time of follow-up and use of antireflux medication were collected., Results: GERD prevalence remained consistently low (≤10%) across all age groups, yet many patients experienced GER symptoms. Histological abnormalities were observed in 80% of adolescents and young adults, including microscopic esophagitis in 50%. BE was diagnosed in 7% before the age of 18, all had GER symptoms. CDH severity, anatomy at the time of CDH correction, alcohol usage, and smoking did not emerge as significant risk factors for GERD., Conclusions: Given the low GERD prevalence in CDH survivors, a symptom-driven approach to diagnosis and follow-up is warranted. We advise long-term follow-up for all adult patients due to the early onset of BE and the limited evidence available. The longitudinal course and impact of GERD on other long-term CDH-related comorbidities should be explored in larger cohorts., Level of Evidence: Not applicable., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently there is a global lack of consensus about the best treatment for asymptomatic congenital pulmonary airway malformation (CPAM) patients. The somatic KRAS mutations commonly found in adult lung cancer combined with mucinous proliferations are sometimes found in CPAM. For this risk of developing malignancy, 70% of paediatric surgeons perform a resection for asymptomatic CPAM. In order to stratify these patients into high- and low-risk groups for developing malignancy, a minimally invasive diagnostic method is needed, for example targeted molecular imaging. A prerequisite for this technique is a cell membrane bound target. The aim of this study was to review the literature to identify potential targets for molecular imaging in CPAM patients and perform a first step to validate these findings.A systematic search was conducted to identify possible targets in CPAM and adenocarcinoma in situ (AIS) patients. The most interesting targets were evaluated with immunofluorescent staining in adjacent lung tissue, KRAS + CPAM tissue and KRAS - CPAM tissue.In 185 included studies, 143 possible targets were described, of which 20 targets were upregulated and membrane-bound. Six of them were also upregulated in lung AIS tissue (CEACAM5, E-cadherin, EGFR, ERBB2, ITGA2 and MUC1) and as such of possible interest. Validating studies showed that MUC1 is a potential interesting target.This study provides an extensive overview of all known potential targets in CPAM that might identify those patients at risk for malignancy and conducted the first step towards validation, identifying MUC1 as the most promising target., Competing Interests: Conflict of interest: C. van Horik reports grants from Sophia Foundation for Scientific Research, outside the submitted work. All other authors have nothing to disclose., (Copyright ©The authors 2023.)

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regimens and suffer unnecessarily because ACDMPV is not always timely recognized and histologic diagnosis is invasive and time consuming. Here, we demonstrate the usefulness of a noninvasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pathophysiological studies have shown that pulmonary vascular development is impaired in fetuses with a congenital diaphragmatic hernia (CDH), leading to a simplified vascular tree and increased vascular resistance. Multiple studies have described prenatal ultrasound parameters for the assessment of the pulmonary vasculature, but none of these parameters are used in daily clinical practice. We provide a comprehensive review of the literature published between January 1990 and February 2022 describing these parameters, and aim to explain the clinical relevance of these parameters from what is known from pathophysiological studies. Prenatal detection of a smaller diameter of the contralateral (i.e. contralateral to the diaphragmatic defect) first branch of the pulmonary artery (PA), higher pulsatility indices (PI), higher peak early diastolic reverse flow values, and a lower vascularization index seem of added value for the prediction of survival and, to a lesser extent, morbidity. Integration within the routine evaluation is complicated by the lack of uniformity of the methods used. To address the main components of the pathophysiological changes, we recommend future prenatal studies in CDH with a focus on PI values, PA diameters and pulmonary vascular branching., (© 2023 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Severe infections with coronaviruses are often accompanied with hyperinflammation, requiring therapeutic strategies to simultaneously tackle the virus and inflammation. By screening a safe-in-human broad-spectrum antiviral agents library, we identified that indomethacin can inhibit pan-coronavirus infection in human cell and airway organoids models. Combining indomethacin with oral antiviral drugs authorized for treating COVID-19 results in synergistic anti-coronavirus activity. Coincidentally, screening a library of FDA-approved drugs identified indomethacin as the most potent potentiator of interferon response through increasing STAT1 phosphorylation. Combining indomethacin with interferon-alpha exerted synergistic antiviral effects against multiple coronaviruses. The anti-coronavirus activity of indomethacin is associated with activating interferon response. In a co-culture system of lung epithelial cells with macrophages, indomethacin inhibited both viral replication and inflammatory response. Collectively, indomethacin is a pan-coronavirus inhibitor that can simultaneously inhibit virus-triggered inflammatory response. The therapeutic potential of indomethacin can be further augmented by combining it with oral antiviral drugs or interferon-alpha., Competing Interests: The authors disclose no conflicts., (© 2023 The Author(s).)

Objectives: To longitudinally evaluate self-reported health status (HS) and quality of life (QoL) in 8- and 12-year-old survivors of congenital diaphragmatic hernia (CDH). We hypothesized that HS would improve with age-as associated health problems tend to decline-whereas QoL would decrease, as the children start to compare themselves with peers., Methods: Self-reported HS and QoL of 133 children born between 1999 and 2013 who had joined our standardized follow-up program were routinely assessed at the ages of 8 and 12 with generic, internationally validated, standardized instruments. Longitudinal evaluation of total and subscale scores was performed using general linear model analyses. In addition, we compared these scores to sex- and age-specific normative data., Results: Between ages 8 and 12, boys born with CDH perceived a decline in HS (mean difference -7.15, P < .001). Self-reported QoL did not change over time in both boys and girls. At both ages, HS was significantly lower than that of healthy peers (effect size = 0.71, P = .001 for boys, and effect size = 0.69, P = .003 for girls), whereas differences in QoL were small., Conclusions: Children born with CDH are at risk for declining HS between 8 and 12 years, but not QoL, compared with healthy peers. Given that children born with CDH tend to grow into deficits, our findings highlight the need for continued somatic and psychological assessments in adolescent and adult CDH survivors., (Copyright © 2023 by the American Academy of Pediatrics.)

Background: Immunocompromised populations, such as organ transplant recipients and patients with inflammatory bowel disease (IBD) receiving immunosuppressive/immunomodulatory medications, may be more susceptible to coronavirus infections. However, little is known about how immunosuppressants affect coronavirus replication and their combinational effects with antiviral drugs., Objective: This study aims to profile the effects of immunosuppressants and the combination of immunosuppressants with oral antiviral drugs molnupiravir and nirmatrelvir on pan-coronavirus infection in cell and human airway organoids (hAOs) culture models., Methods: Different coronaviruses (including wild type, delta and omicron variants of SARS-CoV-2, and NL63, 229E and OC43 seasonal coronaviruses) were used in lung cell lines and hAOs models. The effects of immunosuppressants were tested., Results: Dexamethasone and 5-aminosalicylic acid moderately stimulated the replication of different coronaviruses. Mycophenolic acid (MPA), 6-thioguanine (6-TG), tofacitinib and filgotinib treatment dose-dependently inhibited viral replication of all tested coronaviruses in both cell lines and hAOs. The half maximum effective concentration (EC50) of tofacitinib against SARS-CoV-2 was 0.62 μM and the half maximum cytotoxic concentration (CC50) was above 30 μM, which resulted in a selective index (SI) of about 50. The anti-coronavirus effect of the JAK inhibitors tofacitinib and filgotinib is dependent on the inhibition of STAT3 phosphorylation. Combinations of MPA, 6-TG, tofacitinib, and filgotinib with the oral antiviral drugs molnupiravir or nirmatrelvir exerted an additive or synergistic antiviral activity., Conclusions: Different immunosuppressants have distinct effects on coronavirus replication, with 6-TG, MPA, tofacitinib and filgotinib possessing pan-coronavirus antiviral activity. The combinations of MPA, 6-TG, tofacitinib and filgotinib with antiviral drugs exerted an additive or synergistic antiviral activity. Thus, these findings provide an important reference for optimal management of immunocompromised patients infected with coronaviruses., (© 2023 The Authors. United European Gastroenterology Journal published by Wiley Periodicals LLC on behalf of United European Gastroenterology.)

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Roman Truckenmueller reports a relationship with 300MICRONS GmbH that includes: board membership and equity or stocks. Stefan Giselbrecht reports a relationship with 300MICRONS GmbH that includes: board membership and equity or stocks., (© 2023 The Authors.)

Pulmonary neuroendocrine (NE) cells represent a small population in the airway epithelium, but despite this, hyperplasia of NE cells is associated with several lung diseases, such as congenital diaphragmatic hernia and bronchopulmonary dysplasia. The molecular mechanisms causing the development of NE cell hyperplasia remains poorly understood. Previously, we showed that the SOX21 modulates the SOX2-initiated differentiation of epithelial cells in the airways. Here, we show that precursor NE cells start to develop in the SOX2 + SOX21 + airway region and that SOX21 suppresses the differentiation of airway progenitors to precursor NE cells. During development, clusters of NE cells start to form and NE cells mature by expressing neuropeptide proteins, such as CGRP. Deficiency in SOX2 resulted in decreased clustering, while deficiency in SOX21 increased both the numbers of NE ASCL1 + precursor cells early in development, and the number of mature cell clusters at E18.5. In addition, at the end of gestation (E18.5), a number of NE cells in Sox2 heterozygous mice, did not yet express CGRP suggesting a delay in maturation. In conclusion, SOX2 and SOX21 function in the initiation, migration and maturation of NE cells., (© 2023. The Author(s).)

The lung originates from the ventral foregut and develops into an intricate branched structure of airways, alveoli, vessels and support tissue. As the lung develops, cells become specified and differentiate into the various cell lineages. This process is controlled by specific transcription factors, such as the SRY-related HMG-box genes SOX2 and SOX21 , that are activated or repressed through intrinsic and extrinsic signals. Disturbances in any of these processes during the development of the lung may lead to various pediatric lung disorders, such as Congenital Diaphragmatic Hernia (CDH), Congenital Pulmonary Airway Malformation (CPAM) and Broncho-Pulmonary Dysplasia (BPD). Changes in the composition of the airways and the alveoli may result in reduced respiratory function and eventually lead to chronic lung disorders. In this concise review, we describe different intrinsic and extrinsic cellular processes required for proper differentiation of the epithelium during development and regeneration, and the influence of the microenvironment on this process with special focus on SOX2 and SOX21.

The lung is composed of a highly branched airway structure, which humidifies and warms the inhaled air before entering the alveolar compartment. In the alveoli, a thin layer of epithelium is in close proximity with the capillary endothelium, allowing for an efficient exchange of oxygen and carbon dioxide. During development proliferation and differentiation of progenitor cells generates the lung architecture, and in the adult lung a proper function of progenitor cells is needed to regenerate after injury. Malfunctioning of progenitors during development results in various congenital lung disorders, such as Congenital Diaphragmatic Hernia (CDH) and Congenital Pulmonary Adenomatoid Malformation (CPAM). In addition, many premature neonates experience continuous insults on the lung caused by artificial ventilation and supplemental oxygen, which requires a highly controlled mechanism of airway repair. Malfunctioning of airway progenitors during regeneration can result in reduction of respiratory function or (chronic) airway diseases. Pathways that are active during development are frequently re-activated upon damage. Understanding the basic mechanisms of lung development and the behavior of progenitor cell in the ontogeny and regeneration of the lung may help to better understand the underlying cause of lung diseases, especially those occurring in prenatal development or in the immediate postnatal period of life. This review provides an overview of lung development and the cell types involved in repair of lung damage with a focus on the airway., Competing Interests: The authors declare that the review was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Eenjes, Tibboel, Wijnen and Rottier.)

Mycoplasma pneumoniae and Streptococcus pneumoniae are the most common bacterial causes of pneumonia in children. The clinical characteristics of pneumonia differ significantly between the two bacteria. We aimed to elucidate the differences in pathogenesis between M. pneumoniae and S. pneumoniae by characterizing the respiratory epithelial cell immune response to both pathogens. Using primary human bronchial epithelial cells in air-liquid interface cultures, we observed lower production of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 in response to M. pneumoniae than to S. pneumoniae. In contrast to the differences in proinflammatory cytokine production, Toll-like receptor 2 (TLR2)-mediated signaling in response to M. pneumoniae was stronger than to S. pneumoniae. This difference largely depended on TLR1 and not TLR6. We found that M. pneumoniae, but not S. pneumoniae, also induced signaling of TLR10, a coreceptor of TLR2 that has inhibitory properties. M. pneumoniae-induced TLR10 signaling on airway epithelial cells was partially responsible for low IL-8 production, as blocking TLR10 by specific antibodies increased cytokine production. M. pneumoniae maintained Th2-associated cytokine production by epithelial cells, which concurs with the known association of M. pneumoniae infection with asthma. M. pneumoniae left IL-33 levels unchanged, whereas S. pneumoniae downregulated IL-33 production both under homeostatic and Th2-promoting conditions. By directly comparing M. pneumoniae and S. pneumoniae, we demonstrate that M. pneumoniae avoids induction of proinflammatory cytokine response despite its ability to induce robust TLR2 signaling. Our new findings suggest that this apparent paradox may be partially explained by M. pneumoniae-induced signaling of TLR2/TLR10.

Background: Human seasonal coronaviruses usually cause mild upper-respiratory tract infection, but severe complications can occur in specific populations. Research into seasonal coronaviruses is limited and robust experimental models are largely lacking. This study aims to establish human airway organoids (hAOs)-based systems for seasonal coronavirus infection and to demonstrate their applications in studying virus-host interactions and therapeutic development., Methods: The infections of seasonal coronaviruses 229E, OC43 and NL63 in 3D cultured hAOs with undifferentiated or differentiated phenotypes were tested. The kinetics of virus replication and production was profiled at 33 °C and 37 °C. Genome-wide transcriptome analysis by RNA sequencing was performed in hAOs under various conditions. The antiviral activity of molnupiravir and remdesivir, two approved medications for treating COVID19, was tested., Findings: HAOs efficiently support the replication and infectious virus production of seasonal coronaviruses 229E, OC43 and NL63. Interestingly, seasonal coronaviruses replicate much more efficiently at 33 °C compared to 37 °C, resulting in over 10-fold higher levels of viral replication. Genome-wide transcriptomic analyses revealed distinct patterns of infection-triggered host responses at 33 °C compared to 37 °C temperature. Treatment of molnupiravir and remdesivir dose-dependently inhibited the replication of 229E, OC43 and NL63 in hAOs., Interpretation: HAOs are capable of modeling 229E, OC43 and NL63 infections. The intriguing finding that lower temperature resembling that in the upper respiratory tract favors viral replication may help to better understand the pathogenesis and transmissibility of seasonal coronaviruses. HAOs-based innovative models shall facilitate the research and therapeutic development against seasonal coronavirus infections., Funding: This research is supported by funding of a VIDI grant (No. 91719300) from the Netherlands Organization for Scientific Research and the Dutch Cancer Society Young Investigator Grant (10140) to Q.P., and the ZonMw COVID project (114025011) from the Netherlands Organization for Health Research and Development to R.R., Competing Interests: Declaration of interests No conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 ( TBX4 ), TBX5 , and SRY-box TF 9 ( SOX9 ), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4 , TBX5 , SOX9 , or E1A-associated protein P300 ( EP300 ) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.

Congenital diaphragmatic hernia is a structural birth defect of the diaphragm, with lung hypoplasia and persistent pulmonary hypertension. Aside from vascular defects, the lungs show a disturbed balance of differentiated airway epithelial cells. The Sry related HMG box protein SOX2 is an important transcription factor for proper differentiation of the lung epithelium. The transcriptional activity of SOX2 depends on interaction with other proteins and the identification of SOX2-associating factors may reveal important complexes involved in the disturbed differentiation in CDH. To identify SOX2-associating proteins, we purified SOX2 complexes from embryonic mouse lungs at 18.5 days of gestation. Mass spectrometry analysis of SOX2-associated proteins identified several potential candidates, among which were the Chromodomain Helicase DNA binding protein 4 (CHD4), Cut-Like Homeobox1 (CUX1), and the Forkhead box proteins FOXP2 and FOXP4. We analyzed the expression patterns of FOXP2, FOXP4, CHD4, and CUX1 in lung during development and showed co-localization with SOX2. Co-immunoprecipitations validated the interactions of these four transcription factors with SOX2, and large-scale chromatin immunoprecipitation (ChIP) data indicated that SOX2 and CHD4 bound to unique sites in the genome, but also co-occupied identical regions, suggesting that these complexes could be involved in co-regulation of genes involved in the respiratory system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schilders, Edel, Eenjes, Oresta, Birkhoff, Boerema-de Munck, Buscop-van Kempen, Liakopoulos, Kolovos, Demmers, Poot, Wijnen, Tibboel and Rottier.)

Congenital diaphragmatic hernia (CDH) is a congenital structural anomaly in which the diaphragm has not developed properly. It may occur either as an isolated anomaly or with additional anomalies. It is thought to be a multifactorial disease in which genetic factors could either substantially contribute to or directly result in the developmental defect. Patients with aneuploidies, pathogenic variants or de novo Copy Number Variations (CNVs) impacting specific genes and loci develop CDH typically in the form of a monogenetic syndrome. These patients often have other associated anatomical malformations. In patients without a known monogenetic syndrome, an increased genetic burden of de novo coding variants contributes to disease development. In early years, genetic evaluation was based on karyotyping and SNP-array. Today, genomes are commonly analyzed with next generation sequencing (NGS) based approaches. While more potential pathogenic variants are being detected, analysis of the data presents a bottleneck-largely due to the lack of full appreciation of the functional consequence and/or relevance of the detected variant. The exact heritability of CDH is still unknown. Damaging de novo alterations are associated with the more severe and complex phenotypes and worse clinical outcome. Phenotypic, genetic-and likely mechanistic-variability hampers individual patient diagnosis, short and long-term morbidity prediction and subsequent care strategies. Detailed phenotyping, clinical follow-up at regular intervals and detailed registries are needed to find associations between long-term morbidity, genetic alterations, and clinical parameters. Since CDH is a relatively rare disorder with only a few recurrent changes large cohorts of patients are needed to identify genetic associations. Retrospective whole genome sequencing of historical patient cohorts using will yield valuable data from which today's patients and parents will profit Trio whole genome sequencing has an excellent potential for future re-analysis and data-sharing increasing the chance to provide a genetic diagnosis and predict clinical prognosis. In this review, we explore the pitfalls and challenges in the analysis and interpretation of genetic information, present what is currently known and what still needs further study, and propose strategies to reap the benefits of genetic screening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brosens, Peters, van Weelden, Bendixen, Brouwer, Sleutels, Bruggenwirth, van Ijcken, Veenma, Otter, Wijnen, Eggink, van Dooren, Reutter, Rottier, Schnater, Tibboel and de Klein.)

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR dim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GR D/D ) have previously helped to define the functions of GR monomers and dimers. Since GR D/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GR D+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GR L/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and K d values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Congenital diaphragmatic hernia (CDH) is a structural birth defect characterized by a diaphragmatic defect, lung hypoplasia and structural vascular defects. In spite of recent developments, the pathogenesis of CDH is still poorly understood. CDH is a complex congenital disorder with multifactorial etiology consisting of genetic, cellular and mechanical factors. This review explores the cellular origin of CDH pathogenesis in the diaphragm and lungs and describes recent developments in basic and translational CDH research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Edel, Schaaf, Wijnen, Tibboel, Kardon and Rottier.)

Tracheoesophageal Fistula (TOF) is a congenital anomaly for which the cause is unknown in the majority of patients. OA/TOF is a variable feature in many (often mono-) genetic syndromes. Research using animal models targeting genes involved in candidate pathways often result in tracheoesophageal phenotypes. However, there is limited overlap in the genes implicated by animal models and those found in OA/TOF-related syndromic anomalies. Knowledge on affected pathways in animal models is accumulating, but our understanding on these pathways in patients lags behind. If an affected pathway is associated with both animals and patients, the mechanisms linking the genetic mutation, affected cell types or cellular defect, and the phenotype are often not well understood. The locus heterogeneity and the uncertainty of the exact heritability of OA/TOF results in a relative low diagnostic yield. OA/TOF is a sporadic finding with a low familial recurrence rate. As parents are usually unaffected, de novo dominant mutations seems to be a plausible explanation. The survival rates of patients born with OA/TOF have increased substantially and these patients start families; thus, the detection and a proper interpretation of these dominant inherited pathogenic variants are of great importance for these patients and for our understanding of OA/TOF aetiology.

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1 ) cellular composition, 2 ) response to cigarette smoke exposure, and 3 ) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium, SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium., Competing Interests: EE, MB, AB, GE, FB, Ld, MS, DT, JC, RR No competing interests declared, (© 2021, Eenjes et al.)

Background: Pulmonary hypertension is the major cause of morbidity and mortality in congenital diaphragmatic hernia (CDH). Mutations in several genes that encode signaling molecules of the transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) pathways have previously been associated with CDH. Since studies on the activation of these pathways in CDH are scarce, and have yielded inconsistent conclusions, the downstream activity of both pathways was assessed in the nitrofen-CDH rat model. Methods and Results: Pregnant Sprague-Dawley rats were treated with nitrofen at embryonic day (E) 9.5 to induce CDH in offspring. At E21, lungs were screened for the expression of key factors of both signaling pathways, at both the mRNA transcript and protein levels. Subsequently, paying particular attention to the pulmonary vasculature, increased phosphorylation of SMAD2, and decreased phosphorylation of Smad5 was noted in the muscular walls of small pulmonary vessels, by immunohistochemistry. This was accompanied by increased proliferation of constituent cells of the smooth muscle layer of these vessels. Conclusions: Increased activation of the TGFβ pathway and decreased activation of the BMP pathway in the pulmonary vasculature of rats with experimentally-induced CDH, suggesting that the deregulated of these important signaling pathways may underlie the development of pulmonary hypertension in CDH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mous, Buscop-van Kempen, Wijnen, Tibboel, Morty and Rottier.)

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial-endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air-liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial-endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial-endothelial and alveolar-endothelial models with primary lung epithelial cells.

Background: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with heterozygous variants in the FOXF1 gene or its regulatory region. Patients with ACD/MPV unnecessarily undergo invasive and expensive treatments while awaiting a diagnosis. The aim of this study was to reduce the time to diagnose ACD/MPV by developing a targeted next-generation sequencing (NGS) panel that detects FOXF1 variants., Methods: A FOXF1-targeted NGS panel was developed for detection of mutations and large genomic alterations and used for retrospective testing of ACD/MPV patients and controls. Results were confirmed with Sanger sequencing and SNP array analysis., Results: Each amplicon of the FOXF1-targeted NGS panel was efficiently sequenced using DNA isolated from blood or cell lines of 15 ACD/MPV patients and 8 controls. Moreover, testing of ACD/MPV patients revealed six novel and six previously described pathogenic or likely pathogenic FOXF1 alterations., Conclusion: We successfully designed a fast and reliable targeted genetic test to detect variants in the FOXF1 gene and its regulatory region in one run. This relatively noninvasive test potentially prevents unnecessary suffering for patients and reduces the use of futile and expensive treatments like extra-corporeal membrane oxygenation., Impact: FOXF1-targeted NGS potentially prevents ACD/MPV patients from unnecessary suffering and expensive treatments. FOXF1-targeted NGS potentially reduces the number of misdiagnosis in ACD/MPV patients. Retrospective testing of ACD/MPV patients using FOXF1-targeted NGS revealed six novel pathogenic or likely pathogenic variants.

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy., Competing Interests: AM, TB, SR, DS, Pv, RR, ML, BH No competing interests declared, (© 2021, Mykytyn et al.)

There is increasing evidence that surface curvature at a near-cell-scale influences cell behaviour. Epithelial or endothelial cells lining small acinar or tubular body lumens, as those of the alveoli or blood vessels, experience such highly curved surfaces. In contrast, the most commonly used culture substrates for in vitro modelling of these human tissue barriers, ion track-etched membranes, offer only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically curved track-etched membranes, preserving the mainly spherical geometry of the cells' native microenvironment. The curved membranes were created by a combination of three-dimensional (3D) micro film (thermo)forming and ion track technology. We could successfully demonstrate the formation, the growth and a first characterization of confluent layers of lung epithelial cell lines and primary alveolar epithelial cells on membranes shaped into an array of hemispherical microwells. Besides their application in submerged culture, we could also demonstrate the compatibility of the bioinspired membranes for air-exposed culture. We observed a distinct cellular response to membrane curvature. Cells (or cell layers) on the curved membranes reveal significant differences compared to cells on flat membranes concerning membrane epithelialization, areal cell density of the formed epithelial layers, their cross-sectional morphology, and proliferation and apoptosis rates, and the same tight barrier function as on the flat membranes. The presented 3D membrane technology might pave the way for more predictive barrier in vitro models in future., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Esophageal atresia (EA) and tracheoesophageal fistula (TEF) are relatively frequently occurring foregut malformations. EA/TEF is thought to have a strong genetic component. Not much is known regarding the biological processes disturbed or which cell type is affected in patients. This hampers the detection of the responsible culprits (genetic or environmental) for the origin of these congenital anatomical malformations. Therefore, we examined gene expression patterns in the TEF and compared them to the patterns in esophageal, tracheal and lung control samples. We studied tissue organization and key proteins using immunohistochemistry. There were clear differences between TEF and control samples. Based on the number of differentially expressed genes as well as histological characteristics, TEFs were most similar to normal esophagus. The BMP-signaling pathway, actin cytoskeleton and extracellular matrix pathways are downregulated in TEF. Genes involved in smooth muscle contraction are overexpressed in TEF compared to esophagus as well as trachea. These enriched pathways indicate myofibroblast activated fibrosis. TEF represents a specific tissue type with large contributions of intestinal smooth muscle cells and neurons. All major cell types present in esophagus are present-albeit often structurally disorganized-in TEF, indicating that its etiology should not be sought in cell fate specification., Competing Interests: None of the authors have financial, professional, or personal conflicts of interest, all authors reviewed and approved the final manuscript.

Polymeric membranes are widely applied in biomedical applications, including in vitro organ models. In such models, they are mostly used as supports on which cells are cultured to create functional tissue units of the desired organ. To this end, the membrane properties, e.g., morphology and porosity, should match the tissue properties. Organ models of dynamic (barrier) tissues, e.g., lung, require flexible, elastic and porous membranes. Thus, membranes based on poly (dimethyl siloxane) (PDMS) are often applied, which are flexible and elastic. However, PDMS has low cell adhesive properties and displays small molecule ad- and absorption. Furthermore, the introduction of porosity in these membranes requires elaborate methods. In this work, we aim to develop porous membranes for organ models based on poly(trimethylene carbonate) (PTMC): a flexible polymer with good cell adhesive properties which has been used for tissue engineering scaffolds, but not in in vitro organ models. For developing these membranes, we applied evaporation-induced phase separation (EIPS), a new method in this field based on solvent evaporation initiating phase separation, followed by membrane photo-crosslinking. We optimised various processing variables for obtaining form-stable PTMC membranes with average pore sizes between 5 to 8 µm and water permeance in the microfiltration range (17,000-41,000 L/m 2 /h/bar). Importantly, the membranes are flexible and are suitable for implementation in in vitro organ models.

The prenatal and perinatal environments can have profound effects on the development of chronic inflammatory diseases. However, mechanistic insight into how the early-life microenvironment can impact upon development of the lung and immune system and consequent initiation and progression of respiratory diseases is still emerging. Recent studies investigating the developmental origins of lung diseases have started to delineate the effects of early-life changes in the lung, environmental exposures and immune maturation on the development of childhood and adult lung diseases. While the influencing factors have been described and studied in mostly animal models, it remains challenging to pinpoint exactly which factors and at which time point are detrimental in lung development leading to respiratory disease later in life. To advance our understanding of early origins of chronic lung disease and to allow for proper dissemination and application of this knowledge, we propose four major focus areas: 1) policy and education; 2) clinical assessment; 3) basic and translational research; and 4) infrastructure and tools, and discuss future directions for advancement. This review is a follow-up of the discussions at the European Respiratory Society Research Seminar "Early origins of lung disease: towards an interdisciplinary approach" (Lisbon, Portugal, November 2019)., Competing Interests: Conflict of interest: N.D.J. Ubags has nothing to disclose. Conflict of interest: M.A. Alejandre Alcazar reports grants from Deutsche Forschungsgemeinschaft, Marga and Walter Boll Stiftung and Stiftung Oskar-Helene-Heim, during the conduct of the study. Conflict of interest: S.G. Kallapur has nothing to disclose. Conflict of interest: S. Knapp reports grants from Austrian Science Fund, during the conduct of the study. Conflict of interest: S. Lanone has nothing to disclose. Conflict of interest: C.M. Lloyd has nothing to disclose. Conflict of interest: R.E. Morty has nothing to disclose. Conflict of interest: C. Pattaroni has nothing to disclose. Conflict of interest: N.L. Reynaert reports grants from Lung Foundation Netherlands, during the conduct of the study. Conflict of interest: R.J. Rottier has nothing to disclose. Conflict of interest: H.H. Smits has nothing to disclose. Conflict of interest: W.A.A. de Steenhuijsen Piters has nothing to disclose. Conflict of interest: D.H. Strickland has nothing to disclose. Conflict of interest: J.J.P. Collins reports grants from Lung Foundation Netherlands, during the conduct of the study., (Copyright ©ERS 2020.)

The potential for malignant degeneration is the most common reason for some practitioners to resect asymptomatic congenital pulmonary airway malformations (CPAMs). We aimed to investigate the potential of various immunohistochemical (IHC) and genomic biomarkers to predict the presence of mucinous proliferations (MPs) in CPAM. Archival CPAM tissue samples were re-assessed and underwent IHC analysis using a panel of differentiating markers (TTF1/CDX2/CC10/MUC2/MUC5AC/p16/p53/DICER1). In each sample, intensity of IHC staining was assessed separately in normal lung tissue, CPAM, and MP tissue, using a semiquantitative approach. Likewise, next-generation targeted sequencing of known adult lung driver mutations, including KRAS/BRAF/EGFR/ERBB2, was performed in all samples with MP and in control samples of CPAM tissue without MP. We analyzed samples of 25 CPAM type 1 and 25 CPAM type 2 and found MPs in 11 samples. They were all characterized by strong MUC5AC expression, and all carried a KRAS mutation in the MP and adjacent nonmucinous CPAM tissue, whereas the surrounding normal lung tissue was negative. By contrast, in less than half (5 out of 12) control samples lacking MP, the CPAM tissue also carried a KRAS mutation. KRAS mutations in nonmucinous CPAM tissue may identify lesions with a potential for malignant degeneration and may guide histopathological assessment and patient follow-up., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Diagnosing Alveolar Capillary Dysplasia with Misalignment of the Pulmonary Veins (ACD/MPV) based on a genetic alteration in the FOXF1 gene, is complicated by the poor understanding of the causal relation between FOXF1 variants and the ACD/MPV phenotype. Here, we report the generation of human iPSC lines from two ACD/MPV patients, each carrying a different heterozygous FOXF1 mutation, which enables disease modeling for further research on the effect of FOXF1 variants in vitro. The iPSC lines were generated from skin fibroblasts using the non-integrating Sendai virus. The lines expressed pluripotency genes, retained the heterozygous mutation and were capable of trilineage differentiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM + cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/β-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.

Airway epithelial cells and macrophages participate in inflammatory responses to external noxious stimuli, which can cause epithelial injury. Upon injury, epithelial cells and macrophages act in concert to ensure rapid restoration of epithelial integrity. The nature of the interactions between these cell types during epithelial repair is incompletely understood. We used an in vitro human coculture model of primary bronchial epithelial cells cultured at the air-liquid interface (ALI-PBEC) and polarized primary monocyte-derived macrophages. Using this coculture, we studied the contribution of macrophages to epithelial innate immunity, wound healing capacity, and epithelial exposure to whole cigarette smoke (WCS). Coculture of ALI-PBEC with lipopolysaccharide (LPS)-activated M(GM-CSF) macrophages increased the expression of DEFB4A, CXCL8, and IL6 at 24 h in the ALI-PBEC, whereas LPS-activated M(M-CSF) macrophages only increased epithelial IL6 expression. Furthermore, wound repair was accelerated by coculture with both activated M(GM-CSF) and M(M-CSF) macrophages, also following WCS exposure. Coculture of ALI-PBEC and M(GM-CSF) macrophages resulted in increased CAMP expression in M(GM-CSF) macrophages, which was absent in M(M-CSF) macrophages. CAMP encodes LL-37, an antimicrobial peptide with immune-modulating and repair-enhancing activities. In conclusion, dynamic crosstalk between ALI-PBEC and macrophages enhances epithelial innate immunity and wound repair, even upon concomitant cigarette smoke exposure., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

The mortality and morbidity of patients with congenital diaphragmatic hernia (CDH) is primarily caused by treatment-resistant, persistent pulmonary hypertension. Structural vascular changes, exemplified by extensive muscularization, are already present early in gestation, but the origin of these abnormalities is unknown. Understanding the origin of the vascular defects is important to improve treatment modalities. Here, we show that the distribution of pericytes is different and may thereby potentially initiate the vascular pathology in CDH. Transient inhibition of retinoic acid (RA) signaling early during pregnancy, the basis of the CDH mouse model, led to an increase in the number of pericytes, thereby affecting the angiogenic potential of pericytes in the fetuses. Pericytes of CDH lungs showed reduced proliferation and an increased ACTA2 expression, which indicates that these pericytes are more contractile than in control lung pericytes. This resulted in increased pericyte coverage of pulmonary vessels and reduced expansion of the capillary bed, the earliest pathological sign of the structural changes in CDH. Furthermore, the pericytes had reduced and altered collagen IV deposition in CDH, pointing to a loss of basal membrane integrity between pericytes and endothelial cells. Inhibition of RA signaling in vitro resulted in reduced migration of pericytes, reduced angiogenesis, and loss of collagen IV expression. Importantly, we confirmed our findings in lungs of human CDH patient samples. In summary, inhibition of RA signaling affects the lung pericyte population, leading to increased contractility, reduced pulmonary angiogenesis, and aberrant lung development, as observed in CDH.

Bronchopulmonary dysplasia (BPD), characterized by alveoli simplification and dysmorphic pulmonary microvasculature, is a chronic lung disease affecting prematurely born infants. Pulmonary hypertension (PH) is an important BPD feature associated with morbidity and mortality. In human BPD, inflammation leads to decreased fibroblast growth factor 10 (FGF10) expression but the impact on the vasculature is so far unknown. We used lungs from Fgf10+/- versus Fgf10+/+ pups to investigate the effect of Fgf10 deficiency on vascular development in normoxia (NOX) and hyperoxia (HOX, BPD mouse model). To assess the role of fibroblast growth factor receptor 2b (Fgfr2b) ligands independently of early developmentaldefects, we used an inducible double transgenic system in mice allowing inhibition of Fgfr2b ligands activity. Using vascular morphometry, we quantified the pathological changes. Finally, we evaluated changes in FGF10, surfactant protein C (SFTPC), platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin 2 (α-SMA) expression in human lung samples from patients suffering from BPD. In NOX, no major difference in the lung vasculature between Fgf10+/- and control pups was detected. In HOX, a greater loss of blood vessels in Fgf10+/- lungs is associated with an increase of poorly muscularized vessels. Fgfr2b ligands inhibition postnatally in HOX is sufficient to decrease the number of blood vessels while increasing the level of muscularization, suggesting a PH phenotype. BPD lungs exhibited decreased FGF10, SFTPC and PECAM but increased α-SMA. Fgf10 deficiency-associated vascular defects are enhanced in HOX and could represent an additional cause of morbidity in human patients with BPD., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In this article, the Group Chairs and early career members of the European Respiratory Society (ERS) Paediatric Assembly highlight some of the most interesting findings in the field of paediatrics which were presented at the 2018 international ERS Congress., Competing Interests: Conflict of interest: R. Nenna has nothing to disclose. Conflict of interest: K.A. Hunt reports personal fees from Chiesi and Medimmune, and grants from SLE Ltd, outside the submitted work. Conflict of interest: T. Dassios has nothing to disclose. Conflict of interest: J.J.P. Collins has nothing to disclose. Conflict of interest: R.J. Rottier has nothing to disclose. Conflict of interest: N.M. Liu has nothing to disclose. Conflict of interest: B. Rottier has nothing to disclose. Conflict of interest: M. Goutaki has nothing to disclose. Conflict of interest: B. Karadag has nothing to disclose. Conflict of interest: A. Prayle has nothing to disclose. Conflict of interest: R.M. Fernandes has served on independent data monitoring committees for studies sponsored by Ablynx, and participated in clinical trials sponsored by Janssen. Conflict of interest: G.F. Parisi has nothing to disclose. Conflict of interest: J. Barben has nothing to disclose. Conflict of interest: B. Rubbo has nothing to disclose. Conflict of interest: D. Snijders has nothing to disclose. Conflict of interest: H. Makrinoti has nothing to disclose. Conflict of interest: G. Hall has nothing to disclose. Conflict of interest: M.W. Pijnenburg has nothing to disclose. Conflict of interest: J. Grigg reports personal fees from GSK, Vifor Pharmaceuticals, AstraZeneca and Respiri UK, outside the submitted work.

Aims: Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis., Methods and Results: Using short interference RNA-mediated loss-of-function assays, the function and mechanism of signaling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neovessel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G 0 /G 1 cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of canonical Wnt signaling, non-canonical Wnt/Ca 2+ -mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1., Conclusions: The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2.

Patients with congenital diaphragmatic hernia (CDH) often suffer from severe pulmonary hypertension, and the choice of current vasodilator therapy is mostly based on trial and error. Because pulmonary vascular abnormalities are already present early during development, we performed a study to modulate these pulmonary vascular changes at an early stage during gestation. Pregnant Sprague-Dawley rats were treated with nitrofen at day 9.5 of gestation (E9.5) to induce CDH in the offspring, and subsequently, the phosphodiesterase-5 inhibitor sildenafil and/or the novel prostaglandin-I receptor agonist selexipag (active compound NS-304) were administered from E17.5 until E20.5. The clinical relevant start of the treatment corresponds to week 20 of gestation in humans, when CDH is usually detected by ultrasound. CDH pups showed increased density of air saccules that was reverted after the use of only sildenafil. The pulmonary vascular wall was thickened, and right ventricular hypertrophy was present in the CDH group and improved both after single treatment with sildenafil or selexipag, whereas the combination therapy with both compounds did not have additive value. In conclusion, antenatal treatment with sildenafil improved airway morphogenesis and pulmonary vascular development, whereas selexipag only acted positively on pulmonary vascular development. The combination of both compounds did not act synergistically, probably because of a decreased efficiency of both compounds caused by cytochrome- P450 3A4 interaction and induction. These new insights create important possibilities for future treatment of pulmonary vascular abnormalities in CDH patients already in the antenatal period of life.

We show that hypoxia inducible factor 2α (HIF2α) is highly expressed in patients with pulmonary hypertension (PH). HIF2α is expressed in every patient with congenital diaphragmatic hernia, while only half of the controls express HIF2α. Our data suggest that HIF2α is a link between hypoxia and the development of PH.

In this article, the group chairs of the Paediatric Assembly of the European Respiratory Society (ERS) highlight some of the most interesting findings presented at the 2017 ERS International Congress, which was held in Milan, Italy., Competing Interests: Conflict of interest: J. Grigg reports personal fees from GSK and Vifor Pharmaceuticals (honoraria for advisory boards), outside the submitted work. Conflict of interest: G. Hall reports serving on Vertex MBW and LCI Advisory Boards, outside the submitted work.

Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells, but current methods require large numbers of animals which is unwanted in view of the 3R principle and introduces variation. Moreover, stringent breeding schemes are frequently needed to generate sufficient numbers of genetically modified animals. Current protocols do not incorporate expansion of MTEC, and therefore we developed a protocol to expand MTEC while maintaining their differentiation capacity. MTEC were isolated and expanded using the ROCK inhibitor Y-27632 in presence or absence of the γ-secretase inhibitor DAPT, a Notch pathway inhibitor. Whereas MTEC proliferated without DAPT, growth rate and cell morphology improved in presence of DAPT. ALI-induced differentiation of expanded MTEC resulted in an altered capacity of basal cells to differentiate into ciliated cells, whereas IL-13-induced goblet cell differentiation remained unaffected. Ciliated cell differentiation improved by prolonging the ALI differentiation or by adding DAPT, suggesting that basal cells retain their ability to differentiate. This technique using expansion of MTEC and subsequent ALI differentiation drastically reduces animal numbers and costs for in vitro experiments, and will reduce biological variation. Additionally, we provide novel insights in the dynamics of basal cell populations in vitro.

Advances in antenatal screening have provided evidence that the prevalence of congenital pulmonary airway malformation is higher than previously thought. This means that, on a global scale, more congenital pulmonary airway malformations will be diagnosed and seen by clinicians across many different specialties. This increase poses new controversies and challenges for clinicians in terms of prenatal and postnatal management. Important and exciting research has begun to emerge in the past decade. This Review describes current understanding of the disease, the latest surgical developments, and highlights management issues and controversies, including the rationale and timing of surgical intervention, the choice of surgical approach, and long-term functional outcomes. In doing so, we attempt to provide a balanced management algorithm for readers., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to 'complex-specific' DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2). This tag was biotinylated by the bacterial birA protein and the resulting bioSox2 protein was used to identify associating partners of Sox2 at different phases of lung development in vivo (the Sox2 interactome). Homozygous bioSox2 mice are viable and fertile irrespective of the biotinylation of the bio tag, indicating that the bioSox2 gene is normally expressed and the protein is functional in all tissues. This suggests that partners of Sox2 are most likely able to associate with the bioSox2 protein. BioSox2 complexes were isolated with high affinity using streptavidin beads and analysed by MALDI-ToF mass spectrometry analysis. Several of the identified binding partners are already shown to have a respiratory phenotype. Two of these partners, Wdr5 and Tcf3, were validated to confirm their association in Sox2 complexes. This bioSox2 mouse model will be a valuable tool for isolating in vivo Sox2 complexes from different tissues.

Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterised by a diaphragmatic defect, persistent pulmonary hypertension (PH) and lung hypoplasia. The relative contribution of these three elements can vary considerably in individual patients. Most affected children suffer primarily from the associated PH, for which the therapeutic modalities are limited and frequently not evidence based. The vascular defects associated with PH, which is characterised by increased muscularisation of arterioles and capillaries, start to develop early in gestation. Pulmonary vascular development is integrated with the development of the airway epithelium. Although our knowledge is still incomplete, the processes involved in the growth and expansion of the vasculature are beginning to be unravelled. It is clear that early disturbances of this process lead to major pulmonary growth abnormalities, resulting in serious clinical challenges and in many cases death in the newborn. Here we provide an overview of the current molecular pathways involved in pulmonary vascular development. Moreover, we describe the abnormalities associated with CDH and the potential therapeutic approaches for this severe abnormality., Competing Interests: Conflict of interest: None declared., (Copyright ©ERS 2018.)