Selection of potential targets for stratifying congenital pulmonary airway malformation patients with molecular imaging: is MUC1 the one?

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.

Distinct roles for SOX2 and SOX21 in differentiation, distribution and maturation of pulmonary neuroendocrine cells.

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.

Identification of SOX2 Interacting Proteins in the Developing Mouse Lung With Potential Implications for Congenital Diaphragmatic Hernia.

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.

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/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 Kd 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.

Disease modeling following organoid-based expansion of airway epithelial cells.

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.

SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways.

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.

Histological, immunohistochemical and transcriptomic characterization of human tracheoesophageal fistulas.

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.

Inhibition of retinoic acid signaling induces aberrant pericyte coverage and differentiation resulting in vascular defects in congenital diaphragmatic hernia.

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.

Hypoxia inducible factor 2α (HIF2α/EPAS1) is associated with development of pulmonary hypertension in severe congenital diaphragmatic hernia patients.

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.

Treatment of rat congenital diaphragmatic hernia with sildenafil and NS-304, selexipag's active compound, at the pseudoglandular stage improves lung vasculature.

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.

Differentiated type II pneumocytes can be reprogrammed by ectopic Sox2 expression.

The adult lung contains several distinct stem cells, although their properties and full potential are still being sorted out. We previously showed that ectopic Sox2 expression in the developing lung manipulated the fate of differentiating cells. Here, we addressed the question whether fully differentiated cells could be redirected towards another cell type. Therefore, we used transgenic mice to express an inducible Sox2 construct in type II pneumocytes, which are situated in the distal, respiratory areas of the lung. Within three days after the induction of the transgene, the type II cells start to proliferate and form clusters of cuboidal cells. Prolonged Sox2 expression resulted in the reversal of the type II cell towards a more embryonic, precursor-like cell, being positive for the stem cell markers Sca1 and Ssea1. Moreover, the cells started to co-express Spc and Cc10, characteristics of bronchioalveolar stem cells. We demonstrated that Sox2 directly regulates the expression of Sca1. Subsequently, these cells expressed Trp63, a marker for basal cells of the trachea. So, we show that the expression of one transcription factor in fully differentiated, distal lung cells changes their fate towards proximal cells through intermediate cell types. This may have implications for regenerative medicine, and repair of diseased and damaged lungs.

Sox2 regulates the emergence of lung basal cells by directly activating the transcription of Trp63.

Lung development is determined by the coordinated expression of several key genes. Previously, we and others have shown the importance of the sex determining region Y-box 2 (Sox2) gene in lung development. Transgenic expression of Sox2 during lung development resulted in cystic airways, and here we show that modulating the timing of ectopic Sox2 expression in the branching regions of the developing lung results in variable cystic lesions resembling the spectrum of the human congenital disorder congenital cystic adenomatoid malformation (CCAM). Sox2 dominantly differentiated naive epithelial cells into the proximal lineage irrespective of the presence of Fgf10. Sox2 directly induced the expression of Trp63, the master switch toward the basal cell lineage and induced the expression of Gata6, a factor involved in the emergence of bronchoalveolar stem cells. We showed that SOX2 and TRP63 are coexpressed in the lungs of human patients with type II CCAM. The combination of premature differentiation toward the proximal cell lineage and the induction of proliferation resulted in the cyst-like structures. Thus, we show that Sox2 is directly responsible for the emergence of two lung progenitor cells: basal cells by regulating the master gene Trp63 and bronchoalveolar stem cells by regulating Gata6.

Premature differentiation of vascular smooth muscle cells in human congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterized by the herniation of abdominal organs into the chest cavity. The high mortality and morbidity of CDH patients are primarily caused by the associated pulmonary hypertension (PH), characterized by the thickening of the vascular media and adventitia. The media consist of heterogeneous populations of vascular smooth muscle cells (VSMC), ranging from synthetic to the characteristic contractile cells. VSMCs are influenced by developmental and environmental cues and may play a role in the development of the structural changes observed in CDH patients. Therefore, we hypothesized that the distribution of the VSMC populations may already be different at the origin of CDH development. METHODOLOGY: We analyzed the protein expression of specific markers associated with synthetic and contractile VSMC phenotypes in human lungs at different developmental stages. Next, we compared lungs of premature and term CDH patients, as well as patients with lung hypoplasia due to renal agenesis or PROM, with age-matched controls. RESULTS: Synthetic and contractile VSMCs are distributed in a temporal and spatial specific pattern along the proximodistal axis of the lung. CDH patients have more abundant contractile VSMCs which are also more distally distributed. This different distribution pattern is already observed from 19 weeks of gestation onwards. CONCLUSION: Our data suggest that the more extensive distribution of contractile VSMCs is associated with an early maturation of the pulmonary vasculature, contrasting the concept that CDH might be the result of delayed maturation of the epithelium.

Reversal of pulmonary vascular remodeling in pulmonary hypertensive rats.

Pulmonary hypertension is responsible for significant mortality and morbidity among newborns and infants. The pathology is characterized by pulmonary vascular remodeling with medial hypertrophy and adventitial thickening, leading to decreased gas exchange. Since it is unknown if these abnormalities are reversible, we analyzed these vascular changes in pulmonary hypertensive rats. Exposure of rats to hypobaric hypoxia for 4 weeks induced clinical signs of pulmonary hypertension, such as increased right ventricular systolic pressure, increased right ventricular weight and considerable pulmonary vascular remodeling. The vascular changes were associated with the expression of Non -Muscle Myosin Heavy Chain B in the pre-acinar vessels and an increased expression of alpha Smooth Muscle Actin, Smooth Muscle Myosin Heavy Chain 2 and Calponin in the intra-acinar vessels. The right ventricular systolic pressure and right ventricular weight gradually decreased after specific periods of recovery in normoxia, although this reversal did not reach baseline levels after six weeks at normoxia. However, the cellular changes in the pulmonary vasculature were completely reversed. Development of pulmonary hypertension is associated with an increase of synthetic perivascular cells in the pre-acinar arteries and an aberrant differentiation of perivascular cells in the smallest intra-acinar arteries. These cellular and structural changes in the pulmonary vasculature are completely reversible after recovery in normoxia.