Bulk RNA sequencing of human pediatric lung cell populations reveals unique transcriptomic signature associated with postnatal pulmonary development.

Postnatal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well-documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human postnatal lung development are yet to be fully understood. In this study, we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and nonendothelial mesenchymal cells, along with lung leukocytes) from 1-day-old to 8-yr-old organ donors with no known lung disease. For analysis, we considered the donors in four age groups [less than 30 days old neonates, 30 days to < 1 yr old infants, toddlers (1 to < 2 yr), and children 2 yr and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared with the number of DEG found during infant to toddler- or toddler to older children-transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.NEW & NOTEWORTHY This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.

A novel non-recurrent CNV deletion involving TBX4 and leaving TBX2 intact causes congenital alveolar dysplasia.

Congenital alveolar dysplasia (CAD) belongs to rare lethal lung developmental disorders (LLDDs) in neonates, manifesting with acute respiratory failure and pulmonary arterial hypertension refractory to treatment. The majority of CAD cases have been associated with copy-number variant (CNV) deletions at 17q23.1q23.2 or 5p12. Most CNV deletions at 17q23.1q23.2 were recurrent and encompassed two closely located genes, TBX4 and TBX2. In a few CAD cases, intragenic frameshifting deletions or single-nucleotide variants (SNVs) involved TBX4 but not TBX2. Here, we describe a male neonate who died at 27 days of life from acute respiratory failure caused by lung growth arrest along the spectrum of CAD confirmed by histopathological assessment. Trio-based genome sequencing revealed in the proband a novel non-recurrent ~1.07 Mb heterozygous CNV deletion at 17q23.2, encompassing TBX4 that arose de novo on the paternal chromosome. This is the first report of a larger-sized CNV deletion in a CAD patient involving TBX4 and leaving TBX2 intact. Our results, together with previous reports, indicate that perturbations of TBX4, rather than TBX2, cause severe lung phenotypes in humans.

Human inherited CCR2 deficiency underlies progressive polycystic lung disease.

We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca2+ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.

Diminished TMEM100 Expression in a Newborn With Acinar Dysplasia and a Novel TBX4 Variant: A Case Report.

Acinar dysplasia (AcDys) of the lung is a rare lethal developmental disorder in neonates characterized by severe respiratory failure and pulmonary arterial hypertension refractory to treatment. Recently, abnormalities of TBX4-FGF10-FGFR2-TMEM100 signaling regulating lung development have been reported in patients with AcDys due to heterozygous single-nucleotide variants or copy-number variant deletions involving TBX4, FGF10, or FGFR2. Here, we describe a female neonate who died at 4 hours of life due to severe respiratory distress related to AcDys diagnosed by postmortem histopathologic evaluation. Genomic analyses revealed a novel deleterious heterozygous missense variant c.728A>C (p.Asn243Thr) in TBX4 that arose de novo on paternal chromosome 17. We also identified 6 candidate hypomorphic rare variants in the TBX4 enhancer in trans to TBX4 coding variant. Gene expression analyses of proband's lung tissue showed a significant reduction of TMEM100 expression with near absence of TMEM100 within the endothelium of arteries and capillaries by immunohistochemistry. These results support the pathogenicity of the detected TBX4 variant and provide further evidence that disrupted signaling between TBX4 and TMEM100 may contribute to severe lung phenotypes in humans, including AcDys.

Further refinement of the differentially methylated distant lung-specific FOXF1 enhancer in a neonate with alveolar capillary dysplasia.

Heterozygous SNVs or CNV deletions involving the FOXF1 gene, or its distant enhancer, are causative for 80-90% of cases of alveolar capillary dysplasia with misalignment of pulmonary veins. Recently, we proposed bimodal structure and parental functional dimorphism of the lung-specific FOXF1 enhancer, with Unit 1 having higher activity on the paternal chr16 and Unit 2 on the maternal chr16. Here, we describe a novel unusually sized pathogenic de novo copy-number variant deletion involving a portion of the FOXF1 enhancer on maternal chr16 that implies narrowing Unit 2 to an essential ~ 9-kb segment. Using a restrictase-based assay, we found that this enhancer segment is weakly methylated at ApT adenine, with about twice the frequency of methylation on the maternal versus paternal chr16. Our data provide further insight into the FOXF1 enhancer structure and function.

New insights into the natural history of bronchopulmonary dysplasia from proteomics and multiplexed immunohistochemistry.

Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, n = 3) and "established" BPD (eBPD, n = 3) compared with respective full-term born (n = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood.NEW & NOTEWORTHY We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.

Lung biopsy in the diagnosis and management of chILD.

Children's interstitial and diffuse lung disease (chILD) comprises a large number of diverse entities ranging from disorders of lung development, maturation and function unique in infancy to immune-mediated, environmental, vascular and other conditions overlapping with adult disease. Pathologic evaluation of the lung has played a central role in characterizing many of these disorders, resulting in revised nomenclature and classifications to help guide clinical management(1-4). Technological advancements are rapidly uncovering genetic and molecular underpinnings of these conditions, as well as widening the phenotypes which bridge adult disease, often reducing the perceived need for diagnostic lung biopsy. As such the decision to get a lung biopsy in chILD is frequently for rapid ascertainment of disease in a critically ill child or when clinical presentation, imaging and laboratory studies fail to provide a cohesive diagnosis needed for treatment. While there have been modifications in surgical procedures for lung biopsy that minimize postoperative morbidity, it remains a high-risk invasive procedure, especially in a medically complex patient(5). Thus, it is essential that the lung biopsy be handled properly to maximize diagnostic yield, including close communication between the clinician, radiologist, surgeon, and pathologist before biopsy to determine best sampling site(s) and prioritization of tissue utilization. This review provides an overview of optimal handling and evaluation of a surgical lung biopsy for suspected chILD, with emphasis on specific conditions in which pathologic features play a critical role in providing an integrated diagnosis and guiding management.

Prenatal Detection of a FOXF1 Deletion in a Fetus with ACDMPV and Hydronephrosis.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by the arrest of fetal lung formation, resulting in neonatal death due to acute respiratory failure and pulmonary arterial hypertension. Heterozygous single-nucleotide variants or copy-number variant (CNV) deletions involving the FOXF1 gene and/or its lung-specific enhancer are found in the vast majority of ACDMPV patients. ACDMPV is often accompanied by extrapulmonary malformations, including the gastrointestinal, cardiac, or genitourinary systems. Thus far, most of the described ACDMPV patients have been diagnosed post mortem, based on histologic evaluation of the lung tissue and/or genetic testing. Here, we report a case of a prenatally detected de novo CNV deletion (~0.74 Mb) involving the FOXF1 gene in a fetus with ACDMPV and hydronephrosis. Since ACDMPV is challenging to detect by ultrasound examination, the more widespread implementation of prenatal genetic testing can facilitate early diagnosis, improve appropriate genetic counselling, and further management.

FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Do paternal deletions involving the FOXF1 locus on chromosome 16q24.1 manifest with more severe non-lung anomalies?

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal lung developmental disorder in neonates due to heterozygous loss-of-function of the mesenchymal transcription factor gene, FOXF1. Interestingly, unlike ACDMPV-causing point mutations in FOXF1 that can be inherited from the mother or father, causative copy-number variant (CNV) deletions arise de novo and almost exclusively on chromosome 16 inherited from the mother (n = 50 vs. n = 3). Here, we describe a fourth case of a de novo paternal CNV deletion encompassing FOXF1, its neighboring long non-coding RNA gene FENDRR, and their distant lung-specific enhancer, identified in a 21-week-old fetus with tetralogy of Fallot, gastrointestinal and genitourinary abnormalities, a single umbilical artery, and patchy histopathological findings of ACDMPV in autopsy lung. We also review the ACDMPV-causative CNV deletions detected prenatally and propose that the majority of paternal deletions manifest with more severe additional non-lung abnormalities.

Tyrosine kinase-altered spindle cell neoplasms with EGFR internal tandem duplications.

In this study, we present two extra-renal pediatric spindle cell neoplasms with epidermal growth factor receptor (EGFR) internal tandem duplications (ITD). Histologically, these tumors demonstrated the same histologic features seen in other tyrosine kinase-altered spindle cell neoplasms, with one case showing abundant adipose tissue with cellular fibrous septae resembling lipofibromatosis and the other case showing fascicles of spindled cells resembling infantile fibrosarcoma. There was variable expression of CD34, S100, and SMA, and all cases were negative for panTRK. This case series adds to our molecular understanding of the spectrum of tyrosine kinase-altered spindle cell neoplasms and represents the first reported examples of EGFR ITDs in extra-renal tumors. The presence of EGFR alterations in the absence of gene fusions represents a potential therapeutic target and necessitates a broader testing panel for this group of tumors.

Excess neuropeptides in lung signal through endothelial cells to impair gas exchange.

Although increased neuropeptides are often detected in lungs that exhibit respiratory distress, whether they contribute to the condition is unknown. Here, we show in a mouse model of neuroendocrine cell hyperplasia of infancy, a pediatric disease with increased pulmonary neuroendocrine cells (PNECs), excess PNEC-derived neuropeptides are responsible for pulmonary manifestations including hypoxemia. In mouse postnatal lung, prolonged signaling from elevated neuropeptides such as calcitonin gene-related peptide (CGRP) activate receptors enriched on endothelial cells, leading to reduced cellular junction gene expression, increased endothelium permeability, excess lung fluid, and hypoxemia. Excess fluid and hypoxemia were effectively attenuated by either prevention of PNEC formation, inactivation of CGRP gene, endothelium-specific inactivation of CGRP receptor gene, or treatment with CGRP receptor antagonist. Neuropeptides were increased in human lung diseases with excess fluid such as acute respiratory distress syndrome. Our findings suggest that restricting neuropeptide function may limit fluid and improve gas exchange in these conditions.

Identification of Distinct Inflammatory Programs and Biomarkers in Systemic Juvenile Idiopathic Arthritis and Related Lung Disease by Serum Proteome Analysis.

OBJECTIVE: Recent observations in systemic juvenile idiopathic arthritis (JIA) suggest an increasing incidence of high-mortality interstitial lung disease often characterized by a variant of pulmonary alveolar proteinosis (PAP). Co-occurrence of macrophage activation syndrome (MAS) and PAP in systemic JIA suggests a shared pathology, but patients with lung disease associated with systemic JIA (designated SJIA-LD) also commonly experience features of drug reaction such as atypical rashes and eosinophilia. This study was undertaken to investigate immunopathology and identify biomarkers in systemic JIA, MAS, and SJIA-LD. METHODS: We used SOMAscan to measure ~1,300 analytes in sera from healthy controls and patients with systemic JIA, MAS, SJIA-LD, or other related diseases. We verified selected findings by enzyme-linked immunosorbent assay and lung immunostaining. Because the proteome of a sample may reflect multiple states (systemic JIA, MAS, or SJIA-LD), we used regression modeling to identify subsets of altered proteins associated with each state. We tested key findings in a validation cohort. RESULTS: Proteome alterations in active systemic JIA and MAS overlapped substantially, including known systemic JIA biomarkers such as serum amyloid A and S100A9, and novel elevations in the levels of heat-shock proteins and glycolytic enzymes. Interleukin-18 levels were elevated in all systemic JIA groups, particularly MAS and SJIA-LD. We also identified an MAS-independent SJIA-LD signature notable for elevated levels of intercellular adhesion molecule 5 (ICAM-5), matrix metalloproteinase 7 (MMP-7), and allergic/eosinophilic chemokines, which have been previously associated with lung damage. Immunohistochemistry localized ICAM-5 and MMP-7 in the lungs of patients with SJIA-LD. The ability of ICAM-5 to distinguish SJIA-LD from systemic JIA/MAS was independently validated. CONCLUSION: Serum proteins support a systemic JIA-to-MAS continuum; help distinguish systemic JIA, systemic JIA/MAS, and SJIA-LD; and suggest etiologic hypotheses. Select biomarkers, such as ICAM-5, could aid in early detection and management of SJIA-LD.

Neonatal Hyperoxia Activates Activating Transcription Factor 4 to Stimulate Folate Metabolism and Alveolar Epithelial Type 2 Cell Proliferation.

Oxygen supplementation in preterm infants disrupts alveolar epithelial type 2 (AT2) cell proliferation through poorly understood mechanisms. Here, newborn mice are used to understand how hyperoxia stimulates an early aberrant wave of AT2 cell proliferation that occurs between Postnatal Days (PNDs) 0 and 4. RNA-sequencing analysis of AT2 cells isolated from PND4 mice revealed hyperoxia stimulates expression of mitochondrial-specific methylenetetrahydrofolate dehydrogenase 2 and other genes involved in mitochondrial one-carbon coupled folate metabolism and serine synthesis. The same genes are induced when AT2 cells normally proliferate on PND7 and when they proliferate in response to the mitogen fibroblast growth factor 7. However, hyperoxia selectively stimulated their expression via the stress-responsive activating transcription factor 4 (ATF4). Administration of the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia suppressed ATF4 and thus early AT2 cell proliferation, but it had no effect on normative AT2 cell proliferation seen on PND7. Because ATF4 and methylenetetrahydrofolate dehydrogenase are detected in hyperplastic AT2 cells of preterm infant humans and baboons with bronchopulmonary dysplasia, dampening mitochondrial oxidative stress and ATF4 activation may provide new opportunities for controlling excess AT2 cell proliferation in neonatal lung disease.

Interstitial lung disease in children with Rubinstein-Taybi syndrome.

INTRODUCTION: Rubinstein-Taybi syndrome (RSTS) is a rare genetic syndrome caused primarily by a mutation in the CREBBP gene found on chromosome 16. Patients with RSTS are at greater risk for a variety of medical problems, including upper airway obstruction and aspiration. Childhood interstitial lung disease (ILD) thus far has not been definitively linked to RSTS. Here we present three patients with RSTS who developed ILD and discuss possible mechanisms by which a mutation in CREBBP may be involved in the development of ILD. METHODS: Routine hematoxylin and eosin staining was performed on lung biopsy tissue for histological analysis. Immunofluorescent staining was performed on lung biopsy tissue for markers of fibrosis, surfactant deficiency and histone acetylation. Cases 1 and 2 had standard clinical microarray analysis. Case 3 had whole exome sequencing. Bioinformatics analyses were performed to identify possible causative genes using ToppGene. RESULTS: Computed tomography images in all cases showed consolidated densities overlying ground glass opacities. Lung histopathology revealed accumulation of proteinaceous material within alveolar spaces, evidence of fibrosis, and increased alveolar macrophages. Immunofluorescent staining showed increase in surfactant protein C staining, patchy areas of increased anti-smooth muscle antibody staining, and increased staining for acetylated histone 2 and histone 3 lysine 9. DISCUSSION: Clinical characteristics, radiographic imaging, lung histopathology, and immunofluorescent staining results shared by all cases demonstrated findings consistent with ILD. Immunofluorescent staining suggests two possible mechanisms for the development of ILD: abnormal surfactant metabolism and/or persistent activation of myofibroblasts. These two pathways could be related to dysfunctional CREBBP protein.

Severe delayed hypersensitivity reactions to IL-1 and IL-6 inhibitors link to common HLA-DRB1*15 alleles.

OBJECTIVES: Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, delayed hypersensitivity reaction (DHR). We observed DRESS to inhibitors of interleukin 1 (IL-1) or IL-6 in a small group of patients with Still's disease with atypical lung disease. We sought to characterise features of patients with Still's disease with DRESS compared with drug-tolerant Still's controls. We analysed human leucocyte antigen (HLA) alleles for association to inhibitor-related DHR, including in a small Kawasaki disease (KD) cohort. METHODS: In a case/control study, we collected a multicentre series of patients with Still's disease with features of inhibitor-related DRESS (n=66) and drug-tolerant Still's controls (n=65). We retrospectively analysed clinical data from all Still's subjects and typed 94/131 for HLA. European Still's-DRESS cases were ancestry matched to International Childhood Arthritis Genetics Consortium paediatric Still's cases (n=550) and compared for HLA allele frequencies. HLA association also was analysed using Still's-DRESS cases (n=64) compared with drug-tolerant Still's controls (n=30). KD subjects (n=19) were similarly studied. RESULTS: Still's-DRESS features included eosinophilia (89%), AST-ALT elevation (75%) and non-evanescent rash (95%; 88% involving face). Macrophage activation syndrome during treatment was frequent in Still's-DRESS (64%) versus drug-tolerant Still's (3%; p=1.2×10-14). We found striking enrichment for HLA-DRB1*15 haplotypes in Still's-DRESS cases versus INCHARGE Still's controls (p=7.5×10-13) and versus self-identified, ancestry-matched Still's controls (p=6.3×10-10). In the KD cohort, DRB1*15:01 was present only in those with suspected anakinra reactions. CONCLUSIONS: DRESS-type reactions occur among patients treated with IL-1/IL-6 inhibitors and strongly associate with common HLA-DRB1*15 haplotypes. Consideration of preprescription HLA typing and vigilance for serious reactions to these drugs are warranted.

Multimodality Imaging Evaluation of Fetal Spine Anomalies with Postnatal Correlation.

Congenital anomalies of the spine are associated with substantial morbidity in the perinatal period and may affect the rest of the patient's life. Accurate early diagnosis of spinal abnormalities during fetal imaging allows prenatal, perinatal, and postnatal treatment planning, which can substantially affect functional outcomes. The most common and clinically relevant congenital anomalies of the spine fall into three broad categories: spinal dysraphism, segmentation and fusion anomalies of the vertebral column, and sacrococcygeal teratomas. Spinal dysraphism is further categorized into one of two subtypes: open spinal dysraphism and closed spinal dysraphism. The latter category is further subdivided into those with and without subcutaneous masses. Open spinal dysraphism is an emergency and must be closed at birth because of the risk of infection. In utero closure is also offered at some fetal centers. Sacrococcygeal teratomas are the most common fetal pelvic masses and the prognosis is variable. Finally, vertebral body anomalies are categorized into formation (butterfly and hemivertebrae) and segmentation (block vertebrae) anomalies. Although appropriate evaluation of the fetal spine begins with US, which is the initial screening modality of choice, MRI is increasingly important as a problem-solving tool, especially given the recent advances in fetal MRI, its availability, and the complexity of fetal interventions. Online supplemental material is available for this article. ©RSNA, 2021.

Bronchopulmonary Dysplasia and Pulmonary Hypertension. The Role of Smooth Muscle adh5.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification, airway hyperreactivity, and pulmonary hypertension. In our BPD model, we have investigated the metabolism of the bronchodilator and pulmonary vasodilator GSNO (S-nitrosoglutathione). We have shown the GSNO catabolic enzyme encoded by adh5 (alcohol dehydrogenase-5), GSNO reductase, is epigenetically upregulated in hyperoxia. Here, we investigated the distribution of GSNO reductase expression in human BPD and created an animal model that recapitulates the human data. Blinded comparisons of GSNO reductase protein expression were performed in human lung tissues from infants and children with and without BPD. BPD phenotypes were evaluated in global (adh5-/-) and conditional smooth muscle (smooth muscle/adh5-/-) adh5 knockout mice. GSNO reductase was prominently expressed in the airways and vessels of human BPD subjects. Compared with controls, expression was greater in BPD smooth muscle, particularly in vascular smooth muscle (2.4-fold; P = 0.003). The BPD mouse model of neonatal hyperoxia caused significant alveolar simplification, airway hyperreactivity, and right ventricular and vessel hypertrophy. Global adh5-/- mice were protected from all three aspects of BPD, whereas smooth muscle/adh5-/- mice were only protected from pulmonary hypertensive changes. These data suggest adh5 is required for the development of BPD. Expression in the pulmonary vasculature is relevant to the pathophysiology of BPD-associated pulmonary hypertension. GSNO-mimetic agents or GSNO reductase inhibitors, both of which are currently in clinical trials for other conditions, could be considered for further study in BPD.

Genotype-Phenotype Correlation of Tracheal Cartilaginous Sleeves and Fgfr2 Mutations in Mice.

OBJECTIVES: To characterize tracheal cartilage morphology in mouse models of fibroblast growth factor receptor (Fgfr2)-related craniosynostosis syndromes. To establish relationships between specific Fgfr2 mutations and tracheal cartilaginous sleeve (TCS) phenotypes in these mouse models. METHODS: Postnatal day 0 knock-in mouse lines with disease-specific genetic variations in the Fgfr2 gene (Fgfr2C342Y/C342Y , Fgfr2C342Y/+ , Fgfr2+/Y394C , Fgfr2+/S252W , and Fgfr2+/P253R ) as well as line-specific controls were utilized. Tracheal cartilage morphology as measured by gross analyses, microcomputed tomography (µCT), and histopathology were compared using Chi-squared and single-factor analysis of variance statistical tests. RESULTS: A greater proportion of rings per trachea were abnormal in Fgfr2C342Y/+ tracheas (63%) than Fgfr2+/S252W (17%), Fgfr2+/P253R (17%), Fgfr2+/Y394C (12%), and controls (10%) (P < .001 for each vs. Fgfr2C342Y/+ ). TCS segments were found only in Fgfr2C342Y/C342Y (100%) and Fgfr2C342Y/+ (72%) tracheas. Cricoid and first-tracheal ring fusion was noted in all Fgfr2C342Y/C342Y and 94% of Fgfr2C342Y/+ samples. The Fgfr2C342Y/C342Y and Fgfr2C342Y/+ groups were found to have greater areas and volumes of cartilage than other lines on gross analysis and µCT. Histologic analyses confirmed TCS among the Fgfr2C342Y/C342Y and Fgfr2C342Y/+ groups, without appreciable differences in cartilage morphology, cell size, or density; no histologic differences were observed among other Fgfr2 lines compared to controls. CONCLUSION: This study found TCS phenotypes only in the Fgfr2C342Y mouse lines. These lines also had increased tracheal cartilage compared to other mutant lines and controls. These data support further study of the Fgfr2 mouse lines and the investigation of other Fgfr2 variants to better understand their role in tracheal development and TCS formation. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E1349-E1356, 2021.

Pediatric Cystic Lung Lesions: Where Are We Now?

Pediatric cystic lung lesions have long been a source of confusion for clinicians, radiologists, and pathologists. They encompass a wide spectrum of entities with variable prognostic implications, including congenital lung malformations, pulmonary neoplasms, and hereditary conditions. As our understanding of the developmental and genetic origins of these conditions has evolved, revised nomenclature and classifications have emerged in an attempt to bring clarity to the origin of these lesions and guide clinical management. This review discusses cystic lung lesions and the current understanding of their etiopathogenesis.