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.

Single Cell Multiomics Identifies Cells and Genetic Networks Underlying Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.

Guided construction of single cell reference for human and mouse lung.

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.

LungMAP Portal Ecosystem: Systems-Level Exploration of the Lung.

An improved understanding of the human lung necessitates advanced systems models informed by an ever-increasing repertoire of molecular omics, cellular, imaging, and pathological datasets. To centralize and standardize information across broad lung research efforts we expanded the LungMAP.net website into a new gateway portal. This portal connects a broad spectrum of research networks, bulk and single-cell multi-omics data and a diverse collection of image data that span mammalian lung development, and disease. The data are standardized across species and technologies using harmonized data and metadata models that leverage recent advances including those from the Human Cell Atlas, diverse ontologies, and the LungMAP CellCards initiative. To cultivate future discoveries, we have aggregated a diverse collection of single-cell atlases for multiple species (human, rhesus, mouse), to enable consistent queries across technologies, cohorts, age, disease, and drug treatment. These atlases are provided as independent and integrated queryable datasets, with an emphasis on dynamic visualization, figure generation, re-analysis, cell-type curation, and automated reference-based classification of user-provided single-cell genomics datasets (Azimuth). As this resource grows, we intend to increase the breadth of available interactive interfaces, supported data types, data portals and datasets from LungMAP and external research efforts.

Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates.

Perinatal inflammatory stress is associated with early life morbidity and lifelong consequences for pulmonary health. Chorioamnionitis, an inflammatory condition affecting the placenta and fluid surrounding the developing fetus, affects 25 to 40% of preterm births. Severe chorioamnionitis with preterm birth is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may markedly alter the development of the lung. Here, we used intra-amniotic lipopolysaccharide (LPS) challenge to induce experimental chorioamnionitis in a prenatal rhesus macaque (Macaca mulatta) model that mirrors structural and temporal aspects of human lung development. Inflammatory injury directly disrupted the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single-cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis that was extensively disrupted by perinatal inflammation, leading to a loss of gas exchange surface and alveolar simplification, with notable resemblance to chronic lung disease in newborns. Blockade of the inflammatory cytokines interleukin-1ß and tumor necrosis factor-α ameliorated LPS-induced inflammatory lung injury by blunting stromal responses to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis.

Proteomic Analysis of Human Lung Development.

Rationale: The current understanding of human lung development derives mostly from animal studies. Although transcript-level studies have analyzed human donor tissue to identify genes expressed during normal human lung development, protein-level analysis that would enable the generation of new hypotheses on the processes involved in pulmonary development are lacking. Objectives: To define the temporal dynamic of protein expression during human lung development. Methods: We performed proteomics analysis of human lungs at 10 distinct times from birth to 8 years to identify the molecular networks mediating postnatal lung maturation. Measurements and Main Results: We identified 8,938 proteins providing a comprehensive view of the developing human lung proteome. The analysis of the data supports the existence of distinct molecular substages of alveolar development and predicted the age of independent human lung samples, and extensive remodeling of the lung proteome occurred during postnatal development. Evidence of post-transcriptional control was identified in early postnatal development. An extensive extracellular matrix remodeling was supported by changes in the proteome during alveologenesis. The concept of maturation of the immune system as an inherent part of normal lung development was substantiated by flow cytometry and transcriptomics. Conclusions: This study provides the first in-depth characterization of the human lung proteome during development, providing a unique proteomic resource freely accessible at Lungmap.net. The data support the extensive remodeling of the lung proteome during development, the existence of molecular substages of alveologenesis, and evidence of post-transcriptional control in early postnatal development.

A census of the lung: CellCards from LungMAP.

The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.

YAP regulates alveolar epithelial cell differentiation and AGER via NFIB/KLF5/NKX2-1.

Ventilation is dependent upon pulmonary alveoli lined by two major epithelial cell types, alveolar type-1 (AT1) and 2 (AT2) cells. AT1 cells mediate gas exchange while AT2 cells synthesize and secrete pulmonary surfactants and serve as progenitor cells which repair the alveoli. We developed transgenic mice in which YAP was activated or deleted to determine its roles in alveolar epithelial cell differentiation. Postnatal YAP activation increased epithelial cell proliferation, increased AT1 cell numbers, and caused indeterminate differentiation of subsets of alveolar cells expressing atypical genes normally restricted to airway epithelial cells. YAP deletion increased expression of genes associated with mature AT2 cells. YAP activation enhanced DNA accessibility in promoters of transcription factors and motif enrichment analysis predicted target genes associated with alveolar cell differentiation. YAP participated with KLF5, NFIB, and NKX2-1 to regulate AGER. YAP plays a central role in a transcriptional network that regulates alveolar epithelial differentiation.

Heterogeneity in Human Induced Pluripotent Stem Cell-derived Alveolar Epithelial Type II Cells Revealed with ABCA3/SFTPC Reporters.

Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.

Short-term exposure to intermittent hypoxia leads to changes in gene expression seen in chronic pulmonary disease.

Obstructive sleep apnea (OSA) results from episodes of airway collapse and intermittent hypoxia (IH) and is associated with a host of health complications. Although the lung is the first organ to sense changes in oxygen levels, little is known about the consequences of IH to the lung hypoxia-inducible factor-responsive pathways. We hypothesized that exposure to IH would lead to cell-specific up- and downregulation of diverse expression pathways. We identified changes in circadian and immune pathways in lungs from mice exposed to IH. Among all cell types, endothelial cells showed the most prominent transcriptional changes. Upregulated genes in myofibroblast cells were enriched for genes associated with pulmonary hypertension and included targets of several drugs currently used to treat chronic pulmonary diseases. A better understanding of the pathophysiologic mechanisms underlying diseases associated with OSA could improve our therapeutic approaches, directing therapies to the most relevant cells and molecular pathways.

Glucocorticoid regulates mesenchymal cell differentiation required for perinatal lung morphogenesis and function.

While antenatal glucocorticoids are widely used to enhance lung function in preterm infants, cellular and molecular mechanisms by which glucocorticoid receptor (GR) signaling influences lung maturation remain poorly understood. Deletion of the glucocorticoid receptor gene (Nr3c1) from fetal pulmonary mesenchymal cells phenocopied defects caused by global Nr3c1 deletion, while lung epithelial- or endothelial-specific Nr3c1 deletion did not impair lung function at birth. We integrated genome-wide gene expression profiling, ATAC-seq, and single cell RNA-seq data in mice in which GR was deleted or activated to identify the cellular and molecular mechanisms by which glucocorticoids control prenatal lung maturation. GR enhanced differentiation of a newly defined proliferative mesenchymal progenitor cell (PMP) into matrix fibroblasts (MFBs), in part by directly activating extracellular matrix-associated target genes, including Fn1, Col16a4, and Eln and by modulating VEGF, JAK-STAT, and WNT signaling. Loss of mesenchymal GR signaling blocked fibroblast progenitor differentiation into mature MFBs, which in turn increased proliferation of SOX9+ alveolar epithelial progenitor cells and inhibited differentiation of mature alveolar type II (AT2) and AT1 cells. GR signaling controls genes required for differentiation of a subset of proliferative mesenchymal progenitors into matrix fibroblasts, in turn, regulating signals controlling AT2/AT1 progenitor cell proliferation and differentiation and identifying cells and processes by which glucocorticoid signaling regulates fetal lung maturation.

A comparison of genetic sampling methodologies for candidate-gene analyses.

Much of the recent gains in knowledge regarding the influence of patient genetics on medication pharmacokinetics (drug absorption, distribution, metabolism and elimination) how patients process medications) and pharmacodynamics (drug response) have been attributed to the technologic advances in genetic testing methodologies and the involvement of large clinical data sets and biobanks. Indeed, Genome Wide Association Studies (GWAS) and Phenome Wide Association Studies (PWAS) along with ever-evolving biomedical informatics techniques and the expansion of the -omics sciences (e.g., transcriptomics, metabolomics, proteomics) have brought about unprecedented advances in precision medicine. Although the simpler candidate-gene analysis technique is not considered cutting-edge, it is reliable and important to the translation of pharmacogenomic research and the advancement of precision medicine. Leveraging the knowledge of biological plausibility (i.e., genetic mutation → altered function of protein product → altered drug pharmacokinetics/dynamics) to appropriately select genes for inclusion, the candidate-gene analysis technique does not necessitate large patient cohorts nor extensive multi-gene genetic analysis arrays. It is often the ideal method for clinicians to begin evaluating whether genetic information might improve their pharmacologic treatment strategies for their patients. Having access to specific patient populations and expertise regarding their medical subspecialty, physician scientists can implement a candidate-gene analysis in small cohorts. Even with less than 100 patients, results can often be used to determine whether further investigation is warranted and to inform future studies. Herein, we present a comparison of select contemporary methodologies regarding collection, processing and genotype testing applicable to the efficient implementation of candidate-gene studies.

Integration of transcriptomic and proteomic data identifies biological functions in cell populations from human infant lung.

Systems biology uses computational approaches to integrate diverse data types to understand cell and organ behavior. Data derived from complementary technologies, for example transcriptomic and proteomic analyses, are providing new insights into development and disease. We compared mRNA and protein profiles from purified endothelial, epithelial, immune, and mesenchymal cells from normal human infant lung tissue. Signatures for each cell type were identified and compared at both mRNA and protein levels. Cell-specific biological processes and pathways were predicted by analysis of concordant and discordant RNA-protein pairs. Cell clustering and gene set enrichment comparisons identified shared versus unique processes associated with transcriptomic and/or proteomic data. Clear cell-cell correlations between mRNA and protein data were obtained from each cell type. Approximately 40% of RNA-protein pairs were coherently expressed. While the correlation between RNA and their protein products was relatively low (Spearman rank coefficient rs ~0.4), cell-specific signature genes involved in functional processes characteristic of each cell type were more highly correlated with their protein products. Consistency of cell-specific RNA-protein signatures indicated an essential framework for the function of each cell type. Visualization and reutilization of the protein and RNA profiles are supported by a new web application, "LungProteomics," which is freely accessible to the public.

Association of Genetic Polymorphisms in the Beta-1 Adrenergic Receptor with Recovery of Left Ventricular Ejection Fraction in Patients with Heart Failure.

Two common genetic polymorphisms in the beta-1 adrenergic receptor (ADRB1 Ser49Gly [rs1801252] and Arg389Gly [rs1801253]) significantly affect receptor function in vitro. The objective of this study was to determine whether ADRB1 Ser49Gly and Arg389Gly are associated with recovery of left ventricular ejection fraction (LVEF) in patients with heart failure. Patients with heart failure and baseline LVEF ≤ 40% were genotyped (n = 98), and retrospective chart review assessed the primary outcome of LVEF recovery to ≥ 40%. Un/adjusted logistic regression models revealed that Ser49Gly, but not Arg389Gly, was significantly associated with LVEF recovery in a dominant genetic model. The adjusted odds ratio for Ser49 was 8.2 (95% CI = 2.1-32.9; p = 0.003), and it was the strongest predictor of LVEF recovery among multiple clinical variables. In conclusion, patients with heart failure and reduced ejection fraction that are homozygous for ADRB1 Ser49 were significantly more likely to experience LVEF recovery than Gly49 carriers.

Neonatal Lung Disease Associated with TBX4 Mutations.

Variable lung disease was documented in 2 infants with heterozygous TBX4 mutations; their clinical presentations, pathology, and outcomes were distinct. These findings demonstrate that TBX4 gene mutations are associated with neonatal respiratory failure and highlight the wide spectrum of clinicopathological outcomes that have implications for patient diagnosis and management.

Clinical features related to statin-associated muscle symptoms.

INTRODUCTION: Statins reduce cardiovascular disease risk and are generally well tolerated, yet up to 0.5% of statin-treated patients develop incapacitating muscle symptoms including rhabdomyolysis. Our objective was to identify clinical factors related to statin-associated muscle symptoms (SAMS). METHODS: Clinical and laboratory characteristics were evaluated in 748 statin-treated Caucasians (634 with SAMS and 114 statin-tolerant controls). Information was collected on statin type, concomitant drug therapies, muscle symptom history, comorbidities, and family history. Logistic regression was used to identify associations. RESULTS: Individuals with SAMS were 3.6 times (odds ratio [OR] 3.60, 95% confidence interval [CI] 2.08-6.22) more likely than statin-tolerant controls to have a family history of heart disease. Additional associations included obesity (OR 3.08, 95% CI 1.18, 8.05), hypertension (OR 2.24, 95% CI 1.33, 3.77), smoking (OR 2.08, 95% CI 1.16, 3.74), and statin type. DISCUSSION: Careful medical monitoring of statin-treated patients with the associated coexisting conditions may ultimately reduce muscle symptoms and lead to improved compliance. Muscle Nerve 59:537-537, 2019.

Time-resolved proteome profiling of normal lung development.

Biochemical networks mediating normal lung morphogenesis and function have important implications for ameliorating morbidity and mortality in premature infants. Although several transcript-level studies have examined normal lung development, corresponding protein-level analyses are lacking. Here we performed proteomics analysis of murine lungs from embryonic to early adult ages to identify the molecular networks mediating normal lung development. We identified 8,932 proteins, providing a deep and comprehensive view of the lung proteome. Analysis of the proteomics data revealed discrete modules and the underlying regulatory and signaling network modulating their expression during development. Our data support the cell proliferation that characterizes early lung development and highlight responses of the lung to exposure to a nonsterile oxygen-rich ambient environment and the important role of lipid (surfactant) metabolism in lung development. Comparison of dynamic regulation of proteomic and recent transcriptomic analyses identified biological processes under posttranscriptional control. Our study provides a unique proteomic resource for understanding normal lung formation and function and can be freely accessed at Lungmap.net.

Reconstructing differentiation networks and their regulation from time series single-cell expression data.

Generating detailed and accurate organogenesis models using single-cell RNA-seq data remains a major challenge. Current methods have relied primarily on the assumption that descendant cells are similar to their parents in terms of gene expression levels. These assumptions do not always hold for in vivo studies, which often include infrequently sampled, unsynchronized, and diverse cell populations. Thus, additional information may be needed to determine the correct ordering and branching of progenitor cells and the set of transcription factors (TFs) that are active during advancing stages of organogenesis. To enable such modeling, we have developed a method that learns a probabilistic model that integrates expression similarity with regulatory information to reconstruct the dynamic developmental cell trajectories. When applied to mouse lung developmental data, the method accurately distinguished different cell types and lineages. Existing and new experimental data validated the ability of the method to identify key regulators of cell fate.