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.

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.

Multifunctional Activity-Based Protein Profiling of the Developing Lung.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.

LungMAP: The Molecular Atlas of Lung Development Program.

The National Heart, Lung, and Blood Institute is funding an effort to create a molecular atlas of the developing lung (LungMAP) to serve as a research resource and public education tool. The lung is a complex organ with lengthy development time driven by interactive gene networks and dynamic cross talk among multiple cell types to control and coordinate lineage specification, cell proliferation, differentiation, migration, morphogenesis, and injury repair. A better understanding of the processes that regulate lung development, particularly alveologenesis, will have a significant impact on survival rates for premature infants born with incomplete lung development and will facilitate lung injury repair and regeneration in adults. A consortium of four research centers, a data coordinating center, and a human tissue repository provides high-quality molecular data of developing human and mouse lungs. LungMAP includes mouse and human data for cross correlation of developmental processes across species. LungMAP is generating foundational data and analysis, creating a web portal for presentation of results and public sharing of data sets, establishing a repository of young human lung tissues obtained through organ donor organizations, and developing a comprehensive lung ontology that incorporates the latest findings of the consortium. The LungMAP website (www.lungmap.net) currently contains more than 6,000 high-resolution lung images and transcriptomic, proteomic, and lipidomic human and mouse data and provides scientific information to stimulate interest in research careers for young audiences. This paper presents a brief description of research conducted by the consortium, database, and portal development and upcoming features that will enhance the LungMAP experience for a community of users.

Automated measurement of heterogeneity in CT images of healthy and diseased rat lungs using variogram analysis of an octree decomposition.

BACKGROUND: Assessing heterogeneity in lung images can be an important diagnosis tool. We present a novel and objective method for assessing lung damage in a rat model of emphysema. We combined a three-dimensional (3D) computer graphics method-octree decomposition-with a geostatistics-based approach for assessing spatial relationships-the variogram-to evaluate disease in 3D computed tomography (CT) image volumes. METHODS: Male, Sprague-Dawley rats were dosed intratracheally with saline (control), or with elastase dissolved in saline to either the whole lung (for mild, global disease) or a single lobe (for severe, local disease). Gated 3D micro-CT images were acquired on the lungs of all rats at end expiration. Images were masked, and octree decomposition was performed on the images to reduce the lungs to homogeneous blocks of 2 × 2 × 2, 4 × 4 × 4, and 8 × 8 × 8 voxels. To focus on lung parenchyma, small blocks were ignored because they primarily defined boundaries and vascular features, and the spatial variance between all pairs of the 8 × 8 × 8 blocks was calculated as the square of the difference of signal intensity. Variograms-graphs of distance vs. variance-were constructed, and results of a least-squares-fit were compared. The robustness of the approach was tested on images prepared with various filtering protocols. Statistical assessment of the similarity of the three control rats was made with a Kruskal-Wallis rank sum test. A Mann-Whitney-Wilcoxon rank sum test was used to measure statistical distinction between individuals. For comparison with the variogram results, the coefficient of variation and the emphysema index were also calculated for all rats. RESULTS: Variogram analysis showed that the control rats were statistically indistinct (p = 0.12), but there were significant differences between control, mild global disease, and severe local disease groups (p < 0.0001). A heterogeneity index was calculated to describe the difference of an individual variogram from the control average. This metric also showed clear separation between dose groups. The coefficient of variation and the emphysema index, on the other hand, did not separate groups. CONCLUSION: These results suggest the octree decomposition and variogram analysis approach may be a rapid, non-subjective, and sensitive imaging-based biomarker for characterizing lung disease.

Imaging nicotine in rat brain tissue by use of nanospray desorption electrospray ionization mass spectrometry.

Imaging mass spectrometry offers simultaneous spatially resolved detection of drugs, drug metabolites, and endogenous substances in a single experiment. This is important when evaluating effects of a drug on a complex organ system such as the brain, where there is a need to understand how regional drug distribution impacts function. Nanospray desorption electrospray ionization, nano-DESI, is a new ambient technique that enables spatially resolved analysis of a variety of samples without special sample pretreatment. This study introduces an experimental approach for accurate spatial mapping of drugs and metabolites in tissue sections by nano-DESI imaging. In this approach, an isotopically labeled standard is added to the nano-DESI solvent to compensate for matrix effects and ion suppression. The analyte image is obtained by normalizing the analyte signal to the signal of the standard in each pixel. We demonstrate that the presence of internal standard enables online quantification of analyte molecules extracted from tissue sections. Ion images are subsequently mapped to the anatomical brain regions in the analyzed section by use of an atlas mesh deformed to match the optical image of the section. Atlas-based registration accounts for the physical variability between animals, which is important for data interpretation. The new approach was used for mapping the distribution of nicotine in rat brain tissue sections following in vivo drug administration. We demonstrate the utility of nano-DESI imaging for sensitive detection of the drug in tissue sections with subfemtomole sensitivity in each pixel of a 27 µm × 150 µm area. Such sensitivity is necessary for spatially resolved detection of low-abundance molecules in complex matrices.

High-speed tandem mass spectrometric in situ imaging by nanospray desorption electrospray ionization mass spectrometry.

Nanospray desorption electrospray ionization (nano-DESI) combined with tandem mass spectrometry (MS/MS), high-resolution mass analysis of the fragment ions (m/Δm = 17 500 at m/z 200), and rapid spectral acquisition enabled simultaneous imaging and identification of a large number of metabolites and lipids from 92 selected m/z windows (±1 Da) with a spatial resolution of better than 150 µm. Mouse uterine sections of implantation sites on day 6 of pregnancy were analyzed in the ambient environment without any sample pretreatment. MS/MS imaging was performed by scanning the sample under the nano-DESI probe at 10 µm/s, while higher-energy collision-induced dissociation (HCD) spectra were acquired for a targeted inclusion list of 92 m/z values at a rate of ∼6.3 spectra/s. Molecular ions and their corresponding fragments, separated by high-resolution mass analysis, were assigned on the basis of accurate mass measurement. Using this approach, we were able to identify and image both abundant and low-abundance isobaric and isomeric species within each m/z window. MS/MS analysis enabled efficient separation and identification of isomeric and isobaric phospholipids that are difficult to separate in full-scan mode. Furthermore, we identified several metabolites associated with early pregnancy and obtained the first 2D images of these molecules.

In situ casting and imaging of the rat airway tree for accurate 3D reconstruction.

The use of anatomically accurate, animal-specific airway geometries is important for understanding and modeling the physiology of the respiratory system. One approach for acquiring detailed airway architecture is to create a bronchial cast of the conducting airways. However, typical casting procedures either do not faithfully preserve the in vivo branching angles or produce rigid casts that when removed for imaging are fragile and thus easily damaged. We address these problems by creating an in situ bronchial cast of the conducting airways in rats that can be subsequently imaged in situ using three-dimensional micro-CT imaging. We also demonstrate that deformations in airway branch angles resulting from the casting procedure are small, and that these angle deformations can be reversed through an interactive adjustment of the segmented cast geometry. Animal work was approved by the Institutional Animal Care and Use Committee of Pacific Northwest National Laboratory.

Automated, foot-bone registration using subdivision-embedded atlases for spatial mapping of bone mineral density.

We present an atlas-based registration method for bones segmented from quantitative computed tomography (QCT) scans, with the goal of mapping their interior bone mineral densities (BMDs) volumetrically. We introduce a new type of deformable atlas, called subdivision-embedded atlas, which consists of a control grid represented as a tetrahedral subdivision mesh and a template bone surface embedded within the grid. Compared to a typical lattice-based deformation grid, the subdivision control grid possesses a relatively small degree of freedom tailored to the shape of the bone, which allows efficient fitting onto subjects. Compared with previous subdivision atlases, the novelty of our atlas lies in the addition of the embedded template surface, which further increases the accuracy of the fitting. Using this new atlas representation, we developed an efficient and fully automated pipeline for registering atlases of 12 tarsal and metatarsal bones to a segmented QCT scan of a human foot. Our evaluation shows that the mapping of BMD enabled by the registration is consistent for bones in repeated scans, and the regional BMD automatically computed from the mapping is not significantly different from expert annotations. The results suggest that our improved subdivision-based registration method is a reliable, efficient way to replace manual labor for measuring regional BMD in foot bones in QCT scans.

Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP).

The Human BioMolecular Atlas Program (HuBMAP) aims to create a multi-scale spatial atlas of the healthy human body at single-cell resolution by applying advanced technologies and disseminating resources to the community. As the HuBMAP moves past its first phase, creating ontologies, protocols and pipelines, this Perspective introduces the production phase: the generation of reference spatial maps of functional tissue units across many organs from diverse populations and the creation of mapping tools and infrastructure to advance biomedical research.

Automated platform for high-resolution tissue imaging using nanospray desorption electrospray ionization mass spectrometry.

An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours by precisely controlling the distance between the sample and the nano-DESI probe. This is achieved by mounting the sample holder onto an automated XYZ stage, defining the tilt of the sample plane, and recalculating the vertical position of the stage at each point. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSI QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. Low background noise enables simultaneous detection of lipids and metabolites in the tissue section. High-resolution mass analysis combined with tandem mass spectometry (MS/MS) experiments enabled identification of the observed species. In addition, the high dynamic range (>2000) of the technique allowed us to generate ion images of low-abundance isobaric lipids. A high-spatial resolution image was acquired over a small region of the tissue section revealing the distribution of an abundant brain metabolite, creatine, on the boundary between the white and gray matter. The observed distribution is consistent with the literature data obtained using magnetic resonance spectroscopy.

An optimized approach and inflation media for obtaining complimentary mass spectrometry-based omics data from human lung tissue.

Human disease states are biomolecularly multifaceted and can span across phenotypic states, therefore it is important to understand diseases on all levels, across cell types, and within and across microanatomical tissue compartments. To obtain an accurate and representative view of the molecular landscape within human lungs, this fragile tissue must be inflated and embedded to maintain spatial fidelity of the location of molecules and minimize molecular degradation for molecular imaging experiments. Here, we evaluated agarose inflation and carboxymethyl cellulose embedding media and determined effective tissue preparation protocols for performing bulk and spatial mass spectrometry-based omics measurements. Mass spectrometry imaging methods were optimized to boost the number of annotatable molecules in agarose inflated lung samples. This optimized protocol permitted the observation of unique lipid distributions within several airway regions in the lung tissue block. Laser capture microdissection of these airway regions followed by high-resolution proteomic analysis allowed us to begin linking the lipidome with the proteome in a spatially resolved manner, where we observed proteins with high abundance specifically localized to the airway regions. We also compared our mass spectrometry results to lung tissue samples preserved using two other inflation/embedding media, but we identified several pitfalls with the sample preparation steps using this preservation method. Overall, we demonstrated the versatility of the inflation method, and we can start to reveal how the metabolome, lipidome, and proteome are connected spatially in human lungs and across disease states through a variety of different experiments.

The insulin-like growth factor pathway is altered in spinocerebellar ataxia type 1 and type 7.

Polyglutamine diseases are inherited neurodegenerative disorders caused by expansion of CAG repeats encoding a glutamine tract in the disease-causing proteins. There are nine disorders, each having distinct features but also clinical and pathological similarities. In particular, spinocerebellar ataxia type 1 and 7 (SCA1 and SCA7) patients manifest cerebellar ataxia with degeneration of Purkinje cells. To determine whether the disorders share molecular pathogenic events, we studied two mouse models of SCA1 and SCA7 that express the glutamine-expanded protein from the respective endogenous loci. We found common transcriptional changes, with down-regulation of insulin-like growth factor binding protein 5 (Igfbp5) representing one of the most robust changes. Igfbp5 down-regulation occurred in granule neurons through a non-cell-autonomous mechanism and was concomitant with activation of the insulin-like growth factor (IGF) pathway and the type I IGF receptor on Purkinje cells. These data define one common pathogenic response in SCA1 and SCA7 and reveal the importance of intercellular mechanisms in their pathogenesis.

Spatial distribution of marker gene activity in the mouse lung during alveolarization.

This data is a curated collection of visual images of gene expression patterns from the pre- and post-natal mouse lung, accompanied by associated mRNA probe sequences and RNA-Seq expression profiles. Mammalian lungs undergo significant growth and cellular differentiation before and after the transition to breathing air. Documenting normal lung development is an important step in understanding abnormal lung development, as well as the challenges faced during a preterm birth. Images in this dataset indicate the spatial distribution of mRNA transcripts for over 500 different genes that are active during lung development, as initially determined via RNA-Seq. Images were systematically acquired using high-throughput in situ hybridization with non-radioactive digoxigenin-labeled mRNA probes across mouse lungs from developmental time points E16.5, E18.5, P7, and P28. The dataset was produced as part of The Molecular Atlas of Lung Development Program (LungMAP) and is hosted at https://lungmap.net. This manuscript describes the nature of the data and the protocols for generating the dataset.

Towards High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry Coupled to Shear Force Microscopy.

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 µm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue height were found to be dependent on the tissue type and were in the range of 0-5 µm for lung tissue and 0-3 µm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects. Graphical Abstract ᅟ.

Cell type-resolved human lung lipidome reveals cellular cooperation in lung function.

Cell type-resolved proteome analyses of the brain, heart and liver have been reported, however a similar effort on the lipidome is currently lacking. Here we applied liquid chromatography-tandem mass spectrometry to characterize the lipidome of major lung cell types isolated from human donors, representing the first lipidome map of any organ. We coupled this with cell type-resolved proteomics of the same samples (available at Lungmap.net). Complementary proteomics analyses substantiated the functional identity of the isolated cells. Lipidomics analyses showed significant variations in the lipidome across major human lung cell types, with differences most evident at the subclass and intra-subclass (i.e. total carbon length of the fatty acid chains) level. Further, lipidomic signatures revealed an overarching posture of high cellular cooperation within the human lung to support critical functions. Our complementary cell type-resolved lipid and protein datasets serve as a rich resource for analyses of human lung function.

Hemodynamics Modify Collagen Deposition in the Early Embryonic Chicken Heart Outflow Tract.

Blood flow is critical for normal cardiac development. Hemodynamic stimuli outside of normal ranges can lead to overt cardiac defects, but how early heart tissue remodels in response to altered hemodynamics is poorly understood. This study investigated changes in tissue collagen in response to hemodynamic overload in the chicken embryonic heart outflow tract (OFT) during tubular heart stages (HH18 to HH24, ~24 h). A suture tied around the OFT at HH18 was tightened to constrict the lumen for ~24 h (constriction range at HH24: 15-60%). Expression of fibril collagens I and III and fibril organizing collagens VI and XIV were quantified at the gene and protein levels via qPCR and quantitative immunofluorescence. Collagen I was slightly elevated upstream of the band and in the cushions in banded versus control OFTs. Changes in collagen III were not observed. Collagen VI deposition was elevated downstream of the band, but not overall. Collagen XIV deposition increased throughout the OFT, and strongly correlated to lumen constriction. Interestingly, organization of collagen I fibrils was observed for the tighter banded embryos in regions that also showed increase in collagen XIV deposition, suggesting a potentially key role for collagens I and XIV in the structural adaptation of embryonic heart tissue to hemodynamic overload.

Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung.

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylphosphatidylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murine lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 924 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. This multi-omic view provides a unique resource and deeper insight into normal pulmonary development.

A transcriptome atlas of the mouse brain at cellular resolution.

A genome-wide expression atlas of the nervous system at cellular resolution would be a valuable resource for neurobiology, genetics, developmental biology and medicine. Progress in automation of in situ hybridization makes such an atlas possible. Standardized and computerized annotation of expression patterns will be critical for producing a searchable atlas database that can be accessed through the internet.