Temporal gene expression of mesenchymal cells in the pediatric lung

INTRODUCTION: The newborn lung undergoes vast biochemical and physiological changes during adaptation from the intrauterine to the extrauterine environment. Lung morphogenesis continues from birth into early childhood, mediated by dynamic gene expression and a diversity of pulmonary cell types (Whitsett, JA. et al. Physiol. Rev, 2019). Murine models demonstrate that pulmonary mesenchymal cells exhibit remarkable heterogeneity in function and morphology during development, however, confirmation of their role is lacking in human neonates and early childhood (Guo, M. et al. Nat. Comm, 2019). In addition, many current human genomic studies of lung maturation suffer from limited sample size, limiting their applicability to longitudinal pediatric lung development. Temporal analysis of gene expression aims to bridge this gap, and the most common analytical approach utilizes Short Time-series Expression Miner (STEM) (Ernst, J. & Bar-Joseph, Z. BMC Bioinformatics, 2006). STEM utilizes unique methods to cluster, compare, and visualize short time-series gene expression data. METHODS: Dissociation of lung cells, sorting into enriched populations, and RNA isolation was performed at the Human Tissue Core of the Molecular Atlas of Lung Development Program (Bandyopadhyay, G. et al. Am. J. Physiol. Lung Cell Mol. Physiol, 2018). RNA sequencing (RNAseq) was performed at the University of Rochester Genomics Research Center using the Ilumina NovaSeq6000, and reads were aligned using the Splice Transcript Alignment to a Reference algorithm (STaR). Reads were further normalized using counts per million (CPM) and variance-mean dependence calculated with DESeq as implemented in Bioconductor. Genes not detected in at least 3 time points or exhibiting a minimum fold change of at least 3 across the time series were excluded from further analysis. Time-series analysis was performed with STEM, and profiles were assigned significance by Fisher’s exact test (p\u3c0.05). Genes selected from profiles of interest were functionally enriched using ToppGene Functional Gene Enricher (Chen, J. et al. BMC Bioinformatics, 2007). RESULTS: RNAseq was performed using RNA obtained from pulmonary mesenchymal cells, (n=24, (\u3c1 d/o - 8 y/o, 17 m, 7 f) generating 24.3±5.5 million reads at depth of 10 million reads (48.3±4.6% of genome mapped). CPM normalized expression values for repeat donor time points were averaged and then separated into a younger (n=9, \u3c1 d/o - 1 y/o) and older (n=8, 1 y/o - 8 y/o) group. A total of 17,843 genes passed filtering criteria in the younger group and 17,840 passed in the older group. Using STEM, 16 and 20 profiles were found to be significant in the younger and older group, respectively. 7 profiles in the younger group and 8 profiles in the older group were selected for further functional analysis based on significance and directionality of gene expression changes. Multiple profiles in both groups demonstrated matrix fibroblast associated gene expression increasing in both groups, peaking at 2 years. Next, proliferative fibroblast and cell division associated gene expression decreased from birth to 1 year in the younger group. Detection of multiple mesenchymal- like profiles validates the purity of cells enriched. Additionally, gene expression associated with immune- like pathways increased in both groups. Finally, cell signatures in the older group associated with the Wnt pathway decreased from 1 year until 2 years and then increased from 4 years to 8 years. CONCLUSIONS: In summary, analysis of dynamic gene expression in isolated cells across a time series demonstrates the unique heterogeneity of pulmonary mesenchymal cells throughout adolescence. In addition, increased gene expression associated with immune signatures during pediatric lung development was noted. Further validation and exploration using this technique may advance understanding of the diversity of pulmonary cell types and pathophysiology of pediatric lung disease

Platform dependence of inference on gene-wise and gene-set involvement in human lung development

<p>Abstract</p> <p>Background</p> <p>With the recent development of microarray technologies, the comparability of gene expression data obtained from different platforms poses an important problem. We evaluated two widely used platforms, Affymetrix U133 Plus 2.0 and the Illumina HumanRef-8 v2 Expression Bead Chips, for comparability in a biological system in which changes may be subtle, namely fetal lung tissue as a function of gestational age.</p> <p>Results</p> <p>We performed the comparison via sequence-based probe matching between the two platforms. "Significance grouping" was defined as a measure of comparability. Using both expression correlation and significance grouping as measures of comparability, we demonstrated that despite overall cross-platform differences at the single gene level, increased correlation between the two platforms was found in genes with higher expression level, higher probe overlap, and lower p-value. We also demonstrated that biological function as determined via KEGG pathways or GO categories is more consistent across platforms than single gene analysis.</p> <p>Conclusion</p> <p>We conclude that while the comparability of the platforms at the single gene level may be increased by increasing sample size, they are highly comparable ontologically even for subtle differences in a relatively small sample size. Biologically relevant inference should therefore be reproducible across laboratories using different platforms.</p

Type VI collagen promotes lung epithelial cell spreading and wound-closure.

Basement membrane (BM) is an essential part of the extracellular matrix (ECM) that plays a crucial role in mechanical support and signaling to epithelial cells during lung development, homeostasis and repair. Abnormal composition and remodeling of the lung ECM have been associated with developmental abnormalities observed in multiple pediatric and adult respiratory diseases. Collagen VI (COL6) is a well-studied muscle BM component, but its role in the lung and its effect on pulmonary epithelium is largely undetermined. We report the presence of COLVI immediately subjacent to human airway and alveolar epithelium in the pediatric lung, in a location where it is likely to interact with epithelial cells. In vitro, both primary human lung epithelial cells and human lung epithelial cell lines displayed an increased rate of "wound healing" in response to a scratch injury when plated on COL6 as compared to other matrices. For the 16HBE cell line, wounds remained >5-fold larger for cells on COL1 (p6-fold larger on matrigel (p96% closure at 10 hr). The effect of COL6 upon lung epithelial cell phenotype was associated with an increase in cell spreading. Three hours after initial plating, 16HBE cells showed >7-fold less spreading on matrigel (p4-fold less spreading on COL1 (p<0.01) when compared to COL6. Importantly, the addition of COL6 to other matrices also enhanced cell spreading. Similar responses were observed for primary cells. Inhibitor studies indicated both integrin β1 activity and activation of multiple signaling pathways was required for enhanced spreading on all matrices, with the PI3K/AKT pathway (PI3K, CDC42, RAC1) showing both significant and specific effects for spreading on COL6. Genetic gain-of-function experiments demonstrated enhanced PI3K/AKT pathway activity was sufficient to confer equivalent cell spreading on other matrices as compared to COL6. We conclude that COL6 has significant and specific effects upon human lung epithelial cell-autonomous functions

Expression Profiles of the Mouse Lung Identify a Molecular Signature of Time-to-Birth

A greater understanding of the regulatory processes contributing to lung development could help ameliorate morbidity and mortality in premature infants and identify individuals at risk for congenital and/or chronic lung diseases. Genomics technologies have provided rich gene expression datasets for the developing lung that enable systems biology approaches for identifying large-scale molecular signatures within this complex phenomenon. Here, we applied unsupervised principal component analysis on two developing lung datasets and identified common dominant transcriptomic signatures. Of particular interest, we identify an overlying biological program we term “time-to-birth,” which describes the distance in age from the day of birth. We identify groups of genes contributing to the time-to-birth molecular signature. Statistically overrepresented are genes involved in oxygen and gas transport activity, as expected for a transition to air breathing, as well as host defense function. In addition, we identify genes with expression patterns associated with the initiation of alveolar formation. Finally, we present validation of gene expression patterns across the two datasets, and independent validation of select genes by qPCR and immunohistochemistry. These data contribute to our understanding of genetic components contributing to large-scale biological processes and may be useful, particularly in animal models of abnormal lung development, to predict the state of organ development or preparation for birth

MicroRNA expression profiling defines the impact of electronic cigarettes on human airway epithelial cells

While all forms of tobacco exposure have negative health effects, the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here, we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM, GCLC, GPX2, NQO1 and HO-1. Vaporization of, and/or the presence of nicotine in, eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE, and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A, 140, 126, 374A, 26A-2, 147B, 941 and 589) for further study. We validated increased expression of multiple miRNAs, including miR126, following eCig exposure. We also found significant reduction in the expression of two miR126 target genes, MYC and MRGPRX3, following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells, some of which are epigenetically programmed at the level of miRNA regulation

Fibroblast Growth Factor Receptors Control Epithelial–Mesenchymal Interactions Necessary for Alveolar Elastogenesis

Rationale: The mechanisms contributing to alveolar formation are poorly understood. A better understanding of these processes will improve efforts to ameliorate lung disease of the newborn and promote alveolar repair in the adult. Previous studies have identified impaired alveogenesis in mice bearing compound mutations of fibroblast growth factor (FGF) receptors (FGFRs) 3 and 4, indicating that these receptors cooperatively promote postnatal alveolar formation

Transcriptional Regulation on Aneuploid Chromosomes in Diverse Candida albicans Mutants

Abstract Candida albicans is a diploid fungus and a predominant opportunistic human pathogen. Notably, C. albicans employs reversible chromosomal aneuploidies as a means of survival in adverse environments. We previously characterized transcription on the monosomic chromosome 5 (Ch5) that arises with adaptation to growth on the toxic sugar sorbose in the mutant Sor125(55). We now extend this analysis to the trisomic hybrid Ch4/7 within Sor125(55) and a diverse group of three mutants harboring a single Ch5. We find a similar pattern of transcriptional changes on either type of aneuploid chromosome within these mutants wherein expression of many genes follows chromosome ploidy, consistent with a direct mechanism to regulate genes important for adaptation to growth. In contrast, a significant number of genes are expressed at the disomic level, implying distinct mechanisms compensating for gene dose on monosomic or trisomic chromosomes consistent with maintaining cell homeostasis. Finally, we find evidence for an additional mechanism that elevates expression of genes on normal disomic Ch4 and Ch7 in mutants to levels commensurate with that found on the trisomic Ch4/7b in Sor125(55). Several of these genes are similarly differentially regulated among mutants, suggesting they play key functions in either maintaining aneuploidy or adaptation to growth conditions