[Show abstract][Hide abstract] ABSTRACT: Background:
Amniotic fluid (AF) is a proximal fluid to the fetus containing higher amounts of cell-free fetal RNA/DNA than maternal serum, thereby making it a promising source for identifying novel biomarkers that predict fetal development and organ maturation. Our aim was to compare AF transcriptomic profiles at different time points in pregnancy to demonstrate unique genetic signatures that would serve as potential biomarkers indicative of fetal maturation.
We isolated AF RNA from 16 women at different time points in pregnancy: 4 from 18 to 24 weeks, 6 from 34 to 36 weeks, and 6 from 39 to 40 weeks. RNA-sequencing was performed on cell-free RNA. Gene expression and splicing analyses were performed in conjunction with cell-type and pathway predictions.
Sample-level analysis at different time points in pregnancy demonstrated a strong correlation with cell types found in the intrauterine environment and fetal respiratory, digestive and external barrier tissues of the fetus, using high-confidence cellular molecular markers. While some RNAs and splice variants were present throughout pregnancy, many transcripts were uniquely expressed at different time points in pregnancy and associated with distinct neonatal co-morbidities (respiratory distress and gavage feeding), indicating fetal immaturity.
The AF transcriptome exhibits unique cell/organ-selective expression patterns at different time points in pregnancy that can potentially identify fetal organ maturity and predict neonatal morbidity. Developing novel biomarkers indicative of the maturation of multiple organ systems can improve upon our current methods of fetal maturity testing which focus solely on the lung, and will better inform obstetrical decisions regarding delivery timing.
BMC Medical Genomics 10/2015; 8(1):67. DOI:10.1186/s12920-015-0138-5 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the use of the mucin proteins MUC5B and MUC5AC as prognosis markers for non-small cell lung cancer (NSCLC) carrying epidermal growth factor receptor (EGFR) mutations.
Patients who underwent surgical resection at Nagasaki University Hospital and related facilities in Japan between June 1996 and March 2013.
159 Japanese patients (male: n=103; female: n=56) with NSCLC, who underwent surgical resection (EGFR-mutant type: n=78, EGFR wild type: n=81).
Patients whose tumours expressed MUC5B had significantly longer overall survival and relapse-free survival compared to the MUC5B-negative patients with EGFR mutant NSCLC (p=0.0098 and p=0.0187, respectively). In patients with EGFR wild-type NSCLC, there was no association with MUC5B expression. MUC5AC expression was not different between EGFR mutant and wild-type NSCLC.
Present findings indicate that MUC5B, but not MUC5AC, is a novel prognostic biomarker for patients with NSCLC carrying EGFR mutations but not for patients with NSCLC carrying wild-type EGFR.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
BMJ Open 07/2015; 5(7):e008366. DOI:10.1136/bmjopen-2015-008366 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed LungGENS (Lung Gene Expression iN Single-cell), a web-based bioinformatics resource for querying single-cell gene expression databases by entering a gene symbol or a list of genes or selecting a cell type of their interest. Gene query provides quantitative RNA expression of the gene of interest in each lung cell type. Cell type query returns associated selective gene signatures and genes encoding cell surface markers and transcription factors in interactive heatmap and tables. LungGENS will be broadly applicable in respiratory research, providing a cell-specific RNA expression resource at single-cell resolution. LungGENS is freely available for non-commercial use at https://research.cchmc.org/pbge/lunggens/default.html.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
[Show abstract][Hide abstract] ABSTRACT: Gas exchange after birth is entirely dependent on the remarkable architecture of the alveoli, its formation and function being mediated by the interactions of numerous cell types whose precise positions and functions are controlled by a diversity of signaling and transcriptional networks. In the later stages of gestation, alveolar epithelial cells lining the peripheral lung saccules produce increasing amounts of surfactant lipids and proteins that are secreted into the airspaces at birth. The lack of lung maturation and associated lack of pulmonary surfactant in preterm infants causes respiratory distress syndrome (RDS), a common cause of morbidity and mortality associated with premature birth. At the time of birth, surfactant homeostasis begins to be established by balanced processes involved in surfactant production, storage, secretion, recycling, and catabolism that serve to maintain alveolar homeostasis. Insights from physiology and engineering made in the 20th century enabled survival of newborn infants requiring mechanical ventilation for the first time. Thereafter, advances in biochemistry, biophysics, and molecular biology led to an understanding of the pulmonary surfactant system that made possible exogenous surfactant replacement for treatment of preterm infants. Identification of surfactant proteins, cloning of the genes encoding them, and elucidation of their roles in the regulation of surfactant synthesis, structure, and function has provided increasing understanding of alveolar function and homeostasis in health and disease. This "perspective" seeks to consider developmental aspects of the pulmonary surfactant system and its importance in the pathogenesis of acute and chronic lung diseases related to alveolar homeostasis.
American Journal of Respiratory Cell and Molecular Biology 05/2015; 53(1). DOI:10.1165/rcmb.2015-0128PS · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef-/- mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen.
The Journal of clinical investigation 04/2015; 125(5). DOI:10.1172/JCI79422 · 13.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances in physiology and biochemistry have provided fundamental insights into the role of pulmonary surfactant in the pathogenesis and treatment of preterm infants with respiratory distress syndrome. Identification of the surfactant proteins, lipid transporters, and transcriptional networks regulating their expression has provided the tools and insights needed to discern the molecular and cellular processes regulating the production and function of pulmonary surfactant prior to and after birth. Mutations in genes regulating surfactant homeostasis have been associated with severe lung disease in neonates and older infants. Biophysical and transgenic mouse models have provided insight into the mechanisms underlying surfactant protein and alveolar homeostasis. These studies have provided the framework for understanding the structure and function of pulmonary surfactant, which has informed understanding of the pathogenesis of diverse pulmonary disorders previously considered idiopathic. This review considers the pulmonary surfactant system and the genetic causes of acute and chronic lung disease caused by disruption of alveolar homeostasis.
Annual Review of Pathology Mechanisms of Disease 01/2015; 10(1):371-393. DOI:10.1146/annurev-pathol-012513-104644 · 18.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mucosal surfaces are exposed to the external environment and pathogens and are therefore protected by a secreted layer of mucus rich in mucin glycoproteins, which are the main components of mucus. Mucus provides physical protection and hydration, excludes pathogens, and is a reservoir for antimicrobial molecules. Underlying mucus further protection is provided by epithelial cell surface mucins, which limit microbial adherence and regulate growth and apoptosis. Differentiation of the cells that produce mucins, and expression of mucins and proteins involved in mucin biosynthesis, is regulated by innate and adaptive immunity. Experimental deficiencies in mucins lead to infectious and inflammatory diseases, and mucin gene polymorphisms are associated with disease. Many chronic mucosal inflammatory diseases are characterized by mucus hypersecretion driven by immune and microbial factors, which contributes to pathology and is a target for therapy. In this chapter we describe the nature of this mucosal barrier, its regulation and function, and its involvement in human disease, with particular emphasis on the mucin component of the barrier.
[Show abstract][Hide abstract] ABSTRACT: The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and 'instruct' the professional immune system to protect the lungs from infection and injury.
[Show abstract][Hide abstract] ABSTRACT: The ocular surface epithelia, including the stratified but non-keratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor β receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGFβ signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGFβ restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGFβ inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGFβ in regulating goblet cell differentiation.
Development 11/2014; 141(23). DOI:10.1242/dev.117804 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current treatments for inflammation associated with bronchopulmonary dysplasia fail to show clinical efficacy. Foxm1, a transcription factor of the Forkhead box family, is a critical mediator of lung development and carcinogenesis, but its role in bronchopulmonary dysplasia-associated pulmonary inflammation is unknown. Immunohistochemistry and RNA analysis were used to assess Foxm1 in lung tissue from hyperoxia-treated mice and patients with bronchopulmonary dysplasia. LysM-Cre/Foxm1-/- mice, in which Foxm1 was deleted from myeloid-derived inflammatory cells, including macrophages, monocytes and neutrophils, were exposed to neonatal hyperoxia causing lung injury and remodeling. Measurements of lung function and flow cytometry were used to evaluate effects of Foxm1 deletion on pulmonary inflammation and repair. Increased Foxm1 expression was observed in pulmonary macrophages of hyperoxia-exposed mice and lung tissue from patients with bronchopulmonary dysplasia. After hyperoxia, deletion of Foxm1 from the myeloid cell lineage decreased numbers of interstitial macrophages (CD45+CD11b+Ly6C-Ly6G-F4/80+CD68-) and impaired alveologenesis and lung function. The exaggerated bronchopulmonary dysplasia-like phenotype observed in hyperoxia-exposed LysM-Cre/Foxm1-/- mice was associated with increased expression of neutrophil-derived myeloperoxidase, proteinase 3 and cathepsin-g, all of which are critical for lung remodeling and inflammation. Our data demonstrate that Foxm1 influences pulmonary inflammatory responses to hyperoxia, inhibiting neutrophil-derived enzymes and enhancing monocytic responses that limit alveolar injury and remodeling in neonatal lungs.
American Journal of Respiratory Cell and Molecular Biology 10/2014; 52(5). DOI:10.1165/rcmb.2014-0091OC · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SAM-pointed domain-containing ETS transcription factor (SPDEF) is expressed in normal prostate epithelium. While its expression changes during prostate carcinogenesis (PCa), the role of SPDEF in prostate cancer remains controversial due to the lack of genetic mouse models. In present study, we generated transgenic mice with the loss- or gain-of-function of SPDEF in prostate epithelium to demonstrate that SPDEF functions as tumor suppressor in prostate cancer. Loss of SPDEF increased cancer progression and tumor cell proliferation, whereas over-expression of SPDEF in prostate epithelium inhibited carcinogenesis and reduced tumor cell proliferation in vivo and in vitro. Transgenic over-expression of SPDEF inhibited mRNA and protein levels of Foxm1, a transcription factor critical for tumor cell proliferation, and reduced expression of Foxm1 target genes, including Cdc25b, Cyclin B1, Cyclin A2, Plk-1, AuroraB, CKS1 and Topo2alpha. Deletion of SPDEF in transgenic mice and cultures prostate tumor cells increased expression of Foxm1 and its target genes. Furthermore, an inverse correlation between SPDEF and Foxm1 levels was found in human prostate cancers. The two-gene signature of low SPDEF and high FoxM1 predicted poor survival in prostate cancer patients. Mechanistically, SPDEF bound to, and inhibited transcriptional activity of Foxm1 promoter by interfering with the ability of Foxm1 to activate its own promoter through auto-regulatory site located in the -745/-660 bp Foxm1 promoter region. Re-expression of Foxm1 restored cellular proliferation in the SPDEF-positive cancer cells and rescued progression of SPDEF-positive tumors in mouse prostates. Altogether, SPDEF inhibits prostate carcinogenesis by preventing Foxm1-regulated proliferation of prostate tumor cells. The present study identified novel crosstalk between SPDEF tumor suppressor and Foxm1 oncogene and demonstrated that this crosstalk is required for tumor cell proliferation during progression of prostate cancer in vivo.
[Show abstract][Hide abstract] ABSTRACT: Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.
[Show abstract][Hide abstract] ABSTRACT: Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (ScapΔ/Δ, resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2Δ/Δ, resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/β-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners.
PLoS ONE 05/2014; 9(5):e91376. DOI:10.1371/journal.pone.0091376 · 3.23 Impact Factor