Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of human airway epithelium, large airways, pre and post-mechanical injury

ABSTRACT: Responses of the Human Airway Epithelium Transcriptome to In Vivo Injury; To identify genes participating in repair of the human airway epithelium following injury, we used bronchoscopy and brushing to denude the airway epithelium of healthy individuals, sequentially sampled the same region 7 and 14 days later, and assessed the recovered epithelium for relative levels of gene expression using Affymetrix high-density oligonucleotide microarrays with TaqMan PCR confirmation. Histologic assessment showed that the epithelium was denuded immediately following injury, at 7 days the epithelium was completely covered but partially de-differentiated, and by 14 days there was close to normal proportions of differentiated cells. Gene expression analysis was carried out with both the Affymetrix Microarray Suite 5.0 and Robust Multi-array Average algorithms, applying a multiple test correction to identify bona fide changes in gene expression. At day 7, there were substantial differences in the gene expression pattern compared to the resting epithelium, with a distinctive airway epithelial â??repair transcriptomeâ?? of actively proliferating cells in the process of re-differentiation. The repair transcriptome at 7 days was dominated by genes encoding proteins involved in cell cycle regulation, transcription, signal transduction, metabolism and transport. Interestingly, the majority of cell cycle genes differentially expressed at day 7 belonged to the G2 and M late phases of the cell cycle, suggesting that the proliferating cells are relatively synchronized 1 wk following injury. At 14 days post-injury, the majority of the gene expression changes observed at day 7 were no longer observed, with the expression profile similar to that of resting airway epithelium. Using a class prediction algorithm, a group of 50 genes dominated by cell cycle genes, that represent a human airway epithelial â??repair signatureâ?? was identified. These observations provide a baseline of the functional gene categories participating in the process of normal human airway epithelial repair that can be used in future studies of injury and repair in human airway epithelial diseases. Experiment Overall Design: comparison of gene expression in airway epithelial cells of the large airways, before and after mechanical injury caused by airway brushing

ORGANISM(S): Homo sapiens

SUBMITTER: Jacqueline Salit

PROVIDER: E-GEOD-5372 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Responses of the human airway epithelium transcriptome to in vivo injury.

Heguy Adriana A Harvey Ben-Gary BG Leopold Philip L PL Dolgalev Igor I Raman Tina T Crystal Ronald G RG

Physiological genomics 20061212 2

To identify genes participating in human airway epithelial repair, we used bronchoscopy and brushing to denude the airway epithelium of healthy individuals, sequentially sampled the same region 7 and 14 days later, and assessed gene expression by Affymetrix microarrays with TaqMan RT-PCR confirmation. Histologically, the injured area was completely covered by a partially redifferentiated epithelial layer after 7 days; by 14 days the airway epithelium was very similar to the uninjured state. At d ...[more]

PMID: 17164391

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Project description:Responses of the Human Airway Epithelium Transcriptome to In Vivo Injury To identify genes participating in repair of the human airway epithelium following injury, we used bronchoscopy and brushing to denude the airway epithelium of healthy individuals, sequentially sampled the same region 7 and 14 days later, and assessed the recovered epithelium for relative levels of gene expression using Affymetrix high-density oligonucleotide microarrays with TaqMan PCR confirmation. Histologic assessment showed that the epithelium was denuded immediately following injury, at 7 days the epithelium was completely covered but partially de-differentiated, and by 14 days there was close to normal proportions of differentiated cells. Gene expression analysis was carried out with both the Affymetrix Microarray Suite 5.0 and Robust Multi-array Average algorithms, applying a multiple test correction to identify bona fide changes in gene expression. At day 7, there were substantial differences in the gene expression pattern compared to the resting epithelium, with a distinctive airway epithelial “repair transcriptome” of actively proliferating cells in the process of re-differentiation. The repair transcriptome at 7 days was dominated by genes encoding proteins involved in cell cycle regulation, transcription, signal transduction, metabolism and transport. Interestingly, the majority of cell cycle genes differentially expressed at day 7 belonged to the G2 and M late phases of the cell cycle, suggesting that the proliferating cells are relatively synchronized 1 wk following injury. At 14 days post-injury, the majority of the gene expression changes observed at day 7 were no longer observed, with the expression profile similar to that of resting airway epithelium. Using a class prediction algorithm, a group of 50 genes dominated by cell cycle genes, that represent a human airway epithelial “repair signature” was identified. These observations provide a baseline of the functional gene categories participating in the process of normal human airway epithelial repair that can be used in future studies of injury and repair in human airway epithelial diseases. Keywords: response to airway injury

Project description:Understanding the way in which the airway heals in response to injury is fundamental to dissecting the mechanisms underlying airway disease pathology. Only limited data is available in relation to in vivo characterisation of the molecular features of repair in the airway. This study sought to characterise the dynamic changes in gene expression that are associated with airway repair in response to physical injury. Gene expression changes in the airway wall following bronchial brush biopsy were profiled in anaesthetised sheep. The experimental design featured sequential studies in the same animals (n=8) over the course of a week and yielded data relating to the repair process at 6 hours, and 1, 3 and 7 days after injury. Notable features of the transcriptional response included the early and sustained preponderance of down-regulated genes associated with angiogenesis and immune cell activation, selection and differentiation. Later features of the repair response included the up-regulation of cell cycle genes at d1 and d3, and the later pronounced up-regulation of extracellular matrix-related genes at d3 and d7. It is possible to follow the process of airway wall repair in response to physical injury in the same animal over the course of time. Transcriptional changes featured coordinate expression of functionally related genes in a reproducible manner both within and between animals. This characterisation will provide a foundation against which to assess the perturbations that accompany airway disease pathologies of comparative relevance. Keywords: response to airway injury Each sheep was subjected to a protocol which involved the procedure of endobronchial brush biopsy (BBr) being performed, under anaesthesia, on three occasions, with two separate airway sites being subjected to brushing on each occasion. Two sheep were subjected to BBr seven days, three days and six hours prior to euthanasia, two sheep at seven days, three days and one day prior to euthanasia, two sheep at seven days, one day and six hours prior to euthanasia and two sheep at three days, one day and six hours prior to euthanasia. At post mortem examination (PME) each time point was therefore represented by material derived from six sheep. Airway tissue from a naM-CM-/ve site (from a segment not subjected to BBr) was also collected from each sheep at necropsy.

Project description:In the process of seeking novel lung host defense regulators by analyzing genome-wide RNA sequence data from normal human airway epithelium, we detected expression of POU2AF1, a known transcription co-factor previously thought to be expressed only in lymphocytes. Lymphocyte contamination of human airway epithelial samples obtained by bronchoscopy and brushing was excluded by immunohistochemistry staining, the observation of up-regulation of POU2AF1 in purified airway basal stem/progenitor cells undergoing differentiation and analysis of differentiating single basal cell clones. Lentivirus-mediated up-regulation of POU2AF1 in airway basal cells induced up-regulation of host defense genes, including MX1, IFIT3, IFITM and known POU2AF1 downstream genes HLA-DRA, ID2, ID3, IL6, BCL6. Interestingly, expression of these genes paralleled changes of POU2AF1 expression during airway epithelium differentiation in vitro, suggesting POU2AF1 helps to maintain a "host defense tone" even in pathogen-free condition. Cigarette smoke, a known risk factor for airway infection, suppressed POU2AF1 expression both in vivo in humans and in vitro in human airway epithelial cultures, accompanied by deregulation of POU2AF1 downstream genes. Finally, enhancing POU2AF1 expression in human airway epithelium attenuated the suppression of host defense genes by smoking. Together, these findings suggest a novel function of POU2AF1 as a potential regulator of host defense genes in the human airway epithelium. Methods: Massive parallel RNA sequencing was used to compare the transcriptome of lentivirus mediated POU2AF1 or RFP (control) gene expression in human primary airway epithelial cells (3 samples per group). Uninfected basal cell was used as a further control. Conclusions: The genes up-regulated by POU2AF1 in human airway epithelial cells are mainly related to the intracellular or extracellular anti-pathogen response, suggesting POU2AF1 plays a role in airway epithelial host defense. This Series represents samples complementary to those in GSE60989.

Project description:Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair. Bronchiolar Clara cells undergo phenotypic changes during development and in disease. These changes are poorly described due to a paucity of molecular markers. We used chemical and transgenic approaches to ablate Clara cells, allowing identification of their unique gene expression profile. Flavin monooxygenase 3 (Fmo3), paraoxonase 1 (Pon1), aldehyde oxidase 3 (Aox3), and claudin 10 (Cldn10) were identified as novel Clara cell markers. New and existing Clara cell marker genes were categorized into three classes based on their unique developmental expression pattern. Cldn10 was uniformly expressed in the epithelium at Embryonic Day (E)14.5 and became restricted to secretory cells at E18.5. This transition was defined by induction of Clara Cell Secretory Protein (CCSP). Maturation of secretory cells was associated with progressive increases in the expression of Fmo3, Pon1, Aox3, and Cyp2f2 between late embryonic and postnatal periods. Messenger RNA abundance of all categories of genes was dramatically decreased after naphthalene-induced airway injury, and displayed a sequence of temporal induction during repair that suggested sequential secretory cell maturation. We have defined a broader repertoire of Clara cell-specific genes that allows staging of epithelial maturation during development and repair. 32 samples were isolated and used to screen UniSet Mouse 20K I Bioarrays (total lung RNA Mus musculus). Control RNA was isolated from uninjured mice (N = 4 mice), and RNA was also isolated from mice exposed to naphthalene and recovered for 1, 2, 3, and 6 days (N = 4 mice per timepoint). Additionally, transgenic mice expressing HSV-tk under control of the mouse CCSP promoter were exposed to Ganciclovir (GCV) and recovered for 3, 6, and 9 days (N = 4 mice per timepoint).

Dataset Information

Transcription profiling of human airway epithelium, large airways, pre and post-mechanical injury

Publications

Responses of the human airway epithelium transcriptome to in vivo injury.

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