Unknown,Transcriptomics,Genomics,Proteomics

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Expression levels in strained vs. non-strained Calu-3 lung epithelial cells

ABSTRACT: Ventilator induced lung injury can lead to serious conditions like ARDS which are associated with a high mortality (around 30%, Stapleton et al., Chest, 2005). We hypothesized that changes of expression levels of different genes would lead us to the identification of critical target genes, which might influence the inflammation and outcome associated with this condition. We used human whole genome U133 Plus 2.0 microarrays to detail the changes of gene expression and identified distinct classes of up-regulated genes during this process. Confluent non strained and strained human Calu-3 cells were selected for RNA extraction and hybridization on Affymetrix microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Michael Koeppen

PROVIDER: E-GEOD-34789 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Basal cells within the human airway epithelium constitute the stem/progenitor cells for other epithelial cell types. Basal cells respond to mucosal injury and damage to the airway mucosa in an ordered sequence of spreading, migration, proliferation and phenotype shifting (differentiation) to other needed cell types. However, dynamic gene transcription in the early events of injury and repair has not been examined in these cells. Methodology and findings: Airway epithelial cells were obtained from donated lungs and grown in submersion culture on pliable membranes to obtain a pure population of basal cells. Microarrays were used to assess the transcriptome of basal cells 8 and 24 hr after mechanical injury (MI), or to cyclic stretch (CS) in a Flexcell system (0.5 Hz, 20% distension), or both treatments. We identified 121 signature genes with > 2-fold higher differential expression (DE) 8 hr after MI; expression of nearly all of these genes returned to baseline at 24 hr after injury. In cells subjected to CS, little change in DE was noted at 8 hr, whereas at 24 hr a CS signature of 1430 DE genes were identified. The MI signature was characterized by genes encoding growth factor receptors related to the EGF pathway, IL-6, IL-8 and IL-33, extracellular matrix components, and NF-kB and p38-MAPK signaling pathways, whereas the CS signature was characterized by a broad range of genes that did not identify specific signaling pathways. Combined MI and CS at 8 hr elicited DE of down-regulated genes not seen with either stimulus alone, and at 24 hr elicited DE that was similar to that seen with CS alone. Conclusion and significance: The human airway basal epithelial cell transcription signature in the first hours after MI, after CS, and after both stimuli identifies unique differentially expressed genes and pathways that may be important in the early molecular response and biology to airway injury. Total RNA obtained from primary (AEC) and differentiated (dAEC) human airway epithelial cells subjected to 8 or 24 hours in vitro mechanical or cyclic stretch or both injuries compared to sham control as well as to type of injury. Cells were collected from four donated lungs and cultured separated in submission or air liquid interface condition prior to injury for various durations.

Project description:Acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung injury (ALI). The necessity for mechanical ventilation (MV) renders patients at risk for ventilator induced lung injury (VILI). Exposure to repetitive cyclic stretch (CS) and/or over-inflation exacerbates injury. Reducing tidal volume (VT) is the only therapeutic strategy shown to mitigate morbidity and mortality. Cyclic stretch has been shown to differentially regulate gene expression in part through the activation of mammalian mitogen-activated protein kinase (MAPK). Although these studies have shown both molecular and cellular alterations, no unifying hypothesis to explain MV-induced lung injury has emerged. In the current study, we hypothesized that coordinated expression of cyclic stretch (CS)-responsive genes relies on the presence of common CS-sensitive regulatory elements. To identify CS-responsive genes, we undertook a comparative examination of the gene expression profile of human bronchial epithelial airway (Beas-2B) cells in response to various injurious stimuli involved in the pathogenesis of acute lung injury (ALI)/Ventilator induced lung injury (VILI): cyclic stretch, tumor necrosis factor alpha (TNF-a), and lipopolysaccharide (LPS). Experiment Overall Design: Human Bronchial Epithelial Cells (Beas-B2) cells grown on silicon elastic plates coated with Type I collagen (Flexercell International, McKeesport, PA) were exposed to six regiments for 4 h: 1) control (static, [control]); 2) mechanical stretch (25 PKa, 30 cycles per min, [stretch]); 3) LPS (1 mcg/ml [LPS]); 4) TNF-Î± (20 ng/ml; [TNF]); 5) mechanical stretch plus LPS [LPS+S], and 6) mechanical stretch plus TNF-Î± [TNF+S]. Total RNA (duplicate experiments) was extracted using TRIZOL reagent (as per manufactures specifications) and purified using Qiagen mRNA purification Kit (as per manufacturers specifications). mRNA was hybridized to Affymetrix Human U133plus2.0 chips. Probe based analysis, background reduction, and quantile data normalization was performed in MeV 4.0 of TM4 using Robust Multi-array Average (RMA).

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Expression levels in strained vs. non-strained Calu-3 lung epithelial cells

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