Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human bronchial epithelial cells exposed to cyclic stretch with and without LPS or TNFa

ABSTRACT: 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).

ORGANISM(S): Homo sapiens

SUBMITTER: Claudia Chimisso dos Santos

PROVIDER: E-GEOD-16650 | 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:Engineered cardiac tissues (ECTs) are platforms to investigate cardiomyocyte maturation and functional integration, to evaluate the feasibility of generating implantable tissues for cardiac repair and regeneration, and may be useful models for pharmacology and toxicology bioassays. These ECTs rapidly mature in vitro to acquire the features of functional cardiac muscle and respond to mechanical load with increased proliferation and maturation. ECTs can be generated from various immature cardiac cell sources and little is known regarding the broad changes in regulatory transcript expression that occur in these in vitro tissues during normal maturation and in response to mechanical or pharmacologic interventions. We tested the hypothesis that global ECT gene expression patterns are sensitive to mechanical loading conditions and tyrosine kinase inhibitors, similar to the maturing myocardium. We generated 3D ECTs from day 14.5 rat embryo ventricular cells, as previously published, and then treated constructs after 5 days in culture for 48 hours with mechanical stretch (5%, 0.5 Hz) and/or the p38MAPK (p38 mitogen-activated protein kinase) selective inhibitor BIRB796. RNA was isolated from 3 sets of experiments and assayed using a standard Agilent rat 4x44k V3 microarray and Pathway Analysis software for transcript expression fold changes and changes in regulatory molecules and networks. At the threshold of a 1.5 fold change in expression, mechanical stretch altered 1,559 transcripts, versus 1,411 for BIRB796, and 1,846 for stretch plus BIRB796. As anticipated, top pathways altered in response to these stimuli include Cellular Development, Cellular Growth and Proliferation; Tissue Development; Cell Death, Cell Signaling, and Small Molecule Biochemistry as well as numerous other pathways. Changes in transcript expression were confirmed by quantitative-PCR for selected regulatory molecules. Thus, ECTs display a broad spectrum of altered gene expression in response to mechanical load and/or tyrosine kinase inhibition, reflecting the complex regulation of proliferation, differentiation, and architectural alignment that occurs during ECT maturation and adaptation. This approach can now be used to test the role of individual molecules and pathways on the regulation of ECT maturation and remodeling. 7 and 4 biological replicates with four groups (control, mechanical stretch, BIRB and mechanical stretch with BIRB)

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Transcription profiling of human bronchial epithelial cells exposed to cyclic stretch with and without LPS or TNFa

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