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.
Project description: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. The experiment was performed with 3 replicates.
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:AVICs were exposed to cyclic stretch to examine the role of mechanical stimuli on gene expression AVICs cultured on collagen 1 coated Bioflex were exposed to 14% stretch at 1 hz or static conditions using a Flexcell-5000 14% stretch was the experimental condition while the static condition was the control
Project description:Aortic valve calcification is a significant and serious clinical problem for which there are no effective medical treatments. Individuals born with bicuspid aortic valves, 1-2% of the population, are at the highest risk of developing aortic valve calcification. Aortic valve calcification involves increased levels of calcification and inflammatory genes. Bicuspid aortic valve leaflets experience increased strain. The molecular mechanisms involved in the pathogenesis of calcification of BAVs are not well understood, especially the molecular response to mechanical stretch. HOTAIR is a long non-coding RNA (lncRNA) that has been implicated with cancer but has not been studied in cardiac disease. We have found that HOTAIR levels are decreased in BAVs and in human aortic interstitial cells (AVICs) exposed to cyclic stretch. Reducing HOTAIR levels via siRNA in AVICs results in increased expression of calcification genes.
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-α (20 ng/ml; [TNF]); 5) mechanical stretch plus LPS [LPS+S], and 6) mechanical stretch plus TNF-α [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).
Project description:The aim of the experiment was to determine the effect of cyclic stretch-relaxation ("stretch") on gene expression patterns in normal diploid human bladder smooth muscle cells. Cells plated on silicone elastomer bottomed 6-well culture dishes were grown to ~80% confluence, serum-depleted for 48h and subjected to cyclic stretch-relaxation at 20% elongation for 4h. Cells seeded in stretch plates but not subjected to stretch served as controls. Total RNA was extracted from both groups of cells, reverse-transcribed, biotin-labeled, fragmented and hybridized to HG-U133A. Four biological replicates were generated for each treatment group (non-stretched or stretched).
Project description:We profiled the transcriptomes of 4 human lung cell types that were subjected to control (non-stretch) and 30% tonic stretch conditions for 4 hours and 24 hours. Total RNA extracted from cells under control (time-matched) or 30% stretch conditions (4 hours or 24 hours) performed in technical replicate experiments. human lung alveolar A549 cells, human lung bronchoepithelial 16HBE14o- cells, human fetal lung fibroblasts CCL-153, human juvenile lung fibroblasts CCL-151
Project description:Mechanical stress is a potent regulator of cell growth, contractility and gene regulation. Abnormal uterine distension during pregnancy increases the risk of preterm birth and likely activates crosstalk between multiple signaling networks with protein phosphorylation playing a critical role. Telomerized human uterine smooth muscle cells were exposed to 18% biaxial stretch for 5 min and the phosphoproteome was probed by mass spectrometry. We observed specific phospho-activation of mitogen activated protein kinase at threonine 183 and tyrosine 185, myosin regulatory light chain 9 at threonine 19, and heat shock protein 27 at serine 82. Our analysis revealed protein phosphorylation changes in signaling pathways related to actin cytoskeleton remodeling, activation of the focal adhesion kinase pathway, smooth muscle contraction and mechanistic target of rapamycin activation. These data point to potential mechanistic links between stretch-induced phosphorylation and development of the contractile phenotype in myometrial cells.
Project description:A novel avian-origin H7N9 influenza A virus (IAV) emerged in China in early 2013 causing mild to lethal human respiratory infections. H7N9 originated from multiple reassortment events between avian viruses and carries genetic markers of human adaptation. Determining whether H7N9 induces a host-response closer to human or avian IAV is important to better characterize this emerging virus. Here we compared the human lung epithelial cell response to infection with A/Anhui/01/13 (H7N9) or highly pathogenic avian-origin H5N1, H7N7, or human seasonal H3N2 IAV. Here, polarized confluent monolayers of Calu-3 cells were infected apically with the avian-origin IAVs A/Anhui/01/2013 (H7N9) [Anhui01], A/Netherland/219/2003 (H7N7) [NL219], A/Vietnam/1203/2004 (H5N1) [VN1203], or a human seasonal virus A/Panama/2007/1999 (H3N2) [Pan99] at an MOI of 1. Time-matched mocks were also included using the same cell stock as the rest of the samples. Culture medium (same as what the virus stock is in) was used for the mock infections. Quadruplicate wells were infected for each virus/timepoint. Measured timepoints were 3, 7, 12 and 24 hours post-inoculation and the RNA was used for transcriptional analysis via microarray.
Project description:Rationale: During excessive pressure or overload, cardiac cells are subjected to increased mechanical stress. We investigate how the stress response of cardiac cells to mechanical stress can be compared to genotoxic stresses induced by DNA damaging agents. Methods: Cultures enriched for cardiomyocytes and cultures of fibroblasts were derived from ventricles of neonatal rat hearts. In three independent experiments, both cell cultures were subjected to mechanical stress (cyclic stretch at 60 cycles/min), UV irradiation (10 J/m2), or X-rays (8.5 Gy). Cells were harvested 24 h. after (the onset of) treatment. Total RNA was isolated using an RNeasy® kit (Qiagen). Single stranded cDNA was synthesized from RNA from myocyte-enriched populations (10-14 microgram) and populations of fibroblasts (7.5-14 microgram), using Superscript II reverse transcriptase and T7-oligo(dT)24 primers at 42ºC for 1 h. Double stranded cDNA was obtained by using DNA ligase, DNA polymerase I and RNAse H at 16ºC for 2 h, followed by T4 DNA polymerase at 16ºC for 5 min. cRNA was synthesized from cDNA using the BioArray HighYield® RNA transcript labeling kit (Enzo) in the presence of biotin-labeled CTP and UTP. cRNA was fragmented in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM KAc and 30 mM MgAc, at 94ºC for 35 min. Fragmented cRNA was hybridized to GeneChip RG-U34A arrays (Affymetrix) at 45ºC for 16 h. Arrays were washed and incubated with a streptavidin-phycoerythrin complex. Fluorescent signals were determined with a GeneChip scanner.