Comparison of human aortic valve interstitial cells (AVICs) exposed to cyclic stretch to static samples
ABSTRACT: 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:We explored the hypothesis that Serotonin (5HT) receptor signaling, that can be enhanced with 5HT transporter blockade with Fluoxetine (Fluox), in the aortic valve may vary based upon the biomechanical activity of the aortic valve leaflet. We used Affymetrix microarrays to study gene expression profiling of Porcine Aortic Valves (PAV) incubated under organ culture conditions for 24 hours in either a static state or with 10% cyclic stretch, simulating physiologic leaflet motion. PAV in the bioreactor with or without stretch were exposed to 5HT along or the combination 5HT plus Fluox. Fresh porcine aortic valves were obtained from a local abattoir. The three leaflets were excised from each valve and a rectangular section of tissue 15x10 mm was isolated from the central region of each valve cusp. These samples were randomized and assigned to one of four groups. The experimental groups were: 1) Static conditions with no agents added; 2) Cyclic stretch conditions with no agents added; 3) Static conditions with 5HT plus Fluox added; and 4) Cyclic stretch conditions with 5HT plus Fluox added.
Project description:miRNA-Sequencing was performed on human aortic valve interestitial cells (AVICs) exposed to 14% stretch at 1 hz or static conditions for 24h. Six static control and six samples exposed to cyclic stretch 14% for 24h
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: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:Investigation of whole genome gene expression level changes in hAVICs and hMVICs, compared to human dermal fibroblasts. A three chip assay using total RNA obtained from primary cultured normal valve interstital cells and human dermal fibroblasts was performed. Each chip measures the expression level of 45,033 genes from normal AVICs,MVICs and DFs.
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: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.