Project description:Fluid induced shear stress is widely recognized as an important biophysical signal in cell-cell mechanotransduction. To identify cellular signaling pathways that are regulated by fluid shear stress, we applied the unbiased approach of transcriptional profiling. Our cDNA array analysis detected that 1165 of the 6288 sampled unigenes were significantly affected by pulsatile fluid flow. GenMapp 2.1 analysis revealed pathways of genes regulated by shear stress: angiogenesis, blood vessel morphogenesis, regulation of endothelial cell proliferation and prostaglandin biosynthesis. Individual genes significantly up-/down-regulated by shear stress included vascular endothelial growth factor A (VEGFa), cysteine rich protein 61 (CRY61), platelet derived growth factor, alpha (PDGFa), connective tissue growth factor (CTGF), Neuropilin 1 (NRP1), angiotensin II receptor, type 1a (AGTR1a) and fibroblast growth factor 1 (FGF1). Based on these findings, we hypothesize that fluid shear stress regulated VEGF most likely stimulates MC3T3-E1 cells through autocrine/paracrine release and may provide a powerful recruitment signal for osteoclasts, endothelial cells and/or stem cells during bone remodeling. Keywords: stress response
Project description:Renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in the immortalized renal proximal tubular epithelial cell line. Cells were exposed to laminar fluid shear stress (1.9 dyn/cm2) in a cone-plate device and compared to static controls.
Project description:Pkd1-/- renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in the immortalized Pkd1-/- renal proximal tubular epithelial cell line. Cells were exposed to laminar fluid shear stress (1.9 dyn/cm2) in a cone-plate device and compared to static controls.
Project description:The lymphatic system removes fluid from the interstitial space and returns it to the blood with a tremendous capacity: during inflammation, lymph flow rates can increase dramatically; however, during chronic lymphedema, there is little or no flow. The ability of lymphatic endothelium to sense and actively regulate this function is unknown, and shear stress is likely a key indicator of lymph flow. We profiled gene expression in human dermal microvascular lymphatic endothelial cells exposed to 0, 2 and 20 dyn/cm2 shear stress as representative of chronic lymphedema, normal, and acute inflammatory conditions, respectively. We found important adaptive responses correlated to multiple aspects of lymphatic function. Importantly, shear stress upregulated intracellular water and solute transporters while decreasing cell-cell adhesion and basement membrane components and increasing cell-matrix interactions. This data indicate that during high loading conditions, both passive and active drainage function increases, while conversely when fluid drainage is blocked, transport function is diminished in the lymphatic endothelium. These data demonstrate the first functional-adaptive response of lymphatic endothelium to flow conditions, thus indicating that the lymphatic endothelium plays an active role in regulating their function. Keywords: Shear stress, dose response, cell type comparison Lymphatic endothelial cells were subjected to 0, 2, or 20 dyn/cm2 shear stress; blood endothelial cells were subjected to 0 or 20 dyn/cm2 shear stress. Four samples were used for each cell type/shear level group for a total of 20 samples. Each sample was independently compared to human universal reference RNA via two-color microarray analysis for a total of 20 arrays. In all cases, the experimental samples were labeled with Cy5 dye while the reference RNA was labeled with Cy3.
Project description:The opportunistic pathogen, Staphylococcus aureus, encounters a wide variety of fluid shear levels within the human host, which may play a key role in dictating whether this organism adopts a commensal interaction with the host or transitions to cause disease. Using rotating-wall vessel bioreactors to create a physiologically-relevant, low fluid shear environment, S. aureus was evaluated for cellular responses that could impact its colonization and virulence. S. aureus cells grown in a low fluid shear environment initiated a novel attachment-independent biofilm phenotype and were completely encased in extracellular polymeric substances. Compared to controls, low-shear cultured cells displayed slower growth and repressed virulence characteristics, including decreased carotenoid production, increased susceptibility to oxidative stress, and reduced survival in whole blood. Transcriptional whole genome microarray profiling suggested alterations in metabolic pathways. Further genetic expression analysis revealed the down-regulation of the RNA chaperone Hfq, which parallels low fluid shear responses of certain Gram negative organisms. This is the first study to report an Hfq association to fluid shear in a Gram positive organism, suggesting an evolutionarily conserved response to fluid shear among structurally diverse prokaryotes. Collectively, our results suggest S. aureus responds to a low fluid shear environment by initiating a biofilm/colonization phenotype with diminished virulence characteristics, which could lead to insight into key factors influencing the divergence between infection and colonization during initial host pathogen interaction. Genetic expression profiles of Staphylococcus aureus cultured under low fluid shear conditions was compared to control cultures of S. aureus which was cultured in identical hardware in an orientation disrupting the low fluid shear effect. Samples from the same date of culture were compared (control 21:low 21 and control 30: low 30). S. aureus was cultured for 20 hours in either the low fluid shear or control orientated rotating wall vessel (RWV) bioreactor at which point the cells were removed and RNA extracted. At 20 hours, both cultures were in the same stage of growth (stationary phase) and at this point phenotypic differences between control and low fluid shear cultures were noted.
Project description:The ability of bacteria to sense and respond to mechanical forces has important implications for pathogens during the in vivo infection process, as they experience wide fluid shear fluctuations in the host. However, relatively little is known about how mechanical forces encountered in the infected host drive microbial pathogenesis. We previously demonstrated an inverse relationship between fluid shear-induced responses of classic gastrointestinal disease-causing Salmonella Typhimurium (x3339) and systemic multidrug resistant (MDR) S. Typhimurium (ST313 D23580) when the organisms were cultured under fluid shear forces like those in the intestinal tract and bloodstream. To advance our understanding of how incremental increases in physiological fluid shear impact D23580 pathogenesis phenotypes and transcriptomic responses, we applied dynamic bioreactor technology to introduce and quantify incremental increases in fluid shear during culture. Our data indicate that D23580 responds dynamically to a range of physiological fluid shear levels by altering pathogenesis-related phenotypes (stress responses, host cell colonization) and transcriptomic responses (including genes important for adherence and invasion). These phenotypic and molecular genetic changes directly correlated with incrementally increased fluid shear. This is the first demonstration that incremental changes in fluid shear alter stress responses and gene expression in any ST313 strain and offers new insight into how physiological fluid shear forces encountered by MDR bacteria in the infected host might impact their disease-causing ability in unexpected ways.
Project description:The effect of hemodynamic shear stress on endothelial gene expression was investigated in the porcine iliac arteries. Computational fluid dynamics simulations identified three anatomical regions likely to experience high, medium, and low shear stress. The expression of genes in endothelial cells recovered from these regions were assessed using microarray. Keywords: shear stress, endothelial cell gene expression, in vivo
Project description:The lymphatic system removes fluid from the interstitial space and returns it to the blood with a tremendous capacity: during inflammation, lymph flow rates can increase dramatically; however, during chronic lymphedema, there is little or no flow. The ability of lymphatic endothelium to sense and actively regulate this function is unknown, and shear stress is likely a key indicator of lymph flow. We profiled gene expression in human dermal microvascular lymphatic endothelial cells exposed to 0, 2 and 20 dyn/cm2 shear stress as representative of chronic lymphedema, normal, and acute inflammatory conditions, respectively. We found important adaptive responses correlated to multiple aspects of lymphatic function. Importantly, shear stress upregulated intracellular water and solute transporters while decreasing cell-cell adhesion and basement membrane components and increasing cell-matrix interactions. This data indicate that during high loading conditions, both passive and active drainage function increases, while conversely when fluid drainage is blocked, transport function is diminished in the lymphatic endothelium. These data demonstrate the first functional-adaptive response of lymphatic endothelium to flow conditions, thus indicating that the lymphatic endothelium plays an active role in regulating their function. Keywords: Shear stress, dose response, cell type comparison
Project description:The effect of hemodynamic shear stress on endothelial gene expression was investigated in the porcine iliac arteries. Computational fluid dynamics simulations identified three anatomical regions likely to experience high, medium, and low shear stress. The expression of genes in endothelial cells recovered from these regions were assessed using microarray. Keywords: shear stress, endothelial cell gene expression, in vivo 11 iliac arteries were obtained from 6 animals, total RNA were extracted from high, medium and low shear region. The expression profiles were compared with a reference RNA using dual channel arrays. Samples with low original RNA quality and low hybridization quality were not included.