Project description:We are interested in the role of NOTCH1 and Shear Stress in Aortic Valve Endothelium. Primary human aortic valve endothelium was subjected to 4 conditions in vitro. 1) Control siRNA, No shear stress. 2) NOTCH1 siRNA, No shear stress. 3) Control siRNA, 15 dynes/cm2 shear stress. 4) NOTCH1 siRNA, 15 dynes/cm2 shear stress. Triplicates of each condition were pooled for library perp and sequencing
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 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:Many studies have characterized the results of shear stress changes on cultured endothelial cells in different bioreactor systems. However it is still unclear how an invasive intervention like stent procedure may influence the transcriptional response of endothelium. To study the simultaneous effects of shear stress changes and stent application on endothelial gene expression, we have used an experimental apparatus of laminar flow bioreactor (LFB) system with human cultured endothelial cells exposed or not exposed to stent procedure with different flow conditions. Microarray analysis was evaluated in each experimental protocol.
Project description:The role of shear stress, the frictional force of blood flow, on the endothelium has been well documented. Differences in shear stress can have profound effects on endothelial and blood vessel biology. Endothelial cells (ECs), termed endocardial ECs, line the heart chambers and are exposed to complex shear stress patterns. While it has been demonstrated that shear stress is important for heart development, little has been shown on the role of shear stress on adult ECs. 4D-MRI studies demonstrate regional differences in blood residence time. We sought to determine the effect of regional differences in endocardial shear stress on the endocardial transcriptome using RNA sequencing (RNA-seq) on 3 different regions (apex, mid-ventricle, outflow tract) from 8 adult pigs, for a total of 24 RNA-seq assays.
Project description:Many studies have characterized the results of shear stress changes on cultured endothelial cells in different bioreactor systems. However it is still unclear how an invasive intervention like stent procedure may influence the transcriptional response of endothelium. To study the simultaneous effects of shear stress changes and stent application on endothelial gene expression, we have used an experimental apparatus of laminar flow bioreactor (LFB) system with human cultured endothelial cells exposed or not exposed to stent procedure with different flow conditions. Microarray analysis was evaluated in each experimental protocol. HUVECs (2nd and 5th passage) covered on Thermanox slides were submitted to static, low and physiological (0, 1, 5 and 10 dyne/cm2) shear stress in absence (AS) or presence (PS) of stent in LFB system for 24h. Affymetryx analysis has been performed in duplicate by Consortium for Genomic Technologies (Cogentech; Milan, Italy)