Shear Stress and NOTCH1 Regulate Calcification Related Genes in Human Aortic Valve Endothelium (RNA-Seq)
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ABSTRACT: 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:Many studies have characterised the effect of normal laminar shear stress (LSS) on endothelial responses, however elevated shear stress, as would be experienced overlying a stenotic plaque, has not been studied in depth. Therefore we used transcriptomics and related functional analyses to compare cells exposed to laminar shear stress at 15 or 75 dynes/cm2 for 24 hours (LSS15-normal or LSS75-high shear stress). Human umbilical vein endothelial cells (HUVEC n=4 per flow condition from different batches of pooled donor HUVEC p2) were cultured for 24 hours on gelatin coated slides under laminar shear stress of either 15 dynes/cm2 or 75 dynes/cm2 (LSS15 or LSS75) to assess the effect of high shear stress on endothelial cells. Total RNA was obtained using Qiagen kit and array analysis performed by Service XS (Leiden, Netherlands).
Project description:In this study, we characterized the adaptive response of arterial endothelial cells to acute increases in shear stress magnitude in well-defined in vitro settings. Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (30 ± 15 dynes/cm2) was then applied. The transcriptomics studies using microarray identified genes that were sensitive to the elevated shear magnitude. A significant number of the identified genes in our study are previously unknown as sensitive to shear stress. Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (30 ± 15 dynes/cm2) was then applied. Gene expression at multiple time points was measured using microarray.
Project description:Porcine aortic and aortic valve endothelial cells were exposed to 20 dynes/cm2 steady laminar shear stress with static cultures serving as controls. Total RNA was hybridized to Agilent Human 1 cDNA arrays and processed using the Agilent Feature Extraction Software Keywords = aortic valve Keywords = endothelial Keywords = shear stress Keywords: other
Project description:This study characterizes the response of primary human endothelial cells (human umbilical vein endothelial cells, HUVECs) to the relative shear stress changes that occur during the initiation of arteriogenesis at the entrance regions to a collateral artery network. HUVECs were preconditioned to a baseline level of unidirectional shear of 15 dynes/cm2 for 24 hours. After 24 hours preconditioning, HUVECs were subjected to an arteriogenic stimulus that mimics the shear stress changes observed in the opposing entrance regions into a collateral artery network. The arteriogenic stimulus consisted of a 100% step wise increase in shear stress magnitude to a unidirectional 30 dynes/cm2 in either the same or opposite direction of the preconditioned shear stress. This simulates either the feeding entrance to the collateral artery circuit or the region that drains into the vasculature downstream of an obstruction in a major artery, respectively. In vivo analysis of collateral growth in the mouse hindlimb showed enhanced outward remodeling in the re-entrant (direction reversing) region that reconnects to the downstream arterial tree, suggesting reversal of shear stress direction as a key enhancer of arteriogenesis. Transcriptional profiling using microarray techniques identified that the reversal of shear stress direction, but not an increase in shear stress alone, yielded a broad-based enhancement of the mechanotransduction pathways necessary for the induction of arteriogenesis. Human umbilical vein endothelial cells (HUVECs) were preconditioned to a unidirectional clockwise shear stress of 15 dynes/cm2 for 24 hours. An acute increase in shear stress magnitude to 30 dynes/cm2 in either a clockwise (non-reversed) or counter-clockwise (reversed) direction was applied for 6 hours. An additional preconditioned control culture was maintained under a unidirectional clockwise shear stress of 15 dynes/cm2 and harvested at the same time point, 6 hours post-conditioning. Each condition of reversed, non-reversed, and control was performed in tandem from the same starting cell culture as one replicate. The total experiment consisted of four replicates. Gene transcription was then assessed using microarray expression analysis.
Project description:Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress frequency (2 Hz) was then applied. The transcriptomics studies using microarray identified genes that were sensitive to the elevated shear frequency. keywords: adaptation, shear stress, frequency, microarray, gene expression Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (2 Hz) was then applied. Gene expression at multiple time points was measured using microarray.
Project description:Lymphatic valves are specialized units regularly distributed along collecting vessels that allow unidirectional forward propulsion of the lymph, and its efficient transport from tissues to the bloodstream. Lymphatic endothelial cells that cover lymphatic valve sinuses are subjected to complex flow patterns, due to recirculation of the lymph during the collecting vessel pumping cycle. They also express high levels of FOXC2 transcription factor. We used microarrays to study the transcriptional networks controlled by FOXC2 in human lymphatic endothelial cells subjected to oscillatory shear stress or cultured under static conditions. Human lymphatic endothelial cells were transfected with control or FOXC2 siRNAs and subjected to 24-hour oscillatory shear stress (1 dyn/cm2; 1/4 Hz) or kept under static conditions as a control. RNA were amplified and hybridized on Affymetrix Human Gene 1.0 ST Arrays. The experiment was run twice independently, using each time a different siRNA to knockdown FOXC2, as previously described (Sabine et al, 2012, Dev Cell).
Project description:In this study, we characterized the adaptive response of arterial endothelial cells to acute increases in shear stress magnitude in well-defined in vitro settings. Porcine endothelial cells were preconditioned by a basal level shear stress of 15 ± 15 dynes/cm2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (30 ± 15 dynes/cm2) was then applied. The transcriptomics studies using microarray identified genes that were sensitive to the elevated shear magnitude. A significant number of the identified genes in our study are previously unknown as sensitive to shear stress.
Project description:In order to simulate the effects of shear stress in regions of the vasculature prone to developing atherosclerosis, we subjected human umbilical vein endothelial cells to reversing shear stress, in order to mimic hemodynamic conditions at the wall of the carotid sinus, a site of complex, reversing blood flow and commonly observed atherosclerosis. We compared the effects of reversing shear stress (time-average 1 dyne/cm2, maximum +11 dynes/cm2, minimum -11 dynes/cm2, 1 Hz), arterial steady shear stress (15 dynes/cm2), and low steady shear stress (1 dyne/cm2) in terms of gene expression, cell proliferation, and monocyte adhesiveness. Microarray analysis revealed most differentially expressed genes were similarly regulated by all three shear stress regimens when compared to static culture. Comparisons of the three shear stress regimens to each other allowed identification of 138 genes regulated by low average shear stress and 22 by fluid reversal. Functional assays indicated that low average shear stress induces increased cell proliferation as compared to high shear stress. Reversing shear stress was the only condition that induced monocyte adhesion. Monocyte adhesion was partially inhibited by incubation of the endothelial cells with ICAM-1 blocking antibody. Increased surface heparin sulfate proteoglycan expression was observed in cells exposed to reversing shear stress. When these cells were treated with heparinase III monocyte adhesion was significantly reduced. Our results suggest that low steady shear stress is the major impetus for differential gene expression and cell proliferation, while reversing flow regulates monocyte adhesion.