Project description:Objective: Shear forces play a key role in the maintenance of vessel wall integrity. Current understanding regarding shear-dependent gene expression is mainly based on in vitro or in vivo observations with experimentally deranged shear, hence reflecting acute molecular events in relation to flow. Our objective was to combine computational fluid dynamic (CFD) simulations with global microarray analysis to study flow-dependent vessel wall biology in portions of the entire aorta under physiological conditions. Methods and Results: Animal-specific WSS magnitude and vector direction were estimated using CFD based on aortic geometry and flow information acquired by MRI. Two distinct flow pattern regions were identified in the normal rat aorta; the distal part of the inner curvature being exposed to low WSS and a non-uniform vector direction, and a region along the outer curvature being subjected to markedly higher levels of WSS and a uniform vector direction. Microarray analysis identified numerous novel mechanosensitive genes, including Hand2, trpc4 and slain2, and confirmed well-known ones, such as klf2 and BMP4. Three genes were further validated for protein , including Hand2, which showed higher expression in the endothelium in regions exposed to disturbed flow. Gene ontology analysis revealed an over-representation of genes involved in transcriptional regulation. Microarray analysis of two distinct flow pattern regions were identified in the normal rat aorta. Tissue pieces from groups of five animals were pooled for each region giving total of 28 pools; 14 paired sample pools of high and low wall shear stress.
Project description:Objective: Shear forces play a key role in the maintenance of vessel wall integrity. Current understanding regarding shear-dependent gene expression is mainly based on in vitro or in vivo observations with experimentally deranged shear, hence reflecting acute molecular events in relation to flow. Our objective was to combine computational fluid dynamic (CFD) simulations with global microarray analysis to study flow-dependent vessel wall biology in portions of the entire aorta under physiological conditions. Methods and Results: Animal-specific WSS magnitude and vector direction were estimated using CFD based on aortic geometry and flow information acquired by MRI. Two distinct flow pattern regions were identified in the normal rat aorta; the distal part of the inner curvature being exposed to low WSS and a non-uniform vector direction, and a region along the outer curvature being subjected to markedly higher levels of WSS and a uniform vector direction. Microarray analysis identified numerous novel mechanosensitive genes, including Hand2, trpc4 and slain2, and confirmed well-known ones, such as klf2 and BMP4. Three genes were further validated for protein , including Hand2, which showed higher expression in the endothelium in regions exposed to disturbed flow. Gene ontology analysis revealed an over-representation of genes involved in transcriptional regulation.
Project description:A series of two color gene expression profiles obtained using Agilent 44K expression microarrays was used to examine sex-dependent and growth hormone-dependent differences in gene expression in rat liver. This series is comprised of pools of RNA prepared from untreated male and female rat liver, hypophysectomized (‘Hypox’) male and female rat liver, and from livers of Hypox male rats treated with either a single injection of growth hormone and then killed 30, 60, or 90 min later, or from livers of Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart and killed 30 min after the second injection. The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. A comparison of untreated male liver and untreated female liver liver gene expression profiles showed that of the genes that showed significant expression differences in at least one of the 6 data sets, 25% were sex-specific. Moreover, sex specificity was lost for 88% of the male-specific genes and 94% of the female-specific genes following hypophysectomy. 25-31% of the sex-specific genes whose expression is altered by hypophysectomy responded to short-term growth hormone treatment in hypox male liver. 18-19% of the sex-specific genes whose expression decreased following hypophysectomy were up-regulated after either one or two growth hormone injections. Finally, growth hormone suppressed 24-36% of the sex-specific genes whose expression was up-regulated following hypophysectomy, indicating that growth hormone acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression. For full details, see V. Wauthier and D.J. Waxman, Molecular Endocrinology (2008)
Project description:In order to establish a rat embryonic stem cell transcriptome, mRNA from rESC cell line DAc8, the first male germline competent rat ESC line to be described and the first to be used to generate a knockout rat model was characterized using RNA sequencing (RNA-seq) analysis.
Project description:Oligodendrocytes undergo extensive changes as they differentiate from progenitors into myelinating cells. To better understand the; molecular mechanisms underlying this transformation, we performed a comparative analysis using gene expression profiling of A2B5+; oligodendrocyte progenitors and O4+ oligodendrocytes. Cells were sort-purified ex vivo from postnatal rat brain using flow cytometry. Using Affymetrix microarrays, 1707 transcripts were identified with a more than twofold increase in expression inO4+oligodendrocytes. Many genes required for oligodendrocyte differentiation were upregulated in O4+ oligodendrocytes, including numerous genes encoding; myelin proteins. Transcriptional changes included genes required for cell adhesion, actin cytoskeleton regulation, and fatty acid and; cholesterol biosynthesis. At the O4+ stage, there was an increase in expression of a novel proline-rich transmembrane protein (Prmp). Localized to the plasma membrane, Prmp displays adhesive properties that may be important for linking the extracellular matrix to the; actin cytoskeleton. Together, our results highlight the usefulness of this discovery-driven experimental strategy to identify genes relevant; to oligodendrocyte differentiation and myelination. Experiment Overall Design: Whole brain dissociates were prepared from one litter of 10 male postnatal day 7 rat pups for each of the 5 A2B5 bioligcal replicates and the 4 O4+ bioligical replicates. Total RNA was extracted from single A2B5+ and single O4+ cells sorted directly from postnatal day7 rat whole brain dissociates using flow cytometry.