ABSTRACT
In the arterial circulation, regions of disturbed flow (DF), which are characterized by flow separation and transient vortices, are susceptible to atherogenesis, whereas regions of undisturbed laminar flow (UF) appear protected. Coordinated regulation of gene expression by endothelial cells (EC) may result in differing regional phenotypes that either favor or inhibit atherogenesis. Linearly amplified RNA from freshly isolated EC of DF (inner aortic arch) and UF (descending thoracic aorta) regions of normal adult pigs was used to profile differential gene expression reflecting the steady state in vivo. By using human cDNA arrays, approximately 2,000 putatively differentially expressed genes were identified through false-discovery-rate statistical methods. A sampling of these genes was validated by quantitative real-time PCR and/or immunostaining en face. Biological pathway analysis revealed that in DF there was up-regulation of several broad-acting inflammatory cytokines and receptors, in addition to elements of the NF-kappaB system, which is consistent with a proinflammatory phenotype. However, the NF-kappaB complex was predominantly cytoplasmic (inactive) in both regions, and no significant differences were observed in the expression of key adhesion molecules for inflammatory cells associated with early atherogenesis. Furthermore, there was no histological evidence of inflammation. Protective profiles were observed in DF regions, notably an enhanced antioxidative gene expression. This study provides a public database of regional EC gene expression in a normal animal, implicates hemodynamics as a contributory mechanism to athero-susceptibility, and reveals the coexistence of pro- and antiatherosclerotic transcript profiles in susceptible regions. The introduction of additional risk factors may shift this balance to favor lesion development.
Subject(s)
Aorta/physiology , Endothelium, Vascular/physiology , Gene Expression Regulation , Transcription, Genetic/genetics , Animals , Apoptosis/genetics , Computational Biology , Enzymes/genetics , Gene Expression Profiling , Male , Oxidation-Reduction , Polymerase Chain Reaction/methods , Proteins/genetics , Regional Blood Flow , Reproducibility of Results , SwineABSTRACT
Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.
Subject(s)
Endothelium, Vascular/chemistry , Endothelium, Vascular/metabolism , Gene Expression Profiling/methods , Nanotechnology/methods , Nucleic Acid Amplification Techniques/methods , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/metabolism , Bias , Cell Line , Databases, Genetic , Endothelium, Vascular/cytology , Gene Expression Regulation/genetics , Humans , Internet , Sensitivity and Specificity , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
The completion of the Human Genome Project and ongoing sequencing of mouse, rat and other genomes has led to an explosion of genetics-related technologies that are finding their way into all areas of biological research; the field of biorheology is no exception. Here we outline how two disparate modern molecular techniques, microarray analyses of gene expression and real-time spatial imaging of living cell structures, are being utilized in studies of endothelial mechanotransduction associated with controlled shear stress in vitro and haemodynamics in vivo. We emphasize the value of such techniques as components of an integrated understanding of vascular rheology. In mechanotransduction, a systems approach is recommended that encompasses fluid dynamics, cell biomechanics, live cell imaging, and the biochemical, cell biology and molecular biology methods that now encompass genomics. Microarrays are a useful and powerful tool for such integration by identifying simultaneous changes in the expression of many genes associated with interconnecting mechanoresponsive cellular pathways.
