Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 45
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KGaA, WeinheimISBN: 3-527-30596-315510Gene Expression Profiling in Pulmonaryand Systemic Vascular Cells Exposed to Biomechanical StimuliShwu-Fan Ma, Shui Qing Ye, and Joe G. N. GarciaThe pulmonary and systemic circulations are continuously exposed to bloodborne bioactive agonists, hormones and cellular constituents as well as biophysical forces such as shear stress and cyclic strain. Alterations in gene expression represent important adaptive responses which allow vascular cells to either remodel,form new vessels or progress to programmed cell death.
Differential gene expression in vascular cells and in blood cells, due to gene-gene and gene-environmentinteractions (including local hemodynamics), can be considered the molecular basis of relevant vascular pathogenic processes. Fortunately, the ability to elucidatethe role of specific genes in pathophysiologic pathways relevant to human diseasehas been accelerated by the emergence of new technologies such as cDNA/oligonucleotide microarrays and serial analysis of gene expression (SAGE), which allowhigh throughput gene expression profiling. In this chapter, we provide information on microarray and SAGE techniques in the context of human vascular diseases and the systematic analysis of gene expression of vascular cells, informationwith important implications for our understanding of the mechanisms of vascularhomeostasis and dysfunction.10.1IntroductionThe Human Genome Project (www.ncbi.nlm.nih.gov), well on its way to providinga complete catalogue of all human genes, is arguably the most important biological discovery ever.
Annotating and integrating these rivers of genomic data intobiologically-relevant processes in order to provide useful translational informationis a daunting challenge which will persist for the foreseeable future. This is particularly true for a variety of systemic and pulmonary vascular processes, which areevoked either by rapid-acting chemical and cellular agents or by chronic environmental stimuli. Both normal adaptive and pathobiologic vascular responses tosuch stimuli involve well-orchestrated alterations in gene and protein expressionin vascular cell targets.
It is in this context that the field of functional genomicshas begun to focus on dissecting the molecular basis of cellular function at thelevel of the organ and the tissue. Characterization of genes abnormally expressedProteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E. van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-315610 Gene Expression Profiling and Vascular Cellsin diseased vascular tissues offer the potential for the discovery of genes thatserve as diagnostic markers, prognostic indicators or targets for therapeutic intervention [1]. Historically, methods such as cDNA subtraction [2], and mRNA differential display [3] have been limited by the capacity to analyze only a limited number of genes without quantitative information.
More recently, two technologies, serial analysis of gene expression (SAGE) [4] and gene-specific oligonucleotide [5] orcDNA microarrays [6] now allow researchers to determine the expression patternof thousands of genes simultaneously and have yielded novel insight into thepathophysiology of human diseases. While the full impact of these technologieson vascular diseases has not yet been realized, the potential for their use in thepost-genomic era as diagnostic molecular markers or for identifying novel therapeutic interventions are currently being explored.In this chapter, we will address relevant literature to the genomic-based studiesthat have focused on vascular responses with a particular focus on vascular geneexpression induced by biophysical perturbations. Although both pulmonary andsystemic circulations are exposed to varying level of shear stress, lung endothelialcells also experience variable flow patterns dictated by the unique pulmonary circulation where the distribution of blood flow is non-uniform decreasing from thebase to the apex.
Furthermore, lung capillaries routinely collapse with cessation offlow, a physiologic event in the lung for example, during hypoxic vasoconstriction.Finally, the lung circulation is uniquely exposed to cyclic strain produced in thecourse of normal tidal breathing, an exposure exaggerated when patients with respiratory failure are placed on mechanical ventilation. Thus, it might be predictedthat systemic and pulmonary vascular cells would share similar gene expressionprofiles but also retain phenotypic responses under specific environmental or biophysical conditions unique to their respective vascular beds.10.2Principles of Array, SAGE and Mini SAGE TechnologiesThe basis for gene microarrays (addressed in detail elsewhere, see i.e.
Chapter 6)embodies the core principle of molecular biology with the specific hybridizationbetween labeled-nucleic acids and the complementary immobilized nucleic acidson a matrix [7]. Gene arrays utilize this basic principle to place a collection ofgene-specific nucleic acids on solid supports at defined locations.
With the adaptable nature of the fabrication and hybridization methods, microarrays have becomeone of the most powerful and versatile tools for genomic analysis. Probes such asPCR products, cDNAs or oligonucleotide probes (25- to 70-mers), are roboticallydeposited (e.g. cDNA microarray) or synthesized by photolithographic techniques(oligonucleotide microarray). Microarrays are relatively easy to use and suitable forhigh-throughput applications where expression profiling of hundreds of diseasesamples can be efficiently performed.The SAGE technology involves a short sequence tag (10 bp) from a unique position within each transcript containing sufficient information to uniquely identify10.3 Analysis of Gene Expression Profiling in Vascular Cellsa transcript. The concatenation of tags in a serial fashion allows for increased efficiency of a sequence-based analysis (Fig.
10.1, details of the SAGE techniquefound in two comprehensive reviews [1,8]). Sequences of the concatemers enablecataloging and quantification of the tags, which delineates the identification andthe relative abundance of the transcripts for a given cell line or tissue [4]. Theportability of the SAGE data allows direct comparisons to be made, a difficult taskto accomplish with microarrays given the differences in formats and normalization methodologies. SAGE accurately determines the absolute abundance ofmRNAs and with the potential identification of new genes and alternatively processed transcripts that are unique to a specific cell type.
A significant drawback ofSAGE is the requirement for large amount of input mRNA (2.5–5.0 lg) equivalent to 250 to 500 lg total RNA. We recently utilized a modified method, miniSAGE, to profile gene expressions of human fibroblasts from 1 lg total RNA without additional PCR amplification [9].10.3Analysis of Gene Expression Profiling in Vascular CellsAs early as 1856, Virchow et al. [10] recognized that vascular endothelium mightparticipate as a central component in the atherosclerotic disease process. Ku et al.[11] reported that atherosclerotic lesions often originate near branches, bifurcations and curvatures of arteries, where the laminar flow pattern is disturbed.
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