Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 22
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Microarraygene expression profiles in dilated andhypertrophic cardiomyopathic end-stageheart failure. Physiological Genomics.2002. (in press).Attwood, T. K. The babble of bioinformatics. Science 2000. 290, 471–473.614Principles of cDNA Microarrays as Appliedin Heart Failure ResearchSara Arab, Mansoor Husain, and Peter Liu4.1The Clinical ProblemSymptomatic heart failure (HF) affects 4.7 million patients in the US with approximately 550,000 new cases of heart failure identified annually [1–3] .
Proportionally, there are 428,000 patients in Canada, incurring $ 8 billion in annual hospital costs alone. This burden expected to double in the next 2 decades [4–6].Other forms of cardiovascular disease are plateauing, but the incidence of heartfailure is increasing. The one year mortality rate is between 25–40% [5].
This ispartly the consequence of our success in treating myocardial infarction and sudden deaths, and partly due to the aging population.Meanwhile, cardiac dysfunction often occurs in the patient long before symptoms manifest. Thus the best opportunity to curb the tide of heart failure is likelypresented by intervention early in the disease process [7, 8]. What is requiredtherefore is a concerted effort to identify the underlying pathogenesis of the disease, and the timely application of this information for the development of earlyprognostic indicators and therapies.4.2The Need for a New ParadigmCurrently, only three classes of agents: angiotensin converting enzyme inhibitors,beta blockers and aldosterone antagonists can modify the disease process and alter the natural history of heart failure [6, 7, 9].
These agents can improve survival,reduce hospitalization and enhance quality of life in HF patients. However, manychallenges remain, because the one year mortality is still high in the presence ofstate of art therapy, and increasing numbers of patients in severe heart failurehave a tremendously compromised quality of life. One such challenge is the needto find new targets for the treatment of HF.
The traditional approaches have beenlimited to biochemical analysis of peripheral blood or myocardial biopsies. Manyof the targets to date have been selected by reference to other fields of investigations, including nephrology, hypertension and cancer biology. Other targets derivProteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E. van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-3624 cDNA Microarrays in Heart Failure Researching from previous hypothesis driven research have unfortunately failed to deliverin recent investigations.
Agents that have been studied modulate calcium sensitivity or calcium release, cytokine inhibitors, vasopeptidase inhibitors as well as endothelin antagonists.With the birth of microarray technology, and the near completion of the humangenome [10], we now have an unprecedented opportunity to supplement the hypothesis driven approach with so called “knowledge driven” approach to science,such that genome based discovery takes place side by side with the traditionalroutes of discovery.
Microarrays let us to generate new knowledge without requirering a prior known paradigm. For example, recently, Dr. Josef Penninger and Peter Backx from our Centre have discovered the function of a novel angiotensinconverting enzyme (ACE2). This is partially derived from in silico analysis of genome and microarray databases, illustrating the power of this novel approach todiscovery [11].4.3The Potential Role of the MicroarrayMicroarray technology has the singular power to provide a system based description of a global change of state in specific experimental conditions. This capapability is a great advantage over traditional molecular approaches that expire a singlepathway in a complex network of interacting biological activities (Fig. 4.1).
Theolder approaches metaphorically shine a single beam of light in a darkroom in order to determine the dynamic contents of the room. Microarray allows a systemwide or modular approach to analysis of change in an experimental condition,akin to turning on of multiple lighting systems to illuminate our dark room.The first time investigator may find, that the information derived from microarray technology is overwhelming and difficult to comprehend.
However, as experience accumulates, and the bioinformatics tools become more sophisticated, thestrength of the method to decode changing patterns of gene expression becomesmore apparent. When the experiments are done carefully, the data can be extremely consistent from experiment to experiment. This consistently adds a levelof robustness to the observations beyond that previously available with technologies to study single molecular target techniques.As the information database grows signals are more easily differentiated fromthe noise and the patterns emerge across different model systems. Such capabilities give us insight to the key regulators of a number of these systems. The ability of the bioinformatics system to provide this important insight is continuing toimprove, thus making the additional experiments aimed at deciphering the function for novel gene targets more meaningful.4.4 Strengths of Microarray TechnologyThe complex known pathways contributing to the process of heart failure.
Thetraditional approach of investigating a singlemolecular target is intrinsically limited due tothe network nature of the disease process.Fig. 4.1The microarray will permit the scientist to examine multiple targets simultaneously, to determine potential molecular interactions, andidentify novel members of participating pathways.4.4Strengths of Microarray TechnologyDNA microarray enables researchers for the first time to visualize global patternsof gene expression under different conditions. A long way to express a simplethought. The variation in the expression of a single gene is richer than the allelicvariation in its sequence. Finding the link between the expression variation andphenotypic variation may provide clues to the biological roles of gene(s) to identify the molecular basis of phenotypic variation among cells and individuals.
Togenerate a gene expression profile of a condition (s) is to identify the up- ordown-regulation of gene (s) expression which can be both the cause and effect ina disease state. To study the global changes involving thousands of genes is notpossible without microarray. A large portion of the genome can be interrogated simultaneously to identify the common cluster of genes with similar expression pattern which may reflect a similar function.
The exceptions or unknown membersmay help to identify functionally important novel genes. Moreover, the temporalsequence of gene expression can be followed with microarray technology. Finally,microarray technologies can be used to develop diagnostic tools, either to identifythe markers or to use the global geneexpression information for classifying different stages of the disease state, by using a subset of global candidate genes relevant to the disease (Fig. 4.2). The aggregate database is also useful to develop new63644 cDNA Microarrays in Heart Failure ResearchOne of the potential applications ofmicroarrays in the setting of heart failure isby developing disease relevant “chips” in thefuture, once the relevance of these genes areknown. For example, from a global set of can-Fig.
4.2didate genes, one may ultimately identify 100or so critical genes that are differentially regulated in not only heart failure, but also reflectthe specific stage or etiology of heart failure.drugs based either on novel genes thus identified, or on a knowledge of expression levels of mRNA in different pathological states.A remarkable growth has occured in the source of the chips and the relatedcomponents of microarray techniques such as clone-set, arrayer, scanner, andanalysis software. Technologies are expected to evolve significantly in the next fewyears. New microarray methods combined with bioinformatics will continue toprovide increasing insight into the molecular basis of biological events.4.5Caveats of Using the Microarray TechnologyOn the other hand, microarray technologies have limitations, some of which reflectits relative early stages of development, and others of which relate to basic principlesof good scientific investigation.1.
Proper experimental design. Because microarray techniques are costly and complex, much thought must be given to experimental design if results are to beinterpreted. Experiments must have advanced planning, rigorous controls, anda hypothesis facilitated process that allows focus and subsequent validation. Itis only when traditional hypotheses driven experiments are validated, that onecan begin the exploratory component of the dataset.4.6 Experimental Design2. Ultrahigh quality of RNA is required for reproducible results. This is the mostfundamental requirement for any microarray experiment, otherwise “noise in,noise out”.
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