DNA Microarray Application in Vulnerable plaque research Mehran Haidari PhD Center for Vulnerable Plaque Research Texas Heart Institute
Atherosclerosis and the resulting coronary heart disease represent the most common cause of death in industrialized nations. Although certain key risk factors have been identified, the molecular mechanism responsible for this complex disease and its deadly complications remains as a challenge in the years to come. Rupture of atherosclerotic plaque is the predominant underlying process in the pathogenesis of acute coronary syndromes. Although we have gained a great deal of knowledge on underlying pathology involved in plaque vulnerability to rupture, the exact molecular mechanisms underlying the process is still largely unexplored.
Evolution of genomic and proteomic techniques has opened the door to the world of unknown molecular mechanisms in the body that allowing thorough investigation into susceptibility of certain people / patients to certain outcomes. Investigation of advanced atherosclerosis using the tools for systematic gene and protein expression analysis is a surprisingly neglected area of study and has not been touched widely enough. Only a few numbers of investigators worldwide are actively pursuing this field. (B.C.G Faber, J.A.P Deamen; L.D Adams, Stephen M.Schwartz; M.P. Herman, Uwe Schonbeck; k.J.Haley, Richard T Lee; Timothy A.McCaffrey;L.W.Stanton, R Tyler White;D.Shiffman, Richard M Lawn;Brian K Coombes, ) Schwartz Lee Deamen
During the last half of the 20th century, the analysis of the regulation and function of genes largely Been driven by step-by-step studies of individual genes and proteins. In the past decade, a paradigm shift has emerged in which we are now able to produce large amounts of data about many genes in a highly parallel and rapidly serialized manner. An important tool in this process has been the development of DNA microarray.
Low-throughput methods of gene expression • Northern Blotting, cumbersome, time-consuming • Nuclease protection, at least 10 fold more sensitive • Quantitative RT-PCR, state of the art High-throughput Methods of gene expression Serial Analysis of Gene Expression (SAGE) Rapid Analysis of Gene Expression (RAGE) Representational Difference Analysis (RDA) Suppression Subtractive Hybridization (SSH) Differential screening (plus/minus screening) Differential Display (DD) DNA Microarray =400,000 Northern Blotting
What is DNA Microarray? Mark Schena Patrick O Brown Reverse Northern blotting A large number of genes deposited onto a glass slide (large scale dot blot). The RNA sample is RT with simultaneous incorporation of label, resulting in labeled cDNA. Microarray slides serve as hybridization targets for labeled cDNA.
Basic Steps in Performing a DNA Microarray Experiments 1- Processing cDNA clones to generate print-ready material 2-Printing cDNA clones (or oligonucleotide) onto a substrate 3-Sample RNA isolation 4-Preparation of the probe (e.g. cDNA synthesis and labeling, RT reaction) 5- Hybridization of labeled probe DNA to the DNA arrayed on the substrate 6-Image acquisition, image analysis and data analysis
Microarray Fabrication Technologies In Situ Synthesis of Nucleic Acid (Chip ,GeneChip,oligonucleotide array) 15-20 different 25-mer oligonucleotides Exogenous Deposition of cDNA (cDNA, spotted array) Single DNA fragments, greater 0.5 Kb
When to use Microarray • Analysis of Gene Expression • Monitoring Changes in Genomic DNA • Gene Discovery, Sequencing and Pathway Analysis
Analysis of Gene Expression 1- Different tissues or different developmental states. 2- Normal or diseased states. 3- Exposure to drugs or different physiological conditions.
Micro or Macro Two basic substrates commonly used for cDNA printing are glass and membrane filters. Chemically treated microscope glass slides are the most widely used support. Microarray, Microscope Slide,80000 Spots. Macroarray, Nylon Membrane, 500,-18000 Spots.
RNA Preparation • No difference between total RNA or mRNA • Type of tissue might have profound effect on extraction process. 10 -20 µg of RNA is needed/slide. • Laser captured microdissection (LCM) , incorporation of a PCR step( access to subpopulations cells in vulnerable plaque).
Sample Labeling A single round of transcription is used to generate a labeled cDNA probe (RT-PCR). Most microarray utilize two fluorophores, Cyanine3(Green emission) and Cyanine5 (Red emission). Fluorophores have different size and different ability for incorporation in Cdna.
Affymetrix Genechip Biotinylated cRNA is synthesized from cDNA phycoerthrin linked to avidin is used for labeling. Each sample hybridized separately Hybridization to oligonucleotide is sensitive in detection of single-nucleotide mismatches. Disadvantages Advantages Sequence data required High density chip Oligonucleotid selection rules are not well defined Consistent and uniform geometry Single Nucleotide Polymorphisms(SNP) Expensive Not best target for hybridization No need for maintaining cDNA clones
Normalization First step is during scanning, when sensitivity of detection is adjusted by the laser voltage. Gene expression value can be expressed relative to the expression of housekeeping genes. In the absence of control genes, normalization to the median microarray value is popular. No consensus on Data Analysis( ANOVA), Clustering (categorizing genes according to their pattern of expression).
How Much is Significant??? Analyzed gene changes are often expressed as a fold increase either greater than twofold or less than 0.5 fold (DeRisi). With a large number of microarrays, small changes can be statistically valid. Elcock et al. detected 1.1 fold changes with 95 % confidence interval when each experimental sample was hybridized to seven microarray slides (with two replicate spots for each gene). Derisi et al.Nat Genet 1996:14:457-60
Housekeeping genes These are genes that are expressed constitutively and their level of expression is thought to be stable, regardless of the sample used ( Actin, Cyclophilin, GAPDH). DeRisi used 90 housekeeping genes and found that changes that were <0.5 and > 2.4 were acceptable. Actin is one of the most commonly used housekeeping genes and it has been shown to be downregulated in heat shock experiments. In fact, there is an appreciable amount of literature available to suggest that there is no such thing as housekeeping gene.
Accuracy and Precision DNA microarray represents a developing technology, there remain substantial obstacles in the design and analysis of these microarray. There are no globally accepted rules or standards for performing controlled microarray experiments. A good experiments include more control component then the real comparison.
Principles of Q.C in DNA Microarray Down-Scaling of an experiment makes it generally sensitive to external and internal fluctuation. Rajeevan et al. estimated that 30% of microarray results are false-positive. J.Mol.Diag 2001,3:26-31 Microarray findings should be confirmed, at least by one of the low-throughput gene expression methods. • Replication of each experiments on multiple array. • Dual labeling, swapping the dyes for control and treated sample. • Using a large number of controls on every array.
Controls mRNA from genes that are not homologous to the organism understudy (Arabidopsis). cDNA from the organism with high, medium and low expression represented on the array (sensitivity). Cold DNA (e.g., calf thymus DNA, yeast tRNA) is added to block nonspecific annealing. Spots of DNA from another organism whose mRNA is not represented in the sample (Background). Total genomic DNA or cDNA clones of common contaminant such as E.Coli and yeast are represented in the array to monitor for contamination.
Lack of information The number of genes encoded by the Human genome has been estimated 32,000 - 38,000. Between 21,000 - 27,000 genes are expressed in the cardiovascular system No cDNA Library for Atherosclerotic plaques ESTs from cardiovascular tissues or cell type or from diseased specimens remain limited. Only 5% of total ESTs deposited in GeneBank derived from cardiovascular tissue.
Premature Cardiovascular EST data from most model organisms are almost nonexistent. The construction of cardiovascular gene databases at different stages of pathology cast light on the complex genetic mechanisms underlying disease of cardiovascular system. DNA microarray technology is in infancy DNA microarray in atherosclerosis was not born or at least is premature.
Identification of Gene Potentially Involved in Rupture of Human Atherosclerosis Plaques. Circ Res 2001;89;547554 Deamen The first study dealing with differential gene expression in whole-mount specimens of rupture plaques using macroarray. Mammalian mRNA population 20% Abundant transcript (1000-12000 copies/cell) 25% Medium abundant (100-1000 copies/cell) % 50 small number copies (< 13 copies/cell) Mammalian mRNA encoding proteins that regular cellular behavior are expressed at low abundance. Suppression Subtractive Hybridization (SSH) technique isolates low abundant sequence that might not be isolated by use of microarray technology.
A pool of 3 ruptured plaques was compared with a a pool of advanced but stable plaques. Perilipin was the known gene that up regulated (confirmed by RT-PCR) , 8 of 10 ruptured plaques expressed perilipin while expression was absent in 10 stable plaque. Perilipin is a protein which present on the surface layer of intracellular lipid droplets in adipocyte and prevent lipolysis. They speculated that the increase in perilipin result in increased lipid retention and plaque destabilization. actin was down regulated in ruptured plaques. The down regulation of one gene was not confirmed by RT-PCR.
Prelipin is unlikely to be the sole marker of rupture. The author used only 10% of differentially expressed gene for doing macroarray A large effort at macroarray and then sequencing would have yield more differences. An alternative would be to hybridized the subtractand against a large array. (Stephen M.Schwartz et al.Circ Res 2001:89;471-473) Other alternative is the isolation of cell type-specific genes (LCM) rather than plaque-type-specific genes.
Richard T Lee et al. Treated cultured Human aortic SMC with TNF and used DNA microarray with 8600 genes to monitor the gene expression. Marked increase in eotaxin confirmed with northern blotting. Immunohistochemical analysis demonstrated overexpression of eotaxin and its receptor in the Human atheroma (SMC). Circulation;2000:102:2185-2189
McCaffrey et al. compared transcript profile of fibrous cap vs adjacent media of 13 patients ,using macroarray (membrane 588 known genes). Early growth response gene(Egr-1) was highly expressed in lesion (confirmed by RT-PCR). Many Erg-1 inducible genes including PDGF , TGF- and ICAM-1 were also strongly elevated in the lesion. Immunocytochemistry indicated that Egr-1 was expressed in SMC. ACTIN and GAPDH were use as housekeeping gene. J.C.I 2000,105:653-662
Adams et al. Compared gene expression of media of aorta and vena cava, using cDNA microarray of 4048 known genes. 68 genes had consistent elevation in message expression the aorta. The most differentially gene was Regulator of G Protein Signaling (RGS5). Northern analysis and in situ hybridization were used to confirm the results. Circulation Research 2000.8.623
R.M Lawn et al. examined the response of macrophages to exposure to oxidized LDL, using microarray containing 10000 Human genes. 268 genes were found to be at least twofold up regulated. Orphan nuclear receptors (PPAR, LXR and RXR) and ABC1 were among genes which unregulated after exposure. Real Time -PCR was used to confirm the results. J.B.C 2000:275;48, 37324-37332
L.A Mcintire et al. identified 52 genes with altered expression under shear stress Using DNA microarray in primary human umbilical vein endothelial cells. Significant increases in mRNA levels for 32 and significant decreases in expression for 20 genes were reported. The most enhanced genes were cytocromes P45 1A1 and 1B1 and human prostaglandin transporter. Most dramatically down regulated genes were connective tissue growth factor and endotheline-1. Northern blot analysis confirmed the results obtained on microarray. PNAS2001, 98:8955-8960
Brian K Coombes et al. used DNA macroarray to study the transcriptional response of Endothelial cells to infection with C.Pneumonia. C.Pneumonia infection up regulated m RNA expression for approximately 8% (20) of the genes studies (268). Genes coding for cytokines (IL-1), Chemkines (MCP-1) and cellular growth factor (PDGF) were the most prominently up regulated genes.
Proteomic is the study of the proteom or the entire protein complement of a genom It has been readily apparent that examining changes in the proteom offers insight into Understanding cellular and molecular mechanisms that cannot be obtained through genomic analysis. A recent study analyzing human liver samples determined the correlation coefficient between the amount of m RNA present to the corresponding protein abundance to be 0.48 (Anderson and Seilhamer 1997).
Many genes are expressed constitutively and regulation of their function is at the translational or posttranslational Levels (ApoB ,CFTR, TCR). Several studies have demonstrated selective TnI degradation under Ischemia/reperfusion, partly responsible for contractile dysfunction Observed after myocardial ischemia.( Circ Res.1999;84;9-20) Virtually all known cellular signaling pathways are largely mediated through a complex cascade of reversible protein phosphorylation.
Acute insults to cells lead to alteration in phenotype through rapid posttranslational Modification of proteins, whereas in chronic disease states cotranslational and Posttranslational protein modification occur in concert with altered gene expression. Most proteomic studies in cardiovascular focused in dilated cardiomyopathy and there is no report of proteomic evaluation in vulnerable plaque. Global proteome analysis provides a better representation of the phenotype than does gene expression analysis.
Our research group at the vascular biology laboratory of Center for Vulnerable Plaque Research in Texas Heart Institute is conducting a series of genomic and proteomic experiments to shed light on the possible molecular mechanisms involved in the onset and pathogenesis of atherosclerosis. Differential gene and protein expression of morphologically advance, but stable human atherosclerotic lesions and ruptured human atherosclerotic lesions are examined in a large number of patients in the whole-mount specimens.
Furthermore, Laser Captured Microdissection technology will be employed to evaluate gene and protein expression in different cell populations of atheroma plaques correlated with other markers (such as pH, Temperature, …). Transcript profile of blood monocytes from coronary patients with different presentations and healthy controls will be examined to address the association of gene expression and SNP with coronary risk. We hope these approaches lead to better understanding of the molecular process involved in development and complication of vulnerable plaques.
The lack of information in genomic and particularly proteomic approaches in vulnerable plaque is apparent and this highlights need for genomic and proteomic evaluation of plaque destabilization