This document discusses the use of DNA microarrays in vulnerable plaque research. It provides background on atherosclerosis and identifies DNA microarrays as a tool that can be used to investigate the molecular mechanisms underlying plaque vulnerability. The document outlines the basic steps of a DNA microarray experiment and discusses considerations for experimental design, data analysis, and validation of results. It also summarizes several studies that have used DNA microarrays or related techniques to examine gene expression in atherosclerosis.

1. DNA Microarray
Mehran Haidari
Application in Vulnerable plaque research
Center of Vulnerable Plaque Research
Texas Heart Institute

2. 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.

3. 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, )
Deamen Schwartz Lee

4. 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

5. 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

6. What is DNA Microarray?
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
Reverse Northern blotting
Patrick O Brown
Mark Schena

7. 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

8. 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

9. Analysis of Gene Expression
Monitoring Changes in Genomic
DNA
Gene Discovery, Sequencing and Pathway Analysis
When to use Microarray

10. Analysis of Gene Expression
1- Different tissues or different developmental states
2- Normal or diseased states
3- Exposure to drugs or different physiological conditions

11. 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
Micro or Macro

12. 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)

13. Sample Labeling
Most microarray utilize two fluorophores,
Cyanine3(Green emission) and Cyanine5 (Red emission)
Fluorophores have different size and different ability
for incorporation in cDNA
A single round of transcription is used to generate
a labeled cDNA probe (RT-PCR)

14. Affymetrix Genechip
Biotinylated cRNA is synthesized from cDNA
phycoerthrin linked to avidin is used for labeling
Each sample hybridized separately
Advantages
High density chip
Consistent and uniform geometry
Single Nucleotide Polymorphisms(SNP)
No need for maintaining cDNA clones
Disadvantages
Sequence data required
Oligonucleotid selection rules
are not well defined
Not best target for hybridization
Expensive
Hybridization to oligonucleotide is sensitive in
detection of single-nucleotide mismatches

15. No consensus on Data Analysis( ANOVA), Clustering
(categorizing genes according to their pattern of expression)
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

16. Analyzed gene changes are often expressed as a fold increase
either greater than twofold or less than 0.5 fold (DeRisi)
How Much is Significant???
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

17. Housekeeping genes
These are genes that are expressed constitutively and their level of
expression is thought to be stable, regardless of the sample used (β
Actin, Cyclophilin, GAPDH)
DeRisi used 90 housekeeping genes and found that changes that
were <0.5 and > 2.4 were acceptable
β 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

18. 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
Accuracy and Precision

19. Principles of Q.C in DNA Microarray
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
Rajeevan et al. estimated that 30% of
microarray results are false-positive
Microarray findings should be confirmed, at least
by one of the low-throughput gene expression methods
Down-Scaling of an experiment makes it generally
sensitive to external and internal fluctuation
J.Mol.Diag 2001,3:26-31

20. 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

21. 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
Lack of information
No cDNA Library for Atherosclerotic plaques
Only 5% of total ESTs deposited in GeneBank derived from cardiovascular tissue
ESTs from cardiovascular tissues or cell type
or from diseased specimens remain limited

22. 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
Premature

23. The first study dealing with differential gene expression in whole-mount
specimens of rupture plaques using macroarray
Suppression Subtractive Hybridization (SSH) technique isolates low abundant
sequence that might not be isolated by use of microarray technology
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
Identification of Gene Potentially Involved in Rupture of
Human Atherosclerosis Plaques
Circ Res 2001;89;547554
Deamen

24. 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
β actin was down regulated in ruptured plaques
The down regulation of one gene was not confirmed by RT-PCR
A pool of 3 ruptured plaques was compared with a
a pool of advanced but stable plaques

25. 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
Other alternative is the isolation of cell type-specific genes
(LCM) rather than plaque-type-specific genes
(Stephen M.Schwartz et al.Circ Res 2001:89;471-473)

26. Richard T Lee et al. Treated cultured Human aortic SMC with
TNFα 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

27. 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-β and ICAM-1
were also strongly elevated in the lesion
Immunocytochemistry indicated that Egr-1 was expressed in SMC
β ACTIN and GAPDH were use as housekeeping gene
J.C.I 2000,105:653-662

28. 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

29. 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.
Real Time -PCR was used to confirm the results.
Orphan nuclear receptors (PPARγ, LXR and RXR) and ABC1 were
among genes which unregulated after exposure.
J.B.C 2000:275;48, 37324-37332

30. 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

31. 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.

32. 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).

33. 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.

34. 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.

35. 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.

36. 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.
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, …).
We hope these approaches lead to better understanding of the
molecular process involved in development and complication of
vulnerable plaques.

37. 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