SHAPE Society

1. Search for Genomic and
Proteomic Risk Factors and
Protective Factors
Associated with Coronary
Heart Disease
Mehran Haidari MD, Mohammad Madjid MD, Silvio
Litovsky MD, Ward Casscells MD, James T Willerson
MD, Xiaohong Wu MD, and Morteza Naghavi MD.

2. Background
 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.
 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.
 Investigation of advanced atherosclerosis using the tools for
systematic gene 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.

3. Background
 Several studies used high throughput gene
expression methods to examine the gene expression
of activated human umbilical vein endothelial cells,
vascular smooth muscle cells, and cholesterol-loaded
macrophages with that of non activated cells(Lu kp et al.
BBRC; 1998:253 ;828-833, De Vries CJ et al, JBC 2000;275:23939-
23947; Shiffman D. et al: JBC; 2000 275:37324-37332).These
studies in cell lines revealed differential regulation of
genes involved in leukocytes trafficking, cell cycle
control, and apoptosis. However, expression of these
genes in vivo remains to be determined.

4. Background
 Some groups focused on difference in
gene expression between fatty streaks
and advanced lesions (Hiltumen MO et
al. Current opinion on Lipidology
1999.10:515-519) and intima and media
of human atherosclerotic plaques
(McCaffery TA et al. , JCI;
2000:105;653-662).

5. Background
 Haley et al. (Circulation: 2000;102:2185-2189
) examined differential gene expression from
cultured human aortic smooth muscle cells
treated with TNFα using DNA microarray
technology. The authors reported that Eotaxin
and its receptors, CCR3, were overexpressed
in human atherosclerosis, suggesting that
Eotaxin participates in vascular inflammation.

6. Background
 Faber et al. (8) compared transcript profile of
morphologically advanced, but stable human atherosclerotic
lesions. Using suppression subtractive hybridization (SSH)
technique on whole-mount specimen they overcame the
problem of isolation of low abundant sequences that might
not be isolated by use of microarray technology. They found
25 genes that showed at least a 2-fold difference in
expression.Perilipin was up regulated in ruptured plaques
and the genes coding for fibronectin and immunoglobulin λ
chain were down regulated in ruptured plaques. This was
the first study used high thorough-put method for the gene
expression of ruptured plaque. However, the study suffers
from serious limitations. The number of specimens that they
used was three from ruptured plaques and three from stable
plaques which were pooled for SSH.

7. Objective and hypothesize
 SEARCH FOR GENOMIC AND PROTEOMIC RISK FACTORS AND
PROTECTIVE FACTORS ASSOCIATED WITH CORONARY HEART
DISEASE by screen large number of patients’ blood cells in
different group. We are going to look for unknown genes and protein
in the blood that may predispose possible heart attack. Similarly in
elderly population who have had risk factors for heart attack but
fortunately never experience heart attack, we will be looking for
possible genes and proteins that protect them from having a heart
attack
 Almost every alteration in physiology and pathology of cell is
accompanied by differential gene and protein expression we
hypothesize that the gene expression profile of the monocytes and
neutrophiles are distinct among each group of patients. Thus, by
using DNA microarray technology we can identify different transcript
profile among each groupe of patients which would help us develop
new diagnostic and therapies for coronary heart disease.

8. Design
Study Population:
 This study will be an analytic case-control study and either sex,
18 to 80 years old patients will be recruited in the study. Based
on the criteria of the heart attack and risk factor ( below),
patients will be classified into five groups. First group are
patients who have heart attack with two or more than two of the
risk factors. Second group are patients who have heart attack
but no risk factors. Third group are patients who have two or
more than two risk factors but no heart attack with age over 70
years old. Fouth group are patients who have two or more than
two risk factors but no heart attack with age between 50-70
years old. Fifth group are patients with either sex who have no
heart attack and no risk factors, aged from 50-70 years old.( As
the scheme next).

10. Design
Criteria for Heart Attack:
 Patients who admitted to Hermann and St. Luke’s
Episcopal Hospital with Myocardial infarction or Acute
coronary syndromes.

11. Design
Risk Factor:
We classify the key risk factors include:
 1. Gender and Age
 2.Hyperlipidemia;
 3. Hyperblood pressure;
 4. Smoking;
 5. Physical inactivity;
 6. Obesity and overweight;
 7. Diabetes mellitus;
 8. Adverse dietary pattern.

12. Design
Exclusion criteria:
 Pregnancy
 Breast feeding

13. Design
Sample size:
 In order to obtain necessary information
for evaluation of techniques and
calculation of required sample size, we
need to do pilot study. Fifty patients
from each group will take part in the
study.

14. Experimental Procedures.
Baseline Examination:
 Information about history of smoking,
diabetes mellitus and, general physical
examination, blood pressure, lipid profile,
height and weight, fasting blood glucose will
be obtained from the medical record of
patients. Participants will be asked for
donation of 10 ml of blood (after giving
approved informed consent).

15. Experimental Procedure
Monocytes and Neutrophiles collection and RNA Isolation:
 10ml of whole blood samples will be withdrawed from each
group of patients. 5ml of blood will be used forRNA isolation.
The rest of blood will be saved in the bank and for future protein
analysis.
 The Monocyte and neutrophils isolation will use CD14
(Monocyte marker) coated dynalbeads and CD15 (Neutrophils
markers) dynalbeads(Dynal Biotech). The isolation of mRNA will
be isolated by using Dynalbeads mRNA DIRECT kit(Dynal
Biotech). RNA quantity will be determined by optical density
measurement at 260 nm and 280nm with 260/280 ratio at 1.7-
2.0 as indication of high purity. 1% Agarose gel electrophoresis
will be further used to examine the purity of mRNA.

16. Experimental Procedure
Preparation of Labeled Cellular RNA:
 A total of 5 µg of mRNA will be used for double-stranded
complementary DNA (c DNA) synthesis.
 Double-stranded c DNA will be generated with a c DNA
synthesis kit (Superscript c DNA Synthesis System:Life
Technologies, Giathersburg, DNA)
 The c DNA will be extracted with phenol/chlorofom, ethanol
precipitation and use as a template for in vitro transcription
with biotin-labeled nucleotides (BioArray High Yield RNA
Transcript Labeling Kit: Enzo Diagnostics, Framindale, NY).
 The cellular RNA (c RNA) will be fragmented at 94°C for 35
min in fragmentation buffer and hybridization mix will be
generated by addition of herring sperm DNA (0.1mg/ml)
sodium chloride (1M), Tris-acetate (10 mM) and Tween-20
(0.0001 %). A mixture of three bacterial and phage c RNA
will be included to serve as an internal control for
hybridization efficiency.

17. Experimental procedure
Hybridization of Microarray:
 Aliquots of each sample (5 µg c RNA in 200µl
hybridization mix) will be hybridized to a Genechip
(U133A and U1333B expression probe arrays,
Affymetrix).
 After hybridization, each array will be washed, stain
with streptavidin phycoerythrin (Molecular Probes,
Eugene. OR), rewash, hybridize with biotin –labeled
antistreptavidin phycoerythrin antibodies (Vector
Laboratories, Burlingame, CA), restain with
streptavidin phycoerythrin, scan (Affymetrix 428 Array
Scanner), and will wash according to procedures
developed by manufacturer (Affymetrix).

18. Experimental Procedure
Analysis of Genechip Data:
 Scanned output files will be analyzed using
Genechip 3.3 software (Affymetrix) and the
expression value for each gene will be
determined by calculating the average of
differences (perfect match intensity minus
mismatch intensity) of the probe pairs in use
for that gene.

19. Experimental Procedure
Suppression Subtractive Hybridization
 To isolate low abundant sequences that might not be isolated by
use of microarray technology, Suppression Subtractive
Hybridization (SSH) will be used to detect differentially
expressed sequences.
 The SSH procedure will be performed by using the PCR-
selected c DNA subtraction kit (Clontech) essentially according
to the protocol of the manufacturer.
 The differentially expressed genes will amplified by two rounds
of PCR and The c DNA will be extracted with phenol/chlorofom,
ethanol precipitation and use as a template for in vitro
transcription with biotin-labeled nucleotides (BioArray High Yield
RNA Transcript Labeling Kit: Enzo Diagnostics, Framindale,
NY).

20. Experimental Procedure
Real-Time PCR:
 To confirm any changes in gene
expression in microarray real time PCR
techniques will be used.

21. Experimental Procedure
RNA in Situ Hybridization and
Immunochemistry:
 RNA in Situ Hybridization and
Immunochemistry techniques will be
used to examine any change in the
protein mass of the interested genes.

22. Data Analysis
 We will coorperate with Dr.Fofanov and Dr.
Christoph F. Eick from UH Department of
Computer Science for developing software
and database for further analyze the gene
expression profile from different group of
patients in order to gain further genetic
information that would help us to be able to
develop new diagnostic and therapies for
Coronary Heart Disease.