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.

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.

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.

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).

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.

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 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.

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.

DesignStudy 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).

DesignCriteria for Heart Attack:

Patients who admitted to Hermann and St. Luke’s Episcopal Hospital with Myocardial infarction or Acute coronary syndromes.

DesignRisk 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.

DesignExclusion criteria: Pregnancy Breast feeding

DesignSample 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.

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).

Experimental ProcedureMonocytes 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.

Experimental ProcedurePreparation of Labeled Cellular RNA: A total of 5 g 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 94C 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.

Experimental procedureHybridization of Microarray: Aliquots of each sample (5 g c RNA in 200l

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).

Experimental ProcedureAnalysis 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.

Experimental ProcedureSuppression 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).

Experimental ProcedureReal-Time PCR: To confirm any changes in gene

expression in microarray real time PCR techniques will be used.

Experimental ProcedureRNA 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.

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.