Search for Genomic and Proteomic Risk Factors and Protective Factors Associated with Coronary Heart Disease

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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.

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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.

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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 1998253
  828-833, De Vries CJ et al, JBC
  200027523939-23947 Shiffman D. et al JBC
  2000 27537324-37332).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.

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Background

• Some groups focused on difference in gene
  expression between fatty streaks and advanced
  lesions (Hiltumen MO et al. Current opinion on
  Lipidology 1999.10515-519) and intima and media
  of human atherosclerotic plaques (McCaffery TA et
  al. , JCI 2000105653-662).

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Background

• Haley et al. (Circulation 20001022185-2189 )
  examined differential gene expression from
  cultured human aortic smooth muscle cells treated
  with TNFa 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.

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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.

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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.

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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).

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Design

• Criteria for Heart Attack
• Patients who admitted to Hermann and St. Lukes
  Episcopal Hospital with Myocardial infarction or
  Acute coronary syndromes.

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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.

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Design

• Exclusion criteria
• Pregnancy
• Breast feeding

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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.

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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).

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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.

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Experimental Procedure

• Preparation 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
  SystemLife 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.

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Experimental procedure

• Hybridization of Microarray
• Aliquots of each sample (5 ?g c RNA in 200?l
  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).

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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.

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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).

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Experimental Procedure

• Real-Time PCR
• To confirm any changes in gene expression in
  microarray real time PCR techniques will be used.

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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.

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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.