SHAPE Society

1. Basic Molecular GeneticBasic Molecular Genetic
StudiesStudies
in Atherosclerosisin Atherosclerosis
Methodology, Applications,Methodology, Applications,
and “Future Horizons”and “Future Horizons”

2. Risk factors: e.g.
hypertension, DM,
infection( HSV,CP
), smoking etc
Hyperlipidemia
• Diet
• Familial
lipid deposition
and removal
imbalance
Endothelia
l injury
Release of cytokines, adhesion
molecules vasoactive molecules and
growth factor
Lipid
accumulatio
n and
oxidation
Macrophage and T
cell accumulation
Chronic Active
Inflammation
SMC
proliferation
Intimal
thickening and
fibrous cap
formation
Core formation
Plaque formation
SMC and foam cell apoptosis, active macrophage
infiltration, and matrix degradation
Plaque rupture
A
T
H
E
R
O
S
C
L
E
R
O
S
I
S
A
t
A
G
L
A
N
C
E

3. Molecular genetics
Brief overview of methods to analyse
and study genetics

4. Polymerase chain reaction-PCR
• This is achieved by repeated rounds of three
different steps: heat denaturation of template
DNA,
• Annealing of two convergent oligonucleotide
primers to the opposite strands of the DNA
template
• 5’-3’ extension from each of the annealed primers
using a thermosable DNA polymerase

5. Chain Termination
• This is done to obtain DNA fragments of different
lengths
• For this reaction to occur four standard dNTPs
(dATP, dCTP, DGTP, dTTP) are included in the
reaction mixtures

6. Gel-Electrophoresis and
Autoradiography
• After the chain terminated strands are obtained,
they are run through an agarose gel and usually
stained with Ethidium bromide and visualize the
bands on a u.v transilluminator.
• Electrophoresis makes use of the fact that
molecules like DNA migrate in an electric field
inversely proportional to its molecular weight
towards the positive pole.

7. Gel electrophoresis
• Fragments of DNA are electrophoresed and size of
each fragment is determined

8. Southern Blot for DNA analysis
• The restriction fragments obtained by gel-
electrophoresis are subject to denaturation with
alkali and transferred to a nitrocellulose filter or
nylon membrane by blotting.
• The filter is them incubated under hybridization
conditions with a specific radiolabeled probe
usually generated from a cloned restriction
fragment.

9. • The DNA restriction fragment that is
complementary to the probe hybridizes, and its
location on the filter can be revealed by
autoradiography.

10. RT-PCR
a way to detect mRNA in cells
1. by using Reverse-
Transcriptase enzyme
a cDNA is made on the
template of RNA
2. PCR amplifies the
cDNA derived from
mRNA molecule
3. The amplified cDNA is
detected
mRNA
Standard PCR
primer
Bases
cDNA
Reverse
transcriptase

11. Methods to detect Sequence Variations
• Use of silver stainings to detect Nucleic acids
• Nonradioactive Method for the Detection of
Single-strand Conformational Polymorphisms.
• Temperature Gradient Gel Electrophoresis(TGGE)
for the detection of polymorphic DNA and RNA.
• TGGE in Quanttitative PCR of DNA and RNA
• Direct sequencing of PCR products

12. Analysis of Specific Nucleic
Acids in Complex Mixtures
• Southern Blotting Detects specific DNA
fragments
• Northern Blotting Detects Specific RNAs
• Nuclease Protection is used to Quantitate Specific
RNAs and Map the DNA Regions encoding them

13. Probes
• A probe is a relatively small piece of DNA that is
used to find another piece of DNA.
• In nucleic acid hybridization a DNA probe,
labeled radioactively or non-radioactively, seeks
out and finds complementary DNA In the target
DNA.
• The shortest useful probe is about 20 bases long
and is known as oligonucleotide probe.
• RNA molecules may also be used as probes and
are termed “riboprobes”.

14. Nucleic acid probes are segments of
DNA(/RNA) that :
1.1. have been labeledhave been labeled (with enzymes antigeneic
substrate , chemiluminescent moieties or
radiolabeled) and can bind with high specificityan bind with high specificity
to DNA/ RNA targets
2. probes can be 2 to 1000 bases
3. early DNA probes were labeled with 32 P, now
in most kits radioisotopes are replaced by
enzymes, affinity labeles and chemiluminescent
molecules. ( Enzymes like alkaline phosphatase ,
horseradish peroxidase , affinity labeles like
biotin, digoxigenin)

15. Hybridization
• The process of forming a double stranded DNA
molecule between a probe and a target.
• DNA is double stranded and can be made single
stranded.
• If a large excess of DNA, called a probe, that has
complementarity to a particular sequence of
interest is added to the target, they form a double
stranded structure and this is termed hybridization.

16. Nucleic Acid
Immunocytochemistry
This technique uses nucleic acid-antibody complexes
as probes and nucleic acids as targets. The
aspects of a nucleic acid immunocytochemistry
are as follows:
• Target is DNA or mRNA localized within a cell
• The recognition and detection of target nucleic
acid relies on base pairing(hybridization) of
complementary bases of the target nucleic acid

17. • The recognition and detection of target nucleic
acid relies on base pairing(hybridization) of
complementary bases of the target nucleic acid.
• The antibody moiety complexed to the nucleic
acid portion of the probe serves only as a signal-
generating system reporting that a target has
been found and hybridization has occurred.

18. Fluorescence In Situ
Hybridization (FISH)
• FISH is widely used to determine the
chromosomal map location and the relative
order of genes and DNA sequences within a
chromosomal band.

19. Recombinant DNA Technology
• Recombinant DNA is artificially created DNA for the
purpose of genetic engineering which can be introduced
into appropriate cells to form a clone of such cells.
• It makes use of two enzymes:
• Restriction enzymes: which cut the DNA from any
organism at specific sequences.
• Ligases: which can insert DNA restriction fragments into
replicating DNA molecules producing recombinant DNA.

20. DNA Cloning with Plasmid
Vectors
• Plasmids are extrachromosomal self-replicating DNA
molecules.
• E-coli Plasmids can be engineered for use as cloning
vectors.
• Replication origin(ORI) is a specific DNA sequence of 50-
100 base-pairs that must be present in a plasmid for it to
replicate.
• Cells to be transformed are selected and then under
favorable conditions, i.e;high concentration of certain
divalent cations,1 cell in about 10,000 or more cells
becomes competent to take up foreign DNA.

22. Commercial Potential of
Recombinant DNA
• Genetic Engineering as a man-made entity has
been refined over the 25 years of its existence to
the point where medically and pharmaceutically
important reagents ( for example, insulin, and
interferon) as well as certain vaccines are now
routinely being made on a manufacturing scale.
• Genetic Engineering has been also used in
brewing, fermenting, wine making and other
fields.

23. Genomic analysis
1. DNA sequencing
2. Single molecule sequencing
3. SNP( single nucleotide polymorphism)
4. DNA array technology (Functional
Genomic analysis)

24. DNA sequencing
1. A chain terminators
stops DNA synthesis at
different sites.
2. A strand of DNA is
replicated
3. The chain terminator is
a nucleotide that is
similar but not the
same as original
nucleotide
4. The place of all A's,
T's, C's and G's are
detected by gel-
electrophoresis.

25. Single molecule sequencing
• Uses exonucleases to degrade DNA by cleaving
individual nucleotides from 5’-end of a DNA
molecule and detecting them
• The most common methods use DNA segments
made from nucleotides where four bases have
been differentially tagged with fluorescent labels
• When the tagged nucleotide is clipped, it flows
past a laser-based fluorescence detector

26. Single nucloetide polymorphism
• The most common type of stable genetic
variations, these point mutations can produce
different phenotypes and can be contributory
factors for human disease.
• SNP detections methods are diverse and beyond
the scope of this review.
• One notable approach uses matrix-assisted laser
desorption mass spectroscopy(MALDI-MS) on a
silicon chip for SNP detection; another system
employs electrical circury on siliccon microchips
to produce a fluorescent signal

27. Expressed Sequence Tags
• ESTs are identified using reverse transcriptase
(RT) to create cDNA sequnces from mRNA
present in a cell, allowing genes expressed in
different tissues or environmental conditions to be
easily amplified by PCR for further study
• Because ESTs might be only gene
fragments(typically 3’ or 5’0, they are more easily
generated than entire sequence information

28. Active and In-active mRNA
• Protein expression levels are not correlated with
mRNA expression levels
• Because protein rather than mRNA levels
determine phenotype, there are efforts underway
to investigate this difference by analysing the
translation state of mRNA
• Active and inactive ribosomes can be readily
separated using sucrose gradient centrifugation;
the fractions can then be identified using labeled
cDNA probes and used to interpret data based on
mRNA expression to estimate protein levels

29. DNA arrays
• DNA arrays consists of large number of DNA molecules
spotted in a systematic order on a solid substrate( such as
nylon membrane, glass slide, or silicon chip).
• Depending on the size of each DNA spot on the array,
DNA arrays can be categorized as microarrays(each DNA
spot has a diameter of <250 microns) and
macroarrays( spot diameter of>300 microns).
• When the solid substrate used is small in size, arrays are
also referred to as DNA chips

30. Microarray assays
• Traditional hybridization assays developed in the
1970s utilize flexible membranes such as
nitrocellulose and nylon, radioactivity, and
autoradiography.
• By contrast, microarray or biochip assays utilize
solid surfaces such as glass with fluorescent
labelling and detection, this miniaturized biochip
format has revolutionized biological analysis.
• Advantages of solid surface are it being non-
porous, allows using of small sample volumes,
rapid hybridization kinetics, uniform attachement

31. Microarray Fabrication
• There are three primary technologies used
presently in microarray manufacture include
photolithography, ink-jetting, mechanical
microspotting, and derivatives thereof.

32. • Microarray fabrication technologies
CRITERION PHOTOLITHOG
RAPHY
PIEZOELECTRIC MICROSPOTT
ING
Combinatorail
syntesis
Yes Yes No
Ink-jetting NO Yes No
Surface printing No No Yes
Masks needed Yes NO NO
Sample tracking NO NO Yes
Density(/cm) 244000 10000 6500
Array elements Oligos only Oligos and cDNAs Oligos and
cDNAs
Prototyping cost High Moderate Low

33. • Continued
Criterion photolitography Piezoelectric Microspotting
Gene expression,
mutation
detection
Gene
expression,
mutation
detection
Gene
expression,
mutation
detection
Commercial
vendors
Affymetrix Biodot,
Cartesian,
Incyte, Packard
Instruments,
Protogene,
Rosetta
Cartesian, Genetix,
Hyseq Genometrix,
Gene Machines,
Genetic, Synteni
Microsystems,
Norgen Systems,
Telechem,
Molecular
Dynamics

34. Applications of DNA array
• Gene expression profiling
• De novo gene sequencing
• Gene mutation analysis(SNP)
• Gene mapping and genotyping

35. Main steps in performing a DNA
array experiment
1. Printing target DNA or oligoneculotide onto a
substance,
2. Sample RNA isolation (either total RNA or
mRNA),
3. cDNA synthesis and labeling,
4. Hybridization of the labeled probe cDNA to
target cDNA on the substrate,
5. Imaging of hybridization results and image
analysis

36. Schematic of probe preparation , hybridization,
scanning and immage analysis

37. • cDNA microarray target printing apparatus. Computer-
controlled robotic cantilever arm, capable of moving in XYZ
directional planes, can be armed with up to 16 (two rows of eight)
`quill' print tips on the print head. In one automated print cycle,
the print head dips the quills into a set of target DNA wells
arrayed in 96 well microwell plates; then the print head traverses
the vacuum table and touches the quill tips to each glass slide in
succession ,depositing target DNA; the print head continues to the
wash/dry station where the tips are cleaned twice with water and
dried. This cycle repeats as the print head returns to wet the tips
in the next set of targets, continuing until all targets of a 96 well
microwell plate have been printed. An autoloading mechanism
removes spent microwell plates and can serve up new plates. By
this method, microarray slides can be printed with as many as
15000 precise and discrete cDNA targets

40. Microarray hybridization. This pseudocolored image
represents a portion of a microarray with the reference
probe (normal fibroblasts) in green and
rhabdomyosarcoma in red. The up (red) and down
(green) regulation of several diseased genes are
illustrated. Representative genes of interest are boxed.
Cancer Res. 58(1998)5009-5013

41. red color indicates
overexpression and
green
color reduced
expression

42. • The impact of molecular biology and genetic
research on the discovery of the root causes of a
wide variety of hereditary and acquired diseases
has long been self-evident.
• Now growing research in this field has shown that
conditions like hypertension, arryhtmias, certain
cardiomyopathies, Hyperlipidemia, aortic
aneurysms, myocardial infarction are all
genetically determined.
Molecular Genetics and Its Application
to Understanding Cardiovascular Disease

43. Gene Therapy
• Gene therapy, i.e; to introduce genes in selected cells to
treat genetic or acquired diseases .Recombinant DNA
technology has revolutionized this field.
• Clinical trials are underway to treat a variety of
conditions like cancer, AIDS, familial
hypercholesterolemia, cystic fibrosis, and many other
diseases with this therapy.
• The growing acceptance of gene therapy for
cardiovascular diseases is due to its potential to treat
common , multifactorial disorders like cancer,
atherogenesis, and inflammation.

44. Our Aim
• We intend to induce rupture of atherosclerotic plaque in
apo-e mice by injecting several drugs, which we hope will
cause rupture of the the hemodynamic system and raising
the plaque by altering oxidative stress and alter the
composition of the plaque.
• After achieving this goal we will perform gene- array
study on these specimens and will try to find out which
genes are expressed more in plaques which are ruptured or
are prone to rupture, by comparing our results at the
histopathology lab which will define the structural details
of such vulnerable plaques.

45. • Drugs being used to
induce rupture of
atherosclerotic plaque
in apo-e mice

46. Drugs being used
• LNAME
• Adrenalin
• Cocaine
• Xanthine
• Xanthine Oxidase
• Interleukin-1 beta
• Methionine
• Buthionine Sulfoximine

47. L-NAME
• L-NAME (N-nitro-L-arginine methyl ester), is a negative
effector of nitric oxide synthase.
• Action:By virtue of this action, it blocks the action of
nitric oxide and interferes with the arterial dilatation,
causing more load on the heart and the circulation
• The production of NO is a major contributor to the
endothelium-dependent relaxation in large isolated
arteries, including coronary, mesenteric, pulmonary,
systemic, and cerebral arteries.

48. • All the actions of nitric-oxide are inhibited by L-NAME,
as well as it promotes adhesion of platelets and leukocytes
to the vascular lumen..
• Also the inhibitory effect of NO towards inhibiting platelet
aggregation action of prostacyclin and inhibition of growth
of vascular smooth cells is abolished by L-NAME.

49. Interleukin-1 beta
• Class: Cytokine
• These cytokines are polypeptides produced
by many cell types(but principally activated
lymphocytes and macrophages).
• Their secretion can be stimulated by
endotoxin, immune complexes, toxins,
physical injury, and a variety of
inflammatory processes.

50. • Monocytes are the main source of
secreted IL-1. They express
predominantly IL-1-beta while
human keratinocytes express
large amounts of IL-1-alpha .
Murine macrophages display a
transition from IL-1-beta to IL-1-
alpha production during
maturation of monocytes into
inflammatory macrophages .

51. Action
• Their most important actions in inflammation are
the local effects on endothelium, the systemic
acute-phase reactions, and the effect on
fibroblasts.
• In particular they induce the synthesis and surface
expression of the endothelial adhesion molecules
that mediate leukocyte sticking and increase
surface thrombogenicity of the endothelium.

52. Adrenalin
• Class: Sympathomimetic
• Mech: At low doses, beta effects on the vascular
system predominate, whereas at high doses, alpha
effects are strongest.
• Action: Major actions are on the cardiovascular
system, causes tachycardia, increases
myocardium’s oxygen demand, constricts
arterioles in the skin, mucous membrane and
viscera.

53. • Also causes broncodilation, hyperglycemia,
lipolysis.
• Adverse effects: causes CNS disturbances
including tremor, anxiety, and marked elevation of
blood pressure, and arrythmias.

54. Methionine
• Class: Amino acid
• Metabolism of the amino acid methionine, a
limiting amino acid in the synthesis of many
proteins, affects several biochemical pathways
involving the production of nutrients which are
essential to the optimal functioning of the
cardiovascular, skeletal, and nervous system
Homocysteine is an intermediate product of
methionine metabolism.

55. Action
• Homocysteine has direct cytotoxic effects on
endothelial cells in vitro
• Homocysteine has a direct procoagulant effect on
factors V and X and inhibits protein C activation.
• Additionally, there is exciting preliminary data
linking a flux in the homocysteine pathway with
liver disease, leukemia, psoriasis and other
relatively common disorders. Research is being
conducted currently in many of these areas.

56. • In the past 20+ years, research has shown that
elevated homocysteine, or
hyperhomocysteinaemia, is linked to
atherosclerosis, pregnancies complicated by neural
tube defects, early pregnancy loss and venous
thrombosis.
• Patients with chronic renal disease have a two to
three fold increase in homocysteine levels .

57. Buthionine Sulfoximine
• Class: selective glutathione (GSH) synthase
inhibitor.
• Due to their action causing depletion of
GSH, which is a necessary component of
the natural antioxidant system, BSO causes
oxidative stress, which might cause
hypertension by inactivation and
sequestration of NO(mediated by ROS).

58. Cocaine
• Class: Sympathomimetic
• Mech: Blocks neuronal re-uptake of
norepinephrine, serotonin, and dopamine.
• Action: Stimulates CNS, causes euphoria,
increases motor activity.
• Potentiates the action of cathecholamines,i.e;
tachycardia, hypertension, pupillary dilation and
peripheral vasoconstriction.

59. • Adverse effects:
• Anxiety, causing increased blood pressure and
heart rate, sweating.
• Depression, Like all stimulant drugs, cocaine
stimulation of CNS is followed by a period of
mental depression.

60. Xanthine, Xanthine-Oxidase
• Xanthine oxidase (XO) is a complex enzyme
containing flavins, molybdenum, iron and sulfide
cofactors. The reaction catalyzed by Xanthine
oxidase is shown below:
• XO
• Xanthine + O2 + H2O ------> Uric Acid + H2O2
• XO is thought to be the principal source of free
radical generation via degradation of nucleotides
to the end product, uric acid.

61. • Xanthine can exert lethal effects by
generating free radicals, this has been
proved by several studies, and the effcet of
these free radicals is to depress myocardial
contractility, by virtue of robbing the
myocytes of high energy phospahtes, also it
causes the LDLs to be more prone to be
oxidized.

62. Doses of drugs used
Based on references from articles
from Pub-Med

63. • . Substance: LNAME
Route of Administration: IP
Dose: 20 mg/kg/day for 7 days
Frequency: 7
References: Nitric oxide 2000Apr;4(2):85-93
Life Sci 1995;57(21):1949-61,
Br J Pharmacology:1994oct;113(2):345-8

64. • 2. Substance: adrenalin
Route of Administration: IP
Dose: 1 microgram/kg
Frequency: 7
References: Auton Pharmacol 1998 jun;18(3):149-
155
Life Sci 1982 Apr 26;30(170:1465-72
Can J Cardiol 1990 Mar;6(2):71-4
.

65. • Substance: cocaine
Route of Administration: IP
Dose: 10 mg/kg/day
Frequency: 7
References: Nihon Arukoru Yakubutsu Igakkai Zasshi
1997 Apr;32(2):122-38
J Appl Physiol 1991 Mar;70(3):1323-7
Eur J Invest 1997 Feb;27(2):116-20

66. • 4. Substance: xanthine
Route of Administration: IP
Dose: 0.0012 mM/kg/day for 7 days
Frequency: 7
References: Naunyn Schmiedebergs Arch Pharmacol
1994 Sp;350 (3):227-83
Naunyn Schmiedebergs Arch Pharmacol 1992
Oct;346(4):457-61

67. • 5. Substance: xanthine oxidase
Route of Administration: IP
Dose: 0.035 U/ml/day for 7 days
Frequency: 7
References: Cardiovasc Res 1986 Aug;20(8):597-
603
Mol Cell Biochem 1995 Apr 26;145(2):103-10
Naunyn Schmiedebergs Arch Pharmacol 1994
Sp;350 (3):227-83
Naunyn Schmiedebergs Arch Pharmacol 1992
Oct;346(4):457-61

68. • 6. Substance: IL-1 beta
Route of Administration: IP
Dose: 1ug/mouse/day for 7 days
Frequency:7
Reference: Cancer Biother Radiopharm 1997
Apr:12(2):101-9
Am J Physiol 1995 Apr;268(4 Pt 1):E551-7
Brain Res 1999 Jan 9;815(2):337-48
Pediatr Res 1995 Jun;37(6):714-9
J Neuroendocrinol 1994 Apr;6(2):217-24

69. • Substance: methionine
Route of Administration: IP
Dose: 1280 mg/kg/day for 7 days
Frequency: 7
Reference: Pharmacol Biochem Behav 1999
Sep;64(1):89-93

70. • Substance: Buthionine sulfoximine
Route of Administration: IP
Dose:1 mmol/kg
Frequency: 7
References:
In previous studies doses of 2.5mmol, 7.2mmolI/V,
And 5mmol/kg, and 3mmol/kghave been used, the dose
we are going to use is much lower than that.
Hepatology 2000 Aug;32(2):321-33
J Pharmacol Exp Ther 1998Nov;287(2):515-20

71. Taking Samples and performing
the study

72. 9/11/00 42
Samples:
A. Human carotid atherosclerotic plaque from St.
Luke’s hospital OR: ( endarterectomy results)
B. Ruptured human coronary atherosclerotic
plaque from sudden cardiac death victims with
plaque rupture from Dr Kolgogi (Dr Virmani’s
Lab)
C. Atherosclerotic plaque of Apo-E K/O old mice
(18 months) fed with high fat diet that have been
prompted to rupture using several interventions
( modified Falk procedures).
D. Atherosclerotic plaque of Apo-E K/O 18 months
old mice without being prompted to rupture.
E. Normal C57BL mice artery.

73. Results
• We will perform gene-array on the RNAs
obtained form the ruptures palque to see
which genes are more expressed in the
palque
• We will also compare our results in the
histochemical laboratory

74. 9/10/00 43
Gene array method
 We will use mRNA for probe. RNA will be extracted
according the protocol and then cDNA is made by
RT-PCR. Hybridize with specific probes and
scanned.
 We will use Affymetrix gene array materials.
 The chips for samples As & Bs contain 12000
human genes.
 The chips for samples Cs and Ds contain 19000
mouse genes.
 We will scan the chips with Affymetrix scanner.

75. If we do not find
anything
Very pleasant, at least
we shalll find
something new