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The contribution of arachidonate 5-lipoxygenase to inflammatory response in coronary artery disease

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The contribution of arachidonate 5-lipoxygenase to inflammatory response in coronary artery disease

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THE CONTRIBUTION OF ARACHIDONATE 5-LIPOXYGENASE TO
INFLAMMATORY RESPONSE IN CORONARY ARTERY DISEASE

by

Susanna Vikman

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

December 2010

Copyright 2010 Susanna Vikman

ii
Table of Contents
List of Tables iii
List of Figures iv
Abbreviations v
Abstract vi
Chapter 1.1 Introduction 1
1.2 Background 2
1.3 Historical perspective 3
1.4 Coronary artery disease risk factors 7
1.4.1 Clinical risk factors 7
1.4.2 Environmental risk factors 7
1.4.3 Genetic risk factors 8
1.5 Pathogenesis of atherosclerosis 10
1.5.1 LDLc mediated atherosclerosis 10
1.5.1 Endothelial cell dysfunction 16
1.5.2 Role of inflammation in atherosclerosis 17
1.6 Arachidonic acid, leukotrienes and atherosclerosis 18
1.7 5-lipoxygenase synthesis, regulation and metabolism 22
1.8 Atherosclerosis as a disease of failed endogenous repair 31
Chapter 2. Discussion and Conclusion 35
References 41

iii
List of Tables
Table 1. Important events in the history of the study of the heart 5
Table 2. Genome-wide association studies for CAD 9

iv
List of Figures
Figure 1. Global burden of disease. 3
Figure 2. Cross section of a coronary atherosclerotic plaque 12
Figure 3. Formation of atherosclerotic lesion 15
Figure 4. Oxygenated fatty acid biosynthetic pathway 20
Figure 5. Biosynthetic pathway of leukotrienes 23
Figure 6. Gene expression pattern of ALOX5 25
Figure 7. Mean expression of ALOX5 28
Figure 8. DNA methylation heat map for ALOX5 promoter 29
Figure 9. Interactions between injury and endogenous repair 33

v
Abbreviations
ABC1 transporter ATP- binding cassette-1 transporter
AHA American Heart Association
CABG Coronary artery bypass grafting
CAD Coronary artery disease
CHD Coronary heart disease
CVD Cardiovascular disease
EPC Endothelial progenitor cell
FLAP 5-LO activating protein
HDLc High density lipoprotein cholesterol
ICAM-1 Intercellular adhesion molecule-1
IL-1 Interleukin-1
KLF-2 Kruppel-like factor-2
LDLc Low density lipoprotein cholesterol
Lp(a) Lipoprotein (a)
Lp-PLA
2
Lipoprotein associated phospholipase A
2
5-LO 5-lipoxygenase
LSS Laminar shear stress
LTA
4
Leukotriene A
4

LTB
4
Leukotriene B
4

LTC
4
Leukotriene C
4

LTD
4
Leukotriene D
4

LTE
4
Leukotriene E
4
M-CSF Macrophage colony-stimulating factor
MHC Major histocompatibility complex
MI Myocardial infarction
NO Nitric oxide
NOS3 Nitric oxide synthase 3
PC Progenitor cell
PDGFB Platelet derived growth factor
PGI
2
Prostaglandin I
2
PLA
2
Phospholipase A
2

PUFA Polyunsaturated fatty acid
RT-PCR Reverse transcription polymerase chain reaction
Th
1
Type 1 T-helper lymphocyte
Th
2
Type 2 T-helper lymphocyte
TGF- Transforming growth factor
TNF- Tumor necrosis factor
tPA Tissue plasminogen activator
VCAM-1 Vascular cell adhesion molecule-1
VPC Vascular progenitor cell

vi
Abstract
This study provides a review of the literature on coronary artery disease (CAD) focusing
on the role of the enzyme 5-lipoxygenase (encoded by ALOX5) and in the regulatory
pathways of ALOX5 in different human leukocyte populations. CAD affects millions of
people being the leading cause of death in the world. Disease states contributing to CAD
such as atherosclerosis present a highly complex and multi-factorial pathophysiology.
Inflammation is recognized as being an important component in the development of
CAD. Leukotrienes are potent inflammatory mediators produced from arachidonic acid
through oxidative reaction catalyzed by 5-lipoxygenase (5-LO). Several reports have
shown that leukotrienes are involved in the etiology of CAD. ALOX5 promoter
polymorphism increased carotid artery intima-media thickness and that increased dietary
arachidonic acid intake significantly enhanced the genotype effect. It is becoming
apparent that hexanucleotide repeat variants of the ALOX5 promoter are differentially
methylated and partly regulate ALOX5 expression in human lymphocytes. Homozygous
shorter repeat variant showed higher gene expression than the homozygous wild type
allele carriers in lymphocytes. Since atherosclerosis has an inflammatory component, it is
important to better understand the role of immune response in the development of the
disease. A recent study suggests the possibility of differential mechanism regulating
ALOX5 expression in different human leukocyte populations. A new hypothesis
concerning CAD is also discussed in this study. The recognition of the major risk factors
contributing to CAD and improvements in therapies are critically important.

1
Chapter 1.1 Introduction

The aim of this study was to review the current state of research in the field of coronary
artery disease (CAD). Specific emphasis was given to the role of inflammation in
development of atherosclerosis. Leukotriene pathway was studied in greater detail and its
role in inflammation and in the pathogenesis of CAD. Specific interest was given to the
genes that participate in the biochemical processes converting arachidonic acid to
leukotrienes. Leukotrienes are inflammatory mediators that can modulate the immune
system and contribute to inflammatory reactions such as asthma and atherosclerosis.

This thesis contains a historical perspective of the CAD, starting from the origins of
auscultation, through the discovery of the heart as a driving force behind the blood
circulation in the body, ending up in issues concerning CAD in modern times.

I will discuss risk factors affecting CAD, followed by a short discussion of the
pathogenesis of atherosclerosis, which leads to CAD. The endothelial cell dysfunction is
also discussed, as well as the role of inflammation and its association with CAD. Finally,
I will discuss the role of leukotrienes and their relationship with 5-lipoxygenase (5-LO)
in CAD. 5-LO is encoded by the gene ALOX5. For clarity in the use of nomenclature in
this thesis, ALOX5 is used when referring to the gene and 5-LO when referring to the

2
protein coded by ALOX5. In addition, a new hypothesis of bone marrow derived
progenitors cells in the pathophysiology of CAD will be discussed.

1.2 Background

Cardiovascular diseases (CVD) are disorders that affect the function of blood vessels and
heart, including stroke, CAD, pulmonary embolism and congenital heart disease.
Although the incidence of cardiovascular diseases has been decreasing in the western
countries in the past decades, CVDs still remain the leading cause of death in the world
(Mathers et al., 2006). It has been estimated that CVDs caused 17.5 million deaths
representing about 30% of all deaths worldwide in 2005 (Benjamin et al., 2008).
According to the American Heart Association (AHA), there are about 81 million people
in the United States affected by cardiovascular diseases, and about 1 million people die of
cardiovascular diseases every year (Lloyd-Jones et al., 2010). CAD alone caused
approximately 425 000 deaths in the US in 2006, being the leading cause of death in
United States. There are approximately 17 million people living with a history of
myocardial infarction or angina pectoris or both in the United States (Benjamin et al.,
2008).

3
Figure 1. Global burden of disease. Cardiovascular diseases are responsible for 30% of
the deaths in the world, representing 17.5 million deaths annually (Modified from World
Health Report- making a difference 1999).

1.3 Historical perspective

The history of the study of the heart dates back to ancient times in Egypt, China and India
where a general inspection of the chest and palpation of the pulse was practiced. The

4
auscultation of the heart was first practiced by Hippocrates (460-370 B.C.). Claudius
Galen (A.D. 120) was a Roman physician who treated the Roman emperors and
gladiators, dissected apes and came to the conclusion that the liver was the main organ of
the blood

circulation (Gotto, 1985 p. 8). In the early 17
th
century, William Harvey published De
Motu Cordis, after studying the blood flow in more than 80 animal species. He proposed
that the heart is the driving force behind blood circulation (Silverman and Wooley, 2008).
Floyer introduced the 1-minute pulse watch, the practice of pulse rate measurement, in
1707. In 1816, René Laennec invented the wooden monaural stethoscope to be used
when listening to cardiovascular sound. In 1856, von Kölliker and Müller showed that
heart produced electricity and in 1902 Einthoven invented the modern electrocardiogram,
with 3-lead electrocardiograph (Silverman and Wooley, 2008).

These findings formed the foundation for understanding the pathophysiological symp-
toms of CAD. One of the earliest descriptions of CAD was reported by Leonardo da
Vinci. He made the observation about the waxy fat in the arteries of the heart (Gotto,
1985 p. 9). The first person to use the term arteriosclerosis was Johann Lobstein in 1833.
Karl Huber suggested in 1882 that atheroma, which developed in the coronary arteries,
could eventually lead to myocardial fibrosis (Silverman and Wooley, 2008). Felix
Marchand used the term atherosclerosis describing the disease of the coronary artery

5
intima in 1904 (Gotto, 1985 p. 13). Already in 1912 Stuckey reported dietary interactions
and atheroclerosis. A diet containing egg yolks and brain caused severe arterial lesions
while sunflower seed oil and fish oil did not cause these lesions (Gotto, 1985 p. 14).

Table 1. Important events in the history of the study of the heart.

Name Year Activity/ Discovery
Hippocrates 460-370
B.C.
Auscultation of the heart
Claudius Galen A.D. 120 Believed that liver was the main organ
of blood circulation
Leonardo da Vinci 1452-1519 Gave one of the earliest descriptions of
coronary artery disease
William Harvey 1628 Proposed that the heart is the driving
force behind the blood circulation
John Floyer 1707 Invented 1-minute pulse watch
René Laennec 1816 Invented the wooden monaural
stethoscope
Johann Lobstein 1833 First to use the term arteriosclerosis
Albert Kölliker and Heinrich Müller 1856 Showed that heart produce electricity
Karl Huber 1882 Suggested that atheroma could lead to
myocardial fibrosis
Willem Einthoven 1902 Invented 3-lead electrocardiograph
Felix Marchand 1904 Used the term arteriosclerosis
describing the disease of coronary
artery intima
Stuckey 1912 Reported dietary interactions in
atherosclerosis
A. L. Myasnikov 1958 Reported about cholesterol influence on
atherosclerotic plaque formation
Rene Favoloro 1967 First coronary artery bypass
Russel Ross and John Glomset 1976 "Response to injury"-theory
Andreas Grüntzig 1977 First percutaneous angioplasty

6
By the 1950s, coronary thrombosis, angina pectoris and myocardial infarction had
become more familiar concepts due to the electrocardiographic, clinical and laboratory
findings. Myasnikov reported the relationships between cholesterol, vitamin C, vitamin D
and atherosclerotic plaque formation in 1958 (Myasnikov, 1958). In 1967, the first
coronary artery bypass, a surgical procedure to increase blood flow to cardiac muscle in
veins affected by CAD, was introduced by Rene Favoloro. Percutaneous coronary
angioplasty of the arteries was first performed by Grüntzig in Switzerland in 1977
(Silverman and Wooley, 2008). Percutaneous coronary angioplasty has become a routine
procedure in cardiology to treat stenotic arteries. Angioplasty is less invasive than
coronary artery bypass, since it is performed by inserting a stent from the artery in the
groin. However, coronary artery bypass surgery (CABG) has proven to be superior in
durability and more cost effective in long term. Also CABG has demonstrated lower
death rates in a retrospective study (Hannan et al., 2008). The diagnosis and the
treatment of CAD have advanced in the last decades rapidly. The advances in technology
to treat heart disease and recognition of risk factors have permitted the decrease of CAD
since the 1950s. The Framingham study, which started in 1948, identified several risk
factors for CAD. One of the most important findings of this study was the higher risk of
cigarette smokers to develop CAD (Dawber et al., 1959). Subsequent clinical and
epidemiological studies emphasized the importance of increased serum total cholesterol,
cholesterol bound to low density lipoprotein (LDLc) and apolipoprotein A for the
development of the CAD (Kannel et al., 1971).

7
1.4 Coronary artery disease risk factors

1.4.1 Clinical risk factors

Pathophysiology of CAD has been well studied in detail and the associated clinical risk
factors are well known. The risk of significant cardiovascular event (myocardial
infarction, stroke and angina pectoris) increases with age (Banai et al., 2001). Men are
more susceptible to CAD than women and men develop CAD at younger age than
women (Banai et al., 2001). Diabetes, obesity, dyslipidemia and high blood pressure are
major independent risk factors often found together in patients with metabolic syndrome
(Allison, 2007 p. 695). Other CAD risk factors include elevated C-reactive protein
(CRP), homocysteine, lipoprotein(a) [Lp(a)], and lipoprotein associated phospholipase A
2
(Lp-PLA
2
) levels as well as chronic inflammation (Lopez- Jimenez et al., 2007).
However, managing these risk factors has not been shown to lower the risk of CAD in
intervention studies (Maron et al., 2008).

1.4.2 Environmental risk factors

As mentioned, cigarette smoke is a strong environmental risk factor for CAD. Recently,
air pollution has been reported to be a likely factor contributing to the development of
CAD (Sandhu et al., 2005). High fat diet directly affects blood cholesterol levels and, has

8
been associated with pathogenesis of CAD. Other environmental risk factors are, low
antioxidant intake, infectious agents and physical inactivity (Lusis, 2000; Ross, 1999).
Systemic infections such as oral periodontal disease have been linked to CAD (Ford et al.
2010).

1.4.3 Genetic risk factors

The heritability of CAD has been estimated to be 56-63% (Nora et al., 1980). Relatively
rare mendelian disorders result in accelerated form of atherosclerosis due to mutations in
genes involved in lipid metabolism, including mutations in LDL receptor, ABC1
transporter, and cystathione β-synthetase (Lusis, 2000; Ross, 1999). In attempts to
identify candidate genes associated with the common form of CAD, several genomic loci
have been identified in genome wide studies of different populations (e.g. Pajukanta et
al., 2000; Ozaki et al., 2002). In Table 2. are shown GWAS results for CAD published
after 25
th
of November 2008. Most of these loci reside in the proximity of genes involved
lipid metabolism. Some of these findings have not been replicated across different
populations (Helgadottir et al., 2004). The chromosomal region in the short arm of
chromosome 9 (9p21.3) has been identified in several genome wide association studies
for CAD (Kathiresan et al., 2009; Helgadottir et al., 2007; Samani et al., 2007). However,
this region does not code for a protein and is still under active investigation.

9
Table 2. Genome-wide association studies for CAD published in GWAS catalogue (as of
17
th
of October 2010). Shown only studies published after November 25
th
2008.

Date
First
author
Region Reported Gene(s)
Strongest
SNP-Risk
Allele
Risk Allele
Frequency
in
Controls
P-
value
OR or
beta-
coefficient
and [95%
CI]
3/1/2009 Erdmann 3q22.3 MRAS rs9818870-T 0.15 7 x 10
-
13

1.15 [1.11-
1.19]
12q24.31 HNF1A,C12orf43 rs2259816-T 0.36 5 x 10
-
7

1.08 [1.05-
1.11]
4/1/2009 Tregouet 6q25.3 SLC22A3,LPAL2,LPA 4-SNP
haplotype-2
0.02 4 x 10
-
15

(CCTC)
1.82 [1.57-
2.12]
6q25.3 SLC22A3,LPAL2,LPA 4-SNP
haplotype-1
0.16 1 x 10
-
9

(CTTG)
1.2 [1.13-
1.28]
11/25/2008 Samani 9p21.3 Intergenic rs1333049-
C
0.47 3 x 10
-
19

1.36 [1.27-
1.46]
1p13.3 PSRC1 rs599839-A 0.77 4 x 10
-
9

1.29 [1.18-
1.40]
6q25.1 MTHFD1L rs6922269-
A
0.25 3 x 10
-
8

1.23 [1.15-
1.33]
10q11.21 CXCL12 rs501120-T 0.87 9 x 10
-
8

1.33 [1.20-
1.48]
15q22.33 SMAD3 rs17228212-
C
0.3 2 x 10
-
7

1.21 [1.13-
1.30]
2q36.3 pseudogene rs2943634-
C
0.65 2 x 10
-
7

1.21 [1.13-
1.30]
1q41 MIA3 rs17465637-
C
0.71 1 x 10
-
6

1.2 [1.12-
1.30]
11/25/2008 WTCCC 9p21.3 CDKN2A,CDKN2B rs1333049-
C
0.47 1 x 10
-
13

1.47 [1.27-
1.70]
1q43 Intergenic rs17672135-
T
0.87 2 x 10
-
6

1.43 [1.23-
1.64]
22q12.1 Intergenic rs688034-T 0.31 4 x 10
-
6

1.11 [0.99-
1.25]
16q23.3 Intergenic rs8055236-
G
0.8 6 x 10
-
6

1.91 [1.33-
2.74]
11/25/2008 McPherson NS NS NS NS NS NS

10
1.5 Pathogenesis of atherosclerosis

1.5.1 LDLc mediated atherosclerosis

Fatty streaks develop early in life, and can be found in the arteries of infants. Fatty
streaks develop first in aorta, and later they can also be found in the coronary arteries in
teenagers and in the cerebral arteries of individuals in their early 30s (Allison, 2007 p.
687).

Low density lipoprotein cholesterol (LDLc) is the carrier of cholesterol from liver to the
peripheral tissues (Mayes, 1996). High density lipoprotein cholesterol (HDLc) transports
cholesterol back to the liver to be metabolized. It is also called reverse cholesterol
transport. Under normal conditions LDLc binds to its receptors trough Apolipoprotein
B100 ligand on the plasma membrane. This is followed by internalization of the ligand-
receptor complex via endocytosis. LDLc is processed in the lysosomes and cholesterol is
released to the cytoplasm. Cholesterol synthesis is regulated by the activity of HMG-CoA
reductase, which is inhibited by statins, which are one type of the plasma cholesterol
lowering medication (Mayes, 1996). Cholesterol is an essential structural component of
the cell membrane and is used to produce bile acid, steroid hormones and vitamins in the
body.

11
In the healthy endothelium, the LDLc releases cholesterol esters and gets recycled back
to the cell surface (Berg et al., 2002). When the LDLc levels in the blood are constantly
increased the artery wall endothelium becomes injured. At the site of injury the excess
LDLc crosses the thin layer of endothelial cells and accumulates in the subendothelial
intima and becomes exposed to oxidative enzymes. Oxidized LDLc activates
inflammatory signaling cascades, which lead to immune response (Blackshear and
Kantor, 2007).

The accumulation of fatty streaks in the vascular endothelium causes inflammatory
mononuclear leukocytes and T-lymphocytes to segregate to the site of fatty streaks. As a
consequence, the endothelial cells become dysfunctional in early stages of
atherosclerosis. This leads to the thickening of the subendothelial intima, which is
exacerbated by an inflammatory response mediated trough LDLc (Blankenberg et al.,
2003).

12
Figure 2. Cross section of a coronary atherosclerotic plaque. Arterial plaques consist of
lipid core (CORE), elastic fibres covered with cap of fibrous tissue (CAP). Shoulder
region of the atherosclerotic plaque is vulnerable to rupture and acute atherothrombosis.
Intima (I), media (M) , adventitia (A), internal elastic lamina (IEL) (Taken from Lusis,
2000).

As a response to the inflammatory injury, adhesion molecules and chemokines are
produced. The stickiness of the endothelial cells leads mononuclear leukocytes to adhere
to the endothelium. Mononuclear leukocytes can be found in large numbers in the
developing atheroma (Blackshear and Kantor, 2007). The monocytes infiltrate through
the endothelium to the subintima and differentiate into macrophages. Subintimal
macrophages phagocytose large amounts of oxidized LDLc trough scavenger receptor.
The accumulation of the lipid-filled macrophages (foam cells) is typical for the early
stage atherosclerotic plaque (Blackshear and Kantor, 2007). Histological studies have
shown that the atherosclerotic lesions contain abundance of foam cells, lymphocytes and

13
mast cells (Kovanen et al., 1995). Often layers of smooth muscle cells and connective
tissue are found in complex coronary plaques, suggesting cycles of progression. The
formation of atherosclerotic plaques consequently narrows the lumen of the blood vessel
(Figure 2) and blood flow is affected. The narrow arterial branches and bifurcations could
be more vulnerable to alterations in the blood flow patterns. The curvatures in the arterial
landscape might experience more turbulent hemodynamic shear stress than straight vessel
where the laminar shear stress is more uniform. Indeed, laminar blood flow up-regulates
NOS3, nitric oxide synthetase 3, which is expressed in endothelium and has anti-oxidant,
anti-thrombotic and anti-adhesive properties (Topper et al., 1996). Thus, the laminar
shear stress (LSS) in different parts of the vascular system could explain why atheromas
are more likely to be found in coronary arteries. Moreover, the variation in LSS is
responsible for regulating the expression of flow responsive genes, which could
contribute to dysfunction of endothelial cells (Pajukanta et al., 2007).

As described above, prolonged mechanical and chemical insults to the arterial intima due
to the excessive levels of LDLc leads to several immunologic reactions. First, the
monocytes are segregated to the site of insult, which will migrate to the subintima and
become foam cells. Secondly, T-lymphocytes recognize the leukocyte adhesion
molecules on the endothelial surface, which have been shown to be upregulated by
reduced shear stress both in vivo and in vitro (Topper et al., 1996). The pro-inflammatory
gene expression pattern both in endothelial cells as well as in monocytes and T-

14
lymphocytes is responsible for the increased adherence, migration and proliferation of the
inflammatory cells at the site of lesion. Increased expression of intercellular adhesion
molecule-1 (ICAM-1), platelet-derived growth factor beta polypeptide (PDGFB), and
vascular cell adhesion molecule-1 (VCAM-1) is characteristic for the progression of
atherosclerotic plaque (Blankenberg et al., 2003) (Figure 3). On the other hand,
upregulation of certain genes at the atherosclerosis–resistant sites inhibit the pro-
inflammatory signaling. Activation of a novel transcription factor Kruppel-like factor
(KLF-2) induces atheroprotective functions in vascular endothelial cells. It has been
described to regulate the transcription of genes involved in endothelial inflammation,
thrombosis, hemostasis and vascular tone (Parmar et al., 2006).

15
Figure 3. Formation of atherosclerotic lesion. Complex molecular interactions are
involved in the formation of the atherosclerotic lesion (Taken from Lusis, 2000). The
endothelial expression of leukocyte adhesion molecules (P-selectin, E-selectin, ICAM-1,
VCAM-1, and PCAM-1) increases due to the accumulation of oxidized LDLc to the
intima. In addition, chemotactic molecules and growth factors are produced (MCP-1 and
M-CSF) by the endothelial cells. Monocytes and T-lymphocytes migrate to the site of
injury, and upregulate the expression of proteoglycans and cytokines. Monocytes
proliferate and differentiate to macrophages, which phagocytose large amounts of LDLc
and become foam cells. Nitric oxide synthase 3 (NOS) expression is decreased, which
promotes vasoconstriction, and increased expression of endothelial leukocyte adhesion
molecules (ELAMs). Advanced glycation end products (AGEs) has similar type of effect
with an increase in the permeability of the endothelium.

16
1.5.2 Endothelial cell dysfunction

The endothelium is the single cell layer of specialized cells in the inner surface of the
blood vessels (Banai et al., 2001). It plays an active role in vascular homeostasis and end
organ blood flow. Endothelial cells can release various vasoactive molecules, which
regulate the vascular tone and have anti-thrombotic and anti-inflammatory properties.
Endothelial cells are exposed to various chemical and mechanical stimuli in the blood. In
normal and healthy state, vessels are vasodilated whereas in the case of endothelial
dysfunction blood vessels become vasoconstricted (Shah et al., 2008). The vasodilated
and anti-atherogenic microenvironment is achieved by a release of vasoactive molecules
such as nitric oxide (NO), prostaglandin I
2
(PGI
2
) and tissue plasminogen activator (tPA)
(Badimon et al., 2008). As mentioned above endothelial injury often occurs from high
LSS and oxidized LDL. The constant mechanical and chemical insult leads to altered NO
synthesis by nitric oxide synthase 3 (NOS3), which concurrently promotes
vasoconstriction (Pajukanta et al., 2007). Vasoconstriction can be exacerbated by
expression of other vasoconstricting agents such as angiotensin converting enzyme
(ACE), thromboxane A
2
(TXA
2
) and leukotrienes (LT) (Ross, 1995).

Other antithrombotic and procoagulant factors secreted by endothelial cells are
antithrombin, von Willebrandt factor and interleukins 1, 6 and 8 depending on the type of
immunomodulatory stimulus (Banai et al. 2005). In normal conditions, the endothelium is

17
antithrombotic, antiadhesive and vasodilated. The endothelium becomes only pro –
atherogenic under a constant and chronic biochemical injury such as smoking,
hypertension, diabetes and dyslipidemia. Pro-inflammatory and pro-thrombotic state
leads to atherosclerotic lesion formation. Further, dysfunctional endothelium contributes
to the plaque activation and rupture, which can lead to thrombosis and vasospasm (Lemos
and Rourke, 2008). Prevalence of atheromas is highest in abdominal aorta and coronary
arteries, whereas internal mammary artery or radial artery is very unlikely to be affected
by atherosclerosis. In addition to endothelial dysfunction, differences in vasa vasorum
density or in progenitor cells repair mechanism might contribute to the development of
atherosclerotic lesions (Blackshear and Kantor, 2007).

1.5.3 Role of inflammation in atherosclerosis

Since Ross suggested that injury and inflammation contributes to the development of
CAD (Ross et al., 1976), there has been a special interest in the field of cardiovascular
research to better understand inflammatory pathways. The primary cause for
atherosclerotic lesion initiation is the accumulation of oxidized LDLc in the artery wall,
which leads to secretion of pro-inflammatory molecules. These pro-inflammatory
molecules attract specifically T-lymphocytes and mononuclear leukocytes to the site
(Shah, 2007). The activation of T-lymphocytes and monocytes leads to upregulation of
the surface receptors, which bind selectins and integrins on the endothelial lining of the

18
vessel wall. Increased vascular permeability allows leukocytes to cross the endothelial
wall and become exposed to growth factors, promoting proliferation. Further, the pro-
inflammatory cytokines can influence lipoprotein transfer within the arterial endothelium.
Tumor necrosis factor α (TNF- α), interleukin-1 (IL-1), and macrophage colony-
stimulating factor (M-CSF) can increase the binding of LDL to endothelium and
upregulate expression of the LDL-receptor gene on the cell surface (Ross, 1999). Viral
and bacterial infections have been associated with CAD. Chlamydia pneumoniae,
cytomegalovirus and Helicobacter pylori have been shown to provoke chronic
endothelial inflammation (Shah et al., 2008).

1.6 Arachidonic acid, leukotriene metabolism and atherosclerosis

Recently, dietary polyunsaturated fatty acids (PUFAs) and their metabolites have
gathered a lot of interest in cardiovascular research. PUFAs, such as omega- 6 and
omega- 3 fatty acids are essential lipid metabolites that contribute to several biological
processes such as inflammation and neuronal signaling (Berger and Roberts, 2005).
Specifically, arachidonic acid (ω−6) metabolites such as leukotrienes have been reported
to contribute to inflammatory diseases such as asthma and atherosclerosis (In et al., 1997;
Drazen et al., 1999). The rate limiting enzyme in the oxygenation of arachidonic acid into
leukotrienes is 5-lipoxygenase (5-LO) encoded by ALOX5. A study by Dwyer et al.
showed that a specific ALOX5 promoter allele could lead to increased carotid artery

19
intima-media thickness (CIMT), which is a non-invasive predictive measurement of
cardiovascular risk. Increased dietary arachidonic acid intake significantly enhanced the
genotype effect (Dwyer et al., 2004).

Arachidonic acid (AA), a polyunsaturated omega-6 fatty acid is found abundantly in
brain and muscles. Arachidonic acid is a key component of endothelial cell membrane
(Crawford et al., 1997). Animal products, eggs and nuts are rich in arachidonic acid (Min
and Crawford, 2004). Omega-6 fatty acids are used to make cell membranes, which
consists of phospholipids such as phosphatidylethanolamine, phosphatidylcholine and
phosphatidylinositides. Despite the fact that omega-6 fatty acids are essential building
components in the cell, their end products can be inflammatory. Omega-3 fatty acids
compete for the same rate limiting enzymes and are considered less inflammatory.
Arachidonic acid can be metabolized to prostaglandins, prostacyclins, thromboxanes,
leukotrienes and lipoxins (Murphy et al., 2004).

20
Figure 4. Oxygenated fatty acid biosynthetic pathway (modified from Andreani et al.
2004)

Leukotrienes are potent inflammatory mediators produced from arachidonic acid through
oxidative reaction catalyzed by 5-LO (Murphy et al., 2004). Western diet contains
abundance of arachidonic acid but it can also be cleaved from phospholipids by the
enzymatic activity of phospholipase A (PLA2) (Figure 4). 5-LO catalyzes the first
reaction in the oxygenation of arachidonic acid to produce 5(S)-hydroperoxy-
6,8,11,14,(E,Z,Z,Z)-eicosatetraenoic acid (5-HPETE) (Murphy et al., 2004). The second
reaction in the leukotriene pathway catalyzed by 5-LO, is the dehydration of 5-HPETE to
produce 5(S), 6(S) - oxido 7, 9, 11, 15 (E, E, Z, Z) - eicosatetraenoic acid, leukotriene A4

21
(LTA
4
). Further LTA
4
can be metabolized into several biologically active compounds
leukotriene B
4
(LTB
4
), leukotriene C
4
(LTC
4
), leukotriene D
4
(LTD
4
) and (LTE
4
)

(Mayes,
1996). LTB
4
is produced from LTA
4
through enzymatic reaction with leukotriene A4
hydrolase (LTA
4
H). Leukotriene B
4
has been implicated as mediator of inflammation
since it interacts with structurally specific G-protein coupled receptors on leukocytes.
Activation of these receptors leads to immunological reactions such as chemotaxis,
aggregation of granulocytes and vasodilation (Ford-Hutchinson, 1981; Bray et al., 1981).

An alternative enzymatic route of LTA
4
involves conjugation of glutathione by the
enzyme LTA
4
synthase to produce LTC
4
(Mehrabian and Allayee, 2003) (Figure 5).
LTC
4
can be further metabolized by cleavage of glutamic acid to form LTD
4
. The
catalyst enzyme participating to this reaction is gamma glutamyl transferase. LTD
4
can
form LTE
4
by a specific membrane bound peptidase, which catalyzes the cleavage of
glycine (Ford-Hutchinson et al., 1994). LTC
4
, LTD
4
and LTE
4
are often referred to as
cysteinyl leukotrienes due to the presence of amino acid cysteine in their molecular
structure. Together they form the slow reacting substance of anaphylaxis, a severe acute
multi-system type I hypersensitivity reaction (Austen, 2008). Cysteinyl leukotrienes also
interact with structurally specific G-protein coupled receptors and are responsible for the
bronchoconstriction and vasoconstriction in the lung.

22
1.7 5-Lipoxygenase synthesis, regulation and metabolism

ALOX5 is the human gene coding for 5-LO spanning a 71.89-kb region in chromosome
10. The gene ALOX5 is comprised of 14 exons and produced a mRNA that is 2568 base
pairs in length. 5-lipoxygenase enzyme consists of 674 amino acids forming a 78-kDa
protein (ALOX5, arachidonate 5-lipoxygenase, Gene ID: 240). 5-LO can be activated by
several molecules (Figure 5), the most important activators being ATP, Ca
2+
, leukocyte
factors and phostaditylcholine (Riendeau et al., 1989). When calcium concentration
increases in the cytosol, 5-LO translocates to the nuclear membrane where it binds to 5-
LO activating protein (FLAP) (Murphy et al., 2004).

23
Figure 5. Biosynthetic pathway of leukotrienes. Arachidonic acid (AA) is cleaved from
nuclear membrane phospholipids by cellular phospholipase A (cPLA
2
). When calcium
concentration increases in the cytosol, 5-LO translocates to the nuclear membrane where
it binds to 5-LO activating protein (FLAP). 5-LO catalyzes the first 2 reactions in the
oxygenation of arachidonic acid to produce leukotriene A
4
(LTA
4
), which can be further
metabolized to leukotriene B
4
, leukotriene C
4
(LTC
4
), leukotriene D
4
(LTD
4
), and
leukotriene E
4
(LTE
4
) (Mehrabian and Allayee, 2003).

ALOX5 core promoter was first characterized by Hoshiko et al. (1990).The ALOX5
promoter displays several features that are described to be common in housekeeping
genes. For example, ALOX5 promoter lacks TATAA and CCAAT boxes and it has
multiple GC-rich elements. The ALOX5 promoter contains several tandem repeats of 5’-
GGGCGG-3’ upstream (-179 to -56 from ATG) from the transcription start site (Hoshiko
et al., 1990). These repeat sequences contain transcription factor Sp-1 and Egr-1 binding
sequences. Sp-1 has a zinc finger motif which binds to the consensus sequence 5’- (G/T)

24
GGGCGG (G/A) (G/A) (C/T)-3’. Egr-1 has also a zinc finger binding motif which
recognizes consensus sequence 5'-CGCCCCCGC-3’. Egr-1 has been described to be
involved in the maturation process of hematopoietic stem cells (Friedman, 2007). A study
of the guinea pig ALOX5 promoter found out that cis-acting sequences most likely
regulate 5-LO expression. These cis-acting sequences bind to factors Sp-1, Ab-2, NF-kb
and c-Ha-ras (Chopra et al., 1992).

5-LO is expressed in very low levels in various tissues including kidney, brain and liver.
However it is expressed in much more significant levels in lung and cells of
hematopoietic origin (Figure 6). Especially during hematopoietic cell differentiation and
maturation 5-LO is upregulated (Stoffers et al., 2010). Since 5-LO is expressed at
significant levels only in few cell types, this suggests that its regulation may be cell type
specific.

25
Figure 6. Gene expression profile of arachidonate 5-lipoxygenase (from Su et al., 2004).
Highest levels of 5-LO can be detected in the lung and in the cells of hematopoietic
origin.

26
5-LO expression was reported to be regulated by 1, 25 dihydroxyvitamin D3 (Brungs et
al., 1994). A high dose of 1α, 25-dihydroxyvitamin D3 (calcitriol) resulted in 4-fold
increase of 5-LO mRNA, a 14-fold increase in 5-LO protein, and 38-fold upregulation of
5-LO activity. In the same study, transforming growth factor beta (TGF- β) was identified
to modify the 5-LO activity (Brungs et al., 1994).

ALOX5 promoter polymorphism is involved in the lack of response to 5-LO inhibitor
treatment in asthma patients (Drazen et al., 1999). Drazen et al. described a
pharmacogenetic association between ALOX5 promoter polymorphism and the response
to the anti-asthma treatment (ABT-761). In 2002, it was suggested that ALOX5
transcription is regulated by methylation (Uhl et al., 2002), an addition of a methyl group
to cytosine nucleotides, which serves as an epigenetic mark (Holliday and Pugh, 1975;
Riggs, 1975) In the study by Uhl et al. the expression of 5-LO was increased by the
demethylating agent 5-aza-2’-deoxycytidine (AdC) in U937 (human leukemic monocyte
lymphoma cell line) and HL-60TB (human promyelocytic leukemia cell line) cells. In
U937 and HL-60TB cells treatment with AdC lead to a 4-fold and 23-fold increase
respectively. It is unclear whether the demethylation acts directly on the 5-LO promoter
or if it upregulates the gene expression of certain transcription factors which subsequently
enhance 5-LO expression. However, this experiment used the cell lines U937 and HL-
60TB, which under normal conditions do not exhibit 5-LO activity. Vikman et al. showed
that 5-LO gene expression could at least partially be regulated by differential methylation

27
pattern at the promoter repeats in lymphocytes (Vikman et al. 2009). This work also
showed that the 5-LO promoter is methylated at relatively low levels in leukocytes
isolated from blood. In this experiment, ALOX5, ALOX5AP and LTA4H gene expression
levels were measured by quantitative real time RT-PCR. The relative quantity (RQ)
values of gene expression were compared to methylation patterns in 5-LO promoter
gathered by mass array method with bisulfite treated DNA. Bisulfite conversion involves
sulphonation, deamination and desulphonation of DNA. In this process unmethylated
cytosine nucleotides are converted to uracil, but methylated cytosine residues are
protected (Frommer et al., 1992). The results indicated a significant correlation between
the promoter genotype and ALOX5 gene expression RQ in lymphocytes. Granulocytes
and monocytes showed statistically significant differences in ALOX5 gene expression
(Figure 7). The DNA methylation profiles of ALOX5 promoter in blood leukocytes are
shown in Figure 8.

28
Figure 7. Mean expression levels of ALOX5 (RQ units) in granulocytes (G),
lymphocytes (L) and monocytes (M). Highest levels of ALOX5 expression were seen in
blood granulocytes. Monocytes expressed ALOX5 at relatively low levels. Lymphocytes
expressed ALOX5 in lowest levels compared to the other blood leukocytes.

29
Figure 8. DNA methylation heat map for ALOX5 promoter region by cell type,
granulocytes (G), lymphocytes (L) and monocytes (M). Yellow color indicates less DNA
methylation and blue indicates more DNA methylation. The levels of DNA methylation
are relatively low in blood leukocytes (< 5%).

Another study reported that in old and young mice, the 5-LO and methylation levels in
the heart were differentially expressed (Dzitoyeva et al. 2009). In young mice, the
ALOX5 was expressed at higher levels than in old mice, whereas methylation levels were
lower in young mice than older mice.

30
Methylation may not explain the level of ALOX5 expression in cells that transcribe the
gene at higher levels. Stoffers et al. suggested that ALOX5 expression is regulated
through histone modifications by calcitriol (Stoffers et al., 2010). In this study, Mono
Mac 6 (human monoblastic leukemia cell line) cells treated with calcitriol (1α,25-
dihydroxyvitamin D3) had increased histone H4 K20 monomethylation and histone H3
K36 trimethylation at previously identified vitamin D responsive elements at ALOX5
locus. Combined treatment of calcitriol and transforming growth factor beta (TGF-β)
increased histone H4 acetylation (Stoffers et al 2010).

Recently, efforts have been directed to elucidate the occurrence of transcellular
biosynthesis of leukotrienes in vivo (Sala et al. 2010). In a study ALOX5 -/- knockout
mice, animals were irradiated following transplantation of bone marrow from the LTA
4

hydrolase -/- knockout mice or of the glutathione transferase -/- knockout mice.
Peritoneal fluid content of leukotrienes was measured with mass spectrometry after
induction of inflammatory response by Zymosan. Biologically relevant amounts of LTB
4

and cysteinyl leukotrienes were detected (Zarini et al. 2009). However, transcellular
biosynthesis of leukotrienes was first described by Maclouf et al. (1989). In this
experiment platelets and granulocytes were incubated, and the largest amount of
leukotriene produced was LTC
4
. Since platelets lack the ability to produce 5-LO or LTA
4

hydrolase, but have LTA
4
synthase activity, LTC
4
must have formed through transcellular

31
synthesis. Approximately 60-70 % of the LTA
4
synthesized by granulocytes is not
processed in this cell population but transferred to other cells (Sala et al. 1996).

1.8 Atherosclerosis as a disease of endogenous repair

A novel hypothesis concerning CAD was introduced by Zenovich and Taylor in 2008
(Zenovich et al. 2008). These authors suggested that pathophysiology of CAD could be
associated with endogenous repair mechanisms of the endothelium (Taylor et al. 2008).
For example, in the presence of chronic hypercholesterolemia, the balance between
normal repair mechanism and disease progression could be disturbed. The main
inflammatory pathways contributing to atherosclerosis have been identified, but anti-
inflammatory treatment has not been able to reverse the cascade of chronic inflammation
of CAD. Capacity for endogenous repair decreases with age and mean differences in
bone marrow derived progenitor cells (PCs) have been observed between female and
male patients (Zenovich et al. 2008).

In the mouse ApoE-/- model for atherosclerosis, female and male mice had significant
differences in the percentages of vascular progenitor cells (VPCs) and CD34
+
positive
cells. In male mouse there was a linear correlation between plaque deposition and in the
percentage of VPCs and CD34
+
positive cells. In female mice, endothelial progenitor
cells (EPCs) remained at higher levels compared to the male mice and the mean

32
percentage of VPCs stayed stable during the study (Zenovich et al., 2008). The authors
suggested that in the presence of chronic inflammation, the bone marrow’s capacity to
maintain reparative PCs cells at appropriate levels is challenged. For example, in the case
of endothelial injury, a Th1-type (T helper 1 lymphocyte) inflammatory signaling
activates bone marrow to release detrimental progenitor cells and tissue injury takes
place. In case of Th2-type (T helper 2 lymphocyte) of response reparative cells are
recruited from bone marrow and tissue integrity is restored. If injury is persistent, as in
the case of smoking or dyslipidemia, the balance between Th1-type and Th2-type
cytokine signaling tends to tip towards tissue injury and disease progression.

The progenitor cells have been proposed to contribute to plaque formation. Bone marrow
derived smooth muscle progenitor cells have been reported to contribute to the aortic
allografts in rats (Religa et al., 2002). The smooth muscle cells (SMCs) found in
atherosclerotic plaques are phenotypically different from SMCs in healthy blood vessels.
However, it is yet unclear whether SMCs arise from the pre-existing medial SMCs or
from circulating progenitor cells derived from bone marrow.

33
Figure 9. Interactions between injury and endogenous repair. Endothelial injury leads to
release of inflammatory cytokines. In early stages of the disease inflammatory markers
reach bone marrow and recruit reparative progenitor cells mediated through Th2-type
cytokines. When injury is persistent as in as in the case of smoking or dyslipidemia, the
Th1-type response becomes dominant, which leads to tissue injury and disease
progression (modified from Zenovich et al. 2008).

T-lymphocytes are hematopoietic cells that participate in adaptive immune responses. A
two stage maturation process takes place in thymus where T cells are being stimulated by
cytokines and introduced to antigens. Once they interact with antigen, T cells proliferate

34
and differentiate into helper T cells (CD4+) or cytotoxic effector T cells (CD8+) in
appropriate cytokine environment (Wucherpfennig, 2009).

T helper cells can further differentiate into several effector subpopulations, such as Th1,
Th2, Treg or Th17 cells. T helper cells assist other cells of the immune system to produce
an appropriate immunologic response. Th1 cells activate macrophages and Th2 cells help
B cells for antibody production whereas Treg cells suppress the T-cell responses (Murphy
et al., 2008).

T helper cells, Th1 and Th2 have an important role in the development of autoimmune
diseases. Especially, the ratio of Th1 cells to Th2 cells can increase the risk to develop
inflammatory diseases. The mechanism behind the disturbances in the balance between
Th1 and Th2 is under ongoing investigation.

35
Chapter 2. Discussion and Conclusion

The perception of atherosclerosis has shifted over the last few decades. First,
atherosclerosis was seen as a disease of buildup of different type of lipids in the arteries.
Several clinical and epidemiologic studies have shown that atherosclerosis is a sum of
several risk factors that contribute to the development of the disease. In addition to
classical lipoprotein and cholesterol levels, coronary artery patients might have several
other genetic risk factors that make them more susceptible to plaque development and
rupture, which can lead to myocardial infarction. Despite advances in molecular genetics,
we still lack novel biomarkers for diagnosing early CAD. In addition, predictive tests for
different susceptibility and response to treatment therapy are needed.

It is therefore important to understand the molecular mechanisms driving the
development of atherosclerosis and recognize the environmental and genetic factors that
contribute to the disease. Some of the known risk factors are modifiable such as smoking,
high plasma cholesterol and blood pressure. Non-modifiable risk factors include age,
gender and family background of CAD. The genetic factors contributing to
atherosclerosis have been less understood despite the fact that the heritability of CAD has
been estimated to be 56-63% (Nora et al, 1980).

36
As described earlier, there are several physiological risk factors that could be
differentially regulated due to the genetic polymorphisms involved in inflammatory
pathways. One of the promising genes contributing to inflammation is 5-LO as several
studies have associated 5-LO pathway with higher risk of developing CAD.

The production of 5-lipoxygenase appears to be tightly regulated by variety of
mechanisms. In a cell specific manner, 5-LO production is regulated by methylation and
histone modifications. 5-LO expression is partly regulated by the promoter repeat
(GGGCGG)
n
polymorphism which could act through the binding of transcription factor
and by DNA methylation. It is also suggested that the decreased expression of 5-LO is
controlled by DNA methylation, but other mechanisms regulating 5-LO expression likely
exists. 5-LO is expressed at very low levels in lymphocytes extracted from blood. The 5-
LO promoter is methylated at significantly higher levels in lymphocytes than in
monocytes or granulocytes. 5-LO expression is higher in blood granulocytes compared to
monocytes or lymphocytes. However, 60-70% of LTA
4
produced by granulocytes is
transferred to other cell types via transcellular biosynthesis. Granulocytes are not typical
for atherosclerotic plaque. Therefore more studies are needed on the regulation of 5-LO
in the blood leukocytes. It is unclear whether the transcellular biosynthesis pathway plays
an important role in the inflammatory nature of atherosclerotic plaque. Also the effect of
DNA methylation on 5-LO promoter polymorphism could be more pronounced in tissues
expressing 5-LO at low levels. In myocardial or endothelial tissue, where 5-LO is

37
expressed normally at very low levels and most likely 5-LO loci is highly methylated, the
5-LO promoter repeat polymorphism could lead to more significant differences at protein
levels. Especially in situations where blood flow to the myocardium is decreased, the
expression of 5-LO could exacerbate the myocardial inflammation and tissue necrosis.
The enzyme activity has been reported to be enhanced by transforming growth factor beta
(TGF-β) and vitamin D. The early studies conducted in rabbits by Myasnikov showed an
increase in the atherosclerotic lesion size and progression when vitamin D and cholesterol
were administered simultaneously (Myasnikov, 1958).

Other arachidonic acid metabolites should also be taken into consideration when
evaluating the effect of leukotrienes on atherosclerosis. The alternative pathway to
convert arachidonic acid to prostaglandin, prostacyclin, thromboxane by cyclooxygenase-
1 or -2 (COX-1 or COX-2) or to lipoxin by 15-lipoxygenase could also contribute to the
inflammatory response in atherosclerosis. Aspirin irreversibly inactivates COX-1 and
modifies COX-2 enzymatic activity, which prevents the production of prostaglandins, has
been used as preventive medication against acute cardiovascular events (Weber, 2004).
Prostaglandin, prostacyclin, thromboxane and leukotriene pathway could contribute to
the development of CAD.

In addition to the promoter genotype, the enzymatic activity and expression of 5-LO
appears to be regulated by the availability of two substrates, AA and oxygen. As the

38
contemporary Western diet contains more omega-6 than omega-3 fatty acids, there is
abundance of substrate for 5-LO. As observed in the study by Dwyer et al. (2004) dietary
AA modified the genotype effect on atherosclerosis so that the variant allele (GGGCGG-
repeat deletion or addition) carriers would be protected against atherosclerosis consuming
low AA containing diet. Alternatively homozygotes wild type allele carriers would have
higher risk for developing atherosclerosis with low AA diet. Therefore it is important to
take into consideration the dietary interaction between 5-LO expression and activity
when investigating the role of 5-LO in the pathogenesis of atherosclerosis. On the other
hand, appropriate levels of leukotrienes are most likely protective against infections,
which have been associated with CAD.

To better understand 5-LO involvement in CAD, it would be interesting to determine its
expression in hypoxic conditions in myocytes. During myocardial infarction, it seems
plausible that 5-LO expression is upregulated. To test this, blood serum or plasma should
be collected from control group and from patients during acute myocardial infarction. In
hospitals, patient serum is collected as a standard protocol during myocardial or post-
infarction. 5-LO gene expression levels could be measured from blood granulocytes,
lymphocytes and monocytes. Simultaneously, end products of 5-LO metabolism (LTB
4
,
LTC
4
and prostaglandins) could be measured by enzyme-linked immunosorbent assay.
Despite the fact that 5-LO is not a very specific marker for certain inflammation type,
there could be a specific fraction or pattern of 5-LO metabolites specific for MI.

39
Substrate of 5-LO, arachidonic acid (AA) is abundant in muscle and in brain. Severe
injury to myocardium could lead to release of AA from the tissue to the blood stream. As
a consequence to abundance of substrate, hypoxia and calcium concentration the
secretion of 5-LO would be increased. The change in 5-LO levels or its metabolites could
be detected in blood and used as predictive test for the extent for myocardial injury.

Microarray analysis could be used to obtain information on genes regulated by 5-LO
metabolism end products. For example, human leukocytes could be treated with 5-LO
and the gene expression profile could be compared between treated and non –treated cell
populations. 5-LO could be used as a diagnostic marker for myocardial hypoxia or acute
MI since its expression seems to be altered by oxygen-glucose deprivation (Li et al.,
2009). In addition, as a follow up to this project, it would be important to carry out
studies on histone modifications in the promoter of 5-LO. This would allow greater
insights into the transcriptional regulation of this gene.

The new hypothesis about CAD as a disease of failed endogenous repair is compelling. It
is very likely that the bone marrow derived progenitor cells are involved in the
development of CHD. The proliferative and regenerative capacity of the bone marrow
progenitor cells diminishes with age. In addition, Beerman et al. (2010) reported that the
ratio of committed lineage of the hematopoietic stem cells (HSC) changes during aging.

40
Aging skews the bone marrows potential to produce more myeloid than lymphoid lineage
cells. Whether a similar shift happens to other type of reparative progenitor cells is
unclear. With aging, the dysfunctional endothelial progenitor cells could contribute to the
acceleration of the plaque progression towards stenosis and rupture.

In closing, although the incidence of CVD has been decreasing in the western countries
in the past decades, CVDs still remain the leading cause of death in the world. This study
has provided a review of the literature on coronary artery disease (CAD) focusing on the
role of the enzyme 5-lipoxygenase and in the regulatory pathways of ALOX5 in different
human leukocyte populations. Although advances have been made in recent times
towards a better understanding of CAD and the identification of risk factors contributing
to CAD, many aspects of the pathophysiology of the disease states associated with it
remain elusive. In this work, some potential future avenues for study ALOX5 and its
contribution to CAD were suggested. These and other forthcoming lines of research will
no doubt elucidate additional regulatory pathways leading to CAD, enabling us to
provide better prevention strategies and patient treatment.

41
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Abstract (if available)

Abstract This study provides a review of the literature on coronary artery disease (CAD) focusing on the role of the enzyme 5-lipoxygenase (encoded by ALOX5) and in the regulatory pathways of ALOX5 in different human leukocyte populations. CAD affects millions of people being the leading cause of death in the world. Disease states contributing to CAD such as atherosclerosis present a highly complex and multi-factorial pathophysiology. Inflammation is recognized as being an important component in the development of CAD. Leukotrienes are potent inflammatory mediators produced from arachidonic acid through oxidative reaction catalyzed by 5-lipoxygenase (5-LO). Several reports have shown that leukotrienes are involved in the etiology of CAD. ALOX5 promoter polymorphism increased carotid artery intima-media thickness and that increased dietary arachidonic acid intake significantly enhanced the genotype effect. It is becoming apparent that hexanucleotide repeat variants of the ALOX5 promoter are differentially methylated and partly regulate ALOX5 expression in human lymphocytes. Homozygous shorter repeat variant showed higher gene expression than the homozygous wild type allele carriers in lymphocytes. Since atherosclerosis has an inflammatory component, it is important to better understand the role of immune response in the development of the disease. A recent study suggests the possibility of differential mechanism regulating ALOX5 expression in different human leukocyte populations. A new hypothesis concerning CAD is also discussed in this study. The recognition of the major risk factors contributing to CAD and improvements in therapies are critically important.

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Asset Metadata

Creator Vikman, Susanna (author)

Core Title The contribution of arachidonate 5-lipoxygenase to inflammatory response in coronary artery disease

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2010-12

Publication Date 12/08/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag 5-lipoxygenase,arachidonic acid metabolism,atherosclerosis,coronary artery disease,leukotriene,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Allayee, Hooman (committee member), Joseph, Hacia (committee member)

Creator Email djsuzie@hotmail.com,vikman@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3588

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Legacy Identifier etd-Vikman-4198.pdf

Dmrecord 426799

Document Type Thesis

Rights Vikman, Susanna

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu