77:222 Spring 2001 Free Radicals in Biology and Medicine Page 0

This student paper was written as an

assignment in the graduate course
Free Radicals in Biology and Medicine
(77:222, Spring 2001)

offered by the

Free Radical and Radiation Biology Program

B-180 Med Labs
The University of Iowa
Iowa City, IA 52242-1181
Spring 2001 Term
Instructors:
GARRY R. BUETTNER, Ph.D.
LARRY W. OBERLEY, Ph.D.

with guest lectures from:

Drs. Freya Q . Schafer, Douglas R. Spitz, and Frederick E. Domann

The Fine Print:

Because this is a paper written by a beginning student as an assignment, there

are no guarantees that everything is absolutely correct and accurate.

In view of the possibility of human error or changes in our knowledge due to

continued research, neither the author nor The University of Iowa nor any other
party who has been involved in the preparation or publication of this work
warrants that the information contained herein is in every respect accurate or
complete, and they are not responsible for any errors or omissions or for the
results obtained from the use of such information. Readers are encouraged to
confirm the information contained herein with other sources.

All material contained in this paper is copyright of the author, or the owner of the
source that the material was taken from. This work is not intended as a threat to
the ownership of said copyrights.
M.Wang Atherosclerosis 1 1

Atherosclerosis:
The Past, the Present and the Future

by

Min Wang

B180 Med Lab

The University of Iowa
Iowa City, IA 52242-1181

For 077: 222, Spring 2001

3. May 2001

Abbreviation List:

CuZnSOD copper-zinc superoxide LDL low density lipoprotein

dismutase
ECM extracellular matrix IL-1 interlecukin-1
EDRF endothelium-derived MCP-1 monocyte chemoattractant
releasing factor protein
EGF epidermal growth factor MnSOD manganese superoxide
dismutase
FGF fibroblast growth factor PDGF platelet-derived growth factor
ICAM-1 intracellular adhesion PUFA polyunsaturaed fatty acid
molecule-1
ICAM-2 intracellular adhesion ROS reactive oxygen species
molecule-2
IFN-γ interferon-γ SMC smooth muscle cell
IGF insulin- like growth factor VCAM vascular cell adhesion
molecule
M.Wang Atherosclerosis 2 1

Table of Contents

1. Abstract……………………………………………………………………………. 3

2. Introduction………………………………………………………………………... 3-5

3. Pathogenesis of Atherosclerosis…………………………………………………… 5-12

3.1 Structure of Blood Vessels 6
3.2 Cells Involved in Atherosclerosis 7
a. Endothelial cells 7
b. Smooth muscle cells 8
3.3 Molecules Involved in Atherosclerosis 9
a. Adhesion molecules 10
b. Mitogens and growth factors 11

4. ROS in Atherosclerosis……………………………………………………………. 12-17

4.1 LDL Oxidation 13
4.2 Atherogenic Properties of Oxidized LDL 14
4.3 Effects of Antioxidants on Atherosclerosis 15
a. α-tocopherol 15
b. antioxidant enzyme system 16

5. Future plans for research…………………………………………………………... 17-22

5.1 Hypothesis 18
5.2 Identify genes that may be involved in the signal transduction pathways for 18
atherosclerosis
5.3 Identify proteins that may be disturbed by MnSOD in the atherosclerogenic 22
signal transduction pathways.

6. Summary................................................................................................................... 22
7. References................................................................................................................. 23-25
M.Wang Atherosclerosis 3 1

1. Abstract

Atherosclerosis is a complex and common disease. It is the major cause of heart disease

and stroke, which together remain the leading cause of death in the United States. The

mechanisms of atherosclerogenesis remain unclear. The most attractive hypothesis proposed has

been that atherosclerosis begins because the innermost layer of the artery, the endothelium,

becomes damaged. The interactions between endothelium cells and oxidized low-density

lipoprotein (LDL), and between smooth muscle cells (SMCs) and oxidized LDL are important

parts of atherosclerogenesis. Many molecules are also involved, such as adhesion molecules and

some mitogens including platelet-derived growth factor (PDGF). With the rising regards of the

important roles of free radicals in the process of diseases, many scientists believe that

atherosclerosis is a free radical disease. This paper will discuss the process of atherosclerosis

followed by a proposed plan for future research.

2. Introduction

Atherosclerosis is a disease of the arteries characterized by fatty deposits on the intimal,

or inner, lining. The presence of fatty deposits, called plaques, leads to an important loss of

arterial elasticity with narrowing of the artery as shown in Figure 1. This constriction to smooth

blood- flow ultimately deprives vital organs of their blood supply. Clots may lodge in arteries

supplying the heart, causing myocardial infarction (heart attack), or the brain, causing stroke [1].

Atherosclerosis is the major cause of heart disease and stroke, which together remain the leading

cause of death in the United States [2].

M.Wang Atherosclerosis 4 1

Atherosclerosis is a slow, progressive disease that may start in childhood. In some

people this disease progresses rapidly in their third decade. In others it doesn't become

threatening until they are in their fifties or sixties [3]. The major targets of Atheroscle rosis are

the aorta of the coronary and cerebral arteries. The three basic proceses leading to the formation

of atherosclerotic lesions are: (i) invasión of the artery wall by leukocyes, particularly monocytes

and T- lymphocytes; (ii) smooth muscle cell (SMC) phenotypic modulation, proliferation, and

synthesisi of extracellular matrix; and (iii) intracellular lipoprotein uptake in macrophage and

SMC, and lipid accumulation [4].

Figure 1. The comparison of a normal artery and atheroscletic artery. The figure on the left
is showing a section of an artery (normal). The figure on the right has plaque buildup
(http://adminweb.ucis.dal.ca/cprrc/atherosc.htm).

The most attractive hypothesis proposed has been that atherosclerosis begins because the

innermost layer of the artery, the endothelium, becomes damaged [5]. Three possible causes of

damage to the arterial wall are: elevated levels of cholesterol and triglyceride in the blood, high

blood pressure and tobacco smoke. Because of the damage, over time lipids, cholesterol, fibrin,

platelets, cellular debris, calcium and other substances are deposited in the artery wall. These

substances may stimulate the cells of the artery wall to produce still other substances that result

in further accumulation of cells in the innermost layer of the artery wall where the atherosclerotic
M.Wang Atherosclerosis 5 1

lesions form. These cells accumulate and many of them divide. At the same time, lipoprotein

builds up within and around these cells. They also form connective tissue.

Since Goldstein and Brown’s observation that oxidative modification of low-density

lipoprotein (LDL) leads to foam cell formation, there has been intense interest in the relationship

between oxidative stress and atherosclerosis [6]. With the understanding of the

pathophysiological roles played by reactive oxygen species (ROS) and lipid peroxidation in the

vasculature, there is also accumulating evidence that antioxidant compounds have beneficial

effects in atherosclerosis [7]. In 1980, a factor released by the endothelium upon stimulation

with acetylcholine was discovered, which is called endothelium-derived releasing factor (EDRF)

[8]. Additional investigations have identified nitric oxide as EDRF and have associate

abnormalities of EDRF action with atherosclerosis. This paper introduces the pathogenesis of

atherosclerosis and discusses the role of reactive oxygen species (ROS) in atherosclerosis,

followed by a proposal for future approaches.

3. Pathogenesis of Atherosclerosis

The vascular walls are relatively rich in extracellular matrix (ECM). In the capillaries,

ECM forms the basal lamina, and in the aorta, ECM is the major portion of the media and the

adventitia [9]. The extracellular matrix is the result of the biosynthetic activity of most cells of

the organisms. The selection of the quality and expression of the quantity of these ECM

macromoleculs requires a precise “programming” to be able to determine the differentiation of

the vascular wall cells. And any deviation from this precise “programming” can cause problems.

The development of atherosclerosis is a good example of such deviations from the normal

programming of the biosynthesis of ECM macromolecules. This section concerns two major cell
M.Wang Atherosclerosis 6 1

types of the arterial wall: endothelial cells and smooth muscle cells as well as the extracellular

matrix macromolecules they synthesize. There are also other cell types that are involved in the

process of atherosclerosis such as monocytes, macrophages and platelets and other molecules

such as chemotactic factors. They are beyond the scope of this paper.

3.1 Structure of Blood Vessels

In general, the blood vessel wall is composed of three layers (as shown in Figure 2 [10]):

the intima, media and adventitia. The intima consists of a single layer of endothelial cells lying

on a continuous basement membrane

composed of type IV collagen and structural

glycoproteins. The media is composed of

smooth muscle cells surrounded by a

connective tissue matrix made up of

collagen fibres (type I and type III), elastic

fibres, glycosaminoglycans, and structural

glycoproteins. The adventitia is the outer

layer containing fibroblasts, adipocytes,

mast cells, and an extracellular matrix of

Figure 2: Diagram of the crosssection of
collagen fibres, lipids, and structural
blood vessel walls [10].
glycoproteins.
M.Wang Atherosclerosis 7 1

3.2 Cells Involved in Atherosclerosis

a. Endothelial cells

Although the size of the blood vessels varies extensively from the capillaries to the veins and

arteries, they all have lining the luminal surface a single layer of endothelial cells surported by an

underlying ECM [10]. Morphology of the endothelium varies between large and small vessels.

In large veins and arteries, the endothelium exists as a tightly packed sheet of polygonal cells. In

small venules and capillaries, individual endothelial cells form the vessel through which the

blood cells pass in single file.

The integrity of endothelial cells is related to atherogenesis [11], and this relationship is still

being investigated in many laboratories. It is beyond the shadow of doubt that macromolecules

of the configuration of LDL can gain entrance to the artery wall via the transport vesicles which

traverse the endothelial cells [12]. Therefore, endothelium works as the gate keeper and

regulator of lipoprotein transport. The endothelium cells also have potential to express leukocyte

adhesion molecules, attractant protein, and majoy histocompatibility antigens [13]. Many factors

can induce endothelial cell injury and cause it dysfunction. The results of endothelial cell injury

are increased endothelial permeability, increased leukocyte adhesion, accumulation of

macrophages in subendothelial intima and SMC activation and proliferation [14]. Lipoproteins

(mainly LDL and also VLDL), oxidized lipoprotein, and circulating cells (particularly

monocytes and T- lymphocytes) enter the subendothelial space and form the fatty streaks, then

monocytes transform to macrophages and foam cells in the vessel wall, and eventually results in

complex advanced lesion of atherosclerosis as shown in Figure 3 [15].

M.Wang Atherosclerosis 8 1

b. Smooth muscle cells

Smooth muscle cells are the predominant

cells in the raised fibrous plaque [16]. In a

normal artery, SMC are the major component of

the medial layer, although they may also be

present in the intima in some arterial segments.

When SMC is stimulated to migrate and

proliferate as it is in some pathological

conditions, it becomes much more active

synthetically and more responsive to stimuli

causing cell proliferation. Furthermore, it

changes its morphological appearance. Rough

endoplasmic reticulum, free ribosomes and the

Golgi apparatus become more prominent [17].

The interaction of SMCs with lipoproteins,

especially the LDLs, has also become a very

improtant part of the formation of

atherosclerosis [18]. The arterial SMCs can

take up relatively large quantities of LDLs and

store some of these in vacuoles, perhaps by an

Figure 3. The hypothetical process of atherosclerosis [15].

M.Wang Atherosclerosis 9 1

unregulated, non-receptor scavenger mechanism, whereas the quantity of LDL taken up by

SMCs under usual circumstances is regulated by the LDL receptor system and its “feedback”

mechanisms. Features and functions of the arterial SMC are shown in Figure 4.

Figure 4. Features and functions of the arterial smooth muscle cell [18].

3.3 Molecules Involved in Atherosclerosis

There are a lot of molucules involved in the development of atherosclerosis, as shown in

Figure 5 [18]. This section discusses several important molecular components: the adhesion

molecules and some growth factors, especially the one that is called platelet-derived growth

factor (PDGF).
M.Wang Atherosclerosis 10 1

Figure 5. The athrosclerotic process involves many molecules [18].

a. Adhesion molecules

There are evidence showing that the expression of adhesion molecules is correlated with the

extent of the atherosclerotic lesion [19, 20]. There are several different structual groups of

adhesion factors which have been identified on endothelial cells and which interact with

receptors on leukocytes and platelets. Intracellular adhesion molecule-1 (ICAM-1) and

intracellular adhesion molecule-2 (ICAM-2) are cell surface glycoproteins found on many cell

types. ICAM-1 is inducible on cultured endothelial cells by the inflammatory mediatiors

interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon-γ (IFN-γ) and endotoxin. ICAM-1

can bind lymphocytes, monocytes and neutrophils to endothelium. ICAM-2 is constitutively

expressed on endothelial cells and appears to be a truncated form of ICAM-1. Vascular cell
M.Wang Atherosclerosis 11 1

adhesion molecule (VCAM) is also induced by cytokines and binds selectively to lymphocytes

and to some degree to monocytes, but not to neutrophils [21].

In human samples from autopsies and hearts of atherosclerotic patients, ICAM-1 was

detected in endothelial cells over plaques, intimal vascular smooth muscle cells, and

macrophages. VCAM-1 was detected in luminal endothelial cells in advanced coronary artery

plaques and neovascular endothelial cells at base of plaques, It was also found in focal

endothelial cells of uninvolved vessels with diffused intimal thickening and in macrophages.

These CAMs are likely to mediate leukocyte infiltration [22].

b. Mitogens and growth factors

A number of mitogens and growth factors secreted by endothelial cells and other cells are

decisive factors in the growth and propagation of the atherosclerotic plaque. These mitogens

include platelet-derived growth factor (PDGF), insulin- like growth factor (IGF), epidermal

growth factor (EGF), fibroblast growth factor (FGF) and so on [23]. PDGF will be discussed in

this paper.

PDGF is not only a major mitogen of SMCs, but also is a chemoattractant for SMCs and

monocytes. It is present in three isoforms, which interact with several different cell receptors.

PDGF gene expression is low in the normal vascular wall tissue and high in sites prone to SMC

proliferation such as the intima of atherosclerotic plaques. PDGF is produced by endothelial

cells, smooth-muscle cells and macrophages. It is among a dozen of different growth factors that

stimulate smooth- muscle cells to proliferate and produce large amounts of ECM. PDGF can

bind to the cell- surface PDGF receptor on contractile smooth- muscle cells and switch these cells

to the synthetic smooth- muscle cell type, as shown in Figure 6. As these cells proliferate and

synthesize ECM, they form a fatty streak, which eventually lead to atherosclerosis [24].
M.Wang Atherosclerosis 12 1

Lumen

Endothelium

Intima
Elastic
Lamina

Media

“Contractile” “Synthetic”
PDGF Interconversion Differentiated Undifferentiated
receptor of phenotype SMC SMC

Figure 6. Smooth muscle cell proliferation in a growing atherosclerotic plaque [24].

4. ROS in Atherosclerosis

The study of free radical biology has so grown in recent years that it has generated

dedicated journals such as Free Radicals in Biology and Medicine. The increase in the scientific

literature reflects the opinion that oxidative stress may be involved in the pathogenesis of many

diseases. The three major types of cellular damage resulting from ROS involve lipids, protein

oxidaiton and oxidation of DNA. The oxidative modificaiton of low-density lipoprotein (LDL)

cholesterol was postulated to be a pivatol step in atherogenesis. The most common evidence in

athrosclerotic leision is the fatty streak in which lipids accumulated by SMC, monocytes and

macrophages. The amount of low-density lipoprotein (LDL) oxidation occurs in areas where the

antioxidant concentration is low, such as the arterial wall. This section discusses the possible

roles of lipoprotein in the process of atherosclerosis. We first start out with LDL oxidation.

4.1 LDL Oxidation

M.Wang Atherosclerosis 13 1

The che mical events of LDL oxidation are very complex. Although the exact séquense

of events leading to LDL oxidation in vivo is unknown, initiation begins with either abstraction

of hydrogen atoms from polyunsaturaed fatty acid (PUFA) within LDL by various ROS or by

direct enrichment of the LDL with lipoperoxides from cells. Either mechanism leads to a

loading of LDL with lipoperoxides which change into more reactive intermediates that can

initiate oxidation in neighboring PUFAs or PUFAs in nearby LDL particles [25]. In vitro, the

decomposition of lipoperoxides to more reacive peroxyl radicalsl is dependent on the presence of

transition metals (eg. copper or iron) and can be inhibited by metal chelators.

Importantly, the oxidation process is autocatalytic such that, a single hydrogen

abstraction could lead to oxidation of the entire LDL particle as well as neighboring LDL

particles. This process may be facilitated by LDL’s intrinsic phospholipase A2 activity., which

leaves off oxidized fatty acids from lecithin, generating isolecithin. The oxidized fatty acids

released by phospholipase A2 are more mobile and presumably may help spread the oxidation

process to other areas of the LDL particle [26]. Initially, the oxidation process procedes slowly,

but eventua lly the antioxidant content withiin LDL is depleted and the number of fatty acid

lipoperoxides amplify such that the oxidation process rapidly accelerates (the propagation

phase). Eventually, PUFAs are cleaved into a variety of reactive aldehydes, ketones and other

short chanin fragments (the termination phase). These in turn may bind to apopretein B-100 in

LDL which leads to decreased recognition and binding by the LDL receptor. In addition, new

epitopes are formed that lead to recognition and enhanced uptake of modified LDL by the

scavenger receptor of macrophages. There may in fact be a family of “scavenger” receptors

whose function is to remove modified or altered proteins, including LDL [27].

M.Wang Atherosclerosis 14 1

Oxidation of LDL may be initiated by a number of different mechanisms. However, cell-

mediated oxidation of LDL may be the most relavent to our understanding of LDL oxidation in

vivo. In tissure culture, all the cells normally present in the artery wall, including endothelial

cells, smooth muscle cells, macrophages and lymphocytes can oxidize LDL [28]. The

mechanisms by which cells initiate oxidation of LDL are poorly defined.

Table 1. Mechanisms By Which Oxidized LDL May Be Atherogenic [25].

1 It has enhanced uptake by macrophages leading to cholesteryl ester enrichment and

foam cell formation

2 It is chemotactic for circulating monocytes and T- lymphocytes.

3 It inhibits the motility of tissue macrophages

4 It is cytotoxic.

5 It renders LDL more susceptible to aggregation, which leads to enhanced macrophage

uptake

6 It can alter gene expression of neighboring arterial cells such as induction of MCP-1,
CSF, IL-1 and endothelial expression of adhesion molecules.

7 It can adversely alter coagulation pathways, such as by induction of tissue facotor.

8 It can adversely alter vasomotor properties of coronary arteries

9 It is immunogenic and can elicit autoantibody formation and reactive T-cells.

4.2 Atherogenic Properties of Oxidized LDL

Oxidized LDL takes on a variety of properties that make it more atherogenic than

unmodified LDL. These are outlined in Table 1 [25]. Every early step in the initial stages of

atherosclerosis is the focal adherence of monocytes to the artery wall. This may result from
M.Wang Atherosclerosis 15 1

expression of specific leukocyte adherence molecules such as VCAM-1on endothelial cells.

These adherence proteins are expressed prior to monocyte binding to the endothelium and

accumulate over areas of the artery wall which later become sites of foam cell formation. LDL

that has been minimally oxidized stimulateds the expression and secretion of many different

cytokines. In vitro, adding minimally modified LDL to endothelial and smooth muscle cells

stimulates their secretion of monocyte chemoattractant protein (MCP-1) [29]. This is shown in

figure 7 [30]. Presumably, this cytokine will attract monocytes to the endothelium where they

bind to specific adherence proteins. During LDL oxidation, phosphatidylcholine is formed and

can stimulated expression of the adherence molecule VCAM-1, and is one examp le of the many

products of modified lipoproteins that likely influence monocyte recruitment.

Figure 7. Proposed mechanisms by which oxidized LDL contributes to atherosclerosis [30].

4.3 Effects of Antioxidants on Athe rosclerosis

a. α -tocopherol

Because the above hypothesis implicates oxidatively modified LDL in the pathogenesis

of atherosclerosis, treatment with antioxidant, such as α-tocopherol, could conceivably retard the

progression or actually regress the atherosclerotic lesion [31]. α-tocopherol is a chain-breaking

M.Wang Atherosclerosis 16 1

antioxidant that prevent oxidation by trapping peroxyl free radicals (shown in Figure 8 [32]),

thereby providing first- line protection against lipid peroxidation. In models involving normal

animals fed a high-cholesterol diet, significant inhibition of atherosclerosis with vitamin E

treatment has been shown in monkeys [31]. Moreover, several studies have linked the

atherosclerosis- inducing effect of cigarette smoke with low antioxidant status [33, 34]. Cigarette

smokers can be considered to be under oxidative stree, even if antioxidant concentrations are

normal, as cigarette smoke is extremely high in free radicals. Although the red blood cells and

lipoprotein particles of smokers have similar concentrations of vitamin E compared to

nonsmokers, the lipids of these cells and particles are more susceptible to oxidation.

Supplementation with vitamin E prevents lipid peroxidation in smokers.

Figure 8. Chain reaction of vitamin E (α -tocopherol) with lipid radicals and vitamin C [32].

b. Antioxidant enzyme system

Cells in our body have balanced antioxidant enzyme system to protect from oxidative

damage, as shown in Figure 9 [35].

M.Wang Atherosclerosis 17 1

H2O
G SH
GP x GR
CA T GS S G
O2 + H 2 O H2O 2

S OD PS H PS S G
+ G SH
O2

Figure 9. The antioxidant enzyme system [35].

Manganese superoxide dismutase (MnSOD) and Copper-Zinc superoxide dismutase

(CuZnSOD) both convert superoxide (O 2 •-) to produce hydrogen peroxide (H2 O2 ) during normal

oxygen metabolism and contribute to the redox state of the cell. MnSOD is located in the

mitochondrial matrix whereas CuZnSOD is in the cytosole. It has been shown that MnSOD can

be induced in atherosclerotic arteries, may be as a reply to increased mitochondrial oxidation

[36]. Although in vitro studies showed that CuZnSOD inhibited cell- mediated oxidation of

LDL, CuZnSOD transgenic mice did not reduce the extent of atherosclerotic lesion development

[37]. However, their role in preventing atherosclerosis still needs investigation.

5. Future plans for research

Although there are a huge bunch of papers and books about atherosclerosis, from the

above information, we can see that there are still two important points remaining unclear. First,

the mechanisms of atherogenesis are unclear. We know that LDL oxidation contributes to the

process of atherogenesis, but we don’t know the siganal transduction pathways that are involved

in which LDL is oxidized and accumulated upon the artery walls. Second, the role of antioxidant
M.Wang Atherosclerosis 18 1

enzymes in preventing atherogenesis remains undefined. The following hypothesis aims at

answering these questions.

5.1 Hypothesis

The hypothesis is that using proteomics technique, we will be able to identify proteins

that change expression during atherosclerosis and those proteins most probably are involved in

the signal transduction pathways leading to atherosclerosis; With this technique, we are also be

able to identify proteins that are influenced by MnSOD overexpression and those proteins are

candidates that are disturbed by MnSOD on the atherosclerogenetic signal transduction

pathways. Following steps can be done to prove this hypothesis.

5.2 Identify proteins that may be involved in the signal transduction pathways for

Atherosclerosis.

Specific Aim 1: Set up an animal model suitable for atherosclerosis analysis.

In this study, we will choose mice as the animal model. Mice are fed with normal chow

supplemented with high cholesterol and butter fat for 14 weeks prior to base- line analysis

(supplemented diet group). Blood will be drawn 1 week prior to the end of the experimental

diet, plasma cholesterol is then quantified. Mice are divied into three groups with mean

cholesterol levels not significantly different. One group of mice are killed for analysis of

atherosclerosis (base- line group). Remaining mice are injected intravenously with either

AdMnSOD (AdMnSOD atherosclerotic group) or control Adnull (atherosclerotic control group)

virus. The supplemented diet are continued after vector administration. Blood samples are

collected and centrifuged to obtain plasma, which is stored at –20o C for lipid analyses and at 4 o C

for FPLC. Mice are killed 6 weeks after vector administration for analysis of atherosclerosis.

Control group uses age and sex matched mice and fed with normal diet. Rational: high
M.Wang Atherosclerosis 19 1

cholesterol and butter fat diet can lead to atherosclerosis. And mouse is a classic model for

atherosclerosis analysis [38].

Specific Aim 2: Analysis of atherosclerotic lesions

a. Evaluation of whole aortas

The entire aorta, from junction with the heart to the iliac bifurication, is removed from

mice, cut open and staind with the Oil red O staining solutions as described by Paigen [39], and

examined under a dissecting micorscope. The spots stained with Oil red O solutions are

atherosclerotic lesions. Visual estimation of the area covered by Oil red O staining lesions will

be made.

b. Evaluation of aortic cross sections

The heart and the upper section of

the aorta are removed. Extraneous tissue are

trimmed. The heart and the aortas are

sectioned from the apex to the base of the

heart and beyond into the ascending aorta up

to the aortic arch. Sectioning continues

along the ascending aorta away from the Figure 10. Anatomy of a mouse heart and aorta [39].
Line A represents a line between the tips of the atria;
heart until the valve cusps are no longer Line B represents the beginning of the aortic sinus;
Line C represents the beginning of one cross section of
visible. Every cross-section slide is stained the aorta and Line D represents the end of this cross
section. The average distance between Line C and Line
with Oil red O staining solution and D is about 280 µm.

evaluated for atherosclerotic lesions [39].

Rational: Pathologic properties of atherosclerotic lesions can be measured by evaluating the

extend of lesions along whole aorta and aorta cross-sections.

M.Wang Atherosclerosis 20 1

Specific Aim 3. Analysis of LDL and LDL oxidation

a. LDL purification and analysis of cholesterol levels. LDL are purified from plasma by

sequential untracentrifugations. Protein concentrations in plasma and LDL are determined

according to the method of Lowry [40]. Plasma cholesterol and triglyceride levels are measured

by FPLC gel filtration on two superose 6 columns. The cholesterol concentrations in the FPLC

fractions are determined using an enzymatic assay kit.

Lipoprotein are isolated from plasma samples by sequential untracentrifuagtion. The

plasma samples obtained at various time points during experiments are pooled from mice each of

the AdMnSOD atherosclerotic and Adnull atherosclerotic group. The VLDL, LDL and HDL are

isolated by tube slicing. The lipoprotein frations are analyzed for the lipid and protein contents.

b. Detection of oxidized LDL by ELISA. The oxidized LDL in blood plasma were

detected by ELISA using anti-oxLDL monoclonal antibodies (mAb). Ninety-six well plates are

coated with plasma samples. Primary and secondary antibodies are added consequently. Sulfuric

acid and the optical density (OD) are measured with a microplate reader to determine the

concentration of oxidized LDL.

Rational: LDL oxidation are highly correlated with atherosclerosis.

Specific Aim 4: identify proteins that may involved in the atherosclerosis process.

Among the techniques of functional genomics, both DNA micorarrays and proteomics

hold great promise for the study of complex biological systems with applications in molecular

medicine. These novel and powerful gene expression profiling techniques permit the analysis of

the expression levels of thousands genes simultaneously both in health and disease. DNA

microarray focuses on the mRNA level whereas proteomics is used to analyze global patterns of

gene expression at the protein level. Proteins are frequently the functional molecules and,
M.Wang Atherosclerosis 21 1

therefore, the most likely to reflect differences in gene expression. Some messengers are

transcribed but not translated, and the number of mRNA copies does not necessarily reflect the

number of functional protein molecules. Therefore, we are going to use the proteomics

techniques to approach our hypothesis, to identify proteins involved in atherosclerogenic signal

transduction pathways.

Step 1. Protein seperation with 2D PAGE.

After analyzing the LDL contents and atherosclerotic lesions of the supplemented diet

group of mice, total protein will be isolated from artery walls of the base- line group mice and

control group mice, separated on a high resolution two-dimensional polyacrylamide gel

electrophoresis (2D PAGE). Proteins will be separated both in terms of their isoelectric point

(pI) and molecular weight. This technique was originally described by O’Farrell [41].

Step 2. Protein detection and quantification

The proteins on the gels will be detected by silver nitrate staining and Coomasie Blue

staining. Quantitative fluorescence measurements can be performed with CCD-camera based

systems as well as with laser scanner systems.

Step 3. Protein identification and database construction

Proteins that have detectable expression difference between control group and base- line

group will be identified with Edman peptide sequencing. The peptide sequences will be blasted

against NCBI protein database to identify the corresponding proteins isolated from 2D PAGE

(http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html#protein_databases).

A comprehensive 2D PAGE database for atherosclerosis will be generated based on the

protein expression profile obtained from the above steps. This database will be exceedingly

important for the atherosclerosis research in that it provides a global overview of the proteins
M.Wang Atherosclerosis 22 1

that may be involved in the process of Atherosclerosis. And future research can be directed

based on the information provided by this database.

5.3 Identify proteins that may be disturbed by MnSOD in the atherosclerogenic

signal transduction pathways.

Same steps will be used as described in 5.2. To identify proteins that may be disturbed

by MnSOD in atherosclerogenic signal transduction pathways, AdMnSOD atherosclerotic group,

atherosclerotic control group and control group will be used for 2D PAGE analysis. Again, a

comprehensive 2D PAGE database for effect of MnSOD on atherosclerosis will be generated

based on the protein expression profile obtained from the above steps. Again, this database will

be extremely important in that it provides great information about the effects that MnSOD may

have on atherosclerosis.

6. Summary

Atherosclerosis is a major cause of heart disease and stroke. The mechanisms of

atherosclerogenesis remain unclear. Evidences show that oxidized LDL plays a very important

role in atherosclerogenesis. But the signal transduction pathways that lead to LDL oxidation are

far beyond clear. With the rising regarding of the important roles of free radicals in the process

of diseases, many scientists believe that atherosclerosis is a free radical disease. Antioxidants

may play an important role in preventing or reversing atherosclerosis. But the exact roles of

antioxidant in the process of atherosclerosis are still undefined. Since prometics techniques

provide us a way to profile protein expression both in health and disease, it is a very promising

technique to answer all the above questions. With the generation of a comprehensive

atherosclerosis 2D PAGE database and effects of MnSOD on atherosclerosis database, future

research can be directed based on the important information provided by these databases.
M.Wang Atherosclerosis 23 1

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and Clinical Science. Edinburgh ; New York : Churchill Livingstone. pp 51-56.

2. Kumar V, Cotran RS, Robbins SL. (1997). Blood vessels. In Kumar V, Cotran RS, robbins SL, ed.
Basic Pathology. Philadelphia: W.B.Saunders company; pp 127-128.

3. McGill HC Jr, McMahan CA, Herderick EE, Malcom GT, Tracy RE, Strong JP. (2000). Origin of
atherosclerosis in childhood and adolescence. J Med Chem. 72:1307S-1315S.

4. Campbell JH, Campbell GR. (1997). The cell biology of atherosclerosis--new developments. Cell
Biol. Atheroscler. 27:497-500.

5. Xu Q. (2000). Biomechanical-stress-induced signaling and gene expression in the development of

arteriosclerosis. Trends Cardiovasc. 10:35-41.

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