This student paper was written as an assignment in the ......This student paper was written as an assignment in the graduate course Free Radicals in Biology and Medicine (77:222, Spring

77:222 Spring 2003 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 2003)

offered by the

Free Radical and Radiation Biology Program

B-180 Med Labs The University of Iowa

Iowa City, IA 52242-1181 Spring 2003 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.

RN Rodionov Atherosclerosis page 1 of 21

The roles of free radicals in the

pathogenesis of atherosclerosis by

Roman N Rodionov

3150ML

Department of Internal Medicine

The University of Iowa

Iowa City, IA 52242

For 77:222 Spring 2003

05.07.03

Paper V

Abbreviations

acetylLDL – acetylated low density lipoproteins, ADMA - asymmetric dimethylargenine, ASL –

argininosuccinate lyase, ASS - argininosuccinate synthetase, cGMP – cyclic guanylate monophosphate,

CSF – colony stimulating factor, DDAH - dimethylarginine dimethylaminohydrolase, ECM – extracellular

matrix, HDL – high density lipoproteins, IL-1 – interleukin 1, LDL – low density lipoproteins, MCF –

monocyte colony-stimulating factor, MCP-1 – monocyte chemoattactant protein 1, NMMA – N-

monomethylargenine, NO- nitric oxide, oxLDL – oxidized low density lipoproteins, PUFA –

Polyunsaturated fatty acid, ROS – Reactive oxygen species, TNF-α – tumor necrosis factor alfa,VLDL –

very low density lypoproteins , IDL – intermediate density lipoproteins, ,

SAH – S-adenosylhomocysteine, SAM – S-adenosylmethionine, sGC – soluble guanylate cyclase, PRMT –

protein arginine methyl transferase,


Contents

Contents 2

Abstract 2

Definition 3

Epidemiology and risk factors 3

Clinical manifestations 3

Pathogenesis 3

Roles of free radicals in the pathogenesis of atherosclerosis

LDL oxidation

Redox sensitive gene expression

Role of Nitric oxide in atherosclerosis

5

Antioxidants in atherosclerosis

Proposal of new experiments

Conclusions

References

Abstract Atherosclerosis is the leading cause of death and disability in the developed world. Pathogenesis of

atherosclerosis is becoming better and better understood. Series risk factors for atherosclerosis has been

determined. A lot of research is going on in this area. It is clearly proven now that free radicals are

involved into the pathogenesis of atherosclerosis itself and most of the predisposing risk factors.

Atherosclerosis was shown to be associated with an increased production of reactive oxygen species

(oxidative stress) and decreased bioavailability of nitric oxide. Many different mechanisms how oxidative

stress contributes to atherosclerotic lesions formation and progression have been elucidated. They include,

but not limited to LDL oxidation, decrease expression of “antiatherosclerotic” genes, increase expression of

“proatherosclerotic” genes and decrease in NO bioavailability. The goal of this paper is to review the

different roles of free radicals in the pathogenesis of atherosclerosis and propose several experiments to

further elucidate free radical nature of atherosclerosis.


Definition Atherosclerosis comes from the Greek words athero (meaning gruel or paste) and sclerosis (hardness)

Atherosclerosis is a degenerative disease of large and medium-sized arteries, characterized by intimal

deposition of lipids (formation of atherosclerotic plaques), chronic inflamation and fibrosis and resulting in

narrowing of the arteries, impairment of blood flow and predisposition to thrombosis [combination of

several definitions].

Epidimiology and risk factors Atherosclerosis is the leading cause of death and disability in the developed world [1]. Every 34 second a

person in the USA dies from heart disease. A huge number of epidemiological studies has revealed several

risk factors for atherosclerosis, that are traditionally are divided into groups: modifiable and unmodifiable

(Table 1).

Modifiable risk factors Unmodifiable risk factors

Smoking, obesity, physical inactivity, lipid

disoders, hypertension, insulin resistance

Age, Male gender, Genetics

Table 1 [1] Risk factors for atherosclerosis

Clinical manifestations Although atherosclerosis is a systemic disease distinct clinical manifestation depend on particular

circulatory bed affected. Atherosclerosis of the coronaries arteries commonly causes myocardial infarction

and angina pectoris. Atherosclerosis of the arteries supplying central nervous system frequently provokes

strokes and transient cerebral ischemia. In the peripheral circulation, atherosclerosis causes

imminentclaudication and gangrene and can jeopardize limb viability. Involment of splanchic circulation

can cause mesenteric ischemia. Atherosclerosis can affect kidney directly (e.g., renal stenosis) or as a

frequent site of atheroembolic disease. [1]

Pathogenesis The key processes in atherosclerosis are intimal thickening and lipid accumulation, producing the

characteristic atheromatous plaques. Atherosclerotic plaques have three principal components: (1) cells,

including smooth muscle cells, macrophages, and other leucocytes; (2) connective tissue extracellular

matrix, including collagen, elastic fibers and proteoglycans; and (3) intracellular and extracellular liver

deposits.


Fig 1. “Response to injury hypothesis. [4]

The current concept of atherosclerosis

pathogenesis is called “response to injury

hypothesis” [4].

According to this hypothesis atherosclerosis is

considered as a chronic inflammatory response of

arterial wall, initiated by some form of injury to th

endothelium. Sequence of the events in the

development of atherosclerotic lesion are

illustrated on the Fig. 1

e

ncreased

d

rface,

3. emigrate from media to

dation of

4. smooth muscle cells engulf

5. vely

1. Development of focal regions of chronic

endothelial injury. The main factors that could

induce this injury include, but not limited to

hyperlipidemia, hypertension, smoking,

homocysteine, homodynamic factors, toxins,

viruses, immune reactions.

2. Chronic endothelial injury results in endothelial

dysfunction, which includes decreased NO

production, increase ROS production, i

permeability, increased leukocyte adhesion an

emigration. Lipoproteins (mainly LDL and to

some extent VDL) stick to endothelial su

get oxidized and insudate into the vessel wall.

Smooth muscle cells

intima. Monocytes defferinciate to

macrophages and get activated. Insu

lipids continues

Macrophages and

oxidized lipids and become foam cells

Smooth muscle cells proliferate and acti

synthesize ollagen and proteoglycans. Increase

in both extracellular and intracellular lipid

deposition [4]


Developed plaques are shown on Fig. 2. Plaques could be divided into “vulnerable” and “stable” depending

on their morphology, and dynamic of progression. Vulnerable plaques have thin fibrous cap, large lipid

pool, many inflammatory cells and few smooth muscle cells. On the contrary, stable plaques have thick

fibrous cap, smaller liquid pool, few inflammatory cells and dense extracellular matrix

ig. 2 Evolution and stabilization of the plaque [22]

most of the complications of atherosclerosis. Most

ses

ree Radicals in atherosclerosis ctions in the vasculature. There are many enzyme systems

e should

F

Vulnerable plaque is unstable and is responsible for the

of the component in lipid core are very thrombogenic. Plaque rupture is the main cause of thrombosis in

atherosclerosis. Plaque disruption occurs most frequently where fibrous cap is thinnest and most heavily

infiltrated by foam cells. Macrophage-derived protease enzymes (e.g. collagenases, gelatinases,

stromelysin, metalloelastase and matrilysin) may be involeved. Gradual growth of the plaque cau

ischemia.

FRadicals play a lot of very important fun

responsible for production of free radicals in all the vascular cells. Several antioxidant systems are

responsible for control of free radical levels. Radicals are involved in many signaling pathways. On

think about very precise balance between free radical production and degradation. Any changes of this

balance could contribute to the pathogenesis of cardiovascular diseases.


Atherosclerosis was shown to be associated with increase production of reactive oxygen species and

decreased bioavailability of nitric oxide.

All the common risk factors for atherosclerosis, such as hypercholesterolemia, diabetes, hypertension,

smoking and aging increase production of reactive oxygen species by endothelial, vascular smooth muscle,

and adventitial cells (Fig. 3)

Fig. 3 Risk factors for atherosclerosis cause

production of ROS [3]

Fig. 4 Sources of ROS in vasculature [3]

Many different sources of reactive oxygen species in the vasculature were identified, that include, but not

limited to the following:

Source of free radicals Localization

Lipoxygenases, cyclooxygenases, NADPH oxidase Plasma membrane

Electron transport system Mitochondria

Xantine oxidase, Hemoglobin, Riboflavin,

transitional metals (Fe2+/3+, Cu1+/2+)

Cytosol

Oxidases, Flavoproteins Peroxisomes

Mixed-function oxidase electron transport

cytochromes P-450 and b5

Endoplasmic reticulum

Table 2. Sources of ROS in vasculature

All these enzymes use various substrates as sources of electrons that subsequently reduce molecular oxygen

to form ROS. A 1-electron reduction of leads to production of superoxide, and a 2-electron oxidation of

oxygen leads to formation of hydrogen peroxide. Dismutation of superoxide , by superoxide dismutase can

also lead to formation of hydrogen peroxide. Superoxide can react with NO and form peroxynitrite, which

can react with carbon dioxide and than decompose, producing hydroxyl radical, which plays very important

roles in lipid peroxidation (Fig. 4)


LDL oxidation There are different forms of lipid transport particles in human body. They have been traditionally classified

depending on their size and composition into the following groups: chylomicrons, very low density

lipoproteins (VLDL), low density lipoproteins (LDL), intermediate density lipoproteins (IDL), high density

lipoproteins (HDL). The main lipoproteins that carry cholesterol to peripheral tissues are LDL. These

particle play very important role in the pathogenesis of atherosclerosis, because they are responsible for

lipid accumulation in the vessel wall, and particulary inside the foam cells. The typical LDL particle

consists of a central lipophilic core containing approximately 1600 molecules of cholesteryl ester and 170

molecules of triclyceride. Surrounding this lipid core is a monolayer of approximately 600 free cholesterol

molecules and 700 of phosphatidylcholine. The protein portion of the LDL particle embraces its entire

surface and consists of apolipoprotein -B (apoB). Apo B is a glycosylated protein containing approximately

4500 aminoacid residues [19]. It was shown that native LDLs cannot induce foam-cell formation, because

their uptake is slow and because their receptor could be downregulated. Either acetyl LDL or oxLDL can

induce foam-cell formation because their uptake is rapid and the scavenger receptor is not downregulated in

response to an increase in cellular cholesterol [8] Fig. 5.

Fig 5. Scavenging of native and modified LDL by macrophages. [8]


So, in order to cause foam cell formation LDL should first be modified. LDL oxidation starts after LDL

particle is trapped in the artery wall by binding to extracellular proteoglycans. A number of the species

were proposed to be responsible for oxidation initiation: hydroxyl radical, Fe2+/Fe3+/O2, peroxynitrite,

tyrosyl radical, lypoxygenase and myeloperoxydase (in macrophages) [24]

The precise characterization of LDL oxidation has been problematic, manly because of both the complexity

and heterogeneity of human LDL both amongst individuals and in response of dietary variations. Average

lipid composition of LDL particle is listed in Table 3.

Table 3. Lipid composition of human LDL, [24]

Polyunsaturated fatty acids (PUFA) are the main target for lipid peroxydation. The simplified chemistry of

PUFA oxidation is shown on Fig. 6.

Fig 6. Scheme for lipid peroxidation. [24]


Oxidation makes LDL immunogenic, which stimulates inflammation and increases phagocytosis of LDL

by macrophages

OxLDL are heterogeneous in their composition, metabolism and biological properties. The main toxic

compounds in oxLDL include, but not limited to oxysterols, oxidized fatty acids, lysophospholipids and

sphinolipids [23]. OxLDL have a lot of proatherogenic properties; some of them are listed in Table 4

Some of the proatherogenic functions of ox LDL [8]

Function Ref.

1 Induction of monocyte binding to endothelial cells Watson

2 Increase tissue factor activity Marathe

3 Mimic effects of platelet-activating factor Marathe

4 Increase expression of MCF and MCP-1 Navab

5 Increase expression of VCAM 1 Gimbrone

6 Induce Fas-mediated apoptosis Walsch

7 Induce expression of IL-1 and IL-8 Terkeltaub

8 Inhibit NO release or function Murohara

9 Increase collagen synthesis in smooth muscle cells Jimi

10 Increase intracellular calcium Thorin

11 Activate NFkB Brand

12 Induce expression of type 1 metalloproteinase Rajavashisth

Table 4.proatherogenic effects of oxLDL [8]

Redox sensitive gene expression Eukaryotic cells have evolved a lot of mechanisms to rapidly respond to changes in the enviroment by

altering the expression of genes. There are several redox sensitive transcription factors that play important

role in these responces. There are several redox sensitive regulatory points in the intracellular signaling

pathways in the vascular cells.

1. ROS influence Ca2+ signaling via increasing the concentration of intracellular Ca2+. The exact

mechanism is still unknown – the most probable candidates are inhibition of ATP-dependent Ca2+ pump

and enhanced Ca2+ transport through the Ca2+ channels

2. ROS were shown to cause activation of tyrosine kinases signaling. It is still to be identified whether it is

due to activation of tyrosine kinases or inhibition of the corresponding phosphotases.


3. ROS are able to activate mitogen-activated protein kinases (MAPK). MAPKs play a role in relaying

signals from extracellular stimuli to the cell nucleus. One of the best-characterized functional targets of

the MAPK family is the transient phosphorylation of the transcription factor complex that regulates the

c-fos promoter. Another is the phosphorylation and subsequent activation of the transcriptional

activation domain of c-jun. C-jun and c-fos are components of the redox-sensitive transcription factor

AP-1. AP-1 was shown to be responsible for activation of ICAM-1 and MCP-1 gene expression during

the development of vascular deseases.

4. Another important factor, activated by ROS is NF-kB, which is able to respond directly to the oxidative

stress. NF-kB was shown to regulate a lot of genes, involved in the pathogenesis of atherosclerosis:

TNF-α, IL-1, macrophage CSF, granulocyte CSF, granulocyte-macrophage CSF, MCP-1, tissue factor,

VCAM-1, ICAM-1 E-selectin etc…

5. Peroxisome Proliferator-Activated Receptors (PPARs) belong to the nuclear hormone receptor

superfamily of transcription factors. PPARs are involved in glucose and lipid metabolism and are

implicated in metabolic disoders, predisposing to atherosclerosis, such as dyslipidemia and diabetes.

OxLDL were shown to activate PPARγ-dependent gene expression of CD36, which is the main oxLDL

receptor in macrophages. On the other hand the role of PPARα and PPARγ in mediated anti-

inflammatory responses in the vessel wall has been reported. In particular, PPARα was shown to inhibit

IL-1-induced production of IL-6, prostaglandin and COX-2.

The main redox sensitive pathways of the regulation of gene expression are illustrated in the Fig.7

Fig 7. Redox sensitive regulation of gene expression [7]


The main groups of the genes regulated by ROS include:

1. Adhesion molecules

2. Chemoatractants

3. Matrix metalloproteases.

feration

ole of Nitric oxide in atherosclerosis in the cardiovascular system was discovered in the end of

ric oxide in the response to the certain stimuli: shear stress,

effects.

h

ric oxide were discovered. Thus NO

were proposed. The

elf

ic oxide

tration is

e

4. Genes responsible for proli

5. Cytokines

RThe role of nitric oxide as a signaling molecule

20th centure. Robert F Furchgott, Louis J Ignarro and Ferid Murad received Nobel Prize for their

contribution to this discovery in 1998.

Endothelial cells are able to produce nit

bradykinin, acetylcholine, serotonin etc. NO oxide defuses through the vessel wall causing different

The main one is activation of soluble guanyl cyclase in smooth muscle cells. Activation of sGC causes

increase of intracellular level of cGMP, which in its turn activate cGMP-dependent protein kinases, thic

mediate vasorelaxation via phosphorylation of proteins that regulate intracellular Ca2+ levels [32]

Vasorelaxation reduces ischemia and protects heart from overload.

During last couple decades several other vasoprotective effects of nit

was shown to inhibit platelet aggregation, LDL oxidation, monocyte adhesion and smooth muscle cells

proliferation. Protective effects of nitric oxide in vasculature are shown in Fig. 8.

Several mechanisms for decreased bioavailability of nitric oxide in atherosclerosis

major one is reaction of nitric oxide with superoxide, that results in formation of peroxynitrite, which its

has a lot of toxic effects. Another mechanism is uncoupling of NOS, which results not only in decrease of

nitric oxide production, but also in hyperproduction of superoxide. OxLDL were shown to directly

inactivate nitric oxide or decrease eNOS synthesis and activity in endothelial cells. [24] Another

mechanism for decreased eNOS activity is accumulation in the cells endogenous inhibitors of nitr

synthases. The most important endogenous inhibitors of nitric oxide synthases are asymmetric

dymethylargenine (ADMA) and N-monomethylargenine (NMMA). Increase of ADMA concen

considered to be one of the mechanisms of homocysteine induced endothelial dysfunction. It is proposed

that homocysteine inhibits dimethylarginine dimethylaminohydrolase (DDAH). This enzyme is responsibl

for hydrolysis of asymmetric dimethylargenine ADMA. Inhibition of DDAH results in increase of ADMA,


NOplatelet aggregation

monocyte adhesionsmooth muscle cell

proliferation

LDL oxidation

endothelial cellapoptosis

Endothelial ferritin synthesis ecSOD Synthesis

SOD

2O2•- + 2H+ O2 + H2O2

Fe2+

FerritinFree Fe2+

vasodilatation

Fig 8. Vasoprotective effects nitric oxide. - inhibition; - induction. [author’s slide]

which results in inhibition and uncoupling of eNOS and increased NO availability [21].

Several studies has addressed effects of different antioxidants on NO bioavailability. Protective effects of

antioxidants in atherosclerosis will be discussed below.

Antioxidants and atherosclerosis One can look at atherosclerosis as at the imbalance between oxidant and antioxidant systems in vascular

cells (Fig. 9). There were a lot of human and animal studies of the roles of antioxidants.

Fig. 9 Balance between oxidant and antioxidant systems in vascular cells [6]


Vitamin E

Alfa-tocopherol is the principal lipid-soluble chain breaking antioxidant. It was shown to inhibit lipid

peroxydation, remove and repair oxidized lipids. Scheme of this reaction is shown on the Fig. 9.

GR, Free Radicals in

Table 5. Selected clinical trials of natural antioxidant therapies on atherosclerotic events [31]

Fig. 10 Proposed scheme for removal and repair of oxydised lipids [Buettner

tment and prevention of atherosclerosis have been

Molecular Biology and medicine, Lecture, 2003]

Series of trials for clinical usage of Vitamin E in trea

done. As one can see results of these trials are very controversial Table 5.


Antioxidant enzyme system

There are several antioxidant systems, responsible for protection of vascular cells.

1. SOD.

It was shown that expression of SOD is downregulated in atherosclerosis. One of the mechanisms is decrease

of bioavailability of NO, which upregulates SOD expression in the response to oxidative stress.

2. Glutathione peroxidase

3. Catalase

Expression of antioxidants can be altered by hormones such as AngII, TNF-α or IL-1β.

roposed experiments ent of

therosclerosis. Still the list is not complete. The goal of this chapter is to discuss potential future direction

f the research in this area and propose several experiments to further elucidate the problem.

ubtypes: PPARα, PPARδ and PPARγ. PPARs has been shown to be actively

PAR activity.

a series of cardioprotective and antiatherosclerotic genes [30], we

arget.

pecific aim 1

egulate PPARα receptors?

a) Reporter constructs should be designed to assess PPAR dependent activation of gene expression.

Construct

PPARα consensus site fused with luciferase gene and put in adenovirus. Three constructs should be made.

Each construct will address the question about the activation of different PPAR isoform.

Vascular endothelial cells should be tranfected with the construct or empty adenovirus (control) Efficiency

of the transfection should be controlled.

Cells should be exposed to the source of superoxide (xantine oxidase + hypoxantine)

Changes in luciferase activity should be measured during different time points (every 6 hours for 4 days)

using detection of luciferin bioluminescence from the cell lysate after adding luciferin.

PThis paper has discussed a lot of different way in which free radicals can contribute to the developm

a

o

1. The role of superoxide in modulation of PPARs activity.

PPARs comprise three s

involved in the pathogenesis of atherosclerosis. Several recent reports have shown that OxLDL are able to

activate PPARs. However it haven’t been addressed whether the other ROS could modulate P

As long as PPARα was shown to activate

will choose it as our first t

S

Is superoxide able to downr


Interpretation of results:

If our hypothesis is correct exposure of the cells to superoxide should cause decrease in reporter gene

ctivity. To prove that these changes are superoxide mediated we can show that SOD can reverse them. We

tured from SOD transgenic mouse or PEG-SOD.

ld be

the source of superoxide (xantine oxidase). Expressions of the main inducible genes in response

ereas no changes

nisms of regulation, besides PPARα.

of endothelial

se to superoxide?

ing

e mice have increase level of superoxide

vascular tissues and remarkable endothelial dysfunction. We can use different approaches to address the

Superoxide levels Expression of PPAR

regulated genes

Endothelial dependent

vasodilataion

a

can use cells cul

b) Primary endothelial cells should be cultured from wt mice and PPARα knockout mice. Cells shou

exposed to

to superoxide should be determined (ELISA, Northern Blot, RNase Protection Assay etc). If our hypothesis

is correct, superoxide will decrease expression of PPARα-regulated genes in wt mice, wh

in gene expression will be found in knockouts. This experiment will address the possibility that “PPARα-

regulated” genes have some other mecha

Specific aim 2

Does inactivation of PPARα by superoxide play important role in the development

dysfunction in the respon

The best way to address this question is to use Cu,Zn-SOD knockout mice [29]. The rational for choos

Cu,Zn-SOD as a target is that we are interested in regulation of PPAR, so we need increase of superoxide

in cytoplasm and nucleus. As it has been previously described thes

in

issue, whether this dysfunction is at least partly mediated through inactivation of PPARα.

a) Crossbreed Cu,Zn-SOD knockout mice with mice, overexpressing PPARα (tgPPARα) Compare

three groups Cu,Zn-SOD (-/-),and Cu,Zn-SOD (-/-)tgPPARα . We need to compare superoxide levels (for

example using dihydroethidium fluorescence), expression of PPAR regulated genes (RT-PCR) and

endothelial dependent vasodilataion[29]. If our hypothesis is correct overexpression of PPARα

Predicted results (if the hypothesis is correct):

Cu,Zn-SOD (-/-) Increased Normal impaired

tgPPARα Normal increased normal

Cu,Zn-SOD (-/-

)tgPPARα

Increased Normal or less increased

than in second row

Normal or less impaired

than in the first row

,


2. Mechanisms of decreased availability of nitric oxide by superoxide.

Several mechanisms of decreased availability of nitric oxide by superoxide have been described (see

al tissues

eral arginine analogues were shown to inhibit NOS.

hemical structure of some shown i

genous inhibitor of NOS was shown to be involved in the

e

active site, which is susceptible to

above). However, most of the researchers in this are believe that the list is not complete. For example the

possibility that superoxide can decrease availability of NO via increasing concentration of endogenous

inhibitors of NOS has not been addressed yet.

Nitric Oxide Synthase (NOS) is the main enzyme responsible for nitric oxide synthesis in mamm

is This enzyme uses arginine as a substrate. Sev

C methylarginines is n the Fig. 11.

Fig. 11 Chemical structure of methylargenines [online sourses]

Asymmetric dymethylargenine (ADMA), endo

pathogenesis of several cardiovascular diseases including atherosclerosis [25, 27]. Regulation of ADMA

metabolism is shown on the Fig. 12. DDAH (dimethylargenine deaminohydrolase) is the main enzym

responsible for hydrolysis of ADMA. This enzyme has cysteine in its

oxidation [25]


Fig. 12 Regulation of ADMA and NO synthesis [author’s slide] ADMA – asymmetric dymethylarginine, DDAH –dimethylarginine deaminohydrolase, SAM –

SAH – S-adenosylhomocysteine, PRMT – protein arginine methyl transferase, ASS - argininosucc

argininosuccinate lyase; Black arrow – inhibition; Yellow areas – chemical reactions

Possibility that DDAH could be directly inhibited by superoxide has not been addressed yet.

S-adenosylmethionine,

inate synthetase, ASL –

Hypothesis

Superoxide inhibits DDAH, which decreases ADMA hydrolysis in endothelial cells. Increased ADMA

pecific aim 1.

ecombinant DDAH should be used for this experiment. There are two different ways to measure DDAH

easuring decrease in substrate concentration (ADMA), (2) measuring of product

DDAH activity. As a

ontrol we can use xantine oxidase a lone or hypoxantine alone. To prove that the effect we see is due to

n check whether it can be reversed by adding SOD. ADMA concentration could be

olorimetric assay [28].

level causes inhibition of eNOS and consequently decreased bioavailability of NO

S

Superoxide is able to inhibit DDAH activity in vitro.

R

activity: (1) m

accumulation. DDAH should be incubated with xantine oxidase + hypoxantine in the access of oxygen.

Hopefully we will be able to show dose dependent and time dependent inhibition of

c

superoxide we ca

measured by HPLC. Citruline could be measured using c


Specific aim 2

Superoxide causes increase in ADMA concentration in endothelial cells

Cultured endothelial cell will be exposed to xantine oxidase + hypoxantine. Time dependent and dose

dependent (does of superoxide) accumulation of ADMA will be measured. ADMA will be measured in ce

lysates and in media using HPLC. Incubation the cells will PEG-SOD should reverse ADMA

accumulation.

ll

onclusions atherosclerosis is difficult to underestimate. Since 1900, Cardiovascular disease has been

,500 Americans die from heart

d the

ns were beyond the scope of this review, for example roles of ROS in plaque stability.

CSignificance of

the number 1 killer in the United States for every year but 1918. More than 2

disease each day. Billions of dollars are invested into research of this disease. The more data is acquire

more obvious it becomes how greatly free radicals contribute to the pathogenesis. Elucidation of all these

mechanism will allow finding a lot of potential targets for therapy and prevention.

A lot of questio


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This student paper was written as an assignment in the ......This student paper was written as an assignment in the graduate course Free Radicals in Biology and Medicine (77:222, Spring