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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,
RN Rodionov Atherosclerosis page 2 of 21
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.
RN Rodionov Atherosclerosis page 3 of 21
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.
RN Rodionov Atherosclerosis page 4 of 21
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]
RN Rodionov Atherosclerosis page 5 of 21
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.
RN Rodionov Atherosclerosis page 6 of 21
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)
RN Rodionov Atherosclerosis page 7 of 21
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]
RN Rodionov Atherosclerosis page 8 of 21
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]
RN Rodionov Atherosclerosis page 9 of 21
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.
RN Rodionov Atherosclerosis page 10 of 21
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]
RN Rodionov Atherosclerosis page 11 of 21
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,
RN Rodionov Atherosclerosis page 12 of 21
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]
RN Rodionov Atherosclerosis page 13 of 21
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.
RN Rodionov Atherosclerosis page 14 of 21
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
RN Rodionov Atherosclerosis page 15 of 21
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
,
RN Rodionov Atherosclerosis page 16 of 21
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]
RN Rodionov Atherosclerosis page 17 of 21
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
RN Rodionov Atherosclerosis page 18 of 21
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
RN Rodionov Atherosclerosis page 19 of 21
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