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The Role of Chronic Inflammation in Cardiovascular Disease and its Regulation by Nutrients

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Multiple risk markers for atherosclerosis and cardiovascular disease act in a synergistic way through inflammatory pathways. This article discusses some of the key inflammatory biochemical risk markers for cardiovascular disease; in particular, the role of three basic cell types affected by these risk markers (endothelial cells, smooth muscle cells, and immune cells), the crucial role of inflammatory mediators, nitric oxide balance in cardiovascular pathology, and the use of nutrients to circumvent several of these inflammatory pathways. Most risk markers for cardiovascular disease have a pro-inflammatory component, which stimulates the release of a number of active molecules such as inflammatory mediators, reactive oxygen species, nitric oxide, and peroxynitrite from endothelial, vascular smooth muscle, and immune cells in response to injury. Nitric oxide plays a pivotal role in preventing the progression of atherosclerosis through its ability to induce vasodilation, suppress vascular smooth muscle proliferation, and reduce vascular lesion formation. Nutrients such as arginine, antioxidants (vitamins C and E, lipoic acid, glutathione), and enzyme cofactors (vitamins B2 and B3, folate, and tetrahydrobiopterin) help to elevate nitric oxide levels and may play an important role in the management of cardiovascular disease. Other dietary components such as DHA/EPA from fish oil, tocotrienols, vitamins B6 and B12, and quercetin contribute further to mitigating the inflammatory process.
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Page 32 Alternative Medicine Review Volume 9, Number 1 2004
Cardiovascular/ Inflammation Review
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
Abstract
Multiple risk markers for atherosclerosis and
cardiovascular disease act in a synergistic way
through inflammatory pathways. This article
discusses some of the key inflammatory
biochemical risk markers for cardiovascular
disease; in particular, the role of three basic
cell types affected by these risk markers
(endothelial cells, smooth muscle cells, and
immune cells), the crucial role of inflammatory
mediators, nitric oxide balance in
cardiovascular pathology, and the use of
nutrients to circumvent several of these
inflammatory pathways.
Most risk markers for cardiovascular disease
have a pro-inflammatory component, which
stimulates the release of a number of active
molecules such as inflammatory mediators,
reactive oxygen species, nitric oxide, and
peroxynitrite from endothelial, vascular smooth
muscle, and immune cells in response to injury.
Nitric oxide plays a pivotal role in preventing
the progression of atherosclerosis through its
ability to induce vasodilation, suppress
vascular smooth muscle proliferation, and
reduce vascular lesion formation. Nutrients
such as arginine, antioxidants (vitamins C and
E, lipoic acid, glutathione), and enzyme
cofactors (vitamins B2 and B3, folate, and
tetrahydrobiopterin) help to elevate nitric oxide
levels and may play an important role in the
management of cardiovascular disease. Other
dietary components such as DHA/EPA from
fish oil, tocotrienols, vitamins B6 and B12, and
quercetin contribute further to mitigating the
inflammatory process.
(Altern Med Rev 2004;9(1):32-53)
The Role of Chronic Inflammation in
Cardiovascular Disease and
its Regulation by Nutrients
Henry Osiecki, BSc (Hons), Grad Dip Nutr & Dietetics
Introduction
Multiple risk factors for atherosclerosis
and cardiovascular disease include disordered lipid
profiles, autoimmunity, infection, homocysteine,
asymmetrical dimethylarginine, C-reactive pro-
tein, genetic predisposition, and various metabolic
diseases.1-5 Many risk factors act in a coordinated
or synergistic way through one or two inflamma-
tory pathways. Risk factors appear to act on three
cell types that coordinate their action to influence
cardiovascular dynamics, function, and structure.
These cell types include:
Endothelial cells that line the vascular
lumen. They control the intra- and
transcellular flow of nutrients, hormones,
and immune cells, and regulate vascular
tone and blood flow.6
Smooth muscle cells (SMC) or vascular
smooth muscle cells (VSMC) that
maintain vascular tone and structure.
Immune cells, including monocytes/
macrophages and T lymphocytes, which
defend the endothelium and SMC from
chemical and biological insult.
The disruption or over-expression of the
coordinated activities of these cells can lead to
cardiovascular disease.7-10 Chronic inflammation
Henry Osiecki BSc(Hons), Post Grad. Dip. Nutrition &
Dietetics – author Cancer: A Nutritional Biochemical
Approach, 2003; nutritional consultant to nutritional/
pharmaceutical industry.
Correspondence address: Bioconcepts, 9/783 Kingsford
Smith Dve, Eagle Farm 4009 Australia
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
Alternative Medicine Review Volume 9, Number 1 2004 Page 33
Review Cardiovascular/ Inflammation
is the most common disruptor of the activities of
these cells. Risk factors for cardiovascular disease
that have a pro-inflammatory component include
LDL cholesterol, smoking, elevated blood sugar,
hypertension, diabetes, infection, homocysteine,
ischemia, oxidant damage, interleukin-6, lipopro-
tein (a), high sensitivity C-reactive protein (hs-
CRP), serum intracellular adhesion molecule-1,
and apolipoprotein-B.2,4,5,11-14 In addition, these in-
flammatory risk markers can react synergistically
to increase relative risk (Figure 1). One common
link among these risk factors is the activity and
metabolism of nitric oxide (NO).
Endothelial Cell Function
Endothelial cells play a vital physiologi-
cal role in dividing blood from tissue. These cells
actively inhibit the activation of the hemostatic
mechanism and maintain blood circulation and
fluidity, limit the efflux of cells and protein from
the bloodstream, and participate in the mainte-
nance of normal vasomotor tone.6
Endothelial cells are highly metabolically
active and behave in a similar manner to paracrine
or endocrine gland cells in the release of chemi-
cal mediators.10,15,16 The endothelium generates a
number of active molecules in response to injury
or toxic chemical or oxidant stimuli, such as:
Adhesion molecules, intracellular
adhesion molecule (ICAM-1), vascular
cell adhesion molecule (VCAM-1),
fibronectin, selectins, interleukin-1,
heparin sulfate17
Clotting or coagulation factors17,18 (von
Willebrand Factor, thromboxane,
prostacyclin)
Fibrinolysis factors19,20 (e.g., tissue
plasminogen factor)
Figure 1. Relative and Synergistic Risk among Several Associated Factors
Lipoprotein(a)
Homocysteine
Total Cholesterol
LDL-Cholesterol
Apolipoprotein B
TC:HDL-C Ratio
hs-CRP
hs-CRP + TC:HDL-C Ratio
Relative Risk of Cardiovascular Events According To Several Biochemical Markers
Risk Factor
0 1.0 2.0 4.0 6.0
Relative risk for future cardiovascular events among apparently healthy women in the Women's Health Study
according to baseline values of Lipoprotein(a), Homocysteine, LDL-Cholesterol, Apolipoprotein B, high-
sensitivity C-Reactive Protein (hs-CRP) and TC:HDL-Cholesterol (TC:HDL-C ) ratio.
For consistency, risk estimates and 95% C's are computed for those in the top quartile, as opposed to the
bottom quartile, for each marker.
Page 34 Alternative Medicine Review Volume 9, Number 1 2004
Cardiovascular/ Inflammation Review
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
Components of the renin-angiotensin
system21 (e.g., angiotensin II that acts as a
pro-inflammatory cytokine and augments
the production of reactive oxygen species)
Prostaglandins22-24 (e.g., prostacyclin)
Growth-promoting or angiogenesis factors
(transforming growth factor-beta (TGF-β),
platelet-derived growth factor (PDGF))25
Vascular tone regulators18,25-28 (NO and
endothelin-1)
These biological molecules demonstrate
that the endothelium senses change in the local
milieu, and respond by releasing a variety of
cytokines and chemicals that regulate vascular
smooth muscle relaxation/contraction, vascular
structure, platelet and monocyte function, and
coagulation.26,28,31
The endothelium secretes a number of
vascular-relaxing substances as well as several
vasoconstricting agents (Table 1). However, one
of the most potent endogenous vasodilators is en-
dothelial-derived nitric oxide. NO is a critical
modulator of blood flow and blood pressure,27,32
and opposes the vasoconstricting effects of
endothelin, angiotensin II, serotonin, and norepi-
nephrine.31.33,34 NO also suppresses the prolifera-
tion of vascular smooth muscle.28,35
It was initially thought a continuous basal
synthesis of NO from the vascular endothelium
maintained resting vascular tone. Recent evidence,
however, suggests that NO production is increased
whenever the endothelium is damaged or stressed;
otherwise, only residual synthesis occurs.36 Defi-
ciency or loss of NO activity contributes not only
to increased vascular resistance but to blood ves-
sel medial thickening and/or myointimal hyper-
plasia, thus altering the structure of the vascular
bed (Table 2).27,35,37
A second messenger of internal cellular
communication – cyclic GMP (cGMP), produced
in response to nitric oxide – is a key regulator of
vascular smooth muscle cell contractility, growth,
and differentiation.40 It is implicated in opposing
the pathophysiology of hypertension, cardiac hy-
pertrophy, atherosclerosis, and vascular injury/
restenosis (Figure 2).43,46
Function of Vascular Smooth
Muscle Cells
Vascular smooth muscle cells contribute
to the maintenance of vascular tone. The balance
between stimuli that initiate contraction or dila-
tion is important in providing the elastic recoil
essential for normal functioning of the arteries.33
Contraction of vascular smooth muscle (VSM) can
be initiated by mechanical, electrical, and chemi-
cal stimuli. Passive stretching of VSM can cause
contraction that originates from the smooth muscle
itself. A number of stimuli such as norepineph-
rine, angiotensin II, vasopressin, endothelin-1, and
thromboxane (TXA2) can elicit contraction.33 Each
of these substances binds to specific receptors on
the VSMC or onto endothelial receptors adjacent
to VSM and causes contraction of smooth muscle.
Nitric oxide, epinephrine, and
prostacyclin can induce vasodilation of vascular
smooth muscle.28,47 NO is synthesized by a con-
stitutive form of nitric oxide synthase (NOS) lo-
cated in the endothelial lining of blood vessels and
is a major contributor to regulation of blood pres-
sure and blood flow. However, during hyperten-
sion and in atherogenesis, SMC change pheno-
type from an elastic mode to a secretory mode.48-
50 These activated VSMC secrete and release a
range of growth promoters and chemo-attrac-
tants.51-53 This phenotypic change is crucial to the
mechanical strength of the atheromatous plaque.54
Proliferating SMC can secrete matrix proteins and
thicken the vascular wall. If these proteins are rich
in collagen and elastic fibrils, the structural
strength of the atheroma is assured, as a rich ma-
trix of collagen forms a solid cap over the vascu-
lar lesion.54-56
Lesions, however, that develop and in-
crease in size exhibit increased cholesterol/lipid
deposits and show signs of increased cell death
(particularly SMC death).54,55 SMC can undergo
apoptosis, weakening the vascular wall and caus-
ing aneurisms (Table 3).57,58 The result is a lesion
Alternative Medicine Review Volume 9, Number 1 2004 Page 35
Review Cardiovascular/ Inflammation
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
Table 1. The Balance between Contracting and Dilating Factors
Decreases platelet adhesion and aggregation, as well as
promoting relaxation of vascular smooth muscle. It inhibits
endothelin-1 release.
22-24
A potent vasodilator peptide that protects the vascular
system from oxidative stress.
44
It hyperpolarizes VSM by stimulating the cellular membrane
potassium/calcium pump, thereby preventing smooth
muscle contraction. It is activated by shear pressure
associated with blood flow.
22-24
Also known as endothelium-derived factor, is a vascular
dilator. It also inhibits growth and proliferation of vascular
smooth muscle.
25
A soluble gas that diffuses through water and lipid phases,
it is a potent vasodilator. It is derived from the amino acid
arginine through the action of the enzyme, nitric oxide
synthase (NOS).
29
Its production is influenced by a number
of factors: shear pressure (i.e., hemodynamic shear stress
exerted by viscous drag of flowing blood) and various
bioactive molecules such as estrogen, acetylcholine,
bradykinin, substance P, histamine, insulin, bacterial
endotoxins, adenosine, and thromboxane.
23
There are a number of isoforms of endothelins (ET-1, ET-2,
and ET-3) with a wide range of biological actions. ET-1 is a
potent vasoconstrictor and pressor agent. It is released by
the endothelium. ET-1 release is stimulated by angiotensin
II, antidiuretic hormone, thrombin, cytokines, and reactive
oxygen species. Its release is inhibited by NO, prostacyclin,
and atrial natriuretic peptide.
23,24,30
Activates its own receptor on the VSMC and causes
vasoconstriction.
23
Activates thromboxane receptors.
23
Is a potent vasoconstrictor and pressor agent. It is
produced by the action of angiotensin-converting enzyme
on angiotensin1.
24,31
Quenches NO, thus contributing to vasoconstrictor tone. It
can produce vasoconstriction in its own right. It is produced
during infection, inflammation, or high oxidant stress.
23
Prostacyclin (PGI2)
Adrenomedullin (AM)
Endothelial-derived Hyperpolarizing
Factor (EDHF)
C-type Natriuretic Peptide (CNP)
Nitric Oxide (NO)
Endothelins (ET)
Thromboxane (TXA2)
Prostaglandin H2
Angiotensin II
Superoxide Anion (O2.-)
Endothelial-derived Relaxing Factors
Endothelial-derived Contracting Factors
Page 36 Alternative Medicine Review Volume 9, Number 1 2004
Cardiovascular/ Inflammation Review
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
with a large lipid pool that may weaken and rup-
ture, allowing lipid or atheroma fragments to en-
ter the circulation.59 After rupture, exposure of the
underlying lesion (collagen fragments) to the
blood vessel initiates thrombotic episodes of plate-
let and thrombin aggregation that may lead to or-
gan failure or tissue damage through embolus.
Small ruptures of atheroma plaque frequently re-
seal, incorporating thrombi into the lesion.54,60,61
As this sequence of events
persists, the plaque increases in bulk,
incorporating platelets, which further
stimulates cell proliferation through
the release of platelet-derived growth
factor (PDGF). If the rupture is mas-
sive, this may lead to prothrombotic
stimuli sufficient to occlude the lu-
men of the blood vessel.56,60
The VSM accumulation seen
around an atheroma can be viewed
as a beneficial repair process. Fail-
ure to repair through inhibition of cell
proliferation or stimulation of
apoptosis may reduce VSM accumu-
lation, which can be detrimental as it
increases the risk of plaque rupture.
The Contribution of
Immune Cells –
Monocytes/Macrophages
and T Lymphocytes
In atherosclerosis, macro-
phages are important for intracellu-
lar lipid accumulation and foam cell
formation. Monocytes respond to
chemotactic factors (monocyte
chemo-attractant protein MCP-1),
cytokines, and macrophage growth
factors produced by vascular endo-
thelial cells, smooth muscle cells, and
infiltrated cells, by migrating from
peripheral blood into the arterial in-
tima and differentiating into mac-
rophages. Unquenched intracellular
reactive oxygen species (ROS) in-
duce monocytes to differentiate into
macrophages.62 Macrophages express a variety of
receptors, particularly scavenger receptors, and
take up modified lipoproteins, including oxidized
low-density lipoprotein, beta-very-low-density li-
poprotein, and/or enzymatically degraded low-
density lipoprotein. These cells accumulate cho-
lesterol esters in the cytoplasm, which leads to
foam cell formation in lesion development. In
addition, macrophages and macrophage-derived
Figure 2. The Biochemical Pathway of NO
Activation of cGMP
1
2
3
4
L-Arginine
Nitric oxide (NO)
Guanylate cyclase
cyclic Guanosine
monophosphate
(cGMP)
5' GMP
Enzyme catalyzed reaction
Activates enzyme precursor
Enzyme catalyzed
reaction to produce
cGMP
Enzyme catalyzed
reaction to remove
cGMP
Guanosine
triphosphate
(GTP)
Phosphodiesterase
enzyme (PDE)
Causes muscle
relaxation
Does not cause
muscle relaxation
Alternative Medicine Review Volume 9, Number 1 2004 Page 37
Review Cardiovascular/ Inflammation
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foam cells produce ceroid and advanced glycated
end-products (AGEs) and accumulate these sub-
stances in their cytoplasm. Extracellularly gener-
ated AGEs are taken up by macrophages via re-
ceptors for AGEs. Most foam cells die in loco
because of apoptosis and some foam cells escape
from the lesions into peripheral blood. Macro-
phages also play multifaceted roles in inducing
plaque rupture, blood coagulation, and fibrinoly-
sis via the production of various enzymes, activa-
tors, inhibitors, and bioactive mediators. During
the development of atherosclerosis, macrophages
interact with vascular endothelial cells, medial
smooth muscle cells, and infiltrated inflammatory
cells, particularly T cells and dendritic cells.63,64
Activation of endothelial cells causes
blood monocytes and T lymphocytes to stick to
the luminal surface of the endothelium. Monocytes
squeeze through the junction between the endo-
thelial cells and enter the sub-endothelium, which
is between the endothelium and the internal elas-
tic lamina. Normally, the single endothelial layer
lies almost directly over the internal elastic lamina.
However, in the initial development of an athero-
matous lesion, monocytes/macrophages fill this
potential space.54,64 Oxidized lipids/cholesterol that
Table 2. Activity of NO
NO Deficiency
Impairs endothelial vasodilatation.
23,38,45
Increases vascular resistance.
27,38
Contributes to vascular medial thickening
and/or myointimal hyperplasia.
Accelerates vascular lesions by
increasing platelet aggregation and
immune cell migration to the lesion.
Contributes to abnormal vasomotor tone
and ischemic conditions.
102
Contributes to the initiation and
progression of atherosclerosis.
143
Increases oxidant stress and vascular
injury. Excess superoxide anion binds
with NO to form peroxynitrite.
39,41,42
NO Actions
Induces vasodilatation.
29,81
Reduces blood pressure.
27,32,38
Suppresses proliferation of
vascular smooth muscle.
28,29,35
Reduces lesion formation after
vascular injury.
29
Inhibits interaction of circulating
immune cells with the vascular wall
by inhibiting adhesion molecule
activation and expression.
Prevents platelet aggregation or
thrombus formation.
27,29
Prevents the progression of
atherosclerosis.
Induces or activates guanylate
cyclase, thus increasing cellular
cGMP (Figure 2) in SMC and
inducing muscle relaxation.
23
Disrupts free radical and oxidant-
mediated reactions. Binds with
super oxide anion.
23,39
Page 38 Alternative Medicine Review Volume 9, Number 1 2004
Cardiovascular/ Inflammation Review
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may be present in the lesion are scavenged by
macrophages, as they form toxic foam cells. In
human atherosclerotic lesions, many of the mac-
rophage foam cells also contain ceroid – an in-
soluble polymer formed by oxidation of mixtures
of lipid and protein.63 Figure 3 summarizes the
interactions of monocytes/macrophages with
modified/oxidized LDL.
Further recruitment of monocytes and
macrophages can occur by the release of cytokines
from the endothelium and VSM as part of the in-
flammatory cycle.54,63,64 These cells attempt to re-
move apoptotic cell debris, although the presence
of modified or oxidized LDL may hamper this de-
bris removal,65,66 resulting in the recruitment of
more inflammatory cells and the subsequent re-
lease of Fas-L (a death-inducing ligand) and death
of surrounding or adjacent neutrophils, monocytes,
and activated VSMC.58,67 As a result, the athero-
matous plaque core becomes rich in macrophages
as the plaque ages.
Once activated, macrophages can over-
express the production of matrix-degrading en-
zymes (matrix metalloproteins; MMPs) and pro-
thrombin.68 This process also acti-
vates SMC and increases the produc-
tion of excessive ROS that induce
oxidative modification of LDLs.69,70
A vicious cycle ensues of endothe-
lial cell activation or dysfunction that
induces the expression of VCAM-1
and monocyte chemo-attractant pro-
teins (MCP-1), leading to increased
monocyte/macrophage recruitment
into the intima.64
Oxidative stress also de-
creases the expression of endothelial
nitric oxide synthase (eNOS) by en-
dothelial cells.71 As eNOS limits
monocyte/macrophage-endothelial
cell interaction, the loss of eNOS or
its decreased expression results in
formation of a macrophage-rich
atheroma. This results in a soft
plaque that increases the risk of un-
stable angina, thrombosis, and acute
myocardial infarction.72,73
Macrophages and T lym-
phocytes can also produce NO through an induc-
ible nitric oxide synthase mechanism (iNOS).41,74
The excess NO can react with the superoxide an-
ion to produce peroxynitrite, a very aggressive free
radical species that can induce cellular apoptosis,
cellular mitochondrial dysfunction, and lipid
peroxidation.41,42
Three basic isoforms of NOS enzymes
have been identified that generate NO from the
amino acid arginine:41,74-76
Endothelial nitric oxide synthase (eNOS)
– a constitutive NOS which is Ca++/
calmodulin-dependent77,78
Neuronal isoforms (nNOS), which are the
normal constituents of healthy cells and
neurons
Inducible isoforms (iNOS), which are not
normally expressed by vascular tissue but
by immune cells
Table 3. Properties of SMC in Advanced Plaques54-58,60
1. Poor proliferation
2. Early senescence
3. Increased apoptosis (programmed cell death)
4. Increased cellular DNA damage in VSMC
5. Increased sensitivity to oxidized lipids/cholesterol
and peroxynitrite, resulting in induced plaque;
VSMC death while leaving normal VSMC in the
artery unaffected
6. Inflammatory cells adjacent to the plaque
can kill plaque VSMC.
7. Apoptotic VSMC release pro-inflammatory
cytokines and membrane bound micro-particles
into the circulation, which can initiate a
pro-coagulant cascade as well as recruiting
monocytes and macrophages to the surrounding area.
Alternative Medicine Review Volume 9, Number 1 2004 Page 39
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Inducible NOS is calcium-independent
and is stimulated by cytokines such as interferon-
gamma and interleukin-1β.41,77,78 iNOS-derived
NO plays an important role in numerous physi-
ological and pathophysiological conditions (e.g.,
blood pressure regulation, inflammation, and in-
fection).79,80
eNOS and nNOS generate NO, but NO
generation from these two isoforms can have op-
posing roles in the process of ischemic injury.
While increased NO production from nNOS in
neurons can cause neuronal injury, endothelial NO
production from eNOS can decrease ischemic in-
jury by inducing vasodilation.76
Nitric Oxide: Its Clinical Relevance
Many studies suggest NO is a potent,
endogenous anti-atherogenic molecule that
suppresses key processes in atherosclerosis (Table
2).29,39,81 As mentioned previously, nitric oxide is
produced through the action of the enzyme nitric
oxide synthase on the amino acid arginine to
produce nitric oxide and citrulline.23,82,83 The
cofactors required for this reaction include vitamin
B3 (a cofactor for nicotinamide adenine
dinucleotide phosphate),41,84 vitamin B2 (a cofactor
for flavin adenine dinucleotide),41,84
tetrahydrobiopterin (BH4),77,84,85 and calmodulin
(a calcium-ion modulator).41,84
Tetrahydrobiopterin stabilizes NO syn-
thase and facilitates the binding to L-arginine (Fig-
ure 4). Under conditions when intracellular con-
centration of tetrahydrobiopterin is reduced, NO
synthase generates superoxide anions instead of
NO.84 Under physiological conditions there is a
balance between endothelial production of NO and
oxygen-derived free radicals.
Once synthesized, NO diffuses across the
endothelial cell membrane and enters the vascu-
lar smooth muscle cells where it activates the pro-
duction of the second cellular system cGMP (Fig-
ure 2).23 Once activated, this messenger system
plays numerous roles such as controlling vascular
tone and platelet and mitochondrial function.27,86
Figure 3. Monocyte/Macrophage and Foam Cell Formation
Vessel wall
LDL
HDL inhibits
adhesion molecule
expression
HDL inhibits
oxidation of LDL
Monocyte
NO PG
1
2
LDL Vessel Lumen
Adhesion
molecules
Cytokines
Macrophage
MCP-1
HDL promote cholesterol efflux
Foam cell
Modified LDL
Growth factors
metalloproteinases
Cell proliferation
matrix degradation
NO PG
1
2
Page 40 Alternative Medicine Review Volume 9, Number 1 2004
Cardiovascular/ Inflammation Review
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Decreased production of NO, or decreased
sensitivity to the action of NO, has consistently
been shown to impair endothelial-dependent
vasodilation, contributing to the pathogenesis of
atherogenesis.22,23,31,34,71 Many risk factors interfere
with or are associated with endothelium-dependent
vasodilation, including hyperlipidemia,
hypertension, types 1 and 2 diabetes, cigarette
smoking, hyperhomocysteinemia, infection,
inflammation, low birth weight, insulin resistance,
Figure 4. The Role of Tetrahydrobiopterin in Production of NO41,84,85
De Novo Pathway
GTP-Cyclohydrolase
7,8-Dihydroneopterintriphosphate
6-Pyruvoyl tetrahydrobiopterin
6-Pyruvoyl Tetrahydrophorin
Sepiapterin reductase
Tetrahydrobiopterin
(BH4)
Salvage Pathway
Sepiapterin
Sepiapterin reductase
7,8-Dihydroneopterin
Dihydrofolate reductase
(high dose folate
can replace BH4)
Vitamins
B2 and B3
GTP
eNOS
L-arginine NO
NO
cGMP
Relaxation
ONOO BH4
O2
O2
H2O2
HO2+O2
PAF
Stimulation of NO production by
acetylcholine, bradykinin, estrogen,
serotonin, substance P
Ischemic-reperfusion
injury exacerbates
radical formation and
decreases BH4
O2
auto-oxidation
Tetrahydrobiopterin (BH4) acts as a cofactor for the action of NOS activity. BH4 is susceptible to auto-oxidation
with the resultant production of superoxide radicals. The superoxide anion produced can react with NO to
produce peroxynitrite (ONOO-) and thus reduce NO activity. Superoxide anion is reduced to hydrogen peroxide
(H2O2) by the enzyme superoxide dismutase, which is a copper- or selenium-dependent enzyme.
Adapted from: M Viljoen et al. Geneeskunde, The Medicine Journal 2001;43(8)
Endothelial Cell
Smooth Muscle Cell
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hypercholesterolemia, chronic kidney disease,
microalbuminuria, AGEs, age-related vascular
changes, and a family history of heart disease.5,6,86-91
Excessive production of NO can also con-
tribute to vascular cell pathology, as excessive NO
can disrupt mitochondrial function and ATP pro-
duction,92 indirectly initiate apoptosis,93 and lead
to formation of the peroxynitrite radical and other
cytotoxic substances.42,94 These negative effects
may be due to timing of release, duration of ac-
tion, and concentration of NO at a particular cel-
lular point as well as the oxidative state within its
area of activity. 95
Mechanisms Involved in Decreased
Nitric Oxide Levels
Deficiency in Cofactor Vitamins B3
and B2 and Tetrahydrobiopterin
(BH4)A decreased intake of the cofactor or an
increased requirement of BH4, due to, for ex-
ample, diabetes, smoking, or hypercholester-
olemia, may cause cofactor deficiencies.89 In ad-
dition, oxidant stress increases BH4 destruction.71
In either case, deficiency of these cofactors,
whether a relative demand deficiency or local tis-
sue deficiency, can result in decreased NO pro-
duction and impaired endothelial vasodilation. In
clinical situations, abnormalities in BH4 metabo-
lism have been implicated in the endothelial dys-
function observed in hypertension, reperfusion
injury, homocysteinemia, hypercholesterolemia,
and smoking.71,96,97 Vitamin C and folic acid are
important in stabilizing and maintaining intracel-
lular levels of BH4.98-100
Decreased or Increased Nitric Oxide
Synthase Enzyme Expression and
Activity
Hyperglycemia causes increased eNOS
expression with a concomitant increase in super-
oxide anion production, resulting in NO inactiva-
tion.39 Chronic inflammation or bacterial endo-
toxins can increase the synthesis of iNOS and in-
duce hypotension by excessive production of
NO.41,79,80 In advanced atherosclerosis, reduced
expression of eNOS enzyme has been observed,
possibly due to the action of oxidized LDLs.35,101
Increased Endogenous Nitric Oxide
Synthase Inhibitors
Two of the most potent endogenous in-
hibitors of NOS are asymmetric dimethylarginine
(ADMA) and symmetrical dimethylarginine
(SDMA).102,103 These two endogenous inhibitors
are synthesized from methylated arginine-rich pro-
teins.102,104 ADMA is further metabolized to cit-
rulline and methylamines by the action of the en-
zyme dimethylarginine dimethylaminohydrolase
(DDAH), of which two isoforms (DDAH I and
II) have been identified.103-105 Therefore, inhibition
or modulation of DDAH will have a profound ef-
fect on plasma ADMA levels. Oxidized LDL cho-
lesterol, hyperglycemia, and oxidant stress can
cause a decline in DDAH activity.35,36,106-108
Elevated levels of endogenous ADMA are
predictive of vascular lesion formation.103,105,109
Plasma elevation of ADMA has been observed in
the following disease states: chronic renal fail-
ure,105,110 hypercholesterolemia,105 congestive heart
failure,105,110 hypertension,111 atherosclerosis,105
homocysteinemia,112,113 Raynauds disease,114 and
in situations resulting in oxidative stress102 – to-
bacco smoking, aging, diabetes, and insulin resis-
tance.35,106-108,115,116 These are the common risk fac-
tors associated with atherosclerosis and coronary
artery diseases.
The administration of L-arginine and vi-
tamin E has been shown to improve endothelium-
dependent vascular function in subjects with high
ADMA levels.110,117,118
Decreased Nitric Oxide
Bioavailability
Nitric oxide can react with superoxide
anions to produce peroxynitrite anions, thus
quenching the biological effects of NO.39 In con-
ditions associated with oxidative stress, such as
hypercholesterolemia and glucocorticoid excess,
NO production may be high but inactivated, re-
sulting in impairment of endothelial-dependent
Page 42 Alternative Medicine Review Volume 9, Number 1 2004
Cardiovascular/ Inflammation Review
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
vasodilation.38,39,119 Quenching free radicals with
lipoic acid,120-124 coenzyme Q10,125 quercetin,128.129
vitamins C and E,130-132 superoxide dismutase,131
and glutathione133-138 results in the reduction of NO
degradation and maintenance of endothelial func-
tion.88,139
Decreased Vascular Smooth Muscle
Sensitivity to Nitric Oxide
Diabetes and hyperglycemia-induced
hypo-responsiveness in vascular smooth muscle
may be overcome by increasing the activity of
guanylate cyclase, the enzyme that increases the
synthesis of cGMP, the second cellular messen-
ger system stimulated by NO.140
Furthermore, this impaired vasodilation
in response to NO derived from vascular endo-
thelium or organic nitrates in vascular smooth
muscle may be related to increased degradation
of the second messenger cyclic guanosine mono-
phosphate by type 5 phosphodiesterase.40
Several common cardiovascular risk fac-
tors or disease states impair nitric oxide synthesis
as well as its activity.22,71,141-143 Therefore, it is not
surprising that NO is a major player in cardiovas-
cular physiology.
Increasing Levels of Nitric Oxide
Fortunately, some of the risk factors noted
above can be managed by increasing the synthe-
sis and activity of NO by:
Supplementing with arginine (as it has
been shown to compete with ADMA) to
prevent the inhibition of eNOS by this
endogenous inhibitor. It normalizes
endothelial vasodilation in
hypercholesterolemic/hypertensive, and
hyperhomocysteinemic patients.97,116
Supplementing with antioxidants to
reduce the oxidative stress strongly
implicated in endothelial dysfunction.
Vitamins C and E, lipoic acid, glutathione,
and superoxide dismutase can increase the
bioavailability of NO, reduce oxidative
stress, and increase DDAH activity.35,144,145
Ensuring nutrient cofactors – vitamins B2
and B3 and tetrahydrobiopterin – are
available to activate NOS. High-dose folic
acid can be a substitute for
tetrahydrobiopterin.81,96,99,116
Auxiliary Nutrients to Reduce
Cardiovascular Risk
The most important factor determining
plaque stability is the plasma level of atherogenic
LDL particles.146 Increased levels of these particles
cause endothelial dysfunction with impaired va-
sodilation capacity and heightened vasoconstric-
tion, as well as inducing and maintaining inflam-
matory infiltration of the plaque, impairing the
strength of the fibrous cap, and facilitating aggre-
gation and coagulation.146
Lipid-lowering treatments (e.g.,
tocotrienols,147-149 and supplemental DHA/EPA and
omega-3 rich diets150-153) can decrease the risk of
plaque rupture and subsequent thrombogenicity,
as well as normalize the impaired endothelial func-
tion in hypercholesterolemic patients.154
Furthermore, lipid lowering diminishes
inflammation and macrophage accumulation, as
well as increases interstitial collagen accumula-
tion in atheroma, resulting in an increase in a
plaque’s mechanical stability.112,155 Thus, a de-
crease in lipid levels, along with modification of
other risk factors, has the potential to become a
cornerstone for treatment of acute coronary syn-
dromes, in addition to being an effective treatment
in primary and secondary prevention of coronary
heart disease.146
The presence of oxidized LDL in athero-
sclerotic lesions supports the contention that oxi-
dant stress is a contributing factor to atheroscle-
rosis.156-158 As a corollary, antioxidants that can
inhibit LDL oxidation may be regarded as anti-
atherogenic. This concept is supported by animal
studies showing that antioxidants such as probucol,
butylated hydroxytoluene, tocotrienols, and alpha-
tocopherol can slow the progression of atheroscle-
rosis.147-149,158 Epidemiological and clinical data
indicate a protective role of dietary antioxidants
against cardiovascular disease, including vitamin
E, beta-carotene, and vitamin C.159-164 Likewise,
Alternative Medicine Review Volume 9, Number 1 2004 Page 43
Review Cardiovascular/ Inflammation
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
basic research studies on
LDL oxidation have demon-
strated a protective role for
antioxidants, present either
in the aqueous environment
of LDL or associated with
the lipoprotein itself.158
Quercetin has been
shown to be inversely asso-
ciated with mortality from
coronary heart dis-
ease159,165,166 by inhibiting the
expression of
metalloproteinase 1 (MMP-
1), thus inhibiting the disrup-
tion of atherosclerotic
plaques and contributing to
plaque stabilization.
Lipoic acid plays a
crucial role in preventing
atherosclerosis. It induces
the production of NO and
inhibits the activation of
monocyte chemo-attractant
protein-1.120,144,167-169 It also
improves NO-mediated va-
sodilation in diabetic pa-
tients.170,171
Hyperhomocysteinemia
is an inflammatory risk fac-
tor for cardiovascular dis-
ease for which nutritional supplementation is in-
dicated.100,172 High levels of homocysteine induce
sustained injury of arterial endothelial cells and
proliferation of arterial smooth muscle cells, and
enhance expression/activity of key participants in
vascular inflammation, atherogenesis, and vulner-
ability of the established atherosclerotic plaque.173
Other effects of homocysteine include impaired
generation and decreased bioavailability of NO,
interference with transcription factors and signal
transduction, oxidation of LDLs, and decreased
endothelium-dependent vasodilation.173
Reduction of homocysteine by vitamins
B6 and B12 and folate is crucial in reducing car-
diovascular risk and oxidant stress associated with
elevated plasma levels.172,173 Folate reduces plasma
homocysteine levels and enhances eNO synthesis
and shows anti-inflammatory activity.100 It stimu-
lates endogenous BH4 (a cofactor necessary for
eNO synthesis). BH4, in turn, enhances NO gen-
eration and augments arginine transport into the
cells. Folic acid increases the concentration of
omega-3 PUFAs, which also enhance eNO syn-
thesis.100 Vitamin C augments eNO synthesis by
increasing intracellular BH4 and stabilization of
BH4.98,99 The ability of folate to augment eNO
generation is independent of its capacity to lower
plasma homocysteine levels.100
Table 4. The effects of ROS on Endothelium and VSMC
Reactive Oxygen Species63
Impair vascular function by injuring endothelial and VSMC membranes
React with NO, activating it23
Oxidize tetrahydrobiopterin, the cofactor for NOS
Peroxidize low density lipoproteins (LDL) to oxidized LDLs, which in
turn upregulates adhesion molecules on endothelial cells and PDGF
receptors on SMC, resulting in SMC proliferation and extracellular
matrix synthesis
Stimulate the synthesis of asymmetric dimethylarginine (ADMA),
inhibiting NOS activity or expression71
Inhibit guanylate cyclase, leading to a decrease in cGMP, which
decreases the action of NO on SMC
In the vasculature promote the expression of receptors and chemotactic
agents to facilitate the migration of inflammatory cells to the
development of an atheroma172
ROS are generated within the vessel wall by several mechanisms, including a vascular type of a
NAD (P) H oxidase.126 Mechanical stress, environmental factors, cytokines, low-density
lipoproteins (LDL), and exposure to catalytic metal ions can stimulate ROS formation. Their
ability to modify LDL, react with endothelial-derived nitric oxide subsequently forming
peroxynitrite, and to amplify the expression of various genes important for leucocyte recruitment
within the arterial wall are the basis of the oxidant injury theory of atherosclerosis. In animal
studies, antioxidant therapy (probucol, butylated hydroxytoluene, N’, N’-diphenylenediamide,
vitamin E, superoxide dismutase) have been successfully used to prevent fatty streak formation,
and to restore impaired nitric oxide-dependent vaso relaxation.127
Page 44 Alternative Medicine Review Volume 9, Number 1 2004
Cardiovascular/ Inflammation Review
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
Discussion
From a physiological point of view, the
major contributors to atherosclerotic plaque for-
mation include macrophage accumulation, smooth
muscle cell activation, endothelial cell activation,
oxidative stress giving rise to altered blood rheol-
ogy and vascular tone, and plaque build-up. This
process leads to basically two forms of plaque –
stable and unstable. Unstable plaques are charac-
terized by a thin fibrous cap overlying a macro-
phage/lipid-rich core, while stable fibrous plaques
have a solid cap of collagen, elastin fibrils, and
smooth muscle cells over the lipid lesion. As dis-
cussed earlier, regional macrophages and activated
smooth muscle cells over-express matrix-degrad-
ing enzymes (such as collagenases), and
prothrombotic
molecules68
contribute to
the progres-
sion of the ath-
erosclerotic le-
sion. These
atherosclerotic
lesions also
produce ex-
cess ROS that
induce oxida-
tive modifica-
tion of LDLs
and further en-
dothelial dys-
function
(Table
4).26,28,69,70
These pro-
cesses can
contribute to
plaque insta-
bility and
thrombogenicity,
resulting in the
onset of acute
coronary
events. Rec-
ognizing that
atherosclerosis is a multi-factorial inflammatory
process lends to the assumption that anti-inflam-
matory drugs and nutrients might mitigate the dis-
ease. It is interesting to note that many drugs used
in the treatment of cardiovascular risk factors have
anti-inflammatory properties by acting as antioxi-
dants. The following are examples: angiotensin
converting enzyme (ACE) inhibitors,200 inhibitors
of VCAM-1 (e.g., fibrates such as gemfibrozil),201
inhibitors of inflammatory cytokine release (e.g.,
aspirin),202 and lipid-lowering drugs (HMGCoA-
reductase inhibitors).203 All of these prevent lipo-
protein oxidation and NO quenching. Similarly,
nutrients with anti-inflammatory and antioxidant
activity can contribute to the treatment of athero-
sclerosis.
Figure 5. Common Risk Factors, Oxidative Stress, and
Endothelial Dysfunction
Hypertension
Diabetes Smoking LDL Homocysteine
Estrogen
deficienc
y
Endothelial cells and
vascular smooth muscle
Endothelial Dysfunction
Apoptosis
Leukocyte
adhesion
Lipid
deposition
Vasoconstriction
VSMC
growth
Thrombosis
O2
H2O2
A
b
n
o
r
m
a
l
N
O
m
e
t
a
b
o
l
i
s
m
Adapted From: Secades JJ, Frontera G. Meth Find Exp Clin Pharmacol 1995;17(Suppl B):1-54.
Alternative Medicine Review Volume 9, Number 1 2004 Page 45
Review Cardiovascular/ Inflammation
Copyright©2004 Thorne Research, Inc. All Rights Reserved. No Reprint Without Written Permission
Table 5. Inflammation and Atherosclerosis – A Summary of Pathophysiology and
Potential Nutrient Interventions
Inflammation and its Actions
Inflammation may determine plaque
stability:154,174
- Unstable plaques have increased
leucocytic infiltrates
- T cells and macrophages
predominate rupture sites
- Cytokines and metalloproteins
influence both stability and degradation of
the fibrous cap
Inflammation increases the release of
oxidant free radicals, which can lead
to:71,158
- Apoptosis
- Leucocyte adhesion
- Lipid oxidation and deposition
- Vascular constriction
- VSMC growth and matrix deposition
- Thrombosis and platelet aggregation
- Impaired NO metabolism
- Cell phenotype change
- Vascular leakage
Inflammation may be heightened by:
- Improper balance between omega-3 and -6
fatty acids. Excess omega-6 fatty acids
increases inflammatory response.
- Exposure to trans fatty acids
- Hyperglycemia, diabetes, smoking,
chronic infection
- Ischemic conditions
- Advanced glycated end products
- Hyperhomocysteine
- Hormonal imbalance
- LDL oxidation
Processes that Modify Inflammatory Activity
Lipid lowering may reduce plaque
inflammation by:112,154
- Decreasing macrophage numbers
- Decreasing the expression of
collagenolytic enzymes (MMPs)
- Increasing interstitial collagen
- Decreasing the expression of E-selectin
- Reducing calcium deposits
Lipid lowering can be achieved by:175-186
- Dietary modification
- Supplementation with fish oil or omega-3
fatty acids (DHA/EPA)150-153,182
- Increasing the intake of fiber181,183
- Supplementing with niacin and
vitamin C185
- Statins
- Tocotrienols147-149
Maintaining NO and oxidant
balance by:82,83,186-191,208
- Supplementing with arginine,97,110,116,117
tetrahydrobiopterin,187-190
vitamins B2, B3, and C and folic acid, which
maintain NO synthesis89,99,100,110,117
- Supplementing with antioxidant nutrients:
vitamins C and E,130-132,163,193-195
tocotrienols,145,149 quercetin,128,129,159,165,166
CoQ10,167 lipoic acid, 120-123,170,171
superoxide dismutase,131 and
glutathione.133-138 These antioxidants
inhibit LDL oxidation, potentiate NO and
prostacyclin synthesis, attenuate cell
mediated LDL oxidation, inhibit agonist
induced monocyte adhesion, decrease
endothelial expression of adhesion
molecules, reduce the proliferation of
smooth muscle cells, and inhibit platelet
aggregation.196-199
Page 46 Alternative Medicine Review Volume 9, Number 1 2004
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It can now be hypothesized that
atherosclerosis may be an inflammatory disease
that contributes to derangement of the vascular NO
metabolic pathway and to increased oxidant stress.
Most risk factors directly or indirectly influence
this derangement and thus contribute to the
expression of adverse cardiovascular symptoms
(Figure 5). Fortunately, many nutrient factors can
modify these risks and improve quality outcomes
(Table 5).
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... The difference in the concentrations of L-glutamic acid could be as a result of climatic, geographical, chemical, and variety factors [25]. The levels of L-glutamic acid in the vegetables are below the recommended nutrition intake (500-3000 mg/kg body weight) [26]. ...
... Levels of L-citrulline, L-arginine, and Lglutamic acid in the nuts (table IV) were below the recommended nutrition intake (RNI). The RNI's are; Lcitrulline (3-6 g/kg body weight), [23] L-arginine (5-10 g/kg body weight) [24] and L-glutamic acid (0.5-3 g/kg body weight) [26]. The levels of the three amino acids are slightly lower than the levels reported in literature. ...
Article
Full-text available
Background: L-citrulline, L-arginine, and L-glutamic acid are amino acids which are vital in the human body. L-citrulline boosts immunity, combats sarcopenia, detoxifies the liver, and enhances male fertility. L-arginine boosts internal production of nitric oxide, prevents abnormal blood clotting and accelerates healing of wounds. L-glutamic acid cleanses the central nervous system and improves overall brain health. Deficiency of these amino acids can lead to accumulation of ammonia and impact negatively to the nervous systems of the human body. This study aimed to identify and determine the levels of L-citrulline, L-arginine, and L-glutamic acid in selected fruits, vegetables, nuts, and seeds sold in markets in Nairobi City County, Kenya using Liquid Chromatography- Mass Spectrometry (LC-MS). Materials and Methods: Data was collected from 28 selected samples and data analysis was done using Statistical Analysis Software (SAS) version 9.4. Results: LC-MS showed the presence of both L-arginine and L-citrulline in most fruits, vegetables, and nuts while L-glutamic acid was present in the seeds. The levels of the amino acids in the fruits and vegetables were in the following range: L-citrulline (0.65- 19.41 mg/100g) in the button mushroom, cucumber, pumpkin, amaranthus, and kales, (3.16-3.79 mg/100g) in the watermelons and (1.57-10.21 mg/100g) in the nuts. L-arginine was in the range; 1.73 - 16.48 mg/100g in the amaranthus, kales, button mushroom, butternut squash, and cucumber, 5.44-6.56 mg/100g in the watermelons and 0.93-10.73 mg/100g in the nuts and L-glutamic acid (0.013-0.28 mg/100g) in the seeds of pumpkin, butternut, and watermelons. Conclusion: The results showed that locally available vegetables, fruits and nuts are rich in L-citrulline and L-arginine.
... Interleukins are involved in the chronic inflammatory response that is typical of atherosclerosis (Khan et al., 2014;Osiecki, 2004). The initiation and further development of many types of malignancy are also due to chronic inflammation. ...
... Research on identical twins revealed that ARHL development could be attributed to not only genetic factors but also environmental factors, including noise exposure, lack of exercise, uncontrolled calorie intake, oxidative stress due to reactive oxygen species (ROS), such as nitric oxide (NO), and systemic immune dysfunctions (Christensen et al., 2001;Karlsson et al., 1997). These immune disorders are accompanied by autoimmune diseases and age-related chronic inflammation, termed inflammaging (Tu and Friedman, 2018), which has been closely linked to not only type 2 diabetes (Grant and Dixit, 2013), cardiovascular disease (Osiecki, 2004), and Alzheimer's disease (Mészáros et al., 2020), but also ARHL (Watson et al., 2017). Therefore, a better understanding of immunosenescence that coexists with inflammaging (Heinrich and Helling, 2012;Xia et al., 2016), as well as oxygen stress as an inducer of inflammaging (Casciaro et al., 2017), is essential to overcome ARHL. ...
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We investigated the association between cellular immunity and age-related hearing loss (ARHL) development using three CD4⁺ T cell fractions, namely, naturally occurring regulatory T cells (Treg), interleukin 1 receptor type 2-expressing T cells (I1R2), and non-Treg non-I1R2 (nTnI) cells, which comprised Treg and I1R2-deleted CD4⁺ T cells. Inoculation of the nTnI fraction into a ARHL murine model, not only prevented the development of ARHL and the degeneration of spiral ganglion neurons, but also suppressed serum nitric oxide, a source of oxidative stress. Further investigations on CD4⁺ T cell fractions could provide novel insights into the prevention of aging, including presbycusis.
... Our hypothesis had biological plausibility, although it was not robustly supported by the results. Specifically, vitamin E is a lipid-soluble antioxidant that preserves endothelial function 52 . Also, vitamin E increases intracellular free magnesium that protects against vascular damage caused by magnesium deficiency 53 . ...
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Plasma fibrinogen predicts cardiovascular and nonvascular mortality. However, there is limited population-based evidence on the association between fibrinogen levels and dietary intakes of micronutrients possibly associated with inflammation status. Data were taken from the ENRICA study, conducted with 10,808 individuals representative of the population of Spain aged ≥ 18 years. Nutrient intake (vitamin A, carotenoids, vitamin B6, vitamin C, vitamin D, vitamin E, magnesium, selenium, zinc and iron) was estimated with a validated diet history, and plasma fibrinogen was measured under appropriate quality checks. Statistical analyses were performed with linear regression and adjusted for main confounders. The geometric means of fibrinogen (g/L) across increasing quintiles of nutrient intake were 3.22, 3.22, 3.22, 3.16, and 3.19 (p-trend = 0.030) for vitamin E; 3.23, 3.22, 3.20, 3.19, and 3.19 (p-trend = 0.047) for magnesium; and 3.24, 3.22, 3.19, 3.21, and 3.19 (p-trend = 0.050) for iron. These inverse associations were more marked in participants with abdominal obesity and aged ≥ 60 years, but lost statistical significance after adjustment for other nutrients. Although dietary intakes of vitamin E, magnesium and iron were inversely associated with fibrinogen levels, clinical implications of these findings are uncertain since these results were of very small magnitude and mostly explained by intake levels of other nutrients.
... In parallel, the cardioprotective potential of dietary flavonoids such as chrysin, quercetin, kaempferol and apigenin has been evinced to be associated with the reduction of adhesion molecules expression in human aortic endothelial cells (Lotito and Frei 2006). Quercetin has been identified to interrupt atherosclerotic plaques and suppress metalloproteinase-1 expression for tumbling the risk of CHD (Osiecki 2004). Though several flavonoids have been recorded to possess cardioprotective effect, the correlation between consumption of flavonoidrich foods and risk of CVD is still inconsistent. ...
Chapter
Flavonoids constitute a large group of plant phenolic metabolites with diverse structural compounds exhibiting multiple biological activities. Flavonoids have been used over centuries in folk medicine for tackling various human ailments and promoting the human health. With centuries old historical background, flavonoids still hold the valour to be captivated by researchers and clinicians for reframing the current medications to recuperate the equilibrium in human health. In this context, a vast range of biological activities of flavonoids has been documented by various research groups. These findings unwind the multi-targeting potential of flavonoids in various clinical conditions, which hints the ability of flavonoids to gratify the need of current treatment strategy mandating the handling of other complications accompanying a diseased condition. Moreover, the ubiquitous dietary sources of flavonoids underscore their innocuous nature as well as the likelihood to be used in clinical settings. This also enlightens that daily consumption of flavonoids from various dietary sources could act as better nutraceuticals for nourishing the health and assist the risk management of many complications. With this, a comprehensive overview on therapeutic applications of multipotent flavonoids has been provided in this chapter.
... Activated macrophages produce numerous inflammatory mediators, such as cytokines (tumor necrosis factor-α: TNF-a), chemokines (CCL2), and nitric oxide (NO), which are highly toxic for microorganisms and can also be harmful to surrounding healthy tissues and lead to aberrant inflammation [3,4]. For example, excessive or chronic NO production reacts with superoxide anions to become peroxynitrite, which is a very aggressive-free radical species, leading to the induction of mitochondrial dysfunction and cytotoxicity of the surrounding tissues [5][6][7]. And CCL2, which belongs to the C-C class of chemokines, is a critical modulator of inflammation, regulating macrophage recruitment during wound healing, infections, and autoimmune diseases [8]. ...
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Squalene (SQ), a natural precursor of many steroids, can inhibit tumor progression and decrease serum cholesterol levels. However, it is difficult to discern the effect of highly active molecules in the treatment of diseases because not enough active compounds reach the site of pathology in crowded biosystems. Therefore, it is necessary to design artificial probes that work effectively within crowded systems. In this study, to facilitate cell penetration, the ethylene glycol moiety (used as a probe) was chemically added to SQ to form 2-(2-hydroxyethoxy)-3-hydroxysqualene (HEHSQ). HEHSQ was prepared from 2,3-epoxysqualene and characterized by Rf, FT-IR, ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry. We then evaluated the anti-inflammatory effects of SQ and HEHSQ on lipopolysaccharide- (LPS-) stimulated RAW264.7 murine macrophages. To determine the effect of SQ and HEHSQ on the viability of RAW264.7 cells, an MTT assay was performed. To quantify the anti-inflammatory effect of SQ and HEHSQ, we measured nitric oxide (NO) production, gene expression, and secretion of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and chemokine C-C motif chemokine 2 (CCL2) in LPS-stimulated RAW264.7 cells using an in vitro inflammatory model. 2,3-Epoxysqualene was prepared according to a reported methodology. The reaction of 2,3-epoxysqualene and ethylene glycol in 2-propanol produced 49% HEHSQ. MTT results showed that 10 and 100 µg/mL HEHSQ treatment decreased cell viability, whereas SQ treatment (1–100 µg/mL) did not have any effect on viability. SQ (100 µg/mL) and HEHSQ (1 µg/mL) treatment significantly reduced the production of LPS-stimulated NO and decreased the expression and secretion of proinflammatory TNF-α and CCL2. Therefore, our results suggested that the anti-inflammatory effects of HEHSQ are 100 times higher than that of unmodified SQ. To the best of our knowledge, this study has demonstrated for the first time that HEHSQ can be potentially used as a safe alternative treatment to anti-inflammatory drugs. 1. Introduction Inflammation is a protective response against trauma, infection, and tissue injury [1]. Acute inflammation usually subsides in a short while. However, if acute inflammation persists or the agent causing the inflammation is not eliminated, then acute inflammation can progress to a chronic stage. Chronic inflammation is a long-term phenomenon, causing tissue destruction and organ dysfunction. Furthermore, chronic inflammation is associated with various diseases including atherosclerosis, cardiovascular diseases, and arthritis [2]. Chronic inflammation is considered an underlying cause of several diseases, and thus, it is important to prevent such inflammation. Macrophages play a critical role during inflammation. Following tissue injury or infection first, the responder macrophages are activated and exhibit an inflammatory phenotype [2]. Activated macrophages produce numerous inflammatory mediators, such as cytokines (tumor necrosis factor-α: TNF-a), chemokines (CCL2), and nitric oxide (NO), which are highly toxic for microorganisms and can also be harmful to surrounding healthy tissues and lead to aberrant inflammation [3, 4]. For example, excessive or chronic NO production reacts with superoxide anions to become peroxynitrite, which is a very aggressive-free radical species, leading to the induction of mitochondrial dysfunction and cytotoxicity of the surrounding tissues [5–7]. And CCL2, which belongs to the C-C class of chemokines, is a critical modulator of inflammation, regulating macrophage recruitment during wound healing, infections, and autoimmune diseases [8]. Therefore, chemokines are involved in a variety of inflammatory conditions, both acute and chronic [9]. Thus, modulation of macrophage activation could be a good strategy to prevent chronic inflammation. Today, nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly prescribed drugs worldwide. These display anti-inflammatory and analgesic effects; however, long-term use of NSAIDs causes undesirable side effects including gastrointestinal irritation, high blood pressure, and liver problems [10]. Therefore, complementary medicine that has anti-inflammatory properties and is derived from natural products has gained attention. Squalene (SQ), a natural precursor of many steroids, is so named because of its presence in shark liver oil. It is also prepared from the microalgae Aurantiochytrium [11]. Because it is not toxic to humans, SQ has been extensively used as a dietary supplement [12, 13], an adjuvant in therapeutic applications [14], and for alleviating oxidative stress [15]. Moreover, our previous study revealed that the high SQ-producing microalgal strain (Aurantiochytrium) had an anti-inflammatory effect on lipopolysaccharide- (LPS-) stimulated mouse macrophage RAW264.7 cells in an in vitro inflammatory model [16]. Unfortunately, to date, attempts to improve the pharmacological effects of SQ have not met with much success. One of the factors limiting the pharmacological activity of SQ is its insolubility in polar solvents such as methanol, tetrahydrofuran, and dimethyl sulfoxide (DMSO); therefore, it cannot permeate cell membranes and reach tissues in a crowded environment. In general, however, cells and tissues of living organisms make up a multimolecular system wherein diverse types of molecules get compartmentalized. Therefore, it is not often possible to use highly active molecules in the treatment of diseases, such as cancer, because enough active compounds cannot reach the site of pathology in crowded biosystems. Therefore, to improve pharmacological activity, artificial probes must be designed that effectively work within the crowded tissue architecture. In the present study, we enhanced the pharmacological activity of SQ by increasing its bioavailability. The ethylene glycol moiety (used as a probe) was selectively introduced into SQ, creating HEHSQ, to facilitate tissue permeability. Also, we investigated the anti-inflammatory effect of SQ and HEHSQ on LPS-stimulated RAW264.7 murine macrophages. 2. Materials and Methods 2.1. Characterization of Squalene All solvents used in the study were of reagent-grade quality and used without further purification unless otherwise noted. 2,3-Epoxysqualene was prepared according to a previously described methodology [17]. All column chromatography was undertaken using Merck silica gel 60 as the solid support. HEHSQ was characterized by Rf, FT-IR, ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry. The infrared (IR) spectrum was measured on a JASCO FT-IR-4100. NMR spectra were recorded on a Bruker Avance-400 spectrometer operated at 400 MHz and a JEOL RESONANCE ECX-100 spectrometer operated at 100 MHz at room temperature (20°C) in the Fourier transform mode. ¹H NMR spectra were reported in δ units, parts per million (ppm), and calibrated relative to the signal for residual chloroform (7.26 ppm) in chloroform-d1 (CDCl3). ¹³C NMR data were reported in ppm relative to CDCl3 (77.16 ppm) and obtained with 1H decoupling. High-resolution mass spectrometric analyses were undertaken using an electrospray ionization time of flight based as a reserpine (m/z 609.2812) matrix on a JEOL JMS-100CS instrument. 2.2. Sample Preparation for In Vitro Study SQ was purchased from Fujifilm Wako Co, Ltd. (Tokyo, Japan). SQ and HEHSQ were dissolved in DMSO to obtain the respective stock solutions, which were then diluted in cell culture medium for the in vitro experiment. 2.3. Preparation of LPS LPS (Escherichia coli O111:B4) was purchased from EMD Millipore Co. (Billerica, MA, USA). A total of 5 mg of LPS was dissolved in 2 mL of phosphate-buffered saline without divalent cations (PBS (−)) and stored at −80°C in the dark until subsequent use. 2.4. Culture of RAW264.7 Cells Murine macrophage-like RAW264.7 cells were purchased from RIKEN BioResource Center (RCB0535, RIKEN BRC, Tsukuba, Japan). RAW264.7 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin at 37°C in a humidified incubator containing 5% CO2. Cells were seeded onto 96-well plates at a density of 2.0 × 10⁵ cells per well and were incubated at 37°C for 24 h. 2.5. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay Cell viability and mitochondrial activity were determined using the MTT assay to check the cytotoxicity of SQ (1, 10, and 100 µg/mL) and HEHSQ (1, 10, and 100 µg/mL). RAW264.7 cells were seeded at 2 × 10⁵ cells/mL in 96-well plates (BD BioCoat, USA) and incubated for 24 h. Then, the cells were treated with SQ or HEHSQ for 24 h. A solution of 5 mg/mL MTT dissolved in PBS was added (10 µL/well) and incubated for another 24 h. The resulting MTT formazan was dissolved in 100 µL of 10% sodium dodecyl sulfate (w/v), and the absorbance was measured using a microtiter plate reader (Dainippon Sumitomo Pharma Co., Ltd., Japan). The absorbance values were normalized to that of the culture medium, and viability was calculated as a percentage (%) of the untreated cells. 2.6. Measurement of NO Production To evaluate the effect of SQ and HEHSQ on NO production in LPS-stimulated RAW264.7 cells, the Griess reaction was used following the methodology described in a previous study [16]. Briefly, RAW264.7 cells were seeded at 2 × 10⁵ cells/mL in 96-well plates (BD BioCoat) and incubated for 24 h. The cells were treated with SQ (1, 10, and 100 µg/mL) or HEHSQ (1, 10, and 100 µg/mL) at 37°C for 24 h. After the treatment, LPS solution (final concentration: 1 µg/mL) was added to each well and incubated for 12 h at 37°C. Then, the cell supernatant was mixed at a 1 : 1 ratio with the Griess reagent (1% sulfanilic acid and 0.1% N-(1-naphthyl) ethylene diamine dihydrochloride in 2.5% phosphoric acid). After 10 min incubation in the dark, the absorbance was measured at 540 nm using a microtiter plate reader (Dainippon Sumitomo Pharma Co., Ltd., Japan), and nitrite concentration was determined by dilution of sodium nitrite (NaNO2, Fujifilm Wako Co, Ltd., Tokyo, Japan) as a standard. 2.7. RNA Isolation from RAW264.7 Cells RAW264.7 cells were seeded at 3.7 × 10⁵ cells/mL in a 10 cm² dish (BD BioCoat) and incubated at 37°C for 24 h. The RAW264.7 cells were then treated with SQ (100 μg/mL) or HEHSQ (1 μg/mL) at 37°C for 24 h. After the treatment, LPS solution (final concentration: 1 μg/mL) was added to each well and incubated for a further 12 h at 37°C. Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan) following the manufacturer’s instructions as per a previous study [16]. 2.8. Measurement of Proinflammatory Cytokine and Chemokine Using Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Real-time RT-PCR was performed to evaluate the effect of SQ or HEHSQ on proinflammatory cytokine and chemokine expression in RAW264.7 cells. The TaqMan probe (Thermo Fisher Scientific, USA) was used for the quantification of gene expression. Using a superscript III reverse transcriptase kit (Thermo Fisher Scientific), a complementary DNA (cDNA) solution was synthesized following the manufacturer’s instructions. For quantification of transcript amounts, TaqMan real-time RT-PCR amplification reactions were performed using the Applied Biosystems 7500 Fast Real-Time System (Thermo Fisher Scientific). All primer sets and the TaqMan Universal PCR Master Mix were obtained from Thermo Fisher Scientific. Specific primers for actin beta (Mm02619580_g1), tumor necrosis factor α (TNF-α) (Mm00443258_m1), C-C motif chemokine 2 (CCL2) (Mm00441242_m1), and iNOS (Mm00440502_m1) were used. 2.9. Enzyme-Linked Immunosorbent Assay (ELISA) to Measure TNF-α and CCL2 The effects of SQ or HEHSQ on cytokine production in RAW 264.7 cells were determined by ELISA. In the assay, cells (3.7 × 10⁵ cells/mL) were seeded in a 10 cm² dish (BD BioCoat) and treated with SQ (100 μg/mL) or HEHSQ (1 μg/mL) at 37°C for 24 h. After the treatment, LPS solution (final concentration: 1 μg/mL) was added to each well and incubated for 12 h at 37°C. The cell supernatants were collected by centrifugation at 1000 × for 5 min, and the levels of TNF-α and CCL2 were determined using commercial ELISA kits (Proteintech Group, Japan) according to the manufacturer's instructions. 2.10. Statistical Analysis All results are expressed as mean ± standard deviation (SD), and statistical evaluation was performed using the one-way ANOVA followed by post hoc Ryan-Einot-Gabriel-Welsch multiple range test. Differences were considered statistically significant when the value was less than 0.05. 3. Results 3.1. Preparation and Identification of 2-(2-Hydroxyethoxy)-3-hydroxysqualene 2,3-Epoxysqualene (Figure 1(a)) (31 mg, 71 mmol) and ethylene glycol (2.4 mL, 43 mmol) were dissolved in 2-propanol (9.6 mL). The mixture was heated to 80°C for 6 h while being stirred and then cooled to room temperature. H2O (15 mL) and ethyl acetate (30 mL) were added, and the organic layer washed with saturated brine and H2O and dried over Na2SO4. The solvent was evaporated following column chromatography on silica gel, eluting with hexane:ethyl acetate (5 : 1) produced HEHSQ (Figure 1(b)) (17 mg, 49%) as a colorless oil. Rf = 0.08 (hexane:ethyl acetate = 4 : 1). IR (CHCl3): 3451, 2977, 2932, 2877, 1452, 1381, 1219, 1083, 909, 670 cm⁻¹. ¹H NMR (400 MHz, CDCl3): δ 5.20–5.08 (m, 5H), 3.75–3.68 (m, 2H), 3.55–3.46 (m, 3H), 2.29 (m, 1H), 2.12–1.95 (m, 16H), 1.68 (s, 3H), 1.62 (s, 3H), 1.60 (br s, 12H), 1.52–1.37 (m, 5H), 1.15 (s, 3H), 1.14 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl3): δ 135.1, 135.1, 134.9, 134.8, 131.2, 124.7, 124.4, 124.3, 124.3 (2C), 77.8, 76.0, 62.4, 62.2, 39.7, 39.7 (2C), 36.8, 29.7, 28.6 (2C), 26.7, 26.7, 26.6, 25.7, 21.6, 19.8, 17.7, 16.0 (3C), 16.0 ppm. HRMS (ESI): m/z 511.4131 (calculated for C32H56NaO3 [M + Na]⁺ 511.4127, Δ + 0.4 mmu). (a)
... Chronic inflammation is a maladaptive response that involves active inflammation and tissue damage. This persistent inflammation is associated with long-term human disease risk including allergy (293), cancer (294) and cardiovascular disease (295). ...
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Poor maternal nutrition during pregnancy is detrimental to fetal development and adversely affects long-term health by increasing the risk of chronic diseases, such as neuropsychiatric disorders. Previous studies show that maternal obesity may influence offspring behaviour such as anxiety/stress in adult life. This may be due to altered development of the hypothalamo-pituitaryadrenal (HPA) axis. This may be exacerbated by obesity in adult life which independently influences behaviour and HPA axis function. The mechanisms behind the effect of obesity to impact neuropsychiatric disorders is unclear, however increased inflammation found in obese individuals may be inducing permanent changes to HPA function. This study investigated the effects of maternal and postnatal obesity on behaviour, HPA axis function in young and mature adult mouse offspring, and assessed neuroinflammation as a potential mechanism. In this study, female C57BL/6 mice were fed either an obesogenic high-fat diet (HF; 45% kcal fat) or control diet (C; 7% kcal fat) 6 weeks before mating, throughout pregnancy and lactation. Offspring were fed C or HF diet from weaning onwards. Maternal care and pup anxiety were assessed on postnatal day 7 via pup retrieval and ultrasonic vocalisations (USVs) during maternal separation. In 15 and 52 week-old offspring, anxiety was assessed by open field (OF) and elevated plus maze (EPM) and memory was assessed by novel object recognition (NOR). Corticosterone and ACTH concentrations (basal and area under curve [AUC]) were measured during a 30 minute restraint test. Analysis of neuroinflammation was performed via immunohistochemistry and mRNA levels of genes associated with HPA axis function and inflammation. A maternal obesogenic HF diet was associated with poor maternal care and anxiety in males from 1 week of age, and subtle changes to anxiety persisted into young and mature adulthood. Postnatal obesity was associated with decreased and increased anxiety at 15 and 52 weeks of age respectively, and memory was impaired at 15 but not 52 weeks of age in males. Changes in anxiety and memory were associated with HPA dysregulation and microglial activation in the brain at 15 but not 52 weeks of age in males. In female offspring, changes in anxiety and memory were only observed at 52 weeks of age due to postnatal and maternal obesity respectively. Anxiety, but not memory, in females corresponded to changes in HPA regulation, but not inflammation at this age. Maternal obesity, in addition to further postnatal obesity, subtly exacerbates some effects of anxiety and the stress response which is seen primarily in male, but not female, offspring at multiple ages. Overall, the effect of maternal obesity is sex-specific and age-dependent. These data are a novel addition to the existing literature on the effects of maternal obesity on HPA axis function and behaviour, particularly due to the additional assessment of further postnatal obesity. Further analysis of the role of inflammation during obesity at different stages of the life course will enhance our understanding of the risk of neuropsychiatric disorders to future generations.<br/
... Quercetin was also shown to decrease the level of proinflammatory mediators such as TNF-α, IL-6, MIP-1α, and P-selectin in murine RAW264.7 macrophages [108]. Further, quercetin treatment can potentially disrupt atherosclerotic plaques through the inhibition of matrix metalloproteinase 1 [109]. Soy isoflavone administration reduced the risk of chronic inflammation-mediated cardiovascular disease by reducing the endothelial production of TNF-α in a mouse model [110]. ...
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