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Prostaglandins, Leukotrienes and Essential Fatty Acids 79 (2008) 109115

Contents lists available at ScienceDirect

Prostaglandins, Leukotrienes and


Essential Fatty Acids
journal homepage: www.elsevier.com/locate/plefa

Basic mechanisms behind the effects of n-3 fatty acids on


cardiovascular disease
Marika Massaro b,c, Egeria Scoditti b,c, Maria Annunziata Carluccio b,c, Raffaele De Caterina a,b,
a
b
c

G. dAnnunzio University, 66013 Chieti, Italy


C.N.R. Institute of Clinical Physiology, Pisa and Lecce, Italy
University of Lecce, Ecotekne, Lecce, Italy

a r t i c l e in fo

Keywords:
n-3 fatty acids
Anti-inammatory lipid derivatives
Adhesion molecules
Endothelium
Endothelial activation
Atherosclerosis
Cyclooxygenase-2
Plaque angiogenesis
Metalloproteinases
Plaque rupture

abstract
The epidemiological association between high intakes of n-3 fatty acids (FA) and decreased morbidity
and mortality from cardiovascular disease (CVD) can be explained by two main basic mechanisms:
(a) an effect on atherothrombosis, and (b) an effect on cardiac arrhythmias. These mechanisms probably
reect different benecial inuences of n-3 FA on cardiovascular biology. Effects on atherothrombosis
include the modulation of the expression of pro-atherogenic genes (e.g., endothelial leukocyte adhesion
molecules, inammatory cytokines and cyclooxygenase (COX)-2) and the hepatic synthesis of very low
density lipoproteins (VLDL), and are slow in onset, requiring incorporation into cell membrane
phospholipids, and usually doses in humans in the order of 3 g/day or higher. Effects on cardiac
arrhythmias include complex interactions with ion channels (sodium, potassium and calcium channels),
typically requiring the presence of free FA in extracellular uids and usually occurring with lower doses
(around 1 g/day) of nutritional or pharmacological intake. We have focused most of our research effort
in unraveling the pathophysiological background of protection by n-3 FA from atherothrombosis. As the
result of incorporation of n-3 FA in the sn-2 position predominantly of the phosphatidyl ethanolamine
pool in the inner leaet of the plasma membrane, n-3 FA appear on the one hand to increase the
production of bioactive lipid mediators (protectins and resolvins) affecting cytokine-induced signal
transduction; and on the other hand to directly interfere with the generation of reactive oxygen species
(mostly hydrogen peroxide), directly responsible for the activation of the transcription factor nuclear
factor (NF)-kB, which controls the expression of a variety of pro-inammatory and pro-atherogenic
genes, including those encoding for interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)a, vascular
cell adhesion molecule-1 (VCAM-1), E-selectin, and COX-2. The upstream-direct or indirect-inhibition of
cytokine- and other atherogenic trigger-induced signaling pathway may involve interference with the
activation of protein kinase (PK) C isoforms and NADP(H) oxidase. Such interference may also explain
the blunt anti-inammatory effect of n-3 FA in many experimental models and clinical conditions
of inammation. All together, these mechanisms may provide an integrated view of how n-3 FA may
affect CVD.
& 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Atherosclerosis is the underlying cause of most cardiovascular
diseases (CVD), including coronary artery disease, ischemic gangrene, abdominal aortic aneurysms, and many cases of heart failure
and stroke. This group of diseases constitutes the main cause of
death in the Western world today. The World Health Organization
expects CVD to be the main killer worldwide within 15 years, due

 Corresponding author at: Institute of Cardiology and Center of Excellence on


Aging, G. dAnnunzio UniversityChieti, C/o Ospedale SS. Annunziata, Via dei
Vestini, 66013 Chieti, Italy. Tel.: +390 871 41512; fax: +390 871 402817.
E-mail address: rdecater@unich.it (R. De Caterina).

0952-3278/$ - see front matter & 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plefa.2008.09.009

to the accumulation of several metabolic risk factors, including


obesity and diabetes, in both the Western world and developing
countries. These facts underscore the need for an intensied
research effort in parallel to putting increased preventive
measures into action for CVD.
In atherosclerosis, the combined action of frequently coexisting
risk factors, such as dyslipidemia, hypertension, hyperglycemia
and its consequences, induce the gradual thickening of the arterial
wall to form an atherosclerotic plaque, occasionally resulting in
the narrowing of the artery lumen. This may cause the chronic
onset of organ ischemia, most commonly affecting the heart
and the brain. Plaques can however also abruptly rupture,
causing thrombosis, the main underlying cause of myocardial
infarction or stroke. Intensive studies of the cellular and molecular

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M. Massaro et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 79 (2008) 109115

mechanisms that underlie atherogenesis have led to a general


agreement on the pathogenesis of this process, recognizing the
involvement of chronic inammation at every step of the process,
from its onset, to its progression, to its ultimate clinical manifestations.
Most risk factors indeed act by promoting atherogenesis, by inducing
or intensifying such underlying inammatory processes.
The recent recognition that diet strategically affects the
majority of modiable risk factors for CVD has further highlighted
the importance of a correct dietary approach to ght atherosclerosis [1], emphasizing the recently appreciated antiinammatory properties of specic essential fatty acids (FA), mostly
the n-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA), as extremely valuable preventive or therapeutic means.
Since the early studies in Greenland Eskimos, many studies
have conrmed the association between n-3 FA and decreased risk
of CVD [2]. Of particular interest are two recent meta-analyses,
suggesting that EPA and DHA signicantly reduce the rates of
cardiac and sudden death, and possibly stroke also for modest
consumption of sh (12 servings/week), thus remarking that the
benets of sh intake exceed the potential health risks from
methylmercury, dioxins, polychlorinated biphenyls and other
environmental contaminants [3,4].
Because of this renewed interest on n-3 FA cardioprotective
potential, this review will focus on summarizing the most recent
mechanistic ndings on the anti-inammatory, cardioprotective
and lipid-regulating effects exerted by n-3 FA.

2. Metabolism n-3 FA
Usually esteried in the sn-2 position of the phospholipid n-6
FA arachidonic acid (AA) as well as the n-3 FA EPA and DHA, can
be released through the action of phospholipase A2 and metabolized through orthodox and unorthodox pathways (Fig. 1).
The orthodox pathway involves reactions catalyzed by
cyclooxygenase (COX) and lipoxygenases (LO) to eicosanoids,
including prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT), which are mediators of a vast number of biologic

effects. AA is the precursor of the prostanoids of the 2-series


(including PGI2 (prostacyclin) and TXA2), whereas EPA is the
precursor of prostanoids of the 3-series (PGI3 and TXA3).
Increasing the content of n-3 FA in the diet causes a partial
substitution of the FA of the n-6 series, especially decreasing the
relative proportions of AA in cell membrane phospholipids. This
causes a net decrease in the production of prostanoids (because n3 FA are worse substrates for the metabolizing enzymes) and
favors the synthesis of generally less biologically active prostanoids, especially TXA3, which, contrary to AA-derived TXA2, has
minimal platelet-aggregating and vasoconstricting activity. The
results of these changes in eicosanoid production are vasodilatation and inhibition of platelet aggregation. In leukocytes and
monocytes, AA and EPA are substrates of 5-LO for the synthesis of
LT. LTB4, derived from AA, has potent chemotactic and other
leukocyte-activating properties, whereas sulphido-peptide LT
(LTC4, LTD4, LTE4) have vasoconstrictive effects and can increase
vascular permeability. Through 5-LO, EPA gives rise to LT of the
5-series, namely LTB5, LTC5, LTD5, and LTE5, which have weaker
pro-inammatory and vasoconstrictive activities than those of the
4-series. On the contrary, in endothelial cells (EC), AA and EPA are
precursors of the almost equipotent PGI2 and PGI3, respectively,
both endowed with anti-aggregating and vasodilating properties.
In addition to these metabolic pathways, the existence and the
benecial contribution of an unorthodox generation of alternative eicosanoid derivatives of n-3 FA has been recently
appreciated. Such alternative compounds are typically produced
during the resolution of self-limited inammation. These compounds were rst identied by Serhan et al. with the trivial name
of resolvins (resolution phase interaction products), to emphasize
their original isolation and production during the resolution phase
of acute inammation and to signify the frequent contribution of
the transcellular biosynthesis of these new mediators. The
production of resolvins is mediated by the serial combined
activities of acetylated COX-2 (or cytochrome P-450 monoxygenase) and 5-LO on EPA, to produce the E-series resolvins (Resolvin
E1 and 2 or RvE1 and RvE2), and on DHA, to produce the
17R D-series resolvins (RvD1 through RvD4). Upon tissue

Stimulus
PLA2

Tissue specific
isomerase

PGE2
PGF2

LTB4
5,6-EET
8,9-EET
11,12-EET
14,15-EET

(acetylated)

GSH
LTC4

PGI3
PGA3
PGF3
TXA3
Resolvin E1, E2

Resolvin D1, 2, 3, 4

NITRIC OXIDE

COX-1
COX-2

15-LOX

lipoxygenase

LTA4

PGI2

PGD2

5-LOX
12-LOX
15-LOX

HPETE

PGH2

TXA2

EPA
DHA

5-LOX

PGG2

P450 monooxygenase
epoxygenase

COX-1
COX-2

cycloxygenase

AA

5-LOX
12-LOX
15-LOX
LTA5

GSH

LTB5 LTC5
LTD5
LTE5

LTD4
LTE4

10,17Sdocosatriene
(neuroprotectin
D1)

NITROLIPIDS?

Fig. 1. Metabolism of n-3 FA versus n-6 FA by COX and lipooxygenases. FA fatty acid(s); PGH2 prostaglandin H2; PGI2 prostacyclin; TXA2 thromboxane A2;
PGD2 prostaglandin D2; PGE2 prostaglandin E2; PGF2a prostaglandin F2a; HPETE hydroperoxy eicosatetraenoic acids; EET epoxyeicosatrienoic acids;
LT leukotrienes; PGI3 prostaglandin I3; PGA3 prostaglandin A3; TXA3 thromboxane A3; PGF3a prostaglandin F3a; GSH glutathione; PLA2 phospholipases
A2; AA arachidonic acid; EPA eicosapentaenoic acid; DHA docosahexaenoic acid.

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specialization, DHA can be also metabolized by 15-LO to produce


the 17S D-series resolvins, with potent anti-inammatory activities [5] (Fig. 1).
In addition, the formation of another set of compounds, formed
by the nitration of unsaturated FA, called nitrolipids, has been
shown to occur in vivo and to have potent biological actions. Such
derivatives are known as nitro-FA (NO2-FA) and display both
cGMP-independent and receptor-dependent signaling actions, as
well as robust electrophilic reactivity. Recent data indicate that
NO2-FA can signal predominantly via nitric oxide(NO)-independent mechanisms, acting via electrophilic and receptor-mediated
reactions to stimulate smooth muscle relaxation, block platelet
activation, inhibit human neutrophil function, superoxide generation, nuclear factor (NF)-kB activation, integrin and immunoglobulin expression, thus globally suppressing inammation [6]
(Fig. 1).

3. Biological and molecular effects of n-3 FA on cardiovascular


diseases
The reduced morbidity and mortality from CVD by n-3 FA can
be explained by two main basic mechanisms: (a) the suppression
of cardiac arrhythmias and (b) reduced atherogenesis, occurring
through the modulation of specic atherothrombotic risk factors,
decreased platelet aggregation, reduced plasma triglycerides and
blood pressure, and, above all, the regulation of systemic and local
inammation underlying plaque inception, progression and
instability.
3.1. Suppression of cardiac arrhythmias
Several epidemiological studies have supported an important
antiarrhythmic effect by n-3 FA, including sudden death, arrhythmic coronary heart disease death and atrial brillation [7].
However, recent ndings also support the view that such
protective effect by n-3 FA occur not only in patient with prior
myocardial infarction, but-importantly-also at a population level
in primary prevention, for a sh intake 4300 g/week [8]. There
are several mechanisms able to explain how n-3 FA may prevent
arrhythmias. It is long known that n-3 FA inhibit voltage-gated
sodium channels, resulting in a longer relative refractory period
and in an increased voltage required for membrane depolarization, which reduces heart rate [9,10]. Furthermore, n-3 FA also
maintain the integrity of L-type calcium channels, thus preventing
cytosolic calcium overload during periods of ischemic stress [11].
On the other hand, n-3 FA may indirectly contribute to the
decrease in the heart rate by improving left ventricular efciency
and reducing blood pressure [12].
3.2. Decreased platelet aggregation
n-6 FA are precursors for the 2-series eicosanoids, which have
a wide range of potential effects on metabolic pathways relevant
to thrombosis. In fact, although the AA derivative PGI2 is a potent
vasodilator, TXA2 stimulates platelet aggregation and produces
vasoconstriction, and 5-LO metabolites, LT, have been linked to
inammation and atherogenesis [13]. Consumption of EPA and
DHA lower tissue levels of AA by inhibiting its synthesis and by
taking its place in membrane phospholipids [14]. EPA-derived
3-series eicosanoids are typically less vasoconstrictive and
produce less platelet aggregation than those made from AA [14].
Fish oil could, therefore, be considered as positive conditioners
of the thromboxaneprostaglandin balance. In general, however,
the reduction in platelet function by EPA and DHA, when

111

measurable, is limited in extent. In addition, many described


effects of sh oil intervention on surrogate in vitro end point
hemostatic indices, such as hemostatic factors, were variable and
overall inconsistent [15].
3.3. Reduced triglycerides synthesis
Although the importance of plasma triglycerides in atherogenesis remains controversial [16], recent data reveal an enhancement of inammatory response by the endothelium exposed to
triglyceride-rich lipoproteins [17], thus re-proposing a role of
these unmodied lipoproteins as possible critical contributors to
inammation in atherogenesis. One of the most consistent and
best recognized anti-atherogenic properties of n-3 FA is in the
reduction of fasting and post-prandial serum triglycerides and
free FA (FFA) [18]. Such effect occurs by a reduction in the
synthesis of triglycerides, and hence the secretion rates of hepatic
very low density lipoproteins (VLDL), through the interference
with most of the transcription factors that control the expression
of enzymes responsible for both triglyceride assembly and FA
oxidation. It was shown, for example, that n-3 FA reduce the
expression of the sterol regulatory element-binding proteins
(SREBP), transcription factors that regulate cholesterol-, FA-, and
triglyceride-synthesizing enzymes [19]. This effect seems to be
due to EPA- and DHA-induced inhibition of liver X receptor alpha/
retinoid X receptor alpha (LXRa/RXRa) heterodimer binding to the
promoter of the SREBP-1c gene [20]. Similarly well documented is
the increase by n-3 FA of the mitochondrial and peroxisomal
FA b-oxidation rates. This effect is mediated by n-3 FA activation
of the peroxisome proliferator-activated receptor (PPAR)a, a
ligand-activated transcription factor [21] that up-regulates the
expression of acyl-coenzyme A oxidase, the rate-limiting enzyme
in the b-oxidation pathway [22,23]. Finally, a possible effect by
n-3 FA on the farnesoid X receptor (FXR) activity was also
postulated. FXR is a nuclear receptor for bile acids that plays a
central role in lipid homeostasis [24]. Studies in hepatoma cell
lines have demonstrated that the activation of FXR suppresses the
gene expression of hepatic lipase and apo CIII, and induces apo CII
and VLDL-receptor gene expression, all of which may contribute
to the triglyceride-lowering action of FXR agonists [25]. Notably,
mice lacking of a functional FXR protein had a pro-atherogenic
serum lipoprotein prole, including elevated triglycerides. Since
DHA is a ligand for FXR, it was postulated that a mechanism for
the triglyceride-lowering effects by DHA may involve FXR-induced
changes in gene expression [25].
3.4. Antihypertensive effects
The antihypertensive effect is viewed as another potential
cardioprotective effect by n-3 FA. By a combined analysis of
36 randomized older trials, sh-oil intake (median dose 3.7 g/day
EPA plus DHA) has been found to reduce systolic blood pressure
by 2.1 mmHg and diastolic blood pressure by 1.6 mmHg [26].
These ndings have been corroborated by those obtained in a
large international cross-sectional epidemiologic study conducted
on 4680 middle age men and women, in which n-3 FA intake was
found to be inversely related to blood pressure, both in
hypertensive and in non-hypertensive subjects [27]. At least two
mechanisms may account for this effect. Firstly, incorporation of
EPA and DHA into membrane phospholipids was shown to
increase systemic arterial compliance [28]. Secondly, EPA and
DHA seem to improve endothelial function by enhancing the
release of NO [29]. This effect seems to be due to the lipid and
structural modication of caveolae, plasma membrane microdomains that act as regulators of endothelial nitric oxide synthase

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(eNOS) activity [30]. Such effects are however quantitatively quite


modest.

3.5. Modulation of inammatory steps in early


and late atherogenesis
It is now widely recognized that atherogenesis is a process
largely overlapping the classical inammatory reaction. In its
initial stages, atherogenesis is indeed featured by the intimal
recruitment of selected populations of leukocytes, especially
monocytes and some T-lymphocytes, and, secondarily, by the
gradual accumulation of lipids, at the beginning inside the
monocytes, with the consequent foam cell generation, and then
extracellularly, after the occurrence of apoptosis and/or necrosis
of the same foam cells. Monocyte recruitment is therefore
considered a crucial event in the onset of atherosclerosis [31]
and many efforts have been made to identify agents able to reduce
the endothelial de novo expression of adhesion molecule for
leukocytes. Using human EC in culture activated by cytokines as
an in vitro model of early atherogenesis, we assessed the effects of
various FA on the surface expression of several adhesion
molecules, and subsequently characterized mechanisms and
functional relevance of such effects. DHA andto a lesser
extentEPA, when added to cultured EC hours to days before
the stimulation with cytokines, and long enough to allow a
signicant incorporation of this FA in cell membrane phospholipids, signicantly inhibited the expression of both vascular cell
adhesion molecule(VCAM)-1 and E-selectin after stimulation with
virtually any stimulus able to elicit the coordinated expression of
such genes [32]. The inhibition of adhesion molecule expression
occurred in a range of n-3 FA concentrations compatible with
nutritional supplementation of these FA to a normal Western diet,
positively related to the extent of n-3 FA incorporation into total
cell lipids, and were inversely related to the content of n-6 FA [32].
This effect was not limited to the expression of transmembrane
molecules involved in leukocyte recruitment, but appeared to
occur also for other cytokine-activated products, such as the
soluble proteins macrophage-colony stimulating factor (M-CSF),
interleukin(IL)-6 and IL-8, and was accompanied by a functional
counterpart, i.e., a reduced monocyte adhesion to cytokineactivated endothelium [32]. Experiments following the fate of
14
C-labelled DHA into cell phospholipids showed a signicant
incorporation of DHA into the phosphatidyl ethanolamine pool,
i.e., in a specic and not particularly abundant phospholipid pool,
likely in the inner plasma membrane, and therefore in a possibly
strategic position to alter intracellular signal transduction pathways. However, whether n-3 FA protective effect occurs directly,
for example by quenching cytokine-elicited reactive oxygen
species (ROS), which act as second messengers in endothelial
activation [33], or indirectly, through the production of metabolically active oxidized products, still remain under scrutiny.
For example, our data do not exclude the possibility that products
of n-3 FA oxidation might inhibit cytokine-induced VCAM-1
expression by a PPARa-dependent mechanism [34].

3.6. Modulation of angiogenesis and plaque stability: effects on


COX-2 expression
A recent study by Thies et al. has highlighted a potential role of
n-3 FA in decreasing the risk of atherosclerotic plaques rupture in
patients awaiting carotid endarterectomy [35]. In this study,
plaques from patients taking sh oil featured a net incorporation
of n-3 FA into plaque lipids, and this correlated with a reduced
macrophage inltration and thicker brous caps compared with

plaques from patients assuming sunower oil-enriched control


capsules [35].
In recent years, supportive experimental evidence has indicated that plaque neovascularization and exacerbated release of
metalloproteinases (MMP) by the activated endothelium and
macrophages play a pathogenetic role in plaque progression [36]
and instabilization [37]. In an attempt to nd out the targetable
mechanistic basis of the plaque-stabilizing effect by n-3 FA, we
postulated an anti-angiogenic activity and a reduction of MMP
release by sh oil as putative mechanisms. We therefore
investigated the effects of n-3 FA rstly on in vitro angiogenesis
models and then on the MMP release by macrophages. In
agreement with what observed by Kanayasu [38] and Tsujii [39],
we found that the exposure of EC to DHA reduced the tube-like
network formation in collagen matrix (Matrigel) in vitro (Fig. 2A).
As postulated by many authors and conrmed by Jones et al. [40],
we inferred a role for COX-2 activity in the orchestration of
angiogenesis by EC, since cell treatment with NS-398, an inhibitor
of COX-2 activity, abolished such vascular organization. It was
therefore plausible that DHA exerts an anti-angiogenic effect at
least in part by reducing stimulated COX-2 expression. To conrm
this hypothesis, we recently observed that treatment of EC with
DHA before PMA stimulation highly reduced COX-2 expression
and activity [41].
The release of MMP is also regulated by COX-2-derived
prostaglandins [42]. In the attempt to fully explain the ndings
on plaque instabilization [35], we tested the effect of DHA on the
release of MMP by human macrophages in culture. We observed
that cell treatment with DHA for 48 h before stimulation resulted
in a signicant inhibition of MMP-9 release (Fig. 2B) [43], thus
potentially having a plaque-stabilizing effect [35].

3.7. The modulation of NF-kB activation as the common


denominator for the anti-inammatory and anti-atherogenic
effect by n-3 FA
Adhesion molecules, soluble cytokines, COX-2 and MMP-9,
which are all reduced in their expression by DHA, are structurally
and functionally different molecules that share however common
features, namely the inducibility by pro-inammatory and proatherogenic stimuli and the presence of at least one consensus
binding site for the transcription factor NF-kB. For this reason, in
an attempt at elucidating the mechanism of n-3 FA action, we
focused our interest on whether and how DHA might interfere
with the pathway leading to NF-kB activation.
Active NF-kB complex are dimers of various combinations of
the Rel family polypeptides, consisting of p50, p52, c-Rel, v-Rel,
p65, and Rel B [44]. In resting cells, NF-kB is retained in the
cytoplasm by binding to one of the inhibitory IkB proteins (IkBa,
IkBb, IkBe, p105, and p100), which blocks the nuclear translocation of NF-kB [44]. NF-kB is activated in response to a wide
variety of pro-inammatory and pro-angiogenic stimuli each
promoting the dissociation of IkB through its phosphorylation,
followed by ubiquitination and degradation. Thus, the unmasking
of the nuclear localization sequence of NF-kB allows NF-kB to
enter the nucleus and bind to kB-regulatory elements. The
phosphorylation of IkB is catalyzed by an IkB kinase(IKK) complex
[44]. The core of the IKK complex consists of a heterodimer of
IKKa and IKKb, and two IKKg subunits. IKKa and IKKb mediate the
phosphorylation of IkB, whereas IKKg links the core to the
upstream signaling molecules [44]. Also the activation of the IKK
complex is dependent on phosphorylation, and multiple upstream
kinases, some of which are redox-sensitive, have been suggested
to act as IKK kinases [44]. Many agents that activate mitogen
activated protein kinase (MAPK) also induce an overproduction of

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40
30

20

1 = Control
2 = VEGF + PMA
3 = VEGF + PMA
+ DHA

10
0

Matrigel

Branching
points per field

M. Massaro et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 79 (2008) 109115

1 = U937, no-stimulated control


2 = MDM

Zymography

3 = MDM + 5 mol/L DHA


4 = MDM + 50 mol/L DHA
5 = MDM + 5 mol/L EPA

6 = MDM + 50 mol/L EPA


7 = MDM + 50 mol/L AA

Fig. 2. DHA inhibits endothelial cell tube formation and MMP-9 release by macrophages. (A) Human umbilical vein endothelial cells were treated with 25 mmol/L
docosahexaenoic acid (DHA) for 48 h and, after enzymatic detachment, plated on Matrigel-coated 48 well-plates and stimulated with 20 ng/mL vascular endothelial growth
factor (VEGF) plus 10 ng/mL phorbol myristate acetate (PMA). After incubation at 37 1C for 4 h, cells showed tube formation. The quantication of tube formation was done
by counting the number of branching points in microscopic photographs. The bar graph represents data from 3 independent experiments with two-wells per condition and
23 elds per well. *po0.05 vs. stimulated control. (B) Gelatin zymography analysis of the effect of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and
arachidonic acid (AA) on the metalloproteinases (MMPs) release in the culture medium by monocyte derived macropages (MDM). MDM monolayers were treated with FA at
the indicated concentrations, in RPMI-1640 containing 5% fetal calf serum for 48 h. Medium was then removed and cells incubated in serum-free medium containing 0.1%
human albumin. After 24 h, media were collected and analyzed by gelatin zymography. EPA and DHA but not AA treatment signicantly reduced the intensity of the band
corresponding to MMP-9. The gelatinolytic activity corresponding to MMP-2 was negligible under these culture conditions.

ROS besides activating NF-kB, suggesting that a cross-talk occurs


between these pathways (Fig. 3). However the exact site of action
of these MAPK along the pathway leading to NF-kB activation, as
well as the site of interference by DHA with ROS production, likely
triggered by the activation of nicotinamide adenine-dinucleotide(phosphate)[NAD(P)H] oxidase (Fig. 3), are still elusive.
By transient transfection experiments using full-length human
COX-2 promoter constructs, we showed that DHA inhibited
promoter activity independent of the pro-inammatory stimuli
used, and that the DHA inhibitory effect was abrogated only when
promoter sequences were lacking, by deletion or site-mutation,
the functional NF-kB sites. This, together with the observed
reduced activation of NF-kB and the reduced nuclear translocation of p65, suggested an interference by DHA with the activated
cytoplasmic signaling pathway leading to NF-kB activation [41].
It is well known that n-3 FA, modifying lipid composition, can
alter membrane lipid microdomains, such as lipid rafts and
caveolae, involved in the compartmentalization, modulation, and
integration of cell signaling components [45]. In our experimental
conditions, DHA accumulates in membrane phospholipids. Therefore, in an attempt at exploring which molecular target(s)
upstream of NF-kB was (were) affected by DHA, we rst focused
our attention on the NAD(P)H oxidase, an enzyme system
producing ROS involved in the activation of NF-kB by IL-1 [46],
the assembly and activation of which is potentially sensitive to
membrane phospholipid composition [47]. We demonstrated that
DHA reduces the p47phox subunit membrane translocation and
hence NAD(P)H oxidase activity and intracellular ROS production
[41]. Since PKC activities are involved in NAD(P)H oxidase
activation and COX-2 expression [48], and since PKCe activity is
specically involved in the activation of NF-kB, we next explored

the possibility that PKC could be another molecular switch


affected by DHA membrane conditioning. We monitored membrane translocation of the main PKC isoforms in EC stimulated by
PMA and IL-1 in the presence of DHA. All such isoforms were
activated by PMA, as demonstrated by their translocation to
plasma membrane; but only the translocation of PKCe was
reduced by DHA treatment [41]. We therefore concluded that
DHA, possibly modifying the plasma membrane lipid composition
and hence membrane microdomain organization, inhibits at least
two molecular switches involved in NF-kB activation: NAD(P)H
oxidase and PKCe activities. DHA might do this as precursor of
various anti-inammatory lipid mediators [5]. To verify if any of
these lipid derivatives is involved in the observed DHA inhibitory
effect, we nally evaluated the reversion of DHA protective effect
in the presence of chemical and/or molecular inhibitors of several
polyunsaturated fatty acid-metabolizing enzymes such as 5-, 12-,
and 15-LO and the cytochrome P450 system. We observed that
only the inhibition of 15-LO partially reverted DHA inhibitory
effect, thus suggesting the involvement of resolvins in mediating,
at least in part, the anti-inammatory effects by DHA [41]. An
integrative molecular model of dietary n-3 FA interference with
the IL-1 signaling pathway leading to adhesion molecules and
COX-2 induction in EC is proposed in Fig. 3.

4. Conclusions and perspectives


The vascular endothelium plays a key role in the progression
of CVD. On the other hand, n-3 FA have emerged as an effective
tool in primary and secondary prevention of CVD and some
forms of cancer. Although they exert multiple actions [49], the

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M. Massaro et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 79 (2008) 109115

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IL-1
D HA

gp91
p22
Rac

p67

p47

p
p

IL-1 RI
AcP

Tollip

NADPH
oxidase

D HA

IL-1 RI
MyD88

IRAKs

PKC

+ +
ROS
(H2O2, O2.-)

TRAF 1-6

+
+

TAK-1/TAB2

ERK1/2

NIK

DHAoxDHA

15-LOX

IKK- - -

PPAR activation

IB,

DHA 10,17S-docosatriene

NF-B activation

Nucleus

Adhesion molecules
COX-2

Fig. 3. Proposed integrative molecular model of dietary omega-3 fatty


acid interference with IL-1 signaling pathways leading to adhesion molecules
and COX-2 induction in endothelial cells. IL-1 binds to the IL-1 receptor type I
(IL-1RI), which heterodimerizes with the IL-1 receptor accessory protein
(IL-1RAcP). The IL-1R-associated kinases (IRAK) are then recruited and associated
by the adapter proteins myeloid differentiation factor (MyD) 88 and Tollinteracting protein (Tollip). The signaling pathway also includes the production
of reactive oxygen species (ROS) (H2O2) through the activation of NAD(P)H oxidase
by IRAK activation, as well as the activation of protein kinase C (PKC), both
contributing to NF-kB activation. DHA, by interfering with the production of ROS
(through the inhibition of p47phox translocation and/or the scavenging of ROS by its
multiple double bonds), would prevent the formation of H2O2, thus limiting the
downstream cascade leading to COX-2 gene expression. Furthermore, DHA reduces
PKCe activation, thus inhibiting ERK1/2 activation, also leading to NF-kB activation
and COX-2 expression. Finally cytokine-induced ROS could attack DHA and
transform it into an oxidized derivative able activate PPARa and thus further
inhibit NF-kB through a mechanism of trans-repression [50]. TRAF, TNF
receptor-associated factor; TAK-1, TGFb-activated kinase 1; TAB-2, TAK1-binding
protein 2; NIK, NF-kB-inducing kinase; IKK, IkB kinase; PPARa, peroxisomeproliferators activated receptor. Modied from Ref. [41].

transcriptional control of several endothelial pro-inammatory


genes, including those encoding for adhesion molecules, chemokines and other soluble cytokines in the endothelium, likely
plays an important role. The recent ndings showing inhibition of
COX-2 expression and activity by n-3 FA allows a deeper
understanding of the therapeutic potential of these nutrients,
being COX-2 overexpression pathogenetically involved in several
inammatory/degenerative diseases besides atherosclerosis
(from rheumatoid arthritis to inammatory bowel disease and
possiblyAlzheimers disease). Most or all such effects of n-3 FA,
involving a blunted activation of inammatory genes in response
to activating stimuli, appear to have the inhibition of NF-kB
activation as a common denominator. Such inhibition appears to
be the nal result of an interference by n-3 FA with early signaling
events in response to inammatory stimuli.
In general, therefore, n-3 FA appear to nely tune the response
of our genes to dangerous environmental challenges (nutrigenomics), by curbing physiological responses without abrogating
them totally. By decreasing the endothelial responsiveness to proinammatory, pro-atherogenic and pro-angiogenic stimuli, n-3 FA
appear to favorably impact molecular events not targeted by any
other drugs or interventions, thus allowing their proposition in a
therapeutic role complementary to those of already implemented
pharmacologic treatments in several inammatory diseases,
including atherosclerosis.

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