Circulation Research. 2001;88:12-21
(Circulation Research. 2001;88:12.)
© 2001 American Heart Association, Inc.
Interactions Between Nitric Oxide and Lipid Oxidation Pathways
Implications for Vascular Disease
Valerie B. O’Donnell,
Bruce A. Freeman
From the Wales Heart Research Institute (V.B.O’D.), University of
Wales College of Medicine, Heath Park, Cardiff, Wales, UK; Departments of
Anesthesiology, Biochemistry, and Molecular Genetics (B.A.F.), and the Center
for Free Radical Biology, University of Alabama at Birmingham, Birmingham,
Ala.
Correspondence to Bruce A. Freeman, PhD, Department of Anesthesiology, 946 Tinsley Harrison Tower, 619 S 19th St, University of Alabama at Birmingham, Birmingham, AL 35233-6810. E-mail bruce.freeman@ccc.uab.edu or o-donnellvb{at}cardiff.ac.uk
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Abstract
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Abstract—Nitric
oxide (
·NO) signaling pathways
and lipid
oxidation reactions are of central importance in both
the maintenance
of vascular homeostasis and the progression
of vascular disease.
Because both of these pathways involve
free radical species that can
also react together at extremely
fast rates, convergent interactions
between these pathways are
expected. Biochemical and cell biology
studies have defined
multiple interactions of
·NO with oxidizing lipids that
could lead
to either vascular protection or potentiation of
inflammatory vascular
injury. For example, low levels of
·NO
generated
by endothelial nitric oxide synthase can terminate
propagating
lipid radicals and inhibit lipoxygenases, reactions that
would
be protective. Alternatively, if generated at elevated levels,
for
example, after inducible nitric oxide synthase expression in
inflammation,
·NO can be converted to
prooxidant species, such as peroxynitrite
(ONOO
–)
and nitrogen dioxide
(
·NO
2), that can
potentiate inflammatory
injury to vascular cells. Finally, both
enzymatic and nonenzymatic
lipid oxidation reactions can influence
·NO bioactivity
by directly scavenging
·NO or altering the induction
and
catalytic activity of nitric oxide synthase enzymes. In
this review, we
summarize the biochemical interactions between
·NO
and lipid oxidation reactions and
discuss the recognized and
potential roles of these reactions in the
vasculature.
Key Words: eicosanoid signaling • lipid • nitric oxide • oxygen • free radical
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Introduction
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The free radical
species
·NO is an endogenously generated
mediator
of smooth muscle relaxation and inhibitor of
platelet/leukocyte
activation that is essential for maintenance of
vascular homeostasis.
In many vascular pathologies, altered
·NO generation
rates, often coupled with
accelerated
·NO removal through
poorly
understood pathways, leads to impaired
·NO
signaling
and secondary generation of toxic
·NO-derived
species.
1 2 3 4 5 6
Reaction of
·NO with
O
2·-, yielding
peroxynitrite
(ONOO
–), accounts for a major
part of the accelerated
·NO
removal
2 3 4 5 6
but is not the only mechanism involved,
because endothelium-derived
relaxing factor (EDRF) activity
is often incompletely restored by
O
2·-
scavengers.
7 8 9
This suggests that the reaction of
·NO
with other
biochemical "sinks" can also account for enhanced rates
of EDRF
consumption. Further evidence for alterations in
·NO
metabolism in vascular disease is
provided by observations of
the tyrosine oxidation/nitration product
3-nitrotyrosine (NO
2-tyr)
and elevations in
inducible nitric oxide synthase (iNOS or NOS2)
activity. For
example, both NO
2-tyr and NOS2 expression are
consistently
elevated clinically (transplant coronary artery disease,
atherosclerotic
lesions, cardiac allograft rejection, and myocardial
inflammation)
and in animal models of vascular disease
(hypercholesterolemia-induced
atherosclerosis, balloon-injured
arteries, ischemic heart injury,
and myocardial inflammation)
(reviewed in Reference 10
10 ; see
also References 11
11 –17).
Increased lipid oxidation is a characteristic feature of
inflammatory vascular diseases and has been suggested to sometimes play
a causative role, although this has not been conclusively
proven.18 19 20 21 22 23 24
The candidate mechanisms that generate oxidized lipids in vivo are
numerous and include metal-dependent Fenton oxidation, enzyme-catalyzed
oxidation by lipoxygenase (LOX) or myeloperoxidase (MPO), reaction with
hypochlorous acid (HOCl), cell-dependent oxidation via a diversity of
O2·- and
H2O2-generating oxidases,
and, finally, oxidation by ·NO-derived
reactive species (eg,
·NO2, nitryl
chloride [NO2C], and
ONOO–).25 26 27 28 29 30 31 32
In particular, support for a pathogenic role of LOX-catalyzed lipid
oxidation in vivo in atherogenesis includes the observations that
functional 15-LOX and its products are present in human and rabbit
lesions,21 33 34
disruption of the mouse 12/15-LOX gene diminishes atherosclerosis in
apoE-deficient mice, and inhibition of 15-LOX prevents development of
atherosclerosis in cholesterol-fed
rabbits.35 36 In
contrast, targeted overexpression of rabbit macrophage 15-LOX prevents
diet-induced
atherosclerosis.37
In the vasculature, nitric oxide
(·NO) and lipid oxidation signaling
pathways can potentially interact at several levels. Because of the
diversity of the biochemical pathways involved, an understanding of how
these processes might impact on vascular homeostasis is important. In
this review, we summarize current knowledge of how lipid oxidation
pathways and ·NO-derived species interact
at a chemical and cellular level and describe what is known about how
these interactions might influence disease
progression.
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Reactions of NO With Purified Lipids, Lipoproteins, and Membranes
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NO potently inhibits lipid oxidation in a variety of in
vitro
model systems, including unsaturated free fatty acid emulsions,
phosphatidylcholine
liposomes or LDL oxidized by
Cu
2+, azo initiators of
LOO
· formation,
ONOO
–, endothelial cells, or
macrophages.
30 31 38 39 40 41 42
This is primarily a consequence of
·NO
reacting
with lipid-derived radicals (eg,
L
·, LO
·, and
LOO
·)
via diffusion-limited rates
(10
9 to 10
11
[mol/L]
–1 ·
s
–1)
to terminate lipid peroxidation propagation
reactions.
38 40 43 44 45 46 47 48 49 50
During the reaction of LOO
· with
·NO, two molecules of
·NO are consumed as
the primary organic
peroxynitrite (LOONO) intermediate rapidly
decomposes
(t
1/2=0.2 to 0.6 second) to secondary radical
species
that reacts
further.
38 For example,
LOONO can decompose to
caged radicals
[LO
·
·NO
2], which can
either terminate
after rearrangement of
LO
· to an epoxide,
L(O)NO
2, or
dissociate and react with additional
·NO
(Figure 1
).
Alternatively, LOONO can hydrolyze to LOOH and
NO
2–. The
small
molecular radius and hydrophobicity of
·NO
facilitates
partitioning into hydrophobic membrane
compartments,
51 thus
enhancing
the efficacy of
·NO to terminate
lipid peroxidation.
The fast rate of reaction between
LOO
· and
·NO
(2
x10
9
[mol/L]
–1 · s
–1),
compared with
-tocopherol (1 to 5
x10
5
[mol/L]
–1 · s
–1,
with rate depending on the alkyl chain length
and charge
characteristics of the LOO
· species),
allows
·NO to spare tocopherol during
lipid peroxidation and
predicts that steady-state
·NO concentrations of 30 nmol/L
will
outcompete endogenous
-tocopherol concentrations (20 µmol/L)
for
termination of
LOO
·.
38
In addition, the reduction
of topheroxyl radical by ascorbate is less
effective at preventing
lipid oxidation–induced tocopherol loss than
the reaction
of
·NO with
LOO
·.
52
These comparisons underscore
the profound capacity of
·NO for antioxidant reactions
in the
vasculature. It is emphasized that these properties of
·NO
can be manifested only if alternative
prooxidative reactions
(eg,
·NO reaction
with O
2·- to yield
ONOO
–)
do not predominate. NO-mediated
termination of lipid radicals
can also limit secondary lipid
oxidation–mediated processes
that are involved in vascular injury,
including nuclear factor-
B(NF-
B)
activation, the linkage of
vascular cell adhesion molecule-1
(VCAM-1) gene expression with NF-
B
activation, lung injury secondary
to intestinal ischemia, pulmonary
epithelial cell oxygen injury,
and the cytotoxicity of
H
2O
2 and alkyl
hydroperoxides.
53 54 55 56 57 58 59
Finally,
·NO can undergo reactions with
O
2 and/or
O
2·- to yield
oxidizing and nitrating species
that also cause
·NO-dependent cytotoxicity in vitro
through
inducing lipid oxidation.
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Figure 1. Fate of the termination product of NO and lipid peroxyl radicals. The initial product, an organic peroxynitrite (1), rapidly decomposes by several mechanisms including hydrolysis to form lipid hydroperoxides (2) and via a caged radical pair (3). The caged radical pair, nitrogen dioxide and alkoxyl radical, can either terminate after rearrangement (one possible rearrangement is shown) (4) or dissociate and react further, eg, with additional molecules of nitric oxide (5).
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Peroxynitrite-Induced Lipid Oxidation and Nitration
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Peroxynitrite is unique as a lipid oxidant, because it
mediates
peroxidation of unsaturated fatty acids in the absence of
transition
metal
catalysts.
29 Peroxynitrite
is more than two orders of
magnitude more potent than
H
2O
2 in catalyzing lipid
oxidation
in vitro and, in contrast to transition metal catalysts,
mediates
LDL oxidation even in the presence of lipophilic
antioxidants.
60 In vitro,
ONOO
– oxidizes diverse classes of lipids
(eg,
purified fatty acids, neutral lipids and phospholipids, and
lipophilic
antioxidants and LDL lipids) forming conjugated diene,
malondialdehyde,
lipid peroxide, lipid hydroxide,
F
2-isoprostane, and oxysterol
products.
29 44 50 60 61 62 63
In the case of LDL, this results in an LDL
derivative recognized by the
macrophage scavenger
receptor.
64 In addition to
oxidizing lipids, ONOO
– mediates linoleate
and
LDL cholesteryl linoleate nitration to the derivative
LNO
265
(also A. Bloodsworth and B.A. Freeman, unpublished data, 2001).
This
reaction proceeds via either hydrogen abstraction by
·NO
2 or addition
mechanisms involving
NO
2+
(Figure 2
). A role for
ONOO
– in
initiating lipid oxidation in atherosclerosis
has been
suggested
66 ; however, this
requires that the barrier
of competing reactions with thiols be
overcome, which are present
intracellularly at concentrations of up to
10 mmol/L
(
k=5.9
x10
3
[mol/L]
–1·s
–1
for
ONOO
– reaction with
cysteine).
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Figure 2. Mechanisms of peroxynitrite oxidation and nitration of unsaturated lipids. Peroxynitrite oxidizes lipids by hydrogen abstraction (1), probably via nitrogen dioxide formation. Nitration could then occur via either hydrogen abstraction (2) or an addition reaction of a nitrosonium-like intermediate (3). In the case of hydrogen abstraction, a conjugated diene product is expected. With addition reactions, rearrangement of the double bonds would not occur.
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Reactions of the ·NO Metabolites ·NO2 and NO2Cl With Unsaturated Lipids
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Several reactive nitrogen species derived from
·NO oxidize
and nitrate unsaturated fatty
acids and their methyl/ethyl esters
in vitro. Nitrogen dioxide will
both oxidize and nitrate unsaturated
lipids, with nitration occurring
by hydrogen abstraction and
addition
reactions.
28 65 67 68 69 70 71 72 73 74
These reactions
result in formation of a complex mixture of products
including
nitrated lipid derivatives and alkylnitrites, including those
shown
in
Figure 3
.
75
Nitration of methyl linoleate and linolenate
by
·NO
2 proceeds via
initial hydrogen abstraction to
form a carbon-centered alkyl radical,
which at low oxygen tensions
combines with
·NO
2 to form
allylic nitro compounds
(Figure
2
). The yield of oxygen-containing lipid products
(eg, LOOH,
LOH, etc) formed by
·NO
2 oxidation thus
depends on the
concentration of O
2 that will
facilitate peroxidation
reactions.
28 At high
O
2 concentrations, for example, in lung lining
fluid,
·NO
2 will
predominantly mediate lipid oxidation. Conversely,
at low
O
2 tension (eg, within an inflamed hypoxic organ
or microvessel),
nitration reactions may preferentially occur. Somewhat
analogous
to
·NO,
·NO
2 can also react
at diffusion-limited
rates with peroxyl and alkoxyl radicals, leading
to inhibition
of peroxidation and formation of novel N-containing lipid
derivatives.
46 76
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Figure 3. Nitrated lipids formed from nitrogen dioxide reaction with ethyl linoleate. Extensive reactions and rearrangements are possible during these reactions, with known products (see Reference 76 for details of formation pathways) including alkyl nitrites (LONO) and nitrolipids (LNO2), shown.
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The oxidation of nitrite
(NO2–) by
MPO-derived hypochlorous acid (HOCl) will yield nitryl chloride
(NO2Cl),77 78
which, in purified LDL, depletes ß-carotene and -tocopherol,
initiates lipid oxidation, forms
3-nitrotyrosine,27 and can
yield an LDL particle similar to that found in foam cells. The direct
oxidation of NO2– by
MPO+H2O2 yields
·NO2 and also
oxidizes LDL lipids to a proatherogenic
particle.79 80 81
Oxidation and nitration of membrane lipids by MPO may be operative in
atherogenesis, because products of MPO activity are found in vascular
lesions25 82 ;
however, this has not been conclusively proven. Finally, acidification
of NO2– forms
nitrous acid (HONO), which decomposes to nitrosating and nitrating
species including N2O3
and ·NO2. Reaction
of ethyl linoleate with HONO yields several nitrated lipids, including
nitroalkenes and nitroalcohols, whereas reaction of lipid
hydroperoxides (LOOH) with HONO forms
nitroepoxylinoleate65 75
(Figure 4). Formation of HONO is favored at pH<4; therefore,
if these reactions are to occur in the vasculature, they will require
acidic microenvironments, for example, in the phagolysosomes of
neutrophils or macrophages.
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Figure 4. Products of nitrous acid reaction with ethyl linoleate and linoleate hydroperoxide. A, Nitrosation/nitration of ethyl linoleate by nitrous acid proceeds via similar pathways to nitrogen dioxide–dependent reactions. However, as a result of the low pH required for these reactions (typically <4.0), alkyl nitrite compounds are not stable and either hydrolyze to alcohols or lose HNO2 via acid catalysis, forming the species shown. B, Nitrosation of lipid hydroperoxides by HONO forms nitroepoxy derivatives and likely proceeds via the LOONO intermediate shown in Figure 1. The two possible rearrangements are shown.
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NO-Derived Reactive Species Modulate the Activity and Expression of Lipid Oxidation Enzymes
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Enzymes such as LOX, prostaglandin endoperoxide H
synthase (PGHS),
and cytochrome P450 (CYP) that oxidize lipids to
bioactive eicosanoids
play critical signaling roles in the regulation
of vascular
cell function and inflammatory responses and are
ubiquitously
expressed by virtually all vascular cells under both
physiological
and inflammatory conditions
(Figure 5
). Generally, lipid oxidation
by these enzymes
involves formation of enzyme-bound radical
intermediates, including
lipid alkyl (L
·) and peroxyl
(LOO
·)
radical species. Free peroxyl or
alkyl radicals react with
·NO
at
diffusion-limited rates. Thus, reaction of
·NO with
enzyme-bound lipid radicals will
modulate rates of eicosanoid
product formation and decrease
bioavailable concentrations of
·NO, as
discussed
below.
46 47 48 49
In addition to lipid
radicals, these enzymes form several other
intermediates that
can react with NO during turnover, including amino
acid or porphyrin
radicals and various redox states of iron. In the
next sections,
the known interactions of
·NO with the various enzyme
intermediates
formed during catalysis are discussed.
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Figure 5. Localization of lipid oxidation enzymes in the vasculature. LOX indicates lipoxygenase; PGHS, prostaglandin endoperoxide H synthase; TXS, thromboxane synthase; PGIS, prostacyclin synthase; and CYP, cytochrome P450.
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Prostaglandin Endoperoxide H Synthase
(PGHS)
Prostaglandins are generated via arachidonate
oxygenation by PGHS, of which there are both constitutive (PGHS-1:
stomach, gut, kidney, and platelets) and inducible (PGHS-2: fibroblasts
and macrophages) isoforms. Under inflammatory conditions, both NOS2 and
PGHS-2 expression is upregulated in tandem by proinflammatory cytokines
including interleukin-1 (IL-1) and tumor necrosis factor, indicating
that high levels of both prostaglandin and
·NO will be produced in concert in
vivo.83 84 85 86 87 88 89 90 91 92
Reactive nitrogen species have multiple effects on PGHS
activity. Purified PGHS-1 is not significantly inhibited by
·NO; however, in several cell types,
including endothelial cells and platelets,
·NO highly stimulates prostaglandin
production.93 94 95 96
In other cell types, ·NO suppresses
lipopolysaccharide (LPS)-induced PGHS-2 expression, resulting in
apparent enzyme
inhibition.97 98
Finally, in NOS2 knockout mice, less urinary prostaglandin
E2 is found, although platelets from these
animals generate more thromboxane B2 in
vitro.99
NO can interact in several ways with PGHS, by forming an
Fe-nitrosyl complex, acting as a peroxidase-reducing substrate,
directly terminating the catalytic tyrosyl radical in the enzyme active
site, and theoretically, by termination of enzyme-bound lipid
radicals93 100 101 102
(Figure 6). Formation of the nitrosyl complex of PGHS by
·NO is favored for the ferrous form,
whereas the
Kd for
formation of the ferric ·NO complex is
very high, being 0.92 mmol/L for
·NO.102
Because the ferrous enzyme is not involved in enzyme catalysis and
nonbiological (mmol/L) concentrations of
·NO would be required for significant
metal center reaction to occur, nitrosyl complex formation is unlikely
to be a mechanism of PGHS activity modulation by
·NO in vivo. Although termination of
tyrosyl radicals by ·NO proceeds at
essentially diffusion-limited rates, it is intriguing that PGHS is not
more readily inhibited in vitro by ·NO.
Possible explanations are that ·NO-tyrosyl
radical reactions are readily reversible, or by acting as a
peroxidase-reducing substrate, ·NO
alternatively contributes to enzyme
activation.93 103
Peroxynitrite is also an oxidizing-peroxidase substrate for both PGHS-1
and PGHS-2, suggesting that ·NO could
activate prostaglandin synthesis under inflammatory conditions where
O2·- production is
abundant.104 105
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Figure 6. Potential sites of NO interaction with PGHS. The cyclooxygenase and peroxidase catalytic cycles of PGHS are shown. Tyr indicates tyrosine; LOOH, lipid hydroperoxide; LOH, lipid hydroxide; AA, arachidonate; and NO2-Tyr, nitrotyrosine.
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Lipoxygenases (LOX)
Lipoxygenases are non-heme iron–containing enzymes
that catalyze oxidation of arachidonate or linoleate to bioactive lipid
hydroperoxides. In mammalian cells, at least three isoforms are known
with the best characterized, 5-LOX, found mainly in
leukocytes.106 12-LOX
isoforms are present in platelets and
monocytes.107 108
15-LOX is expressed in reticulocytes during maturation into
erythrocytes, where it plays a central role in intracellular membrane
degradation. In human monocytes, expression of 15-LOX is induced by
IL-4 and
IL-13.109 110 A
role for 15-LOX in the initiation and progression of atherosclerosis
has also been suggested by the observation of 15-LOX products at
elevated levels in atherosclerotic
lesions.21 22 34 111
Lipoxygenases contain a single non-heme iron that alternates
between Fe2+ and
Fe3+ during catalysis. Resting enzyme
predominantly exists as the reduced form, requiring oxidation by
hydroperoxides before dioxygenation can occur. Inhibition of LOX
(soybean, rabbit and human 15-LOX, and human platelet 12-LOX) by
·NO has been
reported43 112 113 114 115
and was proposed to result from formation of an Fe-nitrosyl complex
with the ferrous enzyme. However, metal center reaction only occurs at
high and nonphysiological ·NO
concentrations,116 117 118 119
making this pathway of LOX inhibition unlikely. Rather, tissue LOX
inhibition results from a termination reaction between
·NO and the enzyme-bound lipid peroxyl
radical
(EredLOO·),43
which would be expected to occur at nanomolar concentrations of
·NO present in vivo. After this reaction,
dissociation and hydrolysis of the organic peroxynitrite (LOONO) gives
LOOH and NO2– as
products
(Figure 7). Because the LOX catalytic cycle is not completed,
reoxidation of the enzyme-bound iron is
required.43 Thus, because
·NO reaction occurs after
O2 insertion into the fatty acid substrate, the
LOX product profile is unchanged, the rate of product generation is
suppressed, and ·NO is
consumed.
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Figure 7. Reaction of NO with enzyme-bound lipid peroxyl radicals formed during lipoxygenase turnover. Ered indicates inactive native ferrous enzyme; Eox, active ferric enzyme; LH, unsaturated lipid substrate; and LOOH, lipid hydroperoxide product. Reaction of NO with enzyme-bound lipid peroxyl radicals suppresses lipid oxidation by forcing the enzyme to undergo an activation step during each catalytic cycle.
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Cytochrome P450 (CYP)
CYP enzymes are a ubiquitously expressed family
of heme proteins that play major roles in xenobiotic metabolism and
lipid oxidation. Nonhepatic CYP arachidonate metabolites also act as
intracellular signaling molecules in vascular tissue. For example, the
CYP4A product 20-hydroxyeicosatetraenoic acid (20-HETE) is a
potent vasoconstrictor whose generation in vascular smooth muscle cells
is inhibited by
·NO.120
A second product, 11,12-EET, is produced by endothelial cells, avidly
esterified into endothelial phospholipid pools, and mediates vascular
relaxation, possibly accounting for a component of the presently
undescribed endothelium-derived hyperpolarizing factor
activity.121 122 123
Preformed EETs in endothelial membranes can influence vascular function
by altering membrane characteristics, ion transport, or lipid-dependent
signaling pathways.124 For
example, one isomer, 5,6-EET, mediates vasodilation by either
increasing ·NO production through
stimulating Ca2+ influx into endothelial
cells125 or by directly
activating smooth muscle Kca
channels.121 126
NO has been shown to inhibit the CYP enzymes thromboxane synthase and
prostacyclin synthase in vitro. This can have a significant effect on
vascular function, in that these enzymes generate thromboxane and
prostacyclin, eicosanoid mediators that are central in regulation of
platelet aggregation and smooth muscle tone in
vivo.127 Formation of
nitrosyl complexes has been observed for some CYP isoforms; however,
the detailed mechanisms by which ·NO
interacts with CYP have not been
elucidated.
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Catalytic ·NO
Consumption by Lipid Oxidation Enzymes
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PGHS and LOX catalyze
·NO
consumption via reaction with
intermediates formed during enzyme
turnover, a reaction first
confirmed using purified enzymes and
isolated vascular cells,
including platelets and
monocytes
43 103
(also M.J. Coffey and
V.B. O’Donnell, unpublished data, 2001).
In these cell
models, the rates and amounts of
·NO consumed are high,
relative to
expected rates and amounts of
·NO
generated,
suggesting that these reactions might play a role in both
physiological
and pathological
·NO removal
in vascular cells.
Studies of soybean LOX-1, purified rabbit reticulocyte
15-LOX, human 15-LOX in murine fibroblast PA317 cells, and porcine
leukocyte 12-LOX in monocytes have shown that the reaction of
·NO with
EredLOO· results in
turnover-dependent ·NO
consumption43 (also
M.J. Coffey and V.B. O’Donnell, unpublished data, 2001). This
scavenging of ·NO effectively prevents
activation of purified or monocyte soluble guanylyl cyclase
(sGC), indicating that enzyme-bound lipid radicals can compete with the
heme of sGC for ·NO binding and thus
attenuate the bioactivity of ·NO in
mammalian cells
(Figure 8A).43
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Figure 8. Schematic diagram showing consequences of PGHS and lipoxygenase consumption of NO in monocytes and platelets. PGH2 indicates prostaglandin H2; AA, arachidonate; LH, unsaturated lipid substrate; and LOOH, lipid hydroperoxide.
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Through acting as a peroxidase-reducing substrate,
·NO is also consumed rapidly by both
purified PGHS-1 plus arachidonate and by the A23187 or
thrombin-activated PGHS-1 activity of human
platelets.103 Rates of
·NO removal by platelets are fast enough
to deplete micromolar ·NO levels and
potently prevent ·NO-dependent activation
of platelet sGC, thus causing platelets to overcome the antiaggregatory
effects of ·NO
(Figure 8B). This reveals a second novel proaggregatory
function for PGHS-1 in addition to its generation of proaggregatory
eicosanoids—specifically, catalytic consumption of the antiaggregatory
species ·NO.
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Lipid Oxidation Products Regulate NO
Bioactivity
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Although
·NO production
regulates the induction and activity
of lipid oxidation enzymes, their
eicosanoid products can conversely
modulate rates of cellular
·NO production. In platelets,
activation
of nitric oxide synthase is inhibited by aspirin
or indomethacin, an
effect that is overcome by addition of thromboxane
A
2.
128
In the murine macrophage cell line J774, induction of NOS2
by LPS is
inhibited by
indomethacin,
129 inferring
involvement
of PGHS products. Activation of LOX also leads to increases
in
NOS2 expression. For example, the nonspecific LOX inhibitor
nordihydroguaiaretic
acid prevents induction of NOS2 in myocytes or
smooth muscle
by IL-1 or LPS,
respectively.
130 131
In addition, isolated
peritoneal macrophages from 12-LOX knockout mice
display 50%
less
NO
2–+NO
3-
generation after interferon-
/LPS
challenge.
132 Finally,
oxidized LDL can have opposing effects on
·NO
bioactivity, either through
lysolecithin-dependent impairment
of endothelium-dependent arterial
relaxation or by causing induction
of
NOS2.
133 134
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Conclusions: Implications for Vascular Disease
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NO and
·NO-derived
reactive species interact with lipid
oxidation pathways via multiple
mechanisms in vitro that are
only recently being revealed. Because both
processes are central
to vascular regulation, an understanding of the
particular interactions
that are involved in pathogenesis of vascular
disease in vivo
is important. A role for
·NO acting as an antioxidant
in vivo by
inhibiting proatherogenic lipid oxidation is suggested,
because
increasing
·NO bioactivity through
l-arginine
supplementation
has been successful in attenuating vascular
dysfunction in
hypercholesterolemic
rabbits.
135 136 137
In
humans, results have been mixed, with intravenous infusion of
l-arginine
acutely
improving coronary vasodilation but having no effect
on microvascular
endothelial function in patients with
hypercholesterolemia.
138 An
alternative successful strategy has been to lower steady-state
concentrations
of
·NO-inactivating
reactive oxygen species in animal
models, via supplementation with
antioxidant enzymes and oxidant
scavengers.
139 140 141 142 143 144
Several isoforms of PGHS and LOX are upregulated
in both clinical and
experimental cases of vascular
disease,
21 34 145 146 147 148 149 150 151
with inhibition of these enzymes
normalizing blood pressure in some
cases.
150 152 153 154
Our
observations of catalytic
·NO
consumption by PGHS and
LOX indicate novel mechanisms by which these
enzymes might contribute
to blood pressure regulation, in addition to
the generation
of vasoactive prostanoids. These are the first
demonstrations
of controlled
·NO removal
by regulated catalytic processes
in mammalian cells. Recent studies
using iNOS knockout mice
demonstrated altered urinary prostaglandin
E
2 levels, confirming
direct interactions
between
·NO signaling and PGHS pathways
in
vivo.
99 Finally, the
reactions of
·NO and
·NO-derived
species (eg,
·NO
2 or
ONOO
–) with oxidizing lipids
leads to
generation of novel nitrated lipid derivatives. If
formed in
atherosclerotic lesions, these species will either
act as novel
·NO donors or possess distinct signaling
properties
similar to eicosanoids (A. Bloodsworth, V.B. O’Donnell,
and
B.A. Freeman, unpublished data, 2001).
Currently, knowledge regarding interactions between lipid
oxidation pathways and ·NO is mainly from
in vitro and animal model studies. Although great progress has been
made at that level, a challenge for the future is to more incisively
define which reactions are involved in the maintenance of vascular
homeostasis and the initiation and progression of clinical vascular
diseases.
|
Acknowledgments
|
|---|
This work was supported by the British Heart
Foundation (V.B.O’D.)
and National Institutes of Health Grants
RO1-HL64937, RO1-HL58115,
and P6-HL58418 (B.A.F). V.B.O’D. is a
Wellcome Trust RCD
Fellow.
|
Footnotes
|
|---|
Original received September 13, 2000; revision received October
26, 2000; accepted November 10, 2000.
|
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Abstract
Introduction