Uncoupling of Endothelial Nitric Oxidase Synthase by Hypochlorous Acid: Role of
NAD(P)H Oxidase−Derived Superoxide and Peroxynitrite
Jian Xu, Zhonglin Xie, Richard Reece, David Pimental and Ming-Hui Zou
Arterioscler Thromb Vasc Biol. 2006;26:2688-2695; originally published online October 5,
2006;
doi: 10.1161/01.ATV.0000249394.94588.82
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Atherosclerosis and Lipoproteins
Uncoupling of Endothelial Nitric Oxidase Synthase by
Hypochlorous Acid
Role of NAD(P)H Oxidase–Derived Superoxide and Peroxynitrite
Jian Xu, Zhonglin Xie, Richard Reece, David Pimental, Ming-Hui Zou
Objective—The aim of the present study is to determine whether hypochlorous acid (HOCl), the major oxidant of
leukocyte-derived myeloperoxidase (MPO), oxidizes the zinc-thiolate center of endothelial nitric oxide synthase (eNOS)
and uncouples the enzyme.
Methods and Results—Exposure of purified recombinant eNOS to HOCl (ⱖ100 ␮mol/L) released zinc and disrupted the
enzyme-active eNOS dimers. In parallel with increased detections of both O2䡠⫺ and ONOO⫺, clinically relevant
concentrations of HOCl disrupted eNOS dimers in cultured human umbilical vein endothelial cells (HUVEC) at
concentration 10- to 100-fold lower than those required for recombinant eNOS. In HUVEC, HOCl increased the
translocation of both p67phox and p47phox of NAD(P)H oxidase and the phosphorylation of atypical protein kinase C-␨.
Further, genetic or pharmacological inhibition of either NAD(P)H oxidase– derived O2䡠⫺ or PKC-␨ or NOS abolished the
effects of HOCl on eNOS dimers. Consistently, HOCl increased both O2䡠⫺ and ONOO⫺ and eNOS dimer oxidation in
isolated mouse aortas from C57BL/6 but less in those of gp91phox knock-out mice. Finally, in human carotid
atherosclerotic arteries, eNOS predominantly existed as monomers in parallel with increased staining of both MPO and
3-nitrotyrosine.
Conclusions—We conclude that HOCl uncouples eNOS by ONOO⫺ generated from PKC-␨– dependent NAD(P)H
oxidase. (Arterioscler Thromb Vasc Biol. 2006;26:2688-2695.)
Key Words: hypochlorite 䡲 endothelial dysfunction 䡲 NAD(P)H oxidase 䡲 peroxynitrite
C
ardiovascular diseases are frequently associated with
vascular inflammation even in the early stage, characterized by leukocyte and platelet adherence to the vascular
wall.1,2 There is evidence that leukocyte-derived myeloperoxidase (MPO) is a potent inducer for vascular injury. MPO
from adhered leukocytes is bound to vascular endothelium3
and is transcytosed to its luminal side4 where it can catalytically consume nitric oxide (NO) to reduce NO bioactivity.5,6
In cardiovascular diseases, MPO is found to be both present
and active in human atherosclerotic lesions.7 Under physiological conditions, HOCl is the major product of leukocyte
protein MPO.8 HOCl is able to cause protein oxidation. For
example, immunostainings for HOCl-modified proteins are
shown to be significantly elevated within and outside of
endothelial cells of human atherosclerotic lesions,9 suggesting that HOCl-mediated protein oxidation occurs in atherosclerosis. In addition, clinically relevant concentrations of
HOCl are shown to induce endothelial cell apoptosis,10
oxidize LDL,11 and impair NO bioavailability.12 However,
the targets and mechanism(s) by which HOCl contributes to
vascular injury remained elusive.
See page 2585
A key determinant of endothelial biology is the cell redox
state, and a key molecule that mediates endothelial function is
NO.13,14 NO is a free radical gaseous molecule and is
synthesized by the action of the enzymes nitric oxide synthases (NOS). The catalytic mechanisms of NOS involve
flavin-mediated electron transport from the C-terminal–
bound NADPH to the N-terminal heme center where oxygen
is reduced and incorporated into the guanidine group of
L-arginine giving rise to NO and L-citrulline. All three NOS
require an active dimer to release NO, and regulation of the
dimeric state of the enzyme complex has been shown to be an
important regulatory step in the function of all NOS isoforms.
L-arginine and tetrahydrobiopterin (BH4) are critical factors
in maintaining the dimeric state of the enzyme, which is
crucial in regulating how electrons are transferred within the
enzyme complex. In the setting of limited L-arginine or BH4
levels, the enzyme functions in an “uncoupled” state in which
NADPH-derived electrons are added to molecular O2 rather
than L-arginine, with O2䡠⫺ as products. This phenomenon was
first recognized in purified nNOS but also extended to eNOS.
Original received June 23, 2006; final version accepted September 15, 2006.
From the Section of Endocrinology and Diabetes, Department of Medicine (J.X., Z.X., R.R., M.-H.Z.), University of Oklahoma Health Sciences Center,
Oklahoma City; and the Division of Cardiology, Department of Medicine (D.P.), Boston University School of Medicine, Boston, Mass.
Correspondence to Ming-Hui Zou, MD, PhD, Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health
Sciences Center, BSEB 325, 941 Stanton L. Young Blvd, Oklahoma City, OK 73104. E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000249394.94588.82
2688
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Xu et al
O2䡠⫺ generated by eNOS has been implicated in a variety of
experimental and clinical vascular disease states including
diabetes, cigarette smoking, hypertension, chronic nitrate
tolerance, and overt atherosclerosis.15 For example, chronic
overexpression of eNOS does not inhibit but accelerates
atherosclerosis under hypercholesterolemia, suggesting that
eNOS might not release vasoprotective NO but O2䡠⫺ in vivo.15
How eNOS becomes uncoupled remained poorly understood.
The present study was aimed to study how HOCl, the
principle oxidant from leukocyte-derived MPO, triggers
eNOS oxidation and uncouples the enzyme.
Methods and Materials
Materials
Human umbilical vein endothelial cells (HUVECs) and cell culture
media were obtained from Cascade Biologics. Polyclonal or monoclonal antibodies against eNOS (Transduction Laboratories), p47phox,
and p67phox were obtained from Santa Cruz Biotechnology. The
antibody against 3-NT was from the Upstate Biotechnology, Inc.
Dihydroethidium (DHE) was purchased from Molecular Probes.
Calcium ionophore A23187 was obtained from Sigma Chemical Co.
Bovine recombinant eNOS obtained from Cayman Chemical, was
further purified using a 2⬘,5⬘-ADP-Sepharose 4B column and calmodulin-Sepharose affinity column as described previously.16,17
Human Carotid Artery Atherosclerotic Plaques
Human carotid artery atherosclerotic plaques were obtained from
sixteen consecutive, not previously examined, surgical inpatients
enlisted to undergo carotid endarterectomy for extracranial highgrade internal carotid artery stenosis (⬎70% luminal narrowing).
The degree of luminal narrowing was determined by repeated
Doppler echography and intraarterial cerebral angiography using the
criteria of the North American Symptomatic Carotid Endarterectomy
Trial (NASCET). Carotid artery endarterectomy was conducted in
standard fashion. Once removed, the plaque sample obtained from
each patient was divided into two parts, ie, “low stenosis” and “high”
stenosis areas, according to the degree of carotid artery stenosis. The
samples were immediately either frozen in liquid nitrogen (LN2) or
formalin fixed for further analysis. The study was reviewed and
approved by the institutional review board of the University of
Tennessee Medical Center at Knoxville.
Mice and Aortas Preparation
Female gene knock-out mice (gp91phox⫺/⫺) and the genetic controls
C57BL/6J mice, 10 weeks of age, were obtained from the Jackson
Laboratory (Bar Harbor, Me). Mice were housed in temperaturecontrolled cages with a 12-hour light-dark cycle and given free
access to water and normal chows. Mouse aortas were removed
immediately after being euthanized with inhaled isoflurane and
cervical dislocation. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee.
The isolated aortas were treated with HOCl as reported.12 Briefly,
isolated mice aortas were removed, cleared of connective tissue, and
immersed in warm PSS (37°C) for equilibration for 90 minutes.
Subsequently, vessel segments were incubated for 30 minutes in
control PSS or in PSS containing HOCl. After extensive washings
with PSS, DHE (10 ␮mol/L) was then added to the PSS for 30
minutes followed by an additional 30 minutes in control PSS. The
segments were subjected to cryotome tissue dissection for immunochemistry (for 3-NT staining) or fluorescent microscopy (for DHE
staining) as previously described.18,19 The staining signal was quantified using AlphaEaseFC software (Alpha Innotech) on the image
taken under the same amplification and exposure time.
Cell Treatment
Confluent HUVECs were exposed to HOCl for 15 minutes at
concentrations indicated. The residual HOCl was removed by exten-
HOCl Oxidizes eNOS Dimers
2689
sive wash with PBS buffer. HOCl-treated HUVEC were further kept
in culture media at 37°C for 30 minutes to 2 hours. The concentrations of HOCl were determined spectrophotometrically in 0.1 mol/L
NaOH (E292 nm⫽350 mol/L cm⫺1).
Assay of eNOS Dimer/Monomer
The total eNOS expression was detected by using Western blots.
eNOS dimers and monomers were assayed by using low temperature
SDS-PAGE, as described previously.16
Detection of Zinc Release
Zinc release was measured by using PAR assay as described
previously.16
Detection of Reactive Oxygen Species
Intracellular O2䡠⫺ was assessed by using DHE fluorescence. Cells were
incubated with DHE (1 ␮mol/L) for 30 minutes, followed by fluorescent
measurement under Em/Ex⫽480/580 nm using Synergy HT MultiDetection Microplate Reader (BioTek Instrument, Inc). O2䡠⫺ generated
by recombinant eNOS was assayed by using the DHE fluorescence/
high-performance liquid chromatography (HPLC) assay19 but with
modification. Briefly, recombinant eNOS (10 ␮g) was incubated with
DHE (0.5 ␮mol/L) in assay buffer (Tris-HCI buffer, pH 7.4; calmodulin: 1 ␮g/mL; L-arginine: 0.5 mmol/L) on ice for 30 minutes, followed
by methanol extraction. HPLC was applied to separate and quantify
oxyethidium (product of DHE with O2䡠⫺) and ethidium (product of DHE
auto-oxidation) by column C-18 (mobile phase: gradient acetonitrile and
0.1% trifluoroacetic acid).19 O2䡠⫺ production was determined by the
conversion of DHE into oxyethidine.
Separation of Cytosolic and Membrane Fractions
Cytosolic and membrane fractions of cells were prepared as described previously.20
Adenovirus Infection
Confluent HUVECs were infected with either adenovirus encoding
Cu/Zn SOD (SOD-1), p67phox dominant negative (p67phox-DN), or
PKC-␨ dominant negative (PKC-␨-DN), as described previously.21
Adenovirus encoding green fluorescence protein (GFP) was used as
a control.
Assay of Nitric Oxide Synthase Activity
The NO synthesizing activity of eNOS was determined by quantifying the rate of the conversion of [3H] L-arginine to [3H] L-citrulline
in the presence or absence of calcium as previously described.22
Assay of Nitric Oxide Bioactivity
NO bioactivity was determined using cyclic GMP (cGMP) ELISA
kit from R&D System, Inc.
Statistical Analysis
Results were analyzed with a 2-way ANOVA. Values are expressed
as mean⫾SEM for n assays. A probability value of ⬍0.05 is
considered statistically significant.
Results
HOCl Disrupts eNOS Dimers, Releases Zinc,
Inhibits the NO-Synthetic Activity, and Induces
O2䡠ⴚ Production in Purified Recombinant eNOS
We had demonstrated that ONOO⫺, formed by O2䡠⫺ and NO
at a diffusion-controlled rate, oxidizes the zinc-thiolate cluster of eNOS and uncouples the enzyme.16 The high reactivity
of the zinc-thiolate center with ONOO⫺ is likely attributable
to the highest charge-to-atomic radius ratio of zinc, which
maintains partial cationic character even in a tetra-coordinate
complex like zinc-thiolate clusters of eNOS.16 Thus, anionic
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Arterioscler Thromb Vasc Biol.
December 2006
(final concentration from 1 ␮mol/L to 1 mmol/L) for 30
minutes. As shown in Figure 1A (stripped and gray bars),
HOCl (100 ␮mol/L) significantly increased zinc release from
both human and bovine eNOS. In parallel, HOCl
(ⱖ100 ␮mol/L) dissociated the eNOS dimers into monomers
(Figure 1B) and suppressed the conversion of 3H-arginine
into 3H-citrulline (Figure 1A, solid bar). However, low
concentrations of HOCl (ⱕ10 ␮mol/L) had no effect on
eNOS (Figure 1A, solid bar). Taken together, these results
suggest that HOCl at concentrations higher than 100 ␮mol/L,
is able to disrupt eNOS dimers, release zinc, and uncouple the
enzyme.
HOCl Inhibits eNOS Activity and NO
Bioavailability in Intact HUVECs
Figure 1. HOCl oxidizes the zinc-thiolate cluster of eNOS and
disrupts eNOS dimers. A, HOCl increases zinc release from the
purified bovine (stripped bar) or human (gray bar) recombinant
eNOS (n⫽4, *P⬍0.05). HOCl dose-dependently inhibits the NO
synthetic activity of bovine recombinant eNOS (solid bar, n⫽3,
*P⬍0.05), or eNOS from HUVEC (open bar, n⫽3, *P⬍0.05). B,
HOCl dissociates eNOS dimers into eNOS monomers in purified
recombinant eNOS. The blot is a representative of 6 experiments (n⫽6, *P⬍0.05).
oxidants, such as ONOO⫺, can oxidize the coordinated thiols
in the process of releasing zinc and forming disulfide bonds.
Under reducing conditions (␤-mercaptoethanol), the disulfide
bonds between eNOS monomers are broken, which result in
dissociation of eNOS dimers into monomer. Thus, the dimer/
monomer ratios can be used as a marker for the redox status
of eNOS.16
Like ONOO⫺, hypochlorite (OCl⫺) is an anionic oxidant.
Thus, it was interesting to investigate the effects of HOCl on
eNOS dimer stability. To this end, purified human or bovine
eNOS were exposed to different concentrations of HOCl
We next determined whether HOCl inhibited A23187induced NO release. Confluent HUVECs were exposed to
HOCl for 15 minutes at concentrations indicated. After that,
HUVECs were washed with media and further incubated for
2 hours. At the end of the 2 hour incubation, eNOS activity
was assayed by measuring the conversion of 3H-arginine into
3
H-citrulline. As shown in Figure 1A (white bars), HOCl
dose-dependently inhibited eNOS activity.
We next determined whether HOCl impaired the NO
bioavailability, as measured by the cGMP contents. HOCl at
10 ␮mol/L, which did not alter the basal levels of cGMP,
significantly suppressed the A23187-upregulated cGMP production (17.5⫾1.2 versus 6.0⫾0.7 pmol/mg protein, vehicle
versus HC-treated, n⫽3).
HOCl Disrupts eNOS Dimers and Enhances O2䡠ⴚ
Production in Intact HUVECs
We next determined whether HOCl oxidized eNOS dimers in
intact cells. As shown, HOCl (1 to 100 ␮ mol/L)
concentrations-dependent dissociated eNOS dimers into
monomers under reducing condition (Figure 2A). Interestingly, low concentrations of HOCl (1 to 10 ␮mol/L), which
had no effect on eNOS dimers in purified recombinant eNOS
(Figure 1B), significantly reduced the levels of eNOS dimers,
as seen by the decreased ratios of dimer/monomer in
HUVECs (Figure 2A). In addition, HOCl significantly inFigure 2. ONOO⫺-dependent eNOS oxidation in HUVEC exposed to HOCl. A, HOCl
dose-dependently dissociates the eNOS
dimer in HUVECs. The blot is a representative of 6 blots from 6 independent experiments. B, HOCl induces the production of
O2䡠⫺ in HUVECs (n⫽6, *P⬍0.05) which can
be prevented in the presence of L-NAME
(1 mmol/L) (n⫽6, P⬎0.5 versus control). C,
L-NAME attenuates HOCl-induced eNOS
dimer dissociation in HUVECs. Adenoviral
overexpression of Cu-Zn-SOD, but not by
the adenoviral infection control GFP, abolishes HOCl-induced eNOS oxidation in
HUVECs. The blot is a representative of 6
blots from 6 independent experiments. D,
HOCl increases 3-NT-positive proteins.
Notably, HOCl-enhanced 3-NT is attenuated
by either L-NAME (1 mmol/L) or adenoviral
overexpression of Cu-Zn-SOD. The blot is a
representative of 6 blots from 6 independent
experiments.
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Xu et al
creased the detection of O2䡠⫺ (Figure 2B). However, HOClenhanced O2䡠⫺ was abolished by L-NAME (1 mmol/L; Figure
2B), suggesting that O2䡠⫺ by HOCl was eNOS-dependent.
Notably these concentrations of HOCl were at least 10-fold
lower than those required in purified enzyme. These results
suggest that the effects of low concentrations of HOCl
observed in HUVECs were unlikely attributable to its direct
effects but might be mediated by (a) factor(s) generated after
being exposed to HOCl.
We next established the time-dependence of HOCl on
eNOS dimers in HUVECs. Unlike in purified eNOS, the
maximal effects of HOCl on eNOS dimers in HUVECs were
seen at 2 hour incubation (data not shown), which was in
parallel with an inhibition on eNOS activity (Figure 1A, open
bar). These results further support that HOCl uncoupled
eNOS by releasing (an) endogenous factor(s) in HUVECs.
Dissociation of eNOS Dimers in Cells Exposed to
HOCl: Evidence of ONOOⴚ-Induced eNOS Dimer
Oxidation in HUVECs
Our previous studies16 had demonstrated that ONOO⫺ generated by high glucose exposure oxidizes eNOS dimers into
monomers under reducing condition. We next investigated
whether ONOO⫺ was involved in HOCl-enhanced eNOS
dimer oxidation. Because ONOO⫺ is formed by simultaneous
generation of O2䡠⫺ and NO,23 inhibition of either O2䡠⫺ or NO
production attenuates the formation of ONOO⫺. To this end,
HUVECs were exposed to HOCl (1 to 10 ␮mol/L) either in
the presence of a NOS inhibitor, L-NAME (1 mmol/L) or in
cells overexpressing Cu/ZnSOD (to prevent O2䡠⫺). Indeed,
either inhibition of NOS with L-NAME or SOD overexpression attenuated HOCl-enhanced eNOS dimer oxidation (Figure 2C). Because neither O2䡠⫺ nor NO at low concentrations
alone cause eNOS dimer oxidation,24,25 these results suggest
that ONOO⫺ might be involved in HOCl-induced eNOS
oxidation.
Exogenous NO has been reported to induce eNOS dimer
dissociation through S-nitrosylation of the zinc-binding cysteine.25 We next determined whether HOCl increased
S-nitrosylation of eNOS. Using the same biotin-switch method,26 we found that HOCl (1 ␮mol/L to 1 mmol/L) did not
alter the levels of S-nitrosylated eNOS (data not shown).
HOCl Enhances the Formation of ONOOⴚ
in HUVECs
We next determined whether HOCl increased the production
of ONOO⫺. ONOO⫺ has a half-life less than 1 second at pH
7.4 and is unstable.23 Instead, ONOO⫺ is an important oxidant
for protein tyrosine nitration, and 3-NT is considered as a
“footprint” for ONOO⫺ production.24 Thus, we determined
whether HOCl increased the formation of 3-NT in HUVECs.
Exposure of HUVECs to HOCl significantly increased the
detection of 3-NT-postive proteins (Figure 2D, shown only
the major band). In addition, either overexpression of Cu/ZnSOD or L-NAME significantly reduced the levels of 3-NT in
HUVECs, implying that HOCl released ONOO⫺ (Figure 2D).
HOCl Induces NAD(P)H Oxidase-Derived O2䡠ⴚ
Anions in HUVECs
Several enzymes including NAD(P)H oxidases, xanthine
oxidase, mitochondria, or cyclooxygenases could be the
HOCl Oxidizes eNOS Dimers
2691
potential sources of O2䡠⫺ leading to eNOS uncoupling.27 We
next determined the sources of O2䡠⫺ in HOCl-treated
HUVECs. As shown in Figure 3A, HOCl significantly
enhanced O2䡠⫺ in HUVEC. Inhibition of mitochondrial respiration with either rotenone (5 ␮mol/L) or antimycin A
(1 mmol/L) did not alter HOCl-enhanced O2䡠⫺ release (data
not shown), excluding mitochondria as the sources of O2䡠⫺. In
contrast, apocynin (1 ␮mol/L), a selective inhibitor which
blocks NAD(P)H oxidase assembly and then its activity,
suppressed HOCl-enhanced O2䡠⫺ (Figure 3A), suggesting
NAD(P)H oxidase might be involved.
Further evidence for NAD(P)H oxidase as the source of
O2䡠⫺ came from genetic inhibition of NAD(P)H oxidase.
Adenoviral overexpression of the dominant negative mutant
of p67phox (p67phox-DN) abolished HOCl-enhanced O2䡠⫺ release (Figure 3A) and eNOS dimer dissociation (Figure 3B,
upper panel). Taken together, these results suggest that
NAD(P)H oxidase was the initial source of O2䡠⫺, which
resulted in the formation of ONOO⫺ and consequent eNOS
uncoupling.
HOCl Enhances Translocation of Cytosolic p47phox
and p67phox Into Membrane
NAD(P)H oxidase activation requires the translocation of
p67phox and p47phox subunits from cytosolic fractions into
membranes.28,29 We next investigated whether HOCl activated NAD(P)H oxidase in HUVECs, by measuring the
translocation of its subunits, p47phox and p67phox, from cytosolic into membrane fractions. As shown in Figure 3C (panel
left), HOCl significantly increased the amount of both p47phox
and p67phox in membrane fractions (Figure 3C), suggesting
that HOCl might increase the activation of NAD(P)H oxidase
in HUVECs.
HOCl-Activated NADP(H) Oxidase
Is PKC-␨–Dependent
We next determined how HOCl activated NAD(P)H oxidase
in HUVEC. PKC-␨ was reported to activate NADPH oxidase.30 We next determined whether inhibition of PKC-␨
attenuated HOCl-induced NADPH oxidase activation. As
shown in Figure 3C (panel right), inhibition of PKC-␨ with its
pseudosubstrate (PKC-␨-PS, 10 ␮mol/L) abolished HOClinduced translocation of both p47phox and p67phox into membranes, suggesting that HOCl might activate PKC-␨ resulting
in the translocation of p47phox and p67phox and activation of
NAD(P)H oxidase. Further, HOCl dose-dependently increased the phosphorylation of PKC- ␨ (Figure 3D). Moreover, overexpression of PKC-␨-DN mutant attenuated HOClinduced eNOS dimer dissociation (Figure 3B, bottom panel).
Taken together, these results indicate that HOCl activated the
NAD(P)H oxidases by activating PKC-␨.
Increased Myeloperoxidase Stainings Are
Associated With Increased eNOS Oxidation in
Human Atherosclerotic Plaques
We further determined whether or not HOCl contributed to
the status of eNOS dimers/monomers and oxidant stress in
human atherosclerosis. To determine the contributions of
HOCl in the development of atherosclerosis, we measured the
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Arterioscler Thromb Vasc Biol.
December 2006
Figure 3. HOCl activates NAD(P)H oxidase
and increases O2䡠⫺ in HUVECs. A, HOClinduced production of O2䡠⫺ (n⫽6, *P⬍0.05)
is blocked in the presence of the NAD(P)H
oxidase inhibitor, apocynin (10 ␮mol/L;
n⫽6, P⫽0.2), or by adenoviral overexpression of the NADPH oxidase subunit, p67phox
dominant negative mutants (p67-DN). B,
Overexpression of p67phox dominant negative mutants of NAD(P)H oxidases (upper
panel) or PKC-␨-dominant negative mutant
(bottom panel) abolishes HOCl-induced
eNOS dimer dissociation. The blot is a representative of 6 blots from 6 independent
experiments. C, HOCl enhances the translocation of the vascular NAD(P)H oxidase
subunits p67phox and p47phox from the
cytosol into the plasma membrane (left
panel), which is PKC-␨– dependent (right
panel). The blot is a representative of 6
blots from 6 independent experiments
(n⫽6, *P⬍0.05). D, HOCl increases the
phosphorylation of PKC-␨ in HUVECs (n⫽6,
*P⬍0.05). The blot is a representative of 6
blots from 6 independent experiments.
levels of MPO (the major enzyme releasing HOCl), 3-NT,
vascular cell adhesion molecule (VCAM)-1, as well as eNOS
dimer/monomers in human carotid atherosclerotic plagues
with either low or high stenosis. As shown in Figure 4A, the
antibody stainings against human MPO, 3-NT, and VCAM-1
were significantly elevated in the arterial tissues with high
Figure 4. Increased myeloperoxidase is associated with eNOS oxidation in human atherosclerosis. eNOS predominantly existed as monomers in
human aortic atherosclerotic plaques, which correlated with increased MPO, 3-nitrotyrosine, and VCAM-1 staining. A, MPO, 3-nitrotyrosine, and
VCAM-1 staining are increased in aortic atherosclerotic plaques. B, Increased eNOS monomerization in arterial tissues obtained from high stenosis,
but eNOS remains as dimers in the arterial tissues with low stenosis (n⫽16 *P⬍0.01). Plaques are obtained from patient who had undergone carotid
endarterectomy for extracranial high-grade internal carotid artery stenosis.
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Xu et al
HOCl Oxidizes eNOS Dimers
2693
Figure 5. NAD(P)H oxidase-dependent
O2䡠⫺ and ONOO⫺ by HOCl in vivo. A,
HOCl increases both O2䡠⫺ (DHE staining)
and 3-NT in aorta only from the
C57BL/6J control (n⫽10) but not in
those of the gp91phox knock-out mice
(n⫽10). B, Uric acid, the ONOO⫺ scavenger, blocks the dimer dissociation of
eNOS from mice (n⫽3, P⬍0.05) aorta
incubated with HOCl (10 ␮mol/L) for 1
hour. The blot is a representative of 3
blots from 3 independent experiments.
C, Proposed scheme of HOCl-induced
eNOS uncoupling: low concentrations of
HOCl enhance NADPH oxidase assembly
and translocation to the plasma membrane on endothelial cells, through activation of PKC-␨. The activated NADPH
oxidase generates O2䡠⫺ which further
enhances ONOO⫺ formation, resulting in
eNOS uncoupling, a state with enhanced
generation of O2䡠⫺ and attenuation of NO
production. Scavenging O2䡠⫺, interruption
of NADPH oxidase assembly, or inhibition of ONOO⫺ formation can prevent
HOCl-induced eNOS oxidation and
uncoupling. Direct interaction of HOCl
with eNOS, an alternative mechanism, is
indicated for high concentration of HOCl. All these events may eventually result in zinc release and uncoupling of eNOS, which might
lead to endothelial dysfunction and atherosclerosis.
stenotic areas, compared with the arterial tissues from low
stenosis. In parallel, the amounts of eNOS dimers were
significantly reduced, and eNOS predominantly existed as
eNOS monomers in the arterial tissues with high stenosis.
Instead, eNOS remained as dimers in the arterial tissues with
low stenosis (Figure 4B). Theses results suggest that HOCl
from MPO might increase oxidant stress and eNOS oxidation
in human atherosclerotic tissues.
HOCl Increases O2䡠ⴚ and 3-NT in Isolated
Thoracic Aorta From C57BL/6J Control But Not
in Those of gp91phox Knock-Out Mice
We next determined whether HOCl oxidized eNOS dimers by
activating NAD(P)H oxidases in isolated aortas. gp91phox
(NOX-2) is one of the catalytical subunits of NADPH oxidase
in vascular tissues, and several analogues of gp91phox such as
NOX-1 and NOX-4 have been identified in vascular tissues.27
We first determined whether NAD(P)H oxidase is required
for HOCl increased O2䡠⫺ and ONOO⫺ in isolated mouse aortas
ex vivo. To this end, mouse aortas isolated from both
wild-type C57BL/6J mice and gp91phox knock-out mice were
exposed to either vehicle (PBS buffer) or HOCl at concentrations indicated. As shown in Figure 5A, HOCl significantly increased both O2䡠⫺ and 3-NT in the wild-type mice but
less in those of the gp91phox knock-out mice, suggesting that
vascular NAD(P)H oxidase was required for HOCl-enhanced
O2䡠⫺ and ONOO⫺.
We further assayed whether HOCl dissociated eNOS
dimers, and if so, the contribution of endogenous ONOO⫺. As
shown in Figure 5B, HOCl significantly decreased the ratios
of eNOS dimers to eNOS monomers, indicating the disruption of the zinc-thiolate center of eNOS. Further, uric acid
(50 ␮mol/L), which is a potent scavenger for ONOO⫺ but not
for HOCl, significantly attenuated HOCl-enhanced dissocia-
tion of eNOS dimers. Becuase uric acid did not alter the
eNOS dimer/monomer ratios in normal tissues (Figure 5B),
these results strongly suggest that HOCl oxidizes eNOS by
HOCl-enhanced ONOO⫺. Taken together, these data suggest
that HOCl at 1 to 10 ␮mol/L activated vascular NADPH
oxidase to produce O2䡠⫺ and ONOO⫺ on reaction with NO
resulting in eNOS oxidation. Thus, HOCl might serve as a
“kindling” oxidant to turn eNOS into an O2䡠⫺ producing
enzyme.
Discussion
In the present study, we have for the first time presented
evidence demonstrating that HOCl, the major oxidant from
leukocyte-derived MPO, is a potent inducer for eNOS uncoupling and 3-NT in the development of cardiovascular diseases. We found that high concentrations of HOCl directly
oxidize the zinc-thiolate center of eNOS. Most importantly,
we found that clinically relevant concentrations of HOCl
uncouple eNOS and increases 3-NT via endogenous O2䡠⫺ and
ONOO⫺, likely via NAD(P)H oxidase stimulation. The key
evidence can be summarized as follows: First, the concentrations required for eNOS oxidation in intact HUVEC were 10
times less than those used in recombinant eNOS in vitro;
Second, the effect of HOCl required at least 30 minutes
preincubation, suggesting an endogenous process was required for HOCl; Third, low concentrations of HOCl increased O2䡠⫺ release and overexpression of SOD but not
catalase (not shown) attenuated HOCl-induced eNOS oxidation in intact cells. These data suggest that O2䡠⫺ generated by
HOCl was required for HOCl-induced eNOS oxidation;
Fourth, inhibition of flavin-protein or L-NAME, a nonselective NOS inhibitor, abolished the effects of HOCl, suggesting
that NO was required for HOCl-induced eNOS oxidation as
well; Fifth, HOCl increased the detection of 3-NT, a hallmark
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2694
Arterioscler Thromb Vasc Biol.
December 2006
of ONOO⫺, suggesting that HOCl might oxidize eNOS by
releasing endogenous ONOO⫺; Sixth, HOCl increased both
O2䡠⫺ and 3-NT in isolated aortas of wild-type mice but
increased much less in NAD(P)H oxidase gp91phox KO
mice, indicating that HOCl activated NADPH oxidase ex
vivo. Moreover, HOCl oxidized eNOS dimer in isolated
aortas from wild types but significantly less in those of
gp91phox KO mice, implying that NAD(P)H oxidase-derived
O2䡠⫺ and ONOO⫺ were the initial sources for eNOS dimer
oxidation. Finally, uric acid, a potent scavenger for ONOO⫺,
but not for HOCl, significantly prevented HOCl-induced
eNOS oxidation, confirming that HOCl via ONOO⫺ oxidizes
eNOS in isolated aortas ex vivo. Therefore, our results
suggest that HOCl might disrupt the zinc-thiolate center of
eNOS via endogenous reactive nitrogen species, likely
ONOO⫺.
We have further demonstrated that HOCl can upregulate
O2䡠⫺ release by activating NAD(P)H oxidase. This is a novel
finding, and the key evidence can be summarized as follows:
First, HOCl significantly increased the detection of O2䡠⫺ in
intact HUVECs, and inhibition of NAD(P)H oxidase with
apocynin abolished the effects of HOCl on eNOS; Second,
overexpression of the NAD(P)H oxidase subunit p67phox-DN
suppressed HOCl-induced O2䡠⫺ and eNOS oxidation; Third,
low concentrations of HOCl significantly increased the translocation of the NAD(P)H oxidase p67phox and p47phox; Fourth,
HOCl increased O2䡠⫺ and ONOO⫺ in the aortas obtained from
the wild-type animals but less in the gp91phox KO mice,
suggesting that NAD(P)H oxidase was involved in HOClincreased O2䡠⫺ release.
Increased formation of 3-NT has been found in over
hundreds of diseases and is considered the footprint of
ONOO⫺ in vivo.31 Recently, this conception has been challenged because HOCl is found to generate 3-NT in the
presence of nitrite.32 In addition, MPO, which can catalyze
3-NT in the presence of nitrite and hydrogen peroxide (H2O2),
has been considered as a major source of 3-NT in vivo
because the levels of 3-NT are significantly reduced in MPO
knockout mice.33 In the present study, we have provided
compelling evidence that MPO-mediated 3-NT might be
ONOO⫺-dependent. In HUVECs, we found that HOCl significantly increased both O2䡠⫺ and 3-NT in HUVECs, which
were inhibited by either overexpression of SOD or inhibition
of NOS with L-NAME. These data strongly suggest that
HOCl increased 3-NT via ONOO⫺. Although MPO is able to
generate 3-NT using hydrogen peroxide and nitrite as substrates,32 it is very unlikely that subcultured HUVECs contain
MPO. In addition, overexpression of SOD, which increases
H2O2 to promote MPO-dependent 3-NT,32 actually decreased
instead of increased the HOCl-enhanced 3-NT in HUVECs,
suggesting the 3-NT is not likely to be generated by MPO.
Other possibilities of 3-NT include a chemical reaction of
HOCl with nitrite.32 This reaction requires millimolar concentrations of both HOCl and nitrite, which is unlikely in
vivo. Enhanced formation of ONOO⫺ was shown to disrupt
eNOS dimers and reduce NO production. Cardiovascular risk
factors such as oxidized lipids and hyperglycemia increase
O2䡠⫺ generation in endothelium by several different pathways,
and O2䡠⫺ is known to inactivate NO and produce ONOO⫺.
ONOO⫺ can disrupt eNOS protein dimers through oxidation
and displacement of the zinc metal ion. Overall, our data
strongly suggest that HOCl might increase 3-NT via ONOO⫺,
both of which could be from the activated NAD(P)H oxidase
and the uncoupled eNOS. Thus, decrease in 3-NT in MPO
KO mice33 might be attributable to a decrease of HOCltriggered ONOO⫺ in the MPO-KO mice. Our data strongly
suggest that ONOO⫺ is a principle source of 3-NT in vivo.
Taken together, our data strongly suggest that HOCl is an
important inducer for vascular endothelial dysfunction, and
clinically relevant concentrations of HOCl increase O2䡠⫺ and
ONOO⫺ via vascular NAD(P)H oxidase, which generates a
“kindling” oxidant, ONOO⫺, resulting in eNOS uncoupling
and endothelial dysfunction both in vitro and in vivo. Because
vascular inflammation as well leukocyte adherence occurs
even in the early stage of numerous cardiovascular diseases,
our results suggest that HOCl-induced eNOS uncoupling
might represent a common pathological pathway in the
development of vascular diseases including hypertension,
diabetes, ischemic injury, and atherosclerosis.
Sources of Funding
This work was supported by the NIH grants (HL079584, HL080499,
and HL074399), a Research Award from the American Diabetes
Association, a Research Award from the Juveniles Diabetes Research Foundation, a grant from Oklahoma Center for Science and
Technology (OCAST), and the funds from the Paul H. Doris Eaton
Travis Chair Funds in Endocrinology of the University of Oklahoma
Health Sciences Center.
Disclosures
None.
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