Bradykinin-Induced Dilation of Human Coronary Arterioles
Requires NADPH Oxidase–Derived Reactive Oxygen Species
Brandon T. Larsen, Aaron H. Bubolz, Suelhem A. Mendoza,
Kirkwood A. Pritchard Jr, David D. Gutterman
Downloaded from http://atvb.ahajournals.org/ by guest on June 16, 2017
Objective—Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor in human coronary arterioles
(HCAs). H2O2 mediates bradykinin (BK)-induced vasodilation and reduces bioavailability of epoxyeicosatrienoic acids
(EETs); however, the cellular and enzymatic source of H2O2 is unknown.
Methods and Results—NADPH oxidase expression was determined by immunohistochemistry. Superoxide and H2O2
production was assayed in HCAs and human coronary artery endothelial cells (HCAECs) using dihydroethidium and
dichlorodihydrofluorescein histofluorescence, respectively. Superoxide was quantified by HPLC separation of dihydroethidium products. Diameter changes of HCAs were measured by videomicroscopy. NADPH oxidase subunits Nox1,
Nox2, Nox4, p22, p47, and p67 were each expressed in HCA endothelium. In HCAs or HCAECs incubated with
dihydroethidium and dichlorodihydrofluorescein, BK induced superoxide and H2O2 formation, which was inhibited by
gp91ds-tat or apocynin but not by gp91scram-tat or rotenone. HPLC analysis confirmed that BK specifically induced
superoxide production. Gp91ds-tat reduced vasodilation to BK but not to papaverine. 14,15-EEZE (an EET antagonist)
further reduced the residual dilation to BK in the presence of gp91ds-tat, but had no effect in the presence of
gp91scram-tat, suggesting that NADPH oxidase– derived ROS modulate EET bioavailability.
Conclusion—We conclude that endothelial NADPH oxidase is a functionally relevant source of H2O2 that mediates
agonist-induced dilation in the human heart. (Arterioscler Thromb Vasc Biol. 2009;29:739-745.)
Key Words: oxidant stress 䡲 endothelium 䡲 coronary circulation 䡲 neurohumoral control of circulation
䡲 hypertension– basic studies
H
2O2
is a reactive oxygen species (ROS) that functions as
an endothelium-derived hyperpolarizing factor (EDHF)
in some circulatory beds, including HCAs.1– 4 EDHF is
particularly important in the microcirculation, where vascular
resistance is regulated. As an EDHF, H2O2 may compensate
to maintain adequate perfusion in states of heightened oxidative stress such as cardiovascular disease, where nitric oxide
(NO)-mediated vasodilation is impaired. In addition to its
direct vasodilator properties, H2O2 can modulate bioavailability of EETs, cytochrome P450 (CYP)-derived metabolites of
arachidonic acid that also function as EDHFs.5
H2O2 arises by enzymatic or spontaneous dismutation of the
superoxide anion. Superoxide is generated from numerous intracellular sources, including mitochondria,4 CYPs,6 and
NADPH oxidases.7 The canonical Nox2-containing NADPH
oxidase was originally identified in phagocytes, where it mediates the microbicidal respiratory burst; however, Nox2 as well as
alternative NADPH oxidases containing Nox1 or Nox4 are now
recognized as an important source of ROS in vascular tissue as
well.8 Although the pathological role of NADPH oxidase is well
defined by its heightened participation in a variety of vascular
diseases,7,9 its possible contribution to physiological vascular
stimuli is less clear, especially in the human heart.
This study was conducted to examine the putative role of
NADPH oxidase in mediating dilation to BK in HCAs. Published data indicate that both flow-induced3 and BK-induced5
dilation of HCAs are sensitive to catalase, implicating a role for
H2O2 in these responses. Evidence points to the mitochondrial
electron transport chain as a critical source of ROS in response
to shear stress,4 but the enzymatic source of H2O2 in response to
BK is not known. The present study provides the first evidence
that NADPH oxidase produces ROS in the human coronary
microcirculation and that this enzyme is critical to the mechanism of dilation to BK, a physiological vasomotor agonist.
Specifically, we examined whether NADPH oxidase is (1)
expressed in HCAs, (2) responsible for BK-induced ROS
production, (3) required for BK-induced dilation, and (4) responsible for oxidative inhibition of the EET-mediated component of
BK-induced dilation.
Methods
For complete details of immunohistochemistry, fluorescence microscopy, HPLC, and videomicroscopy protocols, as well as a listing of
Received April 29, 2008; revision accepted January 28, 2009.
From the Cardiovascular Center (B.T.L., A.H.B., S.A.M., K.A.P., D.D.G.), the Departments of Surgery (K.A.P.) and Medicine (D.D.G.), Medical
College of Wisconsin, and Veterans Administration Medical Center (D.D.G.), Milwaukee, Wisconsin.
Correspondence to David D. Gutterman, MD, Senior Associate Dean for Research, Medical College of Wisconsin, 8701 Watertown Plank Road,
Milwaukee, WI 53226. E-mail [email protected]
© 2009 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
739
DOI: 10.1161/ATVBAHA.108.169367
740
Arterioscler Thromb Vasc Biol
May 2009
materials, please refer to the online supplement at http://atvb.
ahajournals.org.
Table.
Patient demographics (nⴝ55)
Tissue Acquisition
Sex, male
30 (55)
Fresh right atrial appendages were obtained as discarded surgical
specimens from patients undergoing cardiopulmonary bypass procedures, as reported previously.3,10 All tissue acquisition procedures
and experimental protocols were approved by the appropriate Institutional Review Boards. Demographic data and diagnoses were
obtained at the time of surgery.
Age, y (mean⫾SD)
68⫾14
Characteristics
n (%)
Surgical procedure
Coronary artery bypass graft
37 (67)
Valve replacement
Mitral
19 (35)
Immunohistochemistry
Aortic
13 (24)
Immunohistochemistry was performed to visualize NADPH oxidase
subunit expression as reported previously.11
Tricuspid
Superoxide and H2O2 Detection by
Fluorescence Microscopy
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Fluorescence detection of superoxide and H2O2 in HCAECs or HCAs
was performed using dihydroethidium (DHE) and 2⬘,7⬘-dichlorodihydrofluorescein diacetate (DCFH), respectively.3 Briefly, HCAECs or
HCAs were incubated with gp91ds-tat (5⫻10⫺5 mol/L, a specific
peptide inhibitor of NADPH oxidase12), gp91scram-tat (5⫻10⫺5 mol/L,
a scrambled control peptide12), apocynin (3⫻10⫺3 mol/L, an NADPH
oxidase inhibitor13), catalase (1000 U/mL), rotenone (10⫺6 mol/L, a
mitochondrial Complex I inhibitor4), or vehicle. HCAECs or HCAs
were then loaded with DCFH and DHE and exposed to BK (10⫺6
mol/L). Fluorescence detection of H2O2 and superoxide was performed
by krypton-argon laser excitation at 488 and 585 nm while recording
emission at 523 nm and 610 nm, respectively.
HPLC Measurement of Superoxide
HPLC separation and quantification of oxidized DHE products were
performed as described previously.14 Briefly, HCAs were incubated
with the NADPH oxidase inhibitor apocynin (3⫻10⫺3 mol/L) or
vehicle. DHE was then added and HCAs were incubated with BK
(10⫺6 mol/L) or vehicle. DHE metabolites were then extracted,
separated by HPLC, and quantified.14
Measurement of HCA Dilation
by Videomicroscopy
Dilation of HCAs was observed by videomicroscopy as described
previously.10 Briefly, isolated HCAs were cannulated, pressurized,
equilibrated, and constricted with KCl (50 mmol/L) to assess viability.
Only vessels that constricted ⬎30% were used for subsequent experiments.3,10,15 After washing, vessels were constricted 30% to 50% with
endothelin-1 (ET-1, 5⫻10⫺10 to 10⫺9 mol/L). Although published data
indicate little contribution of NO or prostacyclin to BK-induced dilation
of HCAs,15 all experiments were performed in the presence of N␻-nitro⫺4
L-arginine methyl ester (10
mol/L, an NO synthase inhibitor) and
indomethacin (10⫺5 mol/L, a cyclooxygenase inhibitor) to eliminate any
residual confounding effects of these vasodilators. Cumulative concentrations of BK (10⫺10 to 10⫺6 mol/L) were then added and steady state
diameters were measured. After washing, gp91ds-tat (5⫻10⫺5 mol/L)
or gp91scram-tat peptide (5⫻10⫺5 mol/L) was introduced both in the
superfusate and intraluminally under a brief period of flow. In other
vessels, 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE, 10⫺5 mol/L, an
EET antagonist16) was coadministered with gp91ds-tat or gp91scramtat. After equilibration, HCAs were again constricted with ET-1, and
cumulative concentrations of BK were again applied. At the end of each
experiment, papaverine (10⫺4 mol/L, an endothelium-independent vasodilator) was added to determine the maximum diameter.
Statistical Analyses
HPLC data, maximum percent vasodilation (%MD), EC50, and
fluorescence intensities were evaluated using paired or unpaired
Student t tests, whenever applicable. To compare concentration–
response relationships, a 2-factor repeated measures ANOVA was
used. When a significant difference was observed between curves
(P⬍0.05), responses at individual concentrations were compared
3 (5)
Other
2 (4)
Underlying diseases/risk factors
Coronary artery disease
40 (73)
Hypertension
33 (60)
Hypercholesterolemia
25 (45)
Atrial fibrillation
22 (40)
Diabetes mellitus
20 (36)
Tobacco use
15 (27)
Congestive heart failure
9 (16)
Myocardial infarction
3 (5)
using a Holm-Sidak multiple comparison test. Multiple stepwise
regression analyses were used to detect the influence of underlying
diseases, age, and gender on vasodilation at various concentrations.
All analyses were performed using SigmaStat, version 3.1. Statistical
significance was defined as P⬍0.05. All data are described as
mean⫾SEM; n⫽number of patients or experiments.
Results
Atrial appendages were obtained from 55 patients, yielding
135 HCAs with a mean internal diameter of 164⫾7 ␮m.
Patient demographic information is summarized in the Table.
Expression of NADPH Oxidase Subunits
To investigate whether NADPH oxidase may be a source of
ROS in HCAs, immunohistochemistry was used to evaluate
expression of NADPH oxidase subunits. As shown in Figure
1, the catalytic subunits Nox1, Nox2, and Nox4 as well as the
regulatory subunits p22, p47, and p67 are each expressed in
the endothelium. Nonspecific staining was not observed in
the absence of primary antibodies or in the presence of
preimmune isotype control antibodies. Consistent with these
findings, Western blot analysis of HCAEC protein demonstrated expression of Nox2, Nox4, and p22 (supplemental
Figure II). These findings provide indirect evidence that
NADPH oxidase may be a source of ROS in HCAs.
Bradykinin-Induced Reactive Oxygen Species
Production in HCAECs
It was recently reported that the endothelium is required for
BK-induced ROS production in intact HCAs5; however, it is
not known whether ROS actually originate from endothelial
cells in this context, nor has the enzymatic source of ROS
been determined. To assess whether endothelial NADPH
oxidase is required for ROS production in response to BK,
superoxide and H2O2 formation was assayed by DHE and
DCFH histofluorescence, respectively. In HCAECs, superox-
Larsen et al
NADPH Oxidase in Human Coronary Arterioles
741
Figure 1. Expression of NADPH oxidase
subunits in human right atrial appendage.
Representative images after immunohistochemical staining. A positive reaction is
indicated by the formation of a brown
pigment. A through F, Nox1, Nox2, Nox4,
p22, p47, and p67 are each expressed in
HCA endothelium (red arrows), and to a
variable degree in other cell types (n⫽4
each). G and H, Staining was absent
when primary antibody was omitted or
replaced by a preimmune isotype control.
n indicates number of patients.
Bar⫽25 ␮m.
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ide and H2O2 were increased by BK (fluorescence ratio
1.80⫾0.18 and 1.87⫾0.17, n⫽14 for both, respectively,
P⬍0.05), as shown in Figure 2. BK-induced ROS production
was inhibited by apocynin (1.04⫾0.03 and 0.97⫾0.04, n⫽6,
respectively, P⬍0.05 versus BK) but not by rotenone
(1.68⫾0.12 and 1.93⫾0.16, n⫽4, respectively, P⫽not significant [ns] versus BK), suggesting that ROS originate from
NADPH oxidase and not the mitochondrial proximal electron
transport chain. Importantly, catalase completely blocked the
increase in DCFH but not DHE fluorescence, indicating that
DCFH fluorescence was specific for H2O2. Taken together,
these results suggest that BK induces endothelial ROS production through activation of NADPH oxidase.
cence was performed on intact HCAs. As shown in Figure 3, BK
increased superoxide and H2O2 formation (fluorescence ratio
1.52⫾0.10 and 2.24⫾0.31, n⫽9 for both, respectively,
P⬍0.05). Importantly, BK-induced ROS production was completely inhibited by gp91ds-tat (0.89⫾0.05 and 0.98⫾0.06,
n⫽4, respectively, P⬍0.05 versus BK) or apocynin (0.90⫾0.18
and 0.84⫾0.24, n⫽5, respectively, P⬍0.05 versus BK) but not by
gp91scram-tat (1.40⫾0.15 and 1.96⫾0.29, n⫽4, respectively,
P⫽ns versus BK). Importantly, gp91ds-tat and apocynin similarly
affected baseline and BK-induced fluorescence, indicating that
nonspecific effects of apocynin, if any, did not significantly influence ROS production. Taken together, these results suggest that
NADPH oxidase is an important source of microvascular ROS.
Bradykinin-Induced Reactive Oxygen Species
Production in HCAs
Quantification of Bradykinin-Induced
Superoxide Production
To further assess whether NADPH oxidase is required for
BK-induced ROS production, DHE and DCFH histofluores-
Although DHE histofluorescence suggests that BK induces
superoxide formation, this method has an important limita-
Figure 2. Contribution of NADPH oxidase to
BK-induced ROS production in HCAECs. A, Representative histofluorescence images of HCAECs
incubated with DHE and DCFH to visualize superoxide and H2O2 production, respectively, in the
presence of apocynin, rotenone, or catalase.
Bar⫽25 ␮m. B, BK-induced ROS production
requires NADPH oxidase, as evidenced by the inhibitory effect of apocynin on the ratio of DHE and
DCFH fluorescence intensity (I) versus baseline (Io)
(n⫽4 to 6 for each group). n indicates number of
experiments. *P⬍0.05 vs vehicle, †P⬍0.05 vs
BK ⫹ vehicle.
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Arterioscler Thromb Vasc Biol
May 2009
Figure 3. Contribution of NADPH oxidase to
BK-induced ROS production in intact HCAs. A,
Representative histofluorescence images of HCAs
incubated with DHE and DCFH to visualize superoxide and H2O2 production, respectively, in the
presence of gp91ds-tat, gp91scram-tat, or apocynin. Bar⫽100 ␮m. B, BK-induced ROS production
requires NADPH oxidase, as evidenced by the inhibitory effect of gp91ds-tat or apocynin on the
DHE and DCFH fluorescence ratio (n⫽4 to 5 for
each treatment group). n indicates number of
patients. *P⬍0.05 vs vehicle, †P⬍0.05 vs BK ⫹
vehicle.
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tion. When oxidized, DHE forms 2 fluorescent products
(2-hydroxyethidium, 2-OH-E⫹, and ethidium, E⫹) with overlapping emission spectra; however, only 2-OH-E⫹ is specific
for superoxide.17 To specifically assess and quantify superoxide, we separated oxidized products of DHE from isolated
HCAs using HPLC. As seen in Figure 4, 2-OH-E⫹ formation
increased in the presence of BK (36.2⫾14.3 versus 24.6⫾4.6
pmol/␮g protein, ratio 1.52⫾0.19 versus control, n⫽6,
P⬍0.05). In contrast, BK did not increase E⫹ production.
Apocynin had little effect on baseline production of 2-OH-E⫹
(or E⫹, data not shown) but attenuated the increase in
2-OH-E⫹ in response to BK (24.1⫾6.8 pmol/␮g protein, ratio
1.05⫾0.12 versus BK alone, n⫽6, P⬍0.05). This suggests
that BK does indeed stimulate superoxide production in an
NADPH oxidase– dependent manner.
Contribution of NADPH Oxidase to
Bradykinin-Induced Dilation
To determine whether NADPH oxidase is a functionally
relevant source of H2O2, HCA vasodilation was measured
using videomicroscopy. As shown in Figure 5, BK elicited a
concentration-dependent dilation (% maximum dilation [MD]
93⫾3 and ⫺logEC50 7.8⫾0.3, n⫽10) that was inhibited by
gp91ds-tat (%MD 49⫾5 and ⫺logEC50 6.7⫾0.1, n⫽5,
P⬍0.05 versus BK alone) but not by gp91scram-tat (%MD
82⫾3 and ⫺logEC50 7.7⫾0.3, n⫽5, P⫽ns versus BK alone).
Interestingly, BK-induced dilation was not influenced by sex,
age, surgical procedure, or underlying disease. Dilation to
papaverine was not altered in the presence of either gp91dstat or gp91scram-tat, indicating that these peptides do not
directly impair vascular smooth muscle cell (VSMC) vasodilator capacity. Taken together, these results suggest that
NADPH oxidase is required for BK-induced dilation.
NADPH Oxidase–Derived H2O2 Reduces the
EET-Mediated Component of BK-Induced Dilation
Figure 4. Specific HPLC detection and quantification of superoxide. A, Relative production of 2-OH-E⫹, as calculated using
the area under the respective peaks. B, Estimate of microvascular 2-OH-E⫹ content, calculated using a standard curve from
authentic 2-OH-E⫹ and protein content. BK induced superoxide
formation in an NADPH oxidase– dependent manner. *P⬍0.05 vs
vehicle. n indicates number of patients.
Previous studies indicate that BK-induced dilation is predominantly mediated by H2O2; however, a prominent residual
dilation occurs in the presence of catalase that is mediated by
EETs.5 This secondary mechanism is masked by an inhibitory
effect of H2O2 on CYP enzymatic activity, which reduces
EET production; however, the source of ROS in this context
is unknown. To determine whether NADPH oxidase– derived
H2O2 reduces the EET-mediated component of BK-induced
dilation, EEZE was coadministered with gp91ds-tat or
gp91scram-tat. Interestingly, the residual dilation to BK in
the presence of gp91ds-tat was markedly inhibited by EEZE
(%MD 9⫾3 versus 45⫾6 and ⫺logEC50 6.5⫾0.1 versus
6.7⫾0.1 with gp91ds-tat alone, n⫽5, P⬍0.05, Figure 5).
Larsen et al
NADPH Oxidase in Human Coronary Arterioles
743
Figure 5. Contribution of NADPH oxidase to
BK-induced dilation. A, BK-induced dilation of
HCAs is reduced by gp91ds-tat but not by
gp91scram-tat (n⫽10, 5, and 5, respectively). B,
Papaverine-induced dilation is not reduced by
gp91ds-tat or gp91scram-tat (n⫽6, 3, and 3,
respectively). C, BK-induced dilation is not
reduced by EEZE in the presence of gp91scramtat (n⫽5). D, The residual dilation to BK in the
presence of gp91ds-tat is inhibited by EEZE (n⫽5).
†P⬍0.05 vs vehicle curve, *P⬍0.05 vs vehicle at
specific dose. n indicates number of patients.
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However, there was no effect of EEZE in the presence of
gp91scram-tat. These results suggest that EETs mediate
dilation to BK when NADPH oxidase is inhibited, and that
NADPH oxidase– derived H2O2 modulates the bioavailability
or action of EETs.
Discussion
This study is the first to directly investigate a role for NADPH
oxidase in the human coronary microcirculation. The novel
findings of the present study are 4-fold. First, NADPH
oxidase is expressed in HCAs. Second, BK induces endothelial ROS production in an NADPH oxidase– dependent manner. Third, NADPH oxidase is required for BK-induced
dilation. Fourth, an EET-mediated component of BK-induced
dilation is unmasked when NADPH oxidase is inhibited.
Taken together, these data suggest that NADPH oxidase is a
functionally relevant source of ROS that may modulate
vasomotor tone not only by producing H2O2, but also by
modulating EET bioavailability.
NADPH Oxidase and Agonist-Induced
ROS Production
Vascular NADPH oxidase activity is induced by several
physiological stimuli. The contribution of NADPH oxidase to
angiotensin II (AngII)-induced ROS production in VSMCs is
well established, where it plays a role in AngII-dependent
hypertension and VSMC hypertrophy.18 –20 NADPH oxidase
is also activated by mechanical stimulation,21 and its activation may play a role in atherogenesis in areas of oscillatory
shear stress.22
Despite evidence that BK induces H2O2 production in the
coronary circulation,2,5 little is known about the source of
ROS in response to this agonist. BK induces H2O2 release
from porcine coronary microvessels in a manner that requires
the endothelium; however, the enzymatic source of H2O2 in
this model has not yet been characterized.2 The present study
suggests that NADPH oxidase in endothelial cells may be a
source of ROS in response to BK. Inhibition of NADPH
oxidase reduces BK-induced dilation, indicating that this
enzyme complex is a functionally relevant mediator of
vasodilation. Although gp91ds-tat does not completely abolish this response, the magnitude of inhibition is similar to the
published inhibitory effect of catalase on BK-induced dilation
in these vessels.5
It is a somewhat unexpected finding that NADPH oxidase
mediates a vasodilator response to BK in light of substantial
evidence implicating this enzyme as an effector of AngII
signaling and impaired NO-dependent vasodilation.19,20 Animal studies indicate that AngII-induced NADPH oxidase
activity acutely impairs endothelium-dependent vasodilation23 and chronically promotes VSMC hypertrophy18 and
hypertension.12,23 It is not known how AngII and BK elicit
opposing vascular effects through activation of the same
enzyme; however, the present study suggests that critical
agonist-dependent or cell-dependent differences in NADPH
oxidase activation occur. Whereas NADPH oxidase in
VSMCs serves as an effector of AngII to promote vasoconstriction and hypertrophy, NADPH oxidase in endothelial
cells may serve as an effector of BK to modulate acute
vasodilatory function. It is also possible that NADPH oxidase
may have differential roles in normal versus diseased vessels.
In nondiseased vessels where NO-mediated dilation is significant,10 activation of NADPH oxidase may have a net
constricting effect by reducing NO bioavailability. In contrast, diseased HCAs mediate dilation via EDHFs and not via
NO10; therefore, activation of NADPH oxidase to generate
H2O2 may have a net dilating effect. If so, the ratio of
NADPH oxidase activation in endothelial cells versus
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Arterioscler Thromb Vasc Biol
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VSMCs may be an important factor in the regulation of
cardiovascular parameters such as blood pressure and tissue
perfusion.
It is widely recognized that the ratio of BK to AngII plays
an important role in blood pressure regulation. BK receptor
B2 knockout mice exhibit elevated basal blood pressure as
well as an exaggerated blood pressure response to AngII,24
suggesting that BK dampens the vasoconstrictive effects of
AngII. Angiotensin I converting enzyme (ACE) inhibitors
exploit this relationship between BK and AngII. ACE catalyzes not only AngII formation from its inactive precursor,
angiotensin I, but also inactivates BK. Importantly, the
beneficial cardiovascular effects of ACE inhibitors have been
attributed not only to reduced AngII bioavailability, but also
in part to enhanced BK bioavailability.25 The present study
suggests an intriguing mechanism that may underlie the
antihypertensive effect of ACE inhibitors; namely, that they
decrease blood pressure not only by suppressing AngIIinduced NADPH oxidase activity in VSMCs, but also by
promoting NAPDH oxidase activation in endothelial cells
through enhanced BK bioavailability. Although ACE inhibitors inhibit NADPH oxidase activity in human mononuclear
leukocytes26 and an AngII receptor blocker reduces AngIIinduced activation of NADPH oxidase in rats,23 additional
studies will be necessary to define the effect of ACE
inhibitors on endothelial and smooth muscle NAPDH oxidase
in humans.
Potential Study Limitations
An important limitation of the present study is the lack of
quantification of H2O2. Although HPLC of DHE metabolites
may be used to quantify microvascular superoxide, similar
methods for quantification of H2O2 have not yet been
developed. We previously detected flow-induced H2O2 production from HCAs by electron spin resonance4; however,
accurate quantification of H2O2 is not possible by this method
because of the limited number of arterioles that can be
isolated from a patient sample. Fortunately, animal models
are not fraught with the limitation of tissue availability.
Indeed, BK (10⫺6 mol/L) induces endothelial release of H2O2
from porcine coronary microvessels at an approximate concentration of 1.5⫻10⫺6 mol/L.2 Although the concentration
of H2O2 released from HCAs is unknown, the present study
suggests that it may be similar, if not higher. Catalase5 and
gp91ds-tat each inhibit HCA dilation to 10⫺6 mol/L BK by
⬇40%, a response that requires exogenous addition of H2O2
at a concentration of ⬇10⫺5 mol/L3, indirectly suggesting
that BK induces H2O2 production in HCAs in the micromolar
range.
An additional limitation of this study is a lack of direct
EET measurements. Although numerous attempts were made
to quantify EETs released from HCAs using HPLC or liquid
chromatography electrospray-ionization mass spectrometry,
the sensitivity of currently available methods is insufficient to
consistently and reproducibly measure EETs from such small
samples. We were also unable to demonstrate EET production from HCAECs, a finding consistent with the observation
that endothelial cells rapidly lose CYP expression and activity in culture.27 An alternative explanation is that HCAs may
not, in fact, produce EETs. Although this possibility cannot
be ruled out, we believe it is less likely, as CYPs and the
EET-metabolizing soluble epoxide hydrolase enzyme are
robustly expressed in HCA endothelium,11 suggesting that
HCAs are capable of producing and metabolizing EETs.
Indeed, vasodilation in the presence of gp91ds-tat is blocked
by EEZE, indirectly suggesting that EETs are produced when
NADPH oxidase is inhibited. This finding is consistent with
published data indicating that EET production is sensitive to
H2O2.11
The present study indicates that Nox1, Nox2, and Nox4 are
each expressed in HCAs; however, their relative physiological importance in this model is unknown. BK-induced dilation is reduced by gp91ds-tat, suggesting that Nox2 mediates
this response; however, other Nox isoforms may also contribute to this response, as gp91ds-tat cross-reactivity is
theoretically possible.12 Nox4 in particular may contribute to
H2O2-mediated vasodilation, as Nox4 produces much more
H2O2 than superoxide.28 Highly specific inhibitors will therefore be necessary to determine whether Nox1 or Nox4 are
also functional in HCAs.
An additional limitation of this study is the lack of heart
tissue from healthy subjects, as this is rarely obtainable.
However, this limitation is offset by the unique advantage of
studying NADPH oxidase in the clinically-relevant context of
chronic cardiovascular disease and its risk factors, conditions
that cannot be adequately mimicked in animal models. Recent
evidence indicates that NADPH oxidase activity may be
elevated in cardiovascular disease,7 rendering an evaluation
of NADPH oxidase critical in the diseased human heart.
Clinical Implications
With the expanding body of evidence implicating NADPH
oxidase in the pathogenesis of atherosclerosis, hypertension,
and inflammation, pharmacological inhibition of this enzyme
has emerged as a potential therapeutic approach for these
diseases.7 This study suggests a potential limitation of this
approach, as NADPH oxidase inhibitors may acutely reduce
agonist-induced vasodilation. On the other hand, the acutely
detrimental effects of NADPH oxidase inhibition, if any, may
be partially offset by enhanced EET bioavailability. H2O2 is
a proinflammatory and proatherosclerotic molecule,29 but
EETs have antiinflammatory and vasculoprotective effects,30
similar to NO. Although NADPH oxidase inhibition affects
human coronary microvascular function in vitro, additional
studies will be necessary to understand the global effects of
NADPH oxidase inhibitors in vivo.
Conclusions
The present study supports a role for NADPH oxidase as a
modulator of vasomotor tone in the human coronary microcirculation. NADPH oxidase is a functionally relevant source
of ROS that not only mediates agonist-induced vasodilation,
but may also reduce EET bioavailability. NADPH oxidase
activity may therefore influence coronary vascular resistance
and myocardial perfusion, but its physiological importance in
vivo remains to be determined.
Larsen et al
Acknowledgments
The authors thank the Division of Cardiothoracic Surgery at the
Medical College of Wisconsin, the Cardiothoracic Surgery Group of
Milwaukee, the Cardiovascular Surgery Associates of Milwaukee,
the Midwest Heart Surgery Institute, and the Wisconsin Heart Group
for providing surgical specimens, and Dr John R. Falck (UT
Southwestern, Dallas, Texas) for providing EEZE.
Sources of Funding
This work was supported by an American Heart Association Fellowship Award (to B.T.L.), a Veterans Administration Merit Award
(to D.D.G.), and the NIH (HL68769) (to D.D.G.).
Disclosures
None.
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Bradykinin-Induced Dilation of Human Coronary Arterioles Requires NADPH Oxidase−
Derived Reactive Oxygen Species
Brandon T. Larsen, Aaron H. Bubolz, Suelhem A. Mendoza, Kirkwood A. Pritchard, Jr and
David D. Gutterman
Arterioscler Thromb Vasc Biol. 2009;29:739-745; originally published online February 12,
2009;
doi: 10.1161/ATVBAHA.108.169367
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Larsen BT, et al.
Supplement Material
Expanded Materials and Methods
Immunohistochemistry:
Immunohistochemistry was performed to visualize
NADPH oxidase subunit expression in human atrial tissue as reported previously.1
Briefly, right atrial appendages were fixed for 24 hours in zinc formalin buffer,
embedded in paraffin, and sliced into 4 µm sections. Sections were immunolabeled with
a goat anti-human polyclonal antibody against Nox1 (Santa Cruz), goat anti-human
polyclonal antibody against Nox2 (Santa Cruz), goat anti-human polyclonal antibody
against Nox4 (Santa Cruz), a rabbit anti-human polyclonal antibody against p22 (Santa
Cruz), a mouse anti-human monoclonal IgG1 antibody against p47 (BD Biosciences), or a
mouse anti-human monoclonal IgG2b antibody against p67 (BD Biosciences) (1:25, 1:75,
1:25, 1:75, 1:50, and 1:50 dilutions, respectively). Immunostains were visualized using
an avidin-biotin horseradish peroxidase system (Vectastain Universal Quick Kit, Vector
Laboratories). Additional experiments were performed on sections where the primary
antibody was omitted2 or replaced with a preimmune isotype control antibody to assess
for nonspecific binding.
Western blotting: Western blotting was performed on protein isolated from
cultured HCAECs by standard methods using the same Nox2, Nox4, or p22 antibodies
that were used for immunohistochemical studies. Briefly, HCAECs were homogenized
in ice-cold lysis buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% deoxycholic acid,
0.1% SDS, 0.5% NP40) supplemented with a protease inhibitor cocktail (Roche) and
centrifuged at 12,000 g for 10 min at 4 °C. Protein samples (10 µg) were subjected to
1
Larsen BT, et al.
Supplement Material
10% SDS-PAGE, transferred to PVDF membranes, blotted for Nox2 (1:200 dilution),
Nox4 (1:200 dilution), or p22 (1:200 dilution), and visualized by a horseradish
peroxidase-based detection system (ECL-Plus western blotting kit, VWR International).
Superoxide and H2O2 detection by fluorescence microscopy: Human coronary
artery endothelial cell (HCAEC) cultures were seeded and grown in an endothelial cellspecific growth medium according to the manufacturer’s protocol (EGM-2-MV BulletKit,
Cambrex). Dishes containing HCAECs or human coronary arterioles (HCAs) were filled
with HEPES buffer containing Nω-nitro-L-arginine methyl ester (L-NAME, 10-4 mol/L,
a nitric oxide [NO] synthase inhibitor) and indomethacin (10-5 mol/L, a cyclooxygenase
inhibitor) (pH 7.4 at 37°C). After equilibration at 37°C for 60 min, gp91ds-tat (5x10-5
mol/L, a specific peptide inhibitor of NADPH oxidase complex formation3), gp91scramtat (5x10-5 mol/L, a corresponding scrambled control peptide that does not inhibit
NADPH oxidase3), apocynin (3x10-3 mol/L, an NADPH oxidase inhibitor4), catalase
(1000 U/ml), rotenone (10-6 mol/L, an inhibitor of Complex I of the mitochondrial
electron transport chain5), or vehicle was added to the dishes, and HCAECs or HCAs
were loaded for 30 min in the dark with the fluorescence probes 2’,7’dichlorodihydrofluorescein diacetate (DCFH, 5x10-6 mol/L, Molecular Probes) and
dihydroethidium (DHE, 5x10-6 mol/L, Molecular Probes).6 After loading the probes,
HCAECs or HCAs were exposed to BK (10-6 mol/L) or vehicle for 10 min, rinsed in
fresh HEPES, and mounted on glass slides in Fluorescence Mounting Medium (Dako
Corp.). Fluorescence detection of H2O2 and superoxide was performed by krypton-argon
laser excitation at 488 and 585 nm while recording emission at 523 nm and 610 nm,
2
Larsen BT, et al.
Supplement Material
respectively. All fluorescence studies on vessels from a single patient were performed on
the same day with identical microscope settings for all experiments.
Images were
obtained with a laser scanning imaging system mounted on an inverted microscope using
a 20x objective lens. Images were obtained on a computer with the software program
MetaMorph (Universal Imaging Corp.) and analyzed with ImageJ (NIH). Fluorescence
intensity was normalized as the relative change in intensity from baseline (100%).
HPLC measurement of superoxide: Separation and quantification of oxidized
DHE products were performed by adapting an established high-performance liquid
chromatography (HPLC) cell culture-based method7 for use in intact microvascular tissue.
DHE was prepared in DMSO under argon gas and stored at -20ºC until use. Single
isolated HCAs were incubated for 60 min at 37ºC with the NADPH oxidase inhibitor
apocynin (3x10-3 mol/L) or vehicle in HEPES buffer consisting of (in mol/L) 138 NaCl, 4
KCl, 1.6 CaCl2, 1.2 MgSO4, 0.026 Na2ETDA, 1.2 KH2PO4, 10 HEPES, and 6 glucose).
DHE (10-5 mol/L) was then added and HCAs were incubated 30 min with BK (10-6
mol/L) or vehicle. HCAs were washed with fresh buffer and homogenized on ice in a 1:1
v/v solution of HPLC-grade methanol and 10-3 mol/L HCl. Homogenates were
centrifuged at 15,000 g for 20 min at 4ºC. The pellet was reserved for protein
determination, and the supernatant was diluted 3:10 in 1 mol/L HCl and subjected to
HPLC.7 DHE metabolites were separated on a C18 reverse-phase column (Partisil ODS-3
250 X 4.5 mm, Alltech Associates, Deerfield, IL) by a linear solvent gradient from 10%
to 70% CH3CN in H2O over 46 min and monitored by electrochemical detection at 280
mV. A representative HPLC chromatogram is shown in Supplementary Figure S1, below.
3
Larsen BT, et al.
Supplement Material
Microvascular content of DHE products was then calculated from HPLC chromatograms
using the area under the respective peaks and a standard curve derived from authentic
standards at known concentrations. Finally, DHE product content was normalized for
protein content.
Measurement of HCA dilation by videomicroscopy:
Internal diameter
measurements were performed on isolated, pressurized HCAs by videomicroscopy as
reported previously.2,
6, 8-12
Briefly, isolated HCAs were cannulated on glass
micropipettes and secured in an organ chamber containing a physiological saline solution
(PSS) consisting of (in mol/L) 123 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 16 NaHCO3,
0.026 Na2ETDA, 1.2 KH2PO4, and 11 glucose. The preparation was transferred to the
stage of an inverted microscope (magnification 200X, Olympus CK2) coupled to a CCD
video camera (WV-BL200, Panasonic), video monitor (Panasonic), and calibrated
videomicrometer (VIA-100K, Boeckeler Instruments Inc., resolution = 0.4 µm). The
vessels were slowly pressurized to 60 mmHg and incubated without intraluminal flow for
60 min. Bath temperature was maintained at 37°C and was bubbled continuously with
21% O2, 5% CO2, 74% N2 gas to maintain pH = 7.40 ± 0.05, PO = 140 ± 10 mmHg.
2
After the initial equilibration period, vessels were constricted with KCl (50
mmol/L final concentration) to assess viability. Vessels that constricted >30% were used
for subsequent experiments.2, 6, 8-10, 12 After washing with fresh buffer to restore the
concentration of K+ in the bath to baseline (4.7 mol/L), endothelin-1 (ET-1, 5x10-10 ~ 10-9
mol/L) was added to constrict the vessel by 30-50% of its diameter observed at the time
of initial pressurization to 60 mmHg. While published data indicates little contribution of
4
Larsen BT, et al.
Supplement Material
NO or prostacyclin to BK-induced dilation of HCAs,9 all experiments were performed in
the presence of L-NAME (10-4 mol/L) and indomethacin (10-5 mol/L) to eliminate any
residual confounding effects of these vasodilators. Cumulative concentrations of BK (1010
~ 10-6 mol/L) were then added to the bath and steady state diameter measurements
were taken 3 min. after each application. After washing the vessels with PSS, gp91ds-tat
(5x10-5 mol/L) or gp91scram-tat peptide (5x10-5 mol/L) was introduced both in the
superfusate and intraluminally under a brief period of flow, flow was stopped, and the
vessels were incubated for 30 min. HCAs were again constricted with ET-1 in the same
manner, followed by a second application of the same BK concentrations. At the end of
each experiment, papaverine (10-4 mol/L, an endothelium-independent vasodilator) was
added to determine the maximum internal diameter for normalization of dilator responses.
Agonist-induced dilations are expressed as a percent, with 100% dilation representing the
change from the constricted diameter to the maximal diameter obtained by addition of
papaverine. All drugs were added directly to the bath, and the change in bath volume
over the course of experiments was less than 1%.
Materials: Gp91ds-tat and gp91scram-tat peptides were synthesized according to
their respective amino acid sequences as originally described3 using an Applied
Biosystems 432A solid-phase peptide synthesizer that employs FMOC chemistry coupled
with HBTU activation. Peptide identity and purity was monitored by mass spectrometry.
Following their synthesis, each peptide was dissolved in PSS containing 5 mmol/L acetic
acid as described previously.3 EEZE was a kind gift from Dr. John R. Falck (University
of Texas Southwestern, Dallas, Texas). Catalase isolated from bovine liver was obtained
5
Larsen BT, et al.
Supplement Material
from Calbiochem.
HCAECs were obtained from Cambrex, DCFH and DHE were
obtained from Molecular Probes, and all other chemicals were obtained from Sigma
Chemical Co. DCFH and DHE were dissolved in DMSO. Apocynin and EEZE were
prepared in ethanol.
The final concentration of ethanol present in the bath during
vasodilator studies was 0.05%.
Vehicle control studies indicated that the final
concentration of ethanol had no effect on basal tone or function of arterioles.
Indomethacin was dissolved in 20 mmol/L Na2CO3. All other chemicals were dissolved
in distilled water. All concentrations represent final molar concentrations (mol/L) in the
organ chamber.
6
Larsen BT, et al.
Supplement Material
Supplementary Results
Supplementary Figure S1. Representative electrochemical signal at 280 mV of
oxidized DHE products from single isolated HCAs. As indicated by the blue squares and
red circles, two distinct peaks with elution times of 17 and 19 min comigrated with
authentic 2-OH-E+ and E+ standards, respectively.
7
Larsen BT, et al.
Supplement Material
A
B
Nox2
75 kD
Nox4
70 kD
(+) control
HCAECs
(+) control
C
HCAECs
p22
22 kD
(+) control
Supplementary Figure S2.
HCAECs
Expression of NADPH oxidase subunits in
HCAECs. A-C, Representative Western blots of protein isolated from HCAECs. While
immunohistochemical staining of atrial appendages suggests that NADPH oxidase is
expressed in the endothelium of the human coronary circulation, Western blots were
performed on protein isolated from cultured HCAECs to determine whether NADPH
oxidase proteins are indeed present in these cells. As seen above, Nox2, Nox4, and p22
are each expressed in HCAECs.
8
Larsen BT, et al.
Supplement Material
Reference List
(1) Larsen BT, Miura H, Hatoum OA, Campbell WB, Hammock BD, Zeldin DC,
Falck JR, Gutterman DD. Epoxyeicosatrienoic and dihydroxyeicosatrienoic acids
dilate human coronary arterioles via BKCa channels: implications for soluble
epoxide hydrolase inhibition. Am J Physiol Heart Circ Physiol. 2006;290:H491H499.
(2) Miura H, Wachtel RE, Loberiza FR, Jr., Saito T, Miura M, Nicolosi AC,
Gutterman DD. Diabetes mellitus impairs vasodilation to hypoxia in human
coronary arterioles: reduced activity of ATP-sensitive potassium channels. Circ
Res. 2003;92:151-8.
(3) Rey FE, Cifuentes ME, Kiarash A, Quinn MT, Pagano PJ. Novel competitive
inhibitor of NAD(P)H oxidase assembly attenuates vascular O(2)(-) and systolic
blood pressure in mice. Circ Res. 2001;89:408-14.
(4) Hamilton CA, Brosnan MJ, Al-Benna S, Berg G, Dominiczak AF. NAD(P)H
oxidase inhibition improves endothelial function in rat and human blood vessels.
Hypertension. 2002;40:755-62.
(5) Liu Y, Zhao H, Li H, Kalyanaraman B, Nicolosi AC, Gutterman DD.
Mitochondrial sources of H2O2 generation play a key role in flow-mediated
dilation in human coronary resistance arteries. Circ Res. 2003;93:573-80.
(6) Miura H, Bosnjak JJ, Ning G, Saito T, Miura M, Gutterman DD. Role for
hydrogen peroxide in flow-induced dilation of human coronary arterioles. Circ
Res. 2003;92:e31-e40.
(7) Zhao H, Joseph J, Fales HM, Sokoloski EA, Levine RL, Vasquez-Vivar J,
Kalyanaraman B. Detection and characterization of the product of hydroethidine
and intracellular superoxide by HPLC and limitations of fluorescence. Proc Natl
Acad Sci U S A. 2005;102:5727-32.
(8) Miura H, Wachtel RE, Liu Y, Loberiza FR, Jr., Saito T, Miura M, Gutterman DD.
Flow-induced dilation of human coronary arterioles: important role of Ca(2+)activated K(+) channels. Circulation. 2001;103:1992-8.
(9) Miura H, Liu Y, Gutterman DD. Human coronary arteriolar dilation to bradykinin
depends on membrane hyperpolarization: contribution of nitric oxide and Ca2+activated K+ channels. Circulation. 1999;99:3132-8.
(10) Miura H, Gutterman DD. Human coronary arteriolar dilation to arachidonic acid
depends on cytochrome P-450 monooxygenase and Ca2+-activated K+ channels.
Circ Res. 1998;83:501-7.
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(11) Terata K, Miura H, Liu Y, Loberiza F, Gutterman DD. Human coronary arteriolar
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Heart Circ Physiol. 2000;279:H2620-H2626.
(12) Miller FJ, Jr., Dellsperger KC, Gutterman DD. Pharmacologic activation of the
human coronary microcirculation in vitro: endothelium-dependent dilation and
differential responses to acetylcholine. Cardiovasc Res. 1998;38:744-50.
10