Cardiovascular Research 71 (2006) 363 – 373

www.elsevier.com/locate/cardiores

Perivascular adipose tissue promotes vasoconstriction:

The role of superoxide anion

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Yu-Jing Gao a,*, Kumiko Takemori a, Li-Ying Su a, Wen-Sheng An a, Chao Lu a,
Arya M. Sharma b, Robert M.K.W. Lee a
a
Smooth Muscle Research Program and Department of Anaesthesia, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5
b
Department of Medicine, McMaster University, Hamilton, Ontario, Canada

Received 29 September 2005; received in revised form 8 March 2006; accepted 14 March 2006
Available online 21 March 2006
Time for primary review 28 days

Abstract

Objectives: Recent studies have demonstrated that perivascular adipose tissue (PVAT) releases vascular relaxation factor(s). In this study, we
examined if PVAT releases other vasoactive factors in response to perivascular nerve activation by electrical field stimulation (EFS).
Methods and results: In Wistar-Kyoto rats, rings of superior mesenteric artery (MA) with intact PVAT (PVAT (+)) showed a greater
contractile response to EFS than rings with PVAT removed (PVAT ( )). Superoxide dismutase (SOD) reduced the contractile response to
EFS more in PVAT (+) MA than in PVAT ( ) MA. Inhibitors of NAD(P)H oxidase and cyclooxygenase exerted a greater inhibition on EFS-
induced contraction in PVAT (+) MA than in PVAT ( ) MA. Inhibitors of tyrosine kinase (tyrphostin A25) and MAPK/ERK (U 0126)
attenuated EFS-induced contraction in PVAT (+) MA in a concentration-related manner, while inactive forms of these inhibitors (tyrphostin
A1 and U 0124) did not inhibit the response. Exogenous superoxide augmented the contractile response to EFS and to phenylephrine in
PVAT ( ) MA, and this augmentation was blunted by inhibition of tyrosine kinase and MAPK/ERK. EFS increased superoxide generation in
isolated PVAT and PVAT (+)/( ) MA, which was attenuated by NAD(P)H oxidase inhibition. RT-PCR showed the mRNA expression of
p67phox subunit of NAD(P)H oxidase and immunohistochemical staining confirmed its localization in the adipocytes of PVAT.
Conclusion: These results show that PVAT enhances the arterial contractile response to perivascular nerve stimulation through the production
of superoxide mediated by NAD(P)H oxidase, and that this enhancement involves activation of tyrosine kinase and MAPK/ERK pathway.
D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Keywords: Adipose tissue; Contraction; MAPK; Mesenteric artery; Perivascular nerve stimulation; Superoxide anion

1. Introduction presence of PVAT. By virtue of its location and its ability to

Despite the fact that perivascular adipose tissue (PVAT)
surrounds almost all systemic blood vessels, the possible role
of PVAT in modulating vascular function had received little
attention. In studies with isolated vessels, PVAT is routinely
removed. Although Solitis and Cassis [1] demonstrated that
the contractile response of rat aorta to norepinephrine was
attenuated in the presence of PVAT, they attributed this
attenuation to increased uptake of this monoamine in the
* Corresponding author. Tel.: +1 905 521 2368x75433; fax: +1 905 523
1224.
E-mail address: gaoyu@mcmaster.ca (Y.-J. Gao).
0008-6363/$ - see front matter D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2006.03.013
produce a host of vasoactive substance [2,3], PVAT has the
potential to regulate vascular function, but the specific role of
PVAT in this regulation remains largely unknown. Recent
studies including ours have shown that PVAT releases
relaxation factor(s) in rat aorta [4,5], rat mesenteric artery
[6], and human internal thoracic artery [7]. We have further
shown that the ability of the aortic PVAT to release this
relaxation factor was impaired in obese rats induced by
prenatal exposure to nicotine [8]. Although the principle of
this putative relaxation factor remains to be determined,
these findings strongly suggest that PVAT indeed plays an
important physiological/pathological role in modulating
vascular function.
364 Y.-J. Gao et al. / Cardiovascular Research 71 (2006) 363 – 373
The modulation of vascular function by PVAT may not
be limited to the secretion of a relaxing factor. Most of the
regulation in biological system is composed of a balance
between acting and counteracting factors. For example,
vascular endothelial cells have the potential to produce both
relaxing factors (e.g. nitric oxide, prostacyclin and endo-
thelium-derived hyperpolarizing factor) and contracting fac-
tors (e.g. endothelins and endothelium-derived contracting
factor), depending on the type of stimulation. Furthermore,
epidemiological studies have shown that accumulation of
body fat (obesity) is a significant etiologic factor for
hypertension [9,10], which is characterized by an increased
peripheral resistance due to enhanced vasoconstriction. The
finding that PVAT releases relaxation factor(s) does not
provide explanation to this association between obesity and
hypertension, although many other factors could be in-
volved. Here we report that PVAT also enhances the
contractile response elicited by perivascular nerve activation
by electrical field stimulation (EFS), and that this enhance-
ment involves activation of tyrosine kinase and MAPK/
ERK pathway by superoxide generated in PVAT.
2. Methods
2.1. Animals
Male Wistar-Kyoto rats (WKY) at 25– 32 weeks of age
were obtained from the rat colony maintained at the
McMaster University Central Animal Facilities. This study
conforms with the guidelines of the Canadian Council on
Animal Care and was approved by the Animal Care
Committee of McMaster University.
2.2. Contractility study
The procedure for the preparation of superior mesenteric
artery (MA) rings has been described in our previous reports
[11,12]. Briefly, the rat was euthanized by an overdose of
sodium pentobarbital (60 mg/kg, i.p.), and the MA was
collected in oxygenated physiological salt solution (PSS)
with the following composition (in mM): NaCl, 119; KCl,
4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; CaCl2, 1.6;
glucose, 11, at 4 -C. Paired MA rings (4mm long), one with
PVAT intact (PVAT (+)) and the other with PVAT removed
(PVAT ( )), were prepared from each artery. The average
weight of PVAT attached to MA was 3.9 T 0.6 mg (n = 16).
PVAT removal was carried out with fine scissors under a
dissection microscope. Caution was taken not to damage the
adventitial layer during the dissection. To examine the
integrity of the adventitial layer after PVAT removal, some
rings were fixed in 10% formaldehyde and embedded in
paraffin. Cross sections of MA were stained either with
Gomori’s trichrome to measure adventitial layer thickness,
or with silver stain to estimate nerve ending density [13].
The dimension of adventitia was analyzed with the software
of Image-Pro Plus (Silver Spring, MD, USA), and the
density of silver staining in the adventitia was estimated
with the software of ImageJ (NIMH, MD, USA).
A computerized myograph system was used to study the
contraction and relaxation response of the MA. After
equilibration for at least 60 min, the arterial rings were
challenged with 60 mM KCl twice at an interval of 30 min.
Contractile response was elicited with EFS (0.85 ms, 150 V,
a 10-s train, at 2, 6, 12 and 20 Hz at an interval of 3– 5 min)
as described previously [14]. Contractile responses to EFS
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and phenylephrine were expressed as a percentage of KCl
contraction. Relaxation response to sodium nitroprusside
was tested in MA rings precontracted with phenylephrine
(1 AM for PVAT ( ), and 3 AM for PVAT (+), to generate an
equivalent pre-contraction level in these arteries), and
expressed as a percentage of the precontraction value.
To study the role of superoxide anion, one pair of PVAT
(+) and PVAT ( ) MA rings were treated with superoxide
dismutase (SOD, 200 U/ml, a superoxide scavenger en-
zyme), and the other pair treated with solvent served as
control, for 2 h before exposure to EFS. In another set of
experiment, pyrogallol, a superoxide generator, was added to
PVAT ( ) MA 2– 5 min prior to EFS/phenylephrine, to study
the direct effects of exogenous superoxide on the contractile
response. Arterial preparations were incubated with inhib-
itors of nitric oxide synthase (N N-nitro-l-arginine, l-NNA),
NAD(P)H oxidase (apocynin and diphenyleneiodonium
chloride (DPI)), cyclooxygenase (diclofenac), xanthine
oxidase (allopurinol), cytochrome P450 monooxygenase
(17-octadecynoic acid), tyrosine kinase (tyrphostin A25),
and MAPK/ERK (U 0126) for 25 min before exposure to
EFS. The effects of tyrphostin A1 and U 0124, the inactive
forms of tyrphostin A25 and U 0126, were also tested to
exclude non-specific effects of these inhibitors. Recovery of
response to EFS was tested 60 min after washout of SOD or
30 min after washout of other enzyme inhibitors. The
inhibitory effect of SOD, apocynin, DPI, and diclofenac was
expressed as a percentage of the control. The involvement of
a-adrenoceptor in EFS-induced contraction was tested with
a-adrenoceptor antagonist (prazosin hydrochloride, 1 AM),
and the neural origin of the contraction was verified by
tetrodotoxin (1 AM), a sodium channel blocker of nerve
endings, 15 min prior to EFS.
2.3. Superoxide production by PVAT with lucigenin-
enhanced chemiluminescence
Superoxide production by a 4-mm-long segment of MA
with and without PVAT and by PVAT alone was measured
with lucigenin (5 AM)-enhanced chemiluminescence
(recorded every minute for 8 min by a luminometer: T-20/
20, USA) [15]. The average weight of isolated PVAT was
2.3 T 0.2 mg (n = 6). Superoxide level was measured before
and immediately after EFS (12 Hz). In some experiments, the
effects of NADH (10 AM, 25 min) to stimulate superoxide
production and the effects of DPI (100 AM, 25 min) to inhibit
 Y.-J. Gao et al. / Cardiovascular Research 71 (2006) 363 – 373
EFS- and NADH-induced superoxide production were
examined. Chemiluminescence with the respective back-
ground subtracted was expressed as units/min. We had
established with our preliminary experiments that EFS with
buffer alone did not induce detectable level of superoxide
production under this condition (data not shown).
2.4. Visualization of superoxide production by adipocytes
detected with fluoresent dye dihydroethidium (DHE)
Blocks of PVAT were embedded in Optimal Cutting
Temperature compound (OCT from Tissue-Tek\) and flash
frozen in liquid nitrogen immediately after EFS stimulation
(0 Hz or 12 Hz). Each frozen section (30 Am thick) was
covered with 3 AM DHE on top and a cover slip was applied
[16]. Slides were incubated in a light-protected humidified
chamber at 37 -C for 30 min, and were viewed with a
fluorescence microscope (Olympus 1X81, Japan. Excita-
tion: 488 nm; emission: 610 nm). In a separate group of
experiments, tissues were incubated with SOD (600 U/ml,
60 min) prior to EFS. Images were obtained using the
software of Image-Pro Plus (Silver Spring, MD, USA).
2.5. Immunohistochemistry staining for NAD(P)H oxidase
subunit p67phox in the adipocytes of PVAT
PVAT was fixed in 10% formaldehyde and embedded in
paraffin. Sections (4 –6 Am in thickness) were treated in
citrate buffer for antigen retrieval (120 -C for 6 min). All the
following staining steps were carried out in humidified
chamber and manufacture-recommended procedures (Santa
Cruz Biotechnology, USA) were followed. Briefly, endog-
enous peroxidase activity was blocked with 1% hydrogen
peroxide, followed by incubation with 1.5% blocking
serum. Rabbit polyclonal anti-p67phox antibody was used
in a 1:50 dilution overnight at 4 -C. The second antibody,
biotinylated goat anti-rabbit, was applied for 2 h at room
temperature in 1:200 dilutions with PBS containing 0.5%
blocking serum. Slides were then exposed to avidin and
biotinylated horseradish peroxidase (1:50 dilution, 1 h),
followed by staining with a solution containing 3,3V-
diaminobenzidine tetrahydrochloride for 5– 8 min. Slides
treated only with the 2nd antibody were used as negative
control. Hematoxylin was used as a counter-stain.
2.6. Detection of p67phox mRNA from adipocytes of PVAT by
reverse-transcriptase polymerase chain reaction (RT-PCR)
Total RNAs of adipocytes isolated from PVAT were
prepared as described by Engeli et al. [17] with TRIzol
reagent (Invitrogen). RT-PCR was carried out using the
RETRO script (Ambion, USA), according to the manufac-
turer’s instructions. One microgram of total RNA was
reverse-transcribed by MMLV-RT at 44 -C for 1 h, 92 -C
for 10 min. Using complementary DNA, PCR amplification
was performed with gene-specific primers for p67phox
365
(forward, 5V-CAGTTCAAGCTGTTTGCCTG-3V; reverse,
5V-TTCTTGGCCAGCTGAGCCAC-3V) [18]. The conditions
were 40 cycles of denaturation at 94 -C (1 min), annealing at
65 -C (1 min), and extension at 72 -C (1 min), followed by a
further 7-min extension. The purified p67phox PCR product was
confirmed by sequencing on an ABI PRISM 3100 Genetic
Analyzer (Applied Biosystems, CA, USA).
2.7. Chemicals
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Allopurinol, apocynin, diclofenac, DPI, lucigenin, l-
NNA, polyethelene glycol (PEG), prazosin hydrochloride,
pyrogallol, SOD-PEG, sodium nitroprusside, and tetrodo-
toxin were purchased from Sigma, USA; tyrphostin A 25,
tyrphostin A1, U 0126, and U 0124 from Calbiochem, USA,
and DHE from Molecular Probes (Eugene, Oregon, USA).
DPI, apocynin, tyrphostine A25, tyrphostin A1, U 0126, and
U 0124 were dissolved in dimethyl sulfoxide (DMSO).
DHE stock solution was made in nitrogen-purged DMSO
and diluted in filtered PSS. All other agents were dissolved
in deionized water and prepared fresh before use. Polyclonal
antibodies for p67phox and ABC staining system were
purchased from Santa Cruz Biotechnology (California,
USA). TRIzol reagent was purchased from Invitrogen
(Burlington, ON, Canada), and RETROscript was from
Ambion (Austin, TX, USA).
2.8. Statistical analysis
Results were expressed as mean T standard error of the
mean (SEM) where n represents the number of rats.
Statistical analysis was performed by one-way analysis of
variance (ANOVA) followed by post hoc t test for
concentration-dependent or frequency-dependent effects,
or by Student’s t-test for comparison between MA with
and without PVAT, using the software of SigmaStat (SPSS,
Inc, Chicago, USA). The differences were considered
significant when P  0.05.
3. Results
3.1. Effects of dissection on adventitia and nerves
Dissection did not affect the integrity of adventitia
(thickness in Am, 35.6 T 5.6 for PVAT (+) rings, and
32.1 T 4.4 for PVAT ( ) rings, n = 6 for each; P = 0.84) and
the density of the nerves (in arbitrary units, 71.3 T 7.5 for
PVAT (+) rings, 77.6 T 2.9 for PVAT ( ) rings; n = 7 for
each; P = 0.45).
3.2. PVAT on force generation by the arteries
Maximum force generated in response to 60 mM KCl
was not different between PVAT (+) and PVAT ( ) MA
rings (in grams: 0.75 T 0.08, 0.67 T 0.08 for PVAT (+) and
366 Y.-J. Gao et al. / Cardiovascular Research 71 (2006) 363 – 373

( P > 0.05). The contraction of the artery to KCl was not

affected by SOD in both PVAT (+) and PVAT ( ) MA (in
grams, for before and after SOD treatment, respectively;
PVAT (+) MA: 0.79 T 0.08 and 0.75 T 0.09, P = 0.51; PVAT
( ) MA: 0.74 T 0.11 and 0.7 T 0.08, P = 0.49; n = 6 for each).
PEG did not affect the contraction to EFS (data not shown).

3.5. Effects of inhibition of superoxide-producing enzymes

and of tyrosine kinase-MAPK/ERK pathway on the con-
traction to perivascular nerve stimulation

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Treatment with NAD(P)H oxidase inhibitor (apocynin
(100 AM) and DPI (10 AM)) and cyclooxygenase inhibitor
(diclofenac, 10 AM) attenuated the contraction to EFS (12
Hz) more in the PVAT (+) MA than in PVAT ( ) MA (Fig. 3A
and C). Tyrosine kinase inhibitor tyrphostin A25 and MAPK/
ERK inhibitor U 0126, but not the inactive forms of these
enzymes (tyrphostin A1 and U 0124), concentration-depen-

Fig. 1. (A and B) Typical recording of contractile response to KCl (60 mM)

in MA with (PVAT (+)) and without PVAT (PVAT ( )). Maximal force
developed was similar in both preparations. (C and D) Typical contractile
response of PVAT (+) and PVAT ( ) arteries to electrical field stimulation
(EFS), and the results are summarized in (E). *P < 0.05 versus PVAT ( )
arteries (n = 9 rats, 30 – 32 weeks).

PVAT ( ) MA, respectively, n = 14 for each, P = 0.45),

although the onset of contraction was slower in PVAT (+)
MA than in PVAT ( ) MA (Fig. 1A, B). Relaxation
response to sodium nitroprusside (10 AM) in phenylephrine-
precontracted arteries was similar in these rings (in % of
pre-contraction: 90 T 3.3 in PVAT (+) MA and 91 T 3.8 in
PVAT ( ) MA, n = 5 for each, P = 0.9).

3.3. Contraction to perivascular nerve stimulation

EFS elicited a frequency-dependent contractile response

in both PVAT (+) and PVAT ( ) MA, but the amplitude of
contraction was higher in PVAT (+) than in PVAT ( ) MA
(Fig. 1C, D, E). The contraction to EFS was abolished by
tetrodotoxin (1 AM) and by prazosin (1 AM) (data not shown).

3.4. Effects of SOD on the contraction to perivascular nerve

stimulation

Incubation with SOD-PEG (200 units/ml) markedly

reduced the contraction to EFS in PVAT (+) MA, and
removal of SOD partially restored the response (Fig. 2A –
D). In PVAT ( ) MA, SOD also reduced the contraction
response to EFS, but the reduction was less prominent as
compared with that of PVAT (+) MA (Fig. 2D and E).
Furthermore, contractile response became similar between
PVAT (+) and PVAT ( ) MA after incubation with SOD
Fig. 2. (A and B) Typical recording showing the inhibition of contractile
response to electrical field stimulation (EFS) in PVAT (+) arteries by
superoxide dismutase (SOD, 200 U/ml). (C) Recovery of the response to
EFS after washout of SOD (1 h). (D) A summary of results of SOD
treatment on PVAT (+) and PVAT ( ) arteries. (E) A summary of inhibition
(%) by SOD in PVAT (+) and PVAT ( ) MA. *P < 0.05, **P < 0.01 versus
control arteries (n = 6 rats, 30 – 32 weeks).
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Fig. 3. Effects of inhibitors of superoxide-producing enzymes (A) and of tyrosine kinase-MAPK/ERK pathway (B) on the contraction to perivascular nerve
stimulation by EFS (12 Hz) in PVAT (+)/( ) MA, and a summary of inhibition (%) by superoxide-producing enzymes in PVAT (+)/( ) MA (C). NAD(P)H
oxidase inhibitor (apocynin (APO) and DPI (10 AM)), cyclooxygenase inhibitor (diclofenac, DICL) attenuated the contraction more in PVAT (+) than in PVAT
( ) MA. Tyrosine kinase inhibitor (tyrphostin A25, Tyr A25) and MAPK/ERK inhibitor (U 0126) attenuated the contraction in PVAT (+) MA but not in PVAT
( ) MA. The inactive forms of Tyr A25 and U 0126 (tyrphostin A1 (TyrA1) and U 0124) did not inhibit the contraction. NO synthase inhibitor l-NNA (100
AM) enhanced the contraction in PVAT (+)/( ) MA (D). All MA rings were with intact endothelium. *P < 0.05, **P < 0.01 as compared with respective control;
# #
P < 0.01 as compared with the response to EFS without l-NNA (n = 4 – 9 rats; 25 – 27 weeks).

dently attenuated the contraction in PVAT (+) MA, but
showed no effect in PVAT ( ) MA (Fig. 3B). Inhibition of
xanthine oxidase with allopurinol, and cytochrome P450
monooxygenase with 17-octadecynoic acid did not affect the
contraction in either PVAT (+) or PVAT ( ) MA, and these
compounds did not inhibit the contraction to KCl (data not
shown).
3.6. Effects of nitric oxide synthase inhibitor (L-NNA) on the
contraction to perivascular nerve stimulation
Incubation of endothelium-intact MA with l-NNA (100
AM, 30 min) did not affect the basal tension, but enhanced
the contractile response to EFS (12 Hz) in both PVAT (+)
and PVAT ( ) MA to similar extents. The contraction to
EFS was greater in PVAT (+) MA than in PVAT ( ) either
in the presence or absence of l-NNA (Fig. 3D).
3.7. Effects of exogenous superoxide on the contraction to
perivascular nerve stimulation (12 Hz) and to phenyleph-
rine (0.3 lM) in PVAT ( ) MA
Treatment of the arteries with pyrogallol (a superoxide
generator; 10 and 30 AM), which did not contract the
arteries, enhanced the contraction to EFS and to phenyl-
ephrine (Fig. 4A –C). Inhibitors of tyrosine kinase (tyrphos-
368 Y.-J. Gao et al. / Cardiovascular Research 71 (2006) 363 – 373
Fig. 4. Augmentation of contractile response to EFS (12 Hz, A) or to
phenylephrine (0.3 AM, B) by pyrogallol (PYG) in PVAT ( ) arteries. (C)
Summary of the enhancement of contraction to EFS and to phenylephrine
by PYG (n = 9 rats). **P < 0.01 versus control response. (D) Effects of
inhibitors of tyrosine kinase (tyrphostin A25, Tyr A25) and MAPK/ERK (U
0126) on PYG (30 AM)-induced enhancement of EFS-caused contraction in
PVAT ( ) MA. Inactive forms of Tyr A25 (tyrphostin A1, Tyr A1) and U
0126 (U 0124) did not affect the enhancement of EFS-induced contraction
by PYG (n = 4 – 9 rats, 25 – 27 weeks) *P < 0.05, **P < 0.01 versus control.
tin A25, 30 AM) and MAPK/ERK (U 0126, 1 AM) inhibited
the enhancing effects of pyrogallol on EFS-induced
contraction. The inactive form of these inhibitors (tyrphostin
A1 and U 0124) did not inhibit the enhancement by
pyrogallol at the same concentrations (Fig. 4D).
3.8. Immunohistochemical staining for p67phox and detection
of p67phox mRNA by RT-PCR
The cytoplasm and cell membrane of the adipocytes in
the PVAT were positively stained by anti-p67phox antibody
(Fig. 5A), showing the presence of this subunit of NAD(P)H
oxidase. Negative controls did not show any non-specific
staining (Fig. 5B). Expression of p67phox mRNA in PVAT
was also detected by RT-PCR (Fig. 5C) and the specificity
of PCR product was confirmed by nucleotide sequencing
(DNA Data Bank of Japan, accession number: M32011).
3.9. EFS-enhanced superoxide production by PVAT
Basal production of superoxide was greater in PVAT (+)
MA than in PVAT ( ) MA. EFS (12 Hz) increased the
production of superoxide in PVAT ( ) MA slightly, but a
significant increase was found in MA with intact PVAT (Fig.
6A). In isolated PVAT, NADH (a stimulator of NAD(P)H
oxidase) and EFS (12 Hz) enhanced superoxide production,
and this enhancement was attenuated by DPI (a NAD(P)H
oxidase inhibitor) (Fig. 6B). The production of superoxide
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was reduced by chemical scavenger tiron (10 mM) and by
SOD (200 units/ml; data not shown). EFS-induced super-
oxide production by adipocytes of PVAT was confirmed in
frozen sections of PVAT stained with DHE (Fig. 7A, dark
red fluorescent staining of nuclei of adipocytes), while
adipocytes in control tissue (0 Hz) only showed a weak
fluorescence (Fig. 7B). The increase in fluorescence
intensity by EFS was greatly reduced by pretreatment with
SOD-PEG (1000 units/ml, 60 min, 37 -C) prior to EFS
stimulation (data not shown).
4. Discussion
The novel finding of the present study is that PVAT
enhances vasoconstriction in response to perivascular nerve
activation by EFS, and this enhancement may involve the
release of superoxide by PVAT and the activation of tyrosine
kinase and MAPK/ERK pathway. To the best of our
knowledge, this is the first report to show that the presence
of PVAT enhances vasoconstriction, which, therefore, adds
new insights to the current understanding about the role that
PVAT may play in the regulation of vascular function.
We have established that the presence of PVAT did not
affect the ability of the arteries to contract or to relax,
because maximal contraction in response to KCl and
relaxation response to sodium nitroprusside were similar
between PVAT (+) and PVAT ( ) MA. The slower onset of
KCl-induced contraction in PVAT (+) than in PVAT ( ) MA
was probably due to the concomitant release of PVAT-
generated relaxing factor, which suppressed the develop-
ment of contraction to KCl through opening potassium
channels [4,6,7], but eventually its effect was rendered
ineffective by high concentration of extracellular KCl, as
shown in previous studies [19,20]. Adventitia, which is
located between PVAT and medial smooth muscle, contains
nerve endings and plays a crucial role in mediating vascular
response to perivascular nerve stimulation [21]. However,
the difference between PVAT (+) and PVAT ( ) MA in their
responses to EFS was not due to potential damage of the
adventitial tissue during the removal of PVAT, because the
dimensions of adventitia and the density of the nerve
staining were comparable between PVAT (+) and PVAT ( )
MA. We have also established that the contractile response
of the MA to EFS was through the activation of perivascular
sympathetic nerves and was mediated by a1-adrenoceptors,
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Fig. 5. Immunohistochemical staining of NAD(P)H oxidase subunit p67phox and its mRNA expression in the PVAT of mesenteric artery. Cytoplasm and cell
membrane of the adipocytes were positively stained as indicated with arrowheads (A). No positive staining was found in the negative control by omitting the
primary antibody in the staining process (B). Expression of P67phox mRNA in PVAT (C). Data are representative of four rats (30 – 32 weeks).

because tetrodotoxin and prazosin abolished this response,
which is consistent with previous report [22]. Since the
presence of PVAT attenuated contractile response of blood
vessels to several exogenously applied agonists [4,6 – 8], the
enhancement of the contractile response to EFS by PVAT
appears to be a specific phenomenon associated with
perivascular nerve stimulation.
It is well known that endothelium, smooth muscles and
adventitia are local sources of superoxide [23 – 25], and
NAD(P)H oxidase is the main superoxide-producing en-
zyme [26]. However, it is not known whether PVAT, which
surrounds most of the systemic vessels, also contributes to
vascular superoxide production. Our study showed that
PVAT is another source of vascular superoxide in addition to
vessel wall components, because (1) PVAT (+) MA
generated more superoxide than PVAT ( ) MA, (2) isolated
PVAT also produced superoxide, (3) EFS enhanced super-
oxide production by PVAT and the origin of superoxide
from adipocytes in PVAT was confirmed by fluorescent
labeling with DHE. We further found that the main enzyme
responsible for superoxide production by PVAT was
probably NAD(P)H oxidase, because superoxide production
by PVAT was inhibited by the inhibitor of this enzyme (DPI)
and enhanced by the stimulator of this enzyme (NADH).
Localization of NAD(P)H oxidase subunit p67phox in the
cytoplasm and cell membrane of the adipocytes of PVAT
370 Y.-J. Gao et al. / Cardiovascular Research 71 (2006) 363 – 373

stimulation, mimicking the presence of PVAT. Contractile

response to phenylephrine was also enhanced by pyrogallol
in PVAT ( ) MA. Xanthine oxidase and cytochrome P450
monooxygenase, which have also been shown to play a role
in vascular superoxide production [29,30], did not seem to
be involved in superoxide production in PVAT because
inhibitors of these enzymes did not show any effects on
EFS-induced contraction. Cyclooxygenase inhibition re-
duced the contraction to EFS more in PVAT (+) MA than
in PVAT ( ) MA, indicating its involvement in superoxide

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generation [31] in PVAT, and/or its involvement in signal
transduction pathway of superoxide-induced enhancement
of contraction, as shown in vascular tissue [32,33], although
we cannot rule out the possibility that suppression of
vasoconstrictive prostanoid could be involved.
The mechanisms for superoxide-induced enhancement of
contraction to EFS were investigated. A recent report had
found that superoxide potentiated vasoconstriction in rat
mesenteric arteries to a1-adrenoceptor agonist, but the

Fig. 6. (A) Superoxide production by PVAT (+) and PVAT ( ) MA before

and after EFS (12 Hz). MA with or without PVAT generated a basal level of
superoxide, and EFS enhanced this production. **P < 0.01 versus PVAT ( )
MA and #P < 0.05 versus basal condition. (B) EFS (12 Hz) and NADH
(10 AM) stimulated superoxide production in isolated PVAT, and this
stimulation was attenuated by DPI (100 AM) (n = 6 – 8 rats, 30 – 32 weeks).

was shown by our immunohistochemical staining. Expres-

sion of p67phox mRNA was also detected from adipocytes
isolated from PVAT, and the PCR product was confirmed by
sequencing. We had selected p67phox subunit among all the
subunits of NAD(P)H oxidase because the expression of this
subunit in mouse adipose tissue was the most abundant [27].
Expression of gp91phox mRNA was also detected in the
mesenteric PVAT (Gao et al., unpublished data). This is the
first report, to the best of our knowledge, showing that
NAD(P)H oxidase subunit is present in the adipocytes of
PVAT and contributes to vascular superoxide production.
We further showed that superoxide generated by PVAT is
involved in the enhanced contraction to EFS of PVAT (+)
MA, based on the following findings. Firstly, enzyme
scavenger for superoxide (SOD-PEG) exerted greater
attenuation of EFS-induced contraction in PVAT (+) MA
than in PVAT ( ) MA. PEG, a hydrophilic polymer
associated with SOD, did not reduce EFS-induced contrac-
tion, although it attenuated oxidative stress in an animal
model of spinal injury through stabilization of disrupted
membrane [28]. Secondly, NAD(P)H oxidase inhibitors Fig. 7. Fluorescent images showing superoxide production by adipocytes in
mesenteric PVAT, detected with dihydroethedium (DHE). PVAT was
(apocynin and DPI) reduced EFS-induced contraction more
labeled with DHE (3 AM), which gives red fluorescence when oxidized
in PVAT(+) MA than in PVAT ( ) MA . Thirdly, pyrogallol, by superoxide. EFS (12 Hz) increased the density of DHE-positive
a superoxide-generating compound, potentiated the contrac- fluorescent nuclei (A), as compared with control (0 Hz) adipocytes in
tion of PVAT ( ) MA in response to perivascular nerve PVAT (B). Data are representative of four rats (30 – 32 weeks).
 Y.-J. Gao et al. / Cardiovascular Research 71 (2006) 363 – 373
mechanisms for this enhancement were not addressed [34].
We investigated the involvement of tyrosine kinase and
MAPK/ERK in PVAT-enhanced contraction to EFS because
activation of MAPK/ERK pathway by superoxide was
involved in stretch-induced enhancement of contraction of
bovine coronary artery [35]. We found that inhibitors of
tyrosine kinase (tyrphostin A25) and MAPK/ERK (U 0126)
concentration dependently attenuated the contraction to EFS
in PVAT (+) MA, but not in PVAT ( ) MA, suggesting their
involvement in the enhanced contractile response to EFS in
PVAT (+) MA. The inhibition seemed to be specific because
the inactive forms of these inhibitors (tyrphostin A1 and U
0124) did not affect the contractile response. In PVAT ( )
MA, these inhibitors of tyrosine kinase and MAPK/ERK
(but not their inactive forms) also attenuated the enhance-
ment of EFS-induced contraction by superoxide generator
pyrogallol, further suggesting that potentiation of the
contraction to EFS by PVAT-derived superoxide was
mediated through activation of tyrosine kinase and
MAPK/ERK pathway. It should be noted that some tyrosine
kinase inhibitor such as genistein and its inactive form
daidzein also possess antioxidant property [36 –38]. In our
preliminary experiments we did find that daidzein attenu-
ated contractile response to EFS. We also found that SB
202474, the inactive form of SB 203580 (a MAPK/P38
kinase inhibitor) attenuated the response to EFS.
It is known that superoxide may function as messenger
molecules in signal transduction cascades in some physi-
ological processes [39,40], but the mechanisms for activa-
tion of tyrosine kinase and MAPK/ERK pathway to
promote vasoconstriction to EFS as shown in this study
are not clear. Superoxide facilitates synaptic neurotrans-
mission in central nervous system [41], but whether this
also happens in peripheral synaptic or neuromuscular
transmission in perivascular nerves remains to be studied.
Although superoxide may also induce vasoconstriction
directly at certain concentration [33], it does not seem to
be the case because pyrogallol, a superoxide generating
compound, enhanced the contraction to EFS and to
phenylephrine at the concentration which did not show
any contraction by itself.
Another possibility for superoxide to enhance vasocon-
striction is the inactivation of endothelial nitric oxide (NO)
[32]. Inhibition of NO synthase by l-NNA, which did not
affect basal tension, greatly increased the contractile
response to EFS in endothelium-intact MA either with or
without PVAT, suggesting that endothelial NO indeed plays
a role in antagonizing EFS-induced contraction. The fact
that PVAT (+) MA still showed a higher response to EFS
than PVAT ( ) MA in the presence of l-NNA suggests that
inactivation of NO by superoxide is not responsible for the
enhanced response to EFS in PVAT (+) MA. NO may serve
as a neurotransmitter in rat mesenteric arteries [42,43], but
further studies had already shown that neuronal NO did not
participate in EFS-stimulated vasomotor response of WKY
MA [44,45]. Therefore, inactivation of either endothelial or
371
neuronal NO by PVAT-generated superoxide did not appear
to be responsible for the enhanced contractile response to
EFS in PVAT (+) MA.
Our finding that PVAT-derived superoxide enhances
EFS-induced contraction may have physiological signifi-
cance, because PVAT is ubiquitous in almost all systemic
blood vessel, and vascular tone is mainly under the control
of sympathetic innervations. An alteration in PVAT
property such as the amount of superoxide produced may
affect local sympathetic control of vascular tone. This
Downloaded from https://academic.oup.com/cardiovascres/article/71/2/363/277463 by guest on 07 September 2020
finding may also have clinical relevance because obesity is
a well-known risk factor for the development of hyperten-
sion [46 –48], and both the quantity and the quality of
PVAT were altered in obese animals [8]. A recent report on
human and mice have shown that subcutaneous adipocytes
from obese individual produced more reactive oxygen
species than that from the normal and thus lead to systemic
oxidative stress in obesity [27]. Therefore, although
enhanced superoxide production by PVAT in obesity has
not been reported, systemic and/or perivascular oxidative
stress in obesity may enhance vasoconstriction to activation
of sympathetic innervations.
Taken together, our results suggest that in rat MA, EFS
induces vasoconstriction through the release of norepineph-
rine from perivascular nerves and simultaneously stimulates
superoxide generation from PVAT and arteries, and that the
superoxide generated from PVAT potentiates vasoconstric-
tion to norepinephrine, resulting in a greater contraction to
EFS in PVAT (+) MA than in PVAT ( ) MA. Our finding
that PVAT enhances vasoconstriction to perivascular nerve
activation, in conjunction with the finding that PVAT
releases a relaxing factor, suggests that PVAT may play a
dual regulatory role in the modulation of vascular function,
and this enhancement may be part of the mechanisms for
obesity-associated hypertension.
Acknowledgement
We thank Mr. Nathan Ni and Mr. Josh Peachey for their
technical assistance, Dr A.K. Grover for the use of his
luminometer, and Dr W.G. Foster for the use of his
fluorescence microscopy. This study is supported by the
Heart and Stroke Foundation of Ontario, Canada, and the
Canadian Institutes of Health Research. Dr. Gao is
supported by the New Investigator Award funded jointly
by the Canadian Institutes of Health Research and the
Canadian Hypertension Society.
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