Am J Physiol Heart Circ Physiol 312: H896 –H906, 2017.
First published February 24, 2017; doi:10.1152/ajpheart.00684.2016.
RESEARCH ARTICLE
Integrative Cardiovascular Physiology and Pathophysiology
Short-term regular aerobic exercise reduces oxidative stress produced by acute
high intraluminal pressure in the adipose microvasculature
Austin T. Robinson,1,2,3 Ibra S. Fancher,1,5 Varadarajan Sudhahar,4,7 Jing Tan Bian,1 Marc D. Cook,2,3
Abeer M. Mahmoud,1,2,3 Mohamed M. Ali,1,3 Masuko Ushio-Fukai,4 Michael D. Brown,2,3 Tohru Fukai,4,7
and Shane A. Phillips1,3,6
1
Submitted 11 October 2016; accepted in final form 17 February 2017
Robinson AT, Fancher IS, Sudhahar V, Bian JT, Cook MD,
Mahmoud AM, Ali MM, Ushio-Fukai M, Brown MD, Fukai T,
Phillips SA. Short-term regular aerobic exercise reduces oxidative
stress produced by acute high intraluminal pressure in the adipose
microvasculature. Am J Physiol Heart Circ Physiol 312: H896 –
H906, 2017. First published February 24, 2017; doi:10.1152/
ajpheart.00684.2016.—High blood pressure has been shown to
elicit impaired dilation in the vasculature. The purpose of this
investigation was to elucidate the mechanisms through which high
pressure may elicit vascular dysfunction and determine the mechanisms through which regular aerobic exercise protects arteries
against high pressure. Male C57BL/6J mice were subjected to 2 wk
of voluntary running (~6 km/day) for comparison with sedentary
controls. Hindlimb adipose resistance arteries were dissected from
mice for measurements of flow-induced dilation (FID; with or
without high intraluminal pressure exposure) or protein expression
of NADPH oxidase II (NOX II) and superoxide dismutase (SOD).
Microvascular endothelial cells were subjected to high physiological laminar shear stress (20 dyn/cm2) or static condition and
treated with ANG II ⫹ pharmacological inhibitors. Cells were
analyzed for the detection of ROS or collected for Western blot
determination of NOX II and SOD. Resistance arteries from exercised
mice demonstrated preserved FID after high pressure exposure,
whereas FID was impaired in control mouse arteries. Inhibition of
ANG II or NOX II restored impaired FID in control mouse arteries.
High pressure increased superoxide levels in control mouse arteries
but not in exercise mouse arteries, which exhibited greater ability to
convert superoxide to H2O2. Arteries from exercised mice exhibited
less NOX II protein expression, more SOD isoform expression, and less
sensitivity to ANG II. Endothelial cells subjected to laminar shear stress
exhibited less NOX II subunit expression. In conclusion, aerobic exercise
prevents high pressure-induced vascular dysfunction through an improved redox environment in the adipose microvasculature.
NEW & NOTEWORTHY We describe potential mechanisms contributing to aerobic exercise-conferred protection against high intravascular pressure. Subcutaneous adipose microvessels from exercise
mice express less NADPH oxidase (NOX) II and more superoxide
dismutase (SOD) and demonstrate less sensitivity to ANG II. In
Address for reprint requests and other correspondence: S. A. Phillips, Dept.
of Physical Therapy, Univ. of Illinois at Chicago, 1919 W. Taylor St. (AHSB),
Rm. 746 (M/C 898), Chicago, IL 60612 (e-mail: [email protected]).
H896
microvascular endothelial cells, shear stress reduced NOX II but did
not influence SOD expression.
Listen to this article’s corresponding podcast at https://ajpheart.
podbean.com/e/exercise-averts-high-pressure-induced-vasculardysfunction/.
hypertension; endothelium; microcirculation; exercise; oxidative
stress
(CVD) is the number one cause of
death in the United States (35). Hypertension is the number one
risk factor for CVD (35), and endothelial dysfunction also
precedes the development of CVD (23). However, the temporal
relation between high blood pressure and endothelial dysfunction has not been not fully elucidated (7, 38, 42). Regular
aerobic exercise lowers the risk of CVD and is beneficial for
blood pressure and endothelial cell health (45). However, acute
exercise bouts paradoxically increase the risk of cardiovascular
events in sedentary individuals, in part through a transient
increase in blood pressure and endothelial dysfunction (2, 9,
15). Extensive work characterizing subcutaneous adipose resistance arteries has been done in humans (22, 24, 40), demonstrating that endothelium-dependent vasodilation in these
arteries correlates with clinical measures of peripheral vascular
function (6), that high intraluminal pressure elicits endothelial
dysfunction in these arteries (9, 40), and that regular exercise
may be protective against said endothelial dysfunction (16, 40).
High blood pressure is associated with excessive oxidative
stress in the endothelium. Primary sources of oxidative stress
include NADPH oxidase II (NOX II) and mitochondrial ROS.
The local renin-angiotensin system (RAS) plays a role in both of
these pathways (8, 11, 46, 47). In adipose resistance arteries,
blockade of the local RAS through angiotensin type 1 receptor
(AT1R) blockade or angiotensin-converting enzyme (ACE) inhibition prevents both increased superoxide (O⫺
2 ) levels and impairment of endothelium-dependent dilation after high pressure (11).
Uncoupled endothelial nitric oxide synthase (eNOS) in the presence of tetrahydrobiopterin oxidation is also a source of superoxide (28). Recent findings suggest that exercise leads to an increase
CARDIOVASCULAR DISEASE
http://www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Department of Physical Therapy, University of Illinois at Chicago, Chicago, Illinois; 2Department of Kinesiology and
Nutrition, University of Illinois at Chicago, Chicago, Illinois; 3Integrative Physiology Laboratory, University of Illinois at
Chicago, Chicago, Illinois; 4Departments of Medicine (Section of Cardiology) and Pharmacology, Center for Cardiovascular
Research, University of Illinois at Chicago, Chicago, Illinois; 5Division of Pulmonary, Critical Care, Sleep, and Allergy,
Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; 6Division of Endocrinology, Diabetes, and
Metabolism, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; and 7Jesse Brown Veterans Affairs
Medical Center, Chicago, Illinois
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
METHODS
Exercise protocol. Twelve- to sixteen-week-old male C57BL/6J
mice (obtained from Jackson Laboratory, Bar Harbor, ME) were used
for this study. Mice ran on an exercise wheel (12.7-cm diameter)
placed inside their cage for 2 wk. The wheels were instrumented with
a tachometer, composed of a ring magnet on the wheel axle and a Hall
effect sensor (magnetic flux sensor) on the wheel apparatus base to
count the number of revolutions. The analog signal from the Hall
effect sensor was acquired continuously using an analog-to-digital
converter and data logger (DI-710-ULS, Dataq Instruments, Akron,
OH). WinDaq Data Acquisition and Playback Software was used to
determine distance ran based on wheel revolutions and wheel diameter. One revolution corresponds to 40 cm; thus, the revolution count
was converted to running distance by multiplying by 40 cm.
Mice ran an average of 6 km every 24 h, similar to previously
reported values (5, 29). Mice were acclimatized to the running wheels
for 48 h before being given permanent access to the unlocked wheels.
Mice ran on the wheels during the nocturne (2000 to 0800 hours) and
were on a 12:12-h light-dark light cycle. Mice were fed normal mouse
chow ad libitum. Mice were anesthetized with isoflurane (5%) and,
except for where otherwise stated, euthanized for experimentation
within 2 h of their last exercise bout. Mice were not fasted at the time
of death. Control mice weighed 29.4 ⫾ 0.54 g, whereas exercised
mice weighed 28.0 ⫾ 0.71 g. Animal experiments were conducted in
accordance with the University of Illinois at Chicago’s Animal Care
and Use Committee (ACC no. 15-037) and National Institutes of
Health (NIH) guidelines.
Resistance artery FID. Resistance artery FID was performed as
previously described (24, 40). For further detail, refer to Fig. 1.
Briefly, bilateral hindlimb subcutaneous (inguinal) fat pads were
removed from mice and placed in cold (4°C) HEPES buffer. After
dissection of vessel arcades, single resistance arteries (internal diameter: 86 ⫾ 5.45 ␮m) were cannulated on glass micropipettes (internal
diameters of ~20 to 40 ␮m) in an organ chamber filled with Krebs
A
B
Pressure grad (cmH2O)
Diameter (μm)
Shear (dyn/cm2)
Δ10
50.85 ± 3.44
2.88 ± 0.21
Δ20
54.93 ± 3.67
6.73 ± 0.45
Δ40
63.14 ± 4.18
15.48 ± 1.02
Δ60
74.06 ± 5.02
27.32 ± 1.83
Δ100
78.47 ± 5.22
48.09 ± 3.20
Fig. 1. Schematic of the isolated vessel preparation used to generate flow-induced dilation (FID). Flow is generated by manipulating pressure gradients.
Cannulated resistance arteries are equilibrated at 60 cmH2O (44 mmHg) intraluminal pressure for 30 min. Endothelin-1 (ET-1; ~120 pM) is applied into the
external bathing solution in the organ chamber in which the resistance artery is cannulated. Pressure gradients are crated to generate flow. A: to generate flow,
one of the Krebs-filled reservoirs is moved up (a) cm and the other Krebs-filled reservoir is simultaneously moved down (a’) cm from the 60-cm position. The
pressure gradient between the two reservoirs is equal to (a ⫹ a’) cmH2O. a and a’ are equal in magnitude and opposite in direction. Thus, the sum of b and c
will always result in an average pressure of 60 cmH2O (b ⫽ 60 ⫹ a cm and c ⫽ 60 – a’ cm). Furthermore, the total pressure within the system is constant, 120
cmH2O. B: table of the corresponding resistance artery diameters (means ⫾ SE) and shear stress values at the different pressure gradients (⌬10, ⌬20, ⌬40, ⌬60,
and ⌬100 cm H2O) Shear stress was calculated from the flow rate in the vessel lumen and the diameter of the vessels using the following equation described
by Ahn et al. (1): ␶ ⫽ 4(␮Q)/(␲r3), where ␮ is viscosity (0.015 g·cm⫺1·s⫺1), Q is the volumetric flow rate {[⌬P ⫻ r4]/[8/␲ ⫻ l)], where l is length}, r is the
internal radius of the vessel (in ␮m converted to cm), and ⌬P is the change in the pressure gradient (in cmH2O converted to Pa).
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
in antioxidant mechanisms that prevent oxidative stress and endothelial dysfunction in the face of high pressure (9, 40).
Isolated adipose resistance arteries from exercise-trained
individuals produce less O⫺
2 after high intraluminal pressure
compared with resistance arteries from sedentary individuals
and are protected from high pressure-induced vasodilator dysfunction (9, 40). Furthermore, H2O2 appears to be the mediator
of preserved vasodilation in adipose resistance arteries from
exercise-trained adults (9, 40). This is of physiological relevance because O⫺
2 reduces nitric oxide (NO·) bioavailability
and contributes to eNOS uncoupling. However, O⫺
2 is also
converted to H2O2 via spontaneous dismutation or enzymatic
conversion by SOD, whereby H2O2 can act as an alternative
vasodilator in times of reduced NO· bioavailability (43). In
agreement, regular exercise has been shown to increase SOD in
the aortic endothelium (41) and vascular smooth muscle (17).
Exogenous SOD has been shown to rescue resistance artery
flow-induced dilation (FID) after high intraluminal pressure
(25). Thus, regular exercise-induced increases in SOD expression would seem to be a likely mechanism for reduced O⫺
2 and
improved H2O2-mediated vasodilator function in response to
high intraluminal pressure after exercise training. Therefore, we
hypothesized that regular exercise would preserve vascular function after exposure to high intraluminal pressure through increased
SOD expression and improved H2O2-mediated dilation. We also
hypothesized exercise would lead to a reduction in NOX II
expression and that shear stress is the primary mediator of chronic
exercise-related adaptations in regard to NOX II and SOD expression. This investigation is important in that the findings will
elucidate the mechanisms through which regular exercise may
protect the microvascular endothelium from high pressure, thus
facilitating maintenance of microvascular perfusion.
H897
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
A
%Vasodilation
B
CON n=14
100
*
*
60
*
40
20
*
*
10
20
40
60
80
40
20
0
ET-1
100
10
20
40
60
100
Pressure Gradient cm H2O
C
100
BSL
80
BSL
HILP
60
Pressure Gradient cm H2O
HILP
60
40
20
0
Control
EX n=11
100
BSL
HILP
80
0
ET-1
%Vasodilation
Fig. 2. High intraluminal pressure (HILP) impairs
FID in resistance arteries from control mice but
not exercised mice. A: high pressure (HILP) significantly reduced resistance artery FID compared
with baseline (BSL) in control mouse arteries.
*P ⬍ 0.01 at ⌬10, 20, 40, 60, and 100 cmH2O. B:
there was no difference in resistance artery FID
after HILP compared with BSL in exercise mouse
arteries. Vasodilation to papaverine was similar
between resistance arteries from control and exercised mice. C: there was no influence of HILP
on the papaverine response. Data are presented as
means ⫾ SE.
Life Sciences (East Farmingdale, NY). NSC23766 was obtained from
Tocris Bioscience (Bristol, UK). All other chemicals were obtained
from Sigma-Aldrich (St. Louis, MO).
Resistance artery ANG II dose responses. Resistance artery vasoconstriction to ANG II doses was performed as previously described
(37). Resistance arteries maintained at an intraluminal pressure of 60
cmH2O (44 mmHg) were exposed to incremental doses of ANG II
(10⫺10⫺10⫺6 M) for 3 min each. Minimal internal diameters were
recorded for each dose. After the dose response, vessels were maximally dilated using papaverine (100 ␮M).
Resistance artery fluorescence. Superoxide and H2O2 levels were
measured as previously described (24, 40). Briefly, arteries were
maintained at 37°C in 20-ml aerated organ chambers bathed in Krebs
solution at an equilibration pressure of 60 cmH2O (44 mmHg) for 30
min or exposed to HILP (110 mmHg), as described above. Superoxide
levels were determined using 5 ␮M dihydroethidium (DHE). H2O2
levels were assessed using 1 ␮M 2=,7=-dichlorodihydrofluorescein
diacetate (DCF-DA). Both dyes were purchased from Thermo Fisher
Scientific (Waltham, MA). After incubation with dyes, vessels were
exposed to flow (⌬60 cmH2O) 30 min. Prepared vessels were then
excised from the glass micropipettes and subsequently mounted on
slides with Dako fluorescent mounting medium (Dako, Carpinteria,
CA). Mounted vessels were then immediately examined via fluorescent microscopy (Eclipse 80i, Nikon). Acquired images were analyzed for fluorescence intensity in arbitrary units using NIH ImageJ
software. Fluorescence was measured three times along different
lengths of each vessel. Background intensities were subtracted, and
fluorescence was normalized to vessel size to account for autofluorescence (34). Buthionine sulfoxamine (BOS; 1 mM) was used as a
positive control to induce O⫺
2 and H2O2 production in resistance
arteries. A combination treatment of the SOD mimetic tiron (1 mM)
and PEG-Cat (500 U/ml) was used for the negative control.
Human adipose microvascular endothelial cells. Experiments were
performed on isolated human adipose microvascular endothelial cells
(HAMECs) obtained from ScienCell (Cedro, Carlsbad, CA).
HAMECs were grown in ScienCell endothelial cell growth medium
(ECM-1007, PRF) supplemented with 5% FBS, EC growth supple-
Exercise
Papaverine 10-4M
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
solution. The remaining arteries were snap frozen in liquid nitrogen
for molecular biology experiments described below. Resistance artery
diameter was monitored by placing the organ chamber on the stage of
an inverted microscope attached to a real time video-measuring
apparatus (model VIA-100, Boeckeler). Arteries were maintained at
37°C, continuously perfused with warm Krebs buffer, and aerated
with a gas mixture of 21% O2 and 5% CO2.
After an intraluminal pressure of 60 cmH2O (44 mmHg) was
maintained for 30 min, arteries were constricted 40 – 60% with endothelin-1 (ET-1; 125 ⫾ 5 pmol for control mice and 115 ⫾ 3 pmol for
exercised mice, not significant, means ⫾ SE). FID was produced
using two Krebs solution-filled reservoirs to generate pressure gradients of ⌬10, ⌬20, ⌬40, ⌬60, and ⌬100 cmH2O. Pressure gradients are
generated by moving the reservoirs in equal and opposite directions.
Diameters were taken after 3 min of exposure to each pressure
gradient and then moved to create the next pressure gradient. For the
high pressure model, high intraluminal pressure (HILP; 150 cmH2O
or 110 mmHg) was administered for 45 min followed by 15 min of
reequilibration at 60 cmH2O, similar to previously published protocols (2, 9, 11). FID was measured in the absence and presence of the
NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 100 ␮M),
H2O2 scavenger polyethylene glycol-catalase (PEG-Cat; 500 U/ml),
AT1R blocker losartan (50 ␮M), NOX II inhibitors VAS2870 (2 ␮M)
and NSC23766 (10 ␮M), and SOD inhibitor zinc-diethyldithiocarbamate (Zn-DDC; 1 mM), which were added in randomized order
either 30 min before preconstriction with ET-1 or coincubated during
HILP. The maximal diameter of every vessel (endothelium-independent vasodilation) was determined with papaverine (100 ␮M) at the
end of each round of FID. FID was calculated as the percent change
from ET-1-constricted diameter relative to the maximal diameter
measured at rest before ET-1 constriction. Three control male
C57BL/6J mice were used for experiments to determine if impaired
FID occurred in another vascular bed. Six resistance arteries (n ⫽ 6)
were obtained, one from each gracilis muscle of the three mice. The
skeletal muscle resistance arteries were 63.9 ⫾ 9.7 ␮m
(means ⫾ SD). In agreement with our previous work, we noted that
skeletal muscle resistance arteries (32, 37) were smaller than adipose
resistance arteries (11, 24, 40). VAS2870 was obtained from Enzo
%Vasodilation
H898
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
100
*
*
-40
*
-60
-10
-9
-8
-7
-6
20
D
CON n=6
HILP
HILP + VAS2870
80
100
*
*
40
0
ET-1
*
*
10
20
40
60
10
20
40
60
100
100
Pressure Gradient cm H2O
CON n=6
HILP
HILP + NSC23766
80
*
60
20
*
*
Pressure Gradient cm H2O
%Vasodilation
100
*
40
Angiotensin II log [M]
C
*
*
60
0
ET-1
-80
%Vasodilation
CON n=7
HILP
HILP + Losartan
80
-20
%Vasodilation
% Vasoconstriction
B
CON n=5
EX n=4
0
*
*
60
*
40
*
20
0
ET-1
Fig. 3. Impaired FID after HILP in resistance
arteries from control mice involves the local
renin-angiotensin system (RAS) and NADPH
oxidase II (NOX II). A: exercise mouse arteries
were significantly less sensitive to ANG IIinduced constriction compared with control
(CON) mouse arteries. *P ⬍ 0.01 at 10⫺6⫺10⫺8
M. B: blockade of the angiotensin type 1 receptor
(AT1R) with losartan (HILP ⫹ losartan) restored
FID in control mouse arteries. *P ⬍ 0.01 at ⌬10,
20, 40, 60, and 100 cmH2O. C: blockade of NOX
II with VAS2870 (HILP ⫹ VAS2870) restored
FID in control mouse arteries. *P ⬍ 0.01 at ⌬10,
20, 40, 60, and 100 cmH2O. D: blockade of NOX
II though inhibition of RAC1 guanine exchange
factor (GEF) with NSC23766 (HILP ⫹
NSC23766) restored FID in control mouse arteries. *P ⬍ 0.01 at ⌬ 20, 40, 60, and 100 cmH2O.
Data are presented as means ⫾ SE.
10
20
40
60
100
Pressure Gradient cm H2O
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Processor, Cole Palmer) in 60 ␮l RIPA buffer (Sigma) containing 3⫻
protease and 3⫻ phosphatase inhibitor cocktails (Thermo Fisher
Scientific). After treatment with shear and pharmacological agents,
cells were washed twice with cold PBS and lysed with 250 ␮l of 3⫻
protease and phosphatase inhibitor cocktail. Lysates were then centrifuged at 17,000 rcf at 4°C for 20 min. Protein determination of
tissue and cell lysates was determined using a bicinchoninic acid
assay (Pierce, Thermo Fisher Scientific). Tissue and cell supernatants
were subsequently heated at 95°C for 5 min at a 2:1 with a 5%
␤-mercaptoethanol 2⫻ Laemmli sample buffer.
Western blot determination of protein expression. For Western blot
experiments, 20 ␮g of protein were loaded into each well of Bio-Rad
10% gels. Samples were processed by SDS-PAGE (time varied
depending on molecular weight of target proteins at 200 V) and
transferred to polyvinylidene difluoride membranes (0.22 ␮m, BioRad). Proteins were wet transferred at 4°C from 70 to 90 min at
90-110 V depending on the molecular weight of the target protein.
Membranes were blocked with Li-Cor blocking buffer (Lincoln, NE)
for 2 h at room temperature. Membranes were incubated with primary
antibodies for gp91phox and p47phox (1:1,000, Cell Signaling Technologies), GAPDH (1:5000, Cell Signaling Technologies), actin (1:
2,000, Thermo Fisher), SOD I (1:3,300, R&D Systems), and SOD II
and SOD III (1:1,000, Santa Cruz Biotechnology) in Li-Cor blocking
buffer (0.2% Tween 20). Secondary antibodies (1:15,000) were incubated for 2 h in Li-Cor blocking buffer (0.2% Tween 20 and 0.01%
SDS). Membranes were washed in Tris-buffered saline-Tween 20
(4 ⫻ 5 min) after antibody incubations. Membranes were transferred
to Tris-buffered saline after the last wash and imaged within 1 h using
a Li-Cor Odyssey scanner. Boxes were manually placed around each
band of interest, which returned near-infrared fluorescent values of
raw intensity with intralane background subtracted using Odyssey
analytic software (27, 31).
Statistics. All FID and ANG II dose-response comparisons were
analyzed using two-way repeated-measures ANOVA using vessel
ment, 100 U/ml penicillin, and 100 ␮g/ml streptomycin at 37°C in a
humidified incubator (5% CO2).
Shear stress experiments. HAMECs were grown to 80 –90% confluence. High physiological laminar shear stress (20 dyn/cm2 for 24 h)
was generated using a previously described modified cone and plate
design (3, 39). This level of shear stress has been used in several
exercise mimetic models (14, 26). The cone used in these studies is
designed to fit into a 20 ⫻ 100-mm tissue culture dish. Magnetism to
rotate the cone is provided by magnets within a modified Thermo
Scientific Super-Nuova multiplace stirrer (Thermo Fisher Scientific).
The angle between the cone and plate is negligible (~0.5°), thus
facilitating uniform flow over all HAMECs on the culture dish.
HAMECs were switched to serum-free media before shear stress
experiments to prevent bubbling. Control (static) HAMECs were also
switched to serum-free media. All shear experiments were conducted
under sterile conditions. Passages 5⫺8 were used for all experiments.
Cellular fluorescence. HAMECs were grown to ~90% confluence
on coverslips pretreated for 24 h with 2% bovine plasma fibronectin
in basal media. Once the desired confluence was met, cells were
incubated with the O⫺
2 indicator DHE (5 ␮M) and cotreated with or
without losartan (50 ␮M), NOX II inhibitors VAS2870 (2 ␮M) and
NSC23766 (10 ␮M), or the SOD mimetic tiron (1 mM) for 30 min.
For the final 15 min of incubation, ANG II (400 nM) was added, and
for the final 10 min, the nucleic acid stain Hoechst 33342 (1 ␮M) was
added. Cells were then washed twice using cold PBS and mounted on
slides with DAKO fluorescent mounting medium. Mounted slips were
then examined via fluorescent microscopy (Eclipse 80i, Nikon). Acquired images were analyzed for fluorescence intensity in arbitrary
units using NIH ImageJ software. Random sequences of 10 cells were
chosen from each treatment of each passage. This ensured that cases
from no single passage disproportionately affected the data. These
experiments were repeated four times.
Sample preparation for immunoblot analysis. Resistance arteries
were homogenized and sonicated (Qsonica Q55 Sonicator Ultrasonic
A
H899
H900
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
A 100
B
100
EX n=3
HILP
100
60
40
20
80
%Vasodilation
%Vasodilation
80
60
40
20
10
20
40
60
*
80
*
60
40
*
20
0
0
0
ET-1
EX n=5
HILP
HILP + Zn-DDC
HILP + VAS2870
HILP + Losartan
%Vasodilation
C
EX n=3
HILP
ET-1
100
Pressure Gradient cm H2O
10
20
40
60
ET-1
100
Pressure Gradient cm H2O
10
20
40
60
100
Pressure Gradient cm H2O
50
60cm
HILP
150cm H2O
50
n=5
40
30
Exercise
60 cm H2O
Fluorescence
60 cm H2O
Fluorescence
B
Control
*
20
10
0
Resistance artery FID and fluorescence. Baseline resistance
artery FID was similar in vessels obtained from control and
exercise mice (Fig. 2, comparison not shown). After exposure
to high intraluminal pressure, FID was reduced in control
C
150cm H2O
40
30
20
10
Control
60 cm H2O
50
n=4
60cm
HILP
Pressure
DHE (5μM)
Neg Control
Pos Control
D
150cm H2O
n=4
40
30
20
10
Exercise
60 cm H2O
150cm H2O
50
*
n=4
40
30
20
10
0
0
0
Pressure
E
RESULTS
Fluorescence
A
samples in each condition were needed to satisfy power (1 ⫺ ␤) set at
0.80.
Fluorescensce
diameter as a covariate followed by pairwise comparisons using
Bonferroni’s adjustment (treatment ⫻ time). One-way ANOVA with
Tukey’s post hoc test was used to compare HAMEC fluorescent data.
Western blot and resistance artery fluorescence data were compared
using t-tests. ␣ was set at ⬍0.05. With power (1 ⫺ ␤) set at 0.80 and
␣ at ⬍0.05 and assuming 20% differences in treatment conditions
with 10% SD, it was calculated that five samples were needed for each
condition allowing for two pairwise comparisons (adjusted ␣ 0.025).
In some cases, the effect size was adequately large that only four
Pressure
Pressure
DCF-DA (1μM)
Neg Control
Pos Control
Fig. 5. Differential oxidative stress levels after HILP in resistance arteries from control and exercised mice. A: HILP evoked a significant increase in O⫺
2 levels
compared with BSL (60 cmH2O) in control mouse arteries. *P ⬍ 0.05. B: in exercise mouse arteries, HILP exposure did not evoke a significant increase in O⫺
2
levels. C: in control mouse arteries, HILP did not evoke a significant increase in H2O2 levels compared with BSL. D: in exercise mouse arteries, HILP elicited
a significant increase in H2O2 levels. *P ⬍ 0.05. E: data are presented as means ⫾ SE. Buthionine sulfoxamine (BOS; 1 mM) increased and tiron (1 mM) ⫹
polyethylene glycol-catalase (PEG-Cat; 500 U/ml) decreased dihydroethidium (DHE) and 2=,7=-dichlorodihydrofluorescein diacetate (DCF-DA) signals,
indicating that these fluorophores are sensitive to increased ROS.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 4. Effect of ANG II, NOX II, and SOD blockade with HILP on FID in resistance arteries from exercised mice. A: there was no difference in resistance artery
FID after HILP compared with HILP ⫹ blockade of AT1Rs (HILP ⫹ losartan) in exercise mouse resistance arteries. B: there was no difference in resistance
artery FID after HILP compared with HILP ⫹ NOX II inhibition via VAS2870 (HILP ⫹ VAS2870) in exercise mouse resistance arteries. C: blockade of
endogenous SOD with zinc-diethyldithiocarbamate (HILP ⫹ Zn-DDC) reduced FID compared with HILP alone in exercise mouse arteries. *P ⬍ 0.01 at ⌬20,
60, and 100 cmH2O. Data are presented as means ⫾ SE.
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
B
Pre HILP
0
Post HILP
0
*
-20
*
*
Δ %Dilation
Δ %Dilation
A
*
-40
L-NAME n=7
PEG-Cat n=8
-60
10
20
40
*
- 20
*
100
Fig. 6. Preserved FID after HILP in resistance
arteries from exercised mice involves a phenotypic switch from nitric oxide (NO·)-mediated
dilation to H2O2-mediated dilation. A: in the basal
condition, N G -nitro- L -arginine methyl ester
(L-NAME) reduced FID significantly more so
than PEG-Cat in exercise mouse arteries. *P ⬍
0.01 at ⌬20, 40, 60, and 100 cmH2O. B: after
exposure to high pressure, PEG-Cat reduced FID
significantly more so than L-NAME. *P ⬍ 0.01 at
⌬20, 40, 60, and 100 cmH2O. C: in the BSL
condition, a combination blockade of L-NAME ⫹
PEG-Cat resulted in a greater reduction in vasodilation than L-NAME alone. Significant main
effect for condition: F ⫽ 3.13, P ⬍ 0.01. D: after
HILP, a combination blockade of L-NAME ⫹
PEG-Cat resulted in a greater reduction in vasodilation than PEG-Cat alone. Significant main
effect for condition: F ⫽ 6.64, P ⬍ 0.05. These
findings indicate a switch from NO·-mediated
dilation at rest to H2O2-mediated dilation after
exposure to HILP. Data are presented as
means ⫾ SE.
*
L-NAME n=7
PEG-Cat n=11
- 60
60
*
- 40
10
Pressure Gradient cm H2O
20
40
60
100
Pressure Gradient cm H2O
C
D
100
100
L-NAME n=11
PEG-Cat + L-NAME n=4
80
60
%Vasodilation
%Vasodilation
At baseline, FID in arteries from exercised mice was significantly reduced by both L-NAME and PEG-Cat, but the reduction via L-NAME was significantly greater than that of PEGCat (P ⬍ 0.01 at 20, 40, 60, and 100 cmH2O; Fig. 6A). In
contrast, the preserved FID in exercise mouse arteries after
exposure to HILP was also significantly reduced by PEG-Cat
and L-NAME, but the reduction in the presence of PEG-Cat
was significantly greater than that of L-NAME (P ⬍ 0.01 at 20,
40, 60, and 100 cmH2O; Fig. 6B). In the baseline condition,
combination blockade of L-NAME ⫹ PEG-Cat resulted in a
greater reduction in vasodilation than L-NAME alone (Fig. 6C,
main effect for condition F ⫽ 3.13, P ⬍ 0.01). After high
intraluminal pressure, combination blockade of L-NAME ⫹
PEG-Cat resulted in a greater reduction in vasodilation than
PEG-Cat alone (Fig. 6D, main effect for condition F ⫽ 6.64,
P ⬍ 0.05).
Resistance artery protein expression. Resistance arteries
obtained from exercised animals expressed lower protein levels
of the gp91phox NOX II subunit compared with arteries from
control mice (P ⬍ 0.05; Fig. 7A). Resistance arteries obtained
from exercised animals also expressed lower protein levels of
the p47phox NOX II subunit compared with arteries from
control mice (P ⬍ 0.01; Fig. 7B). In contrast, exercise mouse
arteries expressed more SOD I (P ⬍ 0.01; Fig. 7C), SOD II
(P ⬍ 0.01; Fig. 7D), and SOD III protein (P ⬍ 0.05; Fig. 7E).
HAMEC fluorescence and protein expression. HAMECs
exposed to ANG II exhibited significantly greater O⫺
2 levels
compared with HAMECs maintained in the control condition.
AT1R blockade with losartan, NOX II inhibition with
VAS2870 or NSC23766, and treatment with exogenous SOD
mimetic tiron all prevented increased O⫺
2 (P ⬍ 0.01; Fig. 8). In
HAMECs exposed to high shear stress, there was less protein
expression of the gp91phox and p47phox NOX II subunits than in
static cells (P ⬍ 0.01 for both; Fig. 9, A and B). High shear
stress did not influence SOD I or SOD II protein expression
30
Condition, F=3.13, P = 0.004
20
10
PEG-Cat n=11
80
PEG-Cat + L-NAME n=4
60
30
Condition, F=6.64, P = 0.023
20
10
0
0
ET-1
10
20
40
60
100
Pressure Gradient cm H2O
ET-1
10
20
40
60
100
Pressure Gradient cm H2O
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
mouse arteries (P ⬍ 0.01 at 10, 20, 40, 60, and 100 cmH2O;
Fig. 2A). FID was preserved after high intraluminal pressure in
exercise mouse arteries (Fig. 2B). Vasodilation to papaverine
was similar between resistance arteries from control and exercise mice, and there was no influence of high intraluminal
pressure on the papaverine response (Fig. 2C).
Vasoreactivity to ANG II was greater in control mouse
arteries compared with exercise mouse arteries (P ⬍ 0.01 at
10-6⫺10-8 M; Fig. 3A). Impaired FID in control mouse arteries
exposed to HILP was restored by AT1R blockade with losartan
(P ⬍ 0.01 at 10, 20, 40, 60, and 100 cmH2O; Fig. 3B). FID in
control mouse arteries exposed to HILP was also restored with
NOX II inhibition via VAS2870 (P ⬍ 0.01 at 10, 20, 40, 60,
and 100 cmH2O; Fig. 3C) and NSC23766 (P ⬍ 0.01 at 20, 40,
60, and 100 cmH2O; Fig. 3D). The addition of losartan or
VAS2870 did not influence FID after high pressure exposure in
exercise mouse arteries (Fig. 4, A and B). FID after high
intraluminal pressure exposure was significantly reduced by
the endogenous SOD inhibitor Zn-DDC in resistance arteries
obtained from exercised mice (P ⬍ 0.01 at 20, 60, and 100
cmH2O; Fig. 4C).
High pressure resulted in increased O⫺
2 levels in control
mice arteries compared with baseline pressure (P ⬍ 0.01; Fig.
5A). In contrast, exercise mouse arteries exposed to high
pressure did not exhibit significantly greater O⫺
2 levels compared with exercise mouse arteries maintained at baseline
pressure (Fig. 5B). H2O2 levels were similar in control mouse
arteries exposed to high pressure and control mouse arteries
maintained at normal pressure (Fig. 5C). Arteries obtained
from exercise mouse arteries exposed to high pressure exhibited significantly greater H2O2 levels compared with exercise
mouse arteries maintained at 60 cmH2O (P ⬍ 0.01; Fig. 5D).
The fluorescence control experiments indicated that the positive control BOS increased superoxide and H2O2 production in
resistance arteries, whereas the application of tiron and PEGCat reduced the signal for superoxide and H2O2 (Fig. 5E).
H901
H902
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
A
B
gp91phox
p47phox
GAPDH
GAPDH
(AU)
n=4
1.0
phox
(AU)
n=8
1.0
0.5
*
P47
gp91
phox
*
1.5
1.5
0.5
0.0
0.0
CON
EX
EX
D
E
SOD I
SOD II
SOD III
GAPDH
β-Actin
GAPDH
*
1.5
SOD II (AU)
SOD I (AU)
n=7
1.0
0.5
1.5
*
n=8
SOD III (AU)
1.5
1.0
0.5
0.0
0.0
CON
n= 4
1.0
0.5
*
0.0
EX
CON
EX
CON
EX
Fig. 7. Differential expression of NOX II subunits and SOD isoforms in resistance arteries from control and exercised mice. A: there was a significant reduction
in gp91phox expression in exercise mouse arteries compared with control mouse arteries. *P ⬍ 0.05. B: there was a significant reduction in p47phox expression
in exercise mouse arteries compared with control mouse arteries. *P ⬍ 0.01. C: there was a significant increase in SOD I expression in exercise mouse arteries
compared with control mouse arteries. *P ⬍ 0.01. D: there was a significant increase in SOD II expression in resistance arteries from exercise mouse arteries
compared with control mouse arteries. *P ⬍ 0.01. E: there was a significant increase in SOD III expression in in exercise mouse arteries compared with control
mouse arteries. *P ⬍ 0.05. Data are presented as means ⫾ SE.
DISCUSSION
(Fig. 9, C and D). Endothelial cells do not express SOD III (19,
33). All blots were normalized to ␤-actin or GAPDH.
In additional FID experiments, skeletal muscle (gracilis)
resistance arteries obtained from control mice displayed reduced FID after high pressure exposure (P ⬍ 0.01 at 20, 40, 60,
and 100 cmH2O; Fig. 10). FID was preserved after high
intraluminal pressure in adipose resistance arteries from a
mouse that was regularly exercised but withheld from exercise
for 24 h (Fig. 11).
Con
Ang II
30
*
Ang II +
Losartan
Ang II +
VAS2870
Ang II +
NSC23766
Ang II +
Tiron
†
CON
Ang II
Fluorescence
Fig. 8. Effects of ANG II on superoxide levels in
human adipose microvascular endothelial cells
(HAMECs). In HAMECs, treatment with ANG II
(400 nM) evoked a significant increase in O⫺
2 levels.
*P ⬍ 0.05. Combined treatment with ANG II ⫹
losartan (AT1R blocker), VAS2870 or NSC23766
(NOX II inhibitors), or tiron (SOD mimetic) significantly reduced the ANG II-induced increase in O⫺
2
in HAMECS. †P ⬍ 0.05. Data are presented as
means ⫾ SE.
The main findings from this study are that high intraluminal
pressure induced resistance artery vasodilator dysfunction in a
RAS/NOX II-dependent fashion in control mice, whereas arteries from exercised mice were protected from this occurrence. An illustrative summary of the primary findings is
shown in Fig. 12.We have previously shown that ANG II plays
a role in high pressure-induced vascular function (10, 11), and
20
Ang II + Losartan
Ang II + VAS2870
Ang II + NSC23766
10
0
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Ang II + Tiron
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
C
CON
H903
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
gp91phox
A
B
C
P47 phox
D
SOD I
SOD II
GAPDH
0.4
*
0.2
0.0
0.8
n=4
1.0
*
0.8
0.6
0.4
0.2
0.0
n=4
0.6
0.4
0.2
0.0
No shear
No Shear High Shear
No shear High shear
n=4
1.0
SOD II (AU)
P47phox (AU)
gp91phox (AU)
1.0
0.8
SOD I (AU)
n=4
1.0
High shear
0.8
0.4
0.2
0.0
No shear
High shear
the local RAS has been well described elsewhere (4, 36). Yet,
the molecular means through which regular exercise dampens
local RAS-induced endothelial dysfunction remains poorly
understood. Illustrating the role of local RAS losartan restored
impaired FID in control mouse resistance arteries after high
pressure exposure. This finding is in agreement with previous
reports of impaired vasodilator function in ex vivo resistance
arteries exposed to increased intraluminal pressure being
rescued via ACE inhibitors or AT1R blockade (9, 11, 37).
Control mouse arteries also displayed greater vasoconstrictive sensitivity to ANG II compared with exercise mouse
arteries. In addition, exercise mouse arteries displayed protection against local activation of the RAS, as indicated by
the preserved dilation after HILP and no impact of AT1R
blockade (Fig. 4).
The role of NOX II-induced oxidative stress was indicated
by high pressure resulting in increased O2⫺ levels in control
mouse arteries and restoration of FID by treatment with NOX
II inhibition. In contrast, exercise mouse arteries were not
impacted by NOX II blockade after HILP (Fig. 4). Furthermore, exercise mouse arteries did not exhibit increased O⫺
2
(Fig. 6) and were also found to express significantly less
protein for NOX II subunits gp91phox and p47phox (Fig. 7),
which have been shown to play a critical role in O⫺
2 generation
in endothelial cells (21, 30). Collectively, these findings may
partially explain the preserved FID after high pressure exposure in exercise mouse arteries. We did not assess NO· due to
limited tissue availability. However, the finding that L-NAME
reduced vasodilation after high intraluminal pressure, coupled
with reduced NOX II expression and O⫺
2 levels, suggests that
exercise mouse arteries had greater relative NO· bioavailability. However, NO· was not the predominate vasodilator after
high pressure in resistance arteries from exercised mice.
The preserved dilation in resistance arteries from exercised
mice was accompanied by a phenotypic switch from primarily
NO·-dependent to H2O2-dependent dilation. This was indicated
by the reduced FID after H2O2 scavenging with PEG-Cat, by
the reduced FID after inhibition of endogenous SOD with
Zn-DDC, and, finally, by the increased H2O2 fluorescence after
high pressure exposure. While the reduction in preserved
dilation with catalase in exercise mouse arteries supports previous findings (9, 40), we show here, for the first time, that the
blockade of endogenous SOD also abrogates the preserved
FID. These findings suggest that exercise mouse arteries maintained FID after high pressure due to greater SOD activity/
expression and a resultant increase in the conversion of O⫺
2 to
H2O2. In agreement, exercise mouse arteries were found to
100
BSL
BSL
CON n=6
HILP
*
*
*
60
80
%Vasodilation
80
%Vasodilation
24 hr no exercise
n=2
100
*
40
HILP
60
40
20
20
0
0
ET-1
10
20
40
60
100
Pressure Gradient cm H2O
Fig. 10. HILP impairs FID in skeletal muscles in resistance arteries from
control mice. HILP significantly reduced FID compared with BSL in control
mouse arteries. *P ⬍ 0.01 at ⌬20, 40, 60, and 100 cmH2O. Data are presented
as means ⫾ SE.
ET-1
10
20
40
60
100
Pressure Gradient cm H2O
Fig. 11. HILP does not impair FID in resistance arteries from exercised mice
after 24 h of wheel removal. There was no difference in resistance artery FID
after HILP compared with BSL in resistance arteries from exercised mice who
did not run for 24 h. Data are presented as means ⫾ SE.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 9. Expression of NOX II subunits and SOD isoforms in HAMECs exposed to laminar shear stress. A: exposure to high shear stress in HAMECs reduced
gp91phox protein expression compared with HAMECs raised under static conditions. *P ⬍ 0.05. B: in HAMECs, exposure to high shear stress reduced p47phox
expression compared with HAMECs raised under static conditions. *P ⬍ 0.01. C and D: there was no effect of high shear stress in HAMECs compared with
cells grown under static conditions in regard to SOD I expression (C) or SOD II expression (D). Data are presented as means ⫾ SE.
H904
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
express significantly more SOD protein compared with control
mouse arteries. This finding is also similar to previously
reported regular exercise-driven adaptations in other microvascular depots (41, 44).
We previously reported PEG-Cat reactivity was largely
unaffected, whereas the response to L-NAME was largely
reduced, after high pressure exposure in resistance arteries
obtained from sedentary humans. These findings suggest impaired dilation in sedentary humans is due to impaired NO·
bioavailability without a concomitant phenotypic switch to
rescue vasodilation (9, 40). We did not denude vessels in this
particular study to determine if our findings were solely attributable to the endothelium, although our group has done this in
the past (9). We were also not able to distinguish if the
molecular changes made in the vascular wall were specific to
the endothelium as isolating the endothelium and quantifying
protein expression solely from this vascular tunic was not
feasible. As a substitution, we used HAMECs. We hypothesized that ANG II is the primary mediator of high pressureinduced oxidative stress and endothelial dysfunction. To directly assess the effect of ANG II on the endothelium, we used
ANG II treatment in HAMECs. Laminar shear stress was used
to mimic exercise.
Treatment with ANG II in HAMECs resulted in increased
O⫺
2 levels. Treatment with losartan prevented the ANG IImediated increase in O⫺
2 . These findings provide additional
evidence suggesting that the local RAS is an upstream mediator of oxidative stress in response to high pressure. NOX II
inhibition also reduced the ANG II-mediated increases in O⫺
2,
indicating that NOX II is a primary downstream mediator of
local RAS. The finding that HAMECs exposed to shear stress
expressed significantly lower protein of the gp91phox and
p47phox NOX II subunits indicates that increased shear stress
during exercise bouts could be the primary stimulus for reduced NOX II-dependent O⫺
2 . Contrary to our initial hypothesis, SOD protein expression in HAMECs was unaffected by
laminar shear stress. This finding could be due to several
reasons, including that SOD changes in resistance arteries were
primarily due to changes in vascular smooth muscle. Alternatively, paracrine factors released during whole body acute
exercise may play a role in regulating endothelial SOD expression in the adipose microvasculature (10, 18, 48). Nonetheless,
treatment with exogenous tiron also prevented ANG II-mediated increases in O⫺
2 levels in HAMECs, suggesting that
exercise-induced increases in SOD could play a role in combating oxidative stress after local activation of the RAS in the
adipose microcirculation. A limitation to the HAMEC model
was that static cells were used as a comparison with high
physiological shear, whereas a low shear stress model may
have served as a more appropriate control to model in vivo
conditions.
Additional experiments (n ⫽ 2) were performed on arteries
obtained from an exercised mouse whose wheel was removed
for 24 h before death. Endothelium-dependent dilation was
preserved after high pressure exposure, despite no exercise
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
Fig. 12. Schematic of high pressure/ANG II-induced vascular oxidative stress and exercise-induced adaptation. A: high pressure increases ANG II, which acts
on AT1R (1). AT1R stimulation results in the assembly of the NOX II complex, which is composed of several subunits including membrane-bound gp91phox and
⫺
p22phox and cytosolic p47 phox, p67 phox, and the GTPase Rac1 (2). Active NOX II produces O⫺
2 (3), O2 scavenges the vasodilator NO· to form peroxynitrite (4),
or SOD converts O⫺
2 to H2O2, which can serve as an alternative vasodilator in place of NO· (5). The deleterious effects of ANG II-induced oxidative stress were
inhibited by administration of pharmacological agents including the AT1R blocker losartan (6), NOX II inhibitor VAS2870 (7), and the RAC GEF inhibitor
NSC23766 (8). Endogenous SOD activity was blocked with Zn-DDC in resistance arteries from exercised mice (9). B: regular exercise reduced expression of
NOX II subunits. C: regular exercise increased the expression of SOD isoforms.
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION
4.
5.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS
We thank Sang Joon Ahn and Heather Grimm for technical assistance.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants 2NIH-R01-HL-070187-11 (to T. Fukai), R01-HL-116976 (to T. T.
Fukai and M. Ushio-Fukai), K23-HL-085614 (to S. A. Phillips), R01-HL095701 (to S. A. Phillips), and R01-HL-095701- 03S1 (to A. T. Robinson);
Department of Veterans Affairs Merit Review Grant 2I01BX001232-05 (to T.
Fukai), and American Heart Association Scientist Development Grant
15SDG25700406 (to V. Sudhahar).
13.
14.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
15.
16.
A.T.R. conceived and designed research; A.T.R., V.S., M.C., A.M.M., and
M.A. performed experiments; A.T.R. analyzed data; A.T.R., I.S.F., J.T.B.,
M.D.B., T.F., and S.A.P. interpreted results of experiments; A.T.R. prepared
figures; A.T.R. drafted manuscript; A.T.R., I.S.F., V.S., J.T.B., M.C., A.M.M.,
M.A., M.U.-F., M.D.B., T.F., and S.A.P. edited and revised manuscript;
A.T.R., I.S.F., V.S., J.T.B., M.C., A.M.M., M.A., M.U.-F., M.D.B., T.F., and
S.A.P. approved final version of manuscript.
17.
18.
REFERENCES
1. Ahn SJ, Fancher IS, Bian JT, Zhang CX, Schwab S, Gaffin R, Phillips
SA, Levitan I. Inwardly rectifying K⫹ channels are major contributors to
flow-induced vasodilatation in resistance arteries. J Physiol. In press.
doi:10.1113/JP273255.
2. Beyer AM, Durand MJ, Hockenberry J, Gamblin TC, Phillips SA,
Gutterman DD. An acute rise in intraluminal pressure shifts the mediator
of flow-mediated dilation from nitric oxide to hydrogen peroxide in human
arterioles. Am J Physiol Heart Circ Physiol 307: H1587–H1593, 2014.
doi:10.1152/ajpheart.00557.2014.
3. Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, Jo H. Shear
stress stimulates phosphorylation of endothelial nitric-oxide synthase at
19.
20.
21.
Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol
Chem 277: 3388 –3396, 2002. doi:10.1074/jbc.M108789200.
Campbell DJ. Clinical relevance of local renin angiotensin systems. Front
Endocrinol (Lausanne) 5: 113, 2014. doi:10.3389/fendo.2014.00113.
Chinsomboon J, Ruas J, Gupta RK, Thom R, Shoag J, Rowe GC,
Sawada N, Raghuram S, Arany Z. The transcriptional coactivator
PGC-1alpha mediates exercise-induced angiogenesis in skeletal muscle.
Proc Natl Acad Sci USA 106: 21401–21406, 2009. doi:10.1073/pnas.
0909131106.
Dharmashankar K, Welsh A, Wang J, Kizhakekuttu TJ, Ying R,
Gutterman DD, Widlansky ME. Nitric oxide synthase-dependent vasodilation of human subcutaneous arterioles correlates with noninvasive
measurements of endothelial function. Am J Hypertens 25: 528 –534,
2012. doi:10.1038/ajh.2012.8.
Dharmashankar K, Widlansky ME. Vascular endothelial function and
hypertension: insights and directions. Curr Hypertens Rep 12: 448 –455,
2010. doi:10.1007/s11906-010-0150-2.
Doughan AK, Harrison DG, Dikalov SI. Molecular mechanisms of
angiotensin II-mediated mitochondrial dysfunction: linking mitochondrial
oxidative damage and vascular endothelial dysfunction. Circ Res 102:
488 –496, 2008. doi:10.1161/CIRCRESAHA.107.162800.
Durand MJ, Dharmashankar K, Bian JT, Das E, Vidovich M,
Gutterman DD, Phillips SA. Acute exertion elicits a H2O2-dependent
vasodilator mechanism in the microvasculature of exercise-trained but
not sedentary adults. Hypertension 65: 140 –145, 2015. doi:10.1161/
HYPERTENSIONAHA.114.04540.
Durand MJ, Lombard JH. Low-dose angiotensin II infusion restores
vascular function in cerebral arteries of high salt-fed rats by increasing
copper/zinc superoxide dimutase expression. Am J Hypertens 26: 739 –
747, 2013. doi:10.1093/ajh/hpt015.
Durand MJ, Phillips SA, Widlansky ME, Otterson MF, Gutterman
DD. The vascular renin-angiotensin system contributes to blunted vasodilation induced by transient high pressure in human adipose microvessels.
Am J Physiol Heart Circ Physiol 307: H25–H32, 2014. doi:10.1152/
ajpheart.00055.2014.
Farb MG, Bigornia S, Mott M, Tanriverdi K, Morin KM, Freedman
JE, Joseph L, Hess DT, Apovian CM, Vita JA, Gokce N. Reduced
adipose tissue inflammation represents an intermediate cardiometabolic
phenotype in obesity. J Am Coll Cardiol 58: 232–237, 2011. doi:10.1016/
j.jacc.2011.01.051.
Farb MG, Tiwari S, Karki S, Ngo DT, Carmine B, Hess DT, Zuriaga
MA, Walsh K, Fetterman JL, Hamburg NM, Vita JA, Apovian CM,
Gokce N. Cyclooxygenase inhibition improves endothelial vasomotor
dysfunction of visceral adipose arterioles in human obesity. Obesity
(Silver Spring) 22: 349 –355, 2014. doi:10.1002/oby.20505.
Feairheller DL, Park JY, Rizzo V, Kim B, Brown MD. Racial differences in the responses to shear stress in human umbilical vein endothelial
cells. Vasc Health Risk Manag 7: 425–431, 2011. doi:10.2147/VHRM.
S22435.
Franklin NC, Ali M, Goslawski M, Wang E, Phillips SA. Reduced
vasodilator function following acute resistance exercise in obese women.
Front Physiol 5: 253, 2014. doi:10.3389/fphys.2014.00253.
Franklin NC, Robinson AT, Bian JT, Ali MM, Norkeviciute E,
McGinty P, Phillips SA. Circuit resistance training attenuates acute
exertion-induced reductions in arterial function but not inflammation in
obese women. Metab Syndr Relat Disord 13: 227–234, 2015. doi:10.1089/
met.2014.0135.
Fukai T, Siegfried MR, Ushio-Fukai M, Cheng Y, Kojda G, Harrison
DG. Regulation of the vascular extracellular superoxide dismutase by
nitric oxide and exercise training. J Clin Invest 105: 1631–1639, 2000.
doi:10.1172/JCI9551.
Fukai T, Siegfried MR, Ushio-Fukai M, Griendling KK, Harrison DG.
Modulation of extracellular superoxide dismutase expression by angiotensin II and hypertension. Circ Res 85: 23–28, 1999. doi:10.1161/01.RES.
85.1.23.
Fukai T, Ushio-Fukai M. Superoxide dismutases: role in redox signaling,
vascular function, and diseases. Antioxid Redox Signal 15: 1583–1606,
2011. doi:10.1089/ars.2011.3999.
Fuster JJ, Ouchi N, Gokce N, Walsh K. Obesity-induced changes in
adipose tissue microenvironment and their impact on cardiovascular disease. Circ Res 118: 1786 –1807, 2016. doi:10.1161/CIRCRESAHA.115.
306885.
Görlach A, Brandes RP, Nguyen K, Amidi M, Dehghani F, Busse R.
A gp91phox containing NADPH oxidase selectively expressed in endo-
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
being performed at least 24 h before the artery was obtained
(Fig. 11). These findings are in agreement with our previous
human studies (4, 18). Taken together, these findings suggest
preserved endothelium-dependent dilation after high pressure
in exercisers is due to chronic exercise adaptations. The exact
time course for this regular exercise-conferred protection remains to be elucidated. Additional experiments performed in
skeletal muscle (gracilis m.) resistance arteries suggest impaired FID after high intraluminal pressure may be systemic
(Fig. 10). However, our findings are of particular relevance to
the adipose tissue microcirculation. Impaired adipose tissue
perfusion may contribute to CVD risk by promoting inflammation through the secretion of proinflammatory adipokines
(20). Prior studies have shown that inflammation modulates
endothelial dysfunction in human adipose resistance arteries
(12, 13). Our findings suggest regular exercise may play a role
in preventing this impaired perfusion after high pressure exposure.
Clinical implications. The collective findings presented here
elucidate a damaging pathway elicited by high pressure involving local activation of the local RAS and activation of NOX II,
which results in excess oxidative stress in the vascular wall.
Our results suggest regular exercise protects against high
pressure-induced microvascular dysfunction by creating a favorable vascular redox environment in the adipose microcirculation. Regular exercise may protect against high pressure by
creating a favorable redox environment, thus lessening the risk
for cardiovascular events.
H905
H906
22.
23.
24.
25.
27.
28.
29.
30.
31.
32.
33.
34.
35.
thelial cells is a major source of oxygen radical generation in the arterial
wall. Circ Res 87: 26 –32, 2000. doi:10.1161/01.RES.87.1.26.
Goslawski M, Piano MR, Bian JT, Church EC, Szczurek M, Phillips
SA. Binge drinking impairs vascular function in young adults. J Am Coll
Cardiol 62: 201–207, 2013. doi:10.1016/j.jacc.2013.03.049.
Green DJ, Jones H, Thijssen D, Cable NT, Atkinson G. Flow-mediated
dilation and cardiovascular event prediction: does nitric oxide matter?
Hypertension 57: 363–369, 2011. doi:10.1161/HYPERTENSIONAHA.
110.167015.
Grizelj I, Cavka A, Bian JT, Szczurek M, Robinson A, Shinde S,
Nguyen V, Braunschweig C, Wang E, Drenjancevic I, Phillips SA.
Reduced flow-and acetylcholine-induced dilations in visceral compared
with subcutaneous adipose arterioles in human morbid obesity. Microcirculation 22: 44 –53, 2015. doi:10.1111/micc.12164.
Huang A, Sun D, Kaley G, Koller A. Superoxide released to high
intra-arteriolar pressure reduces nitric oxide-mediated shear stress- and
agonist-induced dilations. Circ Res 83: 960 –965, 1998. doi:10.1161/01.
RES.83.9.960.
Kim JS, Kim B, Lee H, Thakkar S, Babbitt DM, Eguchi S, Brown
MD, Park JY. Shear stress-induced mitochondrial biogenesis decreases
the release of microparticles from endothelial cells. Am J Physiol Heart
Circ Physiol 309: H425⫺H433, 2015. doi:10.1152/ajpheart.00438.2014.
Kinoshita-Kikuta E, Kinoshita E, Matsuda A, Koike T. Tips on
improving the efficiency of electrotransfer of target proteins from Phos-tag
SDS-PAGE gel. Proteomics 14: 2437–2442, 2014. doi:10.1002/pmic.
201400380.
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM,
Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to
uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin
Invest 111: 1201–1209, 2003. doi:10.1172/JCI200314172.
Laufs U, Werner N, Link A, Endres M, Wassmann S, Jürgens K,
Miche E, Böhm M, Nickenig G. Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation 109: 220 –226, 2004. doi:10.1161/01.CIR.
0000109141.48980.37.
Li JM, Shah AM. Mechanism of endothelial cell NADPH oxidase
activation by angiotensin II. Role of the p47phox subunit. J Biol Chem
278: 12094 –12100, 2003. doi:10.1074/jbc.M209793200.
Mahmood T, Yang PC. Western blot: technique, theory, and trouble
shooting. N Am J Med Sci 4: 429 –434, 2012. doi:10.4103/1947-2714.
100998.
Mahmoud AM, Szczurek MR, Blackburn BK, Mey JT, Chen Z,
Robinson AT, Bian JT, Unterman TG, Minshall RD, Brown MD,
Kirwan JP, Phillips SA, Haus JM. Hyperinsulinemia augments endothelin-1 protein expression and impairs vasodilation of human skeletal
muscle arterioles. Physiol Rep 4: e12895, 2016. doi:10.14814/phy2.12895.
https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd⫽Retrieve&db⫽
PubMed&list_uids⫽27796268&dopt⫽Abstract.
Marklund SL. Expression of extracellular superoxide dismutase by human cell lines. Biochem J 266: 213–219, 1990. doi:10.1042/bj2660213.
Monici M. Cell and tissue autofluorescence research and diagnostic
applications. Biotechnol Annu Rev 11: 227–256, 2005. doi:10.1016/
S1387-2656(05)11007-2.
Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, de Ferranti S, Després JP, Fullerton HJ, Howard VJ, Huffman MD, Judd SE, Kissela BM, Lackland DT, Lichtman JH, Lisabeth
LD, Liu S, Mackey RH, Matchar DB, McGuire DK, Mohler ER III,
Moy CS, Muntner P, Mussolino ME, Nasir K, Neumar RW, Nichol G,
Palaniappan L, Pandey DK, Reeves MJ, Rodriguez CJ, Sorlie PD,
Stein J, Towfighi A, Turan TN, Virani SS, Willey JZ, Woo D, Yeh
RW, Turner MB; American Heart Association Statistics Committee
and Stroke Statistics Subcommittee. Heart disease and stroke statistics–
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
2015 update: a report from the American Heart Association. Circulation
131: e29 –e322, 2015. doi:10.1161/CIR.0000000000000152.
Paul M, Poyan Mehr A, Kreutz R. Physiology of local renin-angiotensin
systems. Physiol Rev 86: 747–803, 2006. doi:10.1152/physrev.00036.
2005.
Phillips SA, Pechman KR, Leonard EC, Friedrich JL, Bian JT, Beal
AG, Basile DP. Increased ANG II sensitivity following recovery from
acute kidney injury: role of oxidant stress in skeletal muscle resistance
arteries. Am J Physiol Regul Integr Comp Physiol 298: R1682–R1691,
2010. doi:10.1152/ajpregu.00448.2009.
Puddu P, Puddu GM, Zaca F, Muscari A. Endothelial dysfunction in
hypertension. Acta Cardiol 55: 221–232, 2000. doi:10.2143/AC.55.4.
2005744.
Reinhart-King CA, Fujiwara K, Berk BC. Physiologic stress-mediated
signaling in the endothelium. Methods Enzymol 443: 25–44, 2008. doi:
10.1016/S0076-6879(08)02002-8.
Robinson AT, Franklin NC, Norkeviciute E, Bian JT, Babana JC,
Szczurek MR, Phillips SA. Improved arterial flow-mediated dilation after
exertion involves hydrogen peroxide in overweight and obese adults
following aerobic exercise training. J Hypertens 34: 1309 –1316, 2016.
doi:10.1097/HJH.0000000000000946.
Rush JW, Turk JR, Laughlin MH. Exercise training regulates SOD-1
and oxidative stress in porcine aortic endothelium. Am J Physiol Heart
Circ Physiol 284: H1378 –H1387, 2003. doi:10.1152/ajpheart.00190.
2002.
Shimbo D, Muntner P, Mann D, Viera AJ, Homma S, Polak JF, Barr
RG, Herrington D, Shea S. Endothelial dysfunction and the risk of
hypertension: the multi-ethnic study of atherosclerosis. Hypertension 55:
1210 –1216, 2010. doi:10.1161/HYPERTENSIONAHA.109.143123.
Shimokawa H. Hydrogen peroxide as an endothelium-derived hyperpolarizing factor. Pflugers Arch 459: 915–922, 2010. doi:10.1007/s00424010-0790-8.
Thengchaisri N, Shipley R, Ren Y, Parker J, Kuo L. Exercise training
restores coronary arteriolar dilation to NOS activation distal to coronary
artery occlusion: role of hydrogen peroxide. Arterioscler Thromb Vasc
Biol 27: 791–798, 2007. doi:10.1161/01.ATV.0000258416.47953.9a.
Thompson PD, Franklin BA, Balady GJ, Blair SN, Corrado D, Estes
NA III, Fulton JE, Gordon NF, Haskell WL, Link MS, Maron BJ,
Mittleman MA, Pelliccia A, Wenger NK, Willich SN, Costa F; American Heart Association Council on Nutrition, Physical Activity, and
Metabolism; American Heart Association Council on Clinical Cardiology; American College of Sports Medicine. Exercise and acute cardiovascular events placing the risks into perspective: a scientific statement
from the American Heart Association Council on Nutrition, Physical
Activity, and Metabolism and the Council on Clinical Cardiology. Circulation 115: 2358 –2368, 2007. doi:10.1161/CIRCULATIONAHA.107.
181485.
Vecchione C, Carnevale D, Di Pardo A, Gentile MT, Damato A, Cocozza
G, Antenucci G, Mascio G, Bettarini U, Landolfi A, Iorio L, Maffei A,
Lembo G. Pressure-induced vascular oxidative stress is mediated through
activation of integrin-linked kinase 1/betaPIX/Rac-1 pathway. Hypertension
54: 1028 –1034, 2009. doi:10.1161/HYPERTENSIONAHA.109.136572.
Widder JD, Fraccarollo D, Galuppo P, Hansen JM, Jones DP, Ertl G,
Bauersachs J. Attenuation of angiotensin II-induced vascular dysfunction
and hypertension by overexpression of thioredoxin 2. Hypertension 54:
338 –344, 2009. doi:10.1161/HYPERTENSIONAHA.108.127928.
Woods D, Sanders J, Jones A, Hawe E, Gohlke P, Humphries SE,
Payne J, Montgomery H. The serum angiotensin-converting enzyme and
angiotensin II response to altered posture and acute exercise, and the
influence of ACE genotype. Eur J Appl Physiol 91: 342–348, 2004.
doi:10.1007/s00421-003-0993-1.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00684.2016 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on June 18, 2017
26.
EXERCISE AVERTS HIGH PRESSURE-INDUCED VASCULAR DYSFUNCTION