Nitro-Oleic Acid Inhibits

Angiotensin II–Induced Hypertension

Jifeng Zhang,* Luis Villacorta,* Lin Chang,* Zhenzhen Fan, Milton Hamblin, Tianqing Zhu,
Chen S. Chen, Marsha P. Cole, Francisco J. Schopfer, Cheri X. Deng, Minerva T. Garcia-Barrio,
Ying-Hong Feng, Bruce A. Freeman, Y. Eugene Chen

Rationale: Nitro-oleic acid (OA-NO2) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant
vasculoprotective properties in vivo. OA-NO2 exerts cell signaling actions as a result of its strong electrophilic
nature and mediates pleiotropic cell responses in the vasculature.
Objective: The present study sought to investigate the protective role of OA-NO2 in angiotensin (Ang) II–induced
hypertension.
Methods and Results: We show that systemic administration of OA-NO2 results in a sustained reduction of Ang
II–induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting
hypertension established by Ang II infusion. OA-NO2 significantly inhibits Ang II contractile response as
compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type
1 receptor (AT1R)-mediated signaling because vascular contraction by other G-protein– coupled receptors is not
altered in response to OA-NO2 treatment. From the mechanistic viewpoint, OA-NO2 lowers Ang II–induced
hypertension independently of peroxisome proliferation-activated receptor (PPAR)␥ activation. Rather, OA-
NO2, but not OA, specifically binds to the AT1R, reduces heterotrimeric G-protein coupling, and inhibits IP3
(inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor.
Conclusions: These results demonstrate that OA-NO2 diminishes the pressor response to Ang II and inhibits
AT1R-dependent vasoconstriction, revealing OA-NO2 as a novel antagonist of Ang II–induced hypertension. (Circ
Res. 2010;107:540-548.)
Key Words: nitroalkenes 䡲 hypertension 䡲 angiotensin II 䡲 angiotensin II type 1 receptor
䡲 peroxisome proliferation-activated receptor-␥

he nitrate-nitrite (NO2⫺)–nitric oxide (NO) pathway1 is

T emerging as an important mediator of cell signaling and
bioenergetics events, with therapeutic implications in blood
flow regulation and tissue responses to hypoxia.2,3 Nitroalk-
ene derivatives of unsaturated fatty acids represent the con-
vergence of such NO pathway with lipids4 because they are
formed by NO and NO2⫺-dependent redox reactions.1 Thus,
they constitute a subset of molecules in the emerging physi-
ological pool of the nitric oxide derived metabolome5,6 that
control the physiological homeostasis by coordinating adap-
tive responses.7,8 At present, the mechanisms underlying the
production of nitrated lipids under biological conditions,
specific structural isomer distributions, nitrated fatty acid-
susceptible proteome, and detailed biological signaling ac-
tions are incompletely characterized.6 It is agreed that under
conditions in which O2 tension is low (eg, ischemia and
anoxia), the balance between peroxyl radical formation and
coupling with 䡠NO2 shifts toward nitration reactions.9,10 Thus,
nitroalkenes are significantly increased in the heart after
reperfusion in an ischemia/reperfusion injury model,11 and
these species are abundantly generated in the mitochondria of
hearts exposed to ischemic preconditioning.12,13
Nitroalkenes transduce signaling reactions by covalently
modifying nucleophilic protein targets and altering their
structure and function.6 For instance, nitro derivatives of
linoleic and oleic (OA-NO2) acids are activators of peroxi-
some proliferator-activated receptor (PPAR)␥,14,15 form co-
valent adducts with the p65 subunit of nuclear factor ␬B,16

Original received February 5, 2010; revised received June 8, 2010; accepted June 9, 2010. In May 2010, the average time from submission to first
decision for all original research papers submitted to Circulation Research was 14.6 days.
From the Cardiovascular Center (J.Z., L.V., L.C., M.H., T.Z., Y.E.C.); and Department of Biomedical Engineering (Z.F., C.X.D.), College of
Engineering, University of Michigan; Department of Pharmacology & Chemical Biology (C.S.C., M.P.C., F.J.S., B.A.F.), University of Pittsburgh, Pa;
Cardiovascular Research Institute (M.T.G.-B.), Morehouse School of Medicine, Atlanta, Ga; and Department of Pharmacology (Y.-H.F.), Uniformed
Services University of the Health Sciences, Bethesda, Md.
*These authors contributed equally to this work.
Correspondence to Dr Y. Eugene Chen, Cardiovascular Center, MSRB III 7200, University of Michigan Medical Center, 1150 W Medical Center Dr,
Ann Arbor, MI 48109. E-mail echenum@umich.edu or to Dr Bruce A. Freeman, Department of Pharmacology & Chemical Biology, E1340 Thomas E.
Starzl Biomedical Science Tower, 200 Lothrop St, University of Pittsburgh, Pittsburgh, PA 15213. E-mail freerad@pitt.edu
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.218404

540
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 Zhang et al
and activate the Keap1/Nrf2 antioxidant response, reactions
that promote adaptive responses in vascular cells.17,18 These
derivatives of NO-mediated redox reactions represent an
inflammatory-resolving adaptive response.7 In the context of
the vasculature, OA-NO2 inhibits neointimal hyperplasia19
and exerts vasorelaxation of preconstricted aortic rings.20 For
these reasons, and because of the well-established vasorelax-
ant properties of pharmacological doses of nitrite,21 it is
plausible to suggest that nitroalkenes will have a significant
impact in the regulation of blood pressure and vascular tone.
Herein, we determined whether a nitroalkene derivative
ameliorate angiotensin (Ang) II–induced hypertension. As the
major component of the renin–angiotensin–aldosterone sys-
tem, deregulation of Ang II production plays a critical role in
the pathogenesis of hypertension.22 Thus, inhibition of the
catalytic activity of the angiotensin converting enzyme
(ACE) or competitive inhibition of Ang II type 1 receptor
(AT1R) ligand binding by angiotensin receptor blockers
(ARBs) are 2 prevalent treatment options for hypertension.23
Also, molecular targets of nitroalkenes, such as PPAR␥ and
the AT1R reported herein, reflect common signaling targets
that influence the progression of hypertension and other
cardiovascular and metabolic diseases.24
Methods
Ang II–Induced Hypertension Model
Blood pressure (BP) in mice was measured by radiotelemetry. Mice
were subjected to subcutaneous implantation of osmotic minipumps
for delivery of OA-NO2 or oleic acid (OA) at a concentration
supporting an infusion rate of 5 mg/kg per day, and Ang II was
administered at an infusion rate of 500 ng/kg per minute.
In Vivo Infusion for Blood Pressure Tracings and
In Vitro Vessel Contraction Analysis
OA or OA-NO2 and the PPAR␥ inhibitor GW9662 were delivered
via the jugular vein. BP recordings were monitored before and after
Ang II infusion (10 ␮g/mL, infusion rate: 1 ␮L/min) using a 1.4F
microtip catheter sensor inserted into the right carotid artery.
Second-order mesenteric arteries from SD rats were mounted onto a
myograph. Vessels were pretreated with OA-NO2 or OA at
2.5 ␮mol/L and 5 ␮mol/L for 10 minutes and then contracted with
Ang II, phenylephrine (PE), or endothelin-1 (ET-1), respectively.
Analysis of OA-NO2 Binding to the AT1R
The transalkylation reaction of AT1R-bound OA-NO2 was per-
formed according to the methodology described previously.13
An expanded Methods section is available in the Online Data
Supplement at http://circres.ahajournals.org.
Results
OA-NO2 Reduces Ang II–Induced Hypertension
To determine whether OA-NO2 plays a role on Ang II–
mediated hypertension, we delivered OA-NO2 (5 mg/kg per
day), as well as its nonnitrated counterpart, OA, systemically
via osmotic minipumps in C57/BL6 mice. Systemic delivery
of OA and OA-NO2 did not affect baseline systolic or
diastolic BP recordings measured by radiotelemetry during
the first week of the analysis as compared with polyethylene
glycol–treated group that served as vehicle control (Figure
1A and 1C). Subsequently, mice were further treated with
Ang II, supporting an infusion rate of 500 ng/kg per minute
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Nitro-Oleic Acid and Blood Pressure 541
Non-standard Abbreviations and Acronyms
ACE angiotensin-converting enzyme
Ang II angiotensin II
ARB angiotensin receptor blocker
AT1R angiotensin II type 1 receptor
BME ␤-mercaptoethanol
BP blood pressure
ET-1 endothelin-1
GPCR G-protein– coupled receptor
IP3 inositol-1,4,5-trisphosphate
NO nitric oxide
NO2ⴚ nitrate
OA oleic acid
OA-NO2 nitro-oleic acid
PPAR␥ peroxisome proliferator-activated receptor-␥
PE phenylephrine
for 2 additional weeks. Under these conditions, OA-NO2 but
not OA significantly reduced Ang II– dependent increases in
systolic (⬇15 mm Hg reduction) and diastolic BP
(⬇12 mm Hg reduction) compared to control OA- or vehicle-
treated animals (Figure 1B and 1D), without any apparent
changes in the heart rate (Online Figure I). These results
strongly indicate that OA-NO2 treatment has a significant
impact on Ang II–induced hypertension through a prolonged
and sustained reduction of BP.
We then monitored BP in isoflurane-anesthetized mice by
in vivo tracing recordings on short-term infusion of Ang II,
which results in a rapid increase in systemic BP. OA and
OA-NO2 (1 mmol/L, infusion rate: 1 ␮L/min) administered
before Ang II delivery (10 ␮g/mL, infusion rate: 1 ␮L/min) did
not alter basal BP recordings (Figure 2A and 2B). Rather,
OA-NO2, but not similar concentrations of the native OA,
significantly prevented BP elevation after short-term infusion
of Ang II (Figure 2C). To determine whether OA-NO2
actually reduces the pressor response to Ang II, we then
administered OA-NO2 to mice 3 days after Ang II osmotic
minipump implantation (Figure 2D and 2E). Under these
experimental conditions, OA-NO2 but not OA results in a
dose-dependent reduction of BP to the preexisting Ang
II–induced hypertension (Figure 2F). Taken together, these
results demonstrate that OA-NO2 diminishes the pressor
response to Ang II in vivo, regardless of whether the
nitroalkene is administered before of after Ang II challenge.
OA-NO2 Specifically Inhibits Ang
II–Induced Vasoconstriction
To determine the specificity of OA-NO2 effects on Ang
II–induced hypertension, we evaluated whether OA-NO2 may
result in changes of vascular contractility. In isolated second-
order rat mesenteric vessels, the presence of OA-NO2
(2.5 ␮mol/L) resulted in ⬇50% reduction of Ang II–mediated
vasoconstriction at 10⫺7 mol/L (corresponding to ⬇70% of
the contractile response induced by 50 mmol/L KCl), whereas
5 ␮mol/L OA-NO2 but not OA almost completely abolishes
542 Circulation Research August 20, 2010

Figure 1. OA-NO2 inhibits Ang II–induced hypertension. Mice implanted with osmotic pumps adjusted to deliver 5 mg/kg per day
OA-NO2 (red) or OA (blue) and vehicle control (black) were infused with Ang II at a pressor rate of 500 ng/kg per minute. Systolic (A)
and diastolic (C) blood pressure was measured by radiotelemetry. Data are shown as means⫾SEM (n⫽6 in each group). *P⬍0.05.
OA-NO2–treated mice showed significantly lower systolic (⬇15 mm Hg reduction) (B) and diastolic pressure (⬇12 mm Hg reduction) (D)
after Ang II challenge than did OA and vehicle-treated mice as determined both at the night and day time periods. Data in B and D are
shown as means⫾SEM of measurements collected before (3 days) and after Ang II (12 days) (n⫽6 in each group). *P⬍0.05.

contraction induced by Ang II (Figure 3A and Online Figure
IV). Thus, OA-NO2 treatment dose-dependently reduced Ang
II– dependent vasoconstriction. Interestingly, vasoconstric-
tion elicited by signaling through other members of the
G-protein– coupled receptor (GPCR) family, such as by
treatment with PE and ET-1, was not affected on OA-NO2
treatment at any concentration given (Figure 3B and 3C;
Online Figure IV). These results are clearly indicative that
OA-NO2 reduces vasoconstriction by specifically interfering
with Ang II signaling in the vasculature.
OA-NO2 Reduction of Ang
II–Induced Hypertension Is a
PPAR␥-Independent Phenomenon
Because (1) OA-NO2 activates PPAR␥,14,25 (2) PPAR␥ acti-
vators downregulate AT1R in vascular cells,26,27 and (3)
certain ARBs inhibit AT1R through PPAR␥ activity,28 –30 we
assessed whether PPAR␥ activation by OA-NO2 contributes
to the observed reduction of Ang II–induced hypertension in
vivo.28,29 To this aim, we first monitored BP by tracing
recordings on stimulation with Ang II in response to the
treatment with the PPAR␥ antagonist GW9662 (Figure 4).
Efficient inhibition of PPAR␥ by GW9662 was directly
demonstrated monitoring PPAR␥ transcriptional activity by
means of EGFP expression in the aorta of 3⫻PPRE-driven
EGFP transgenic reporter mice (Online Figure V). Inhibition
of PPAR␥ alone does not result in basal BP reduction in vivo
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(Figure 4A and 4B). Moreover, GW9662 administration (10
mg/kg) compared to vehicle treated mice at the same infusion
rate did not attenuate BP increases after Ang II challenge
(Figure 4 and Online Figure II). Of importance, pretreatment
with OA-NO2 but not equimolar concentrations of OA
persistently reduced approximately ⬇10 mm Hg of Ang
II–mediated BP elevation in the presence or absence of
GW9662 (Figure 4C). Further experiments were performed to
determine whether PPAR␥ activation by OA-NO2 affects
already established hypertension. To this aim, we compared
the BP lowering effect of OA-NO2 versus rosiglitazone after
preincubation with GW9662 in Ang II–induced hypertensive
mice as described above. In response to rosiglitazone treat-
ment, BP reduction (⬇7mmHg) was efficiently antagonized
by GW9662 (Online Figure II), whereas OA-NO2 delivery in
the presence of GW9662 results in a dose-dependent BP
reduction comparable to the lack of the PPAR␥ antagonist
(Online Figure III). Next, we determine the contractile
response of mesenteric rings in response to partial inhibition
of Ang II–mediated vasoconstriction by OA-NO 2
(2.5 ␮mol/L) after treatment with GW9662 (10 ␮mol/L). As
shown in Figure 4D, OA-NO2 similarly diminished Ang
II– dependent vasoconstriction with or without PPAR␥ antag-
onism by GW9662 treatment. This series of experiments
indicate that PPAR␥ activation by OA-NO2 is not the major
mechanism involved in OA-NO2 antagonism of both Ang
II–mediated hypertension and vasoconstriction.
 Zhang et al Nitro-Oleic Acid and Blood Pressure 543

Figure 2. OA-NO2 inhibits the pressor response to Ang II. BP was monitored on treatment with OA (A) and OA-NO2 (B) (10 mg/kg)
before Ang II infusion (10 ␮g/mL, infusion rate: 1 ␮L/min). All drugs were delivered via jugular vein administration starting at the time
indicated by the arrows. BP tracings were recorded by insertion of a microtip catheter sensor into the right carotid artery. C, Systolic
BP was averaged for a 10 minutes period before and after Ang II infusion (10 ␮g/mL, infusion rate: 1 ␮L/min). Data are shown as
means⫾SEM (n⫽6 in each group). OA-NO2 significantly reduced the pressor response to Ang II infusion compared to OA. P⬍0.05 (OA-
NO2 vs OA after Ang II infusion). Hypertension was developed in mice in 3 days after implantation of osmotic pumps to infuse Ang II at
a pressor rate of 500 ng/kg per minute. OA (D) or OA-NO2 (E) were then delivered to mice via the jugular vein at increasing concentra-
tions as indicated by the arrows. BP tracings were recorded as described above. F, Data in D and E are summarized as systolic BP
reduction (in mm Hg) after either OA or OA-NO2 treatment as compared to maximal systolic pressure in Ang II hypertensive mice.
OA-NO2 treatment but not OA dose-dependently reduced established hypertension on Ang II delivery. Data are shown as means⫾SEM
(n⫽4 in each group). *P⬍0.05.

OA-NO2 Forms Covalent Adducts With AT1R
Nitroalkenes are highly electrophilic and react with cellular
nucleophiles via a reversible Michael addition reaction.6
Thus, we hypothesized that OA-NO2 could directly modify
nucleophilic residues of AT1R and impact the propagation of
downstream signaling events. A covalent interaction between
OA-NO2 and AT1R was revealed by treatment of HEK293
cells overexpressing AT1R with OA-NO2 or OA. Following
immunoprecipitation of AT1R from cell lysates, a transalky-
lation reaction of AT1R-bound OA-NO2 permitted the detec-
tion and quantification of OA-NO2 adduction by HPLC-
MS/MS (Online Methods). HEK293 cells treated with
increasing concentrations of OA-NO2 revealed significantly
increased levels of AT1R-adducted OA-NO2 compared with
control cells (Figure 5A). OA-NO2 bound to the AT1R,
quantified as a ␤-mercaptoethanol (BME) derivative after
nucleophilic exchange of OA-NO2 from the AT1R to BME,13
revealed a ⬇20-fold increase of adducted OA-NO2 in AT1R-
expressing cells when compared to controls (Figure 5C). The
MS/MS-induced fragmentation of recovered BME-OA-NO2
adducts gave product ions and ion intensities similar to a
synthetic BME-OA-NO2 standard and BME-[13C18]-OA-
NO2 used as an internal control (Figure 5B). Thus, the
presence of BME-exchangeable OA-NO2 demonstrates the

Figure 3. OA-NO2 reduces AT1R-dependent ves-

sel contraction. Second-grade mesenteric artery
rings were mounted into a myograph as described
in the Online Data Supplement. The mesenteric
artery rings were preincubated for 10 minutes with
2.5 or 5 ␮mol/L either OA or OA-NO2 as indicated.
Vascular contraction was induced by serial addi-
tion of increasing concentrations of Ang II (A), PE
(B), or ET-1 (C) to the myograph. Contractile
response was quantified and shown in the figure
as percentage of the vessel contraction force
afforded by 50 mmol/L KCl. OA-NO2 dose-
dependently reduced Ang II–induced vasoconstric-
tion but not that of PE or ET-1. Data are shown as
means⫾SEM, n⫽6 in each group, *P⬍0.05 and
**P⬍0.01 (OA-NO2 vs OA or vehicle (0.1%
ethanol-treated arteries).

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544 Circulation Research August 20, 2010
by serial addition of increasing concentrations of Ang II to the bath in the myograph. Contractile response was quantified and shown in
the figure as percentage of the vessel contraction force afforded by 50 mmol/L KCl. OA-NO2 reduced Ang II–induced vasoconstriction
independent of the PPAR␥ antagonist GW9662. Data are shown as means⫾SEM (n⫽6 in each group). *P⬍0.05.
direct adduction of the AT1R by OA-NO2. Moreover, immu-
noprecipitation of AT1R from HEK293 cells transfected with
Flag-tagged AT1R after treatment with biotin-labeled OA-
NO2, showed a dose-dependent increase in biotin-OA-NO2–
adducted to the AT1R compared with biotin-OA–treated
controls (Figure 5D). Thus, 2 independent approaches
showed that AT1R is a relevant cellular target of OA-NO2
alkylation.
OA-NO2 Does Not Interfere With Ang II Binding
to the Receptor
Next, we sought to determine whether the adduction of the
AT1R by OA-NO2 interferes with Ang II binding. Unlike the
ARB losartan, that inhibits Ang II binding to AT1R, neither
OA-NO2 nor OA affected binding of radio-labeled Ang II to
its receptor (Figure 5E). Furthermore, neither OA-NO2 nor
OA influenced the displacement of [125I]Ang II bound to
AT1R in ligand-receptor binding competition analyses using
nonradioactive Ang II (Figure 5F). This indicates that OA-
NO2 adduction of the AT1R does not affect Ang II binding
and represents a novel manner of AT1R antagonism that is
distinct from competitive ARBs.31
OA-NO2 Interferes With G-Protein Coupling to
the AT1R and Prevents Ang II–Mediated
Contractile Responses in Vascular Smooth
Muscle Cells
Because OA-NO2 adduction of the AT1R does not affect Ang
II binding, we next examined whether OA-NO2 affects AT1R
coupling to the heterotrimeric G-protein (G␣q11). To this aim,
we treated HEK293 cells overexpressing HA-tagged AT1R,
G␣q11-EGFP, G␤1, and HA-tagged G␥2 with OA-NO2 or OA
and determined G␣q11 coupled to the AT1R following immu-
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Figure 4. OA-NO2 reduces Ang II–in-
duced hypertension independently of
PPAR␥ activation. BP in mice was moni-
tored with a microtip catheter sensor
inserted into the right carotid artery by
arteriotomy on treatment with OA (A) and
OA-NO2 (B) (10 mg/kg) delivered via jugu-
lar vein administration after infusion of the
PPAR␥ inhibitor GW9662 (10 mg/kg). A
and B show representative BP recordings
of n⫽6 in each treatment before and after
Ang II infusion (10 ␮g/mL), delivered at an
infusion rate of 1 ␮L/min. C, OA-NO2
delivery results in a significant reduction
of Ang II–induced hypertension
(⬇10 mm Hg) independently of PPAR␥
inhibition by GW9662. Data are depicted
as systolic BP increase (in mm Hg) after
Ang II infusion as compared to baseline
systolic pressure and are shown as
means⫾SEM (n⫽6 in each group).
P⬍0.01 (OA-NO2 vs OA in either
GW9662-treated or DMSO vehicle
control-treated group after Ang II infu-
sion). D, The mesenteric artery rings were
preincubated for 10 minutes with
10 ␮mol/L GW9662 and then with
2.5 ␮mol/L either OA or OA-NO2 as indi-
cated. Vascular contraction was induced
noprecipitation of G␣q11-EGFP and immunodetection of
HA-tagged AT1R by Western blot. OA-NO2 but not OA
significantly reduced AT1R coupled to the immunoprecipi-
tated G␣q11 after 1 minute stimuli with Ang II (100 nmol/L)
(Figure 6A and 6B). Taken together, adduction of OA-NO2
with the AT1R does not affect Ang II binding but reduces
G-protein coupled to the AT1R. Finally, the impact of
OA-NO2 on the contractile response of vascular smooth
muscle cells (VSMCs) downstream of AT1R reaction was
evaluated, where Ang II–triggered second messenger mobi-
lization was measured. Competitive radioreceptor analysis
showed that OA-NO2, but not OA, reduced Ang II–induced
intracellular inositol-1,4,5-trisphosphate (IP3) mobilization
(Figure 6C). Accordingly, we show a dose-dependent inhibi-
tion of Ang II–mediated increase in intracellular calcium
mobilization ([Ca2⫹]i) on OA-NO2 treatment, as determined
by fluorescence imaging of fura-2 (Figure 6D). Pretreatment
of VSMC with 2.5 ␮mol/L OA-NO2 significantly reduced the
ratio of fura-2 fluorescent emission induced by Ang II (75%),
whereas 1 ␮mol/L losartan completely inhibited Ang II–
mediated increases in [Ca2⫹]i. By comparison, OA had no
impact in Ang II–induced [Ca2⫹]i increase (Figure 6D,
Online Figure VI, and Online Movies I through III).
Discussion
Early studies deciphering the physiological and/or pharma-
cological roles of nitrated fatty acids indicate that nitro
derivatives of fatty acids exert relevant biological effects in
the vasculature32–34 (also reviewed35). More recent data, using
cardiovascular models of disease, strongly support the notion
that nitroalkenes serve as novel therapeutic tools associated
with vascular and cardiac tissue damage.11,16,19
 Zhang et al Nitro-Oleic Acid and Blood Pressure 545

Figure 5. OA-NO2 forms covalent

adducts with AT1R but does not affect
Ang II binding to the receptor. A,
HEK293 cells were transfected with 500
ng of FLAG-tagged AT1R plasmid
(pcDNA3.1-AT1R) vs control plasmid
(pcDNA3.1) and treated with OA or
OA-NO2 as indicated. Immunoprecipitated
AT1R was subjected to a BME-based
transnitroalkylation reaction13 as detailed
in the Online Data Supplement. Adducted
OA-NO2 to AT1R was released as BME-
OA-NO2 and quantified using mass spec-
trometry. The chromatographic profile
shows a dose-dependent increase of
OA-NO2 after immunoprecipitation against
AT1R and verified by coelution with
[13C18]OA-NO2 internal standard (bottom).
B, The fragmentation pattern (top) of
AT1R-derived OA-NO2 (5 ␮mol/L treat-
ment) adducted to BME was confirmed by
comparison with that of [13C18]OA-NO2
(bottom). Enhanced levels of the ion
products in the peaks and comparison to
the internal standard confirmed the struc-
ture for the proposed adducts formed
after transnitroalkylation. The data shown
are a representative set of 3 independent
experimental repetitions. C, Quantification
of the AT1R-derived BME adducts after
nucleophilic exchange of OA-NO2 from
the AT1R to BME reveals a significant
increase of adducted OA-NO2 to the AT1R
(270 vs 13 pmol/L BME-OA-NO2, respec-
tively, after 5 ␮mol/L OA-NO2 treatment).
D, HEK293 cells were similarly transfected
with the pcDNA3.1-AT1R plasmid and
treated with biotin-labeled OA-NO2 or
OA for 3 hours at the indicated concen-
trations. Biotin-labeled adducts were visu-
alized by Western blot after immunopre-
cipitation of the Flag-AT1R. OA-NO2 dose-
dependently increased the covalent
adduction to the immunoprecipitated
AT1R. The data shown are a representative set of 3 independent experimental repetitions. E, Competition binding assays were per-
formed using confluent VSMC pretreated with 2.5 ␮mol/L OA or OA-NO2 for 10 minutes and then incubated for 30 minutes with 0.3
nmol/L 125I-[Sar1,Ile8]Ang II and different concentrations of nonradiolabeled Ang II as indicated. Data are shown as percentages of max-
imal binding with 125I-[Sar1,Ile8]-Ang II. F, Similarly, VSMCs treated with different doses of OA or OA-NO2, as indicated, were then incu-
bated for 30 minutes at 37°C with 0.1 nmol/L 125I-[Sar1,Ile8]-Ang II. The ARB losartan (1 ␮mol/L) served as a positive control. In E and
F, radioactivity was measured using a ␥-counter, and values are expressed as means⫾SEM (n⫽4). The experiments were performed 3
times with similar results.

In the present study, we provide compelling evidence
supporting that OA-NO2 reduces Ang II–mediated hyperten-
sion in a model of Ang II infusion in mice. Systemic delivery
of pharmacological doses of OA-NO2 but not OA resulted in
a prolonged and sustained reduction of BP throughout the 14
days of Ang II infusion. The differences in BP in the OA-NO2
group as compared to OA or vehicle group were more
pronounced during the first week of Ang II infusion, when BP
raised and remained highest, and persisted significantly lower
during the second week of Ang II infusion, when physiolog-
ical compensatory mechanisms might occur to reduce BP in
response to a long-term hypertensive challenge.36 Thus, our
long-term radiotelemetry analysis confirmed that OA-NO2
diminished the pressor response to Ang II rather than simply
delay Ang II– dependent hypertension. Furthermore, compar-
ative analysis with the inclusion of the OA-treated group, as
the nonnitrated counterpart for OA-NO2 also indicates that
OA alone at the dosage used in the present study does not
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reduce Ang II-mediated hypertension in contrast to previous
studies indicating that OA or related species might be
hypotensive.37 Thus, at an infusion rate of 5 mg/kg per day,
reduction of Ang II–mediated hypertension is afforded by the
nitrated but not free form of OA.
Under our experimental conditions, Ang II infusion did not
result in significant changes of free nitroalkenes in plasma
(data not shown), indicating that chronic infusion of Ang II
might not generate sufficient systemic reactions to be de-
tected at the serum level of these hypertensive mice. It has
been previously shown that proinflammatory stimuli in-
creases fatty acid nitration in macrophages.38 In vivo, ni-
troalkene production is abundantly generated in the mito-
chondria of the heart after ischemia/reperfusion injury.12,13
Therefore, it seems critical to determine the local production
of nitroalkenes in response to certain types of injury and on
specific subcellular compartments locally in tissues. From the
present study, it cannot be excluded that local generation of
546 Circulation Research August 20, 2010

Figure 6. OA-NO2 uncouples G␣q11 to

the AT1R and inhibits Ang II–induced
IP3 production and Ca2ⴙ mobilization in
VSMC. A, HEK293 cells cultured in 6-well
plates were cotransfected with HA-tagged
AT1R (0.3 ␮g), G␣q11-EGFP (0.3 ␮g), G␤1
(0.1 ␮g), and G␥2 (0.1 ␮g) plasmids and
treated with either OA, OA-NO2 (2.5 ␮mol/
L), or losartan (1 ␮mol/L) 10 minutes
before stimulation with Ang II (100 nmol/L)
for 1 minute. Immunoprecipitated G␣q11
using a GFP antibody was subjected to
deglycosylation before Western blot anal-
ysis for detection of HA-tagged AT1R.
OA-NO2 but not OA reduced AT1R cou-
pled to the immunoprecipitated G␣q11.
The data shown are representative of 3
independent experiments. B, Western
blots were quantified using the ImageJ
software after densitometric scanning of
the films and expressed as relative ratio of
AT1R vs G␣q11 (n⫽3). P⬍0.05, OA-NO2 vs
OA in Ang II–treated cells; P⬍0.05, Ang
II–treated vs vehicle control cells (Con). C,
VSMCs were treated with either
2.5 ␮mol/L OA-NO2 or OA and then
pulsed with 100 nmol/L Ang II for 45 minutes. IP3 production was monitored by radioreceptor assays. Ang II induced a 2-fold increase
of intracellular IP3, whereas OA-NO2, but not OA, blocked Ang II–mediated IP3 production (n⫽6). P⬍0.05, OA-NO2 vs OA in Ang
II–treated cells; P⬍0.05, Ang II–treated vs vehicle control cells (Con). D, VSMCs were similarly treated with either OA-NO2 or OA
(2.5 ␮mol/L) and then pulsed with Ang II (100 nmol/L) in the presence of fura-2. Control experiments were performed with losartan-
pretreated (1 ␮mol/L) cells. Emission ratio between 340 and 380 nm was used to calculate intracellular calcium concentration as
described in Online Data Supplement. The data shown depict a representative time tracing of 6 independent experiments. The ratio–
time traces were generated by averaging 3 typical cells in the field of view, as described in Online Figure VI and Online Movies I
through III.

nitrated fatty acids, for instance, within the vasculature, might
occur in response to Ang II.39
In ex vivo vasoconstriction assays, OA-NO2 inhibited Ang
II–mediated vascular contractility in an AT1R-dependent
fashion. Specificity for the AT1R relies on the observations
that OA-NO2 dose-dependently inhibited Ang II–mediated
vessel contraction but did not reduced vascular contractility
induced by vasoconstrictors signaling through other GPCR,
such as the observed ET-1 and PE. Activation of PPAR␥ by
OA-NO2 is a well-established mechanism that could account
for the reduction of Ang II–induced hypertension and vascu-
lar contractility by OA-NO2. A reverse relationship between
AT1R signaling and PPAR␥ has been recognized because
genetic polymorphisms in the PPAR␥ locus cause hyperten-
sion40 and PPAR␥ agonists improve hypertension and vascu-
lar function.41 Regardless, whereas pharmacological inhibi-
tion of PPAR␥ efficiently blunts rosiglitazone effects on BP,
it did not reverse OA-NO2 reduction of Ang II–mediated
hypertension. Moreover, experimental animal models clearly
indicate that PPAR␥ regulates vascular contractility mediated
not only by AT1R but also by other GPCR.42,43 Taking into
account that OA-NO2 specifically reduced Ang II–mediated
vascular contractility but did not interfere with the contractile
properties of ET-1 and PE, it can be concluded that PPAR␥
activation by OA-NO2 is not the major mechanism involved
in OA-NO2 effects on Ang II–induced hypertension.
Notably, we demonstrate by 2 independent experimental
approaches that OA-NO2 binds to the AT1R. Quantitative
analysis of the adduction of OA-NO2 to the AT1R was
demonstrated by the nucleophilic exchange of nitroalkenes
from adducted proteins to BME, as extensively described.13
Downloaded from http://circres.ahajournals.org/ at TOKYO U MED LIB DQ56580 on May 25, 2015
The transnitroalkylation reaction of OA-NO2 from AT1R
adduction to BME revealed a dose-dependent increase of
bound OA-NO2 to the AT1R. These results were further
confirmed by immunoprecipitation analysis of AT1R and
detection of biotinylated OA-NO2. However, adduction of the
AT1R by OA-NO2 did not interfere with Ang II binding to its
receptor, suggesting that the molecular interactions between
OA-NO2 and AT1R are unlikely similar to typical competi-
tive inhibitors of AT1R.44,45
Crystal structures for some proteins of the seven trans-
membrane family, to which the AT1R belongs, show that
monomers associate through a lipophilic interface with lim-
ited protein–protein contacts.46 It is becoming increasingly
evident that lipid/membrane structures interact with GPCR,
exerting posttranslational modification within the GPCR.47 It
is likely that these protein/lipid interactions48,49 may serve as
regulatory mechanisms with diverse impact on downstream
signaling actions.47 Thus, AT1R adduction by OA-NO2 may
result in an allosteric inhibition by adducting to the hydro-
phobic interface in a fashion reminiscent of the A2A adeno-
sine receptor antagonist.50 This could promote an inactive
state of the receptor, thus uncoupling Ang II binding from
downstream activation.47 Further studies will be aimed at
dissecting the molecular mechanisms by which electrophilic
fatty acid derivatives reduce Ang II–mediated hypertension,
including biochemical and crystallographic approaches, thus
aiding in the design of a new generation of AT1R antagonists
with increased selectivity.
In summary, this is the first demonstration that OA-NO2
has a long-lasting impact on hypertension induced by Ang II
infusion and exerts a significant BP-lowering effect on
 Zhang et al
preexisting hypertension. OA-NO2 antagonizes Ang II sig-
naling through formation of stable adducts with the AT1R,
leading to a reduced vasoconstriction via uncoupling
G-protein signaling and subsequently reducing second mes-
senger mobilization.
Sources of Funding
This work was supported, in part, by NIH grants HL68878 and
HL089544 (to Y.E.C.), HL58115 and HL64937 (to B.A.F.), and
CA116592 (to C.X.D.); American Heart Association Grants
0835237N (to J.Z.) and 09SDG2110143 (to L.C.); and American
Diabetes Association grant 7-08-JF-52 (to F.J.S.). Y.E.C. is an
Established Investigator of American Heart Association (Grant
0840025N).
Disclosures
B.A.F. acknowledges financial interest in Complexa Inc.
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Novelty and Significance
What Is Known?
● Nitroalkenes are nitric oxide derivatives of unsaturated fatty acids
with pleiotropic biological activities of relevance for metabolic and
cardiovascular diseases.
● Nitro-oleic acid (OA-NO2) protects against vascular lesion, athero-
sclerosis, cardiac ischemia/reperfusion injury, and diabetes in
experimental animal models.
What New Information Does This Article Contribute?
● OA-NO2 reduces blood pressure and vasoconstriction in response to
angiotensin (Ang) II.
● OA-NO2 binds Ang II receptor (AT1R), but it does not interfere with Ang
II binding to the receptor unlike typical competitive angiotensin
receptor blockers, thus defining a novel mechanism for antagonism.
●
OA-NO2 and rosiglitazone reduce established hypertension through
different pathways.
● Inhibition of AT1R by OA-NO2 uncouples downstream G-protein
signaling and calcium mobilization.
We demonstrate for the first time that OA-NO2 has a long-lasting
impact on hypertension induced by Ang II infusion in an animal
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model where systemic delivery of pharmacological doses of
OA-NO2, but not its parental fatty acid, oleic acid, results in a
prolonged and sustained reduction of blood pressure. We show
that OA-NO2 lowers blood pressure on preexisting hypertension
and inhibits vascular contractility in an AT1R-dependent fashion.
OA-NO2 shows specificity for the AT1R, because OA-NO2
dose-dependently inhibits Ang II–mediated vessel contraction
but does not reduce vascular contractility induced by alternative
vasoconstrictors, such as endothelin-1 and phenylephrine, sig-
naling through other G-protein coupled receptors. We describe
that OA-NO2 lowers Ang II–induced hypertension independent of
peroxisome proliferation-activated receptor-␥ activation. Rather,
OA-NO2 binds directly to the AT1R without interfering with Ang
II binding, suggesting that the molecular interactions between
OA-NO2 and AT1R are likely to differ from angiotensin receptor
blockers competitive inhibition. OA-NO2 reduces heterotrimeric
G-protein coupling to the AT1R and inhibits downstream signal-
ing. This novel regulatory mechanism of the AT1R by OA-NO2
could inform the design of a new generation of AT1R antagonists
with increased selectivity.
 SUPPLEMENTAL MATERIAL

Nitro-oleic acid inhibits angiotensin II-induced hypertension

Zhang: Nitro-oleic acid and blood pressure.

Jifeng Zhang1*, Luis Villacorta1*, Lin Chang1*, Zhenzhen Fan2, Milton Hamblin1, Tianqing Zhu1, Chen
S. Chen3, Marsha P. Cole3, Francisco J. Schopfer3, Cheri X. Deng2, Minerva T. Garcia-Barrio4, Ying-
Hong Feng5, Bruce A. Freeman2,#, Y. Eugene Chen1,#

1
Cardiovascular Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109
2
Department of Biomedical Engineering, College of Engineering, University of Michigan, Ann Arbor,
Michigan 48109
3
Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania
15213
4
Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, GA 30310
5
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD
20814

*These authors contributed equally to this work

#To whom correspondence may be addressed:

Dr. Y. Eugene Chen
Cardiovascular Center, MSRB III 7200,
University of Michigan Medical Center,
1150 W. Medical Center Dr,
Ann Arbor, MI 48109
Tel: 734-763-7838, Fax: 734-936-2641
echenum@umich.edu

Dr. Bruce A. Freeman

Department of Pharmacology & Chemical Biology,
E1340 Thomas E. Starzl Biomedical Science Tower,
200 Lothrop St,
University of Pittsburgh, Pittsburgh, PA 15213
Tel: 412-648-9319, Fax: 412-648-2229
freerad@pitt.edu

S1
DETAILED METHODS

Animals

Male C57BL/6J mice, 8-10 weeks old, were housed in a temperature-controlled animal facility

with a 12h:12h light-dark cycle and free access to water and rodent chow. The study protocol was

approved by the University of Michigan Committee on Use and Care of Animals.

Blood Pressure Measurements

Blood pressure (BP) was measured by radiotelemetry as previously described in detail1. Briefly,

mice were anesthetized with isoflurane and placed on a controlled warming pad (37°C). The catheter is

attached to a combination pressure transducer, transmitter, and battery, all encapsulated in an

implantable microminiaturized electronic monitor (PA-C10, Data Sciences International, St. Paul, MN).

After preparation of the left common carotid artery, arteriotomy was performed using the bent tip of a

25g needle allowing the 0.4 mm catheter to pass into the vessel toward the heart. The transducer was

secured within the abdominal cavity allowing stable long-term pressure recordings. Telemetry

measurements of pulsatile arterial BP were a continuous 24-h/day, 7-days/week readouts of systolic,

diastolic and mean arterial pressure and heart rate. Baseline BP measurements were initiated after 1

week of postsurgery recovery and subsequent OA, OA-NO2 and PEG-vehicle control delivery (see

below).

Angiotensin II (Ang II)-induced hypertension model and nitroalkene delivery in vivo

After baseline BP recordings were performed (1 week), mice were again anesthetized and

subjected to subcutaneous implantation of osmotic mini-pumps (model 2004, Alzet) for delivery of

nitro-oleic acid (OA-NO2) or oleic acid (OA). We adjusted the stock concentration of synthetic OA-NO2

in the pump to provide an infusion rate of 5mg/kg/day. During the infusion regime, blood analyses

S2
revealed a plasma concentration within the nanomolar range as recently demonstrated2. This

concentration must be considered in the context that the infused OA-NO2 is also metabolized to other

derivatives not specifically determined here but extensively studied in3, 4. After an additional week, a

second osmotic pump (model 2002, Alzet), containing Ang II (Sigma) at a concentration supporting an

infusion rate of 500 ng/kg/min, was implanted subcutaneously on a side of the back of each mouse.

Depicted in Figure 1 are the mean values (n=6) of individual systolic and diastolic BP recordings every

4 h. Heart rate determinations are also shown in Online Figure I.

Short-term nitroalkene delivery in vivo before and after Ang II-induced hypertension and BP

recordings.

For short-term delivery of OA, OA-NO2, Ang II and the PPARγ antagonist GW9662, BP

measurements were determined as previously described5. Briefly, after preparation of the right carotid

artery, a 1.4F micro-tip catheter sensor (model SPR-671, Millar Instruments Inc, Houston, TX) was

inserted. BP was measured for a period of 10 min after steady-state levels were reached using the data-

acquisition system Powerlab 8/30 and chart software (ADInstruments). The drugs were administrated

via jugular vein by a GENIE Plus Infusion Syringe Pump (Kent Scientific, Torrington, CT, USA). OA

or OA-NO2 (1 μmol/L) were delivered at an infusion rate of 1 μL/min (equivalent to a final

concentration of 10 mg/kg) and the PPARγ inhibitor, GW9662 (0.1mmol/L) at an infusion rate of 1

μL/min (equivalent to a to a final concentration of 10 mg/kg), all delivered before Ang II infusion (10

μg/mL, infusion rate: 1 μL/min) as indicated by the arrows in Figures 2A and 2B and Figure 4A and

4B).

For short-term delivery of OA, OA-NO2, the PPARγ agonist rosiglitazone and antagonist

GW9662, in the preexisting hypertension model, mice were first implanted with Ang II minipumps

S3
(model 2002, Alzet) supporting an infusion time of 14 days and a rate of 500 ng/kg/min, as described

above. 3 days after Ang II delivery, systolic BP in mice stably rose to 150mmHg (Figure 2D and 3E).

Increasing concentrations of OA or OA-NO2 were then administered to anesthetized mice via jugular

vein delivery supporting a final dosages of 1.25, 2.5, 5, 10 and 20 mg/kg (Figure 2D to 2F). Under

similar experimental conditions, increasing doses of OA-NO2 or rosiglitazone (final concentrations of

1.25, 2.5, 5, 7.5, 12.5 and 20 mg/kg) were delivered in the presence or absence of the PPARγ antagonist,

GW9662 (10 mg/kg) (Online Figure II and Online Figure III). As described in other experimental

models of hypertension6, 7, rosiglitazone reduced preexisting hypertension, which herein was afforded by

Ang II infusion (Online Figure IIA) In response to rosiglitazone treatment, maximal reduction of BP

reached ∼7 mmHg and GW9662 efficiently antagonized this effect (Online Figure II). In a similar

approach, when OA-NO2 is delivered into hypertensive mice reduction of BP is dose-dependent (Online

Figure IIIA) and pretreatment with GW9662 cannot antagonize this reduction (Online Figure IIIB).

Thus, OA-NO2 equally inhibits Ang-II-induced hypertension in the presence or absence of GW9662

(Online Figure IIIC).

Mesenteric artery contraction and relaxation assays

Sprague-Dawley rats (200-250 g) were sacrificed through i.p. injection of urethane (1 g/kg) and

the mesenteric artery isolated and dissected from the animal and placed on ice in a physiological saline

solution (PSS) consisting of 118 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.18 mmol/L

Mg2SO4, 1.18 mmol/L KH2PO4, 24.9 mmol/L NaHCO3, 10 mmol/L glucose, and 0.03 mmol/L EDTA

before being mounted in the myograph. After the connective tissue was dissected away from the vessels,

second-order mesenteric arteries (2 mm long) were prepared and mounted onto a computer-controlled,

myograph (Multiwire Myograph System. Model 610M, DMT) that was connected to a force transducer

S4
to measure the wall tension developed by the vessel. A computer-assisted normalization protocol was

then performed to set the pretension units to 100 mmHg for each vessel. After the normalization

procedure was complete, the second-order mesenteric rings were balanced for 1h, and then challenged

three times with a high-potassium solution (KPSS, PSS with 50 mmol/L KCl) to determine whether they

were viable. Only segments that demonstrated a contraction in response to KPSS were used for further

studies. After a washout period, vessels were pretreated with OA-NO2 or OA at 5 µmol/L and 2.5

µmol/L and then 10 min contracted with increasing concentrations of Ang II, endothelin-1 (ET-1) and

phenylephrine (PE) as shown in Figure 3. Data acquisition was performed using the DMT

Normalization Module (AD Instruments). Data are shown as mean ± SD, n=6 in each group, *P < 0.05

and **P < 0.01 (OA-NO2 vs. OA or vehicle (0.1% ethanol)-treated arteries). Trace analysis of the

contractile curve was determined using Chart v5 (AD Instruments) and representative contractile curves

in response to Ang II, PE and ET-1 are shown in Online Figure IV.

Generation of PPRE-EGFP transgenic mice and determination of PPARγ transcriptional activity

in the aorta.

The PPRE-EGFP construct contains three tandem consensus PPRE sites, consisting in the

following sequence: AGGACAAAGGTCA, followed by a mini promoter of the thymidine kinase (TK)

gene coupled to the gene encoding enhanced green fluorescent protein (EGFP). The PPRE-EGFP

transgene was also flanked by two pieces of chicken HS4 insulator at each side to avoid positional effect

of the transgene expression (Online Figure VA). The construct was tested in HEK293 and COS-1 cells

before generation of transgenic mice to verify the integrity of the PPRE-EGFP construct. Our

preliminary studies demonstrated that co-transfection with any PPAR isoform expression vector plus

S5
treatment of corresponding ligand increased the EGFP expression in the cell culture system (data not

shown).

Generation of transgenic founder lines was subsequently accomplished using standard techniques

of microinjection, which was performed at the Transgenic Animal Model Core of the University of

Michigan. Briefly, PPRE-EGFP construct was microinjected into (C57BL/6 X SJL)F2 mouse eggs and

surgically transferred to recipients. We obtained a total of 81 offspring (F0) from the microinjection. 5

out of 81 are transgene positive as screened by genomic PCR. The F1 generation was subsequently

obtained by cross breeding with C57 mice. All 5 lines showed germline transmission for PPRE-EGFP

transgene. Line #62 was used for the current experiments and positive trangene expression was further

confirmed by genotyping with the F2 generation (Online Figure VB) using the primers, EGFP-F2: 5’-

CGCCCAGCGTCTTGTCATTG-3’ and EGFP-R2: 5’-ACGCCGTAGGTCAGGGTGGTC-3’

To determine PPAR transcriptional activity in the vasculature in vivo, male PPRE-EGFP

transgenic mice were administered via oral gavage with rosiglitazone (5 mg/kg) with or without the

PPARγ antagonist GW9662 (10 mg/kg). After 20 h, the aortas were harvested, homogenized and

subjected to Western blot analysis with an anti-GFP antibody (Living Colors, Clontech). EGFP

expression as a measure of PPAR transcriptional activity was robustly increased in the aortic tissue in

response to rosiglitazone treatment. Coadministration of GW9662 significantly attenuated rosiglitazone-

dependent PPAR activity (Online Figure VC).

Detection and quantification of AT1R modification by OA-NO2

HEK293 cells were either transfected with control plasmid (pcDNA3.1) or plasmid encoding

FLAG-tagged-AT1R using lipofectamine 2000 (Invitrogen). 24 h after transfection, cells were starved in

DMEM containing 0.1% fatty acid-free bovine serum albumin (BSA) for 24 h, and then incubated with

S6
either 2 μmol/L or 5 μM OA-NO2 for 2 h. Cells were then harvested and lysed using

immunoprecipitation buffer (1% Triton X-100, 10% glycerol, 150 mmol/L NaCl, 10 mmol/L HEPES, 1

mmol/L EDTA) supplemented with Protease inhibitor cocktail (Roche) and phosphatase inhibitors. The

cell lysates were briefly sonicated and clarified by centrifugation at 14000 g for 10 min and 3.5 mg of

total lysates were subjected to immunoprecipitation with anti-FLAG M2 monoclonal antibody-

conjugated agarose (Sigma) overnight. After immunoprecipitation, the beads were washed with washing

buffer (50 mmol/L Tris HCl, 150 mmol/L NaCl, pH 7.4) three times and then eluted by competition with

3 X FLAG peptide (Sigma). The trans-nitroalkylation reaction was carried out in 50 mM phosphate

buffer pH 7.4 in the presence of [13C18]-OA-NO2 internal standard (final 0.5 ng/ml) and β-

mercaptoethanol (BME) (500 mmol/L final) for 2 h at 37°C. The reaction was stopped by addition of

1% formic acid. The trans-nitroalkylated BME-OA-NO2 (transfer of OA-NO2 from protein to BME) was

then further detected and quantified using tandem mass spectrometry3 (see below).

Quantification of BME-OA-NO2 adducts

Quantitative determination of the different BME-adducted molecules was performed through

analysis in the multiple reaction monitoring (MRM) scan mode using a 4000 QTrap triple quadrupole

mass spectrometer (Applied Biosystems, Framingham, MA) as previously reported3. Briefly, BME-

adducts were detected in the negative ion mode by monitoring ions that undergo the M-/[M - BME]-

transition. Thus, the transitions used were as follows: BME-OA-NO2, m/z 404.4/326.3; BME-[13C18]-

OA-NO2, m/z 422.4/344.3 (Figure 5B). The declustering potential was -50 V and the collision energy

was set at -17 for BME adducts. Zero grade air was used as source gas, and nitrogen was used in the

collision chamber. Data were acquired and analyzed using Analyst 1.4.2 software (Applied Biosystems,

Framingham, MA). Quantification (Figure 5C) was achieved by comparing peak area ratios between

S7
analytes and their corresponding internal standards and calculating analytes concentration using an

internal standard curve.

AT1R immunoprecipitation and detection of biotin-labeled OA-NO2.

The synthesis of biotinylated OA and OA-NO2 was performed as previously described8, 9.

HEK293 cells were transiently transfected with the Flag-AT1R plasmid (0.5 μg/well). After 24 h of

serum deprivation cells overexpressing AT1R were treated with OA-NO2 and OA in a dose-dependent

manner for 3 h as described in the figure legends (Figure 5D). Cells were then washed with cold PBS

and lysed using Nonidet P-40 buffer. Cell lysates were then precleared using 50 µl of a 50% slurry of

protein G-Sepharose 4 Fast Flow (Sigma) and incubated at 4oC for 1 h with gentle shaking. The

precleared lysates (5 µg) were incubated with an anti-biotin antibody at 4oC overnight and then further

incubated with protein G-Sepharose 4 Fast Flow for 1 h. The precipitates were subjected to SDS-PAGE

and electrotransfered onto nitrocellulose membranes. To detect the direct binding of biotin-labeled OA

or OA-NO2 to AT1R, immunoblotting was done using an anti-biotin HRP-linked antibody (Cell

Signaling).

Gαq11 immunoprecipitation and detection of HA-tagged-AT1R.

For identification of coupled Gαq11 to the AT1R in response to Ang II treatment, HEK293 cells

were transiently transfected with the following plasmids: HA-tagged-AT1R (0.3 μg/well) was obtained

from the Missouri S&T cDNA Resource Center (www.cdna.org); Gαq11-EGFP (0.3 μg/well), Gβ1 (0.1

μg/well) and HA-tagged-Gγ2 (0.1 μg/well) were a generous gift of Dr. Catherine H. Berlot (Weis Center

for Research, Danville, PA)10. EGFP was tagged to the Gαq11 avoiding modification at the N- or C-

termini of Gαq11, important for interaction of the G-protein subunits and the receptor as described

S8
before10. Cells were then were treated with OA-NO2 and OA (2.5 μmol/L) 10 min before the addition of

Ang II (100 nmol/L). After 1 min, cells were washed with cold PBS, lysed and precleared as described

above. The precleared lysates were incubated with an anti-GFP antibody (Living Colors, Clontech) at

4oC overnight and then further incubated with protein G-Sepharose 4 Fast Flow for 1h. The precipitates

were subjected to a deglycosylation step11 using the Protein Deglycosylation Mix (New England

Biolabs) following the manufacturer’s instructions before SDS-PAGE. Detection of associated HA-

tagged-AT1R to the Gαq11 was obtained by immunoblotting using an anti-HA antibody (Cell Signaling)

(Figure 6A). Western blots were quantified using the ImageJ software (NIH, Bethesda, MD) after

densitometric scanning of the films and expressed as relative ratio of AT1R vs. Gαq11 (Figure 6B).

Angiotensin II type 1 receptor binding assays

The AT1R binding assay was performed as described previously12. Briefly, confluent primary

cultured rat aortic smooth muscle cells (RASMC) in 24-well plates were cultured in DMEM

supplemented with 0.1% FBS for 2 h. The cells pretreated with different dose of OA or OA-NO2 for 10

min as indicated in Figure 5F, and then incubated for 30 min at 37°C with 0.1 nmol/L of 125I-[Sar1,Ile8]-

Ang II (Perkin Elmer). Cells were then washed three times with ice-cold PBS containing 0.1% BSA and

lysed in 0.5 N NaOH. The radioactivity count of the lysate was measured by γ-counter. For AT1R

competition binding studies, the cells were pretreated with 2.5 μmol/L of OA or OA-NO2 for 10 min and
125
then incubated for 30 min with 0.3 nmol/L I-[Sar1,Ile8]-Ang II and serial concentrations of the cold

Ang II as indicated in Figure 5E. Cells were harvested and counted with a γ counter.

Inositol-1,4,5-trisphosphate (IP3) radioreceptor assays

S9
 RASMC were cultured in DMEM-F/12 (Invitrogen) with 10% FBS in 6-well plate until reaching

95% confluence. Cells were washed once with PBS and then incubated with DMEM-F/12, 1% FBS

containing 10 mmol/L of LiCl for 30 min. Cells were then pre-treated with 2.5 μmol/L OA-NO2 or OA

for 5 min. 100 nmol/L of Ang II was added to the cells and incubated for 45 min. IP3 was measured by

radioreceptor assay (PerkinElmer) following the manufacturer’s protocol (Figure 6). The experiments

were performed in triplicates and repeated 3 independent times.

Intracellular Calcium Concentration Measurement

Ratiometric fluorescence imaging was performed using the fluorescent indicator dye fura-2 to
2+
obtain the intracellular calcium concentration ([Ca ]i) from the ratio of emissions at 510 nm with 340

nm and 380 nm excitation13. Cells were incubated for 30min at 37°C in culture medium containing 5

μmol/L fura-2 (Invitrogen, Carlsbad, CA), the membrane permeant acetoxymethyl ester form of fura-2

and 0.05% v/v of 10% w/v Pluronic F-127 (Invitrogen, Carlsbad, CA) in water. Excess dye was

removed by washing the cells three times with culture medium. The dish was then placed on a 37°C

heating stage of an inverted microscope (Nikon Eclipse Ti-U, Melville, NY). To perform real-time

imaging, a monochromator (DeltaRAM X™, PTI, Birmingham, NJ) with a 5 nm bandpass was used to

filter light from a 75W xenon lamp alternately between the wavelengths 340 nm and 380 nm, each with

excitation exposure time of 656 ms. The excitation light was directed through a 20x Super Fluor

objective with a numerical aperture of 0.75 (Nikon, Melville, NY), and the subsequently emitted light

from the cells due to fura-2 fluorescence was passed through an appropriate dichroic filter. The resulting

series of grayscale photomicrographs were acquired with a cooled CCD camera (Photometrics

QuantEM, Tucson, AZ) at a 512x512 resolution (Online Figure VI and Online Movies I to III).

S10
 Image analysis was performed using software package Easy Ratio Pro (PTI, Birmingham, NJ).

The background-corrected fluorescence intensity was obtained by subtracting the fluorescence intensity

from an area free of cells. The ratio was calculated as R=F340/F380, where F340 and F380 are the emission

fluorescent intensity with 340 nm and 380 nm excitation respectively. Change in ratio of F340 and F380 is

related to the change in the [Ca2+]i14.

Statistical analysis

Mean ± SEM values were analyzed using Prism (version 4). Statistical comparisons between two

groups were performed by Student’s t test, and among three groups were performed by one-way

ANOVA. Groups were considered significantly different if P values were <0.05.

S11
Supplemental References

1. Whitesall SE, Hoff JB, Vollmer AP, D'Alecy LG. Comparison of simultaneous measurement of

mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. Am J Physiol

Heart Circ Physiol. 2004;286:H2408-2415.

2. Cole MP, Rudolph TK, Khoo NK, Motanya UN, Golin-Bisello F, Wertz JW, Schopfer FJ,

Rudolph V, Woodcock SR, Bolisetty S, Ali MS, Zhang J, Chen YE, Agarwal A, Freeman BA,

Bauer PM. Nitro-fatty acid inhibition of neointima formation after endoluminal vessel injury.

Circ Res. 2009;105:965-972.

3. Schopfer FJ, Batthyany C, Baker PR, Bonacci G, Cole MP, Rudolph V, Groeger AL, Rudolph

TK, Nadtochiy S, Brookes PS, Freeman BA. Detection and quantification of protein adduction

by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives. Free

Radic Biol Med. 2009;46:1250-1259.

4. Rudolph V, Schopfer FJ, Khoo NK, Rudolph TK, Cole MP, Woodcock SR, Bonacci G, Groeger

AL, Golin-Bisello F, Chen CS, Baker PR, Freeman BA. Nitro-fatty acid metabolome: saturation,

desaturation, beta-oxidation, and protein adduction. J Biol Chem. 2009;284:1461-1473.

5. Chang L, Villacorta L, Zhang J, Garcia-Barrio MT, Yang K, Hamblin M, Whitesall SE, D'Alecy

LG, Chen YE. Vascular smooth muscle cell-selective peroxisome proliferator-activated receptor-

gamma deletion leads to hypotension. Circulation. 2009;119:2161-2169.

6. Chan SH, Wu KL, Kung PS, Chan JY. Oral intake of rosiglitazone promotes a central

antihypertensive effect via upregulation of peroxisome proliferator-activated receptor-gamma

and alleviation of oxidative stress in rostral ventrolateral medulla of spontaneously hypertensive

rats. Hypertension. 2010;55:1444-1453.

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7. Ketsawatsomkron P, Pelham CJ, Groh S, Keen HL, Faraci FM, Sigmund CD. Does peroxisome

proliferator-activated receptor-gamma (PPAR gamma) protect from hypertension directly

through effects in the vasculature? J Biol Chem. 2010;285:9311-9316.

8. Cui T, Schopfer FJ, Zhang J, Chen K, Ichikawa T, Baker PR, Batthyany C, Chacko BK, Feng X,

Patel RP, Agarwal A, Freeman BA, Chen YE. Nitrated fatty acids: Endogenous anti-

inflammatory signaling mediators. J Biol Chem. 2006;281:35686-35698.

9. Kang LT, Vanderhoek JY. Synthesis and use of a novel biotinylated probe for the

chemiluminescent detection of proteins that bind 15-hydroxyeicosatetraenoic acid. Anal

Biochem. 1997;250:119-122.

10. Hughes TE, Zhang H, Logothetis DE, Berlot CH. Visualization of a functional Galpha q-green

fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by

mutational activation and by elimination of palmitoylation sites, but not be activation mediated

by receptors or AlF4. J Biol Chem. 2001;276:4227-4235.

11. Zielinska DF, Gnad F, Wiśniewski JR, Mann M. Precision mapping of an in vivo N-

glycoproteome reveals rigid topological and sequence constraints. Cell. 2010;141:897-907.

12. Cui TX, Nakagami H, Nahmias C, Shiuchi T, Takeda-Matsubara Y, Li JM, Wu L, Iwai M,

Horiuchi M. Angiotensin II subtype 2 receptor activation inhibits insulin-induced

phosphoinositide 3-kinase and Akt and induces apoptosis in PC12W cells. Mol Endocrinol.

2002;16:2113-2123.

13. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly

improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.

14. Hong SJ. Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2

myocardiac ventricular cells. Cell Signal. 2002;14:811-817.

S13
 Online Figure I

Online Figure I. Heart rate measurements by telemetry. Mice implanted with osmotic pumps

adjusted to deliver 5 mg/kg/day OA-NO2 (red) or OA (blue) and vehicle control (black) were infused

with Ang II at a pressor rate of 500 ng/kg/min. Heart rate was measured by radiotelemetry. Data are

expressed in heart beats per min and shown as mean ± SEM, n=6 in each group.

S14
 Online Figure II

Online Figure II. Rosiglitazone reduces Ang II-induced hypertension. Hypertension was developed

in mice in 3 days after implantation of osmotic pumps to infuse Ang II at a pressor rate of 500

ng/kg/min. (A) Rosiglitazone was then administered to the mice via the jugular vein at increasing

concentrations as indicated by the arrows. BP tracings were recorded as described above. (B) GW9662

(10 mg/kg) was delivered as a single dose at the time indicated by the arrow before treatment with

rosiglitazone as described in A. (C) Data in A and B are summarized as systolic BP reduction (in

mmHg) after either rosiglitazone or rosiglitazone + GW9662 treatment as compared to maximal systolic

pressure in Ang II hypertensive mice and are shown as mean ± SEM, n=4 in each group, P < 0.05

(rosiglitazone vs. rosiglitazone + GW9662).

S15
 Online Figure III

Online Figure III. OA-NO2 reduces Ang II-induced hypertension independently of PPARγ.

Hypertension was developed in mice in 3 days after implantation of osmotic pumps to infuse Ang II at a

pressor rate of 500 ng/kg/min. (A) OA-NO2 was then delivered to mice via the jugular vein at increasing

concentrations as indicated by the arrows. BP tracings were recorded as described above. (B) GW9662

(10 mg/kg) was delivered as a single dose at the time indicated by the arrow before treatment with OA-

NO2 as described in A. (C) Data in A and B are summarized as systolic BP reduction (in mmHg) after

either OA-NO2 or OA-NO2 + GW9662 treatment as compared to maximal systolic pressure in Ang II

hypertensive mice and are shown as mean ± SEM, n=4 in each group, P < 0.05 (rosiglitazone vs.

rosiglitazone + GW9662).

S16
 Online Figure IV

Online Figure IV. Contraction curves after OA-NO2 treatment in response to Ang II, PE or ET-1.

The mesenteric artery rings were preincubated for 10 min with 2.5 μmol/L or 5 μmol/L of either OA-

NO2, OA or vehicle as indicated. Vascular contraction was induced by serial addition of increasing

concentrations of Ang II (A), PE (B) and ET-1 (C) to the myograph at the time indicated by the arrows.

S17
 Online Figure V

Online Figure V. Generation of PPRE-EGFP transgenic mice model. (A). Schematic diagram of

PPRE-EGFP transgenic mouse structure. A 3x tandem repeat of the consensus PPRE sequence was

inserted upstream of the mini-TK promoter. EGFP coding sequence and a BGH polyadenylation signal

was cloned downstream of mini-TK promoter. The arrows show the genotyping primer sites. (B)

Genomic PCR to genotype the PPRE-EGFP transgenic mice. P: positive control (transgenic construct);

N: negative control (water); Wt: wild type mouse; Tg: transgenic mouse. (C) EGFP expression in PPRE-

EGFP transgenic mice detected by Western blot. Protein was extracted from aortic tissue of PPRE-

EGFP transgenic mice after 20h of treatment via oral gavage with 5mg/kg rosiglitazone or the

combination of rosiglitazone + GW9662 (10 mg/kg).

S18
 Online Figure VI

Online Figure VI. OA-NO2 inhibits Ang II-mediated intracellular calcium efflux in VSMC. Three

cells (left panel) were randomly selected at the 380 nm channel for monitoring calcium efflux induced

by 0.1 µmol/L Ang II (A). Ang II-induced calcium mobilization was dramatically inhibited by 2.5

µmol/L OA-NO2 (B), but not by 2.5 µmol/L OA (C). Right panels show the normalized ratio-time traces

from three typical cells (each of them depicted in different colors) in the field of view for the randomly

selected cells. Calcium mobilization can be visualized for each treatment with Ang II alone or in the

presence of OA-NO2 or OA in Online Movies I to III, respectively. Movies were acquired at a 512x512

resolution with a cooled CCD camera as described above.

S19
 Nitro-Oleic Acid Inhibits Angiotensin II−Induced Hypertension
Jifeng Zhang, Luis Villacorta, Lin Chang, Zhenzhen Fan, Milton Hamblin, Tianqing Zhu, Chen
S. Chen, Marsha P. Cole, Francisco J. Schopfer, Cheri X. Deng, Minerva T. Garcia-Barrio,
Ying-Hong Feng, Bruce A. Freeman and Y. Eugene Chen

Circ Res. 2010;107:540-548; originally published online June 17, 2010;

doi: 10.1161/CIRCRESAHA.110.218404
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2010 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571

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http://circres.ahajournals.org/content/suppl/2010/06/17/CIRCRESAHA.110.218404.DC1.html

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