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Telmisartan inhibits vasoconstriction via PPARg-
dependent expression and activation of endothelial
nitric oxide synthase
Chi Yung Yuen
1
, Wing Tak Wong
1
, Xiao Yu Tian
1
, Siu Ling Wong
1
*
, Chi Wai Lau
1
,
Jun Yu
2
, Brian Tomlinson
2
, Xiaoqiang Yao
1
, and Yu Huang
1
*
1
Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China; and
2
Department
of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, China
Received 11 August 2010; revised 26 November 2010; accepted 9 December 2010; online publish-ahead-of-print 14 December 2010
Time for primary review: 28 days
Aims Telmisartan activates peroxisome proliferator-activated receptor-g (PPARg) in addition to serving as an angiotensin II
type 1 receptor (AT
1
R) blocker. The PPARg activity of telmisartan on resistance arteries has remained largely
unknown. The present study investigated the hypothesis that telmisartan inhibited vascular tension in mouse mesen-
teric resistance arteries, which was attributed to an increased nitric oxide (NO) production through the PPARg-
dependent augmentation of expression and activity of endothelial nitric oxide synthase (eNOS).
Methods
and results
Second-order mesenteric arteries were isolated from male C57BL/6J, eNOS knockout and PPARg knockout mice
and changes in vascular tension were determined by isometric force measurement with a myograph. Expression
and activation of relevant proteins were analysed by Western blotting. Real-time NO production was measured
by confocal microscopy using the dye DAF. Telmisartan inhibited 9,11-dideoxy-11a,9a-epoxymethanoprostaglandin
F
2a
(U46619)- or endothelin-1-induced contractions. An NOS inhibitor, N
G
-nitro-L-arginine methyl ester (L-NAME),
or an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]-oxadizolo[4,3-a]quinoxalin-1-one (ODQ), prevented telmisar-
tan-induced inhibition of U46619 contractions. A PPARg antagonist, GW9662, abolished telmisartan-induced inhi-
bition. Likewise, the PPARg antagonist rosiglitazone attenuated U46619-induced contractions. The effects of
telmisartan and rosiglitazone were prevented by actinomycin-D, a transcription inhibitor. In contrast, losartan, olme-
sartan, and irbesartan did not inhibit contractions. The inhibition was absent in mesenteric arteries from eNOS
knockout or PPARg knockout mice. Telmisartan augmented eNOS expression, phosphorylation, and NO pro-
duction, which were reversed by the co-treatment with GW9662.
Conclusions The present results suggest that telmisartan-induced inhibition of vasoconstriction in resistance arteries is mediated
through a PPARg-dependent increase in eNOS expression and activity that is unrelated to AT
1
R blockade.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Keywords Telmisartan Endothelial nitric oxide synthase PPARg Mesenteric arteries
1. Introduction
The reninangiotensinaldosterone system plays a central role in the
development of hypertension and diabetic vascular disease. Angioten-
sin II (Ang II) induces cellular oxidative stress by activating the Ang II
type 1 receptor (AT
1
R) and NAD(P)H oxidase and subsequent pro-
duction of superoxide anions in both endothelial and vascular smooth
muscle cells. Elevated levels of reactive oxygen species (ROS) lower
the bioavailability of nitric oxide (NO), thus contributing to
endothelial dysfunction.
13
Consequently, AT
1
R blockers (ARBs)
such as losartan represent a major class of anti-hypertensive drugs
which reduce ROS overproduction in the vascular wall through antag-
onizing the AT
1
R.
Peroxisome proliferator-activated receptor-g (PPARg) is a
ligand-activated nuclear transcription factor,
4,5
which regulates adipo-
genesis and glucose metabolism.
6
PPARg is expressed in endothelial
and vascular smooth muscle cells,
7
and has been shown to enhance
insulin sensitivity and mediate anti-inammatory responses.
8
* Corresponding author. Tel: +852 2609 6787; fax: +852 2603 5022, Email: yu-huang@cuhk.edu.hk (Y.H.) and Email: christine_wong@cuhk.edu.hk (S.L.W.)
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: journals.permissions@oup.com.
Cardiovascular Research (2011) 90, 122129
doi:10.1093/cvr/cvq392

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Mutations of PPARg in humans contribute to the pathogenesis of
insulin resistance, diabetes, and hypertension.
9
PPARg agonists thiazo-
lidinediones, such as rosiglitazone, are therapeutic choices for type 2
diabetes acting by enhancing the insulin sensitivity in liver, muscle, and
adipose cells and by modulating lipid metabolism.
10
Activation of
PPARg in endothelial and smooth muscle cells can produce cardio-
protective benet that is independent of insulin sensitivity.
7
Rosiglita-
zone and pioglitazone have been reported to ameliorate endothelial
dysfunction in diabetic mouse by reducing the NAD(P)H oxidase
expression and ROS production,
11,12
while accumulating clinical
reports show that full PPARg agonists are associated with deleterious
side effects, such as congestive heart failure,
13,14
exacerbation of uid
retention, causing oedema and weight gain
15,16
as well as bone frac-
tures in women with type 2 diabetes.
14
Substantial evidence suggests that telmisartan, an ARB approved for
the treatment of hypertension, is a partial PPARg agonist.
17,18
Clinical
studies show that telmisartan is well tolerated and does not cause
oedema and weight gain.
19,20
In addition, telmisartan may be clinically
more effective than losartan in lowering blood pressure in hyperten-
sive patients.
21
On the other hand, telmisartan can activate PPARg
partly due to its highly lipophilic nature
22
and its ability to bind to
the PPARg ligand-binding domain.
17
This unique nature of being a
dual ARB/PPARg agonist makes telmisartan a more promising drug
in treating cardiovascular diseases in a safer manner. However, it
remains unknown how the PPARg property of telmisartan alters vas-
cular reactivity. The present study examined the hypothesis that tel-
misartan could enhance the expression of endothelial nitric oxide
synthase (eNOS) via a PPARg-dependent mechanism to reduce
ligand-induced contractions in mouse mesenteric resistance arteries.
2. Methods
2.1 Animals
The investigation conformed to the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH Publication
No. 85-23, revised 1996). This study was approved by the Experimental
Animal Ethics Committee, the Chinese University of Hong Kong. Male
C57BL/6J mice aged between 12 and 13 weeks and age-matched eNOS
knockout (eNOS KO) mice were supplied by the Laboratory Animal Ser-
vices Center, the Chinese University of Hong Kong. PPARg KO mice were
provided by Dr Jun Yu, the Chinese University of Hong Kong. The animals
were housed in a temperature-controlled room (22+18C) under a 12 h
light/dark cycle and had free access to water and the standard chow diet.
2.2 Artery preparation
Mice were sacriced by CO
2
inhalation. Second-order mesenteric arteries
and aortae were dissected out and placed in a dissecting dish containing
the ice-cold phosphate buffered saline (PBS) in which the adhering con-
nective tissues were carefully removed. The arterial tissues were cut
into 1.01.3 mm rings for tissue culture.
2.3 Tissue culture of arteries
Pre-warmed Dulbeccos modied Eagle medium supplemented with 10%
FBS and 100 U mL
21
penicillin plus 100 mg mL
21
streptomycin was added
into the wells of the 24-well plate. Different concentrations of telmisartan
(0.110 mmol L
21
) or other ARBs (losartan, olmesartan, and irbesartan;
all used at 10 mmol L
21
) were added into the wells. GW9662 (PPARg
antagonist, 300 nmol L
21
) was added in addition to 10 mmol L
21
telmisar-
tan in separate experiments. The plate was gently shaken to allow
thorough mixing of drugs before arteries were transferred to the wells
for a 24 h incubation at 378C.
2.4 Isometric force measurement
Tension changes of the cultured mesenteric arteries and aortae were
recorded as previously described.
23
Briey, the arteries were suspended
by two stainless steel wires in the 5 mL chamber of a Multi Myograph
(Danish, Myo Technology A/S, Denmark) with Krebs solution constantly
bubbled with 95% O
2
5% CO
2
and maintained at 378C. After 30 min
equilibrium, rings were rst contracted by 30 nmol L
21
U46619, and
then relaxed by 3 mmol L
21
acetylcholine (ACh). Arteries with relax-
ations over 80% were regarded as those with intact endothelium. After
several washes, the telmisartan-treated arteries were cumulatively
contracted by U46619 (1300 mmol L
21
) or endothelin-1 (ET-1,
0.330 nmol L
21
). Results were compared between control and 30 min
incubation of L-NAME (100 mmol L
21
) or 1H-[1,2,4]oxadiazolo
[4,3-a]quinoxalin-1-one (ODQ) (5 mmol L
21
). To examine the changes
in NO-mediated endothelium-dependent relaxations, the arteries were
pre-contracted with 3 mmol L
21
phenylephrine and relaxed by cumulative
additions of ACh (3 nmol L
21
1 mmol L
21
) in the presence of
20 mmol L
21
KCl which eliminated the effect from endothelium-derived
hyperpolarizing factors.
2.5 Western blotting
Western blotting was performed as described elsewhere.
24
Mouse aortae
harvested upon a 24 h incubation protocol were snap-frozen in liquid nitro-
gen. The tissues were homogenized in ice-cold radio-immunoprecipitation
assay lysis buffer containing a cocktail of protease inhibitors (leupetin,
1 mg mL
21
; aprotonin, 5 mg mL
21
; PMSF, 100 mg mL
21
; sodiumorthovana-
date, 1 mmol L
21
; ethylene glycol tetraacetic acid, 1 mmol L
21
; EDTA,
1 mmol L
21
; NaF, 1 mmol L
21
; and b-glycerolphosphate, 2 mg mL
21
).
The lysates were incubated on ice for 30 min and then centrifuged at
20 000 g for 20 min. The supernatant was collected and analysed for
protein concentration using the Lowry method (Bio-Rad, USA). For each
sample, 25 mg of the total protein was electrophoresed under reducing
conditions through a SDSpolyacrylamide gel. The resolved proteins
were electroblotted on an immobilon-P polyvinylidene diuoride mem-
brane (Millipore, USA) using wet transfer at 100 V for 70 min at 48C. The
membranes were blocked with 1% bovine serum albumin in 0.5%
Tween-20 phosphate buffered saline (PBST) for 60 min before an overnight
incubation with either polyclonal rabbit anti-PPARg antibody (1:1000, Cell
Signaling), polyclonal rabbit anti-eNOS antibody (1:500, BD Transduction
Laboratories), or polyclonal rabbit anti-phosphorylated eNOS antibody
(1:1000, Upstate) at 48C. The membranes were then probed with a horse-
radish peroxidase-conjugated swine anti-rabbit secondary antibody at a
dilution of 1:3000 for 1 h at room temperature. The membranes were
developed with an enhanced chemiluminescence detection system (ECL
reagents; Amersham PharmaciaBiotech, Bucks, UK) and then exposed to
X-ray lms. The signal intensities were quantied using a gel documentation
system (GBOX-CHEM1-HR16, SynGene) and normalized with the house-
keeping protein glyceraldehyde 3-phosphate dehydrogenase.
2.6 Real-time measurement on NO production
in endothelial cells
Human aortic endothelial cells (HAEC) were purchased from Cascade
Biologics (Oregon, USA) and cultured in Medium 200 supplemented
with Low Serum Growth Supplement (Cascade Biologics) until 80% con-
uence upon which they were plated on coverslips. Cells were incubated
with telmisartan (10 mmol L
21
) for 24 h with or without 30 min pre-
treatment of GW9662 (300 nmol L
21
) and then imaged as described
earlier.
25
Briey, cells after 24 h incubation were rinsed in NPSS
(140 mmol L
21
NaCl, 5 mmol L
21
KCl, 1 mmol L
21
CaCl
2
, 1 mmol L
21
MgCl
2
, 10 mmol L
21
glucose, and 5 mmol L
21
HEPES, pH 7.4) and
Telmisartan induces and activates eNOS via PPARg 123

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incubated with 1 mmol L
21
DAF-FM diacetate (4-amino-5-methylamino-
2

,7

-diuorescein, Invitrogen) for 10 min at room temperature. The

cells were then excited at 495 nm with emission wavelength of 515 nm
using the Olympus Fluoview FV1000 laser scanning confocal system
(Olympus America, Inc., Melville, NY, USA) mounted on an inverted
IX81 Olympus microscope. Basal NO levels were determined from the
initial reading and real-time changes in ACh (10 mmol L
21
)-stimulated
NO production were recorded and presented as a ratio of uorescence
relative to the initial intensity (time 0 min, before ACh addition).
2.7 Chronic treatment with telmisartan
C57BL/6J mice were orally treated with either telmisartan
(1 mg kg
21
day
21
) or water for 14 days, after which mesenteric arteries
were dissected for isometric force measurement.
2.8 Chemicals
Actinomycin-D, ODQ and N
G
-nitro-L-arginine methyl ester (L-NAME)
were purchased from Tocris (Avonmouth, UK). 9,11-Dideoxy-
11a,9a-epoxymethanoprostaglandin F
2a
(U46619), Ach, and GW9662
were purchased from Sigma-Aldrich (St Louis, MO, USA). Rosiglitazone
was purchased from GlaxoSmithKline and olmesartan from Pzer. Losar-
tan was purchased from Cayman Chemical (Ann Arbor, MI, USA). Telmi-
sartan was purchased from Changzhou Yabang Pharmaceutical Co., Ltd,
Jiangsu Province, China and irbesartan from Zhejiang Apeloa Jiayuan
Pharmaceutical Co. Ltd, Zhejiang Province, China. ACh and L-NAME
was prepared in distilled water, while other chemicals were dissolved in
dimethyl sulphoxide (DMSO, Sigma).
2.9 Statistical analysis
Data are means +SEM of n experiments and analysed with Students
t-test or two-way ANOVA followed by Bonferroni posthoc tests when
more than two groups were compared. pD
2
is the negative logarithm of
the vasoconstrictor concentration needed to produce half of the
maximal contraction as determined by non-linear regression curve
tting (Graphpad Prism Software, version 4.0). P ,0.05 was considered
statistically signicant.
3. Results
3.1 Telmisartan inhibits U46619-induced
contractions
Telmisartan concentration-dependently inhibited U46619-induced
contractions in the mesenteric arteries from C57BL/6J mice. Telmisar-
tan at 10 mmol L
21
attenuated the contractions (pD
2
: 7.89+0.04 for
control and 7.40+0.07 for telmisartan 10 mmol L
21
, P , 0.001,
Figure 1A, Supplementary material online, Figure 1A and Table 1). In
contrast, losartan, olmesartan, and irbesartan (all used at
10 mmol L
21
) did not alter the U46619-induced contractions (Sup-
plementary material online, Figure 2A). Telmisartan also enhanced
ACh-induced endothelium-dependent relaxations (Supplementary
material online, Figure 3A). Similar ndings were also obtained in the
mouse aortae, in which telmisartan, but not losartan, olmesartan,
and irbesartan, inhibited contractions to U46619 (Supplementary
material online, Figure 4A). Telmsartan also potentiated the
ACh-induced relaxations in the mouse aortae (Supplementary
material online, Figure 4D).
3.2 Inhibitory effect of telmisartan on
contractions is mediated by NO
L-NAME (100 mmol L
21
) abolished the inhibitory effect of telmisartan
(10 mmol L
21
) on U46619-induced contractions (Figure 1B, Sup-
plementary material online, Figure 1B and Table 1). ODQ
(5 mmol L
21
) also prevented telmisartan-induced effect (Supplemen-
tary material online, Figure 2B and Table 1). In addition to U46619, tel-
misartan also suppressed contractions induced by endothelin-1 and
this effect was abrogated by L-NAME (Figure 1C, Table 1). In control
rings without telmisartan treatment, L-NAME and ODQ enhanced
the contractions to U46619 and endothelin-1 (Figure 1B and C).
L-NAME abolished the enhanced ACh-induced relaxations in the
mesenteric arteries (Supplementary material online, Figure 3A) and
reversed the telmisartan-induced inhibition on U46619-induced con-
tractions in the aortae (Supplementary material online, Figure 4B).
Mesenteric arteries from C57BL/6J mice orally treated with telmisar-
tan exhibited smaller U46619-induced contractions compared with
those from vehicle-treated control mice. L-NAME restored and
even potentiated U46619-induced contractions (Supplementary
material online, Figure 5).
3.3 Telmisartan up-regulates eNOS
expression and phosphorylation
Twenty four hour incubation of telmisartan concentration-
dependently up-regulated the eNOS expression (Figure 2A) and its
phosphorylation at Ser
1177
(Figure 2B). The positive role of eNOS
was further veried by eNOS KO mice, in which eNOS protein
was not expressed in aortae (Figure 2C). L-NAME did not enhance
the contractions to U46619 and telmisartan (10 mmol L
21
) failed to
inhibit contractions in mesenteric arteries from the eNOS KO mice
(Figure 2D). In addition, the inhibitory effect of telmisartan to
Figure 1 (A) Telmisartan (0.110 mmol L
21
) concentration-dependently attenuated U46619-induced contractions. Inhibitory effect of telmisartan
(Tel, 10 mmol L
21
) to (B) U46619- and (C) endothelin-1-induced contractions was abolished in the presence of L-NAME (100 mmol L
21
). Data are
from 5 to 6 experiments. *P , 0.05 vs. control;
#
P ,0.05 vs. Tel without L-NAME.
C.Y. Yuen et al. 124

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contractions was prevented by an RNA synthesis inhibitor,
actinomycin-D (10 mmol L
21
) (Figure 3A and Table 1).
3.4 Telmisartan elevates PPARg
expression via PPARg activity
Telmisartan concentration-dependently increased the PPARg
expression (Figure 3B). Telmisartan (10 mmol L
21
)-induced
up-regulation of PPARg expression was prevented by a PPARg antag-
onist GW9662 (300 nmol L
21
). Likewise, rosiglitazone, a PPARg
agonist, also increased the GW9662-sensitve PPARg expression
(Figure 3C). In contrast, losartan (10 mmol L
21
) did not affect the
expression of PPARg (Figure 3C).
3.5 Telmisartan-induced eNOS expression
and activation are PPARg-dependent
GW9662 (300 nmol L
21
) antagonized telmisartan-induced inhibition
on U46619-induced contractions and prevented the potentiation of
ACh-induced relaxations in both mesenteric arteries and aortae,
while GW9662 per se did not modify U46619-induced contractions
and ACh-induced relaxations (Figure 4A and B; Supplementary
material online, Figures 3B, 4C and D; Table 1). Likewise, rosiglitazone
(1 mmol L
21
) attenuated U46619-induced contractions, and this
attenuation was reversed by GW9662 (Supplementary material
online, Figure 2C) and prevented by actinomycin-D (Supplementary
material online, Figure 2D).
Telmisartan (10 mmol L
21
) and rosiglitazone (1 mmol L
21
)-induced
increases in eNOS expression (Figure 4C) and phosphorylation
(Figure 4D) were prevented by GW9662 (300 mmol L
21
). On the
contrary, losartan did not increase eNOS expression and only
caused a slight elevation in eNOS phosphorylation, which was insen-
sitive to GW9662 (Figure 4C and D).
PPARg was not expressed in the PPARg KO mice (Figure 5A). In the
mesenteric arteries of PPARg KO mice, the inhibitory effects of telmi-
sartan (10 mmol L
21
) on U46619-induced contractions were dimin-
ished compared with those in C57BL/6J control mice (Figure 5B).
Telmisartan did not augment eNOS expression (Figure 5C) and phos-
phorylation (Figure 5D) in the aortae of PPARg KO mice.
3.6 Telmisartan increases intracellular NO
levels in cultured endothelial cells
HAEC treated with telmisartan for 24 h exhibited higher
un-stimulated (i.e. basal) NO level, which was prevented by
co-treatment of GW9662 or exposure to L-NAME (Figure 6A,
before ACh addition at 0 min, Figure 6B). Addition of ACh to HAEC
stimulated NO production. The increase in intracellular NO level
was remarkably higher in the telmisartan-treated cells compared
with that of the untreated control. Cells co-treated with GW9662
exhibited a rise of NO level similar to that of the control and acute
L-NAME treatment inhibited NO production (Figure 6A and C).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 1 pD
2
and E
max
(%) of telmisartan-induced
inhibition on contractions
Treatment pD
2
E
max
(%) n
C57BL/6J arteries
U46619 (Control) 7.89+0.04 137.09+3.93 6
Tel 0.1 mmol L
21
7.91+0.04 124.46+0.85 4
Tel 1 mmol L
21
7.72+0.10 123.57+4.98 4
Tel 10 mmol L
21
7.40+0.07*** 113.88+5.17** 6
Control 7.87+0.04 138.03+4.67 6
Tel 10 mmol L
21
7.38+0.08*** 113.09+6.02** 6
Control + L-NAME
100 mmol L
21
8.05+0.05 154.53+4.84 5
Tel + L-NAME 7.99+0.05
###
142.22+4.43
##
6
Control + ODQ
5 mmol L
21
7.95+0.08 189.13+4.69 3
Tel + ODQ 7.86+0.07
##
180.29+4.40
###
3
Control 7.88+0.05 137.65+4.76 6
Tel 10 mmol L
21
7.39+0.08*** 113.72+6.30** 6
Tel + Act 10 mmol L
21
7.68+0.07
#
137.59+5.99
#
5
Control 7.87+0.05 137.32+3.23 6
Tel 10 mmol L
21
7.38+0.07*** 113.73+5.76** 6
GW 300 nmol L
21
7.82+0.06 139.11+9.88 4
GW+ Tel 7.90+0.07
###
142.46+8.89
#
6
eNOS KO mouse arteries
U46619 (Control) 8.36+0.06 217.87+7.05 3
Tel 10 mmol L
21
8.29+0.02 197.14+6.21 3
Control + L-NAME
100 mmol L
21
8.41+0.03 214.87+9.68 3
Tel + L-NAME 8.31+0.03 204.42+4.63 3
PPARg KO mouse arteries
U46619 (Control) 8.34+0.07 163.46+11.06 4
Tel 10 mmol L
21
8.31+0.04 139.76+5.06 4
Signicant difference between control and treatment groups is indicated by *P , 0.05;
**P , 0.01; ***P , 0.001 vs. control;
#
P , 0.05;
##
P , 0.01;
###
P , 0.001 vs. tel. Data
are means +SEM of n experiments.
Figure 2 Western blot showing telmisartan (0.110 mmol L
21
)
concentration-dependently up-regulated (A) eNOS expression and
(B) eNOS phosphorylation at Ser
1177
. (C) Absence of eNOS
expression in the aortae of eNOS knockout (eNOS KO) mice com-
pared with the wild-type (WT) mice. (D) Concentrationresponse
curves for U46619 of telmisartan (Tel, 10 mmol L
21
)-treated mesen-
teric arteries from eNOS KO mice. Data are from 3 to 6 exper-
iments. *P ,0.05 and **P ,0.01 vs. control; ***P ,0.001 vs. WT.
Telmisartan induces and activates eNOS via PPARg 125

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4. Discussion
The present study demonstrated for the rst time that telmisartan
attenuates receptor-dependent contractions in the mesenteric resist-
ance arteries and aortae from C57BL/6J mice via the activation of
PPARg and a subsequent increase in eNOS expression and activity.
The concentration-dependent inhibition of telmisartan on
U46619-induced contractions implies its therapeutic potential in
reducing over-constrictions caused by TP receptor activation in vas-
cular complications of hypertension and diabetes.
26,27
Thiazolinediones, such as rosiglitazone, have been reported to
ameliorate endothelial dysfunction by inhibiting ROS production and
stimulating NO generation via a PPARg-mediated pathway.
11,12
Full
PPARg agonists, pioglitazone, and troglitazone, increase NO pro-
duction by up-regulating eNOS expression and eNOS-Ser
1177
phos-
phorylation in a rabbit model of myocardial infarction,
28
bovine
endothelial cells,
29
and human umbilical vein endothelial cells
(HUVEC).
30
We therefore hypothesized that telmisartan, acting as a
partial agonist of PPARg, could also augment eNOS expression and
activity in a PPARg-dependent manner, thereby inhibiting
Figure 3 (A) The inhibitory effect of telmisartan (Tel, 10 mmol L
21
) was abolished by actinomycin-D (10 mmol L
21
). (B) Telmisartan (0.1
10 mmol L
21
) concentration-dependently increased PPARg expression. (C) Telmisartan (Tel, 10 mmol L
21
) and rosiglitazone (Rosi, 1 mmol L
21
)
increased PPARg expression, which was inhibited by GW9662 (GW, 300 nmol L
21
). Data are from 3 to 6 experiments. *P ,0.05 vs. control;
#
P ,0.01 vs. corresponding groups without GW9662.
Figure 4 (A) Representative traces showing the effect of GW9662
(GW, 300 nmol L
21
) on telmisartan (Tel, 10 mmol L
21
)-induced
inhibitions on contractions to U46619. (B) GW9662 (GW,
300 nmol L
21
) abolished the inhibitory effect of telmisartan (Tel,
10 mmol L
21
) to U46619-induced contractions. (C) Telmisartan
(Tel, 10 mmol L
21
) and rosiglitazone (Rosi, 1 mmol L
21
) elevated
the (C) eNOS expression and (D) the phosphorylation at Ser
1177
,
which were prevented by GW9662 (GW, 300 nmol L
21
). (D) Losar-
tan (10 mmol L
21
) mildly increased the p-eNOS level which was
unaffected by GW9662. Data are from 4 to 6 experiments.
*P , 0.05 and **P , 0.01 vs. control or control without GW9662;
#
P ,0.05,
##
P ,0.01, and
###
P ,0.001 vs. correspondingGW
group.
Figure 5 (A) PPARg deletion was conrmed in the PPARg knock-
out (PPARg KO) mice by Western blot analysis on the mouse
aortae. Telmisartan (Tel, 10 mmol L
21
) neither inhibited (B) the con-
tractions to U46619 in the mesenteric arteries from PPARg KO
mice, nor up-regulated (C) eNOS expression and (D) phosphoryl-
ation in the aortae of these mice. Data are from 3 to 4 experiments.
*P ,0.05 and **P ,0.01 vs. corresponding control.
C.Y. Yuen et al. 126

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vasoconstriction. The present study shows that telmisartan-induced
inhibition on U46619-induced contractions was attributed to the
increase in NO production via an augmentation of eNOS expression
and activity, evidenced by the abolishment of such inhibition by
L-NAME and ODQ, as well as by the absence of the inhibition in
arteries from eNOS KO mouse. Indeed, the potentiation of
U46619- or ET-1-induced contractions by acute treatment of
L-NAME or ODQ already suggests that basal NO can suppress vaso-
constriction, in which the role of NO is further exaggerated by telmi-
sartan treatment. The in vitro studies were supported by chronic oral
administration of telmisartan to C57BL/6J mice, from which mesen-
teric arteries exhibited less U46619-induced contractions, and were
markedly potentiated by L-NAME, indicating a signicant increase of
basal NO after telmisartan treatment. Telmisartan also enhanced
the NO-mediated endothelium-dependent relaxations in both mesen-
teric arteries and aortae resulting from the increase in ACh-stimulated
NO production.
Telmisartan-induced inhibition of contractions was signicantly
attenuated by actinomycin-D, conrming that transcriptional event
was involved. In the present study, telmisartan increased eNOS phos-
phorylation at Ser
1177
as revealed by Western blot analysis on the
mouse aortae, which exhibited similar telmisartan-induced effects
and thus acted as the surrogate for molecular studies in regards to
the very limited protein amount from the second-order mouse
mesenteric arteries. In fact, eNOS is not only regulated at its
expression level, but also its activity modied by phosphorylation
31
and post-translational mechanisms including the interaction of
eNOS with other regulatory proteins.
32,33
Increased eNOS phos-
phorylation may result from an increased eNOS expression by telmi-
sartan and the elevated expression of other eNOS-interacting
proteins. Telmisartan was reported to improve endothelial function
by augmenting the vascular level of tetrahydrobiopterin (BH
4
, an
eNOS cofactor) in aortae of Dahl salt-sensitive rats.
34
In addition, tel-
misartan up-regulates a BH
4
-synthesizing enzyme GTP cyclohydrolase
I, which reduces eNOS uncoupling in diabetic rats.
35
Polikandriotis
et al. showed that rosiglitazone elevates endothelial NO production
by increasing heat shock protein 90 (hsp90) in HUVEC.
30
while
hsp90 was identied to strengthen eNOS activities by promoting
eNOS-Ser
1177
phosphorylation.
36
These observations may explain
part of mechanisms by which telmisartan increases the eNOS activity
in vasculatures.
The critical role of PPARg in the telmisartan-induced effects in the
arteries is pinpointed by the following observations. First, the attenu-
ated U46619-induced contractions and the enhanced ACh-induced
relaxations in the telmisartan-treated arteries, augmentation of
aortic eNOS expression and phosphorylation, and elevated basal
and ACh-stimulated NO levels in HAEC were all reversed by
GW9662 (300 nmol L
21
), a PPARg antagonist applied at a much
Figure 6 Representative images (A) showing the basal (0 min) and ACh (10 mmol L
21
)-stimulated (20 min) NO levels of the HAEC under treat-
ments indicated. Telmisartan-treated cells exhibited elevated (B) basal and (C) ACh-stimulated NO levels which were prevented by GW9662
(300 nmol L
21
). Data are from ve experiments. *P , 0.05 and **P , 0.01 vs. control;
#
P ,0.05 and
##
P , 0.01 vs. Tel;

P , 0.05 vs. GW+ Tel.
Telmisartan induces and activates eNOS via PPARg 127

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lower concentration in the present study to avoid its non-specic
antagonism on PPARa or PPARd when used in high concentrations
(10 mmol L
21
) as shown in previous reports.
37,38
Second, the inhi-
bition of U46619-induced contractions by telmisartan was markedly
reduced in the mesenteric arteries from PPARg KO mice. It is
reported that interference with PPARg signalling decreases
endothelium-dependent but not endothelium-independent vasodilata-
tion in small cerebral arteries,
39
suggesting that PPARg is likely to act
on the endothelium. We conrmed that the lack of inhibitory effect of
telmisartan in the arteries of PPARg KO mice is due to the absence of
PPARg and therefore the PPARg-dependent eNOS expression and
phosphorylation were not up-regulated. Third, losartan and olmesar-
tan, two classical ARBs, did not exhibit the telmisartan-like effects.
Even irbesartan, an ARB that has been reported to slightly activate
PPARg, failed to modify the U46619-induced contractions. Benson
et al.
17
compared the PPARg activation capability of a variety of
ARBs and found that telmisartan exhibits the strongest PPARg agon-
istic action (.20-fold activation), second by irbesartan which has only
about two to three-fold slight activation, while none of the others
(candesartan, valsartan, olmesartan, eprosartan, and EXP 3174, the
active metabolite of losartan) can activate PPARg. Collectively, the
unique PPARg-stimulating activity of telmisartan is essential in mediat-
ing the anti-contractile effects in the arteries, possibly independent of
AT
1
R antagonism. The exceptional lipophilic nature of telmisartan
allows it to behave as a PPARg agonist beyond its ARB activity.
22
In
fact, telmisartan has the highest lipophilicity index (+3.20) whereas
EXP 3174, an active metabolite of losartan, has the lowest (22.45)
of the ARBs.
40
Telmisartan has a high volume of distribution com-
pared with other ARBs as revealed by a test determining the ability
of telmisartan to penetrate into tissues.
41
It is therefore thought
that telmisartan possesses a greater capacity to penetrate cell mem-
branes and thereby interacts with the nuclear PPARg. More impor-
tantly, molecular modelling reveals that telmisartan binds to helices
H3, H6, and H7 of the key PPARg ligand-binding domain but other
ARBs can only bind to helix H3.
17
Such difference may account for
the substantially different potentials for activation of PPARg
between telmisartan and other ARBs. Telmisartan increased PPARg
expression as its full agonist rosiglitazone did, suggesting a novel posi-
tive feedback mechanism.
In conclusion, besides acting as an ARB, telmisartan uniquely and
robustly activates PPARg. The present study demonstrates that telmi-
sartan inhibits vasoconstriction through increased NO production
arising from the elevated eNOS expression and phosphorylation at
Ser
1177
via a PPARg-dependent mechanism. Compared with other
ARBs, telmisartan, with the dual ARB/PPARg-agonistic properties,
shall be a more promising drug with higher therapeutic value
against vascular disorders associated with hypertension and type 2
diabetes.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Conict of interest: none declared.
Funding
This study was supported by Hong Kong Research Grant Council
(CUHK465308 and 466110), and CUHK Focused Investment Scheme.
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