yuen et al 2011 cardiovascres2011april

8/12/2019 Yuen Et Al 2011 CardiovascRes2011April

1/8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Telmisartan inhibits vasoconstriction via PPARg-

dependent expression and activation of endothelialnitric oxide synthase

Chi Yung Yuen1, Wing Tak Wong1, Xiao Yu Tian1, Siu Ling Wong1*, Chi Wai Lau1,

Jun Yu2, Brian Tomlinson2, Xiaoqiang Yao1, and Yu Huang1*

1Institute 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 2Department

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 (AT1R) 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

F2a(U46619)- or endothelin-1-induced contractions. An NOS inhibitor, NG-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 1R 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 (AT1R) 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, AT1R 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 AT1R.

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-inflammatory responses.8

* Corresponding author. Tel: +852 2609 6787; fax: +852 2603 5022, Email: [email protected] (Y.H.) and Email: [email protected] (S.L.W.)Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: [email protected].

Cardiovascular Research (2011) 90, 122129

doi:10.1093/cvr/cvq392

p

,

j

g
http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/


2/8

Mutations of PPARg in humans contribute to the pathogenesis of

insulin resistance, diabetes, and hypertension.9 PPARgagonists 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 benefit 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 PPARgagonists are associated with deleterious

side effects, such as congestive heart failure,13,14 exacerbation of fluid

retention, causing oedema and weight gain15,16 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 PPARgagonist.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 nature22

and its ability to bind tothe 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 PPARgproperty 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 AnimalsThe investigation conformed to the Guide for the Care and Use of Laboratory

Animalspublished 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 preparationMice were sacrificed by CO2inhalation. Second-order mesenteric arteriesand 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 arteriesPre-warmed Dulbeccos modified Eagle medium supplemented with 10%

FBS and 100 U mL21 penicillin plus 100 mg mL21 streptomycin was added

into the wells of the 24-well plate. Different concentrations of telmisartan

(0.110mmol L21) or other ARBs (losartan, olmesartan, and irbesartan;

all used at 10mmol L21) were added into the wells. GW9662 (PPARg

antagonist, 300 nmol L21

) was added in addition to 10mmol L21

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 measurementTension changes of the cultured mesenteric arteries and aortae were

recorded as previously described.23 Briefly, 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% O25% CO2 and maintained at 378C. After 30 minequilibrium, rings were first contracted by 30 nmol L21 U46619, and

then relaxed by 3mmol L21 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 (1300mmol L21) or endothelin-1 (ET-1,

0.330 nmol L21). Results were compared between control and 30 min

incubation of L-NAME (100mmol L21) or 1H-[1,2,4]oxadiazolo

[4,3-a]quinoxalin-1-one (ODQ) (5mmol L21). To examine the changes

in NO-mediated endothelium-dependent relaxations, the arteries were

pre-contracted with 3mmol L21 phenylephrine and relaxed by cumulative

additions of ACh (3 nmol L21 1mmol L21) in the presence of

20 mmol L21 KCl which eliminated the effect from endothelium-derived

hyperpolarizing factors.

2.5 Western blottingWestern 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,

1mg mL21; aprotonin,5 mg mL21; PMSF,100mg mL21; sodiumorthovana-

date, 1 mmol L21; ethylene glycol tetraacetic acid, 1 mmol L21; EDTA,

1 mmol L21; NaF, 1 mmol L21; and b-glycerolphosphate, 2 mg mL21).

The lysates were incubated on ice for 30 min and then centrifuged at

20 000g for 20 min. The supernatant was collected and analysed for

protein concentration using the Lowry method (Bio-Rad, USA). For each

sample, 25mg of the total protein was electrophoresed under reducing

conditions through a SDSpolyacrylamide gel. The resolved proteins

were electroblotted on an immobilon-P polyvinylidene difluoride 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 phosphatebuffered saline (PBST) for 60 min beforean 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 films. The signal intensities were quantified using a gel documentationsystem (GBOX-CHEM1-HR16, SynGene) and normalized with the house-

keeping protein glyceraldehyde 3-phosphate dehydrogenase.

2.6 Real-time measurement on NO productionin endothelial cellsHuman 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-

fluence upon which they were plated on coverslips. Cells were incubated

with telmisartan (10mmol L21) for 24 h with or without 30 min pre-

treatment of GW9662 (300 nmol L21) and then imaged as described

earlier.25 Briefly, cells after 24 h incubation were rinsed in NPSS

(140 mmol L21

NaCl, 5 mmol L21

KCl, 1 mmol L21

CaCl2, 1 mmol L21

MgCl2, 10 mmol L21 glucose, and 5 mmol L21 HEPES, pH 7.4) and

Telmisartan induces and activates eNOS via PPARg 123

p

,

j

g


3/8

incubated with 1mmol L21 DAF-FM diacetate (4-amino-5-methylamino-

2 ,7-difluorescein, 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 theinitial reading and real-time changes in ACh (10mmol L21)-stimulated

NO production were recorded and presented as a ratio of fluorescence

relative to the initial intensity (time 0 min, before ACh addition).

2.7 Chronic treatment with telmisartanC57BL/6J mice were orally treated with either telmisartan

(1 mg kg21 day21) or water for 14 days, after which mesenteric arteries

were dissected for isometric force measurement.

2.8 ChemicalsActinomycin-D, ODQ and NG-nitro-L-arginine methyl ester (L-NAME)

were purchased from Tocris (Avonmouth, UK). 9,11-Dideoxy-

11a,9a-epoxymethanoprostaglandin F2a (U46619), Ach, and GW9662were purchased from Sigma-Aldrich (St Louis, MO, USA). Rosiglitazone

was purchased from GlaxoSmithKline and olmesartan from Pfizer. 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 analysisData 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. pD2 is the negative logarithm ofthe vasoconstrictor concentration needed to produce half of the

maximal contraction as determined by non-linear regression curve

fitting (Graphpad Prism Software, version 4.0). P , 0.05 was considered

statistically significant.

3. Results

3.1 Telmisartan inhibits U46619-inducedcontractionsTelmisartan concentration-dependently inhibited U46619-induced

contractions in the mesenteric arteries from C57BL/6J mice. Telmisar-

tan at 10mmol L21

attenuated the contractions (pD2: 7.89+0.04 forcontrol and 7.40+0.07 for telmisartan 10mmol L21, P, 0.001,

Figure 1A, Supplementary material online, Figure 1A and Table 1). In

contrast, losartan, olmesartan, and irbesartan (all used at

10mmol L21) 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 findings 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 oncontractions is mediated by NOL-NAME (100mmol L21) abolished the inhibitory effect of telmisartan

(10mmol L21) on U46619-induced contractions (Figure 1B, Sup-

plementary material online, Figure 1B and Table 1). ODQ

(5mmol L21) also prevented telmisartan-induced effect (Supplemen-

tary material online,Figure 2BandTable1). In addition to U46619, tel-

misartan also suppressed contractions induced by endothelin-1 and

this effect was abrogated by L-NAME (Figure1C, 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 andeven potentiated U46619-induced contractions (Supplementary

material online, Figure 5).

3.3 Telmisartan up-regulates eNOSexpression and phosphorylationTwenty four hour incubation of telmisartan concentration-

dependently up-regulated the eNOS expression (Figure 2A) and its

phosphorylation at Ser1177 (Figure 2B). The positive role of eNOS

was further verified 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 (10mmol L21) 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 (0.110mmol L21) concentration-dependently attenuated U46619-induced contractions. Inhibitory effect of telmisartan

(Tel, 10mmol L21) to (B) U46619- and (C) endothelin-1-induced contractions was abolished in the presence ofL-NAME (100mmol L21). 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

p

,

j

g
http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1


4/8

contractions was prevented by an RNA synthesis inhibitor,

actinomycin-D (10mmol L21) (Figure3A and Table1).

3.4 Telmisartan elevates PPARgexpression via PPARg activity

Telmisartan concentration-dependently increased the PPARgexpression (Figure 3B). Telmisartan (10mmol L21)-induced

up-regulation of PPARgexpression was prevented by a PPARgantag-

onist GW9662 (300 nmol L21). Likewise, rosiglitazone, a PPARg

agonist, also increased the GW9662-sensitve PPARg expression

(Figure 3C). In contrast, losartan (10mmol L21) did not affect the

expression of PPARg (Figure3C).

3.5 Telmisartan-induced eNOS expressionand activation are PPARg-dependentGW9662 (300 nmol L21) 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; Table1). Likewise, rosiglitazone

(1mmol L21) attenuated U46619-induced contractions, and this

attenuation was reversed by GW9662 (Supplementary material

online, Figure 2C) and prevented by actinomycin-D (Supplementarymaterial online, Figure 2D).

Telmisartan (10mmol L21) and rosiglitazone (1mmol L21)-induced

increases in eNOS expression (Figure 4C) and phosphorylation

(Figure 4D) were prevented by GW9662 (300mmol L21). On the

contrary, losartan did not increase eNOS expression and only

caused a slight elevation in eNOS phosphorylation, which was insen-

sitive to GW9662 (Figure4Cand D).

PPARg was not expressed in the PPARg KO mice (Figure5A). In the

mesenteric arteries of PPARgKO mice, the inhibitory effects of telmi-

sartan (10mmol L21) on U46619-induced contractions were dimin-

ished compared with those in C57BL/6J control mice (Figure 5B).

Telmisartan did not augment eNOS expression (Figure5C) and phos-phorylation (Figure5D) in the aortae of PPARg KO mice.

3.6 Telmisartan increases intracellular NOlevels in cultured endothelial cellsHAEC 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, Figure6B). 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 acuteL-NAME treatment inhibited NO production (Figure6A and C).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 1 pD2and Emax(%) of telmisartan-induced

inhibition on contractions

Treatment pD2 Emax(%) n

C57BL/6J arteries

U46619 (Control) 7.89+0.04 137.09+3.93 6

Tel 0.1mmol L21 7.91+0.04 124.46+0.85 4

Tel 1mmol L21 7.72+0.10 123.57+4.98 4

Tel 10mmol L21 7.40+0.07*** 113.88+5.17** 6

Control 7.87+0.04 138.03+4.67 6

Tel 10mmol L21 7.38+0.08*** 113.09+6.02** 6

Control + L-NAME

100 mmol L218.05+0.05 154.53+4.84 5

Tel + L-NAME 7.99+0.05### 142.22+4.43## 6

Control + ODQ

5mmol L217.95+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 10mmol L21 7.39+0.08*** 113.72+6.30** 6

Tel + Act 10mmol L21 7.68+0.07# 137.59+5.99# 5

Control 7.87+0.05 137.32+3.23 6

Tel 10mmol L21 7.38+0.07*** 113.73+5.76** 6

GW 300 nmol L21 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 10mmol L21 8.29+0.02 197.14+6.21 3

Control + L-NAME

100 mmol L218.41+0.03 214.87+9.68 3

Tel + L-NAME 8.31+0.03 204.42+4.63 3

PPARgKO mouse arteries

U46619 (Control) 8.34+0.07 163.46+11.06 4

Tel 10mmol L2

1 8.31+0.04 139.76+5.06 4

Significant 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 ofn experiments.

Figure 2 Western blot showing telmisartan (0.1 10mmol L21)

concentration-dependently up-regulated (A) eNOS expression and(B) eNOS phosphorylation at Ser1177. (C) Absence of eNOS

expression in the aortae of eNOS knockout (eNOS KO) mice com-

pared with the wild-type (WT) mice. (D) Concentration response

curves for U46619 of telmisartan (Tel, 10 mmol L21)-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.


p

,

j

g
http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1


5/8

4. Discussion

The present study demonstrated for the first 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-Ser1177 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 andactivity in a PPARg-dependent manner, thereby inhibiting

Figure 3 (A) The inhibitory effect of telmisartan (Tel, 10 mmol L21) was abolished by actinomycin-D (10mmol L21). (B) Telmisartan (0.1

10mmol L21) concentration-dependently increased PPARg expression. (C) Telmisartan (Tel, 10mmol L21) and rosiglitazone (Rosi, 1mmol L21)

increased PPARg expression, which was inhibited by GW9662 (GW, 300 nmol L21). 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 L21) on telmisartan (Tel, 10mmol L21)-induced

inhibitions on contractions to U46619. (B) GW9662 (GW,

300 nmol L21) abolished the inhibitory effect of telmisartan (Tel,

10mmol L21) to U46619-induced contractions. (C) Telmisartan

(Tel, 10mmol L21) and rosiglitazone (Rosi, 1mmol L21) elevated

the (C) eNOS expression and (D) the phosphorylation at Ser1177,

which were prevented by GW9662 (GW, 300 nmol L21). (D) Losar-

tan (10mmol L21) 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) PPARgdeletion was confirmed in the PPARgknock-

out (PPARg KO) mice by Western blot analysis on the mouse

aortae. Telmisartan (Tel, 10 mmol L21) 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

p

,

j

g


6/8

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 significant 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 significantly

attenuated by actinomycin-D, confirming that transcriptional event

was involved. In the present study, telmisartan increased eNOS phos-

phorylation at Ser1177 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 tothe 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 modified by phosphorylation31

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 (BH4, an

eNOS cofactor) in aortae of Dahl salt-sensitive rats.34 In addition, tel-

misartan up-regulates a BH4-synthesizing enzyme GTP cyclohydrolase

I, which reduces eNOS uncoupling in diabetic rats.35 Polikandriotiset al. showed that rosiglitazone elevates endothelial NO production

by increasing heat shock protein 90 (hsp90) in HUVEC.30 while

hsp90 was identified to strengthen eNOS activities by promoting

eNOS-Ser1177 phosphorylation.36 These observations may explain

part of mechanisms by which telmisartan increases the eNOS activity

in vasculatures.

The critical role of PPARgin 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 byGW9662 (300 nmol L21), a PPARg antagonist applied at a much

Figure 6 Representative images (A) showing the basal (0 min) and ACh (10mmol L21)-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 L21). Data are from five 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.


p

,

j

g


7/8

lower concentration in the present study to avoid its non-specific

antagonism on PPARa or PPARd when used in high concentrations

(10mmol L21) 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 PPARgis likely to act

on the endothelium. We confirmed that the lack of inhibitory effect of

telmisartan in the arteries of PPARgKO 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, theunique PPARg-stimulating activity of telmisartan is essential in mediat-

ing the anti-contractile effects in the arteries, possibly independent of

AT1R 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

Ser1177 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 valueagainst vascular disorders associated with hypertension and type 2

diabetes.

Supplementary material

Supplementary material is available at Cardiovascular Researchonline.

Conflict of interest: none declared.

Funding

This study was supported by Hong Kong Research Grant Council(CUHK465308 and 466110), and CUHK Focused Investment Scheme.

References1. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of

oxidant stress. Circ Res 2000;87:840844.

2. Forstermann U. Oxidative stress in vascular disease: causes, defense mechanisms and

potential therapies. Nat Clin Pract Cardiovasc Med2008;5:338349.

3. Fearon IM, Faux SP. Oxidative stress and cardiovascular disease: novel tools give

(free) radical insight. J Mol Cell Cardiol 2009;47:372381.

4. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ et al.

Differential expression and activation of a family of murine peroxisome proliferator-

activated receptors. Proc Natl Acad Sci U S A 1994;91:73557359.

5. Willson TM, Lambert MH, Kliewer SA. Peroxisome proliferator-activated receptor

gamma and metabolic disease. Annu Rev Biochem 2001;70:341367.

6. Rosen ED, Spiegelman BM. PPARgamma: a nuclear regulator of metabolism, differen-

tiation, and cell growth. J Biol Chem 2001;276:3773137734.

7. Duan SZ, Usher MG, Mortensen RM. Peroxisome proliferator-activated

receptor-gamma-mediated effects in the vasculature. Circ Res 2008;102:283294.

8. Chinetti G, Fruchart JC, Staels B. Peroxisome proliferator-activated receptors

(PPARs): nuclear receptors at the crossroads between lipid metabolism and inflam-

mation. Inflamm Res 2000;49:497505.

9. Barroso I, Gurnell M, Crowley VE, Agostini M, Schwabe JW, Soos MAet al. Dominant

negative mutations in human PPARgamma associated with severe insulin resistance,

diabetes mellitus and hypertension. Nature 1999;402:880883.

10. Gervois P, Fruchart JC, Staels B. Drug Insight: mechanisms of action and therapeutic

applications for agonists of peroxisome proliferator-activated receptors.Nat Clin Pract

Endocrinol Metab 2007;3:145156.

11. Hwang J, Kleinhenz DJ, Rupnow HL, Campbell AG, Thule PM, Sutliff RLet al. The

PPARgamma ligand, rosiglitazone, reduces vascular oxidative stress and NADPHoxidase expression in diabetic mice. Vascul Pharmacol 2007;46:456462.

12. Huang PH, Sata M, Nishimatsu H, Sumi M, Hirata Y, Nagai R. Pioglitazone ameliorates

endothelial dysfunction and restores ischemia-induced angiogenesis in diabetic mice.

Biomed Pharmacother2008;62:4652.

13. Home PD, Pocock SJ, Beck-Nielsen H, Gomis R, Hanefeld M, Jones NPet al. Rosigli-

tazone evaluated for cardiovascular outcomes an interim analysis. N Engl J Med2007;

357:2838.

14. Home PD, Pocock SJ, Beck-Nielsen H, Curtis PS, Gomis R, Hanefeld Met al. Rosigli-

tazone evaluated for cardiovascular outco mes in oral agent combination therapy for

type 2 diabetes (RECORD): a multicentre, randomised, open-label trial.Lancet2009;

373:21252135.

15. Cekmen N, Cesur M, Cetinbas R, Bedel P, Erdemli O. Acute pulmonary edema due to

rosiglitazone use in a patient with diabetes mellitus. J Intensive Care Med 2006;21:

4750.

16. Sotiropoulos KB, Clermont A, Yasuda Y, Rask-Madsen C, Mastumoto M, Takahashi J

et al. Adipose-specific effect of rosiglitazone on vascular permeability and proteinkinase C activation: novel mechanism for PPARgamma agonists effects on edema

and weight gain. FASEB J 2006;20:12031205.

17. Benson SC, Pershadsingh HA, Ho CI, Chittiboyina A, Desai P, Pravenec Met al. Identi-

fication of telmisartan as a unique angiotensin II receptor antagonist with selective

PPARgamma-modulating activity. Hypertension 2004;43:9931002.

18. Schupp M, Janke J, Clasen R, Unger T, Kintscher U. Angiotensin type 1 receptor

blockers induce peroxisome proliferator-activated receptor-gamma activity. Circula-

tion 2004;109:20542057.

19. Michel MC, Bohner H, Koster J, Schafers R, Heemann U. Safety of telmisartan in

patients with arterial hypertension : an open-label observational study. Drug Saf

2004;27:335344.

20. Schumacher H, Mancia G. The safety profile of telmisartan as monotherapy or com-

bined with hydrochlorothiazide: a retrospective analysis of 50 studies. Blood Press

Suppl 2008;1:3240.

21. Xi GL, Cheng JW, Lu GC. Meta-analysis of randomized controlled trials comparing

telmisartan with losartan in the treatment of patients with hyp ertension.Am J Hyper-

tens 2008;21:546552.

22. Takai S, Kirimura K, Jin D, Muramatsu M, Yoshikawa K, Mino Yet al. Significance of

angiotensin II receptor blocker lipophilicities and their protective effect against vascu-

lar remodeling. Hypertens Res 2005;28:593600.

23. Wong SL, Leung FP, Lau CW, Au CL, Yung LM, Yao X et al. Cyclooxygenase-2-

derived prostaglandin F2alpha mediates endothelium-dependent contractions in the

aortae of hamsters with increased impact during aging. Circ Res 2009;104:228235.

24. Wong WT, Tian XY, Xu A, Ng CF, Lee HK, Chen ZY et al. Angiotensin II type 1

receptor-dependent oxidative stress mediates endothelial dysfunction in type 2 dia-

betic mice. Antioxid Redox Signal 2010;13:757768.

25. Liu CQ, Leung FP, Wong SL, Wong WT, Lau CW, Lu L et al. Thromboxane prosta-

noid receptor activation impairs endothelial nitric oxide-dependent vasorelaxations:

the role of Rho kinase. Biochem Pharmacol 2009;78:374381.

26. Ishizuka T, Niwa A, Tabuchi M, Nagatani Y, Ooshima K, Higashino H. Involvement of

thromboxane A2 receptor in the cerebrovascular damage of salt-loaded, stroke-

prone rats. J Hypertens 2007;25:861870.

27. Michel F, Simonet S, Vayssettes-Courchay C, Bertin F, Sansilvestri-Morel P,Bernhardt F et al. Altered TP receptor function in isolated, perfused kidneys of

C.Y. Yuen et al.128

p

,

j

g
http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/http://cardiovascres.oxfordjournals.org/cgi/content/full/cvq392/DC1


8/8

nondiabetic and diabetic ApoE-deficient mice. Am J Physiol Renal Physiol 2008;294:

F120129.

28. Yasuda S, Kobayashi H, Iwasa M, Kawamura I, Sumi S, Narentuoya Bet al. Antidiabetic

drug pioglitazone protects the heart via activation of PPAR-gamma receptors,

PI3-kinase, Akt, and eNOS pathway in a rabbit model of myocardial infarction. Am

J Physiol Heart Circ Physiol 2009;296:H15581565.

29. Cho DH, Choi YJ, Jo SA, Jo I. Nitric oxide production and regulation of endothelial

nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evi-

dence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-

dependent and PPARgamma-independent signaling pathways. J Biol Chem 2004;279:

24992506.30. Polikandriotis JA, Mazzella LJ, Rupnow HL, Hart CM. Peroxisome proliferator-

activated receptor gamma ligands stimulate endothelial nitric oxide production

through distinct peroxisome proliferator-activated receptor gamma-dependent

mechanisms. Arterioscler Thromb Vasc Biol 2005;25:18101816.

31. Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJet al. Reciprocal phosphoryl-

ation and regulation of endothelial nitric-oxide synthase in response to bradykinin

stimulation. J Biol Chem 2001;276:1658716591.

32. Garcia-Cardena G, Martasek P, Masters BS, Skidd PM, Couet J, Li S et al. Dissecting

the interaction between nitric oxide synthase (NOS) and caveolin. Functional signifi-

cance of the nos caveolin binding domain in vivo. J Biol Chem 1997;272:2543725440.

33. Kone BC. Protein-protein interactions controlling nitric oxide synthases.Acta Physiol

Scand2000;168:2731.

34. Satoh M, Haruna Y, Fujimoto S, Sasaki T, Kashihara N. Telmisartan improves endo-

thelial dysfunction and renal autoregulation in Dahl salt-sensitive rats.Hypertens Res

2010;33:135142.

35. Wenzel P, Schulz E, Oelze M, Muller J, Schuhmacher S, Alhamdani MS et al.

AT1-receptor blockade by telmisartan upregulates GTP-cyclohydrolase I and pro-

tects eNOS in diabetic rats.Free Radic Biol Med2008;45:619626.

36. Fontana J, Fulton D, Chen Y, Fairchild TA, McCabe TJ, Fujita Net al. Domain mapping

studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate

Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release.

Circ Res 2002;90:866873.

37. Scalera F, Martens-Lobenhoffer J, Bukowska A, Lendeckel U, Tager M,

Bode-Boger SM. Effect of telmisartan on nitric oxideasymmetrical dimethylarginine

system: role of angiotensin II type 1 receptor gamma and peroxisome proliferator

activated receptor gamma signaling during endothelial aging. Hypertension 2008;51:696703.

38. Tian Q, Miyazaki R, Ichiki T, Imayama I, Inanaga K, Ohtsubo Het al. Inhibition of tumor

necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-

talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB

and CCAAT/enhancer-binding protein-beta. Hypertension 2009;53:798804.

39. Beyer AM, Baumbach GL, Halabi CM, Modrick ML, Lynch CM, Gerhold TD et al.

Interference with PPARgamma signaling causes cerebral vascular dysfunction, hyper-

trophy, and remodeling. Hypertension 2008;51:867871.

40. Wienen W, Entzeroth M, Meel JCA, Stangier J, Busch U, Ebner T et al. A Review on

Telmisartan: A Novel, Long-Acting Angiotensin II-Receptor Antagonist. Cardiovascular

Drug Reviews 2000;18:127154.

41. Kurtz TW, Pravenec M. Antidiabetic mechanisms of angiotensin-converting enzyme

inhibitors and angiotensin II receptor antagonists: beyond the renin-angiotensin

system. J Hypertens 2004;22:22532261.


p

,

j

g

yuen et al 2011 cardiovascres2011april

Documents